green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level
TRANSCRIPT
APPLICABILITY OF LIGHT EMITTING DIODE
IRRADIATION IN PHYSIOTHERAPY
Elke Vinck
Thesis submitted in fulfilment of the requirements for the degree of Doctor in Motor Rehabilitation and Physiotherapy
Promotor prof dr D Cambier
Department of Rehabilitation Sciences and Physiotherapy Faculty of Medicine and Health Sciences
Ghent University Academic Year 2005-2006
Promotor
Prof dr D Cambier Ghent University Belgium
Examination Board
Prof dr P Calders Artevelde University College Belgium
Prof dr D Cambier Ghent University Belgium
Prof dr M Cornelissen Ghent University Belgium
Prof dr M De Muynck Ghent University Belgium
Prof dr M Dyson University of London UK
Prof dr P Lievens Free University Brussels Belgium
Prof dr K Peers Catholic University Leuven Belgium
Prof dr G Vanderstraeten Ghent University Belgium
Process Supervisory Board
Prof dr D Cambier Ghent University Belgium
Prof dr M Cornelissen Ghent University Belgium
Prof dr M De Muynck Ghent University Belgium
Prof dr G Vanderstraeten Ghent University Belgium
VII
TABLE OF CONTENTS
GENERAL INTRODUCTION 1
Background 3
Physical characteristics 6
Mechanisms of action 12
Aims and outline 15
PART I WOUND HEALING 25
Chapter 1 Do infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment
27
Chapter 2 Increased fibroblast proliferation induced by light emitting diode and low level laser irradiation
47
Chapter 3 Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level
61
PART II ANALGESIA 73
Chapter 4 Evidence of changes in sural nerve conduction mediated by light emitting diode irradiation
75
Chapter 5 Pain reduction by infrared light emitting diode irradiation a pilot study on experimentally induced delayed-onset muscle soreness in humans
91
GENERAL DISCUSSION 111
Summary 113
Clinical implications and future research directions 118
Final conclusion 123
NEDERLANDSTALIGE SAMENVATTING 129
Do not attempt to do a thing unless you are sure of yourself
but do not relinquish it simply because someone else is not sure of you
(Stewart E White)
IX
ACKNOWLEDGEMENTS
I wish to thank those people who supported me over the years and who helped me to
shape my life and work
First of all I would like to express my gratitude towards my promotor prof dr D
Cambier as without his encouraging words criticism inspiration and unremitting
support I would still be floundering about the contents of chapter 1 Thank you for
your good advice when I needed it the most
The members of the supervisory committee prof dr M Cornelissen prof dr M De
Muynck and prof dr G Vanderstraeten thank you for your assistance and helpful
feedback during the process of formation of this thesis
I also gratefully acknowledge the external members of the examination board prof dr
P Calders prof dr M Dyson prof dr P Lievens and prof dr K Peers for their
constructive reflections which contributed to the improvement of this thesis
I am greatly indebted to my special mentor prof dr J Anders of the Uniformed
Services University of Bethesda Maryland for the research suggestions she made as
well as for her unlimited belief in the value of my work
I wish to thank prof dr L Deridder for providing access to the laboratory of
Histology the Centre of Sports Medicine of the Ghent University Hospital for
allowing me to use their equipment as well as MDB-Laser Belgium for generously
providing the light emitting diode equipment
Sincere appreciation is extended to the volunteers that participated in this study and to
Tom and Roel for their valuable technical assistance in the collection of the data as
well as for their useful input into the research design of the investigation described in
chapter 5
X
Warm thanks go to the colleagues of the department of Human Anatomy
Embryology Histology and Medical Physics for providing the culture medium for the
technical support for the helpful discussions and principally for the amusing pastime
aseptic chats
In addition I also want to thank my colleagues of the associated institute Kinesitherapie
Gent and above all the colleagues of the department of Rehabilitation Sciences and
Physiotherapy Ann Axel Barbara Bart Bert Bihiyga Bruno Carine Damien Els2
Erik Frike Helga Inge James Karlien Kathy Kat(h)leen Koen Marc Martine
Mieke Paris (Nele) Patrick Roland Sophie Stefan Stijn (Veerle) Wiene Wim and
Youri thank you for the organisation and your attendance at many memorable
sidelines such as the survival-weekend the first department-day Fata Revaki our
legendary Thursday-sport activities and Friday-afternoon coffee breaks St Nicholas
visit skiing holiday New Yearrsquos lunch with quiz bowling spinning the introduction of
ldquosubetelyrdquo during our conspicuous presence at WCPT 2003 et cetera We shared many
treasured moments thanks to you a common working day often turned out to be very
pleasant I know that it will be impossible to find a comparable team of colleagues to
work with in the future
I especially want to thank Barbara to remind me on a regularly basis of my deadlines
to listen to my grieves and joy and to be willing to offer me a window-seat in our
office Kurt (although you abandoned at a certain moment) for solving my computer
problems Pascal for assistance with the statistical analyses Lieven for your motivating
interest and finally Fabienne Tine and Kim as loyal and appreciated friends who
worn-out several sports shoesbathing suits to supply in the weekly portion of sports I
needed to remain physically and mentally fit
I also extend my appreciation to my family and friends for their interest in my research
activities permanent mental support for the adoption of the surviving chickens but in
particular for looking after Louka and for the numerous relaxing moments Special
thanks are going to Katelijne and Frank for the delicious hot meals on lonely evenings
XI
Thomas Stijn and Johan thank you for the repeated (mostly futile) attempts to
convince me to do something together Sebastiaan each time during the past few years
when I doubted about the sense of my work it was your ridiculous story about a man
who wanted to invent superglue but instead invented the well-known yellow post-it
which stimulated me to continue my scientific quest
Of course I owe most gratitude to Luc my most devoted supporter Dearest I know
that since august 2004 you lived a solitary life in Dubai Although I think it was
possibly easier not to live under the same roof with me these last stressful months I
am aware that it was very difficult for you not to be able to play with Louka and to
miss some precious months of her life
Louka thank you for your radiant smile and daily baby speeches I am sorry that you
had to miss your daddy I promise that we will be reunited very soon
Elke Vinck
Ghent March 2006
GENERAL INTRODUCTION
General introduction
3
BACKGROUND
The use of light for therapeutic purposes reaches far back in time Current interest for
photomedicine with his its biological and medical effects relies fundamentally on two
major evolutions in the given field (1) the research results regarding the use of
ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in
the light amplification by stimulated emission of radiation (laser)-technology The production
of the first laser the ruby pulsed laser was rapidly succeeded by the development of
the helium-neon laser and other lasers like the argon the neodymium-glass and the
neodymium-yttrium-aluminium-garnet lasers1
As in the mid-1990s semiconductor and diode-based lasers gained popularity the
principally massive gas and dye lasers were rendered obsolete Therapeutic light
technology further continued to evolve and todayrsquos therapeutic light source is as likely
to be a light emitting diode (LED) or polarized light as a semiconductor or diode
laser1
Technological advancement and variation of the light sources necessitate a
concomitant update and revision of research in the respective domains of application
Unfortunately this logical and rational necessity has rarely been fulfilled From a
historical perspective this lack of appropriate research has led to disenchanting
evolutions in the use of light especially in physiotherapy The experience exists in this
medical field that light sources were promoted and commercialised for a vast regimen
of indications without foregoing scientific backup Consequently research developed
often after the commercial introduction in physiotherapy As these investigations
frequently gave rise to conflicting results for certain indications scepticism arose and
the use of the given modality knew a waning popularity for all its indications The final
result of such an inappropriate frame of promotion commercialisation and research is
a growing clinical disuse of a given modality even for motivated indications In view of
the actual increasing interest in LED treatment and based on former ascertainment
one has to state that a literature review for the given source reveals that research
mostly covers only low level laser (LLL) studies23 Although recently a number of
papers can be noted that report on the effects of LEDs and polarized light still
4
numerous source-specific-questions need to be answered as research concerning
mechanisms of action and efficacy of the current light sources remains limited in view
of a substantiated clinical application4-17
The reason for the contemporary light-oriented interest in physiotherapeutic practice
for LED devices is in essence based on several advantages of LED in comparison with
LLL For example the use of LEDs is esteemed to be safer as the delivered power
does not damage tissue LEDs can be made to produce multiple wavelengths thereby
stimulating outright a broader range of tissue types and probes that cover a large
treatment area are available18 In addition from a commercial point of view LEDs are
far more interesting as they are a good deal cheaper than laser diodes and they have a
long life span as these solid devices stand robust handling
As a result of the above-mentioned lack of literature on LED some providers of these
devices have taken for granted that the biological response of tissue to light irradiation
cannot be equated merely to a light source They declare that a given response solely
depends on the extent of absorption of radiated light by the tissue19 Consequently
these providers state that it is acceptable to extrapolate scientific findings of LLL
studies for explaining the mechanisms of action and detailing the efficacy of LED and
other alternative light sources Thus actually without appropriate scientific support
equal biological effects are attributed to LED as to LLL Nevertheless prudence is
called for such an extrapolation firstly because it is irrespective of the mentioned
dissimilarities and by simple projection one ignores a number of physical differences
between LLL and LED (eg coherence and degree of collimation or divergence)
Secondly LLL therapy is still not yet an established and evidence-based clinical tool20
Notwithstanding the historical efforts there still remains a considerable amount of
ignorance scepticism and controversy concerning the use and clinical efficacy of
LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed
parameters for irradiation as well as to the difficult objective measurement of possible
irradiation effects and even to the exceptional range of unsubstantiated indications for
General introduction
5
which light therapy was promoted27-29 A lack of theoretical understanding can also be
responsible for the existing controversies as the evaluation and interpretation of
research results would be simplified largely when the appropriate knowledge about the
mechanisms of light action would be available
LLL literature can undoubtedly be used as basis for research on LED and as a
comparative reference for these given investigations However to guarantee evidence-
based use of LED within physiotherapy the need for specific research in view of an
accurate consumption of LED is definite especially for potential promising clinical
applications in physiotherapy according to LLL literature mainly wound healing and
analgesia3031
Hitherto the most substantial research concerning the use of LED for improvement
of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded
under the influence of prolonged exposure to microgravity (eg during long-term space
flights) and in case of absence of exposure to sunlight such as in submarine
atmospheres they performed wound healing experiments for military application in the
given circumstances3233 In vitro experiments revealed that LED treatment increased
proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA
synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and
osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies
demonstrated therapeutic benefits of LED in speeding the early phase of wound
closure and in changing gene expression in a type 2 diabetic mouse model183234
Human studies noted 50 faster healing of lacerations a return of sensation and
increased tissue granulation as a result of LED irradiation1833
Associates of the Rehabilitation Sciences Research Group of the Ulster University in
Northern Ireland extensively investigated the effectiveness of light in the treatment of
pain The emphasis was laid primarily on the analysis of the effects of various low level
laser light sources35-44 However in the year 2001 two studies gave an account on the
efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46
6
noticed relatively long-lasting neurophysiological effects a significant change of the
nerve conduction characteristics (decrease of the negative peak latency) was mediated
by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a
comparable multisource diode device was ineffective in the management of delayed-
onset of muscle soreness (DOMS)
Despite the major value of these described trials a definitive answer regarding the
ability of LED in influencing wound healing or pain is not forthcoming cardinally
because a number of aspects are not yet investigated Consequently more research is
required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the
formerly mentioned scepticism regarding the effectiveness of a medium and possibly
to the undeserved fall into disuse of the treatment modality which happened in a way
with LLL therapy
PHYSICAL CHARACTERISTICS
This chapter supplies a short but comprehensive review of opto-physics A brief
description of the physical characteristics of the LED source used is essential as the
physical properties of light play an important part in the ultimate efficacy of treatment
According to the International Electrotechnical Commission (IEC 60825-1) an LED
can be defined as
Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by
radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process
of spontaneous emission1947
The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO
preprototype MDB-Laser Belgium) This illustration shows that a probe consists of
32 single LEDs disseminated over a surface of 18 cm2
General introduction
7
Figure 1 LED device and three available probes (infrared red and green)
Three highly monochromatic probes were available each emitting light of a different
wavelength within the above-defined range (table 1)2748 The wavelength of the light
emitted and thus its colour depends on the band gap energy of the materials forming
the p-n junctiona This light property is a key determinant to obtain maximum
photochemical or biological responses as light absorption by tissue molecules is
wavelength specific27 Only by absorbing radiation of the appropriate wavelength
(namely the wavelengths equal to the energy states of the valence electrons)
photoacceptor molecules will be stimulated resulting in a direct photochemical
reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750
8
absorption spectrum of a particular molecule is limited absorption often only occurs
over a waveband range of about 40-60 nm274851 Nevertheless the absorption
spectrum at cell or tissue level is broad because cells are composed of many different
molecules
Besides its influence on the absorption by means of tissue molecules there is a crucial
link between wavelength and penetration depth of the irradiated light Penetration into
tissue decreases as the wavelength shortens hence green light penetrates less than red
light which at his turn penetrates less into tissue than infrared light2748 Detailed
principles of light penetration will be discussed below
The LED device used emits non-coherent light In the 1980s the observed biological
responses after laser irradiation were generally thought to be attributable to the
coherenceb of the light485253 Though currently the clinical and biological significance
of coherence is seriously questioned54 According to several authors coherence does
not play an essential role in laser-tissue interactions firstly as it was proven that both
coherent and non-coherent light clinically show equal efficacy75556 Secondly as
according to some authors almost immediately after transmission of light through the
skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et
al1957 state that both findings are incorrect coherence is not lost in tissue due to the
phenomenon of scattering and non-coherent light is not as efficient as coherent light
This lack of consensus makes it necessary to mention whether or not light is
coherent2758
Further decisive characteristics to accomplish phototherapeutic efficacy are the power
exposure time output mode and beam area Based on these parameters both
irradiancec and radiant exposured can be calculated According to numerous authors
some of these parameters are more crucial than others to determine whether
b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48
c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48
d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248
General introduction
9
absorption of light will lead to a photobiological event192728485455 However the
literature yields several controversial findings as not all authors attribute an equal
importance to a given parameter For example according to Nussbaum et al59
irradiance was the determinant characteristic in the biomodulation of Pseudomonas
aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49
found that in order to stimulate tissue cell proliferation a specific combination of
irradiance and exposure time are more important than the actual radiant exposure Low
et al3940 on the contrary highlighted the critical importance of the radiant exposure in
observing neurophysiological effects Whereas Mendez et al60 reported that both
parameters influence the final results of light therapy
Koutna et al61 even suggested that the output mode of light applications plays a more
prominent role in the treatment outcome than the wavelength of the used light source
Nevertheless this finding could not be confirmed by other research results Besides
more controversial findings have been published regarding the output mode although
the repetition rate in a pulsed mode was considered as an important treatment
parameter several investigations failed to prove its value19272840414461-64
Based on these findings it was opted within the investigations of this doctoral thesis to
irradiate in a continuous mode The remaining dosimetric parameters (wavelength
exposure time and power) depended on the purpose of each investigation they are
described in the respective chapters The data necessary for the calculation of the
radiant exposure for the equipment used in the respective trials are summarized in
table 1
Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High
Infrared 950 80 120 160 Red 660 15 46 80
Green 570 02 42 10
10
The radiant exposure of the used LED can be calculated as follows65
RE =
Radiant Exposure [Jcm2]
T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]
PRE = α S T
α = 006 (continuous mode) or
003 (pulsed mode)
The parameters commented on so far can be considered as the external dosimetry
involving all parameters directly controlled by the operator limited by the apparatus
used Furthermore there is the so-called internal dosimetry referring to (1) several
physical phenomena (reflection transmission scattering and absorption) influencing
the light distribution within the tissue during energy transfer (2) the optical
characteristics of the irradiated tissue as well as (3) the relation between the external
dosimetry and these respective elements5466
This internal dosimetry determines to a considerable extend the penetration of light
into tissue Penetration can be defined as the tissue depth at which the radiant
exposure is reduced to 37 of its original value1948 However this definition only
accounts for the absolute penetration depth resulting in direct effects of light at that
depth In addition there is also a relative penetration depth leading up to effects
deeper in the irradiated tissue and even in certain degree throughout the entire
body1967 These so-called systemic effects can be caused by chemical processes initiated
at superficial levels at their turn mediating effects at a deeper tissue level57
Involvement of several forms of communication in the tissue such as blood circulation
and transport of transmitters or signal substances is possible1967 This means that light
sources with poor absolute penetration do not necessarily give inferior results than
those with a good absolute penetration19
In the same context it should be noted that calculation and even measurement of the
exact light distribution during irradiation is highly complicated principally as tissues
have complex structures and also because the optical properties of tissues vary largely
inter-individual2768
General introduction
11
Studies regarding actual penetration depth of LED light are scarce consequently the
knowledge on the topic of penetration depth of LED light is based on literature
originating from LLL research19 These findings established with various LLL sources
revealed that there is an obvious relation between penetration depth and
wavelength27486769-71
Three final remarks can be made on the dosimetry First of all it should be noted that
partly as a result of the above-mentioned contrasting findings on dosimetry ideal light
source characteristics for effective treatment of various medical applications are not yet
established and probably never really will be28 Therefore in the attempt to offer
sufficient guidelines for correct use of treatment parameters one should always try to
provide detailed description of light source properties used in any trial so the
practitioner can interpret the scientific results adequately and accordingly draw the
correct conclusions for his clinical practice
A second comment is based on the mentioned possible influence of the external and
internal dosimetric parameters on the photobiological effectiveness of light the
intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation
between radiated wavelength and penetration depth19546572 So it should be
emphasized that caution is recommended when comparing research results of light
sources with different wavelengths or other dissimilar dosimetric parameters
A third and final remark considers the extrapolation issue Comparison of the
therapeutic usefulness of the same light source used on different species should occur
cautiously So simply extrapolating the dosage used for one species to another is
inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo
clinical practice is inappropriate So purposive and specific research is the prerequisite
to produce safe and correct use of light as a therapeutic modality27
12
MECHANISMS OF ACTION
In the past decennia several mechanisms of action for biostimulation and pain
inhibition have been proposed and investigated73 Research was primarily based on
studies at the molecular and cellular levels and as a second resort investigations
occurred at the organism level resulting in numerous possible explanatory
mechanisms272858
It is the common view that light triggers a cascade of cellular and molecular reactions
resulting in various biological responses Thus different mechanisms of whom the
causal relationships are very difficult to establish- underlie the effects of light3448557475
To illustrate this complex matter the various mechanisms of action will be summarised
by means of a comprehensive model (fig 2) Detailed discussion about the different
individual components of the proposed model and other effects than those regarding
wound healing or analgesia were not provided as this was beyond the scope of this
general introduction
As depicted in figure 2 exposure to light leads to photon absorption by a
photoacceptor molecule causing excitation of the electronic state or increased
vibrational state of the given molecule275173 This process is followed by primary
photochemical reactions7475 Several key mechanisms have been discussed in the
literature Respiratory chain activation is the central point and can occur by an
alteration in redox properties acceleration of electron transfer generation of reactive
oxygen species (namely singlet oxygen formation and superoxide generation) as well as
by induction of local transient heating of absorbing chromophores192848515576-83 It is
supposed that each of these respective mechanisms plays a part in obtaining a
measurable biological effect It is yet not clear if one mechanism is more prominent
and decisive than another nevertheless recent experimental evidence has revealed that
mechanisms based on changes in redox properties of terminal enzymes of respiratory
chains might be of crucial importance2848517679
The primary mechanisms occurring during light exposure are followed by the dark
reactions (secondary mechanisms) occurring when the effective radiation is switched
General introduction
13
off2851 Activation of respiratory chain components is followed by the initiation of a
complicated cellular signalling cascade or a photosignal transduction and amplification
chain associated with eg changes in the cellular homeostasis alterations in ATP or
cAMP levels modulation of DNA and RNA synthesis membrane permeability
alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87
The sequence of events finally results in a range of physiological effects essential for
the promotion of the wound healing process for supplying analgesia or other
advantageous responses (acceleration of inflammatory processes oedema re-
absorption increased lymph vessel regeneration or increased nerve
regeneration)12181927486188-93
Photostimulation of the wound healing process can be mediated by increased
fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis
and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations
have been especially focussed on fibroblasts but other possible physiological effects
attributing to an accelerated wound healing were also observed suppression and
alteration of undesirable immune processes increased leukocyte activity new
formation of capillaries increased production of growth factors and enzymes while
monocytes and macrophages can provide an enlarged release of a variety of substances
related to immunity and wound healing1619277376
As pain and nociception are even less understood than wound healing the possible
mechanisms in obtaining pain relief by the use of light are less underpinned However
it is established that light therapy influences the synthesis release and metabolism of
numerous transmitter signal substances involved in analgesia such as endorphin nitric
oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these
neuropharmacological effects there is experimental evidence for diminished
inflammation decreased C-fibre activity increased blood circulation and reduced
excitability of the nervous system1927848899
One should be aware that a large amount of research regarding the possible
mechanisms of light action was conducted at the cellular level The described cascade
of reactions at the organism level is possibly even more complex as in contradiction to
14
the in vitro situation in vivo a range of supplementary interactions can influence the
sequence of effects and accordingly the final responses Besides it needs to be
mentioned that this summary did not take into account the origin of the light or the
external dosimetry thus the description is based on investigations performed with
various light sources and different dosages
Figure 2 Model summarizing the identified mechanisms of light action
Secondarymechanisms
Primary mechanisms
Final effects
Trigger
Stimulated wound healing Analgesia
Exposure to light
Photon absorption by photoacceptors
Respiratory chain activation
Accelerated electrontransfer
Reactive oxygen generation
Heating of absorbing chromophores
Altered redox properties
darr inflammation uarr oedema resorption
uarr lymph vessel regenerationuarr blood circulation
Photosignal transduction and amplification chain
uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation
darr C-fibre activity darr nervous excitability neuropharmacological effects
General introduction
15
Regardless of the large number of previous investigations identification of underlying
mechanisms of light action remains an important issue as these are not yet fully
understood and because probably not all mechanisms of action are currently
identified Convincing explanation of the mechanisms in normal as well as in
pathological tissue could banish the existing suspicion concerning the use of light as a
treatment modality2732547678
AIMS AND OUTLINE
The introduction of LED in medicine and in physiotherapy more specifically requires
particular scientific research especially within the fields of its clinical potential
application wound healing and analgesia The above described gaps in literature
regarding the use of LED laid the foundation of this doctoral thesis
Consequently the general purpose of this thesis is to explore a scientific approach for
the supposed biostimulatory and analgesic effect of LED and to formulate an answer
in view of an evidence-based clinical use of this treatment modality
The detailed objectives can be phrased as follows
Aim 1 To assess the biostimulatory effectiveness of LED
irradiation under normal in vitro conditions
Aim 2 To investigate the value of LED treatment to ameliorate
in vitro cell proliferation under conditions of impaired healing
Aim 3 To examine the effectiveness of LED in changing the
nerve conduction characteristics in view of analgesia
Aim 4 To determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting
Part I investigates the influence of LED on wound healing In pursuit of the first aim
chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial
16
measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these
cells needs to be considered as an exponent of the wound healing process as
fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase
and early remodelling100 Secondly an in vivo case study exploring the postulation that
LED irradiation could accelerate and ameliorate the healing of a surgical incision was
described
The results contrasted sharply with the findings of the in vitro part Two fundamental
causes were proposed in order to explain the different biological effect of LED
irradiation observed in vitro and in vivo the used irradiation parameters and evaluation
method
The experiment described in chapter 2 endeavoured to explore these considerations A
similar study was therefore performed but as distinctive characteristics different light
source properties an adapted irradiation procedure and the use of a colorimetric assay
based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the
counting of the cells were used
As stimulation of the wound healing process is virtually mainly indicated under
conditions of impaired healing (resulting in a situation which threatens to become
chronic and debilitating) proper attention for this matter is warranted192855 Besides
the medical consequences the costs involved with impaired healing yield also a socially
relevant problem to tackle Impaired healing will become even more common as the
world population continues to age After all senescence of systems and age-committed
comorbid conditions are commonly the culprits responsible for poor wound healing101
Thus finding cost-effective time-sparing non-invasive and practical treatment
modalities to cure wounds is a necessity
Aiming to assess the biostimulative effects by means of LED in these circumstances a
third study was conducted with respect of the previous results regarding irradiation
parameters and cell proliferation analysis The irradiation experiment described in
chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose
This medium modification serves as a pattern for cell proliferation in diabetic patients
General introduction
17
a population for whom stimulation of the wound healing process is a clinical relevant
feature
In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored
by means of two studies A first investigation (chapter 4) evaluated the influence of LED
on the sensory nerve conduction characteristics of a human superficial peripheral
nerve as a potential explanatory mechanism of pain inhibition by LED which is based
on the putative neurophysiological effects of this treatment modality The experimental
hypothesis postulated that LED generates an immediate decrease in conduction
velocity and increase in negative peak latency In addition it was postulated that this
effect is most prominent immediately after the irradiation and will weaken as time
progresses
The values of nerve conduction velocity and negative peak latency of a baseline
antidromic nerve conduction measurement were compared with the results of five
identical recordings performed at several points of time after LED irradiation
Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory
setting To guarantee an adequate standardized and controlled pain reduction study
there was opted to observe a healthy population with experimentally induced DOMS
Induction of DOMS has been described in a number of studies as a representative
model of musculoskeletal pain and stiffness because it can be induced in a relatively
easy and standardised manner the time course is quite predictable and the symptoms
have the same aetiology and are of transitory nature4445102-105
The treatment as well as the assessment procedure was performed during 4
consecutive days The first day isokinetic exercise was performed to induce pain
related to DOMS Subsequently the volunteers of the experimental group received an
infrared LED treatment and those of the placebo group received sham-irradiation
Evaluation of the effect of the treatment on perceived pain was registered by a visual
analog scale and by a mechanical pain threshold these observations occurred every day
18
prior to and following LED irradiation Eccentricconcentric isokinetic peak torque
assessment took place daily before each treatment
For the analysis of the results three different factors were taken into consideration
time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental)
In completion of this thesis the most prominent findings are summarized and the
clinical implications are discussed The general discussion also includes some future
research directions and a final conclusion
General introduction
19
REFERENCES
1 Enwemeka C (2005) Light is light Photomed Laser Surg 23(2)159 2 Skinner S Gage J Wilce P and Shaw R (1996) A preliminary study of the effects of laser
radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-92 3 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy
in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austriaca 33(3)132-137
4 Stahl F Ashworth S Jandt K and Mills R (2000) Light-emitting diode (LED) polymerisation of dental composites flexural properties and polymerisation potential Biomaterials 21(13)1379-1385
5 Mills R Jandt K and Ashworth S (1999) Dental composite depth of cure with halogen and blue light emitting diode technology Br Dent J 186(8)388-391
6 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
7 Pontinen P Aaltokallio T and Kolari P (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118
8 Schmidt M Reichert K Ozker K Meyer G Donohoe D Bajic D Whelan N and Whelan H (1999) Preclinical evaluation of benzoporphyrin derivative combined with a light-emitting diode array for photodynamic therapy of brain tumors Pediatr Neurosurg 30(5)225-231
9 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
10 Sommer A Pinheiro A Mester A Franke RP and Whelan H (2001) Biostimulatory windows in low-intensity laser activation lasers scanners and NASAs light-emitting diode array systems J Clin Laser Med Sur 19(1)29-33
11 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
12 Whelan H Smits R Buchman E Whelan N Turner S Margolis D Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Philippi A Graf W Hodgson B Gould L Kane M Chen G and Caviness J (2001) Effect of NASA light-emitting diode irradiation on wound healing J Clin Laser Med Sur 19(6)305-314
13 Ojeda A Redondo E Gonzalez Diaz G and Martil I (1997) Analysis of light-emission processes in light-emitting diodes and semiconductor lasers Eur J Phys 18(2)63-67
14 Monstrey S Hoeksema H Saelens H Depuydt K Hamdi M Van Landuyt K and Blondeel P (2002) Conservative approach for deep dermal burn wounds using polarised-light therapy Br J Plast Surg 55(5)420-426
15 Monstrey S Hoeksema H Depuydt K Van Maele G Van Landuyt K and Blondeel P (2002) The effect of polarized light on wound healing Eur J Plast Surg 24377-382
16 Bolton P Dyson M and Young S (1992) The effect of polarized light on the release of growth factors from the U-937 macrophage-like cell line Laser Ther 233-42
17 Stasinopoulos D (2005) The use of polarized polychromatic non-coherent light as therapy for acute tennis elbowlateral epicondylalgia A pilot study Photomed Laser Surg 23(1)66-69
18 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
19 Tuner J Hode L (2004) The laser therapy handbook Tallinn Prima Books AB 20 Allendorf J Bessler M Huang J Kayton M Laird D Nowygrod R and Treat M (1997) Helium-
neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3)340-345
21 Basford J (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8)671-675
20
22 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
23 Lagan K Clements B McDonough S and Baxter G (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1)27-32
24 Schlager A Kronberger P Petschke F and Ulmer H (2000) Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1)39-42
25 Nemeth A J (1993) Lasers and wound healing Dermatol Clin 11(4)783-789 26 Lowe A Walker M OByrne M Baxter G and Hirst D (1998) Effect of low intensity
monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5)291-298
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science
Publishers 29 Basford J (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg
Med 16(4)331-342 30 Baxter G Bell A Allen J and Ravey J (1991) Low level laser therapy Current clinical practice in
Northern Ireland Physiotherapy 77(3)171-178 31 Cambier D and Vanderstraeten G (1997) Low-level laser therapy The experience in flanders
Eur J Phys Med Rehabil 7(4)102-105 32 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D
Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
33 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
34 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
35 Basford J Hallman H Matsumoto J Moyer S Buss J and Baxter G (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6)597-604
36 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol (Lond) 43563
37 Baxter G Allen J Walsh D Bell A and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol (Lond) 446445
38 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
39 Lowe A Baxter G Walsh D and Allen J (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
40 Lowe A Baxter G Walsh D and Allen J M (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Lasers Med Sci 10(4)253-259
41 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
General introduction
21
42 Baxter G Effect of combined phototherapylow intensity laser therapy upon experimental ischaemic pain Potential relevance of experimental design 14th World Congress Physical Therapy Barcelona Spain 2004 Proceedings CD
43 Craig J Barron J Walsh D and Baxter G (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
44 Craig J Barlas P Baxter G Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
45 Glasgow P Hill I McKevitt A Lowe A and Baxter G (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1)33-39
46 Noble J Lowe A Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
47 IEC 60825-1 (1993) International Electrotechnical Commission Safety of laser products Part 1 Equipment classification requirements and users guide
48 Nussbaum E Baxter G and Lilge L (2003) A review of laser technology and light-tissue interactions as a background to therapeutic applications of low intensity lasers and other light sources Phys Ther Rev 831-44
49 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
50 Nakamura S (1998) The roles of structural imperfections in InGaN-based blue light-emitting diodes and laser diodes Science 281956-961
51 Karu Tiina (consulted on 2005 august 22) Cellular mechanism of low-power laser therapy httpwwwtinnitusustiinakarupresentationhtml
52 Mester E Mester A and Mester A (1985) The biomedical effects of laser application Lasers Surg Med 5(1)31-39
53 Moore K Calderhead G (1991) The clinical application of low incident power density 830 nm GaAlAs diode laser radiation in the therapy of chronic intractable pain A historical and optoelectronic rationale and clinical review Int J Optoelectron 6503-520
54 King P (1989) Low level laser therapy A review Lasers Med Sci 4141-148 55 Karu T (1989) Photobiology of low-power laser effects Health Phys 56(5)691-704 56 Simunovic Z (2000) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical
Application of Low Energy-Laser Laser Therapy LLLT Rijeka Vitgraf 57 Hode L and Tuner J (2000) Low level laser therapy (LLLT) contra light emitting diode therapy
(LEDT) - What is the difference Proceedings of SPIE 416690-97 58 Kitchen S and Partridge C (1991) A review of low level laser therapy Part I Background
physiological effects and hazards Physiotherapy 77(3)161-163 59 Nussbaum E Lilge L and Mazzulli T (2003) Effects of low-level laser therapy (LLLT) of 810 nm
upon in vitro growth of bacteria Relevance of irradiance and radiant exposure J Clin Laser Med Sur 21(5)283-290
60 Mendez T Pinheiro A Pacheco M Nascimento P and Ramalho L (2004) Dose and wavelength of laser light have influence on the repair of cutaneous wounds J Clin Laser Med Sur 22(1)19-25
61 Koutna M Janisch R and Veselska R (2003) Effects of low-power laser irradiation on cell proliferation Scripta Medica 76(3)163-172
62 Al-Watban F and Zhang X (2004) The comparison of effects between pulsed and CW lasers on wound healing J Clin Laser Med Sur 22(1)15-18
63 Panjehpour M Overholt B DeNovo R Petersen M and Sneed R (1993) Comparative study between pulsed and continuous wave lasers for photofrin photodynamic therapy Lasers Surg Med 13(3)296-304
64 Dyson M (2005) The significance of pulsing in the stimulation of tissue repair by light A review Lasers Med Sci 20S21
22
65 MDB Laser (2000) Bio Dio (Preprotoype) User guide Belgium Ekeren 66 Fisher J (1992) Photons physiatrics and physicians A practical guide to understanding laser light
interaction with living tissue Part I J Clin Laser Med Sur 10(6)419-426 67 Tuner J and Hode L (2000) Depth of penetration of laser light in tissue Laser Partner
Clinixperience 151-3 68 Anderson R and Parrish J (1981) The optics of human skin J Invest Dermatol 77(1)13-19 69 De Cuyper H and Lambert H (1991) Penetration depth of infrared- and helium-neon-laserlight
An assessment in vivo Eur J Phys Med Rehabil 1(5)133-137 70 Oshiro T Ogata H Yoshida M Tanaka Y Sasaki K and Yoshimi K (1996) Penetration depths
of 830nm diode laser irradiation in the head and neck assessed using a radiographic phanom model and wavelength-specific imaging film Laser Ther 8197-204
71 Kolarova H Ditrichova D and Wagner J (1999) Penetration of the laser light into the skin in vitro Lasers Surg Med 24(3)231-235
72 Chen Q Huang Z Chen H Shapiro H Beckers J and Hetzel F (2002) Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy Photochem Photobiol 76(2)197ndash203
73 Conlan M Rapley J and Cobb C (1996) Biostimulation of wound healing by low-energy laser irradiation A review J Clin Periodontol 23(5)492-496
74 Parrish J (1981) New concepts in therapeutic photomedicine photochemistry optical targeting and the therapeutic window J Invest Dermatol 7745-50
75 Smith K (1981) Photobiology and photomedicine the future is bright J Invest Dermatol 772-7 76 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees D (1989) Systemic effects
of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
77 Polo L Presti F Schindl A Schindl L Jori G and Bertoloni G (1999) Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth Biochem Biophys Res Commun 257(3)753-758
78 Bertoloni G Sacchetto R Baro E Ceccherelli F and Jori G (1993) Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 6328 nm light J Photochem Photobiol B-Biol 18191-196
79 Lubart R Friedmann H Sinykov M and Grossman N (1995) Biostimulation of photosensitized fibroblasts by low incident levels of visible light energy Laser Ther 7101-106
80 Harris D (1991) Biomolecular mechanisms of laser biostimulation J Clin Laser Med Sur 9277-280
81 Grossman N Schneid N Reuveni H Halevy S and Lubart R (1998) 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures involvement of reactive oxygen species Lasers Surg Med 22212-218
82 Oren D Charney D Lavi R Sinyakov M and Lubart R (2001) Stimulation of reactive oxygen species production by an antidepressant visible light source Biol Psychiatry 49464-467
83 Lavi R Shainberg A Friedmann H Shneyvays V Rickover O Eichler M Kaplan D and Lubart R (2003) Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells 278(42)40917-40922
84 Reddy K (2004) Photobiological basis and clinical role of low-intensity lasers in biology and medicine J Clin Laser Med Sur 22(2)141-150
85 Greco M Guida G Perlino E Marra E and Quagliariello E (1989) Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser Biochem Biophys Res Commun 163(3)1428-1434
86 Vacca R Marra E Quagliariello E and Greco M (1993) Activation of mitochondrial DNA replication by He-Ne laser irradiation Biochem Biophys Res Commun 195(2)704-709
87 Vacca R Marra E Quagliariello E and Greco M (1994) Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser Biochem Biophys Res Commun 203(2)991-997
88 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral
General introduction
23
nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
89 Anders J Borke R Woolery S and Van de Merwe W (1993) Low power laser irradiation alters the rate of regeneration of the rat facial nerve Lasers Surg Med 13(1)72-82
90 Rochkind S Nissan M Barr-Nea L Razon N Schwartz M and Bartal A (1987) Response of peripheral nerve to He-Ne laser Experimental studies Lasers Surg Med 7441-443
91 Lievens P (1986) Lasertherapie (deel II) De invloed van laser-bestralingen op het lymfesysteem en de wondheling Ned T Fysiotherapie 96140-142
92 Lievens P (1991) The effect of a combined HeNe and IR laser treatment on the regeneration of the lymphatic sysytem during the process of wound healing Lasers Med Sci 6193-199
93 Lievens P (1991) The effect of IR laser irradiation on the vasomotricity of the lymphatic system Lasers Med Sci 6189-191
94 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin J and Martin P (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1)171-178
95 Bosatra M Jucci A Olliaro P Quacci D and Sacchi S (1984) In vitro fibroblast and dermis fibroblast activation by laser irradiation at low energy An electron microscopic study Dermatologica 168(4)157-162
96 Enwemeka C Rodriquez O Gall N and Walsh N (1990) Morphometrics of collagen fibril populations in HeNe laser photostimulated tendons J Clin Laser Med Sur 847-52
97 Abergel P Lam T Meeker C Castel C Dwyer R and Uitto J (1984) Biostimulation of procollagen production by low energy lasers in human skin fibroblast culturesJ Invest Dermatol 82(4)395
98 Abergel P Lam T Meeker C Dwyer R and Uitto J (1984) Low energy lasers stimulate collagen production in human skin fibroblast cultures Clin Res 32(1)16A
99 Shimoyama N Lijima K Shimoyama M and Mizuguchi T (1992) The effects of helium-neon laser on formalin-induced activity of dorsal horn neurons in the rat J Clin Laser Med Sur 1091-94
100 Kloth L McCulloch J and Feedar J (1990) Wound healing Alternatives in management USA FA Davis Company
101 Lanzafame R (2004) Revolutions and revelations J Clin Laser Med Surg 22(1)1-2 102 Ciccone C Leggin B and Callamaro J (1991) Effects of ultrasound and trolamine salicylate
phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-678 103 Craig J Cunningham M Walsh D Baxter G and Allen J (1996) Lack of effect of transcutaneous
electrical nerve stimulation upon experimentally induced delayed onset muscle soreness in humans Pain 67(2-3)285-289
104 Minder P Noble J Alves-Guerreiro J Hill I Lowe A Walsh D and Baxter G (2002) Interferential therapy Lack of effect upon experimentally induced delayed onset muscle soreness Clin Physiol Funct Imaging 22(5)339-347
105 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
PART I WOUND HEALING
CHAPTER 1
DO INFRARED LIGHT EMITTING DIODES HAVE A
STIMULATORY EFFECT ON WOUND HEALING FROM AN IN
VITRO TRIAL TO A PATIENT TREATMENT
Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Proceedings of SPIE 2002 4903 156-165
Chapter 1
28
ABSTRACT
Variable effects of different forms of light therapy on wound healing have been
reported This preliminary study covers the efficacy of infrared light emitting diodes
(LED) in this domain
Cultured embryonic chicken fibroblasts were treated in a controlled randomised
manner LED irradiation was performed three consecutive days with a wavelength of
950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each
treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2
The results indicated that LED treatment does not influence fibroblast proliferation at
the applied energy density and irradiation frequency (p=0474)
Meanwhile the effects of LED on wound healing in vivo were studied by treating a
surgical incision (6 cm) on the lateral side of the right foot of a male patient The
treatment started after 13 days when initial stitches were removed The same
parameters as the in vitro study were used but the treatment was performed five times
The healing could only be evaluated clinically the irradiated area (26 cm) showed a
more appropriate contraction less discoloration and a less hypertrophic scar than the
control area (34 cm)
The used parameters failed to demonstrate any biological effect of LED irradiation in
vitro although the case study on the other hand illustrated a beneficial effect
Keywords Light Emitting Diodes Fibroblasts Wound healing
From an in vitro trial to a patient treatment
29
INTRODUCTION
Various beneficial effects of lasers and photodiodes at relatively low intensities have
been reported involving treatment of neurological impairments12 treatment of pain3-5
treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the
enhancement of wound healing has been a focus of contemporary research11-16 It
seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing
remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the
basis of a questionnaire about low power laser (LPL) in the current clinical practice in
Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey
into clinical LPL experience in Flanders
Nevertheless there remains a considerable amount of ignorance scepticism and
controversial issues concerning the use and clinical efficacy of LPL even in the domain
of wound healing12152021 This is at least in part a consequence of the inability to
measure and control operating variables related to connective tissue repair and of the
wide range of suitable parameters for irradiation
Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent
characteristics which make their use in a clinical setting problematic including
limitations in wavelength capabilities and beam width The combined wavelength of
light optimal for wound healing cannot be efficiently produced and the size of
wounds which may be treated by LPLs is limited Some companies offer an
alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be
made to produce multiple wavelengths and can have probes with large surface area
allowing treatment of large wounds Still one can not accept this light source as an
alternative for LPL therapy based on the cited advantages without proper investigation
regarding its biostimulatory effects
The effectiveness of this possible alternative for LPLs must be studied in vitro and in
addition in animal models or in humans because the effects of LED at the cellular level
do not necessarily translate to a noticeable effect in vivo The small amount of previous
investigations demonstrate that LED effects are as difficult to isolate162223 as LPL
Chapter 1
30
effects and the results are conflicting just like the results in literature specific on the
use of LPL121520
The purpose of the first part of this study is to examine the hypothesis stating that
LED irradiation can influence fibroblast proliferation Therefore a comparison of the
proliferation from fibroblasts in irradiated and control wells was performed The in vitro
investigation was linked with an in vivo case study This part enquired the assumption if
LED irradiation could accelerate and ameliorate the healing of a surgical incision
IN VITRO INVESTIGATION
MATERIALS AND METHODS
The complete procedure from isolation to proliferation analysis was executed twice
(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of
control dishes The second trial consisted of 27 irradiated and 27 control dishes
Cell isolation and culture procedures
Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation
and disaggregating of the cells occurred with warm trypsin (NV Life Technologies
Belgium) according the protocol described by Ian Freshney (1994)24 The primary
explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies
Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1
Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life
Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies
Belgium) When cell growth from the explants reached confluence cells were detached
with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV
Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the
cells were removed from the culture flasks by trypsinization and counted by
hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc
From an in vitro trial to a patient treatment
31
NV Life Technologies Belgium) The fibroblasts were seeded at a density of
70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture
medium the cells were allowed to attach for 24 hours in a humidified incubator at
37degC
Properties of the Light Emitting Diode
Prior to LED treatment all dishes were microscopically checked to guarantee that the
cells are adherent and to assure that there is no confluence nor contamination The
dishes were divided randomly into the treated or the control group Medium was then
removed by tipping the dishes and aspirating with a sterile pipette Following the
aspiration 2 ml fresh medium was added and treatment started
A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser
Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-
1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32
single LEDrsquos For the treatments in this study an average power of 160 mW at
continuous mode was applied The irradiation lasted 6 minutes resulting in an energy
density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result
of the divergence in function of this distance the surface of the LED (18 cm2) covered
the complete surface of the used petriplates (215 cm2)
After these manipulations 3 ml medium was added to each dish followed by 24 hours
incubation
One LED irradiation was performed daily during three consecutive days according
this procedure Control cultures underwent the same handling during these three days
but were sham-irradiated
Proliferation analysis
After the last treatment a trypsination was performed to detach the cells from the
culture dishes followed by centrifugation Once the cells were isolated from the used
trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts
Chapter 1
32
within this suspension as reflection for the proliferation was quantified by means of a
Buumlrker Chamber or hemocytometry
The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-
Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab
Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on
the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator
using an inverted light microscope counted the number of cells in 25 small squares
In order to calculate the number of cells one should multiply the amount of cells
counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and
the dilution factor (the amount of Trypan blue suspended with the cells 21=3)
Statistical methods
The data were analysed statistical in order to examine the hypothesis that LED
irradiation enhances fibroblast proliferation They were processed as absolute figures
for both trials separately In a second phase the counted cell numbers were converted
in relative figures so the data of both trials could be analysed as the data of one test
These relative figures were obtained by expressing each figure as a percentage from the
highest figure (=100) of that trial and this for each assay separately
A Kolmogorov-Smirnoff test of normality was performed on the data followed by a
Mann-Whitney-U test when the test of normality was significant and otherwise a T-
test Differences were accepted as significant when plt005 For this analysis SPSSreg
100 was used
RESULTS
The descriptive data for both trials are depicted in figure I The mean number of cells
in trial A is higher than in trial B for the controls as for the treated wells There is a
mean difference of 1252500 fibroblasts between the controls and 1223000 between
the irradiated wells of trial A and B The averages of both trials show that in control
cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no
From an in vitro trial to a patient treatment
33
statistically significant difference could be found between the two groups in either trial
nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not
significant for trial A (p=020) nor trial B (p=020) Only the combined data from both
trials were significant (plt001) for normality Further analysis respectively T-test for
the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the
combined data (p=0474) revealed no statistical significant differences
DESCRIPTIVE DATA
1730000181750029530003070000
00E+00
50E+05
10E+06
15E+06
20E+06
25E+06
30E+06
35E+06
40E+06
Trial A Trial A Trial B Trial B
Mea
n n
um
ber
of
cells
Control
Irradiated
Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B
DISCUSSION
Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities
(lt500 mW) in vitro have been analysed by evaluating various factors involving
(pro)collagen production25-27 cell viability2829 growth factor production28 and
myofibroblast formation30 Fibroblast proliferation also is an important factor to
consider In accordance with wound healing fibroblasts fulfil an essential role especially
in the late inflammatory phase and the early granulation phase31 Despite the failure of
some studies to demonstrate beneficial effects of LPL irradiation on fibroblast
proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very
Chapter 1
34
significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -
208 ) Atabey et al35 also revealed a significant increase in cell number two or more
irradiations resulted in an increased fibroblast proliferation Several other studies
confirmed these positive findings25263637
The results of this present in vitro study indicate that LED treatment does not
influence fibroblast proliferation Although the dosimetric parameters (in particular the
arbitrary energy density of 32 Jcm2) used in this study are well within the
recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous
studies about LPL therapy raising enhanced fibroblast proliferation252634-37
Van Breugel et al36 gave a possible explanation for these controversial results
According to them the fibroblast proliferation is not inherent at the energy density
They provide evidence that independent of the energy density the power density and
the exposure time determine the biostimulative effects of LPL irradiation LPL with a
power below 291 mW could enhance cell proliferation while a higher power had no
effect
Some authors also argued that the absorption spectrum of human fibroblasts show
several absorption peaks and pointed out that a wavelength of 950 nm is far above the
highest peak of about 730 nm3638 At longer wavelengths they determined a general
decrease in absorption Despite these results several investigators pose biostimulative
effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or
even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is
ranged from 800 nm to 830 nm principally because of the presence of cytochrome
oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts
at lower wavelengths is restricted by an inferior skin transmission than at higher
wavelengths38
Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting
respective green and red light The 950 nm beam of light was used for its high power
density but according to a range of remarks mentioned above the effects of the two
other probes must be as well evaluated
From an in vitro trial to a patient treatment
35
Another factor one can not ignore is that besides fibroblast proliferation other
processes or morphologic changes were not analysed although several authors have
posed that those changes and processes could be responsible for the biostimulative
effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et
al30 for example described a massive transformation of fibroblasts into myofibroblasts
after LPL treatment These modified fibroblasts play an important role in contraction
of granulation tissue30 A second example is an increased (pro)collagen production
after low power light therapy25-27 which is also considered as a responsible factor for
accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast
proliferation3640
It may be wondered if the light sources mostly LPL in the consulted literature are
representative for the LED used in this study although this LPL literature is often
used for that purpose As in the early days of LPL the stimulative effects upon
biological objects were explained by its coherence the beam emitted by the Bio-Dio on
the contrary produces incoherent light Nowadays contradictory research results are
responsible for a new discussion the clinical and biological significance of coherence
The findings of some authors172341-43 pose that the coherence of light is of no
importance of LPL and its effects although the opposite has also been stated4445
Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be
accounted for the non-enhanced fibroblast proliferation in this trial
Another possible explanation for the absence of biostimulative effect is related to the
moment of analysis of the proliferation The evaluation one day after the last
irradiation did not allow a delayed enhancement of proliferation while it is determined
in numerous investigations that the effects occur more than 24 hours after the last
treatment273746 and that they weaken after a further undefined period of time34
The fluctuation in cell numbers between both trials despite the use of an identical
protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor
reproducibility of their technique In this study the fluctuations are attributable to the
counting of the cells by Buumlrker hemocytometer before seeding According to some
authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large
Chapter 1
36
variability48 and it is often difficult to standardize48 Overestimation of the cell
concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity
was contradicted by Lin et al50 moreover satisfactory correlations with flow-
cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay
for cell counting (MTT)51 were determined
An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it
provides accurate cell counts in a short period of time with less intervention from the
investigator52
These remarks and controversies point out the possible deficiencies from the used
proliferation analyses and the relativity from the obtained results Other analyse
methods and analyses from different cell processes and morphologic changes could be
considered for further investigation
IN VIVO INVESTIGATION
MATERIALS AND METHODS
The effects of LED on wound healing in vivo were studied by treating a postsurgical
incision A male patient received chirurgical treatment for the removal of a cyst
situated approximately 15 cm posterior from the lateral malleolus of his right foot For
removal of the cyst an incision of 6 cm was made The incision was sutured and 12
days after the surgery the stitches were removed Visual inspection demonstrated that
the healing process of the wound proceeded well but not equally over the whole 6
centimetres (figure II) Epithelialization and wound contraction appeared to have
progressed better in the upper part (approximately 3 cm) of the cicatrice than at the
lower part (covered with eschar) No evidence of infection was noted in either part
LED treatment started the 13th day The incision was treated partially the lowest part
(26 cm) with the inferior epithelialization and wound contraction was irradiated the
remaining 34 cm served as control area This control area was screened from radiation
with cardboard and opaque black cling film
From an in vitro trial to a patient treatment
37
The light source destinated for the treatment was the same device used for the in vitro
irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED
output parameters were identical with those applicated in the preceding in vitro
investigation In particular a continuous wave at an average power of 160 mW and 6
minutes of treatment duration corresponding to an energy density of 32 Jcm2 An
equal distance from the probe to the target tissue as from the probe to the culture
medium was respected A plastic applicant of according height guaranteed constant
distance of 06 cm from the surface of the skin
Figure II Surgical incision before the first treatment 13 days after initial stitching
Therapy was performed once a day during five consecutive days repeatedly at the same
time resulting in an extension of the duration of the in vitro therapy with two days
Visual macroscopic observations were accomplished 6 52 and 175 days after the first
treatment
Comparison of the cutaneous sensitivity at the irradiated area and the control area was
accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175
days after the first treatment A control measurement also occurred at the same region
Chapter 1
38
on the left foot The aesthesiometer used in this study consisted of five hand-held
nylon monofilaments with a length of 38 mm and varying diameter
Sensitivity threshold is traced by presenting a monofilament of a certain diameter
vertically to the skin The monofilament bends when a specific pressure has been
reached with a velocity proportional to its diameter Measurements allow mapping
areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified
from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest
filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished
protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to
finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the
filaments
RESULTS
Visual estimation at any point of time after irradiation divulged no occurrence of
problems with dehiscence or infection in either part of the wound During the five
days of therapy the irradiated area looked dryer than the control area After the last
irradiation this was no longer recorded
Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area
From an in vitro trial to a patient treatment
39
Figure III representing the first evaluation six days after the initial treatment
illustrates that the wound healing has evolved slightly in both parts Though the lower
irradiated part remains of inferior quality as regards to epithelialization and wound
contraction In the course of the reparative process the influence of light exposures
were registered At 52 days after the first irradiation beneficial effects of LED
treatment are clearly present (Figure IV)
Figure IV Surgical incision 52 days after initiating LED treatment
The irradiated area (26 cm) showed a more appropriate contracture than the control
area (34 cm) characterized by less discoloration at scar level and a less hypertrophic
scar A similar trend was noticed at a third visual observation 175 days after the initial
treatment At that moment no impairments at cutaneous sensitivity level were stated
and the sensitivity showed no differences between left or right foot nor between the
two areas of the cicatrice
Chapter 1
40
DISCUSSION
The results of this case study indicate that LED had a positive influence on wound
healing in humans as determined by visual observations Many investigators
examining the effects of LPL on wound healing by means of a range of observation
and treatment methods reported accelerated and enhanced wound healing8-10 others
described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and
LED21 studies were unable to repeat these results
The late but beneficial findings in this study seem to be to the credit of LED-therapy
Though several authors establish positive results in an earlier stage of the wound
healing process8-1020 one should question why the differences did not occur at the first
evaluation on day 6 An explanation can be found in the start of the treatment Most
investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so
they influence a first cellular and vascular reaction with the production of chemical
mediators of inflammation resulting in an enhanced collagen production9 tremendous
proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the
time the first treatment in this study took place the traumatized tissue was in an
overlapping stage between an almost finished inflammatory phase and a scarcely
initiated re-epithelialization and wound contraction phase At that moment an infiltrate
of fibroblasts is present So fibroblast proliferation a possible mechanism of the
biostimulative effect had already occurred and could no longer be influenced Growth
factor production and collagen deposition have also decreased at that stage
Granulation tissue formation and fibroplasia in the contrary are initiating by that time
Those prolonged and slow processes with belated results are of significant importance
for the course of the final stage of wound healing and for the outlook of the future
scar31
The experimental findings revealed that the sensitivity of the skin according to the
threshold detection method of Semmes and Weinstein was normal at all the
investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a
good and objective predictor of normal skin sensitivity No other LPL nor LED
studies investigating this quality of the skin were found
From an in vitro trial to a patient treatment
41
CONCLUSION
This study demonstrates that although LED application at the applied energy density
and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to
have beneficial biostimulative effects on wound healing in human skin confirmed by
the favourable re-epithelialization and contracture
These results are discussed in the context of other experimental findings but no
reasonable explanation for this discrepancy could be found The literature on wound
healing after LED treatment in animal models or in humans is presently very limited
and contradictory The diversity in used radiation parameters and the absence of
references on how the wounds were measured or evaluated or what the end point was
for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro
investigations are better standardised nevertheless these results show a number of
conflicts One can conclude that until today the controversial findings are characteristic
for many results obtained with light photobiomodulation
However the postponed favourable results in the case study confirm some facts of the
discussion Namely the short period of incubation 24 hours in the in vitro part of the
study can be responsible for the lack of enhanced fibroblast proliferation It also
confirms that other cell processes and morphologic changes possibly are responsible
for biostimulative effects in vivo other observation methods should be considered for
future in vivo experiments
Despite these remarks we believe that LED application on cutaneous wounds of
human skin is useful with a single flash daily at the dose applied in this study for at
least three days
Furthermore future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Chapter 1
42
ACKNOWLEDGMENTS
The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the
material necessary for this study as well as Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
From an in vitro trial to a patient treatment
43
REFERENCES
1 G Baxter D Walsh J Allen A Lowe and A Bell Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79(2) 227-34 (1994)
2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)
3 J Stelian I Gil B Habot et al Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy J Am Geriatr Soc 40(1) 23-6 (1992)
4 A Honmura A Ishii M Yanase J Obata and E Haruki Analgesic effect of Ga-Al-As diode laser irradiation on hyperalgesia in carrageenin-induced inflammation Lasers Surg Med 13(4) 463-9 (1993)
5 D Cambier K Blom E Witvrouw G Ollevier M De Muynck and G Vanderstraeten The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15 195-200 (2000)
6 G Reddy L Stehno Bittel and C Enwemeka Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-7 (1998)
7 K Moore N Hira I Broome and J Cruikshank The effect of infrared diode laser irradiation on the duration and severity of postoperative pain a double blind trial Laser Therapy 4 145-148 (1992)
8 A Amir A Solomon S Giler M Cordoba and D Hauben The influence of helium-neon laser irradiation on the viability of skin flaps in the rat Br J Plast Surg 53(1) 58-62 (2000)
9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)
10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)
11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)
12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)
13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)
14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)
15 A Schlager P Kronberger F Petschke and H Ulmer Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1) 39-42 (2000)
16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)
17 GD Baxter and J Allen Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone (1994)
18 G Baxter A Bell J Allen and J Ravey Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77(3) 171-178 (1991)
19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)
20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)
Chapter 1
44
21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)
22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)
23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)
24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)
25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)
26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)
27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)
28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)
29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)
30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)
31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)
32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)
33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)
34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)
35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)
36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)
37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)
38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)
39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)
40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)
41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)
42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)
From an in vitro trial to a patient treatment
45
43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)
44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)
45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)
46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)
47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)
48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)
49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)
50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)
51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)
52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)
53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)
54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)
CHAPTER 2
INCREASED FIBROBLAST PROLIFERATION INDUCED BY
LIGHT EMITTING DIODE AND LOW LEVEL LASER
IRRADIATION
Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Lasers in Medical Science 2003 18(2) 95-99
Chapter 2
48
ABSTRACT
Background and Objective As Light Emitting Diode (LED) devices are
commercially introduced as an alternative for Low Level Laser (LLL) Therapy the
ability of LED in influencing wound healing processes at cellular level was examined
Study DesignMaterials and Methods Cultured fibroblasts were treated in a
controlled randomized manner during three consecutive days either with a infrared
LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and
570 nm) and respective power outputs Treatment duration varied in relation to
varying surface energy densities (radiant exposures)
Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all
irradiated cultures in comparison with the controls Green light yielded a significantly
higher number of cells than red (plt0001) and infrared LED light (plt0001) and than
the cultures irradiated with the LLL (plt0001) the red probe provided a higher
increase (plt0001) than the infrared LED probe and than the LLL source
Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation
in vitro This study therefore postulates possible stimulatory effects on wound healing
in vivo at the applied dosimetric parameters
Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low
Level Laser Tetrazolium salt
LED induced increase of fibroblast proliferation
49
INTRODUCTION
Since the introduction of photobiostimulation into medicine the effectiveness and
applicability of a variety of light sources in the treatment of a wide range of medical
conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results
of several investigations are remarkably contradictory This is at least in part a
consequence of the wide range of indications as well as the wide range of suitable
parameters for irradiation and even the inability to measure the possible effects after
irradiation with the necessary objectivity [457] A lack of theoretical understanding
can also be responsible for the existing controversies In fact theoretical understanding
of the mechanisms is not necessary to establish effects though it is necessary to
simplify the evaluation and interpretation of the obtained results As a consequence
the widespread acceptance of especially Low Level Laser (LLL) therapy in the early
seventies is faded nowadays and biostimulation by light is often viewed with scepticism
[8] According to Baxter [49] contemporary research and consumption in
physiotherapy is in particular focused on the stimulation of wound healing Tissue
repair and healing of injured skin are complex processes that involve a dynamic series
of events including coagulation inflammation granulation tissue formation wound
contraction and tissue remodelling [10] This complexity aggravates research within this
cardinal indication
Research in this domain mostly covers LLL studies but the current commercial
availability of other light sources appeals research to investigate as well the effects of
those alternative light sources eg Light Emitting Diode (LED) apparatus
The scarcity of literature on LED is responsible for consultation of literature
originating from LLL studies [11] but it may be wondered if this literature is
representative for that purpose As in the early days of LLL therapy the stimulating
effects upon biological objects were explained by its coherence [1213] while the beam
emitted by LEDrsquos on the contrary produces incoherent light Though the findings of
some scientists [914151617] pose nowadays that the coherence of the light beam is
not responsible for the effects of LLL therapy Given that the cardinal difference
between LED and LLL therapy coherence is not of remarkable importance in
Chapter 2
50
providing biological response in cellular monolayers [5] one may consult literature
from LLL studies to refer to in this LED studies
The purpose of this preliminary study is to examine the hypothesis that LED
irradiation at specific output parameters can influence fibroblast proliferation
Therefore irradiated fibroblasts cultures were compared with controls The article
reports the findings of this study in an attempt to promote further discussion and
establish the use of LED
MATERIALS AND METHODS
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos Isolation and
disaggregation of the cells was performed with warm trypsin according the protocol
described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in
Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1
L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants
reached confluence cells were detached with trypsine and subcultured during 24 hours
in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours
the cells were removed from the culture flasks by trypsinization and counted by Buumlrker
hemocytometry For the experiment cells from the third passage were plated in 96-well
plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a
density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C
during 24 hours
All supplies for cell culture were delivered by NV Life Technologies Belgium except
for Fetal Calf Serum (Invitrogen Corporation UK)
Irradiation sources
In this study two light sources a Light Emitting Diode (LED) device and a Low Level
Laser (LLL) device were used in comparison to control cultures
The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)
LED induced increase of fibroblast proliferation
51
with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400
mW and a frequency range from 0-1500 Hz
The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)
consisted of three wavelengths emitted by separate probes A first probe emitting
green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the
red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third
probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared
light The area of all three probes was 18 cm2 and their frequency was variable within
the range of 0-1500 Hz
Exposure regime
Prior to irradiation the 96-well plates were microscopically verified to guarantee that
the cells were adherent and to assure that there was no confluence nor contamination
Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining
25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation
The 96-well plates were randomly assigned in the treated (LLL or green red or infrared
LEDrsquos) or the control group
For the treatments in this study the continuous mode was applied as well for the LLL
as for the three LED-probes The distance from light source to fibroblasts was 06 cm
LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting
in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant
exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to
exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective
power output of 160 mW 80 mW or 10 mW
After these handlings the remaining medium was removed and new Hanksrsquoculture
medium was added followed by 24 hours of incubation
One irradiation (LLL or LED) was performed daily during three consecutive days
according to the aforementioned procedure Control cultures underwent the same
handling but were sham-irradiated
Chapter 2
52
Determination of cell proliferation
The number of cells within the 96-well plates as a measure for repair [19] was
quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The
colorimetric assay was performed at two different points of time to determine the
duration of the effect of the used light sources
This assay exists of a replacement of Hanksrsquoculture medium by fresh medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as
described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT
solution was substituted by lysing buffer isopropyl alcohol The plates were
temporarily shaken to allow dissolution of the produced formazan crystals After 30
minutes of exposure to the lysing buffer absorbance was measured The absorbance at
400 to 750 nm which was proportional to fibroblast proliferation was determined
using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)
The complete procedure from isolation to MTT assay was executed six times (Trial A
B C D E and F) while it was impossible to irradiate all the investigated number of
wells with the same LED apparatus on one day All the trials included as much control
as irradiated wells but the number of control and irradiated wells in each trial varied
depending on the number of available cells after the second subculturing A further
consequence of the available number of cells is the number of probes examined per
trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E
Incubation period before proliferation analyses numbered 24 hours To investigate if
the stimulatory effect tends to occur immediately after irradiation or after a longer
period of time incubation in trial F lasted 72 hours
An overview of the followed procedures regarding incubation time before proliferation
analysis number of analysed wells for each trial and the number of probes examined
per trial is given in table 1 As a consequence of the differences in procedures followed
and because each trial started from a new cell line the results of the five trials must be
discussed separately
LED induced increase of fibroblast proliferation
53
Statistical analysis
Depending on the amount of groups to be compared within each trial and depending
on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way
ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons Statistical significance for all tests was
accepted at the 005 level For this analysis Statistical Package for Social Sciences 100
(SPSS 100) was used
RESULTS
The results presented in table 1 show that cell counts by means of MTT assay
revealed a significant (plt0001) increase in the number of cells in comparison to their
respective sham-irradiated controls for all the irradiated cultures of trial A B C D
and E except the irradiated groups in trial F
Moreover the results of trial A showed that the effect of the green and red LED probe
was significantly (plt0001) higher than the effect of the LLL probe With regard to the
amount of proliferation the green probe yielded a significantly higher number of cells
than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe
provided a higher increase in cells (plt0001) than the infrared probe
The infrared LED source and the LLL provided a significant (plt0001) higher number
of cells than the control cultures but no statistical significant difference was recorded
between both light sources
The trials A B C D and E regardless of the number of probes used in each trial
were analysed after 24 hours of incubation after the last irradiation The incubation
period of trial F lasted 72 hours
The means of trial F illustrated that the effect was opposite after such a long
incubation The control cultures had significantly (plt0001) more fibroblasts than the
irradiated cultures with the exception of the LED-infrared group that showed a not
significant increase of cells Further analysis revealed that the green probe yielded a
significantly lower number of cells than the red (plt0001) and the infrared probe
(plt0001) and that the red probe provided a higher decrease (plt0001) than the
Chapter 2
54
infrared probe Laser irradiation induced a significant decrease of fibroblasts in
comparison to the infrared irradiated cultures (plt0001) and the control cultures
(p=0001) LED irradiation with the green and the red probe revealed no statistical
significant differences
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups
Absorbency (proportional to the number of fibroblasts)a
Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050
Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis
DISCUSSION
Despite the failure of some studies [223] to demonstrate beneficial effects of laser and
photodiode irradiation at relatively low intensities (lt500mW) on fibroblast
LED induced increase of fibroblast proliferation
55
proliferation this study provides experimental support for a significant increased cell
proliferation Therefore these results confirm previous studies that yielded beneficial
stimulating effect [1152425] Remarkably though is the higher increase noted after
irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general
decrease in absorption at longer wavelengths and concluded that several molecules in
fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445
470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the
closest to one of these peaks
Karu [5] also emphasises that the use of the appropriate wavelength namely within the
bandwidth of the absorption spectra of photoacceptor molecules is an important
factor to consider
In this particular context penetration depth can almost be ignored as virtually all
wavelengths in the visible and infrared spectrum will pass through a monolayer cell
culture [12] The irradiance (Wcm2) on the contrary could have had an important
influence on the outcome of this study The higher increased proliferation by the lower
wavelengths is possibly a result of the lower irradiance of these wavelengths Lower
irradiances are confirmed by other experiments to be more effective than higher
irradiances [111626]
The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as
described by Poumlntinen [17] but in the scope of these results it also needs to be noticed
that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)
the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)
Consequently the results of especially trial A and F must be interpreted with the
necessary caution It is possible that the determined distinction between the used light
sources and the used probes is a result from the various radiant exposures applied
during the treatments of the cultures
Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after
the last irradiation this study was unable to demonstrate a stimulating effect when
analysis was performed 72 hours after the last irradiation Moreover this longer
incubation period even yielded an adverse effect Although a weakening of the
Chapter 2
56
photostimulating influence over time is acceptable it can not explain a complete
inversion Especially in the knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period [2427] In an attempt to illuminate
this finding one can suppose that the circadian response of the cells triggered by the
LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark
The most obvious explanation is even though a decreased vitality and untimely cell
death in the irradiated cell cultures as a result of reaching confluence at an earlier point
of time than the control cultures The cells of a confluent monolayer have the tendency
to inhibit growth and finally die when they are not subcultured in time No other
reasonable explanations could be found for this discrepancy
Photo-modulated stimulation of wound healing is often viewed with scepticism The
real benefits of Light Emitting Diodes if any can only be established by histological
and clinical investigations performed under well controlled protocols Despite these
remarks this study suggests beneficial effects of LED and LLL irradiation at the
cellular level assuming potential beneficial clinical results LED application on
cutaneous wounds of human skin may be assumed useful at the applied dosimetric
parameters but future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Persons in good health rarely require treatment for wound healing as posed by Reddy
et al [13] light has a possible optimal effect under conditions of impaired healing
Postponed wound healing is a time-consuming and often expensive complication
Thus future prospects must remind to examine the therapeutic efficacy of LED on
healing-resistant wounds
LED induced increase of fibroblast proliferation
57
ACKNOWLEDGMENTS
The authors are grateful to Prof Deridder for supplying the laboratory as well as the
material necessary for this investigation and to Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
Chapter 2
58
REFERENCES
1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55
2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32
3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7
4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994
5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998
6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32
7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42
8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5
9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8
10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7
11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8
12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34
13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9
14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18
15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43
16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6
17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475
18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J
Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9
21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601
22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63
LED induced increase of fibroblast proliferation
59
23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9
24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301
25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are
more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37
27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8
28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26
CHAPTER 3
GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES
FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL
Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Photomedicine and Laser Surgery 2005 23(2) 167-171
Chapter 3
62
ABSTRACT
Background and Objective The chronic metabolic disorder diabetes mellitus is an
important cause of morbidity and mortality due to a series of common secondary
metabolic complications such as the development of severe often slow healing skin
lesions
In view of promoting the wound-healing process in diabetic patients this preliminary
in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on
fibroblast proliferation and viability under hyperglycemic circumstances
Materials and Methods To achieve hyperglycemic circumstances embryonic chicken
fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL
glucose LED irradiation was performed on 3 consecutive days with a probe emitting
green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min
resulting in a radiation exposure of 01 Jcm2
Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all
irradiated cultures in comparison with the controls
Conclusion According to these results the effectiveness of green LED irradiation on
fibroblasts in hyperglycemic circumstances is established Future in vivo investigation
would be worthwhile to investigate whether there are equivalent positive results in
diabetic patients
Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes
Fibroblast proliferation under hyperglycemic circumstances
63
INTRODUCTION
The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide
geographic variation in incidence and prevalence generally 11 of the world
population is affected and worldwide it is the twelfth leading cause of death1 Those
figures may be higher for urban regions as well as for industrialized countries Due to
multiple factors involving the aging process of the population and lifestyle changes
(such as reduced physical activity hypercaloric eating habits and concomitant obesity)
these figures may increase in the future2-6 Therefore diabetes mellitus could become
the most common chronic disease in certain regions as stated by Gale it ldquotargets the
rich in poor countries and the poor in rich countriesrdquo6
The harmful disruption of the metabolic equilibrium in diabetes mellitus results in
characteristic end-organ damage that occurs in various combinations and that follows
an unpredictable clinical pathway
Accordingly the major consequence of diabetes mellitus in terms of morbidity
mortality and economic burden principally concerns macroangiopathies or
arteriosclerosis and microangiopathies including nephropathy neuropathy and
retinopathy7-10
One of these devastating consequences which often appears in time is the
development of various skin defects that are frequently resistant to healing and that
tend to be more severe than similar lesions in nondiabetic individuals Diabetes
mellitus even increases the risk of infection by an increased susceptibility to bacteria
and an impaired ability of the body to eliminate bacteria1112
Skin problems are a severe complication in diabetic individuals and require a
comprehensive and appropriate multidisciplinary approach to prevention and
treatment12
Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to
play the most prominent role in the development of diabetic complications With the
development of insulin treatment for type I diabetes and various oral hypoglycemic
agents for type 2 diabetes a reduction in the development of skin defects due to
hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply
Chapter 3
64
waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in
diabetic patients often need special care in comparison to those persons in good
health who rarely require treatment for wound healing1617 Special care is directed
besides of course toward optimal diabetes regulation toward patient education
maximum pressure relief controlling infection recovery of circulation in case of
ischemia and different modalities of intensive wound treatment18
In the last few years various therapies have been introduced with varying success An
example of such a therapy is the photo-modulated stimulation of diabetic lesions In
vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been
subject of extensive investigation1920 Due to contradictory research results LLL-
photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of
light in the domain of wound healing is less time-consuming less expensive less
invasive than many of the other introduced treatment modalities and practical to use
however it seems worthwhile to investigate the value and benefits of a newly
introduced and alternative light source the light emitting diodes (LEDrsquos)
Preliminary research has proved that green LED with particular properties (an
exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01
Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These
results may be of great importance to the diabetic patient because as posed by Reddy et
al light has a possible beneficial effect in the case of impaired healing1617
To obtain insight into the ability of LED to stimulate fibroblast proliferation under
diabetic-specific conditions of impaired healing the proliferation was assessed in
irradiated and control cultures cultivated in medium with a high quantity of glucose
MATERIAL amp METHODS
Cell cultivation
Primary fibroblast cultures were established by outgrowth from 8-day-old chicken
embryos After isolation and disaggregating as described by Freshney (1994)24 the cells
were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10
Fibroblast proliferation under hyperglycemic circumstances
65
fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin
Secondary cultures were initiated by trypsinization followed by plating of the cells in
80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were
disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently
231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third
passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the
cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified
atmosphere at 37deg C to allow them to attach to the bottom of the wells
Light source specifications and illumination procedure
To control adherence of the cells and to assure that there was no confluence or
contamination the 96-well plates were microscopically examined before irradiation
Subsequently the tissue culture plates were randomly assigned for use in the treated
and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium
was aspirated The remaining 25 (50 microl) medium avoided dehydration of the
fibroblasts throughout irradiation
Irradiation was performed with a light emitting diode (LED) device The LED device
(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power
range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable
within the range of 0-1500 Hz
The investigation used the following illumination properties the continuous mode a
distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2
radiant exposure This procedure resulted in an exposure time of 3 min and a power
output of 10 mW Immediately after irradiation the remaining medium was aspirated
and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM
glucose (30 gL) and incubated at 37deg C
Irradiation and medium changes occurred at 1-day intervals so one irradiation was
implemented each 24 h for 3 days in a row and from the first irradiation onwards all
medium renewals occurred with glucose-supplemented Hanksrsquo culture medium
Control cultures were handled in the same manner but were sham-irradiated
Chapter 3
66
Proliferation assay
Fibroblast survival and proliferation were determined by a sensitive and reproducible
colorimetric assay the assay which detects merely living cells and the signal generated
bears a constant ratio to the degree of activation of the fibroblasts and the number of
fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than
only a small subsample26
Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented
Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution
was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during
30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous
solution The optical density of the final solution was measured on an ELtimes800 counter
(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test
wavelength varying from 400 to 750 nm
The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies
for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except
for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)
Data analysis
On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a
Mann-Whitney U test was performed for nonparametrical comparison of the results
Statistical significance for all tests was accepted at the 005 level For this analysis the
Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used
RESULTS
The MTT measurements from each of the 256 control wells and 256 irradiated wells
and the subsequent nonparametrical analysis from the optical densities obtained
disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic
Fibroblast proliferation under hyperglycemic circumstances
67
circumstances after irradiation than in the same circumstances without irradiation (Fig
1)
Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)
DISCUSSION
The outcome of these in vitro experiments based on the above-described light source
properties and the illumination procedure described clearly demonstrated the
stimulatory potential of LED on fibroblast proliferation and the cell viability of
fibroblasts cultured in hyperglycemic medium Preliminary research has already
demonstrated that under these conditions (an exposure time of 3 min a wavelength of
570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this
procedure allowed the highest number of living cells The nature of the light and the
usual questions concerning coherence wavelength power output and radiant
exposures have been discussed previously23
Although these findings confirm the results previously found one cannot ignore the
important methodological difference between previous investigations and the current
study as the cells in this experiment were cultured in hyperglycemic medium2328-30
Absorbency - Proportional to the number of fibroblasts
621 x 10-1 682 x 10-1
0010203040506070809
1
Control Irradiated
Groups
Ab
sorb
ency
Chapter 3
68
After a growth period with normal Hanksrsquo culture medium a necessary step to ensure
normal growth of these secondary subcultures and normal attachment to the bottom
of the wells the Hanksrsquo culture medium was supplemented with glucose
Several earlier studies have established that exposure to glucose concentrations (20-40
mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human
vascular endothelial cell proliferation1531-34 This restraint is more pronounced for
higher glucose15 concentrations and is expressed especially after protracted exposure to
high glucose levels31 A similar restraint was found for cultured fibroblasts by
Hehenberger et al3536 According to some authors however cultured fibroblasts
conversely have been shown to maintain responsiveness to ambient high glucose323738
As there are some ambiguities in literature regarding normal or inhibited growth of
fibroblasts in medium supplemented with glucose39 a pilot study was performed to
determine the amount of glucose necessary to inhibit normal growth after 72 h of
culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study
demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease
of cell viability and to bring about a decline in fibroblast proliferation
This concentration resulted in a remarkable reduction of cell viability and a noteworthy
decrease in the proliferation rate in comparison to control cultures grown in 55 mM
glucose although this concentration is too high to mimic severe diabetic
hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to
investigate the effect of LED in distinct destructive conditions in order to obtain an
incontrovertible result
In addition it is possible that the present investigation needed a higher amount of
glucose to result in a remarkable reduction of proliferation as exposure to glucose was
limited to 72 h and as previous studies revealed that the antiproliferative effect of high
glucose was more pronounced with prolonged exposure with a maximal inhibition
attained by 7-14 days1531
Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo
cell growth are too complex to compare A key question is whether fibroblast
senescence in tissue culture and in the intact organism are similar Cristofalo et al40
Fibroblast proliferation under hyperglycemic circumstances
69
reported that this is not the case as fibroblasts have a finite ability to divide and
replicate but apparently the pathway or the morphologic characteristics leading to the
replicative senescence is not identical in vivo compared to in vitro
Furthermore extrinsic aging related to environmental damage which in diabetic
patients is mainly due to a chronic exposure to high levels of glucose during life is
unachievable in vitro
Unless a number of questions regarding the mechanism according to which LED
stimulates fibroblast proliferation in this particular condition remain unanswered the
results ascertain the potential effects of LED on fibroblast proliferation and viability
CONCLUSION
The current results should be interpreted with caution However these results
demonstrate the effectiveness of green LED irradiation at the above-described light
source properties and the illumination procedure described on cells in hyperglycemic
circumstances
The findings of the present study using an experimental in vitro model indicate that the
use of LED irradiation to promote wound healing in diabetic patients may have
promising future results As the present study establishes the possibility of using LED
irradiation in experimental in vitro situations it would be a worthwhile extension to
perform in vivo investigations to determine whether these in vitro observations were
relevant to the physiological situation and to determine the effect of these LED
properties on human tissue response
ACKNOWLEDGMENTS
The authors are greatly indebted to P Coorevits for assistance with the statistical
analysis and to Professor L Deridder and Ms N Franccedilois of the department of
Human Anatomy Embryology Histology and Medical Physics for providing access to
the laboratory and for helpful discussions
Chapter 3
70
REFERENCES
1 World Health Organisation The world health report of 2002 Statistical Annex 2002 pp 179-201
2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418
3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957
4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538
5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308
6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J
Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot
wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-
glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A
Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood
glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13
14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309
15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501
16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287
17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255
18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412
19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192
20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137
21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675
22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246
23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99
Fibroblast proliferation under hyperglycemic circumstances
71
24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729
27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63
28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301
29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -
progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of
cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA
damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in
culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia
23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar
K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71
36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141
37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588
38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252
39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97
40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619
PART II ANALGESIA
CHAPTER 4
EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION
MEDIATED BY LIGHT EMITTING DIODE IRRADIATION
Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy
Vanderstraetenab and Dirk Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Lasers in Medical Science 2005 20(1) 35-40
Chapter 4
76
ABSTRACT
The introduction of light emitting diode (LED) devices as a novel treatment for pain
relief in place of low-level laser warrants fundamental research on the effect of LED
devices on one of the potential explanatory mechanisms peripheral neurophysiology in
vivo
A randomised controlled study was conducted by measuring antidromic nerve
conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline
measurement and five post-irradiation recordings (2 min interval each) were performed
of the nerve conduction velocity (NCV) and negative peak latency (NPL)
Interventional set-up was identical for all subjects but the experimental group (=32)
received an irradiation (2 min at a continuous power output of 160 mW resulting in a
radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype
MDB-Laser Belgium) while the placebo group was treated by sham irradiation
Statistical analysis (general regression model for repeated measures) of NCV and NPL
difference scores revealed a significant interactive effect for both NCV (p=0003) and
NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical
significant decreased NCV and an increased NPL in the experimental group and a
statistical significant difference between placebo and experimental group at various
points of time
Based on these results it can be concluded that LED irradiation applied to intact skin
at the described irradiation parameters produces an immediate and localized effect
upon conduction characteristics in underlying nerves Therefore the outcome of this in
vivo experiment yields a potential explanation for pain relief induced by LED
Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative
peak latency middot Analgesic effect
Nerve conduction characteristics
77
INTRODUCTION
Since the introduction of photobiostimulation into medicine the light sources used
have advanced technologically and varied in characteristics over the years
Advancement and variation of the sources implicate a concomitant necessity to revise
research results in the respective domains of application Research and clinical
applications in the past particularly focused on the effectiveness of low-level lasers
have shifted now to novel treatment units such as light emitting diode (LED) devices
The efficacy and applicability of LED irradiation within the field of wound healing has
already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED
is not only promoted for its beneficial effects on the wound-healing process it is also
suggested to be potentially effective in the treatment of pain of various aetiology
although this claim has not yet been investigated thoroughly either experimentally or
clinically The putative analgesic effects of LED remain to be further explored
As the basic vehicle of pain is the neuronal system [7] measuring the
neurophysiological effect of LED treatment would be an appropriate experimental
approach to investigate the efficacy of LED on pain inhibition Nerve conduction
studies have become a technique for investigating the neurophysiologic effects of light
therapy [8-9]
Review of literature regarding standard nerve conduction studies revealed that previous
human studies on the influence of various light sources on peripheral nerves have
utilized different methods which hampers a comprehensive comparison In general
this research was performed on the superficial radial nerve [10-13] described by Shin J
Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve
[891315-17] Following the method of Cambier et al [18] the authors of this study
decided to investigate the effect of the light source used on the conduction
characteristics of the sural nerve By investigating this solely sensory nerve interaction
of motor nerve fibres (motor response can easily be provoked by antidromic nerve
stimulation [19]) can be avoided and given the superficial nature of the nerve it should
be sufficiently amenable to the effects of percutaneous LED irradiation
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78
A second major difference between the trials and therefore also hindering an
appropriate comparison between the results is the wide range of used light sources
HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared
multisource treatment unit [15]
With respect to the potential importance of LED irradiation for the treatment of pain
the current investigation was designed to assess the putative neurophysiological effects
of LED on the sensory nerve conduction of the human superficial peripheral sural
nerve and to establish a time course of the supposed phenomenon
The experimental hypothesis postulates that LED generates an immediate decrease in
conduction velocity and increase in negative peak latency In addition it can be
postulated that this effect is most prominent immediately after the irradiation and will
weaken as time progresses
STUDY DESIGN
The study was approved by the Ethical Committee of the Ghent University Hospital
After explanation of the experimental procedure a written informed consent was
obtained from each subject
Subjects
After screening based on a brief medical history excluding subjects with
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever inflammation of the skin) or conditions
that might affect sensory nerve conduction (such as diabetes peripheral neuropathy
radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral
edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40
females (mean age 26plusmn6 years range 18-42 years) participated in this study The body
mass index (BMI) of each subject varied within the normal range (=185-249) [20]
(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo
Nerve conduction characteristics
79
or an experimental group Each group of 32 subjects was composed of 12 males and
20 females
Experimental Procedure and Data Acquisition
In order to be able to quantify the negative peak latency (NPL) (measured from the
start of the stimulus artefact to the peak of the negative portion of the nerve action
potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was
followed
With respect to the known relationship between nerve conduction characteristics and
temperature the ambient temperature was kept constant (23ordmC-26ordmC room
temperature) during the investigation In view of this temperature issue the
standardized protocol started with 10 min of accommodation during which the
subjects rested in prone position on a treatment table
Immediately before this adjustment period the skin over the dorsolateral aspect of the
left calf and foot was cleaned with alcohol to remove surface lipids This preparation of
the treatment area was followed by the placement of the electrodes (TECA
Accessories Oxford Instruments Medical Systems Division Old Woking UK) as
described by Delisa et al [21]
The two-posted (2 cm separation anode distal) surface caption electrode was placed
distal and posterior of the lateral malleolus on the skin covering the sural nerve The
fixation of the earth electrode (Medelec Oxford Instruments Medical Systems
Division Old Woking UK) occurred 12 cm above the caption electrode according to
the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm
above the caption electrode to map the ideal stimulation point To level off
intraindividual variations in the amount of sensory response attributable to the
successive placement of the bipolar stimulator in course of the investigation a two-
posted (2 cm separation cathode distal) bar stimulating electrode was attached at the
point where the maximal response was obtained
This placement of the electrodes allows antidromic stimulation of the sural nerve
Electrophysiological stimulation and recordings were obtained with a Medelec
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80
Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse
of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was
used to produce each evoked sensory response
Baseline measurements of NPL and NCV were immediately followed by treatment of
the subjects according the protocol detailed below Recordings were subsequently
repeated at 2-min intervals over an 8-min period resulting in five recordings (one
immediately after the completion of the treatment and one at 2 4 6 and 8 min after
irradiation) Skin temperature was recorded concomitantly throughout the procedure
at the time of baseline measurement immediately after LED irradiation at the time of
the first recording and consequently at 2-min intervals together with the four final
electrophysiological recordings For this a surface digital C9001 thermometer
(Comark UK) sensitive to temperature changes of 01degC was used at the same point
of LED administration namely at 7 cm above the caption electrode The procedure
was identical for both conditions but subjects in the placebo group received a sham
LED irradiation
Light Characteristics and Irradiation Procedure
Irradiation was administrated with a light emitting diode device (BIO-DIO
preprototype MDB-Laser Belgium) The probe used emitted infrared light with a
wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2
and the frequency was variable within the range of 0-1500 Hz
Preceding baseline measurement the treatment point was marked on the skin overlying
the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-
point between the stimulation and capture electrode The LED probe was held in
contact with the skin perpendicular to the skin surface during the complete irradiation
procedure LED treatment consisted for all subjects of the experimental group out of 2
minutes lasting irradiation The LED was set to deliver a continuous energy density of
107 Jcm2 at a power output of 160 mW These parameters were selected as they are
appropriate for the treatment of pain in a clinical setting First of all because the
Nerve conduction characteristics
81
duration of the treatment is clinically feasible and secondly because the parameters are
within the scope of previously described light source characteristics [1-36915]
Statistics
Although superficial skin temperature did not change significantly in course of the
investigation the influence of the measured skin temperature on NPL and NCV was
taken into account by using a correction factor of respectively 02 msdegC and 147
ms degC All corrections were calculated towards a reference skin temperature of 32degC
Difference scores ie the variation between baseline measurements and each post-
irradiation recording were used as the basis for statistical analysis A General
Regression Model for repeated measures with one within-subjects factor (time 0
min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and
one between-subjects factor (group placebo or LED irradiated) was performed
followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least
Significant Difference) to determine whether any differences between baseline
measurements and post-irradiation recordings were statistically significant
The Statistical package for social sciences (SPSS 110) was used for analysis and
statistical significance for all tests was accepted at the 005 level
RESULTS
Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated
group plotted against time in minutes The values of the irradiated subjects decrease
directly after the irradiation and reach a first low point 2 min after finishing LED
treatment This decrease is followed by a marginal increase at 4 and 6 min and again an
important decrease at 8 min Statistical analysis (general regression model for repeated
measures) of these data indicated a significant interactive effect (P=0003)
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82
Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)
Post hoc LSD further showed significant differences between baseline measurements
and all post-treatment recordings (Table 1) Mutual comparison of the values from the
post-treatment recordings did not reveal any significant difference In addition there
was no significant difference determined in the placebo group in course of time
Table 1 Summary of the influence of LED irradiation on nerve conduction velocity
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001
2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002
4 0111plusmn0377 0647 -0908plusmn1898 0021 0004
6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001
8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve Conduction Velocity
-14
-12
-1
-08
-06
-04
-02
0
02
04
Baseline 0 min 2 min 4 min 6 min 8 min
Time Course
Dif
fere
nce
Sco
re (
m
s)
PlaceboLED
Nerve conduction characteristics
83
A similar representation was used for the results of the NPL Figure 2 reproduces NPL
plotted against time in minutes revealing for the irradiated group an increased latency
with two important peaks one at 4 min and one at 8 min
Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)
Statistical analysis of the mean difference scores again indicated a significant interactive
effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2
showed significant differences between baseline measurements and all post-treatment
recordings of the experimental group The mean difference score of the first post-
treatment recording of this same group (LED irradiated) differed significantly with the
recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED
irradiation As well as the recording 2 min after irradiation which differed significantly
(P=0013) with the 8 min post-treatment recording As observed for the NCV the
NPL of the placebo group did not reveal any significant difference in time course
At the time of the final recording the NCV and NPL mean difference scores of the
irradiated group did not return to their respective baseline values
Negative Peak Latency
-001
0
001
002
003
004
005
006
007
Baseline 0 min 2 min 4 min 6 min 8 min
Time course
Dif
fere
nce
Sco
re (
ms)
PlaceboLED
Chapter 4
84
Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group
significance) revealed statistical differences between the experimental and the placebo
group for NCV as well as for NPL NCV and NPL were statistical significant between
both groups at all points of time except from the NPL recording immediately after
finishing irradiation
DISCUSSION
Notwithstanding the above-mentioned difficulties in comparing results between
different trials on nerve conduction we attempt to discuss the current findings in view
of the results of the previous studies
This investigation revealed that percutaneous LED irradiation at feasible and current
clinical parameters generates measurable and significant changes in human sural nerve
antidromic conduction latency and velocity These results thus support previous
findings of light-mediated nerve conduction latency shifts in vivo [8101218]
although there are several important issues to be discussed
Table 2 Summary of the influence of LED irradiation on negative peak latency
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0004 plusmn0053 0755 0029plusmn0080 0019 0145
2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021
4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004
6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034
8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve conduction characteristics
85
A first comment deals with the progress of the NCV and NPL in function of time As
postulated the NCV decreases significantly immediately after irradiation
corresponding with a significant increased NPL However this effect does not weaken
as time progresses both variables remain significant throughout the 8 min during
observation period
Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the
conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]
although this slight increase in NPL was not significant at any moment Two other
studies [822] with a GaAlAs laser even registered comparable effects over a period of
55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous
studies post-treatment conduction measurements should be extended in time At
present for all studies it remains unclear at what point of time the effect extinguishes
although the interval of time during which LED treatment remains effective is
clinically important when treating pain
Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least
45 min) mediated by a monochromatic multisource infrared diode device although it
needs to be mentioned that this study performed with a comparable light source as the
current investigation revealed a significant decrease of the NPL These inverse results
between the study of Noble et al [15] and the current investigation could be attributed
to the concomitant increase of the skin temperature [15] As it has been well
recognised that a variation in tissue temperature causes a corresponding alteration in
nerve conduction velocities and peak latencies [91523-27] the temperature changes
may indeed provide an explanation for the observed findings In an attempt to analyse
the influence of a direct photobiological effect on sural nerve conduction
characteristics rather than working out the effects based upon thermal mechanisms
the present study corrected the skin temperature towards a reference temperature of
32degC This correction was performed notwithstanding the fact that the superficial skin
temperature did not change significantly before and after LED irradiation as well as
despite the fact that influencing nerve temperature takes place long after affecting skin
temperature [23] and thus being (almost) impossible after 2 min of irradiation
Chapter 4
86
followed by 8 min of registration Introduction of the correction factor implies likewise
that eventual influence on nerve conduction by cooling of the limb due to inactivity as
described by Greathouse et al [11] can be excluded
These facts suggest that temperature changes did not contribute to the demonstrated
effects of LED on nerve conduction Nevertheless the underlying mechanism of the
observed effects remains indistinct
A following remark regarding the fluctuation of NCV and NPL in function of time
considers the fact that both the NCV and the NPL do not change in a constant way up
to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the
increase in NPL display a small though not significant inversion of the effect at 4 and
(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of
fluctuation is to be expected when measuring NCV and NPL besides there is a similar
variation in the placebo groups
Although investigating dose dependency was not intended an additional remark
considers the fact that the use of optimal irradiation parameters is essential to obtain
the observed neurophysiological effect Nevertheless it is impossible to determine
ideal light source characteristics for effective treatment as the range of used
wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency
(pulsed or continuous) are not sufficiently similar between the different studies It can
only be concluded that a pulsing light source [91028] does not provide the postulated
results Radiant exposure exposure time power range and wavelength are not yet
established but based on this study and previously described assays it can be
speculated that the ranges of these parameters are quite large
In comparison with other studies where the number of subjects is 10 or less [8-
1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-
Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number
of subjects (n=32) was investigated in each group In spite of the large investigated
population it should be noted that the magnitude of the described changes in NCV
and NPL can simply be replicated by lowering the temperature of the extremity as the
observed changes are within the expected physiological ranges making the clinical
Nerve conduction characteristics
87
significance of the change questionable (This fact does not implement that the
decrease and the significant changes were temperature mediated)
A key question and meanwhile the initial impetus for future investigation is whether
the measured effects can be extrapolated to the actual nociceptive afferents namely the
myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])
respectively conducting acute and chronic pain The functional testing of these
nociceptive pathways has recently been extensively evaluated The currently accepted
neurophysiological method of assessing nociceptive pathways relies on laser-evoked
potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]
As up till now LEP is not available in this or any surrounding research centre the
investigators of this study had to perform a standard nerve conduction study (assessing
the large myelinated Aβ afferents) Therefore the current and previous beneficial
results of low level light therapy on conduction characteristics of nerves in vivo should
initiate measurements of clinical effectiveness first of all in laboratory settings and
afterward at a clinical level
CONCLUSION
Despite these remarks and the limited knowledge regarding the underlying mechanism
the present findings enable the following conclusions to be drawn LED irradiation at
clinical applied energy densities produces an immediate and localized effect upon
conduction characteristics in underlying nerves More specifically it is proven that
LED treatment lowers the NCV and augments the NPL resulting in a reduced
number of impulses per unit of time Therefore the outcome of this in vivo experiment
assumes that LED possibly induces pain relief
In order to encourage a widespread acceptance for the use of this non-invasive pain-
reducing modality in clinical settings prospective research should establish the precise
relationship between LED and pain relief as well as determine the ideal irradiation
parameters and verify which painful conditions can be treated with this treatment unit
Chapter 4
88
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the
equipment and MDB-Laser Belgium for generously providing the Light Emitting
Diode equipment
Nerve conduction characteristics
89
REFERENCES
1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9
2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)
3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18
4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8
5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43
6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65
7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53
8 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-34
9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6
10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90
11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7
12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5
13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9
14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore
15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5
16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5
17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604
18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200
19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21
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20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083
21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press
22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9
24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9
25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203
26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13
27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12
28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9
29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445
30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8
31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56
CHAPTER 5
PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE
IRRADIATION A PILOT STUDY ON EXPERIMENTALLY
INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS
Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Accepted for publication in Lasers in Medical Science December 2005
Chapter 5
92
ABSTRACT
Objective The present pilot study investigated the analgesic efficacy of light emitting
diode (LED) In view of a standardised and controlled pain reduction study design this
in vivo trial was conducted on experimentally induced delayed-onset muscle soreness
(DOMS)
Design Thirty-two eligible human volunteers were randomly assigned to either an
experimental (n=16) or placebo group (n=16) Immediately following the induction of
muscle soreness perceived pain was measured by means of a visual analog scale (VAS)
followed by a more objective mechanical pain threshold (MPT) measurement and
finally an eccentricconcentric isokinetic peak torque (IPT) assessment The
experimental group was treated with infrared LED at one of both arms the other arm
served as control Irradiation lasted 6 min at a continuous power output of 160 mW
resulting in an energy density of 32 Jcm2 The subjects of the placebo group received
sham irradiation at both sides In post-treatment a second daily assessment of MPT
and VAS took place The treatment and assessment procedure (MPT VAS and IPT)
was performed during 4 consecutive days
Results Statistical analysis (a general linear model followed by post hoc least
significant difference) revealed no apparent significant analgesic effects of LED at the
above-described light parameters and treatment procedure for none of the three
outcome measures However as the means of all VAS and MPT variables disclose a
general analgesic effect of LED irradiation in favour of the experimental group
precaution should be taken in view of any clinical decision on LED
Conclusion Future research should therefore focus on the investigation of the
mechanisms of LED action and on the exploration of the analgesic effects of LED in a
larger randomised clinical trial and eventually in more clinical settings
Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset
muscle soreness middot Musculus biceps brachii
Delayed-onset muscle soreness
93
INTRODUCTION
The analgesic efficacy of light emitting diode (LED) irradiation is recently being
investigated by means of a nerve conduction study on the superficial peripheral sural
nerve [1] It was demonstrated that LED irradiation at clinical applied densities
produces an immediate and localized effect upon conduction characteristics in
underlying nerves More specific LED induces a decreased number of sensory
impulses per unit of time thus possibly inducing pain relief [1]
Given the established influence of this treatment modality on the nerve conduction
velocity and thereby its potential analgesic ability the current investigation was
designed
Studies investigating the efficacy of a therapeutic modality on pain often experience
difficulties regarding standardisation of the population as analysis or comparison of
pain with different aetiologies is almost impossible Therefore we opted to measure the
analgesic effects of LED in a laboratory setting on a sample with experimentally
induced delayed-onset of muscle soreness (DOMS)
Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the
induction exercise and then spreads throughout the muscle [2-4] The correlates of
DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10
[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force
decreased range of motion and in particular muscle pain which is more pronounced
during movement and palpation [8 11] Despite the large volume of research that has
been undertaken to identify the underlying pathophysiology of DOMS the precise
mechanism is not yet universally accepted Several theories such as the torn-tissue
theory the connective tissue damage theory the muscle spasm theory and the
inflammation theory still remain viable though the current opinion states that DOMS
arises from a sequence of events in which several theories occupy an important place
[2 6 12 13]
DOMS has been used as a representative model of musculoskeletal pain and stiffness
in a number of studies [4 7 11 14 15] as it has a number of advantages it can be
induced in a relatively easy and standardised manner in a group of healthy subjects the
Chapter 5
94
time-course is relatively predictable and the symptoms have the same aetiology and are
of transitory nature [14 16] Nevertheless it should be emphasised that the use of this
particular experimental model to test the effectiveness of LED does not mean that this
treatment modality is necessarily advocated for the treatment of DOMS but merely
that it may be helpful in documenting the efficacy of LED in a clinical model of
musculoskeletal pain and stiffness In addition studies based on the induction of
DOMS under carefully controlled laboratory conditions can not replace research
involving actual patients but offer the opportunity to assess the effectiveness of
particular therapeutic interventions and might help to define additional clinical research
[14]
The experimental hypothesis of the current study postulates that infrared LED reduces
pain and muscle sensitivity associated with DOMS
MATERIALS AND METHODS
The study was approved by the ethical committee of the Ghent University Hospital
After providing information regarding the study design and possible consequences
related to participation at the study written informed consent was obtained from each
subject
Subjects
Healthy human volunteers were recruited from the university population Individuals
with any upper limb pathology neurological deficit and recent injury to either upper
extremity or undiagnosed pain were excluded Other exclusion criteria were
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever and inflammation of the skin) or
conditions in which physical exertion is contraindicated (such as cardiovascular deficits
hypertension and respiratory problems)
Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age
23plusmn4 years) were enrolled All subjects were randomly assigned using a random table
Delayed-onset muscle soreness
95
of numbers to the experimental or placebo group Each group of 16 subjects
consisted by stratification of equal numbers of men and women Age height and
weight did not differ significantly between the three groups
All subjects were physically active however none performed on a regular basis any type
of upper body weight-training Subjects were requested to refrain from any form of
strenuous physical activity and they were asked to avoid any form of medication
including anti-inflammatory agents as well as alcohol for 2 days before testing and for
the duration of the study
Overview of experimental design
The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to
induce pain related to DOMS Immediately following induction exercise an initial
assessment of the outcome measures (visual analog scale or VAS mechanical pain
threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the
subjects were treated under blinded conditions according to the randomised group
allocation In post-treatment the MPT was re-recorded and perceived pain was
reassessed with a VAS Contrary to these outcome measures the muscle strength was
only measured in pre-treatment at the one hand because short-term effects of LED
on muscle strength were not postulated and on the other hand because post-
treatment muscle strength can be influenced by too many different physiological
factors related to the pre-treatment measurement On the succeeding days (day 2 3
and 4) the treatment and assessment procedure was similar with approximately 24 h
separating each treatment
In both of the groups the two arms of the participants were included in the study In
the experimental group an equal number of dominant and non-dominant arms were
treated The non-treated arm served as control arm In the placebo group also an equal
number of dominant and non-dominant arms were considered as treated arm and the
other arm was classified in the non-treated group The procedure was identical for
both conditions but the subjects in the placebo group received sham LED irradiation
on both arms
Chapter 5
96
Specific aspects of the experimental design and procedures are detailed below
Pain induction
Muscle soreness was induced in a standardised fashion via a daily calibrated computer-
operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY
USA) Induction occurred separately and in random order in the elbow flexors of both
arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex
Prior to induction of DOMS the subjects were allowed an initial familiarization session
to become comfortable performing maximum voluntary contractions at the required
angular velocities This was immediately followed by determination of the maximum
eccentric and concentric peak torque at an angular velocity of 60degs and 120degs
Subsequently four sessions of eccentricconcentric work were performed with each
arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs
first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg
and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-
range) followed by two sessions at an angular velocity of 120degs again the first time
along an arch of 120deg and followed by the mid-range performance The subjects were
asked to accomplish maximum voluntary contractions during all the sessions Each
session was performed until exhaustion which was defined as the point when the
subject lost 70 of the initial eccentric and concentric peak torque There was a 1-
minute rest between each session This procedure was based on a pilot study and
previously described induction protocols [17-21]
Outcome measures
Outcome measures of subjective pain measurements MPT and muscle strength were
measured in this order on days 1-4 Subjective pain measurements and MPT occurred
immediately prior to and following irradiation whereas muscle strength measurements
only took place before LED treatment
Measurement of subjective pain Perceived muscle soreness was measured
subjectively by means of a 100-mm VAS A series of scales were completed separately
Delayed-onset muscle soreness
97
for each arm pain at rest followed by pain perception associated with full extension of
the arms and finally with maximal flexion of the arms The subjects were not allowed
to compare one VAS result with another
This assessment tool commonly used in measuring experimentally induced pain [22
23] has been found to be a reliable and valid method [24-26]
MPT Tenderness MPT used as a more objective correlate of muscle tenderness
has been demonstrated to be a reliable method to measure experimental induced
muscle soreness [27] This outcome measure was assessed by using a handheld
pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries
South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a
line from the radial insertion of the musculus biceps brachii at the elbow to the
intertubercular groove of the humerus thus resulting in three measure points one at
the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A
pressure of 4Ns was delivered The subjects were instructed to say yes at the exact
moment the pressure perceived became painful Each point was recorded three times
in pre-treatment as well as in post-treatment The average MPT score for each point in
pre- and post-treatment was used for statistical analyses
Muscle strength assessment Eccentric and concentric IPT were measured on the
same computerised dynamometer as was used for the induction of pain and an
identical standardisation procedure regarding positioning was followed
A warm-up session of two maximum voluntary contractions at the required angular
velocities was followed by determination of the eccentric and concentric peak torque
The first session at 60degs consisted of three repetitions followed by a 1-min during
rest and for the second session at 120degs five repetitions were performed The
subjects were instructed to flex and extend the elbow through the entire range of
motion as forcefully and rapidly as possible for each repetition The maximum
eccentric and concentric torque produced during the respective repetitions was used
for statistical analysis
Chapter 5
98
Light source specifications and treatment procedure
Light treatment was applied daily according to group allocation Irradiation occurred
with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The
probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160
mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The
frequency was variable within the range of 0ndash1500 Hz
During the complete irradiation procedure the LED probe was held in contact with
the skin perpendicular to the skin surface and at the exact mid-point between the MPT
mark at 4 cm and the one at 8 cm Light source properties were identical for all
subjects of the experimental group and consisted out of irradiation of 6-min lasting
duration at a continuous power output of 160 mW resulting in an energy density of
32 Jcm2 To conceal the treated side and condition the subjects were blinded to the
treatment status For the experimental condition a probe was held in contact with each
arm but only one of the two probes was attached to the LED device The subjects of
the placebo group received sham irradiation at both sides
The selected parameters are within the scope of previously described light source
characteristics for pain reduction [1 28-30] and they are appropriate for the treatment
of pain in a clinical setting because the duration of the treatment is clinically feasible
Statistical analysis
The three outcome measures were analysed separately For the VAS and MPT
measurements the same procedure was followed a general linear model (GLM) for
repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-
post preceding and following LED irradiation) and one between-subject factor (group
placebo or infrared LED irradiated) was performed If necessary the GLM was
followed by appropriate pairwise comparisons (post hoc least significant difference or
LSD) to determine whether any differences between measurements were statistically
significant A similar model was carried out separately for both the treated and the
control arm
Delayed-onset muscle soreness
99
In contrast to MPT and VAS the muscle strength was analysed differently The peak
torque values recomputed towards body weight of the subjects were statistically
analysed using a GLM for repeated measures This model consisted of one within-
subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo
or infrared LED irradiated) The model was completed twice first for the treated arm
and consequently for the control arm
The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)
was used for analysis and statistical significance for all tests was accepted at the 005
level
RESULTS
Statistical analysis of all variables of the three outcome measures revealed no significant
interactive effects of the main interaction (time times group times pre-post) The means and
standard deviations of the variables for both the treated and the control arm are
outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The
means of all VAS and MPT variables disclose a non-statistical significant general
analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher
MPT values in the irradiated group than in the placebo group The lower VAS rates are
present from day 1 until the last day of the study but they are more clearly present
from day 3 pre-treatment The higher MPT values are present from day 1 post-
irradiation until the last day and they are more visible at 4 cm followed by 12 cm and
finally at 8 cm In addition to the analgesic influence of LED an increased
convalescence of muscle strength was noted It should be remarked that this outcome
is similar for the treated as well as for the control arm of the irradiated group The
findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms
of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively
Graphical presentation of the other variables shows a similar course
Chapter 5
100
Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42
Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36
Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Visual Analog Scale
0
02
04
06
08
1
12
14
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n V
AS
scor
e (a
t re
st)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
101
Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249
Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Mechanical Pain Threshold
0
5
10
15
20
25
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n M
PT
sco
re (
at 4
cm)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Chapter 5
102
Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group
Day 1 Day 2 Day 3 Day 4
Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04
Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04
Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Isokinetic Peak Torque
04
045
05
055
06
065
Day 1 Day 2 Day 3 Day 4
Time course
Mea
n I
PT
sco
re (
ecce
ntr
ic a
t 60
degse
c)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
103
Despite the absence of significant main interaction effects the remaining interactions
as well as the main effects were statistically significant for some variables Only the
significant interactions including the between-subject factor group as well as the main-
effect group will be discussed The other interactions and effects establish the successful
induction of DOMS but are not relevant in view of the postulated hypothesis
The interaction between group and time is significant (p=014) for the VAS in
association with full extension for the control arm Post hoc LSD reveals no difference
between both groups a significant effect over time for both groups is found
Consequently this will not be further evaluated
A second significant interaction (p=0002) is the one among the within-subject factor
pre-post and the between-subject factor group for the MPT at 12 cm for the control arm
Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more
pressure after than before the treatment whereas in the placebo group a not
significant decrease of supported pressure is noted
Finally GLM analysis revealed that at the treated arm the irradiated group tolerates
significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)
DISCUSSION
It has previously been demonstrated that the LED source used might assist in
accelerating wound healing [31] that it has a direct cellular effect [3233] and that it
changes nerve conduction characteristics [1] Nevertheless LED-treated experimental
induced DOMS failed to prove the analgesic efficacy of LED at the above-described
light parameters and treatment procedure The current outcome concurs with other
research that demonstrated a lack of effect of various forms of light therapy on DOMS
[8 11 15] However despite the absence of an apparent and overall definitive finding
the present results cannot exclude favourable effects of LED treatment on pain Since
first of all an isolated statistical significant pre-post difference between groups (control
arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)
revealed that subjects of the irradiated group tolerate more pressure than the subjects
of the placebo group Second of all the overall means identified generally lower VAS
Chapter 5
104
scores higher MPT values and higher peak torques in the irradiated group This
implied that the treated subjects experienced noticeable less pain supported more
pressure on the painful muscle and generated more force than the non-treated
participants However these results are not statistically significant consequently it is
possible that these differences were found by coincidence and that there is no
relationship between the treatment and the described results of the three outcome
measures though it should be mentioned that the absence of significant findings is
more probably attributable to the small sample size involved in this study This
assumption is based on a post hoc power analysis It was calculated that for the small
effect size measured after treatment and for the measured control group event rate a
sample size of 80 subjects in each group was required at α=005 and power=080
(two-sided) to reveal significant results
Another factor conceivably responsible for the lack of solid evidence of the beneficial
effects of LED treatment upon DOMS-associated pain is related to the size of the
treatment effect in relation to the severity of the induced DOMS It is possible that by
using multiple exhaustive sets of exercise severe DOMS were induced which masked
relatively small but apparent treatment effects [4 11] In this same context it is
possible that the results only become significantly different after a prolonged treatment
and follow-up period as previous research noticed that recuperation subsequent to
DOMS induction can last up to 10 days [8]
Although it needs to be stressed that these results are not statistically significant critical
analysis of the overall means leads up to three additional remarks A primary comment
relates to the pre- and post-treatment courses of the results Starting at day 2 a clear
reduction of pain and muscle sensitivity was observed immediately post-treatment
Still one cannot conclude that this is indicative for the analgesic effect of LED
irradiation as a similar decrease in VAS and increase in MPT values was noted in the
treated and the control arm of the placebo group Perhaps this was caused by placebo
effect as reported by Pollo et al [34] the expectation of the participant can easily result
in pain relief but it can only be elucidated by implementation of a control group
Delayed-onset muscle soreness
105
Nevertheless in the current study this particular finding can be most probably
attributed to the physiological effects of the peak torque measurement performed
between the pre- and post-treatment recordings of VAS and MPT on the painful
flexor muscle of the upper arm For the assessment of muscle strength two short
series of alternative concentric and eccentric efforts were performed in succession
involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist
in the removal of inflammatory markers and exudate consequently reducing local
tenderness [4] In addition the force assessment can be considered as a form of active
warming-up resulting in an increased muscle temperature which can reduce muscle
viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness
[3536] thus decreasing the sensitivity of the muscle and moderating pain during
movement In any case the beneficial influence of LED immediately after irradiation
can not be securely interpreted due to the sequential assessment of the outcome
measures
A second additional remark considers the fact that both arms of the irradiated subjects
demonstrated evidence of the beneficial effects of LED as a similar reduction of pain
and muscle sensitivity and higher peak torques were found in course of time at the
treated arm as well as at the control arm of the irradiated subjects This ascertainment
points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]
stated that in case LED works via systemic effects the use of the contralateral side as a
control arm might be ill-advised Thus reinforcing that future research should include a
control group to bring clarification [4 7 16]
Finally it needs to be mentioned that although the extent of DOMS was probably
relatively high for investigating the postulated hypothesis the time-course of the
present study corresponds to that reported by other investigators [2 3 5-10]
Significant time effects in many of the variables revealed that muscle damage was
evident diffuse muscle soreness became progressively worse 24-48 h after DOMS
induction followed by a small amelioration after 72 h [35910] After 72 h the follow-
Chapter 5
106
up was ceased consequently further regain of force and attenuation of pain and
muscle sensitivity could not be evaluated Extending the duration of the assessment
period could be useful in assessing any longer-term effects of LED treatment
particularly because as mentioned above differences between both groups are more
clearly present from day 3 pre-treatment and also because DOMS may last for up to 10
days when induced with the described protocol [715]
Lack of knowledge regarding both the precise mechanism of action of LED and the
specific pathophysiology of DOMS hampers the way to offer a definitive explanation
for the absence of more obvious statistically significant differences Still the small
number of significant findings and the mean values suggest that possible analgesic
effects of infrared LED may not be excluded yet but to be able to estimate the real
value of LED further research is necessary A large-scaled randomised clinical trial
which takes the above-mentioned remarks into consideration should be performed
CONCLUSION
Regardless of the reasons for the absence of statistical significant effects reported here
and although LED may have some potential in the management of pain and functional
impairment associated with DOMS its effectiveness at the applied densities has not
been established
Future research should focus on evaluation of the appropriateness of DOMS as an
experimental model of pain and muscle damage Validation of this model would
enhance the ability to study various modalities for their potential effects on pain and
muscle injuries Besides the mechanisms of LED action are not known thus further
fundamental investigations need to address the underlying mechanism and
physiological basis of pain modulation utilizing LED treatment
Once LED irradiation has finally proven its treatment value in an experimental model
the most important prospect considers establishing the effectiveness of LED to reduce
pain in clinical settings
Delayed-onset muscle soreness
107
ACKNOWLEDGMENTS
The authors would like to thank Mr T Barbe and Mr R Deridder for their technical
assistance in the collection of the data as well as for their valuable input into the
research design Sincere appreciation is extended to the volunteers that participated in
this study and to MDB-Laser (Belgium) for generously providing the light emitting
diode equipment The authors also gratefully recognize Prof Dr G Van Maele for
assistance with the statistical analysis and for helpful discussion
Chapter 5
108
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34 Pollo A Amanzio M Arslanian A Casadio C Maggi G and Benedetti F (2001) Response Expectancies in Placebo Analgesia and Their Clinical Relevance Pain 93(1)77-84
35 Shellock FG and Prentice WE (1985) Warming-up and Stretching for Improved Physical Performance and Prevention of Sports-Related Injuries Sports Med 2(4)267-278
36 Safran MR Seaber AV and Garrett WE (1989) Warm-up and Muscular Injury Prevention an Update Sports Med 8(4)239-249
37 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
38 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
GENERAL DISCUSSION
General discussion
113
SUMMARY
As outlined in the general introduction the overall objective of this doctoral thesis is to
develop the current knowledge about the mechanisms of LED action in view of the
eventual provision of evidence-based support for the clinical use of LED as a
biostimulatory and analgesic treatment modality especially in the field of
physiotherapy
Part I Wound healing
The investigations described in chapter 1 and 2 were conducted to gain insight into the
potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As
fibroblasts are principal cells for biostimulation (in view of growing and dividing in
healing wounds) the influence of LED irradiation on fibroblast proliferation was
assessed1
The first investigation consisted of a pilot study performed in order to evaluate the
appropriateness of the cell isolation technique cell culture protocol and proliferation
analysis as well as to appraise the feasibility of the light source properties and
illumination procedure
Data analysis revealed no statistically significant differences between the infrared LED
irradiated and control petri dishes for the used parameters (table 1) Considering this
outcome other experimental findings disclose that the absence of stimulatory effects of
LED irradiation on fibroblast proliferation can partly be attributed to the use of
inappropriate light source properties However the applied external dosimetric
parameters are well within the recommended spectrum described by previous studies
investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be
excluded that changes in the illumination procedure (such as the use of lower power
shorter exposure times wavelengths with finer coverage of the absorption spectrum of
the irradiated cells and a longer incubation period between the last irradiation and cell
counting) could still result in an increased fibroblast proliferation467 Of equal
importance in interpreting the lack of distinctive results are the imperfections of the
applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails
114
considerable intervention from the investigator compromising the reliability of the
method It is also a time-consuming technique with an insufficient sensitivity for some
purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11
To avoid contamination of the results by these modifiable remarks a similar
experiment (chapter 2) was performed in which wavelength power and output mode of
the infrared LED source were not modified (table 1) only the exposure time was
reduced resulting in a lower radiant exposure In addition the effect of two other
emission spectra was evaluated These probes emitting red and green light had a
shorter wavelength than the infrared LED source and the power was half or a
sixteenth of the power from the infrared probe Consequently the red LED irradiation
occurred with a different exposure time than the infrared one in order to attain the
same radiant exposure (053 Jcm2) With respect to the green LED it was not
endeavoured to achieve the same radiant exposure as 16 min of irradiation is not
feasible for in vitro or clinical application
Finally also an LLL light source was integrated Although it was not attempted to
analyse the effectiveness of LED in comparison to LLL enclosure of this modality was
interesting in order to join in with the available literature covering mostly LLL studies
To bypass the described problems regarding analysis of fibroblast proliferation
counting of the cells was carried out this time by means of a colorimetric MTT assay
This method provides more accurate cell counts in a short period of time and therefore
can be considered as a more reliable alternative to Buumlrker hemocytometer11
MTT assay 24 h after the last irradiation revealed a significantly increased number of
cells in the irradiated wells in comparison to their (respective sham-irradiated) controls
Although the study supplied experimental support for a significantly increased cell
proliferation by all external dosimetric properties based on the results of the
comparative trial with an incubation period of 24 hours irradiation with the green
LED source yielded the highest number of fibroblasts Thus it can be concluded that
the wavelength of the green LED is probably within the bandwidth of the absorption
spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that
General discussion
115
the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast
cultures in vitro612
Table 1 External dosimetric properties summarized for each chapter
Wavelength Power Exposure
time Output mode
Radiant exposure
PART I Chapter 1
In vitro part
LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2
LED-infrared 950 nm 160 mW 1 min continuous 053 Jcm2
LED-red 660 nm 80 mW 2 min continuous 053 Jcm2
LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2
Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2
PART II Chapter 4
LED-infrared 950 nm 160 mW 2 min continuous 107 Jcm2
Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
The next aim of the first part of this doctoral thesis was to explore whether LED
treatment could ameliorate in vitro cell proliferation under conditions of impaired
healing In the pursuit of this aim fibroblasts were cultured in medium supplemented
with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a
pilot study the amount of glucose necessary to inhibit normal growth was determined
In order to attain an important reduction of cell viability and decreased proliferation
rate a relatively high concentration of glucose (1667 mM) was necessary in
comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia
(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity
between both circumstances in vitro limited to 72 h whereas the human tissue of a
diabetic patient in vivo is chronically exposed to glucose
Treatment of the fibroblasts occurred in respect of the previously described results
with the same irradiation parameters and illumination procedure (chapter 2)
Accordingly green LED irradiation labelled as the most appropriate treatment for
116
irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in
the previous study (table 1)
Analysis of the cell proliferation by means of MTT measurements yielded a
significantly higher rate of proliferation in hyperglycaemic circumstances after
irradiation than in the control conditions (ie hyperglycaemic circumstances without
irradiation) Thus this outcome supported the stimulatory potential of green LED
irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a
considerable destructive hyperglycaemic medium
Finally although the results of the in vivo part of chapter 1 were persuasive and
encouraging they will not be further discussed in this summary of part I as it was not
aimed in this doctoral thesis to investigate the wound healing process in vivo However
the results of this case study can be a valuable hold for future in vivo research
The possible clinical implications of these results and future research directions in the
scope of wound healing will be discussed below
Part II Analgesia
In the second part two studies investigated the effects of LED irradiation as a
potential intervention mode in one of the most important fields in physiotherapy
practice analgesia Chapter 4 describes the influence of LED treatment on changing
sensory nerve conduction characteristics of a human superficial peripheral nerve
Altering nerve conduction characteristics may not be the sole beneficial purpose to
attain with LED irradiation in view of analgesia but the advantage of nerve conduction
characteristics is that they are objective measurable physical variables and changes in
these characteristics provide a potential explanatory mechanism of pain inhibition by
LED treatment17
The results showed that percutaneous LED irradiation at feasible clinical parameters
can generate a significant decrease in NCV and increase in NPL for all recordings post-
treatment in comparison to the baseline measurement The data in the placebo group
did not reveal any significant difference in the same course of time Statistical analysis
General discussion
117
revealed significant differences between the experimental and the placebo group for
NCV as well as for NPL at all time-points of observation with exception of the NPL
recording immediately after finishing irradiation
It was also observed that the noted effects did not weaken as time progressed It can
be concluded that post-treatment conduction measurements should be extended in
time which is in accordance with the findings of some previous studies18-21 Clarifying
the point of time at which the effect extinguishes is necessary and clinically relevant
when treating pain by means of LED irradiation Besides obtaining the desired
neurophysiological effects ideally the optimal irradiation parameters should be
applied The most favourable dosimetric properties are not yet determined but based
on this study and previously described assays it can be speculated that the dosimetric
window is quite large
Regardless of these clinically important remarks the present findings allow to draw the
following conclusion LED irradiation at clinically applied densities can generate an
immediate and localized effect upon conduction characteristics in underlying nerves as
LED treatment results in lowering the NCV and augmenting the NPL Therefore the
outcome of this in vivo experiment assumes a potential pain relief by means of LED
treatment and justifies further research regarding its clinical effectiveness in laboratory
settings and at a clinical level
The fourth and final aim was to determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5
illustrates a clinical study observing the effect of LED treatment on a model
comprising experimentally induced DOMS in a healthy population The progress of
pain perception and peak torque was evaluated during 4 consecutive days commencing
on the day of DOMS induction The effect of infrared LED treatment at the light
parameters described (table 1) was assessed with regard to three different factors time
(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures
(VAS MPT and IPT) revealed no significant interactive effects of the main interaction
118
(timegrouppre-post) For the remaining interactions and for the main effects only a
few significant findings were relevant in view of the postulated hypothesis
Notwithstanding the absence of an apparent and overall statistically significant finding
the present results indicate favourable trends of LED treatment on pain as the means
of all VAS and MPT variables show a statistically nonsignificant general analgesic
effect of infrared LED irradiation expressed by lower subjective pain rates and higher
MPT values in the irradiated group In addition to the analgesic influence of LED an
augmented restoration of muscle strength was noted The lack of solid statistically
significant evidence for these beneficial effects of LED treatment upon DOMS-
associated pain can possibly be attributed to the small sample size in this study or even
to the size of the treatment effect in relation to the severity of the induced DOMS as
induction of severe DOMS can mask relatively small but apparent treatment
effects2223 A final possibility is that the results only become significantly different after
a prolonged treatment and follow up period as previous research demonstrated that
recuperation subsequent to DOMS induction can last up to 10 days24
It should also be noted that the described general analgesic effect of LED irradiation
was identical for the treated as well as for the control arm in the irradiated group
proposing that infrared LED might induce systemic effects 2526 However it needs to
be stressed that these results were not statistically significant
Regardless of the absence of statistically significant findings the mean values suggest a
potential role for infrared LED irradiation in the management of pain and functional
impairment associated with DOMS Notwithstanding this postulation future research
is absolutely required to establish the effectiveness of LED treatment to reduce pain as
well at the applied densities as for other dosimetric parameters
CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS
In the course of the past years during the process of the genesis of this thesis
therapeutic physical agents in general and phototherapeutic modalities in particular
became less important as physiotherapeutic modes of treatment than during the
preceding two decades The diminished use of these treatment modalities in the
General discussion
119
physiotherapy practice is to a certain degree a consequence of the controversial
research findings regarding the use of these physical agents This issue of controversy
led to less support for the use of these treatment modalities and a growing scepticism
regarding the effectiveness of these physical agents within the scope of the growing
climate of evidence-based practice A second responsible protagonist for the loss of
popularity of physical agents is linked with the current tendency within physiotherapy
emphasising active remedial therapy The establishment of this development was based
on various experiments mainly performed during the last decade demonstrating that
active treatment modalities are for numerous impairments and disabilities preferable to
more passive forms of therapy In Belgium the prevailing nomenclature which came
into use on 1 May 2002 went along with this tendency In the appendix to the Royal
decree of 14 September 1984 towards settlement of the nomenclature of medicinal
treatments concerning compulsory insurance for medical care and allowances the
personal involvement of the physical therapist during the physiotherapeutic session
was emphasized and it was even defined that massage physical techniques within the
framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal
application can only be remunerated when they are applied supplementarily and not as a sole therapy
This implies that passive treatment modalities should not be used as sole method of
treatment and should always be considered as an adjunct to an active treatment
program This development needs to be applauded in many cases such as various
painful musculoskeletal problems functional instability rehabilitation of neurological
patients re-activation of the elderly population psychomotor rehabilitation
cardiovascular and respiratory convalescence Nevertheless it would be erroneous to
entirely reject physical agents including LED treatment Based on the findings of the
above described experiments it needs to be stressed that for some purposes especially
within the scope of impaired wound healing LED irradiation could be a suitable
therapeutic measure This statement is founded on the results of part I of the present
thesis they provided satisfactory fundamental evidence for the advantageous effects of
LED treatment on a crucial exponent of the wound healing process namely fibroblast
proliferation The beneficial findings are the result of basic in vitro research As it is
120
inaccurate to simply extrapolate these results to the clinical practice the clinical use of
LED irradiation for wound healing needs to be preceded by purposive and specific in
vivo investigations to substantiate these basic research findings27
The case study described in chapter 1 indicates a foundation for further in vivo research
Visual appraisal of the surgical incision revealed (from the 65th day in the course of the
reparative process onwards) that the irradiated area -which initially showed inferior
epithelialization and wound contraction- showed a more appropriate contracture than
the control area characterized by less discoloration at scar level and a less hypertrophic
scar These clear beneficial effects of LED treatment on a human cutaneous wound
can serve as preliminary impetus for further research into the clinical applicability of
LED therapy although this case study is insufficient in order to guarantee a safe
correct and effective use of LED as a therapeutic modality
Despite these remarks it tentatively can be concluded that based on a detailed analysis
of the available data of the present in vitro studies and the given case report in
combination with the small number of previously published human studies the
beneficial effects of LED irradiation at the cellular level are obvious and therefore a
potentially favourable outcome can be assumed in clinical practice28-30 LED-
modulated stimulation of wound healing can be gradually and vigilantly implemented
clinically Nevertheless the real benefits of LED irradiation within the scope of wound
healing can only be established by additional clinical trials as thus far clinical
application and stipulation of dosimetry still occurs on a trial-and-error basis which is
not conducive to a generally accepted clinical use of LED To lend more credibility to
the treatment of wounds by means of LED irradiation and to expel the existing
controversy and scepticism surrounding this topic in vivo investigations on wound
healing using a number of different animal models and adequately controlled human
studies are necessary In addition these studies should be performed preferably on a
population suffering from impaired healing as a consequence of diabetes mellitus or as
a result of any other debilitating reason because as posed by Reddy et al3132 and as
mentioned above light has possible optimal clinical effects in the treatment of healing-
resistant wounds
General discussion
121
Drawing general conclusions and formulating clinical implications for analgesia is
obviously less manifest first of all because only a limited number of possible
mechanisms of action in order to obtain analgesia were highlighted and secondly
because both studies did not come to a joint or complementary conclusion The
outcome of the first study revealed that LED treatment lowers the NCV and augments
the NPL resulting in a slower stimulus conduction and consequently a reduced number
of sensory pulses per unit of time Thus it could be assumed that LED induces pain
relief but the results of the study describing the effect of LED treatment on
experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy
In addition it needs to be emphasised that the first study (chapter 4) measured the effect
of LED irradiation on the large myelinated Aβ afferents A noteworthy question and
meanwhile a stimulus for future investigation is whether the measured effects can be
extrapolated from these sensory nerve fibres to the actual nociceptive afferents
notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of
these nociceptive pathways relies on laser-evoked potentials which selectively activate
Aδ-fibres and C-fibres3334 This technique was presently not available therefore a
standard sensory nerve conduction study was performed
Whereas stimulation of wound healing by means of LED irradiation can be cautiously
implemented in the clinical practice at this stage it is too early to promote LED
irradiation as a treatment modality for pain To make this possible it is essential to
conduct numerous studies with regards to the use of LED in the field of analgesia
Future research should focus on fundamental investigations in order to discover the
underlying mechanisms and physiological basis of pain modulation utilizing LED
treatment Furthermore the evaluation of the appropriateness of DOMS as an
experimental model of pain is an important prospect to consider as validation of this
model would enhance the ability to study various modalities for their potential effects
on pain Irrespective of the difficulties regarding standardisation of the research
population and evaluation of soreness inextricably linked with clinical pain studies the
122
ultimate objective of future research should be the establishment of the effectiveness
of LED irradiation to reduce pain of miscellaneous origin in a clinical setting
Regardless of the encouraging results of the described studies and besides the earlier
proposed specific directions for future research (directed towards wound healing or
pain relief) it is necessary in the interest of the patientrsquos well being and to the
advantage of the prospective clinical use of LED to highlight a few more issues for
future research Therefore one has to deal with some limitations of the performed
investigations A first limitation concerns the fact that only two mechanisms of LED
action were investigated (notably changed fibroblast proliferation and alteration of the
nerve conduction characteristics) So one can conclude that for further and better
understanding of the mechanisms of action it is necessary to perform more basic
research Answering the questions regarding the functioning of LED irradiation will
simplify the evaluation and reinforce the interpretation of the obtained results and
ultimately contribute to a more widespread and well definded acceptance of the use of
LED in clinical settings
A second general limitation of this doctoral thesis is the substantial difference in the
used external dosimetric parameters between the different chapters and even within
one and the same study (illustrated in table 1) this complicates the comparison
between the different trials In each trial the dosimetry was individually ascertained
based on previous studies within the given field As not for every application the same
dosimetry is suggested in literature a range of dosages were used Another important
factor in deciding on the dosimetry was the clinical applicability of the dosage as it is
useless to investigate the appropriateness of a treatment modality at a clinically
unrealistic dose As a result of this limitation the current findings do not fully
contribute to the explanation regarding the ideal parameters one should use although
this was not set as a principal purpose Based on this thesis and previously described
assays it can be speculated that the possible window for these parameters is quite large
the ideal irradiation parameters and proper timing or sequencing of LED irradiation
General discussion
123
for example to the various phases of wound healing and to different painful conditions
are therefore possibly unattainable
The establishment of an appropriate dosimetry should also consist of investigating the
absolute and relative penetration depth of LED irradiation into human tissue This is
less crucial within the scope of wound healing but it is of key importance while
treating deep-seated tissue (eg nerve fibres muscles circulatory components et
cetera)
Finally this thesis only investigated the efficiency of LED in a very limited number of
conditions notebly wound healing and pain Within the scope of physiotherapy and
medicine in general there are numerous other purposes for which LED irradiation is
promoted such as oedema arthritis miscellaneous orthodontic applications seasonal
affective disorder neonatal jaundice photodynamical therapy et cetera2835-41
In summary additional work on establishing proper dosimetry and identifying the
biochemical or photobiologic phenomena that are responsible for improving wound
healing and reducing pain or even other effects within a broader spectrum of
conditions remains to be done in order to answer unreciprocated questions Until that
time the potential clinical usefulness and actual value of LED irradiation for wound
healing and even to a larger extent for analgesia should always be approached with
appropriate professionalism and even caution
FINAL CONCLUSION
LED devices are promoted for clinical use but the currently available scientific
documentation regarding effectiveness of this physical agent is rather scarce Through
providing scientific support for the biostimulatory and analgesic effectiveness of LED
irradiation this doctoral thesis attempted to bridge in some degree this gap
The conducted studies revealed that LED irradiation undeniably has potential
beneficial effects on wound healing and to a lesser degree within the scope of
analgesia However based on the present results it can be corroborated that light
124
therapy in the guise of LED irradiation is not magic but these results can raise some
corrective doubts in fundamental disbelievers and antagonists
Nevertheless we have to join the queue of scientists who have found beneficial results
but cannot elucidate with certainty how this outcome was established Thus although
the present results are encouraging a continuing development and integration of new
knowledge based on further research is necessary in various domains of intervention
Therefore several directions for future investigations were proposed in order to cover
as many existing gaps and to answer the utmost number of remaining questions as
possible Still one ought to be aware not to carry future fundamental research at a
disproportional level and the inevitable quest for mechanisms of LED action should
not hypothecate the potential clinical value implying that at a certain point it should be
appropriate to make the transfer from science to the application of the available
knowledge in clinical practice
The described findings regarding LED irradiation are comparable to the results of
previously published studies performed with other light sources Consequently as
postulated by some LED providers it can be speculated that the biological response of
tissue to light irradiation can probably not be equated merely to a light source but
rather to a broad photo-energy window
General discussion
125
REFERENCES
1 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
2 Abergel R Lyons R Castel J Dwyer R and Uitto J (1987) Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2)127-133
3 Skinner S Gage J Wilce P and Shaw R (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-192
4 Webb C Dyson M and Lewis W (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5)294-301
5 Atabey A Karademir S Atabey N and Barutcu A (1995) The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 1899-102
6 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
7 Ben-Dov N Shefer G Irinitchev A Wernig A Oron U and Halevy O (1999) Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3)372-380
8 Rebulla P Porretti L Bertolini F et al (1993) White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2)128-133
9 Deneys V Mazzon A Robert A Duvillier H and De Bruyere M (1994) Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2)172-177
10 Mahmoud A Depoorter B Piens N and Comhaire F (1997) The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2)340-345
11 Haskard D and Revell P (1984) Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3)319-322
12 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers
13 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of cultured human endothelial cells Diabetes 36(11)1261-1267
14 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA damage in cultured human endothelial cells J Clin Invest 77(1)322-325
15 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7)621-627
16 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4)491-501
17 Kramer J and Sandrin M (1993) Effect of low-power laser and white light on sensory conduction rate of the superficial radial nerve Physiotherapy Canada 45(3)165-170
18 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
19 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
126
21 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
22 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
23 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
24 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
25 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
26 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer
G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
29 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
30 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255
32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287
33 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-28
34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856
35 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
36 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
37 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263
39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64
40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74
General discussion
127
41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press
NEDERLANDSTALIGE SAMENVATTING
Nederlandstalige samenvatting
131
NEDERLANDSTALIGE SAMENVATTING
Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de
tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te
vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie
van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen
Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest
enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is
ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als
op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang
van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve
interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken
Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van
ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen
moeilijk te verantwoorden
Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin
de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere
overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit
methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan
consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in
de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen
laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in
de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag
aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies
inclusief het gebruik van licht
Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat
de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde
doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en
dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht
komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt
geloosd
132
De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo
lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en
gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te
anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te
voorkomen lijkt een gerichte en rationele a priori aanpak conform de
wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen
Te meer daar grondige literatuurstudie leert dat men ten behoeve van de
werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie
zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser
De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de
extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is
geboden De introductie van alternatieve lichtbronnen in de huidige
kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk
weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus
eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood
naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based
gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder
binnen de domeinen van haar potentieel beloftevolle klinische toepassingen
wondheling en analgesie
Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de
wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op
prominente protagonisten van de wondheling de fibroblasten werd getracht het
fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten
zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de
proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd
respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand
waarbij de normale celgroei werd verstoord
In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het
effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische
Nederlandstalige samenvatting
133
data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden
verklaard door het gebruik van een inadequate LED dosering en anderzijds een
methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie
vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de
precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en
inter-tester variabiliteit tot gevolg
In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde
onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de
bestralingsparameters (dosering) De effecten van de drie verschillende LED
golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten
werden hierbij geanalyseerd door middel van een meer betrouwbare en minder
subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-
yl)-25-diphenyl tetrazolium bromide (MTT)
De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir
effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden
tevens een basis voor meer coherente en relevante inzichten aangaande de globale
bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)
Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het
wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend
karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp
rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde
vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De
fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met
extreem hoge concentraties glucose Deze modificatie van het medium staat model
voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische
praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan
vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de
gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en
proliferatie
134
Het tweede deel van dit proefschrift exploreert het domein van het potentieel
analgetisch effect van LED binnen de kinesitherapie aan de hand van twee
fundamentele onderzoeken
In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere
sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele
hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en
een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale
verklaring van een analgetisch effect van het medium zou kunnen belichamen
Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)
meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf
verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen
aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke
significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de
negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele
hypothese
Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend
fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale
pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp
van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische
krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED
behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier
keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED
op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd
(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde
isokinetische krachtmeting en een registratie van de waargenomen spierpijn De
spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met
behulp van een kwantitatieve hand-hold algometer
Analyse van de bekomen data bracht geen significante verschillen tussen de
controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden
Nederlandstalige samenvatting
135
kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED
behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van
de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de
proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene
afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine
proefgroep die werd onderzocht enof door de grootte van het behandeleffect in
verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers
een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up
kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel
absolute omzichtigheid geboden en moet deze visie louter als speculatief worden
beschouwd
Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de
kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de
laatste jaren aanzienlijk afgenomen
De positieve resultaten van de verschillende in vitro studies in het kader van wondheling
vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing
vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel
gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een
belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek
Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie
Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme
onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch
complementair resultaat Verder onderzoek ter exploratie van de mogelijke
onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom
onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op
termijn wetenschappelijk te rechtvaardigen
136
LED tovenarij trend of therapie
LED mag geen magische krachten worden toegemeten maar verdient het lot van een
kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een
wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen
zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke
indicaties
Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds
the darkness has always got there first and is waiting for it
(Terry Pratchett Reaper Man 1991)
Promotor
Prof dr D Cambier Ghent University Belgium
Examination Board
Prof dr P Calders Artevelde University College Belgium
Prof dr D Cambier Ghent University Belgium
Prof dr M Cornelissen Ghent University Belgium
Prof dr M De Muynck Ghent University Belgium
Prof dr M Dyson University of London UK
Prof dr P Lievens Free University Brussels Belgium
Prof dr K Peers Catholic University Leuven Belgium
Prof dr G Vanderstraeten Ghent University Belgium
Process Supervisory Board
Prof dr D Cambier Ghent University Belgium
Prof dr M Cornelissen Ghent University Belgium
Prof dr M De Muynck Ghent University Belgium
Prof dr G Vanderstraeten Ghent University Belgium
VII
TABLE OF CONTENTS
GENERAL INTRODUCTION 1
Background 3
Physical characteristics 6
Mechanisms of action 12
Aims and outline 15
PART I WOUND HEALING 25
Chapter 1 Do infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment
27
Chapter 2 Increased fibroblast proliferation induced by light emitting diode and low level laser irradiation
47
Chapter 3 Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level
61
PART II ANALGESIA 73
Chapter 4 Evidence of changes in sural nerve conduction mediated by light emitting diode irradiation
75
Chapter 5 Pain reduction by infrared light emitting diode irradiation a pilot study on experimentally induced delayed-onset muscle soreness in humans
91
GENERAL DISCUSSION 111
Summary 113
Clinical implications and future research directions 118
Final conclusion 123
NEDERLANDSTALIGE SAMENVATTING 129
Do not attempt to do a thing unless you are sure of yourself
but do not relinquish it simply because someone else is not sure of you
(Stewart E White)
IX
ACKNOWLEDGEMENTS
I wish to thank those people who supported me over the years and who helped me to
shape my life and work
First of all I would like to express my gratitude towards my promotor prof dr D
Cambier as without his encouraging words criticism inspiration and unremitting
support I would still be floundering about the contents of chapter 1 Thank you for
your good advice when I needed it the most
The members of the supervisory committee prof dr M Cornelissen prof dr M De
Muynck and prof dr G Vanderstraeten thank you for your assistance and helpful
feedback during the process of formation of this thesis
I also gratefully acknowledge the external members of the examination board prof dr
P Calders prof dr M Dyson prof dr P Lievens and prof dr K Peers for their
constructive reflections which contributed to the improvement of this thesis
I am greatly indebted to my special mentor prof dr J Anders of the Uniformed
Services University of Bethesda Maryland for the research suggestions she made as
well as for her unlimited belief in the value of my work
I wish to thank prof dr L Deridder for providing access to the laboratory of
Histology the Centre of Sports Medicine of the Ghent University Hospital for
allowing me to use their equipment as well as MDB-Laser Belgium for generously
providing the light emitting diode equipment
Sincere appreciation is extended to the volunteers that participated in this study and to
Tom and Roel for their valuable technical assistance in the collection of the data as
well as for their useful input into the research design of the investigation described in
chapter 5
X
Warm thanks go to the colleagues of the department of Human Anatomy
Embryology Histology and Medical Physics for providing the culture medium for the
technical support for the helpful discussions and principally for the amusing pastime
aseptic chats
In addition I also want to thank my colleagues of the associated institute Kinesitherapie
Gent and above all the colleagues of the department of Rehabilitation Sciences and
Physiotherapy Ann Axel Barbara Bart Bert Bihiyga Bruno Carine Damien Els2
Erik Frike Helga Inge James Karlien Kathy Kat(h)leen Koen Marc Martine
Mieke Paris (Nele) Patrick Roland Sophie Stefan Stijn (Veerle) Wiene Wim and
Youri thank you for the organisation and your attendance at many memorable
sidelines such as the survival-weekend the first department-day Fata Revaki our
legendary Thursday-sport activities and Friday-afternoon coffee breaks St Nicholas
visit skiing holiday New Yearrsquos lunch with quiz bowling spinning the introduction of
ldquosubetelyrdquo during our conspicuous presence at WCPT 2003 et cetera We shared many
treasured moments thanks to you a common working day often turned out to be very
pleasant I know that it will be impossible to find a comparable team of colleagues to
work with in the future
I especially want to thank Barbara to remind me on a regularly basis of my deadlines
to listen to my grieves and joy and to be willing to offer me a window-seat in our
office Kurt (although you abandoned at a certain moment) for solving my computer
problems Pascal for assistance with the statistical analyses Lieven for your motivating
interest and finally Fabienne Tine and Kim as loyal and appreciated friends who
worn-out several sports shoesbathing suits to supply in the weekly portion of sports I
needed to remain physically and mentally fit
I also extend my appreciation to my family and friends for their interest in my research
activities permanent mental support for the adoption of the surviving chickens but in
particular for looking after Louka and for the numerous relaxing moments Special
thanks are going to Katelijne and Frank for the delicious hot meals on lonely evenings
XI
Thomas Stijn and Johan thank you for the repeated (mostly futile) attempts to
convince me to do something together Sebastiaan each time during the past few years
when I doubted about the sense of my work it was your ridiculous story about a man
who wanted to invent superglue but instead invented the well-known yellow post-it
which stimulated me to continue my scientific quest
Of course I owe most gratitude to Luc my most devoted supporter Dearest I know
that since august 2004 you lived a solitary life in Dubai Although I think it was
possibly easier not to live under the same roof with me these last stressful months I
am aware that it was very difficult for you not to be able to play with Louka and to
miss some precious months of her life
Louka thank you for your radiant smile and daily baby speeches I am sorry that you
had to miss your daddy I promise that we will be reunited very soon
Elke Vinck
Ghent March 2006
GENERAL INTRODUCTION
General introduction
3
BACKGROUND
The use of light for therapeutic purposes reaches far back in time Current interest for
photomedicine with his its biological and medical effects relies fundamentally on two
major evolutions in the given field (1) the research results regarding the use of
ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in
the light amplification by stimulated emission of radiation (laser)-technology The production
of the first laser the ruby pulsed laser was rapidly succeeded by the development of
the helium-neon laser and other lasers like the argon the neodymium-glass and the
neodymium-yttrium-aluminium-garnet lasers1
As in the mid-1990s semiconductor and diode-based lasers gained popularity the
principally massive gas and dye lasers were rendered obsolete Therapeutic light
technology further continued to evolve and todayrsquos therapeutic light source is as likely
to be a light emitting diode (LED) or polarized light as a semiconductor or diode
laser1
Technological advancement and variation of the light sources necessitate a
concomitant update and revision of research in the respective domains of application
Unfortunately this logical and rational necessity has rarely been fulfilled From a
historical perspective this lack of appropriate research has led to disenchanting
evolutions in the use of light especially in physiotherapy The experience exists in this
medical field that light sources were promoted and commercialised for a vast regimen
of indications without foregoing scientific backup Consequently research developed
often after the commercial introduction in physiotherapy As these investigations
frequently gave rise to conflicting results for certain indications scepticism arose and
the use of the given modality knew a waning popularity for all its indications The final
result of such an inappropriate frame of promotion commercialisation and research is
a growing clinical disuse of a given modality even for motivated indications In view of
the actual increasing interest in LED treatment and based on former ascertainment
one has to state that a literature review for the given source reveals that research
mostly covers only low level laser (LLL) studies23 Although recently a number of
papers can be noted that report on the effects of LEDs and polarized light still
4
numerous source-specific-questions need to be answered as research concerning
mechanisms of action and efficacy of the current light sources remains limited in view
of a substantiated clinical application4-17
The reason for the contemporary light-oriented interest in physiotherapeutic practice
for LED devices is in essence based on several advantages of LED in comparison with
LLL For example the use of LEDs is esteemed to be safer as the delivered power
does not damage tissue LEDs can be made to produce multiple wavelengths thereby
stimulating outright a broader range of tissue types and probes that cover a large
treatment area are available18 In addition from a commercial point of view LEDs are
far more interesting as they are a good deal cheaper than laser diodes and they have a
long life span as these solid devices stand robust handling
As a result of the above-mentioned lack of literature on LED some providers of these
devices have taken for granted that the biological response of tissue to light irradiation
cannot be equated merely to a light source They declare that a given response solely
depends on the extent of absorption of radiated light by the tissue19 Consequently
these providers state that it is acceptable to extrapolate scientific findings of LLL
studies for explaining the mechanisms of action and detailing the efficacy of LED and
other alternative light sources Thus actually without appropriate scientific support
equal biological effects are attributed to LED as to LLL Nevertheless prudence is
called for such an extrapolation firstly because it is irrespective of the mentioned
dissimilarities and by simple projection one ignores a number of physical differences
between LLL and LED (eg coherence and degree of collimation or divergence)
Secondly LLL therapy is still not yet an established and evidence-based clinical tool20
Notwithstanding the historical efforts there still remains a considerable amount of
ignorance scepticism and controversy concerning the use and clinical efficacy of
LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed
parameters for irradiation as well as to the difficult objective measurement of possible
irradiation effects and even to the exceptional range of unsubstantiated indications for
General introduction
5
which light therapy was promoted27-29 A lack of theoretical understanding can also be
responsible for the existing controversies as the evaluation and interpretation of
research results would be simplified largely when the appropriate knowledge about the
mechanisms of light action would be available
LLL literature can undoubtedly be used as basis for research on LED and as a
comparative reference for these given investigations However to guarantee evidence-
based use of LED within physiotherapy the need for specific research in view of an
accurate consumption of LED is definite especially for potential promising clinical
applications in physiotherapy according to LLL literature mainly wound healing and
analgesia3031
Hitherto the most substantial research concerning the use of LED for improvement
of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded
under the influence of prolonged exposure to microgravity (eg during long-term space
flights) and in case of absence of exposure to sunlight such as in submarine
atmospheres they performed wound healing experiments for military application in the
given circumstances3233 In vitro experiments revealed that LED treatment increased
proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA
synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and
osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies
demonstrated therapeutic benefits of LED in speeding the early phase of wound
closure and in changing gene expression in a type 2 diabetic mouse model183234
Human studies noted 50 faster healing of lacerations a return of sensation and
increased tissue granulation as a result of LED irradiation1833
Associates of the Rehabilitation Sciences Research Group of the Ulster University in
Northern Ireland extensively investigated the effectiveness of light in the treatment of
pain The emphasis was laid primarily on the analysis of the effects of various low level
laser light sources35-44 However in the year 2001 two studies gave an account on the
efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46
6
noticed relatively long-lasting neurophysiological effects a significant change of the
nerve conduction characteristics (decrease of the negative peak latency) was mediated
by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a
comparable multisource diode device was ineffective in the management of delayed-
onset of muscle soreness (DOMS)
Despite the major value of these described trials a definitive answer regarding the
ability of LED in influencing wound healing or pain is not forthcoming cardinally
because a number of aspects are not yet investigated Consequently more research is
required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the
formerly mentioned scepticism regarding the effectiveness of a medium and possibly
to the undeserved fall into disuse of the treatment modality which happened in a way
with LLL therapy
PHYSICAL CHARACTERISTICS
This chapter supplies a short but comprehensive review of opto-physics A brief
description of the physical characteristics of the LED source used is essential as the
physical properties of light play an important part in the ultimate efficacy of treatment
According to the International Electrotechnical Commission (IEC 60825-1) an LED
can be defined as
Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by
radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process
of spontaneous emission1947
The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO
preprototype MDB-Laser Belgium) This illustration shows that a probe consists of
32 single LEDs disseminated over a surface of 18 cm2
General introduction
7
Figure 1 LED device and three available probes (infrared red and green)
Three highly monochromatic probes were available each emitting light of a different
wavelength within the above-defined range (table 1)2748 The wavelength of the light
emitted and thus its colour depends on the band gap energy of the materials forming
the p-n junctiona This light property is a key determinant to obtain maximum
photochemical or biological responses as light absorption by tissue molecules is
wavelength specific27 Only by absorbing radiation of the appropriate wavelength
(namely the wavelengths equal to the energy states of the valence electrons)
photoacceptor molecules will be stimulated resulting in a direct photochemical
reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750
8
absorption spectrum of a particular molecule is limited absorption often only occurs
over a waveband range of about 40-60 nm274851 Nevertheless the absorption
spectrum at cell or tissue level is broad because cells are composed of many different
molecules
Besides its influence on the absorption by means of tissue molecules there is a crucial
link between wavelength and penetration depth of the irradiated light Penetration into
tissue decreases as the wavelength shortens hence green light penetrates less than red
light which at his turn penetrates less into tissue than infrared light2748 Detailed
principles of light penetration will be discussed below
The LED device used emits non-coherent light In the 1980s the observed biological
responses after laser irradiation were generally thought to be attributable to the
coherenceb of the light485253 Though currently the clinical and biological significance
of coherence is seriously questioned54 According to several authors coherence does
not play an essential role in laser-tissue interactions firstly as it was proven that both
coherent and non-coherent light clinically show equal efficacy75556 Secondly as
according to some authors almost immediately after transmission of light through the
skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et
al1957 state that both findings are incorrect coherence is not lost in tissue due to the
phenomenon of scattering and non-coherent light is not as efficient as coherent light
This lack of consensus makes it necessary to mention whether or not light is
coherent2758
Further decisive characteristics to accomplish phototherapeutic efficacy are the power
exposure time output mode and beam area Based on these parameters both
irradiancec and radiant exposured can be calculated According to numerous authors
some of these parameters are more crucial than others to determine whether
b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48
c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48
d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248
General introduction
9
absorption of light will lead to a photobiological event192728485455 However the
literature yields several controversial findings as not all authors attribute an equal
importance to a given parameter For example according to Nussbaum et al59
irradiance was the determinant characteristic in the biomodulation of Pseudomonas
aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49
found that in order to stimulate tissue cell proliferation a specific combination of
irradiance and exposure time are more important than the actual radiant exposure Low
et al3940 on the contrary highlighted the critical importance of the radiant exposure in
observing neurophysiological effects Whereas Mendez et al60 reported that both
parameters influence the final results of light therapy
Koutna et al61 even suggested that the output mode of light applications plays a more
prominent role in the treatment outcome than the wavelength of the used light source
Nevertheless this finding could not be confirmed by other research results Besides
more controversial findings have been published regarding the output mode although
the repetition rate in a pulsed mode was considered as an important treatment
parameter several investigations failed to prove its value19272840414461-64
Based on these findings it was opted within the investigations of this doctoral thesis to
irradiate in a continuous mode The remaining dosimetric parameters (wavelength
exposure time and power) depended on the purpose of each investigation they are
described in the respective chapters The data necessary for the calculation of the
radiant exposure for the equipment used in the respective trials are summarized in
table 1
Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High
Infrared 950 80 120 160 Red 660 15 46 80
Green 570 02 42 10
10
The radiant exposure of the used LED can be calculated as follows65
RE =
Radiant Exposure [Jcm2]
T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]
PRE = α S T
α = 006 (continuous mode) or
003 (pulsed mode)
The parameters commented on so far can be considered as the external dosimetry
involving all parameters directly controlled by the operator limited by the apparatus
used Furthermore there is the so-called internal dosimetry referring to (1) several
physical phenomena (reflection transmission scattering and absorption) influencing
the light distribution within the tissue during energy transfer (2) the optical
characteristics of the irradiated tissue as well as (3) the relation between the external
dosimetry and these respective elements5466
This internal dosimetry determines to a considerable extend the penetration of light
into tissue Penetration can be defined as the tissue depth at which the radiant
exposure is reduced to 37 of its original value1948 However this definition only
accounts for the absolute penetration depth resulting in direct effects of light at that
depth In addition there is also a relative penetration depth leading up to effects
deeper in the irradiated tissue and even in certain degree throughout the entire
body1967 These so-called systemic effects can be caused by chemical processes initiated
at superficial levels at their turn mediating effects at a deeper tissue level57
Involvement of several forms of communication in the tissue such as blood circulation
and transport of transmitters or signal substances is possible1967 This means that light
sources with poor absolute penetration do not necessarily give inferior results than
those with a good absolute penetration19
In the same context it should be noted that calculation and even measurement of the
exact light distribution during irradiation is highly complicated principally as tissues
have complex structures and also because the optical properties of tissues vary largely
inter-individual2768
General introduction
11
Studies regarding actual penetration depth of LED light are scarce consequently the
knowledge on the topic of penetration depth of LED light is based on literature
originating from LLL research19 These findings established with various LLL sources
revealed that there is an obvious relation between penetration depth and
wavelength27486769-71
Three final remarks can be made on the dosimetry First of all it should be noted that
partly as a result of the above-mentioned contrasting findings on dosimetry ideal light
source characteristics for effective treatment of various medical applications are not yet
established and probably never really will be28 Therefore in the attempt to offer
sufficient guidelines for correct use of treatment parameters one should always try to
provide detailed description of light source properties used in any trial so the
practitioner can interpret the scientific results adequately and accordingly draw the
correct conclusions for his clinical practice
A second comment is based on the mentioned possible influence of the external and
internal dosimetric parameters on the photobiological effectiveness of light the
intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation
between radiated wavelength and penetration depth19546572 So it should be
emphasized that caution is recommended when comparing research results of light
sources with different wavelengths or other dissimilar dosimetric parameters
A third and final remark considers the extrapolation issue Comparison of the
therapeutic usefulness of the same light source used on different species should occur
cautiously So simply extrapolating the dosage used for one species to another is
inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo
clinical practice is inappropriate So purposive and specific research is the prerequisite
to produce safe and correct use of light as a therapeutic modality27
12
MECHANISMS OF ACTION
In the past decennia several mechanisms of action for biostimulation and pain
inhibition have been proposed and investigated73 Research was primarily based on
studies at the molecular and cellular levels and as a second resort investigations
occurred at the organism level resulting in numerous possible explanatory
mechanisms272858
It is the common view that light triggers a cascade of cellular and molecular reactions
resulting in various biological responses Thus different mechanisms of whom the
causal relationships are very difficult to establish- underlie the effects of light3448557475
To illustrate this complex matter the various mechanisms of action will be summarised
by means of a comprehensive model (fig 2) Detailed discussion about the different
individual components of the proposed model and other effects than those regarding
wound healing or analgesia were not provided as this was beyond the scope of this
general introduction
As depicted in figure 2 exposure to light leads to photon absorption by a
photoacceptor molecule causing excitation of the electronic state or increased
vibrational state of the given molecule275173 This process is followed by primary
photochemical reactions7475 Several key mechanisms have been discussed in the
literature Respiratory chain activation is the central point and can occur by an
alteration in redox properties acceleration of electron transfer generation of reactive
oxygen species (namely singlet oxygen formation and superoxide generation) as well as
by induction of local transient heating of absorbing chromophores192848515576-83 It is
supposed that each of these respective mechanisms plays a part in obtaining a
measurable biological effect It is yet not clear if one mechanism is more prominent
and decisive than another nevertheless recent experimental evidence has revealed that
mechanisms based on changes in redox properties of terminal enzymes of respiratory
chains might be of crucial importance2848517679
The primary mechanisms occurring during light exposure are followed by the dark
reactions (secondary mechanisms) occurring when the effective radiation is switched
General introduction
13
off2851 Activation of respiratory chain components is followed by the initiation of a
complicated cellular signalling cascade or a photosignal transduction and amplification
chain associated with eg changes in the cellular homeostasis alterations in ATP or
cAMP levels modulation of DNA and RNA synthesis membrane permeability
alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87
The sequence of events finally results in a range of physiological effects essential for
the promotion of the wound healing process for supplying analgesia or other
advantageous responses (acceleration of inflammatory processes oedema re-
absorption increased lymph vessel regeneration or increased nerve
regeneration)12181927486188-93
Photostimulation of the wound healing process can be mediated by increased
fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis
and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations
have been especially focussed on fibroblasts but other possible physiological effects
attributing to an accelerated wound healing were also observed suppression and
alteration of undesirable immune processes increased leukocyte activity new
formation of capillaries increased production of growth factors and enzymes while
monocytes and macrophages can provide an enlarged release of a variety of substances
related to immunity and wound healing1619277376
As pain and nociception are even less understood than wound healing the possible
mechanisms in obtaining pain relief by the use of light are less underpinned However
it is established that light therapy influences the synthesis release and metabolism of
numerous transmitter signal substances involved in analgesia such as endorphin nitric
oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these
neuropharmacological effects there is experimental evidence for diminished
inflammation decreased C-fibre activity increased blood circulation and reduced
excitability of the nervous system1927848899
One should be aware that a large amount of research regarding the possible
mechanisms of light action was conducted at the cellular level The described cascade
of reactions at the organism level is possibly even more complex as in contradiction to
14
the in vitro situation in vivo a range of supplementary interactions can influence the
sequence of effects and accordingly the final responses Besides it needs to be
mentioned that this summary did not take into account the origin of the light or the
external dosimetry thus the description is based on investigations performed with
various light sources and different dosages
Figure 2 Model summarizing the identified mechanisms of light action
Secondarymechanisms
Primary mechanisms
Final effects
Trigger
Stimulated wound healing Analgesia
Exposure to light
Photon absorption by photoacceptors
Respiratory chain activation
Accelerated electrontransfer
Reactive oxygen generation
Heating of absorbing chromophores
Altered redox properties
darr inflammation uarr oedema resorption
uarr lymph vessel regenerationuarr blood circulation
Photosignal transduction and amplification chain
uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation
darr C-fibre activity darr nervous excitability neuropharmacological effects
General introduction
15
Regardless of the large number of previous investigations identification of underlying
mechanisms of light action remains an important issue as these are not yet fully
understood and because probably not all mechanisms of action are currently
identified Convincing explanation of the mechanisms in normal as well as in
pathological tissue could banish the existing suspicion concerning the use of light as a
treatment modality2732547678
AIMS AND OUTLINE
The introduction of LED in medicine and in physiotherapy more specifically requires
particular scientific research especially within the fields of its clinical potential
application wound healing and analgesia The above described gaps in literature
regarding the use of LED laid the foundation of this doctoral thesis
Consequently the general purpose of this thesis is to explore a scientific approach for
the supposed biostimulatory and analgesic effect of LED and to formulate an answer
in view of an evidence-based clinical use of this treatment modality
The detailed objectives can be phrased as follows
Aim 1 To assess the biostimulatory effectiveness of LED
irradiation under normal in vitro conditions
Aim 2 To investigate the value of LED treatment to ameliorate
in vitro cell proliferation under conditions of impaired healing
Aim 3 To examine the effectiveness of LED in changing the
nerve conduction characteristics in view of analgesia
Aim 4 To determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting
Part I investigates the influence of LED on wound healing In pursuit of the first aim
chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial
16
measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these
cells needs to be considered as an exponent of the wound healing process as
fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase
and early remodelling100 Secondly an in vivo case study exploring the postulation that
LED irradiation could accelerate and ameliorate the healing of a surgical incision was
described
The results contrasted sharply with the findings of the in vitro part Two fundamental
causes were proposed in order to explain the different biological effect of LED
irradiation observed in vitro and in vivo the used irradiation parameters and evaluation
method
The experiment described in chapter 2 endeavoured to explore these considerations A
similar study was therefore performed but as distinctive characteristics different light
source properties an adapted irradiation procedure and the use of a colorimetric assay
based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the
counting of the cells were used
As stimulation of the wound healing process is virtually mainly indicated under
conditions of impaired healing (resulting in a situation which threatens to become
chronic and debilitating) proper attention for this matter is warranted192855 Besides
the medical consequences the costs involved with impaired healing yield also a socially
relevant problem to tackle Impaired healing will become even more common as the
world population continues to age After all senescence of systems and age-committed
comorbid conditions are commonly the culprits responsible for poor wound healing101
Thus finding cost-effective time-sparing non-invasive and practical treatment
modalities to cure wounds is a necessity
Aiming to assess the biostimulative effects by means of LED in these circumstances a
third study was conducted with respect of the previous results regarding irradiation
parameters and cell proliferation analysis The irradiation experiment described in
chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose
This medium modification serves as a pattern for cell proliferation in diabetic patients
General introduction
17
a population for whom stimulation of the wound healing process is a clinical relevant
feature
In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored
by means of two studies A first investigation (chapter 4) evaluated the influence of LED
on the sensory nerve conduction characteristics of a human superficial peripheral
nerve as a potential explanatory mechanism of pain inhibition by LED which is based
on the putative neurophysiological effects of this treatment modality The experimental
hypothesis postulated that LED generates an immediate decrease in conduction
velocity and increase in negative peak latency In addition it was postulated that this
effect is most prominent immediately after the irradiation and will weaken as time
progresses
The values of nerve conduction velocity and negative peak latency of a baseline
antidromic nerve conduction measurement were compared with the results of five
identical recordings performed at several points of time after LED irradiation
Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory
setting To guarantee an adequate standardized and controlled pain reduction study
there was opted to observe a healthy population with experimentally induced DOMS
Induction of DOMS has been described in a number of studies as a representative
model of musculoskeletal pain and stiffness because it can be induced in a relatively
easy and standardised manner the time course is quite predictable and the symptoms
have the same aetiology and are of transitory nature4445102-105
The treatment as well as the assessment procedure was performed during 4
consecutive days The first day isokinetic exercise was performed to induce pain
related to DOMS Subsequently the volunteers of the experimental group received an
infrared LED treatment and those of the placebo group received sham-irradiation
Evaluation of the effect of the treatment on perceived pain was registered by a visual
analog scale and by a mechanical pain threshold these observations occurred every day
18
prior to and following LED irradiation Eccentricconcentric isokinetic peak torque
assessment took place daily before each treatment
For the analysis of the results three different factors were taken into consideration
time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental)
In completion of this thesis the most prominent findings are summarized and the
clinical implications are discussed The general discussion also includes some future
research directions and a final conclusion
General introduction
19
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PART I WOUND HEALING
CHAPTER 1
DO INFRARED LIGHT EMITTING DIODES HAVE A
STIMULATORY EFFECT ON WOUND HEALING FROM AN IN
VITRO TRIAL TO A PATIENT TREATMENT
Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Proceedings of SPIE 2002 4903 156-165
Chapter 1
28
ABSTRACT
Variable effects of different forms of light therapy on wound healing have been
reported This preliminary study covers the efficacy of infrared light emitting diodes
(LED) in this domain
Cultured embryonic chicken fibroblasts were treated in a controlled randomised
manner LED irradiation was performed three consecutive days with a wavelength of
950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each
treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2
The results indicated that LED treatment does not influence fibroblast proliferation at
the applied energy density and irradiation frequency (p=0474)
Meanwhile the effects of LED on wound healing in vivo were studied by treating a
surgical incision (6 cm) on the lateral side of the right foot of a male patient The
treatment started after 13 days when initial stitches were removed The same
parameters as the in vitro study were used but the treatment was performed five times
The healing could only be evaluated clinically the irradiated area (26 cm) showed a
more appropriate contraction less discoloration and a less hypertrophic scar than the
control area (34 cm)
The used parameters failed to demonstrate any biological effect of LED irradiation in
vitro although the case study on the other hand illustrated a beneficial effect
Keywords Light Emitting Diodes Fibroblasts Wound healing
From an in vitro trial to a patient treatment
29
INTRODUCTION
Various beneficial effects of lasers and photodiodes at relatively low intensities have
been reported involving treatment of neurological impairments12 treatment of pain3-5
treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the
enhancement of wound healing has been a focus of contemporary research11-16 It
seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing
remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the
basis of a questionnaire about low power laser (LPL) in the current clinical practice in
Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey
into clinical LPL experience in Flanders
Nevertheless there remains a considerable amount of ignorance scepticism and
controversial issues concerning the use and clinical efficacy of LPL even in the domain
of wound healing12152021 This is at least in part a consequence of the inability to
measure and control operating variables related to connective tissue repair and of the
wide range of suitable parameters for irradiation
Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent
characteristics which make their use in a clinical setting problematic including
limitations in wavelength capabilities and beam width The combined wavelength of
light optimal for wound healing cannot be efficiently produced and the size of
wounds which may be treated by LPLs is limited Some companies offer an
alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be
made to produce multiple wavelengths and can have probes with large surface area
allowing treatment of large wounds Still one can not accept this light source as an
alternative for LPL therapy based on the cited advantages without proper investigation
regarding its biostimulatory effects
The effectiveness of this possible alternative for LPLs must be studied in vitro and in
addition in animal models or in humans because the effects of LED at the cellular level
do not necessarily translate to a noticeable effect in vivo The small amount of previous
investigations demonstrate that LED effects are as difficult to isolate162223 as LPL
Chapter 1
30
effects and the results are conflicting just like the results in literature specific on the
use of LPL121520
The purpose of the first part of this study is to examine the hypothesis stating that
LED irradiation can influence fibroblast proliferation Therefore a comparison of the
proliferation from fibroblasts in irradiated and control wells was performed The in vitro
investigation was linked with an in vivo case study This part enquired the assumption if
LED irradiation could accelerate and ameliorate the healing of a surgical incision
IN VITRO INVESTIGATION
MATERIALS AND METHODS
The complete procedure from isolation to proliferation analysis was executed twice
(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of
control dishes The second trial consisted of 27 irradiated and 27 control dishes
Cell isolation and culture procedures
Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation
and disaggregating of the cells occurred with warm trypsin (NV Life Technologies
Belgium) according the protocol described by Ian Freshney (1994)24 The primary
explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies
Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1
Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life
Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies
Belgium) When cell growth from the explants reached confluence cells were detached
with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV
Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the
cells were removed from the culture flasks by trypsinization and counted by
hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc
From an in vitro trial to a patient treatment
31
NV Life Technologies Belgium) The fibroblasts were seeded at a density of
70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture
medium the cells were allowed to attach for 24 hours in a humidified incubator at
37degC
Properties of the Light Emitting Diode
Prior to LED treatment all dishes were microscopically checked to guarantee that the
cells are adherent and to assure that there is no confluence nor contamination The
dishes were divided randomly into the treated or the control group Medium was then
removed by tipping the dishes and aspirating with a sterile pipette Following the
aspiration 2 ml fresh medium was added and treatment started
A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser
Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-
1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32
single LEDrsquos For the treatments in this study an average power of 160 mW at
continuous mode was applied The irradiation lasted 6 minutes resulting in an energy
density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result
of the divergence in function of this distance the surface of the LED (18 cm2) covered
the complete surface of the used petriplates (215 cm2)
After these manipulations 3 ml medium was added to each dish followed by 24 hours
incubation
One LED irradiation was performed daily during three consecutive days according
this procedure Control cultures underwent the same handling during these three days
but were sham-irradiated
Proliferation analysis
After the last treatment a trypsination was performed to detach the cells from the
culture dishes followed by centrifugation Once the cells were isolated from the used
trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts
Chapter 1
32
within this suspension as reflection for the proliferation was quantified by means of a
Buumlrker Chamber or hemocytometry
The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-
Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab
Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on
the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator
using an inverted light microscope counted the number of cells in 25 small squares
In order to calculate the number of cells one should multiply the amount of cells
counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and
the dilution factor (the amount of Trypan blue suspended with the cells 21=3)
Statistical methods
The data were analysed statistical in order to examine the hypothesis that LED
irradiation enhances fibroblast proliferation They were processed as absolute figures
for both trials separately In a second phase the counted cell numbers were converted
in relative figures so the data of both trials could be analysed as the data of one test
These relative figures were obtained by expressing each figure as a percentage from the
highest figure (=100) of that trial and this for each assay separately
A Kolmogorov-Smirnoff test of normality was performed on the data followed by a
Mann-Whitney-U test when the test of normality was significant and otherwise a T-
test Differences were accepted as significant when plt005 For this analysis SPSSreg
100 was used
RESULTS
The descriptive data for both trials are depicted in figure I The mean number of cells
in trial A is higher than in trial B for the controls as for the treated wells There is a
mean difference of 1252500 fibroblasts between the controls and 1223000 between
the irradiated wells of trial A and B The averages of both trials show that in control
cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no
From an in vitro trial to a patient treatment
33
statistically significant difference could be found between the two groups in either trial
nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not
significant for trial A (p=020) nor trial B (p=020) Only the combined data from both
trials were significant (plt001) for normality Further analysis respectively T-test for
the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the
combined data (p=0474) revealed no statistical significant differences
DESCRIPTIVE DATA
1730000181750029530003070000
00E+00
50E+05
10E+06
15E+06
20E+06
25E+06
30E+06
35E+06
40E+06
Trial A Trial A Trial B Trial B
Mea
n n
um
ber
of
cells
Control
Irradiated
Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B
DISCUSSION
Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities
(lt500 mW) in vitro have been analysed by evaluating various factors involving
(pro)collagen production25-27 cell viability2829 growth factor production28 and
myofibroblast formation30 Fibroblast proliferation also is an important factor to
consider In accordance with wound healing fibroblasts fulfil an essential role especially
in the late inflammatory phase and the early granulation phase31 Despite the failure of
some studies to demonstrate beneficial effects of LPL irradiation on fibroblast
proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very
Chapter 1
34
significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -
208 ) Atabey et al35 also revealed a significant increase in cell number two or more
irradiations resulted in an increased fibroblast proliferation Several other studies
confirmed these positive findings25263637
The results of this present in vitro study indicate that LED treatment does not
influence fibroblast proliferation Although the dosimetric parameters (in particular the
arbitrary energy density of 32 Jcm2) used in this study are well within the
recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous
studies about LPL therapy raising enhanced fibroblast proliferation252634-37
Van Breugel et al36 gave a possible explanation for these controversial results
According to them the fibroblast proliferation is not inherent at the energy density
They provide evidence that independent of the energy density the power density and
the exposure time determine the biostimulative effects of LPL irradiation LPL with a
power below 291 mW could enhance cell proliferation while a higher power had no
effect
Some authors also argued that the absorption spectrum of human fibroblasts show
several absorption peaks and pointed out that a wavelength of 950 nm is far above the
highest peak of about 730 nm3638 At longer wavelengths they determined a general
decrease in absorption Despite these results several investigators pose biostimulative
effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or
even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is
ranged from 800 nm to 830 nm principally because of the presence of cytochrome
oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts
at lower wavelengths is restricted by an inferior skin transmission than at higher
wavelengths38
Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting
respective green and red light The 950 nm beam of light was used for its high power
density but according to a range of remarks mentioned above the effects of the two
other probes must be as well evaluated
From an in vitro trial to a patient treatment
35
Another factor one can not ignore is that besides fibroblast proliferation other
processes or morphologic changes were not analysed although several authors have
posed that those changes and processes could be responsible for the biostimulative
effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et
al30 for example described a massive transformation of fibroblasts into myofibroblasts
after LPL treatment These modified fibroblasts play an important role in contraction
of granulation tissue30 A second example is an increased (pro)collagen production
after low power light therapy25-27 which is also considered as a responsible factor for
accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast
proliferation3640
It may be wondered if the light sources mostly LPL in the consulted literature are
representative for the LED used in this study although this LPL literature is often
used for that purpose As in the early days of LPL the stimulative effects upon
biological objects were explained by its coherence the beam emitted by the Bio-Dio on
the contrary produces incoherent light Nowadays contradictory research results are
responsible for a new discussion the clinical and biological significance of coherence
The findings of some authors172341-43 pose that the coherence of light is of no
importance of LPL and its effects although the opposite has also been stated4445
Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be
accounted for the non-enhanced fibroblast proliferation in this trial
Another possible explanation for the absence of biostimulative effect is related to the
moment of analysis of the proliferation The evaluation one day after the last
irradiation did not allow a delayed enhancement of proliferation while it is determined
in numerous investigations that the effects occur more than 24 hours after the last
treatment273746 and that they weaken after a further undefined period of time34
The fluctuation in cell numbers between both trials despite the use of an identical
protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor
reproducibility of their technique In this study the fluctuations are attributable to the
counting of the cells by Buumlrker hemocytometer before seeding According to some
authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large
Chapter 1
36
variability48 and it is often difficult to standardize48 Overestimation of the cell
concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity
was contradicted by Lin et al50 moreover satisfactory correlations with flow-
cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay
for cell counting (MTT)51 were determined
An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it
provides accurate cell counts in a short period of time with less intervention from the
investigator52
These remarks and controversies point out the possible deficiencies from the used
proliferation analyses and the relativity from the obtained results Other analyse
methods and analyses from different cell processes and morphologic changes could be
considered for further investigation
IN VIVO INVESTIGATION
MATERIALS AND METHODS
The effects of LED on wound healing in vivo were studied by treating a postsurgical
incision A male patient received chirurgical treatment for the removal of a cyst
situated approximately 15 cm posterior from the lateral malleolus of his right foot For
removal of the cyst an incision of 6 cm was made The incision was sutured and 12
days after the surgery the stitches were removed Visual inspection demonstrated that
the healing process of the wound proceeded well but not equally over the whole 6
centimetres (figure II) Epithelialization and wound contraction appeared to have
progressed better in the upper part (approximately 3 cm) of the cicatrice than at the
lower part (covered with eschar) No evidence of infection was noted in either part
LED treatment started the 13th day The incision was treated partially the lowest part
(26 cm) with the inferior epithelialization and wound contraction was irradiated the
remaining 34 cm served as control area This control area was screened from radiation
with cardboard and opaque black cling film
From an in vitro trial to a patient treatment
37
The light source destinated for the treatment was the same device used for the in vitro
irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED
output parameters were identical with those applicated in the preceding in vitro
investigation In particular a continuous wave at an average power of 160 mW and 6
minutes of treatment duration corresponding to an energy density of 32 Jcm2 An
equal distance from the probe to the target tissue as from the probe to the culture
medium was respected A plastic applicant of according height guaranteed constant
distance of 06 cm from the surface of the skin
Figure II Surgical incision before the first treatment 13 days after initial stitching
Therapy was performed once a day during five consecutive days repeatedly at the same
time resulting in an extension of the duration of the in vitro therapy with two days
Visual macroscopic observations were accomplished 6 52 and 175 days after the first
treatment
Comparison of the cutaneous sensitivity at the irradiated area and the control area was
accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175
days after the first treatment A control measurement also occurred at the same region
Chapter 1
38
on the left foot The aesthesiometer used in this study consisted of five hand-held
nylon monofilaments with a length of 38 mm and varying diameter
Sensitivity threshold is traced by presenting a monofilament of a certain diameter
vertically to the skin The monofilament bends when a specific pressure has been
reached with a velocity proportional to its diameter Measurements allow mapping
areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified
from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest
filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished
protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to
finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the
filaments
RESULTS
Visual estimation at any point of time after irradiation divulged no occurrence of
problems with dehiscence or infection in either part of the wound During the five
days of therapy the irradiated area looked dryer than the control area After the last
irradiation this was no longer recorded
Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area
From an in vitro trial to a patient treatment
39
Figure III representing the first evaluation six days after the initial treatment
illustrates that the wound healing has evolved slightly in both parts Though the lower
irradiated part remains of inferior quality as regards to epithelialization and wound
contraction In the course of the reparative process the influence of light exposures
were registered At 52 days after the first irradiation beneficial effects of LED
treatment are clearly present (Figure IV)
Figure IV Surgical incision 52 days after initiating LED treatment
The irradiated area (26 cm) showed a more appropriate contracture than the control
area (34 cm) characterized by less discoloration at scar level and a less hypertrophic
scar A similar trend was noticed at a third visual observation 175 days after the initial
treatment At that moment no impairments at cutaneous sensitivity level were stated
and the sensitivity showed no differences between left or right foot nor between the
two areas of the cicatrice
Chapter 1
40
DISCUSSION
The results of this case study indicate that LED had a positive influence on wound
healing in humans as determined by visual observations Many investigators
examining the effects of LPL on wound healing by means of a range of observation
and treatment methods reported accelerated and enhanced wound healing8-10 others
described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and
LED21 studies were unable to repeat these results
The late but beneficial findings in this study seem to be to the credit of LED-therapy
Though several authors establish positive results in an earlier stage of the wound
healing process8-1020 one should question why the differences did not occur at the first
evaluation on day 6 An explanation can be found in the start of the treatment Most
investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so
they influence a first cellular and vascular reaction with the production of chemical
mediators of inflammation resulting in an enhanced collagen production9 tremendous
proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the
time the first treatment in this study took place the traumatized tissue was in an
overlapping stage between an almost finished inflammatory phase and a scarcely
initiated re-epithelialization and wound contraction phase At that moment an infiltrate
of fibroblasts is present So fibroblast proliferation a possible mechanism of the
biostimulative effect had already occurred and could no longer be influenced Growth
factor production and collagen deposition have also decreased at that stage
Granulation tissue formation and fibroplasia in the contrary are initiating by that time
Those prolonged and slow processes with belated results are of significant importance
for the course of the final stage of wound healing and for the outlook of the future
scar31
The experimental findings revealed that the sensitivity of the skin according to the
threshold detection method of Semmes and Weinstein was normal at all the
investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a
good and objective predictor of normal skin sensitivity No other LPL nor LED
studies investigating this quality of the skin were found
From an in vitro trial to a patient treatment
41
CONCLUSION
This study demonstrates that although LED application at the applied energy density
and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to
have beneficial biostimulative effects on wound healing in human skin confirmed by
the favourable re-epithelialization and contracture
These results are discussed in the context of other experimental findings but no
reasonable explanation for this discrepancy could be found The literature on wound
healing after LED treatment in animal models or in humans is presently very limited
and contradictory The diversity in used radiation parameters and the absence of
references on how the wounds were measured or evaluated or what the end point was
for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro
investigations are better standardised nevertheless these results show a number of
conflicts One can conclude that until today the controversial findings are characteristic
for many results obtained with light photobiomodulation
However the postponed favourable results in the case study confirm some facts of the
discussion Namely the short period of incubation 24 hours in the in vitro part of the
study can be responsible for the lack of enhanced fibroblast proliferation It also
confirms that other cell processes and morphologic changes possibly are responsible
for biostimulative effects in vivo other observation methods should be considered for
future in vivo experiments
Despite these remarks we believe that LED application on cutaneous wounds of
human skin is useful with a single flash daily at the dose applied in this study for at
least three days
Furthermore future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Chapter 1
42
ACKNOWLEDGMENTS
The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the
material necessary for this study as well as Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
From an in vitro trial to a patient treatment
43
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2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)
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9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)
10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)
11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)
12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)
13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)
14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)
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16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)
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19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)
20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)
Chapter 1
44
21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)
22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)
23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)
24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)
25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)
26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)
27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)
28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)
29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)
30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)
31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)
32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)
33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)
34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)
35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)
36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)
37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)
38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)
39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)
40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)
41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)
42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)
From an in vitro trial to a patient treatment
45
43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)
44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)
45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)
46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)
47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)
48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)
49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)
50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)
51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)
52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)
53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)
54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)
CHAPTER 2
INCREASED FIBROBLAST PROLIFERATION INDUCED BY
LIGHT EMITTING DIODE AND LOW LEVEL LASER
IRRADIATION
Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Lasers in Medical Science 2003 18(2) 95-99
Chapter 2
48
ABSTRACT
Background and Objective As Light Emitting Diode (LED) devices are
commercially introduced as an alternative for Low Level Laser (LLL) Therapy the
ability of LED in influencing wound healing processes at cellular level was examined
Study DesignMaterials and Methods Cultured fibroblasts were treated in a
controlled randomized manner during three consecutive days either with a infrared
LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and
570 nm) and respective power outputs Treatment duration varied in relation to
varying surface energy densities (radiant exposures)
Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all
irradiated cultures in comparison with the controls Green light yielded a significantly
higher number of cells than red (plt0001) and infrared LED light (plt0001) and than
the cultures irradiated with the LLL (plt0001) the red probe provided a higher
increase (plt0001) than the infrared LED probe and than the LLL source
Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation
in vitro This study therefore postulates possible stimulatory effects on wound healing
in vivo at the applied dosimetric parameters
Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low
Level Laser Tetrazolium salt
LED induced increase of fibroblast proliferation
49
INTRODUCTION
Since the introduction of photobiostimulation into medicine the effectiveness and
applicability of a variety of light sources in the treatment of a wide range of medical
conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results
of several investigations are remarkably contradictory This is at least in part a
consequence of the wide range of indications as well as the wide range of suitable
parameters for irradiation and even the inability to measure the possible effects after
irradiation with the necessary objectivity [457] A lack of theoretical understanding
can also be responsible for the existing controversies In fact theoretical understanding
of the mechanisms is not necessary to establish effects though it is necessary to
simplify the evaluation and interpretation of the obtained results As a consequence
the widespread acceptance of especially Low Level Laser (LLL) therapy in the early
seventies is faded nowadays and biostimulation by light is often viewed with scepticism
[8] According to Baxter [49] contemporary research and consumption in
physiotherapy is in particular focused on the stimulation of wound healing Tissue
repair and healing of injured skin are complex processes that involve a dynamic series
of events including coagulation inflammation granulation tissue formation wound
contraction and tissue remodelling [10] This complexity aggravates research within this
cardinal indication
Research in this domain mostly covers LLL studies but the current commercial
availability of other light sources appeals research to investigate as well the effects of
those alternative light sources eg Light Emitting Diode (LED) apparatus
The scarcity of literature on LED is responsible for consultation of literature
originating from LLL studies [11] but it may be wondered if this literature is
representative for that purpose As in the early days of LLL therapy the stimulating
effects upon biological objects were explained by its coherence [1213] while the beam
emitted by LEDrsquos on the contrary produces incoherent light Though the findings of
some scientists [914151617] pose nowadays that the coherence of the light beam is
not responsible for the effects of LLL therapy Given that the cardinal difference
between LED and LLL therapy coherence is not of remarkable importance in
Chapter 2
50
providing biological response in cellular monolayers [5] one may consult literature
from LLL studies to refer to in this LED studies
The purpose of this preliminary study is to examine the hypothesis that LED
irradiation at specific output parameters can influence fibroblast proliferation
Therefore irradiated fibroblasts cultures were compared with controls The article
reports the findings of this study in an attempt to promote further discussion and
establish the use of LED
MATERIALS AND METHODS
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos Isolation and
disaggregation of the cells was performed with warm trypsin according the protocol
described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in
Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1
L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants
reached confluence cells were detached with trypsine and subcultured during 24 hours
in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours
the cells were removed from the culture flasks by trypsinization and counted by Buumlrker
hemocytometry For the experiment cells from the third passage were plated in 96-well
plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a
density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C
during 24 hours
All supplies for cell culture were delivered by NV Life Technologies Belgium except
for Fetal Calf Serum (Invitrogen Corporation UK)
Irradiation sources
In this study two light sources a Light Emitting Diode (LED) device and a Low Level
Laser (LLL) device were used in comparison to control cultures
The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)
LED induced increase of fibroblast proliferation
51
with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400
mW and a frequency range from 0-1500 Hz
The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)
consisted of three wavelengths emitted by separate probes A first probe emitting
green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the
red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third
probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared
light The area of all three probes was 18 cm2 and their frequency was variable within
the range of 0-1500 Hz
Exposure regime
Prior to irradiation the 96-well plates were microscopically verified to guarantee that
the cells were adherent and to assure that there was no confluence nor contamination
Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining
25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation
The 96-well plates were randomly assigned in the treated (LLL or green red or infrared
LEDrsquos) or the control group
For the treatments in this study the continuous mode was applied as well for the LLL
as for the three LED-probes The distance from light source to fibroblasts was 06 cm
LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting
in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant
exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to
exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective
power output of 160 mW 80 mW or 10 mW
After these handlings the remaining medium was removed and new Hanksrsquoculture
medium was added followed by 24 hours of incubation
One irradiation (LLL or LED) was performed daily during three consecutive days
according to the aforementioned procedure Control cultures underwent the same
handling but were sham-irradiated
Chapter 2
52
Determination of cell proliferation
The number of cells within the 96-well plates as a measure for repair [19] was
quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The
colorimetric assay was performed at two different points of time to determine the
duration of the effect of the used light sources
This assay exists of a replacement of Hanksrsquoculture medium by fresh medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as
described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT
solution was substituted by lysing buffer isopropyl alcohol The plates were
temporarily shaken to allow dissolution of the produced formazan crystals After 30
minutes of exposure to the lysing buffer absorbance was measured The absorbance at
400 to 750 nm which was proportional to fibroblast proliferation was determined
using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)
The complete procedure from isolation to MTT assay was executed six times (Trial A
B C D E and F) while it was impossible to irradiate all the investigated number of
wells with the same LED apparatus on one day All the trials included as much control
as irradiated wells but the number of control and irradiated wells in each trial varied
depending on the number of available cells after the second subculturing A further
consequence of the available number of cells is the number of probes examined per
trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E
Incubation period before proliferation analyses numbered 24 hours To investigate if
the stimulatory effect tends to occur immediately after irradiation or after a longer
period of time incubation in trial F lasted 72 hours
An overview of the followed procedures regarding incubation time before proliferation
analysis number of analysed wells for each trial and the number of probes examined
per trial is given in table 1 As a consequence of the differences in procedures followed
and because each trial started from a new cell line the results of the five trials must be
discussed separately
LED induced increase of fibroblast proliferation
53
Statistical analysis
Depending on the amount of groups to be compared within each trial and depending
on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way
ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons Statistical significance for all tests was
accepted at the 005 level For this analysis Statistical Package for Social Sciences 100
(SPSS 100) was used
RESULTS
The results presented in table 1 show that cell counts by means of MTT assay
revealed a significant (plt0001) increase in the number of cells in comparison to their
respective sham-irradiated controls for all the irradiated cultures of trial A B C D
and E except the irradiated groups in trial F
Moreover the results of trial A showed that the effect of the green and red LED probe
was significantly (plt0001) higher than the effect of the LLL probe With regard to the
amount of proliferation the green probe yielded a significantly higher number of cells
than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe
provided a higher increase in cells (plt0001) than the infrared probe
The infrared LED source and the LLL provided a significant (plt0001) higher number
of cells than the control cultures but no statistical significant difference was recorded
between both light sources
The trials A B C D and E regardless of the number of probes used in each trial
were analysed after 24 hours of incubation after the last irradiation The incubation
period of trial F lasted 72 hours
The means of trial F illustrated that the effect was opposite after such a long
incubation The control cultures had significantly (plt0001) more fibroblasts than the
irradiated cultures with the exception of the LED-infrared group that showed a not
significant increase of cells Further analysis revealed that the green probe yielded a
significantly lower number of cells than the red (plt0001) and the infrared probe
(plt0001) and that the red probe provided a higher decrease (plt0001) than the
Chapter 2
54
infrared probe Laser irradiation induced a significant decrease of fibroblasts in
comparison to the infrared irradiated cultures (plt0001) and the control cultures
(p=0001) LED irradiation with the green and the red probe revealed no statistical
significant differences
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups
Absorbency (proportional to the number of fibroblasts)a
Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050
Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis
DISCUSSION
Despite the failure of some studies [223] to demonstrate beneficial effects of laser and
photodiode irradiation at relatively low intensities (lt500mW) on fibroblast
LED induced increase of fibroblast proliferation
55
proliferation this study provides experimental support for a significant increased cell
proliferation Therefore these results confirm previous studies that yielded beneficial
stimulating effect [1152425] Remarkably though is the higher increase noted after
irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general
decrease in absorption at longer wavelengths and concluded that several molecules in
fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445
470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the
closest to one of these peaks
Karu [5] also emphasises that the use of the appropriate wavelength namely within the
bandwidth of the absorption spectra of photoacceptor molecules is an important
factor to consider
In this particular context penetration depth can almost be ignored as virtually all
wavelengths in the visible and infrared spectrum will pass through a monolayer cell
culture [12] The irradiance (Wcm2) on the contrary could have had an important
influence on the outcome of this study The higher increased proliferation by the lower
wavelengths is possibly a result of the lower irradiance of these wavelengths Lower
irradiances are confirmed by other experiments to be more effective than higher
irradiances [111626]
The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as
described by Poumlntinen [17] but in the scope of these results it also needs to be noticed
that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)
the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)
Consequently the results of especially trial A and F must be interpreted with the
necessary caution It is possible that the determined distinction between the used light
sources and the used probes is a result from the various radiant exposures applied
during the treatments of the cultures
Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after
the last irradiation this study was unable to demonstrate a stimulating effect when
analysis was performed 72 hours after the last irradiation Moreover this longer
incubation period even yielded an adverse effect Although a weakening of the
Chapter 2
56
photostimulating influence over time is acceptable it can not explain a complete
inversion Especially in the knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period [2427] In an attempt to illuminate
this finding one can suppose that the circadian response of the cells triggered by the
LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark
The most obvious explanation is even though a decreased vitality and untimely cell
death in the irradiated cell cultures as a result of reaching confluence at an earlier point
of time than the control cultures The cells of a confluent monolayer have the tendency
to inhibit growth and finally die when they are not subcultured in time No other
reasonable explanations could be found for this discrepancy
Photo-modulated stimulation of wound healing is often viewed with scepticism The
real benefits of Light Emitting Diodes if any can only be established by histological
and clinical investigations performed under well controlled protocols Despite these
remarks this study suggests beneficial effects of LED and LLL irradiation at the
cellular level assuming potential beneficial clinical results LED application on
cutaneous wounds of human skin may be assumed useful at the applied dosimetric
parameters but future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Persons in good health rarely require treatment for wound healing as posed by Reddy
et al [13] light has a possible optimal effect under conditions of impaired healing
Postponed wound healing is a time-consuming and often expensive complication
Thus future prospects must remind to examine the therapeutic efficacy of LED on
healing-resistant wounds
LED induced increase of fibroblast proliferation
57
ACKNOWLEDGMENTS
The authors are grateful to Prof Deridder for supplying the laboratory as well as the
material necessary for this investigation and to Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
Chapter 2
58
REFERENCES
1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55
2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32
3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7
4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994
5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998
6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32
7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42
8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5
9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8
10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7
11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8
12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34
13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9
14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18
15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43
16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6
17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475
18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J
Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9
21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601
22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63
LED induced increase of fibroblast proliferation
59
23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9
24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301
25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are
more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37
27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8
28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26
CHAPTER 3
GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES
FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL
Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Photomedicine and Laser Surgery 2005 23(2) 167-171
Chapter 3
62
ABSTRACT
Background and Objective The chronic metabolic disorder diabetes mellitus is an
important cause of morbidity and mortality due to a series of common secondary
metabolic complications such as the development of severe often slow healing skin
lesions
In view of promoting the wound-healing process in diabetic patients this preliminary
in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on
fibroblast proliferation and viability under hyperglycemic circumstances
Materials and Methods To achieve hyperglycemic circumstances embryonic chicken
fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL
glucose LED irradiation was performed on 3 consecutive days with a probe emitting
green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min
resulting in a radiation exposure of 01 Jcm2
Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all
irradiated cultures in comparison with the controls
Conclusion According to these results the effectiveness of green LED irradiation on
fibroblasts in hyperglycemic circumstances is established Future in vivo investigation
would be worthwhile to investigate whether there are equivalent positive results in
diabetic patients
Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes
Fibroblast proliferation under hyperglycemic circumstances
63
INTRODUCTION
The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide
geographic variation in incidence and prevalence generally 11 of the world
population is affected and worldwide it is the twelfth leading cause of death1 Those
figures may be higher for urban regions as well as for industrialized countries Due to
multiple factors involving the aging process of the population and lifestyle changes
(such as reduced physical activity hypercaloric eating habits and concomitant obesity)
these figures may increase in the future2-6 Therefore diabetes mellitus could become
the most common chronic disease in certain regions as stated by Gale it ldquotargets the
rich in poor countries and the poor in rich countriesrdquo6
The harmful disruption of the metabolic equilibrium in diabetes mellitus results in
characteristic end-organ damage that occurs in various combinations and that follows
an unpredictable clinical pathway
Accordingly the major consequence of diabetes mellitus in terms of morbidity
mortality and economic burden principally concerns macroangiopathies or
arteriosclerosis and microangiopathies including nephropathy neuropathy and
retinopathy7-10
One of these devastating consequences which often appears in time is the
development of various skin defects that are frequently resistant to healing and that
tend to be more severe than similar lesions in nondiabetic individuals Diabetes
mellitus even increases the risk of infection by an increased susceptibility to bacteria
and an impaired ability of the body to eliminate bacteria1112
Skin problems are a severe complication in diabetic individuals and require a
comprehensive and appropriate multidisciplinary approach to prevention and
treatment12
Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to
play the most prominent role in the development of diabetic complications With the
development of insulin treatment for type I diabetes and various oral hypoglycemic
agents for type 2 diabetes a reduction in the development of skin defects due to
hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply
Chapter 3
64
waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in
diabetic patients often need special care in comparison to those persons in good
health who rarely require treatment for wound healing1617 Special care is directed
besides of course toward optimal diabetes regulation toward patient education
maximum pressure relief controlling infection recovery of circulation in case of
ischemia and different modalities of intensive wound treatment18
In the last few years various therapies have been introduced with varying success An
example of such a therapy is the photo-modulated stimulation of diabetic lesions In
vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been
subject of extensive investigation1920 Due to contradictory research results LLL-
photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of
light in the domain of wound healing is less time-consuming less expensive less
invasive than many of the other introduced treatment modalities and practical to use
however it seems worthwhile to investigate the value and benefits of a newly
introduced and alternative light source the light emitting diodes (LEDrsquos)
Preliminary research has proved that green LED with particular properties (an
exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01
Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These
results may be of great importance to the diabetic patient because as posed by Reddy et
al light has a possible beneficial effect in the case of impaired healing1617
To obtain insight into the ability of LED to stimulate fibroblast proliferation under
diabetic-specific conditions of impaired healing the proliferation was assessed in
irradiated and control cultures cultivated in medium with a high quantity of glucose
MATERIAL amp METHODS
Cell cultivation
Primary fibroblast cultures were established by outgrowth from 8-day-old chicken
embryos After isolation and disaggregating as described by Freshney (1994)24 the cells
were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10
Fibroblast proliferation under hyperglycemic circumstances
65
fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin
Secondary cultures were initiated by trypsinization followed by plating of the cells in
80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were
disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently
231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third
passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the
cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified
atmosphere at 37deg C to allow them to attach to the bottom of the wells
Light source specifications and illumination procedure
To control adherence of the cells and to assure that there was no confluence or
contamination the 96-well plates were microscopically examined before irradiation
Subsequently the tissue culture plates were randomly assigned for use in the treated
and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium
was aspirated The remaining 25 (50 microl) medium avoided dehydration of the
fibroblasts throughout irradiation
Irradiation was performed with a light emitting diode (LED) device The LED device
(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power
range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable
within the range of 0-1500 Hz
The investigation used the following illumination properties the continuous mode a
distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2
radiant exposure This procedure resulted in an exposure time of 3 min and a power
output of 10 mW Immediately after irradiation the remaining medium was aspirated
and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM
glucose (30 gL) and incubated at 37deg C
Irradiation and medium changes occurred at 1-day intervals so one irradiation was
implemented each 24 h for 3 days in a row and from the first irradiation onwards all
medium renewals occurred with glucose-supplemented Hanksrsquo culture medium
Control cultures were handled in the same manner but were sham-irradiated
Chapter 3
66
Proliferation assay
Fibroblast survival and proliferation were determined by a sensitive and reproducible
colorimetric assay the assay which detects merely living cells and the signal generated
bears a constant ratio to the degree of activation of the fibroblasts and the number of
fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than
only a small subsample26
Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented
Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution
was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during
30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous
solution The optical density of the final solution was measured on an ELtimes800 counter
(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test
wavelength varying from 400 to 750 nm
The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies
for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except
for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)
Data analysis
On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a
Mann-Whitney U test was performed for nonparametrical comparison of the results
Statistical significance for all tests was accepted at the 005 level For this analysis the
Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used
RESULTS
The MTT measurements from each of the 256 control wells and 256 irradiated wells
and the subsequent nonparametrical analysis from the optical densities obtained
disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic
Fibroblast proliferation under hyperglycemic circumstances
67
circumstances after irradiation than in the same circumstances without irradiation (Fig
1)
Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)
DISCUSSION
The outcome of these in vitro experiments based on the above-described light source
properties and the illumination procedure described clearly demonstrated the
stimulatory potential of LED on fibroblast proliferation and the cell viability of
fibroblasts cultured in hyperglycemic medium Preliminary research has already
demonstrated that under these conditions (an exposure time of 3 min a wavelength of
570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this
procedure allowed the highest number of living cells The nature of the light and the
usual questions concerning coherence wavelength power output and radiant
exposures have been discussed previously23
Although these findings confirm the results previously found one cannot ignore the
important methodological difference between previous investigations and the current
study as the cells in this experiment were cultured in hyperglycemic medium2328-30
Absorbency - Proportional to the number of fibroblasts
621 x 10-1 682 x 10-1
0010203040506070809
1
Control Irradiated
Groups
Ab
sorb
ency
Chapter 3
68
After a growth period with normal Hanksrsquo culture medium a necessary step to ensure
normal growth of these secondary subcultures and normal attachment to the bottom
of the wells the Hanksrsquo culture medium was supplemented with glucose
Several earlier studies have established that exposure to glucose concentrations (20-40
mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human
vascular endothelial cell proliferation1531-34 This restraint is more pronounced for
higher glucose15 concentrations and is expressed especially after protracted exposure to
high glucose levels31 A similar restraint was found for cultured fibroblasts by
Hehenberger et al3536 According to some authors however cultured fibroblasts
conversely have been shown to maintain responsiveness to ambient high glucose323738
As there are some ambiguities in literature regarding normal or inhibited growth of
fibroblasts in medium supplemented with glucose39 a pilot study was performed to
determine the amount of glucose necessary to inhibit normal growth after 72 h of
culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study
demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease
of cell viability and to bring about a decline in fibroblast proliferation
This concentration resulted in a remarkable reduction of cell viability and a noteworthy
decrease in the proliferation rate in comparison to control cultures grown in 55 mM
glucose although this concentration is too high to mimic severe diabetic
hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to
investigate the effect of LED in distinct destructive conditions in order to obtain an
incontrovertible result
In addition it is possible that the present investigation needed a higher amount of
glucose to result in a remarkable reduction of proliferation as exposure to glucose was
limited to 72 h and as previous studies revealed that the antiproliferative effect of high
glucose was more pronounced with prolonged exposure with a maximal inhibition
attained by 7-14 days1531
Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo
cell growth are too complex to compare A key question is whether fibroblast
senescence in tissue culture and in the intact organism are similar Cristofalo et al40
Fibroblast proliferation under hyperglycemic circumstances
69
reported that this is not the case as fibroblasts have a finite ability to divide and
replicate but apparently the pathway or the morphologic characteristics leading to the
replicative senescence is not identical in vivo compared to in vitro
Furthermore extrinsic aging related to environmental damage which in diabetic
patients is mainly due to a chronic exposure to high levels of glucose during life is
unachievable in vitro
Unless a number of questions regarding the mechanism according to which LED
stimulates fibroblast proliferation in this particular condition remain unanswered the
results ascertain the potential effects of LED on fibroblast proliferation and viability
CONCLUSION
The current results should be interpreted with caution However these results
demonstrate the effectiveness of green LED irradiation at the above-described light
source properties and the illumination procedure described on cells in hyperglycemic
circumstances
The findings of the present study using an experimental in vitro model indicate that the
use of LED irradiation to promote wound healing in diabetic patients may have
promising future results As the present study establishes the possibility of using LED
irradiation in experimental in vitro situations it would be a worthwhile extension to
perform in vivo investigations to determine whether these in vitro observations were
relevant to the physiological situation and to determine the effect of these LED
properties on human tissue response
ACKNOWLEDGMENTS
The authors are greatly indebted to P Coorevits for assistance with the statistical
analysis and to Professor L Deridder and Ms N Franccedilois of the department of
Human Anatomy Embryology Histology and Medical Physics for providing access to
the laboratory and for helpful discussions
Chapter 3
70
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2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418
3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957
4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538
5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308
6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J
Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot
wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-
glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A
Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood
glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13
14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309
15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501
16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287
17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255
18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412
19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192
20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137
21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675
22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246
23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99
Fibroblast proliferation under hyperglycemic circumstances
71
24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729
27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63
28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301
29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -
progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of
cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA
damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in
culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia
23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar
K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71
36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141
37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588
38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252
39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97
40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619
PART II ANALGESIA
CHAPTER 4
EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION
MEDIATED BY LIGHT EMITTING DIODE IRRADIATION
Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy
Vanderstraetenab and Dirk Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Lasers in Medical Science 2005 20(1) 35-40
Chapter 4
76
ABSTRACT
The introduction of light emitting diode (LED) devices as a novel treatment for pain
relief in place of low-level laser warrants fundamental research on the effect of LED
devices on one of the potential explanatory mechanisms peripheral neurophysiology in
vivo
A randomised controlled study was conducted by measuring antidromic nerve
conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline
measurement and five post-irradiation recordings (2 min interval each) were performed
of the nerve conduction velocity (NCV) and negative peak latency (NPL)
Interventional set-up was identical for all subjects but the experimental group (=32)
received an irradiation (2 min at a continuous power output of 160 mW resulting in a
radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype
MDB-Laser Belgium) while the placebo group was treated by sham irradiation
Statistical analysis (general regression model for repeated measures) of NCV and NPL
difference scores revealed a significant interactive effect for both NCV (p=0003) and
NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical
significant decreased NCV and an increased NPL in the experimental group and a
statistical significant difference between placebo and experimental group at various
points of time
Based on these results it can be concluded that LED irradiation applied to intact skin
at the described irradiation parameters produces an immediate and localized effect
upon conduction characteristics in underlying nerves Therefore the outcome of this in
vivo experiment yields a potential explanation for pain relief induced by LED
Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative
peak latency middot Analgesic effect
Nerve conduction characteristics
77
INTRODUCTION
Since the introduction of photobiostimulation into medicine the light sources used
have advanced technologically and varied in characteristics over the years
Advancement and variation of the sources implicate a concomitant necessity to revise
research results in the respective domains of application Research and clinical
applications in the past particularly focused on the effectiveness of low-level lasers
have shifted now to novel treatment units such as light emitting diode (LED) devices
The efficacy and applicability of LED irradiation within the field of wound healing has
already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED
is not only promoted for its beneficial effects on the wound-healing process it is also
suggested to be potentially effective in the treatment of pain of various aetiology
although this claim has not yet been investigated thoroughly either experimentally or
clinically The putative analgesic effects of LED remain to be further explored
As the basic vehicle of pain is the neuronal system [7] measuring the
neurophysiological effect of LED treatment would be an appropriate experimental
approach to investigate the efficacy of LED on pain inhibition Nerve conduction
studies have become a technique for investigating the neurophysiologic effects of light
therapy [8-9]
Review of literature regarding standard nerve conduction studies revealed that previous
human studies on the influence of various light sources on peripheral nerves have
utilized different methods which hampers a comprehensive comparison In general
this research was performed on the superficial radial nerve [10-13] described by Shin J
Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve
[891315-17] Following the method of Cambier et al [18] the authors of this study
decided to investigate the effect of the light source used on the conduction
characteristics of the sural nerve By investigating this solely sensory nerve interaction
of motor nerve fibres (motor response can easily be provoked by antidromic nerve
stimulation [19]) can be avoided and given the superficial nature of the nerve it should
be sufficiently amenable to the effects of percutaneous LED irradiation
Chapter 4
78
A second major difference between the trials and therefore also hindering an
appropriate comparison between the results is the wide range of used light sources
HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared
multisource treatment unit [15]
With respect to the potential importance of LED irradiation for the treatment of pain
the current investigation was designed to assess the putative neurophysiological effects
of LED on the sensory nerve conduction of the human superficial peripheral sural
nerve and to establish a time course of the supposed phenomenon
The experimental hypothesis postulates that LED generates an immediate decrease in
conduction velocity and increase in negative peak latency In addition it can be
postulated that this effect is most prominent immediately after the irradiation and will
weaken as time progresses
STUDY DESIGN
The study was approved by the Ethical Committee of the Ghent University Hospital
After explanation of the experimental procedure a written informed consent was
obtained from each subject
Subjects
After screening based on a brief medical history excluding subjects with
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever inflammation of the skin) or conditions
that might affect sensory nerve conduction (such as diabetes peripheral neuropathy
radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral
edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40
females (mean age 26plusmn6 years range 18-42 years) participated in this study The body
mass index (BMI) of each subject varied within the normal range (=185-249) [20]
(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo
Nerve conduction characteristics
79
or an experimental group Each group of 32 subjects was composed of 12 males and
20 females
Experimental Procedure and Data Acquisition
In order to be able to quantify the negative peak latency (NPL) (measured from the
start of the stimulus artefact to the peak of the negative portion of the nerve action
potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was
followed
With respect to the known relationship between nerve conduction characteristics and
temperature the ambient temperature was kept constant (23ordmC-26ordmC room
temperature) during the investigation In view of this temperature issue the
standardized protocol started with 10 min of accommodation during which the
subjects rested in prone position on a treatment table
Immediately before this adjustment period the skin over the dorsolateral aspect of the
left calf and foot was cleaned with alcohol to remove surface lipids This preparation of
the treatment area was followed by the placement of the electrodes (TECA
Accessories Oxford Instruments Medical Systems Division Old Woking UK) as
described by Delisa et al [21]
The two-posted (2 cm separation anode distal) surface caption electrode was placed
distal and posterior of the lateral malleolus on the skin covering the sural nerve The
fixation of the earth electrode (Medelec Oxford Instruments Medical Systems
Division Old Woking UK) occurred 12 cm above the caption electrode according to
the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm
above the caption electrode to map the ideal stimulation point To level off
intraindividual variations in the amount of sensory response attributable to the
successive placement of the bipolar stimulator in course of the investigation a two-
posted (2 cm separation cathode distal) bar stimulating electrode was attached at the
point where the maximal response was obtained
This placement of the electrodes allows antidromic stimulation of the sural nerve
Electrophysiological stimulation and recordings were obtained with a Medelec
Chapter 4
80
Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse
of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was
used to produce each evoked sensory response
Baseline measurements of NPL and NCV were immediately followed by treatment of
the subjects according the protocol detailed below Recordings were subsequently
repeated at 2-min intervals over an 8-min period resulting in five recordings (one
immediately after the completion of the treatment and one at 2 4 6 and 8 min after
irradiation) Skin temperature was recorded concomitantly throughout the procedure
at the time of baseline measurement immediately after LED irradiation at the time of
the first recording and consequently at 2-min intervals together with the four final
electrophysiological recordings For this a surface digital C9001 thermometer
(Comark UK) sensitive to temperature changes of 01degC was used at the same point
of LED administration namely at 7 cm above the caption electrode The procedure
was identical for both conditions but subjects in the placebo group received a sham
LED irradiation
Light Characteristics and Irradiation Procedure
Irradiation was administrated with a light emitting diode device (BIO-DIO
preprototype MDB-Laser Belgium) The probe used emitted infrared light with a
wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2
and the frequency was variable within the range of 0-1500 Hz
Preceding baseline measurement the treatment point was marked on the skin overlying
the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-
point between the stimulation and capture electrode The LED probe was held in
contact with the skin perpendicular to the skin surface during the complete irradiation
procedure LED treatment consisted for all subjects of the experimental group out of 2
minutes lasting irradiation The LED was set to deliver a continuous energy density of
107 Jcm2 at a power output of 160 mW These parameters were selected as they are
appropriate for the treatment of pain in a clinical setting First of all because the
Nerve conduction characteristics
81
duration of the treatment is clinically feasible and secondly because the parameters are
within the scope of previously described light source characteristics [1-36915]
Statistics
Although superficial skin temperature did not change significantly in course of the
investigation the influence of the measured skin temperature on NPL and NCV was
taken into account by using a correction factor of respectively 02 msdegC and 147
ms degC All corrections were calculated towards a reference skin temperature of 32degC
Difference scores ie the variation between baseline measurements and each post-
irradiation recording were used as the basis for statistical analysis A General
Regression Model for repeated measures with one within-subjects factor (time 0
min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and
one between-subjects factor (group placebo or LED irradiated) was performed
followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least
Significant Difference) to determine whether any differences between baseline
measurements and post-irradiation recordings were statistically significant
The Statistical package for social sciences (SPSS 110) was used for analysis and
statistical significance for all tests was accepted at the 005 level
RESULTS
Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated
group plotted against time in minutes The values of the irradiated subjects decrease
directly after the irradiation and reach a first low point 2 min after finishing LED
treatment This decrease is followed by a marginal increase at 4 and 6 min and again an
important decrease at 8 min Statistical analysis (general regression model for repeated
measures) of these data indicated a significant interactive effect (P=0003)
Chapter 4
82
Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)
Post hoc LSD further showed significant differences between baseline measurements
and all post-treatment recordings (Table 1) Mutual comparison of the values from the
post-treatment recordings did not reveal any significant difference In addition there
was no significant difference determined in the placebo group in course of time
Table 1 Summary of the influence of LED irradiation on nerve conduction velocity
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001
2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002
4 0111plusmn0377 0647 -0908plusmn1898 0021 0004
6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001
8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve Conduction Velocity
-14
-12
-1
-08
-06
-04
-02
0
02
04
Baseline 0 min 2 min 4 min 6 min 8 min
Time Course
Dif
fere
nce
Sco
re (
m
s)
PlaceboLED
Nerve conduction characteristics
83
A similar representation was used for the results of the NPL Figure 2 reproduces NPL
plotted against time in minutes revealing for the irradiated group an increased latency
with two important peaks one at 4 min and one at 8 min
Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)
Statistical analysis of the mean difference scores again indicated a significant interactive
effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2
showed significant differences between baseline measurements and all post-treatment
recordings of the experimental group The mean difference score of the first post-
treatment recording of this same group (LED irradiated) differed significantly with the
recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED
irradiation As well as the recording 2 min after irradiation which differed significantly
(P=0013) with the 8 min post-treatment recording As observed for the NCV the
NPL of the placebo group did not reveal any significant difference in time course
At the time of the final recording the NCV and NPL mean difference scores of the
irradiated group did not return to their respective baseline values
Negative Peak Latency
-001
0
001
002
003
004
005
006
007
Baseline 0 min 2 min 4 min 6 min 8 min
Time course
Dif
fere
nce
Sco
re (
ms)
PlaceboLED
Chapter 4
84
Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group
significance) revealed statistical differences between the experimental and the placebo
group for NCV as well as for NPL NCV and NPL were statistical significant between
both groups at all points of time except from the NPL recording immediately after
finishing irradiation
DISCUSSION
Notwithstanding the above-mentioned difficulties in comparing results between
different trials on nerve conduction we attempt to discuss the current findings in view
of the results of the previous studies
This investigation revealed that percutaneous LED irradiation at feasible and current
clinical parameters generates measurable and significant changes in human sural nerve
antidromic conduction latency and velocity These results thus support previous
findings of light-mediated nerve conduction latency shifts in vivo [8101218]
although there are several important issues to be discussed
Table 2 Summary of the influence of LED irradiation on negative peak latency
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0004 plusmn0053 0755 0029plusmn0080 0019 0145
2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021
4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004
6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034
8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve conduction characteristics
85
A first comment deals with the progress of the NCV and NPL in function of time As
postulated the NCV decreases significantly immediately after irradiation
corresponding with a significant increased NPL However this effect does not weaken
as time progresses both variables remain significant throughout the 8 min during
observation period
Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the
conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]
although this slight increase in NPL was not significant at any moment Two other
studies [822] with a GaAlAs laser even registered comparable effects over a period of
55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous
studies post-treatment conduction measurements should be extended in time At
present for all studies it remains unclear at what point of time the effect extinguishes
although the interval of time during which LED treatment remains effective is
clinically important when treating pain
Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least
45 min) mediated by a monochromatic multisource infrared diode device although it
needs to be mentioned that this study performed with a comparable light source as the
current investigation revealed a significant decrease of the NPL These inverse results
between the study of Noble et al [15] and the current investigation could be attributed
to the concomitant increase of the skin temperature [15] As it has been well
recognised that a variation in tissue temperature causes a corresponding alteration in
nerve conduction velocities and peak latencies [91523-27] the temperature changes
may indeed provide an explanation for the observed findings In an attempt to analyse
the influence of a direct photobiological effect on sural nerve conduction
characteristics rather than working out the effects based upon thermal mechanisms
the present study corrected the skin temperature towards a reference temperature of
32degC This correction was performed notwithstanding the fact that the superficial skin
temperature did not change significantly before and after LED irradiation as well as
despite the fact that influencing nerve temperature takes place long after affecting skin
temperature [23] and thus being (almost) impossible after 2 min of irradiation
Chapter 4
86
followed by 8 min of registration Introduction of the correction factor implies likewise
that eventual influence on nerve conduction by cooling of the limb due to inactivity as
described by Greathouse et al [11] can be excluded
These facts suggest that temperature changes did not contribute to the demonstrated
effects of LED on nerve conduction Nevertheless the underlying mechanism of the
observed effects remains indistinct
A following remark regarding the fluctuation of NCV and NPL in function of time
considers the fact that both the NCV and the NPL do not change in a constant way up
to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the
increase in NPL display a small though not significant inversion of the effect at 4 and
(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of
fluctuation is to be expected when measuring NCV and NPL besides there is a similar
variation in the placebo groups
Although investigating dose dependency was not intended an additional remark
considers the fact that the use of optimal irradiation parameters is essential to obtain
the observed neurophysiological effect Nevertheless it is impossible to determine
ideal light source characteristics for effective treatment as the range of used
wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency
(pulsed or continuous) are not sufficiently similar between the different studies It can
only be concluded that a pulsing light source [91028] does not provide the postulated
results Radiant exposure exposure time power range and wavelength are not yet
established but based on this study and previously described assays it can be
speculated that the ranges of these parameters are quite large
In comparison with other studies where the number of subjects is 10 or less [8-
1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-
Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number
of subjects (n=32) was investigated in each group In spite of the large investigated
population it should be noted that the magnitude of the described changes in NCV
and NPL can simply be replicated by lowering the temperature of the extremity as the
observed changes are within the expected physiological ranges making the clinical
Nerve conduction characteristics
87
significance of the change questionable (This fact does not implement that the
decrease and the significant changes were temperature mediated)
A key question and meanwhile the initial impetus for future investigation is whether
the measured effects can be extrapolated to the actual nociceptive afferents namely the
myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])
respectively conducting acute and chronic pain The functional testing of these
nociceptive pathways has recently been extensively evaluated The currently accepted
neurophysiological method of assessing nociceptive pathways relies on laser-evoked
potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]
As up till now LEP is not available in this or any surrounding research centre the
investigators of this study had to perform a standard nerve conduction study (assessing
the large myelinated Aβ afferents) Therefore the current and previous beneficial
results of low level light therapy on conduction characteristics of nerves in vivo should
initiate measurements of clinical effectiveness first of all in laboratory settings and
afterward at a clinical level
CONCLUSION
Despite these remarks and the limited knowledge regarding the underlying mechanism
the present findings enable the following conclusions to be drawn LED irradiation at
clinical applied energy densities produces an immediate and localized effect upon
conduction characteristics in underlying nerves More specifically it is proven that
LED treatment lowers the NCV and augments the NPL resulting in a reduced
number of impulses per unit of time Therefore the outcome of this in vivo experiment
assumes that LED possibly induces pain relief
In order to encourage a widespread acceptance for the use of this non-invasive pain-
reducing modality in clinical settings prospective research should establish the precise
relationship between LED and pain relief as well as determine the ideal irradiation
parameters and verify which painful conditions can be treated with this treatment unit
Chapter 4
88
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the
equipment and MDB-Laser Belgium for generously providing the Light Emitting
Diode equipment
Nerve conduction characteristics
89
REFERENCES
1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9
2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)
3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18
4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8
5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43
6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65
7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53
8 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-34
9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6
10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90
11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7
12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5
13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9
14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore
15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5
16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5
17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604
18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200
19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21
Chapter 4
90
20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083
21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press
22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9
24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9
25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203
26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13
27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12
28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9
29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445
30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8
31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56
CHAPTER 5
PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE
IRRADIATION A PILOT STUDY ON EXPERIMENTALLY
INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS
Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Accepted for publication in Lasers in Medical Science December 2005
Chapter 5
92
ABSTRACT
Objective The present pilot study investigated the analgesic efficacy of light emitting
diode (LED) In view of a standardised and controlled pain reduction study design this
in vivo trial was conducted on experimentally induced delayed-onset muscle soreness
(DOMS)
Design Thirty-two eligible human volunteers were randomly assigned to either an
experimental (n=16) or placebo group (n=16) Immediately following the induction of
muscle soreness perceived pain was measured by means of a visual analog scale (VAS)
followed by a more objective mechanical pain threshold (MPT) measurement and
finally an eccentricconcentric isokinetic peak torque (IPT) assessment The
experimental group was treated with infrared LED at one of both arms the other arm
served as control Irradiation lasted 6 min at a continuous power output of 160 mW
resulting in an energy density of 32 Jcm2 The subjects of the placebo group received
sham irradiation at both sides In post-treatment a second daily assessment of MPT
and VAS took place The treatment and assessment procedure (MPT VAS and IPT)
was performed during 4 consecutive days
Results Statistical analysis (a general linear model followed by post hoc least
significant difference) revealed no apparent significant analgesic effects of LED at the
above-described light parameters and treatment procedure for none of the three
outcome measures However as the means of all VAS and MPT variables disclose a
general analgesic effect of LED irradiation in favour of the experimental group
precaution should be taken in view of any clinical decision on LED
Conclusion Future research should therefore focus on the investigation of the
mechanisms of LED action and on the exploration of the analgesic effects of LED in a
larger randomised clinical trial and eventually in more clinical settings
Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset
muscle soreness middot Musculus biceps brachii
Delayed-onset muscle soreness
93
INTRODUCTION
The analgesic efficacy of light emitting diode (LED) irradiation is recently being
investigated by means of a nerve conduction study on the superficial peripheral sural
nerve [1] It was demonstrated that LED irradiation at clinical applied densities
produces an immediate and localized effect upon conduction characteristics in
underlying nerves More specific LED induces a decreased number of sensory
impulses per unit of time thus possibly inducing pain relief [1]
Given the established influence of this treatment modality on the nerve conduction
velocity and thereby its potential analgesic ability the current investigation was
designed
Studies investigating the efficacy of a therapeutic modality on pain often experience
difficulties regarding standardisation of the population as analysis or comparison of
pain with different aetiologies is almost impossible Therefore we opted to measure the
analgesic effects of LED in a laboratory setting on a sample with experimentally
induced delayed-onset of muscle soreness (DOMS)
Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the
induction exercise and then spreads throughout the muscle [2-4] The correlates of
DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10
[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force
decreased range of motion and in particular muscle pain which is more pronounced
during movement and palpation [8 11] Despite the large volume of research that has
been undertaken to identify the underlying pathophysiology of DOMS the precise
mechanism is not yet universally accepted Several theories such as the torn-tissue
theory the connective tissue damage theory the muscle spasm theory and the
inflammation theory still remain viable though the current opinion states that DOMS
arises from a sequence of events in which several theories occupy an important place
[2 6 12 13]
DOMS has been used as a representative model of musculoskeletal pain and stiffness
in a number of studies [4 7 11 14 15] as it has a number of advantages it can be
induced in a relatively easy and standardised manner in a group of healthy subjects the
Chapter 5
94
time-course is relatively predictable and the symptoms have the same aetiology and are
of transitory nature [14 16] Nevertheless it should be emphasised that the use of this
particular experimental model to test the effectiveness of LED does not mean that this
treatment modality is necessarily advocated for the treatment of DOMS but merely
that it may be helpful in documenting the efficacy of LED in a clinical model of
musculoskeletal pain and stiffness In addition studies based on the induction of
DOMS under carefully controlled laboratory conditions can not replace research
involving actual patients but offer the opportunity to assess the effectiveness of
particular therapeutic interventions and might help to define additional clinical research
[14]
The experimental hypothesis of the current study postulates that infrared LED reduces
pain and muscle sensitivity associated with DOMS
MATERIALS AND METHODS
The study was approved by the ethical committee of the Ghent University Hospital
After providing information regarding the study design and possible consequences
related to participation at the study written informed consent was obtained from each
subject
Subjects
Healthy human volunteers were recruited from the university population Individuals
with any upper limb pathology neurological deficit and recent injury to either upper
extremity or undiagnosed pain were excluded Other exclusion criteria were
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever and inflammation of the skin) or
conditions in which physical exertion is contraindicated (such as cardiovascular deficits
hypertension and respiratory problems)
Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age
23plusmn4 years) were enrolled All subjects were randomly assigned using a random table
Delayed-onset muscle soreness
95
of numbers to the experimental or placebo group Each group of 16 subjects
consisted by stratification of equal numbers of men and women Age height and
weight did not differ significantly between the three groups
All subjects were physically active however none performed on a regular basis any type
of upper body weight-training Subjects were requested to refrain from any form of
strenuous physical activity and they were asked to avoid any form of medication
including anti-inflammatory agents as well as alcohol for 2 days before testing and for
the duration of the study
Overview of experimental design
The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to
induce pain related to DOMS Immediately following induction exercise an initial
assessment of the outcome measures (visual analog scale or VAS mechanical pain
threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the
subjects were treated under blinded conditions according to the randomised group
allocation In post-treatment the MPT was re-recorded and perceived pain was
reassessed with a VAS Contrary to these outcome measures the muscle strength was
only measured in pre-treatment at the one hand because short-term effects of LED
on muscle strength were not postulated and on the other hand because post-
treatment muscle strength can be influenced by too many different physiological
factors related to the pre-treatment measurement On the succeeding days (day 2 3
and 4) the treatment and assessment procedure was similar with approximately 24 h
separating each treatment
In both of the groups the two arms of the participants were included in the study In
the experimental group an equal number of dominant and non-dominant arms were
treated The non-treated arm served as control arm In the placebo group also an equal
number of dominant and non-dominant arms were considered as treated arm and the
other arm was classified in the non-treated group The procedure was identical for
both conditions but the subjects in the placebo group received sham LED irradiation
on both arms
Chapter 5
96
Specific aspects of the experimental design and procedures are detailed below
Pain induction
Muscle soreness was induced in a standardised fashion via a daily calibrated computer-
operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY
USA) Induction occurred separately and in random order in the elbow flexors of both
arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex
Prior to induction of DOMS the subjects were allowed an initial familiarization session
to become comfortable performing maximum voluntary contractions at the required
angular velocities This was immediately followed by determination of the maximum
eccentric and concentric peak torque at an angular velocity of 60degs and 120degs
Subsequently four sessions of eccentricconcentric work were performed with each
arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs
first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg
and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-
range) followed by two sessions at an angular velocity of 120degs again the first time
along an arch of 120deg and followed by the mid-range performance The subjects were
asked to accomplish maximum voluntary contractions during all the sessions Each
session was performed until exhaustion which was defined as the point when the
subject lost 70 of the initial eccentric and concentric peak torque There was a 1-
minute rest between each session This procedure was based on a pilot study and
previously described induction protocols [17-21]
Outcome measures
Outcome measures of subjective pain measurements MPT and muscle strength were
measured in this order on days 1-4 Subjective pain measurements and MPT occurred
immediately prior to and following irradiation whereas muscle strength measurements
only took place before LED treatment
Measurement of subjective pain Perceived muscle soreness was measured
subjectively by means of a 100-mm VAS A series of scales were completed separately
Delayed-onset muscle soreness
97
for each arm pain at rest followed by pain perception associated with full extension of
the arms and finally with maximal flexion of the arms The subjects were not allowed
to compare one VAS result with another
This assessment tool commonly used in measuring experimentally induced pain [22
23] has been found to be a reliable and valid method [24-26]
MPT Tenderness MPT used as a more objective correlate of muscle tenderness
has been demonstrated to be a reliable method to measure experimental induced
muscle soreness [27] This outcome measure was assessed by using a handheld
pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries
South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a
line from the radial insertion of the musculus biceps brachii at the elbow to the
intertubercular groove of the humerus thus resulting in three measure points one at
the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A
pressure of 4Ns was delivered The subjects were instructed to say yes at the exact
moment the pressure perceived became painful Each point was recorded three times
in pre-treatment as well as in post-treatment The average MPT score for each point in
pre- and post-treatment was used for statistical analyses
Muscle strength assessment Eccentric and concentric IPT were measured on the
same computerised dynamometer as was used for the induction of pain and an
identical standardisation procedure regarding positioning was followed
A warm-up session of two maximum voluntary contractions at the required angular
velocities was followed by determination of the eccentric and concentric peak torque
The first session at 60degs consisted of three repetitions followed by a 1-min during
rest and for the second session at 120degs five repetitions were performed The
subjects were instructed to flex and extend the elbow through the entire range of
motion as forcefully and rapidly as possible for each repetition The maximum
eccentric and concentric torque produced during the respective repetitions was used
for statistical analysis
Chapter 5
98
Light source specifications and treatment procedure
Light treatment was applied daily according to group allocation Irradiation occurred
with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The
probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160
mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The
frequency was variable within the range of 0ndash1500 Hz
During the complete irradiation procedure the LED probe was held in contact with
the skin perpendicular to the skin surface and at the exact mid-point between the MPT
mark at 4 cm and the one at 8 cm Light source properties were identical for all
subjects of the experimental group and consisted out of irradiation of 6-min lasting
duration at a continuous power output of 160 mW resulting in an energy density of
32 Jcm2 To conceal the treated side and condition the subjects were blinded to the
treatment status For the experimental condition a probe was held in contact with each
arm but only one of the two probes was attached to the LED device The subjects of
the placebo group received sham irradiation at both sides
The selected parameters are within the scope of previously described light source
characteristics for pain reduction [1 28-30] and they are appropriate for the treatment
of pain in a clinical setting because the duration of the treatment is clinically feasible
Statistical analysis
The three outcome measures were analysed separately For the VAS and MPT
measurements the same procedure was followed a general linear model (GLM) for
repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-
post preceding and following LED irradiation) and one between-subject factor (group
placebo or infrared LED irradiated) was performed If necessary the GLM was
followed by appropriate pairwise comparisons (post hoc least significant difference or
LSD) to determine whether any differences between measurements were statistically
significant A similar model was carried out separately for both the treated and the
control arm
Delayed-onset muscle soreness
99
In contrast to MPT and VAS the muscle strength was analysed differently The peak
torque values recomputed towards body weight of the subjects were statistically
analysed using a GLM for repeated measures This model consisted of one within-
subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo
or infrared LED irradiated) The model was completed twice first for the treated arm
and consequently for the control arm
The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)
was used for analysis and statistical significance for all tests was accepted at the 005
level
RESULTS
Statistical analysis of all variables of the three outcome measures revealed no significant
interactive effects of the main interaction (time times group times pre-post) The means and
standard deviations of the variables for both the treated and the control arm are
outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The
means of all VAS and MPT variables disclose a non-statistical significant general
analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher
MPT values in the irradiated group than in the placebo group The lower VAS rates are
present from day 1 until the last day of the study but they are more clearly present
from day 3 pre-treatment The higher MPT values are present from day 1 post-
irradiation until the last day and they are more visible at 4 cm followed by 12 cm and
finally at 8 cm In addition to the analgesic influence of LED an increased
convalescence of muscle strength was noted It should be remarked that this outcome
is similar for the treated as well as for the control arm of the irradiated group The
findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms
of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively
Graphical presentation of the other variables shows a similar course
Chapter 5
100
Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42
Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36
Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Visual Analog Scale
0
02
04
06
08
1
12
14
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n V
AS
scor
e (a
t re
st)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
101
Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249
Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Mechanical Pain Threshold
0
5
10
15
20
25
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n M
PT
sco
re (
at 4
cm)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Chapter 5
102
Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group
Day 1 Day 2 Day 3 Day 4
Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04
Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04
Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Isokinetic Peak Torque
04
045
05
055
06
065
Day 1 Day 2 Day 3 Day 4
Time course
Mea
n I
PT
sco
re (
ecce
ntr
ic a
t 60
degse
c)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
103
Despite the absence of significant main interaction effects the remaining interactions
as well as the main effects were statistically significant for some variables Only the
significant interactions including the between-subject factor group as well as the main-
effect group will be discussed The other interactions and effects establish the successful
induction of DOMS but are not relevant in view of the postulated hypothesis
The interaction between group and time is significant (p=014) for the VAS in
association with full extension for the control arm Post hoc LSD reveals no difference
between both groups a significant effect over time for both groups is found
Consequently this will not be further evaluated
A second significant interaction (p=0002) is the one among the within-subject factor
pre-post and the between-subject factor group for the MPT at 12 cm for the control arm
Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more
pressure after than before the treatment whereas in the placebo group a not
significant decrease of supported pressure is noted
Finally GLM analysis revealed that at the treated arm the irradiated group tolerates
significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)
DISCUSSION
It has previously been demonstrated that the LED source used might assist in
accelerating wound healing [31] that it has a direct cellular effect [3233] and that it
changes nerve conduction characteristics [1] Nevertheless LED-treated experimental
induced DOMS failed to prove the analgesic efficacy of LED at the above-described
light parameters and treatment procedure The current outcome concurs with other
research that demonstrated a lack of effect of various forms of light therapy on DOMS
[8 11 15] However despite the absence of an apparent and overall definitive finding
the present results cannot exclude favourable effects of LED treatment on pain Since
first of all an isolated statistical significant pre-post difference between groups (control
arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)
revealed that subjects of the irradiated group tolerate more pressure than the subjects
of the placebo group Second of all the overall means identified generally lower VAS
Chapter 5
104
scores higher MPT values and higher peak torques in the irradiated group This
implied that the treated subjects experienced noticeable less pain supported more
pressure on the painful muscle and generated more force than the non-treated
participants However these results are not statistically significant consequently it is
possible that these differences were found by coincidence and that there is no
relationship between the treatment and the described results of the three outcome
measures though it should be mentioned that the absence of significant findings is
more probably attributable to the small sample size involved in this study This
assumption is based on a post hoc power analysis It was calculated that for the small
effect size measured after treatment and for the measured control group event rate a
sample size of 80 subjects in each group was required at α=005 and power=080
(two-sided) to reveal significant results
Another factor conceivably responsible for the lack of solid evidence of the beneficial
effects of LED treatment upon DOMS-associated pain is related to the size of the
treatment effect in relation to the severity of the induced DOMS It is possible that by
using multiple exhaustive sets of exercise severe DOMS were induced which masked
relatively small but apparent treatment effects [4 11] In this same context it is
possible that the results only become significantly different after a prolonged treatment
and follow-up period as previous research noticed that recuperation subsequent to
DOMS induction can last up to 10 days [8]
Although it needs to be stressed that these results are not statistically significant critical
analysis of the overall means leads up to three additional remarks A primary comment
relates to the pre- and post-treatment courses of the results Starting at day 2 a clear
reduction of pain and muscle sensitivity was observed immediately post-treatment
Still one cannot conclude that this is indicative for the analgesic effect of LED
irradiation as a similar decrease in VAS and increase in MPT values was noted in the
treated and the control arm of the placebo group Perhaps this was caused by placebo
effect as reported by Pollo et al [34] the expectation of the participant can easily result
in pain relief but it can only be elucidated by implementation of a control group
Delayed-onset muscle soreness
105
Nevertheless in the current study this particular finding can be most probably
attributed to the physiological effects of the peak torque measurement performed
between the pre- and post-treatment recordings of VAS and MPT on the painful
flexor muscle of the upper arm For the assessment of muscle strength two short
series of alternative concentric and eccentric efforts were performed in succession
involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist
in the removal of inflammatory markers and exudate consequently reducing local
tenderness [4] In addition the force assessment can be considered as a form of active
warming-up resulting in an increased muscle temperature which can reduce muscle
viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness
[3536] thus decreasing the sensitivity of the muscle and moderating pain during
movement In any case the beneficial influence of LED immediately after irradiation
can not be securely interpreted due to the sequential assessment of the outcome
measures
A second additional remark considers the fact that both arms of the irradiated subjects
demonstrated evidence of the beneficial effects of LED as a similar reduction of pain
and muscle sensitivity and higher peak torques were found in course of time at the
treated arm as well as at the control arm of the irradiated subjects This ascertainment
points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]
stated that in case LED works via systemic effects the use of the contralateral side as a
control arm might be ill-advised Thus reinforcing that future research should include a
control group to bring clarification [4 7 16]
Finally it needs to be mentioned that although the extent of DOMS was probably
relatively high for investigating the postulated hypothesis the time-course of the
present study corresponds to that reported by other investigators [2 3 5-10]
Significant time effects in many of the variables revealed that muscle damage was
evident diffuse muscle soreness became progressively worse 24-48 h after DOMS
induction followed by a small amelioration after 72 h [35910] After 72 h the follow-
Chapter 5
106
up was ceased consequently further regain of force and attenuation of pain and
muscle sensitivity could not be evaluated Extending the duration of the assessment
period could be useful in assessing any longer-term effects of LED treatment
particularly because as mentioned above differences between both groups are more
clearly present from day 3 pre-treatment and also because DOMS may last for up to 10
days when induced with the described protocol [715]
Lack of knowledge regarding both the precise mechanism of action of LED and the
specific pathophysiology of DOMS hampers the way to offer a definitive explanation
for the absence of more obvious statistically significant differences Still the small
number of significant findings and the mean values suggest that possible analgesic
effects of infrared LED may not be excluded yet but to be able to estimate the real
value of LED further research is necessary A large-scaled randomised clinical trial
which takes the above-mentioned remarks into consideration should be performed
CONCLUSION
Regardless of the reasons for the absence of statistical significant effects reported here
and although LED may have some potential in the management of pain and functional
impairment associated with DOMS its effectiveness at the applied densities has not
been established
Future research should focus on evaluation of the appropriateness of DOMS as an
experimental model of pain and muscle damage Validation of this model would
enhance the ability to study various modalities for their potential effects on pain and
muscle injuries Besides the mechanisms of LED action are not known thus further
fundamental investigations need to address the underlying mechanism and
physiological basis of pain modulation utilizing LED treatment
Once LED irradiation has finally proven its treatment value in an experimental model
the most important prospect considers establishing the effectiveness of LED to reduce
pain in clinical settings
Delayed-onset muscle soreness
107
ACKNOWLEDGMENTS
The authors would like to thank Mr T Barbe and Mr R Deridder for their technical
assistance in the collection of the data as well as for their valuable input into the
research design Sincere appreciation is extended to the volunteers that participated in
this study and to MDB-Laser (Belgium) for generously providing the light emitting
diode equipment The authors also gratefully recognize Prof Dr G Van Maele for
assistance with the statistical analysis and for helpful discussion
Chapter 5
108
REFERENCES
1 Vinck E Coorevits P Cagnie B Muynck MD Vanderstraeten G and Cambier D (2005) Evidence of changes in sural nerve conduction mediated by light emitting diode irradiation Lasers Med Sci DOI101007s10103-005-0333-2
2 Cheung K Hume PA and Maxwell L (2003) Delayed Onset Muscle Soreness - Treatment Strategies and Performance Factors Sports Med 33(2)145-164
3 MacIntyre DL Reid WD and McKenzie DC (1995) Delayed muscle soreness The inflammatory response to muscle injury and its clinical implications Sports Med 20(1)24-40
4 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
5 Clarkson PM and Tremblay I (1988) Exercise-induced muscle damage repair and adaptation in humans J Appl Physiol 65(1)1-6
6 Cleak MJ and Eston RG (1992) Delayed onset muscle soreness mechanisms and management J Sports Sci 10(4)325-341
7 Craig JA Cunningham MB Walsh DM Baxter GD and Allen JM (1996) Lack of Effect of Transcutaneous Electrical Nerve Stimulation Upon Experimentally Induced Delayed Onset Muscle Soreness in Humans Pain 67(2-3)285-289
8 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
9 Ebbeling CB and Clarkson PM (1989) Exercise-Induced Muscle Damage and Adaptation Sports Med 7(4)207-234
10 Tiidus PM and Ianuzzo CD (1983) Effects of Intensity and Duration of Muscular Exercise on Delayed Soreness and Serum Enzyme-Activities Med Sci Sports Exerc 15(6)461-465
11 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
12 Armstrong RB (1984) Mechanisms of exercise-induced delayed onset muscular soreness a brief review Med Sci Sports Exerc 16(6)529-538
13 Rodenburg JB Steenbeek D Schiereck P and Bar PR (1994) Warm-up stretching and massage diminish harmful effects of eccentric exercise Int J Sports Med 15(7)414-419
14 Ciccone CD Leggin BG and Callamaro JJ (1991) Effects of ultrasound and trolamine salicylate phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-675 discussion 675-678
15 Craig J Barlas P Baxter D Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
16 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
17 Dvir Z (2003) Isokinetics muscle testing interpretation and clinical applications Churchill Livingstone Edinburgh
18 Lambert MI Marcus P Burgess T and Noakes TD (2002) Electro-Membrane Microcurrent Therapy Reduces Signs and Symptoms of Muscle Damage Med Sci Sports Exerc 34(4)602-607
19 Sumida K Greenberg M and Hill J (2003) Hot gel packs and reduction of delayed-onset muscle soreness 30 minutes after treatment J Sport Rehabil 12221-228
20 Tiidus PM and Shoemaker JK (1995) Effleurage massage muscle blood flow and long-term post-exercise strength recovery Int J Sports Med 16(7)478-483
21 Zhang J Clement D and Taunton J (2000) The efficacy of Farabloc an electromagnetic shield in attenuating delayed-onset muscle soreness Clin J Sport Med 10(1)15-21
22 Fitzgerald GK Rothstein JM Mayhew TP and Lamb RL (1991) Exercise-Induced Muscle Soreness After Concentric and Eccentric Isokinetic Contractions Phys Ther 71(7)505-513
Delayed-onset muscle soreness
109
23 Nosaka K and Clarkson PM (1997) Influence of Previous Concentric Exercise on Eccentric Exercise-Induced Muscle Damage J Sports Sci 15(5)477-483
24 Jensen MP Karoly P and Braver S (1986) The Measurement of Clinical Pain Intensity - a Comparison of 6 Methods Pain 27(1)117-126
25 Price DD Mcgrath PA Rafii A and Buckingham B (1983) The Validation of Visual Analog Scales as Ratio Scale Measures for Chronic and Experimental Pain Pain 17(1)45-56
26 Revill SI Robinson JO Rosen M and Hogg MIJ (1976) Reliability of a Linear Analog for Evaluating Pain Anaesthesia 31(9)1191-1198
27 Nussbaum EL and Downes L (1998) Reliability of Clinical Pressure-Pain Algometric Measurements Obtained on Consecutive Days Phys Ther 78(2)160-169
28 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
29 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
30 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118
31 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in biomedical optics and imaging 3(28 Proceedings of SPIE 4903)156-165
32 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18(2)95-99
33 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2005) Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level J Photomed and Laser Surg 23(2)167-171
34 Pollo A Amanzio M Arslanian A Casadio C Maggi G and Benedetti F (2001) Response Expectancies in Placebo Analgesia and Their Clinical Relevance Pain 93(1)77-84
35 Shellock FG and Prentice WE (1985) Warming-up and Stretching for Improved Physical Performance and Prevention of Sports-Related Injuries Sports Med 2(4)267-278
36 Safran MR Seaber AV and Garrett WE (1989) Warm-up and Muscular Injury Prevention an Update Sports Med 8(4)239-249
37 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
38 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
GENERAL DISCUSSION
General discussion
113
SUMMARY
As outlined in the general introduction the overall objective of this doctoral thesis is to
develop the current knowledge about the mechanisms of LED action in view of the
eventual provision of evidence-based support for the clinical use of LED as a
biostimulatory and analgesic treatment modality especially in the field of
physiotherapy
Part I Wound healing
The investigations described in chapter 1 and 2 were conducted to gain insight into the
potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As
fibroblasts are principal cells for biostimulation (in view of growing and dividing in
healing wounds) the influence of LED irradiation on fibroblast proliferation was
assessed1
The first investigation consisted of a pilot study performed in order to evaluate the
appropriateness of the cell isolation technique cell culture protocol and proliferation
analysis as well as to appraise the feasibility of the light source properties and
illumination procedure
Data analysis revealed no statistically significant differences between the infrared LED
irradiated and control petri dishes for the used parameters (table 1) Considering this
outcome other experimental findings disclose that the absence of stimulatory effects of
LED irradiation on fibroblast proliferation can partly be attributed to the use of
inappropriate light source properties However the applied external dosimetric
parameters are well within the recommended spectrum described by previous studies
investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be
excluded that changes in the illumination procedure (such as the use of lower power
shorter exposure times wavelengths with finer coverage of the absorption spectrum of
the irradiated cells and a longer incubation period between the last irradiation and cell
counting) could still result in an increased fibroblast proliferation467 Of equal
importance in interpreting the lack of distinctive results are the imperfections of the
applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails
114
considerable intervention from the investigator compromising the reliability of the
method It is also a time-consuming technique with an insufficient sensitivity for some
purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11
To avoid contamination of the results by these modifiable remarks a similar
experiment (chapter 2) was performed in which wavelength power and output mode of
the infrared LED source were not modified (table 1) only the exposure time was
reduced resulting in a lower radiant exposure In addition the effect of two other
emission spectra was evaluated These probes emitting red and green light had a
shorter wavelength than the infrared LED source and the power was half or a
sixteenth of the power from the infrared probe Consequently the red LED irradiation
occurred with a different exposure time than the infrared one in order to attain the
same radiant exposure (053 Jcm2) With respect to the green LED it was not
endeavoured to achieve the same radiant exposure as 16 min of irradiation is not
feasible for in vitro or clinical application
Finally also an LLL light source was integrated Although it was not attempted to
analyse the effectiveness of LED in comparison to LLL enclosure of this modality was
interesting in order to join in with the available literature covering mostly LLL studies
To bypass the described problems regarding analysis of fibroblast proliferation
counting of the cells was carried out this time by means of a colorimetric MTT assay
This method provides more accurate cell counts in a short period of time and therefore
can be considered as a more reliable alternative to Buumlrker hemocytometer11
MTT assay 24 h after the last irradiation revealed a significantly increased number of
cells in the irradiated wells in comparison to their (respective sham-irradiated) controls
Although the study supplied experimental support for a significantly increased cell
proliferation by all external dosimetric properties based on the results of the
comparative trial with an incubation period of 24 hours irradiation with the green
LED source yielded the highest number of fibroblasts Thus it can be concluded that
the wavelength of the green LED is probably within the bandwidth of the absorption
spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that
General discussion
115
the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast
cultures in vitro612
Table 1 External dosimetric properties summarized for each chapter
Wavelength Power Exposure
time Output mode
Radiant exposure
PART I Chapter 1
In vitro part
LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2
LED-infrared 950 nm 160 mW 1 min continuous 053 Jcm2
LED-red 660 nm 80 mW 2 min continuous 053 Jcm2
LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2
Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2
PART II Chapter 4
LED-infrared 950 nm 160 mW 2 min continuous 107 Jcm2
Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
The next aim of the first part of this doctoral thesis was to explore whether LED
treatment could ameliorate in vitro cell proliferation under conditions of impaired
healing In the pursuit of this aim fibroblasts were cultured in medium supplemented
with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a
pilot study the amount of glucose necessary to inhibit normal growth was determined
In order to attain an important reduction of cell viability and decreased proliferation
rate a relatively high concentration of glucose (1667 mM) was necessary in
comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia
(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity
between both circumstances in vitro limited to 72 h whereas the human tissue of a
diabetic patient in vivo is chronically exposed to glucose
Treatment of the fibroblasts occurred in respect of the previously described results
with the same irradiation parameters and illumination procedure (chapter 2)
Accordingly green LED irradiation labelled as the most appropriate treatment for
116
irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in
the previous study (table 1)
Analysis of the cell proliferation by means of MTT measurements yielded a
significantly higher rate of proliferation in hyperglycaemic circumstances after
irradiation than in the control conditions (ie hyperglycaemic circumstances without
irradiation) Thus this outcome supported the stimulatory potential of green LED
irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a
considerable destructive hyperglycaemic medium
Finally although the results of the in vivo part of chapter 1 were persuasive and
encouraging they will not be further discussed in this summary of part I as it was not
aimed in this doctoral thesis to investigate the wound healing process in vivo However
the results of this case study can be a valuable hold for future in vivo research
The possible clinical implications of these results and future research directions in the
scope of wound healing will be discussed below
Part II Analgesia
In the second part two studies investigated the effects of LED irradiation as a
potential intervention mode in one of the most important fields in physiotherapy
practice analgesia Chapter 4 describes the influence of LED treatment on changing
sensory nerve conduction characteristics of a human superficial peripheral nerve
Altering nerve conduction characteristics may not be the sole beneficial purpose to
attain with LED irradiation in view of analgesia but the advantage of nerve conduction
characteristics is that they are objective measurable physical variables and changes in
these characteristics provide a potential explanatory mechanism of pain inhibition by
LED treatment17
The results showed that percutaneous LED irradiation at feasible clinical parameters
can generate a significant decrease in NCV and increase in NPL for all recordings post-
treatment in comparison to the baseline measurement The data in the placebo group
did not reveal any significant difference in the same course of time Statistical analysis
General discussion
117
revealed significant differences between the experimental and the placebo group for
NCV as well as for NPL at all time-points of observation with exception of the NPL
recording immediately after finishing irradiation
It was also observed that the noted effects did not weaken as time progressed It can
be concluded that post-treatment conduction measurements should be extended in
time which is in accordance with the findings of some previous studies18-21 Clarifying
the point of time at which the effect extinguishes is necessary and clinically relevant
when treating pain by means of LED irradiation Besides obtaining the desired
neurophysiological effects ideally the optimal irradiation parameters should be
applied The most favourable dosimetric properties are not yet determined but based
on this study and previously described assays it can be speculated that the dosimetric
window is quite large
Regardless of these clinically important remarks the present findings allow to draw the
following conclusion LED irradiation at clinically applied densities can generate an
immediate and localized effect upon conduction characteristics in underlying nerves as
LED treatment results in lowering the NCV and augmenting the NPL Therefore the
outcome of this in vivo experiment assumes a potential pain relief by means of LED
treatment and justifies further research regarding its clinical effectiveness in laboratory
settings and at a clinical level
The fourth and final aim was to determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5
illustrates a clinical study observing the effect of LED treatment on a model
comprising experimentally induced DOMS in a healthy population The progress of
pain perception and peak torque was evaluated during 4 consecutive days commencing
on the day of DOMS induction The effect of infrared LED treatment at the light
parameters described (table 1) was assessed with regard to three different factors time
(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures
(VAS MPT and IPT) revealed no significant interactive effects of the main interaction
118
(timegrouppre-post) For the remaining interactions and for the main effects only a
few significant findings were relevant in view of the postulated hypothesis
Notwithstanding the absence of an apparent and overall statistically significant finding
the present results indicate favourable trends of LED treatment on pain as the means
of all VAS and MPT variables show a statistically nonsignificant general analgesic
effect of infrared LED irradiation expressed by lower subjective pain rates and higher
MPT values in the irradiated group In addition to the analgesic influence of LED an
augmented restoration of muscle strength was noted The lack of solid statistically
significant evidence for these beneficial effects of LED treatment upon DOMS-
associated pain can possibly be attributed to the small sample size in this study or even
to the size of the treatment effect in relation to the severity of the induced DOMS as
induction of severe DOMS can mask relatively small but apparent treatment
effects2223 A final possibility is that the results only become significantly different after
a prolonged treatment and follow up period as previous research demonstrated that
recuperation subsequent to DOMS induction can last up to 10 days24
It should also be noted that the described general analgesic effect of LED irradiation
was identical for the treated as well as for the control arm in the irradiated group
proposing that infrared LED might induce systemic effects 2526 However it needs to
be stressed that these results were not statistically significant
Regardless of the absence of statistically significant findings the mean values suggest a
potential role for infrared LED irradiation in the management of pain and functional
impairment associated with DOMS Notwithstanding this postulation future research
is absolutely required to establish the effectiveness of LED treatment to reduce pain as
well at the applied densities as for other dosimetric parameters
CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS
In the course of the past years during the process of the genesis of this thesis
therapeutic physical agents in general and phototherapeutic modalities in particular
became less important as physiotherapeutic modes of treatment than during the
preceding two decades The diminished use of these treatment modalities in the
General discussion
119
physiotherapy practice is to a certain degree a consequence of the controversial
research findings regarding the use of these physical agents This issue of controversy
led to less support for the use of these treatment modalities and a growing scepticism
regarding the effectiveness of these physical agents within the scope of the growing
climate of evidence-based practice A second responsible protagonist for the loss of
popularity of physical agents is linked with the current tendency within physiotherapy
emphasising active remedial therapy The establishment of this development was based
on various experiments mainly performed during the last decade demonstrating that
active treatment modalities are for numerous impairments and disabilities preferable to
more passive forms of therapy In Belgium the prevailing nomenclature which came
into use on 1 May 2002 went along with this tendency In the appendix to the Royal
decree of 14 September 1984 towards settlement of the nomenclature of medicinal
treatments concerning compulsory insurance for medical care and allowances the
personal involvement of the physical therapist during the physiotherapeutic session
was emphasized and it was even defined that massage physical techniques within the
framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal
application can only be remunerated when they are applied supplementarily and not as a sole therapy
This implies that passive treatment modalities should not be used as sole method of
treatment and should always be considered as an adjunct to an active treatment
program This development needs to be applauded in many cases such as various
painful musculoskeletal problems functional instability rehabilitation of neurological
patients re-activation of the elderly population psychomotor rehabilitation
cardiovascular and respiratory convalescence Nevertheless it would be erroneous to
entirely reject physical agents including LED treatment Based on the findings of the
above described experiments it needs to be stressed that for some purposes especially
within the scope of impaired wound healing LED irradiation could be a suitable
therapeutic measure This statement is founded on the results of part I of the present
thesis they provided satisfactory fundamental evidence for the advantageous effects of
LED treatment on a crucial exponent of the wound healing process namely fibroblast
proliferation The beneficial findings are the result of basic in vitro research As it is
120
inaccurate to simply extrapolate these results to the clinical practice the clinical use of
LED irradiation for wound healing needs to be preceded by purposive and specific in
vivo investigations to substantiate these basic research findings27
The case study described in chapter 1 indicates a foundation for further in vivo research
Visual appraisal of the surgical incision revealed (from the 65th day in the course of the
reparative process onwards) that the irradiated area -which initially showed inferior
epithelialization and wound contraction- showed a more appropriate contracture than
the control area characterized by less discoloration at scar level and a less hypertrophic
scar These clear beneficial effects of LED treatment on a human cutaneous wound
can serve as preliminary impetus for further research into the clinical applicability of
LED therapy although this case study is insufficient in order to guarantee a safe
correct and effective use of LED as a therapeutic modality
Despite these remarks it tentatively can be concluded that based on a detailed analysis
of the available data of the present in vitro studies and the given case report in
combination with the small number of previously published human studies the
beneficial effects of LED irradiation at the cellular level are obvious and therefore a
potentially favourable outcome can be assumed in clinical practice28-30 LED-
modulated stimulation of wound healing can be gradually and vigilantly implemented
clinically Nevertheless the real benefits of LED irradiation within the scope of wound
healing can only be established by additional clinical trials as thus far clinical
application and stipulation of dosimetry still occurs on a trial-and-error basis which is
not conducive to a generally accepted clinical use of LED To lend more credibility to
the treatment of wounds by means of LED irradiation and to expel the existing
controversy and scepticism surrounding this topic in vivo investigations on wound
healing using a number of different animal models and adequately controlled human
studies are necessary In addition these studies should be performed preferably on a
population suffering from impaired healing as a consequence of diabetes mellitus or as
a result of any other debilitating reason because as posed by Reddy et al3132 and as
mentioned above light has possible optimal clinical effects in the treatment of healing-
resistant wounds
General discussion
121
Drawing general conclusions and formulating clinical implications for analgesia is
obviously less manifest first of all because only a limited number of possible
mechanisms of action in order to obtain analgesia were highlighted and secondly
because both studies did not come to a joint or complementary conclusion The
outcome of the first study revealed that LED treatment lowers the NCV and augments
the NPL resulting in a slower stimulus conduction and consequently a reduced number
of sensory pulses per unit of time Thus it could be assumed that LED induces pain
relief but the results of the study describing the effect of LED treatment on
experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy
In addition it needs to be emphasised that the first study (chapter 4) measured the effect
of LED irradiation on the large myelinated Aβ afferents A noteworthy question and
meanwhile a stimulus for future investigation is whether the measured effects can be
extrapolated from these sensory nerve fibres to the actual nociceptive afferents
notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of
these nociceptive pathways relies on laser-evoked potentials which selectively activate
Aδ-fibres and C-fibres3334 This technique was presently not available therefore a
standard sensory nerve conduction study was performed
Whereas stimulation of wound healing by means of LED irradiation can be cautiously
implemented in the clinical practice at this stage it is too early to promote LED
irradiation as a treatment modality for pain To make this possible it is essential to
conduct numerous studies with regards to the use of LED in the field of analgesia
Future research should focus on fundamental investigations in order to discover the
underlying mechanisms and physiological basis of pain modulation utilizing LED
treatment Furthermore the evaluation of the appropriateness of DOMS as an
experimental model of pain is an important prospect to consider as validation of this
model would enhance the ability to study various modalities for their potential effects
on pain Irrespective of the difficulties regarding standardisation of the research
population and evaluation of soreness inextricably linked with clinical pain studies the
122
ultimate objective of future research should be the establishment of the effectiveness
of LED irradiation to reduce pain of miscellaneous origin in a clinical setting
Regardless of the encouraging results of the described studies and besides the earlier
proposed specific directions for future research (directed towards wound healing or
pain relief) it is necessary in the interest of the patientrsquos well being and to the
advantage of the prospective clinical use of LED to highlight a few more issues for
future research Therefore one has to deal with some limitations of the performed
investigations A first limitation concerns the fact that only two mechanisms of LED
action were investigated (notably changed fibroblast proliferation and alteration of the
nerve conduction characteristics) So one can conclude that for further and better
understanding of the mechanisms of action it is necessary to perform more basic
research Answering the questions regarding the functioning of LED irradiation will
simplify the evaluation and reinforce the interpretation of the obtained results and
ultimately contribute to a more widespread and well definded acceptance of the use of
LED in clinical settings
A second general limitation of this doctoral thesis is the substantial difference in the
used external dosimetric parameters between the different chapters and even within
one and the same study (illustrated in table 1) this complicates the comparison
between the different trials In each trial the dosimetry was individually ascertained
based on previous studies within the given field As not for every application the same
dosimetry is suggested in literature a range of dosages were used Another important
factor in deciding on the dosimetry was the clinical applicability of the dosage as it is
useless to investigate the appropriateness of a treatment modality at a clinically
unrealistic dose As a result of this limitation the current findings do not fully
contribute to the explanation regarding the ideal parameters one should use although
this was not set as a principal purpose Based on this thesis and previously described
assays it can be speculated that the possible window for these parameters is quite large
the ideal irradiation parameters and proper timing or sequencing of LED irradiation
General discussion
123
for example to the various phases of wound healing and to different painful conditions
are therefore possibly unattainable
The establishment of an appropriate dosimetry should also consist of investigating the
absolute and relative penetration depth of LED irradiation into human tissue This is
less crucial within the scope of wound healing but it is of key importance while
treating deep-seated tissue (eg nerve fibres muscles circulatory components et
cetera)
Finally this thesis only investigated the efficiency of LED in a very limited number of
conditions notebly wound healing and pain Within the scope of physiotherapy and
medicine in general there are numerous other purposes for which LED irradiation is
promoted such as oedema arthritis miscellaneous orthodontic applications seasonal
affective disorder neonatal jaundice photodynamical therapy et cetera2835-41
In summary additional work on establishing proper dosimetry and identifying the
biochemical or photobiologic phenomena that are responsible for improving wound
healing and reducing pain or even other effects within a broader spectrum of
conditions remains to be done in order to answer unreciprocated questions Until that
time the potential clinical usefulness and actual value of LED irradiation for wound
healing and even to a larger extent for analgesia should always be approached with
appropriate professionalism and even caution
FINAL CONCLUSION
LED devices are promoted for clinical use but the currently available scientific
documentation regarding effectiveness of this physical agent is rather scarce Through
providing scientific support for the biostimulatory and analgesic effectiveness of LED
irradiation this doctoral thesis attempted to bridge in some degree this gap
The conducted studies revealed that LED irradiation undeniably has potential
beneficial effects on wound healing and to a lesser degree within the scope of
analgesia However based on the present results it can be corroborated that light
124
therapy in the guise of LED irradiation is not magic but these results can raise some
corrective doubts in fundamental disbelievers and antagonists
Nevertheless we have to join the queue of scientists who have found beneficial results
but cannot elucidate with certainty how this outcome was established Thus although
the present results are encouraging a continuing development and integration of new
knowledge based on further research is necessary in various domains of intervention
Therefore several directions for future investigations were proposed in order to cover
as many existing gaps and to answer the utmost number of remaining questions as
possible Still one ought to be aware not to carry future fundamental research at a
disproportional level and the inevitable quest for mechanisms of LED action should
not hypothecate the potential clinical value implying that at a certain point it should be
appropriate to make the transfer from science to the application of the available
knowledge in clinical practice
The described findings regarding LED irradiation are comparable to the results of
previously published studies performed with other light sources Consequently as
postulated by some LED providers it can be speculated that the biological response of
tissue to light irradiation can probably not be equated merely to a light source but
rather to a broad photo-energy window
General discussion
125
REFERENCES
1 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
2 Abergel R Lyons R Castel J Dwyer R and Uitto J (1987) Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2)127-133
3 Skinner S Gage J Wilce P and Shaw R (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-192
4 Webb C Dyson M and Lewis W (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5)294-301
5 Atabey A Karademir S Atabey N and Barutcu A (1995) The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 1899-102
6 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
7 Ben-Dov N Shefer G Irinitchev A Wernig A Oron U and Halevy O (1999) Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3)372-380
8 Rebulla P Porretti L Bertolini F et al (1993) White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2)128-133
9 Deneys V Mazzon A Robert A Duvillier H and De Bruyere M (1994) Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2)172-177
10 Mahmoud A Depoorter B Piens N and Comhaire F (1997) The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2)340-345
11 Haskard D and Revell P (1984) Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3)319-322
12 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers
13 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of cultured human endothelial cells Diabetes 36(11)1261-1267
14 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA damage in cultured human endothelial cells J Clin Invest 77(1)322-325
15 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7)621-627
16 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4)491-501
17 Kramer J and Sandrin M (1993) Effect of low-power laser and white light on sensory conduction rate of the superficial radial nerve Physiotherapy Canada 45(3)165-170
18 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
19 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
126
21 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
22 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
23 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
24 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
25 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
26 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer
G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
29 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
30 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255
32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287
33 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-28
34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856
35 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
36 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
37 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263
39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64
40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74
General discussion
127
41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press
NEDERLANDSTALIGE SAMENVATTING
Nederlandstalige samenvatting
131
NEDERLANDSTALIGE SAMENVATTING
Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de
tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te
vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie
van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen
Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest
enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is
ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als
op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang
van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve
interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken
Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van
ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen
moeilijk te verantwoorden
Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin
de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere
overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit
methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan
consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in
de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen
laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in
de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag
aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies
inclusief het gebruik van licht
Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat
de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde
doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en
dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht
komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt
geloosd
132
De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo
lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en
gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te
anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te
voorkomen lijkt een gerichte en rationele a priori aanpak conform de
wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen
Te meer daar grondige literatuurstudie leert dat men ten behoeve van de
werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie
zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser
De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de
extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is
geboden De introductie van alternatieve lichtbronnen in de huidige
kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk
weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus
eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood
naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based
gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder
binnen de domeinen van haar potentieel beloftevolle klinische toepassingen
wondheling en analgesie
Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de
wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op
prominente protagonisten van de wondheling de fibroblasten werd getracht het
fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten
zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de
proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd
respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand
waarbij de normale celgroei werd verstoord
In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het
effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische
Nederlandstalige samenvatting
133
data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden
verklaard door het gebruik van een inadequate LED dosering en anderzijds een
methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie
vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de
precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en
inter-tester variabiliteit tot gevolg
In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde
onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de
bestralingsparameters (dosering) De effecten van de drie verschillende LED
golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten
werden hierbij geanalyseerd door middel van een meer betrouwbare en minder
subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-
yl)-25-diphenyl tetrazolium bromide (MTT)
De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir
effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden
tevens een basis voor meer coherente en relevante inzichten aangaande de globale
bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)
Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het
wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend
karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp
rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde
vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De
fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met
extreem hoge concentraties glucose Deze modificatie van het medium staat model
voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische
praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan
vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de
gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en
proliferatie
134
Het tweede deel van dit proefschrift exploreert het domein van het potentieel
analgetisch effect van LED binnen de kinesitherapie aan de hand van twee
fundamentele onderzoeken
In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere
sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele
hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en
een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale
verklaring van een analgetisch effect van het medium zou kunnen belichamen
Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)
meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf
verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen
aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke
significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de
negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele
hypothese
Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend
fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale
pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp
van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische
krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED
behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier
keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED
op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd
(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde
isokinetische krachtmeting en een registratie van de waargenomen spierpijn De
spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met
behulp van een kwantitatieve hand-hold algometer
Analyse van de bekomen data bracht geen significante verschillen tussen de
controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden
Nederlandstalige samenvatting
135
kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED
behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van
de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de
proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene
afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine
proefgroep die werd onderzocht enof door de grootte van het behandeleffect in
verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers
een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up
kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel
absolute omzichtigheid geboden en moet deze visie louter als speculatief worden
beschouwd
Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de
kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de
laatste jaren aanzienlijk afgenomen
De positieve resultaten van de verschillende in vitro studies in het kader van wondheling
vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing
vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel
gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een
belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek
Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie
Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme
onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch
complementair resultaat Verder onderzoek ter exploratie van de mogelijke
onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom
onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op
termijn wetenschappelijk te rechtvaardigen
136
LED tovenarij trend of therapie
LED mag geen magische krachten worden toegemeten maar verdient het lot van een
kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een
wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen
zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke
indicaties
Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds
the darkness has always got there first and is waiting for it
(Terry Pratchett Reaper Man 1991)
VII
TABLE OF CONTENTS
GENERAL INTRODUCTION 1
Background 3
Physical characteristics 6
Mechanisms of action 12
Aims and outline 15
PART I WOUND HEALING 25
Chapter 1 Do infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment
27
Chapter 2 Increased fibroblast proliferation induced by light emitting diode and low level laser irradiation
47
Chapter 3 Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level
61
PART II ANALGESIA 73
Chapter 4 Evidence of changes in sural nerve conduction mediated by light emitting diode irradiation
75
Chapter 5 Pain reduction by infrared light emitting diode irradiation a pilot study on experimentally induced delayed-onset muscle soreness in humans
91
GENERAL DISCUSSION 111
Summary 113
Clinical implications and future research directions 118
Final conclusion 123
NEDERLANDSTALIGE SAMENVATTING 129
Do not attempt to do a thing unless you are sure of yourself
but do not relinquish it simply because someone else is not sure of you
(Stewart E White)
IX
ACKNOWLEDGEMENTS
I wish to thank those people who supported me over the years and who helped me to
shape my life and work
First of all I would like to express my gratitude towards my promotor prof dr D
Cambier as without his encouraging words criticism inspiration and unremitting
support I would still be floundering about the contents of chapter 1 Thank you for
your good advice when I needed it the most
The members of the supervisory committee prof dr M Cornelissen prof dr M De
Muynck and prof dr G Vanderstraeten thank you for your assistance and helpful
feedback during the process of formation of this thesis
I also gratefully acknowledge the external members of the examination board prof dr
P Calders prof dr M Dyson prof dr P Lievens and prof dr K Peers for their
constructive reflections which contributed to the improvement of this thesis
I am greatly indebted to my special mentor prof dr J Anders of the Uniformed
Services University of Bethesda Maryland for the research suggestions she made as
well as for her unlimited belief in the value of my work
I wish to thank prof dr L Deridder for providing access to the laboratory of
Histology the Centre of Sports Medicine of the Ghent University Hospital for
allowing me to use their equipment as well as MDB-Laser Belgium for generously
providing the light emitting diode equipment
Sincere appreciation is extended to the volunteers that participated in this study and to
Tom and Roel for their valuable technical assistance in the collection of the data as
well as for their useful input into the research design of the investigation described in
chapter 5
X
Warm thanks go to the colleagues of the department of Human Anatomy
Embryology Histology and Medical Physics for providing the culture medium for the
technical support for the helpful discussions and principally for the amusing pastime
aseptic chats
In addition I also want to thank my colleagues of the associated institute Kinesitherapie
Gent and above all the colleagues of the department of Rehabilitation Sciences and
Physiotherapy Ann Axel Barbara Bart Bert Bihiyga Bruno Carine Damien Els2
Erik Frike Helga Inge James Karlien Kathy Kat(h)leen Koen Marc Martine
Mieke Paris (Nele) Patrick Roland Sophie Stefan Stijn (Veerle) Wiene Wim and
Youri thank you for the organisation and your attendance at many memorable
sidelines such as the survival-weekend the first department-day Fata Revaki our
legendary Thursday-sport activities and Friday-afternoon coffee breaks St Nicholas
visit skiing holiday New Yearrsquos lunch with quiz bowling spinning the introduction of
ldquosubetelyrdquo during our conspicuous presence at WCPT 2003 et cetera We shared many
treasured moments thanks to you a common working day often turned out to be very
pleasant I know that it will be impossible to find a comparable team of colleagues to
work with in the future
I especially want to thank Barbara to remind me on a regularly basis of my deadlines
to listen to my grieves and joy and to be willing to offer me a window-seat in our
office Kurt (although you abandoned at a certain moment) for solving my computer
problems Pascal for assistance with the statistical analyses Lieven for your motivating
interest and finally Fabienne Tine and Kim as loyal and appreciated friends who
worn-out several sports shoesbathing suits to supply in the weekly portion of sports I
needed to remain physically and mentally fit
I also extend my appreciation to my family and friends for their interest in my research
activities permanent mental support for the adoption of the surviving chickens but in
particular for looking after Louka and for the numerous relaxing moments Special
thanks are going to Katelijne and Frank for the delicious hot meals on lonely evenings
XI
Thomas Stijn and Johan thank you for the repeated (mostly futile) attempts to
convince me to do something together Sebastiaan each time during the past few years
when I doubted about the sense of my work it was your ridiculous story about a man
who wanted to invent superglue but instead invented the well-known yellow post-it
which stimulated me to continue my scientific quest
Of course I owe most gratitude to Luc my most devoted supporter Dearest I know
that since august 2004 you lived a solitary life in Dubai Although I think it was
possibly easier not to live under the same roof with me these last stressful months I
am aware that it was very difficult for you not to be able to play with Louka and to
miss some precious months of her life
Louka thank you for your radiant smile and daily baby speeches I am sorry that you
had to miss your daddy I promise that we will be reunited very soon
Elke Vinck
Ghent March 2006
GENERAL INTRODUCTION
General introduction
3
BACKGROUND
The use of light for therapeutic purposes reaches far back in time Current interest for
photomedicine with his its biological and medical effects relies fundamentally on two
major evolutions in the given field (1) the research results regarding the use of
ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in
the light amplification by stimulated emission of radiation (laser)-technology The production
of the first laser the ruby pulsed laser was rapidly succeeded by the development of
the helium-neon laser and other lasers like the argon the neodymium-glass and the
neodymium-yttrium-aluminium-garnet lasers1
As in the mid-1990s semiconductor and diode-based lasers gained popularity the
principally massive gas and dye lasers were rendered obsolete Therapeutic light
technology further continued to evolve and todayrsquos therapeutic light source is as likely
to be a light emitting diode (LED) or polarized light as a semiconductor or diode
laser1
Technological advancement and variation of the light sources necessitate a
concomitant update and revision of research in the respective domains of application
Unfortunately this logical and rational necessity has rarely been fulfilled From a
historical perspective this lack of appropriate research has led to disenchanting
evolutions in the use of light especially in physiotherapy The experience exists in this
medical field that light sources were promoted and commercialised for a vast regimen
of indications without foregoing scientific backup Consequently research developed
often after the commercial introduction in physiotherapy As these investigations
frequently gave rise to conflicting results for certain indications scepticism arose and
the use of the given modality knew a waning popularity for all its indications The final
result of such an inappropriate frame of promotion commercialisation and research is
a growing clinical disuse of a given modality even for motivated indications In view of
the actual increasing interest in LED treatment and based on former ascertainment
one has to state that a literature review for the given source reveals that research
mostly covers only low level laser (LLL) studies23 Although recently a number of
papers can be noted that report on the effects of LEDs and polarized light still
4
numerous source-specific-questions need to be answered as research concerning
mechanisms of action and efficacy of the current light sources remains limited in view
of a substantiated clinical application4-17
The reason for the contemporary light-oriented interest in physiotherapeutic practice
for LED devices is in essence based on several advantages of LED in comparison with
LLL For example the use of LEDs is esteemed to be safer as the delivered power
does not damage tissue LEDs can be made to produce multiple wavelengths thereby
stimulating outright a broader range of tissue types and probes that cover a large
treatment area are available18 In addition from a commercial point of view LEDs are
far more interesting as they are a good deal cheaper than laser diodes and they have a
long life span as these solid devices stand robust handling
As a result of the above-mentioned lack of literature on LED some providers of these
devices have taken for granted that the biological response of tissue to light irradiation
cannot be equated merely to a light source They declare that a given response solely
depends on the extent of absorption of radiated light by the tissue19 Consequently
these providers state that it is acceptable to extrapolate scientific findings of LLL
studies for explaining the mechanisms of action and detailing the efficacy of LED and
other alternative light sources Thus actually without appropriate scientific support
equal biological effects are attributed to LED as to LLL Nevertheless prudence is
called for such an extrapolation firstly because it is irrespective of the mentioned
dissimilarities and by simple projection one ignores a number of physical differences
between LLL and LED (eg coherence and degree of collimation or divergence)
Secondly LLL therapy is still not yet an established and evidence-based clinical tool20
Notwithstanding the historical efforts there still remains a considerable amount of
ignorance scepticism and controversy concerning the use and clinical efficacy of
LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed
parameters for irradiation as well as to the difficult objective measurement of possible
irradiation effects and even to the exceptional range of unsubstantiated indications for
General introduction
5
which light therapy was promoted27-29 A lack of theoretical understanding can also be
responsible for the existing controversies as the evaluation and interpretation of
research results would be simplified largely when the appropriate knowledge about the
mechanisms of light action would be available
LLL literature can undoubtedly be used as basis for research on LED and as a
comparative reference for these given investigations However to guarantee evidence-
based use of LED within physiotherapy the need for specific research in view of an
accurate consumption of LED is definite especially for potential promising clinical
applications in physiotherapy according to LLL literature mainly wound healing and
analgesia3031
Hitherto the most substantial research concerning the use of LED for improvement
of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded
under the influence of prolonged exposure to microgravity (eg during long-term space
flights) and in case of absence of exposure to sunlight such as in submarine
atmospheres they performed wound healing experiments for military application in the
given circumstances3233 In vitro experiments revealed that LED treatment increased
proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA
synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and
osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies
demonstrated therapeutic benefits of LED in speeding the early phase of wound
closure and in changing gene expression in a type 2 diabetic mouse model183234
Human studies noted 50 faster healing of lacerations a return of sensation and
increased tissue granulation as a result of LED irradiation1833
Associates of the Rehabilitation Sciences Research Group of the Ulster University in
Northern Ireland extensively investigated the effectiveness of light in the treatment of
pain The emphasis was laid primarily on the analysis of the effects of various low level
laser light sources35-44 However in the year 2001 two studies gave an account on the
efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46
6
noticed relatively long-lasting neurophysiological effects a significant change of the
nerve conduction characteristics (decrease of the negative peak latency) was mediated
by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a
comparable multisource diode device was ineffective in the management of delayed-
onset of muscle soreness (DOMS)
Despite the major value of these described trials a definitive answer regarding the
ability of LED in influencing wound healing or pain is not forthcoming cardinally
because a number of aspects are not yet investigated Consequently more research is
required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the
formerly mentioned scepticism regarding the effectiveness of a medium and possibly
to the undeserved fall into disuse of the treatment modality which happened in a way
with LLL therapy
PHYSICAL CHARACTERISTICS
This chapter supplies a short but comprehensive review of opto-physics A brief
description of the physical characteristics of the LED source used is essential as the
physical properties of light play an important part in the ultimate efficacy of treatment
According to the International Electrotechnical Commission (IEC 60825-1) an LED
can be defined as
Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by
radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process
of spontaneous emission1947
The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO
preprototype MDB-Laser Belgium) This illustration shows that a probe consists of
32 single LEDs disseminated over a surface of 18 cm2
General introduction
7
Figure 1 LED device and three available probes (infrared red and green)
Three highly monochromatic probes were available each emitting light of a different
wavelength within the above-defined range (table 1)2748 The wavelength of the light
emitted and thus its colour depends on the band gap energy of the materials forming
the p-n junctiona This light property is a key determinant to obtain maximum
photochemical or biological responses as light absorption by tissue molecules is
wavelength specific27 Only by absorbing radiation of the appropriate wavelength
(namely the wavelengths equal to the energy states of the valence electrons)
photoacceptor molecules will be stimulated resulting in a direct photochemical
reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750
8
absorption spectrum of a particular molecule is limited absorption often only occurs
over a waveband range of about 40-60 nm274851 Nevertheless the absorption
spectrum at cell or tissue level is broad because cells are composed of many different
molecules
Besides its influence on the absorption by means of tissue molecules there is a crucial
link between wavelength and penetration depth of the irradiated light Penetration into
tissue decreases as the wavelength shortens hence green light penetrates less than red
light which at his turn penetrates less into tissue than infrared light2748 Detailed
principles of light penetration will be discussed below
The LED device used emits non-coherent light In the 1980s the observed biological
responses after laser irradiation were generally thought to be attributable to the
coherenceb of the light485253 Though currently the clinical and biological significance
of coherence is seriously questioned54 According to several authors coherence does
not play an essential role in laser-tissue interactions firstly as it was proven that both
coherent and non-coherent light clinically show equal efficacy75556 Secondly as
according to some authors almost immediately after transmission of light through the
skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et
al1957 state that both findings are incorrect coherence is not lost in tissue due to the
phenomenon of scattering and non-coherent light is not as efficient as coherent light
This lack of consensus makes it necessary to mention whether or not light is
coherent2758
Further decisive characteristics to accomplish phototherapeutic efficacy are the power
exposure time output mode and beam area Based on these parameters both
irradiancec and radiant exposured can be calculated According to numerous authors
some of these parameters are more crucial than others to determine whether
b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48
c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48
d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248
General introduction
9
absorption of light will lead to a photobiological event192728485455 However the
literature yields several controversial findings as not all authors attribute an equal
importance to a given parameter For example according to Nussbaum et al59
irradiance was the determinant characteristic in the biomodulation of Pseudomonas
aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49
found that in order to stimulate tissue cell proliferation a specific combination of
irradiance and exposure time are more important than the actual radiant exposure Low
et al3940 on the contrary highlighted the critical importance of the radiant exposure in
observing neurophysiological effects Whereas Mendez et al60 reported that both
parameters influence the final results of light therapy
Koutna et al61 even suggested that the output mode of light applications plays a more
prominent role in the treatment outcome than the wavelength of the used light source
Nevertheless this finding could not be confirmed by other research results Besides
more controversial findings have been published regarding the output mode although
the repetition rate in a pulsed mode was considered as an important treatment
parameter several investigations failed to prove its value19272840414461-64
Based on these findings it was opted within the investigations of this doctoral thesis to
irradiate in a continuous mode The remaining dosimetric parameters (wavelength
exposure time and power) depended on the purpose of each investigation they are
described in the respective chapters The data necessary for the calculation of the
radiant exposure for the equipment used in the respective trials are summarized in
table 1
Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High
Infrared 950 80 120 160 Red 660 15 46 80
Green 570 02 42 10
10
The radiant exposure of the used LED can be calculated as follows65
RE =
Radiant Exposure [Jcm2]
T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]
PRE = α S T
α = 006 (continuous mode) or
003 (pulsed mode)
The parameters commented on so far can be considered as the external dosimetry
involving all parameters directly controlled by the operator limited by the apparatus
used Furthermore there is the so-called internal dosimetry referring to (1) several
physical phenomena (reflection transmission scattering and absorption) influencing
the light distribution within the tissue during energy transfer (2) the optical
characteristics of the irradiated tissue as well as (3) the relation between the external
dosimetry and these respective elements5466
This internal dosimetry determines to a considerable extend the penetration of light
into tissue Penetration can be defined as the tissue depth at which the radiant
exposure is reduced to 37 of its original value1948 However this definition only
accounts for the absolute penetration depth resulting in direct effects of light at that
depth In addition there is also a relative penetration depth leading up to effects
deeper in the irradiated tissue and even in certain degree throughout the entire
body1967 These so-called systemic effects can be caused by chemical processes initiated
at superficial levels at their turn mediating effects at a deeper tissue level57
Involvement of several forms of communication in the tissue such as blood circulation
and transport of transmitters or signal substances is possible1967 This means that light
sources with poor absolute penetration do not necessarily give inferior results than
those with a good absolute penetration19
In the same context it should be noted that calculation and even measurement of the
exact light distribution during irradiation is highly complicated principally as tissues
have complex structures and also because the optical properties of tissues vary largely
inter-individual2768
General introduction
11
Studies regarding actual penetration depth of LED light are scarce consequently the
knowledge on the topic of penetration depth of LED light is based on literature
originating from LLL research19 These findings established with various LLL sources
revealed that there is an obvious relation between penetration depth and
wavelength27486769-71
Three final remarks can be made on the dosimetry First of all it should be noted that
partly as a result of the above-mentioned contrasting findings on dosimetry ideal light
source characteristics for effective treatment of various medical applications are not yet
established and probably never really will be28 Therefore in the attempt to offer
sufficient guidelines for correct use of treatment parameters one should always try to
provide detailed description of light source properties used in any trial so the
practitioner can interpret the scientific results adequately and accordingly draw the
correct conclusions for his clinical practice
A second comment is based on the mentioned possible influence of the external and
internal dosimetric parameters on the photobiological effectiveness of light the
intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation
between radiated wavelength and penetration depth19546572 So it should be
emphasized that caution is recommended when comparing research results of light
sources with different wavelengths or other dissimilar dosimetric parameters
A third and final remark considers the extrapolation issue Comparison of the
therapeutic usefulness of the same light source used on different species should occur
cautiously So simply extrapolating the dosage used for one species to another is
inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo
clinical practice is inappropriate So purposive and specific research is the prerequisite
to produce safe and correct use of light as a therapeutic modality27
12
MECHANISMS OF ACTION
In the past decennia several mechanisms of action for biostimulation and pain
inhibition have been proposed and investigated73 Research was primarily based on
studies at the molecular and cellular levels and as a second resort investigations
occurred at the organism level resulting in numerous possible explanatory
mechanisms272858
It is the common view that light triggers a cascade of cellular and molecular reactions
resulting in various biological responses Thus different mechanisms of whom the
causal relationships are very difficult to establish- underlie the effects of light3448557475
To illustrate this complex matter the various mechanisms of action will be summarised
by means of a comprehensive model (fig 2) Detailed discussion about the different
individual components of the proposed model and other effects than those regarding
wound healing or analgesia were not provided as this was beyond the scope of this
general introduction
As depicted in figure 2 exposure to light leads to photon absorption by a
photoacceptor molecule causing excitation of the electronic state or increased
vibrational state of the given molecule275173 This process is followed by primary
photochemical reactions7475 Several key mechanisms have been discussed in the
literature Respiratory chain activation is the central point and can occur by an
alteration in redox properties acceleration of electron transfer generation of reactive
oxygen species (namely singlet oxygen formation and superoxide generation) as well as
by induction of local transient heating of absorbing chromophores192848515576-83 It is
supposed that each of these respective mechanisms plays a part in obtaining a
measurable biological effect It is yet not clear if one mechanism is more prominent
and decisive than another nevertheless recent experimental evidence has revealed that
mechanisms based on changes in redox properties of terminal enzymes of respiratory
chains might be of crucial importance2848517679
The primary mechanisms occurring during light exposure are followed by the dark
reactions (secondary mechanisms) occurring when the effective radiation is switched
General introduction
13
off2851 Activation of respiratory chain components is followed by the initiation of a
complicated cellular signalling cascade or a photosignal transduction and amplification
chain associated with eg changes in the cellular homeostasis alterations in ATP or
cAMP levels modulation of DNA and RNA synthesis membrane permeability
alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87
The sequence of events finally results in a range of physiological effects essential for
the promotion of the wound healing process for supplying analgesia or other
advantageous responses (acceleration of inflammatory processes oedema re-
absorption increased lymph vessel regeneration or increased nerve
regeneration)12181927486188-93
Photostimulation of the wound healing process can be mediated by increased
fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis
and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations
have been especially focussed on fibroblasts but other possible physiological effects
attributing to an accelerated wound healing were also observed suppression and
alteration of undesirable immune processes increased leukocyte activity new
formation of capillaries increased production of growth factors and enzymes while
monocytes and macrophages can provide an enlarged release of a variety of substances
related to immunity and wound healing1619277376
As pain and nociception are even less understood than wound healing the possible
mechanisms in obtaining pain relief by the use of light are less underpinned However
it is established that light therapy influences the synthesis release and metabolism of
numerous transmitter signal substances involved in analgesia such as endorphin nitric
oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these
neuropharmacological effects there is experimental evidence for diminished
inflammation decreased C-fibre activity increased blood circulation and reduced
excitability of the nervous system1927848899
One should be aware that a large amount of research regarding the possible
mechanisms of light action was conducted at the cellular level The described cascade
of reactions at the organism level is possibly even more complex as in contradiction to
14
the in vitro situation in vivo a range of supplementary interactions can influence the
sequence of effects and accordingly the final responses Besides it needs to be
mentioned that this summary did not take into account the origin of the light or the
external dosimetry thus the description is based on investigations performed with
various light sources and different dosages
Figure 2 Model summarizing the identified mechanisms of light action
Secondarymechanisms
Primary mechanisms
Final effects
Trigger
Stimulated wound healing Analgesia
Exposure to light
Photon absorption by photoacceptors
Respiratory chain activation
Accelerated electrontransfer
Reactive oxygen generation
Heating of absorbing chromophores
Altered redox properties
darr inflammation uarr oedema resorption
uarr lymph vessel regenerationuarr blood circulation
Photosignal transduction and amplification chain
uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation
darr C-fibre activity darr nervous excitability neuropharmacological effects
General introduction
15
Regardless of the large number of previous investigations identification of underlying
mechanisms of light action remains an important issue as these are not yet fully
understood and because probably not all mechanisms of action are currently
identified Convincing explanation of the mechanisms in normal as well as in
pathological tissue could banish the existing suspicion concerning the use of light as a
treatment modality2732547678
AIMS AND OUTLINE
The introduction of LED in medicine and in physiotherapy more specifically requires
particular scientific research especially within the fields of its clinical potential
application wound healing and analgesia The above described gaps in literature
regarding the use of LED laid the foundation of this doctoral thesis
Consequently the general purpose of this thesis is to explore a scientific approach for
the supposed biostimulatory and analgesic effect of LED and to formulate an answer
in view of an evidence-based clinical use of this treatment modality
The detailed objectives can be phrased as follows
Aim 1 To assess the biostimulatory effectiveness of LED
irradiation under normal in vitro conditions
Aim 2 To investigate the value of LED treatment to ameliorate
in vitro cell proliferation under conditions of impaired healing
Aim 3 To examine the effectiveness of LED in changing the
nerve conduction characteristics in view of analgesia
Aim 4 To determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting
Part I investigates the influence of LED on wound healing In pursuit of the first aim
chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial
16
measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these
cells needs to be considered as an exponent of the wound healing process as
fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase
and early remodelling100 Secondly an in vivo case study exploring the postulation that
LED irradiation could accelerate and ameliorate the healing of a surgical incision was
described
The results contrasted sharply with the findings of the in vitro part Two fundamental
causes were proposed in order to explain the different biological effect of LED
irradiation observed in vitro and in vivo the used irradiation parameters and evaluation
method
The experiment described in chapter 2 endeavoured to explore these considerations A
similar study was therefore performed but as distinctive characteristics different light
source properties an adapted irradiation procedure and the use of a colorimetric assay
based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the
counting of the cells were used
As stimulation of the wound healing process is virtually mainly indicated under
conditions of impaired healing (resulting in a situation which threatens to become
chronic and debilitating) proper attention for this matter is warranted192855 Besides
the medical consequences the costs involved with impaired healing yield also a socially
relevant problem to tackle Impaired healing will become even more common as the
world population continues to age After all senescence of systems and age-committed
comorbid conditions are commonly the culprits responsible for poor wound healing101
Thus finding cost-effective time-sparing non-invasive and practical treatment
modalities to cure wounds is a necessity
Aiming to assess the biostimulative effects by means of LED in these circumstances a
third study was conducted with respect of the previous results regarding irradiation
parameters and cell proliferation analysis The irradiation experiment described in
chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose
This medium modification serves as a pattern for cell proliferation in diabetic patients
General introduction
17
a population for whom stimulation of the wound healing process is a clinical relevant
feature
In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored
by means of two studies A first investigation (chapter 4) evaluated the influence of LED
on the sensory nerve conduction characteristics of a human superficial peripheral
nerve as a potential explanatory mechanism of pain inhibition by LED which is based
on the putative neurophysiological effects of this treatment modality The experimental
hypothesis postulated that LED generates an immediate decrease in conduction
velocity and increase in negative peak latency In addition it was postulated that this
effect is most prominent immediately after the irradiation and will weaken as time
progresses
The values of nerve conduction velocity and negative peak latency of a baseline
antidromic nerve conduction measurement were compared with the results of five
identical recordings performed at several points of time after LED irradiation
Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory
setting To guarantee an adequate standardized and controlled pain reduction study
there was opted to observe a healthy population with experimentally induced DOMS
Induction of DOMS has been described in a number of studies as a representative
model of musculoskeletal pain and stiffness because it can be induced in a relatively
easy and standardised manner the time course is quite predictable and the symptoms
have the same aetiology and are of transitory nature4445102-105
The treatment as well as the assessment procedure was performed during 4
consecutive days The first day isokinetic exercise was performed to induce pain
related to DOMS Subsequently the volunteers of the experimental group received an
infrared LED treatment and those of the placebo group received sham-irradiation
Evaluation of the effect of the treatment on perceived pain was registered by a visual
analog scale and by a mechanical pain threshold these observations occurred every day
18
prior to and following LED irradiation Eccentricconcentric isokinetic peak torque
assessment took place daily before each treatment
For the analysis of the results three different factors were taken into consideration
time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental)
In completion of this thesis the most prominent findings are summarized and the
clinical implications are discussed The general discussion also includes some future
research directions and a final conclusion
General introduction
19
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General introduction
21
42 Baxter G Effect of combined phototherapylow intensity laser therapy upon experimental ischaemic pain Potential relevance of experimental design 14th World Congress Physical Therapy Barcelona Spain 2004 Proceedings CD
43 Craig J Barron J Walsh D and Baxter G (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
44 Craig J Barlas P Baxter G Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
45 Glasgow P Hill I McKevitt A Lowe A and Baxter G (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1)33-39
46 Noble J Lowe A Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
47 IEC 60825-1 (1993) International Electrotechnical Commission Safety of laser products Part 1 Equipment classification requirements and users guide
48 Nussbaum E Baxter G and Lilge L (2003) A review of laser technology and light-tissue interactions as a background to therapeutic applications of low intensity lasers and other light sources Phys Ther Rev 831-44
49 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
50 Nakamura S (1998) The roles of structural imperfections in InGaN-based blue light-emitting diodes and laser diodes Science 281956-961
51 Karu Tiina (consulted on 2005 august 22) Cellular mechanism of low-power laser therapy httpwwwtinnitusustiinakarupresentationhtml
52 Mester E Mester A and Mester A (1985) The biomedical effects of laser application Lasers Surg Med 5(1)31-39
53 Moore K Calderhead G (1991) The clinical application of low incident power density 830 nm GaAlAs diode laser radiation in the therapy of chronic intractable pain A historical and optoelectronic rationale and clinical review Int J Optoelectron 6503-520
54 King P (1989) Low level laser therapy A review Lasers Med Sci 4141-148 55 Karu T (1989) Photobiology of low-power laser effects Health Phys 56(5)691-704 56 Simunovic Z (2000) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical
Application of Low Energy-Laser Laser Therapy LLLT Rijeka Vitgraf 57 Hode L and Tuner J (2000) Low level laser therapy (LLLT) contra light emitting diode therapy
(LEDT) - What is the difference Proceedings of SPIE 416690-97 58 Kitchen S and Partridge C (1991) A review of low level laser therapy Part I Background
physiological effects and hazards Physiotherapy 77(3)161-163 59 Nussbaum E Lilge L and Mazzulli T (2003) Effects of low-level laser therapy (LLLT) of 810 nm
upon in vitro growth of bacteria Relevance of irradiance and radiant exposure J Clin Laser Med Sur 21(5)283-290
60 Mendez T Pinheiro A Pacheco M Nascimento P and Ramalho L (2004) Dose and wavelength of laser light have influence on the repair of cutaneous wounds J Clin Laser Med Sur 22(1)19-25
61 Koutna M Janisch R and Veselska R (2003) Effects of low-power laser irradiation on cell proliferation Scripta Medica 76(3)163-172
62 Al-Watban F and Zhang X (2004) The comparison of effects between pulsed and CW lasers on wound healing J Clin Laser Med Sur 22(1)15-18
63 Panjehpour M Overholt B DeNovo R Petersen M and Sneed R (1993) Comparative study between pulsed and continuous wave lasers for photofrin photodynamic therapy Lasers Surg Med 13(3)296-304
64 Dyson M (2005) The significance of pulsing in the stimulation of tissue repair by light A review Lasers Med Sci 20S21
22
65 MDB Laser (2000) Bio Dio (Preprotoype) User guide Belgium Ekeren 66 Fisher J (1992) Photons physiatrics and physicians A practical guide to understanding laser light
interaction with living tissue Part I J Clin Laser Med Sur 10(6)419-426 67 Tuner J and Hode L (2000) Depth of penetration of laser light in tissue Laser Partner
Clinixperience 151-3 68 Anderson R and Parrish J (1981) The optics of human skin J Invest Dermatol 77(1)13-19 69 De Cuyper H and Lambert H (1991) Penetration depth of infrared- and helium-neon-laserlight
An assessment in vivo Eur J Phys Med Rehabil 1(5)133-137 70 Oshiro T Ogata H Yoshida M Tanaka Y Sasaki K and Yoshimi K (1996) Penetration depths
of 830nm diode laser irradiation in the head and neck assessed using a radiographic phanom model and wavelength-specific imaging film Laser Ther 8197-204
71 Kolarova H Ditrichova D and Wagner J (1999) Penetration of the laser light into the skin in vitro Lasers Surg Med 24(3)231-235
72 Chen Q Huang Z Chen H Shapiro H Beckers J and Hetzel F (2002) Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy Photochem Photobiol 76(2)197ndash203
73 Conlan M Rapley J and Cobb C (1996) Biostimulation of wound healing by low-energy laser irradiation A review J Clin Periodontol 23(5)492-496
74 Parrish J (1981) New concepts in therapeutic photomedicine photochemistry optical targeting and the therapeutic window J Invest Dermatol 7745-50
75 Smith K (1981) Photobiology and photomedicine the future is bright J Invest Dermatol 772-7 76 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees D (1989) Systemic effects
of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
77 Polo L Presti F Schindl A Schindl L Jori G and Bertoloni G (1999) Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth Biochem Biophys Res Commun 257(3)753-758
78 Bertoloni G Sacchetto R Baro E Ceccherelli F and Jori G (1993) Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 6328 nm light J Photochem Photobiol B-Biol 18191-196
79 Lubart R Friedmann H Sinykov M and Grossman N (1995) Biostimulation of photosensitized fibroblasts by low incident levels of visible light energy Laser Ther 7101-106
80 Harris D (1991) Biomolecular mechanisms of laser biostimulation J Clin Laser Med Sur 9277-280
81 Grossman N Schneid N Reuveni H Halevy S and Lubart R (1998) 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures involvement of reactive oxygen species Lasers Surg Med 22212-218
82 Oren D Charney D Lavi R Sinyakov M and Lubart R (2001) Stimulation of reactive oxygen species production by an antidepressant visible light source Biol Psychiatry 49464-467
83 Lavi R Shainberg A Friedmann H Shneyvays V Rickover O Eichler M Kaplan D and Lubart R (2003) Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells 278(42)40917-40922
84 Reddy K (2004) Photobiological basis and clinical role of low-intensity lasers in biology and medicine J Clin Laser Med Sur 22(2)141-150
85 Greco M Guida G Perlino E Marra E and Quagliariello E (1989) Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser Biochem Biophys Res Commun 163(3)1428-1434
86 Vacca R Marra E Quagliariello E and Greco M (1993) Activation of mitochondrial DNA replication by He-Ne laser irradiation Biochem Biophys Res Commun 195(2)704-709
87 Vacca R Marra E Quagliariello E and Greco M (1994) Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser Biochem Biophys Res Commun 203(2)991-997
88 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral
General introduction
23
nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
89 Anders J Borke R Woolery S and Van de Merwe W (1993) Low power laser irradiation alters the rate of regeneration of the rat facial nerve Lasers Surg Med 13(1)72-82
90 Rochkind S Nissan M Barr-Nea L Razon N Schwartz M and Bartal A (1987) Response of peripheral nerve to He-Ne laser Experimental studies Lasers Surg Med 7441-443
91 Lievens P (1986) Lasertherapie (deel II) De invloed van laser-bestralingen op het lymfesysteem en de wondheling Ned T Fysiotherapie 96140-142
92 Lievens P (1991) The effect of a combined HeNe and IR laser treatment on the regeneration of the lymphatic sysytem during the process of wound healing Lasers Med Sci 6193-199
93 Lievens P (1991) The effect of IR laser irradiation on the vasomotricity of the lymphatic system Lasers Med Sci 6189-191
94 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin J and Martin P (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1)171-178
95 Bosatra M Jucci A Olliaro P Quacci D and Sacchi S (1984) In vitro fibroblast and dermis fibroblast activation by laser irradiation at low energy An electron microscopic study Dermatologica 168(4)157-162
96 Enwemeka C Rodriquez O Gall N and Walsh N (1990) Morphometrics of collagen fibril populations in HeNe laser photostimulated tendons J Clin Laser Med Sur 847-52
97 Abergel P Lam T Meeker C Castel C Dwyer R and Uitto J (1984) Biostimulation of procollagen production by low energy lasers in human skin fibroblast culturesJ Invest Dermatol 82(4)395
98 Abergel P Lam T Meeker C Dwyer R and Uitto J (1984) Low energy lasers stimulate collagen production in human skin fibroblast cultures Clin Res 32(1)16A
99 Shimoyama N Lijima K Shimoyama M and Mizuguchi T (1992) The effects of helium-neon laser on formalin-induced activity of dorsal horn neurons in the rat J Clin Laser Med Sur 1091-94
100 Kloth L McCulloch J and Feedar J (1990) Wound healing Alternatives in management USA FA Davis Company
101 Lanzafame R (2004) Revolutions and revelations J Clin Laser Med Surg 22(1)1-2 102 Ciccone C Leggin B and Callamaro J (1991) Effects of ultrasound and trolamine salicylate
phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-678 103 Craig J Cunningham M Walsh D Baxter G and Allen J (1996) Lack of effect of transcutaneous
electrical nerve stimulation upon experimentally induced delayed onset muscle soreness in humans Pain 67(2-3)285-289
104 Minder P Noble J Alves-Guerreiro J Hill I Lowe A Walsh D and Baxter G (2002) Interferential therapy Lack of effect upon experimentally induced delayed onset muscle soreness Clin Physiol Funct Imaging 22(5)339-347
105 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
PART I WOUND HEALING
CHAPTER 1
DO INFRARED LIGHT EMITTING DIODES HAVE A
STIMULATORY EFFECT ON WOUND HEALING FROM AN IN
VITRO TRIAL TO A PATIENT TREATMENT
Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Proceedings of SPIE 2002 4903 156-165
Chapter 1
28
ABSTRACT
Variable effects of different forms of light therapy on wound healing have been
reported This preliminary study covers the efficacy of infrared light emitting diodes
(LED) in this domain
Cultured embryonic chicken fibroblasts were treated in a controlled randomised
manner LED irradiation was performed three consecutive days with a wavelength of
950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each
treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2
The results indicated that LED treatment does not influence fibroblast proliferation at
the applied energy density and irradiation frequency (p=0474)
Meanwhile the effects of LED on wound healing in vivo were studied by treating a
surgical incision (6 cm) on the lateral side of the right foot of a male patient The
treatment started after 13 days when initial stitches were removed The same
parameters as the in vitro study were used but the treatment was performed five times
The healing could only be evaluated clinically the irradiated area (26 cm) showed a
more appropriate contraction less discoloration and a less hypertrophic scar than the
control area (34 cm)
The used parameters failed to demonstrate any biological effect of LED irradiation in
vitro although the case study on the other hand illustrated a beneficial effect
Keywords Light Emitting Diodes Fibroblasts Wound healing
From an in vitro trial to a patient treatment
29
INTRODUCTION
Various beneficial effects of lasers and photodiodes at relatively low intensities have
been reported involving treatment of neurological impairments12 treatment of pain3-5
treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the
enhancement of wound healing has been a focus of contemporary research11-16 It
seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing
remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the
basis of a questionnaire about low power laser (LPL) in the current clinical practice in
Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey
into clinical LPL experience in Flanders
Nevertheless there remains a considerable amount of ignorance scepticism and
controversial issues concerning the use and clinical efficacy of LPL even in the domain
of wound healing12152021 This is at least in part a consequence of the inability to
measure and control operating variables related to connective tissue repair and of the
wide range of suitable parameters for irradiation
Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent
characteristics which make their use in a clinical setting problematic including
limitations in wavelength capabilities and beam width The combined wavelength of
light optimal for wound healing cannot be efficiently produced and the size of
wounds which may be treated by LPLs is limited Some companies offer an
alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be
made to produce multiple wavelengths and can have probes with large surface area
allowing treatment of large wounds Still one can not accept this light source as an
alternative for LPL therapy based on the cited advantages without proper investigation
regarding its biostimulatory effects
The effectiveness of this possible alternative for LPLs must be studied in vitro and in
addition in animal models or in humans because the effects of LED at the cellular level
do not necessarily translate to a noticeable effect in vivo The small amount of previous
investigations demonstrate that LED effects are as difficult to isolate162223 as LPL
Chapter 1
30
effects and the results are conflicting just like the results in literature specific on the
use of LPL121520
The purpose of the first part of this study is to examine the hypothesis stating that
LED irradiation can influence fibroblast proliferation Therefore a comparison of the
proliferation from fibroblasts in irradiated and control wells was performed The in vitro
investigation was linked with an in vivo case study This part enquired the assumption if
LED irradiation could accelerate and ameliorate the healing of a surgical incision
IN VITRO INVESTIGATION
MATERIALS AND METHODS
The complete procedure from isolation to proliferation analysis was executed twice
(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of
control dishes The second trial consisted of 27 irradiated and 27 control dishes
Cell isolation and culture procedures
Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation
and disaggregating of the cells occurred with warm trypsin (NV Life Technologies
Belgium) according the protocol described by Ian Freshney (1994)24 The primary
explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies
Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1
Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life
Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies
Belgium) When cell growth from the explants reached confluence cells were detached
with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV
Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the
cells were removed from the culture flasks by trypsinization and counted by
hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc
From an in vitro trial to a patient treatment
31
NV Life Technologies Belgium) The fibroblasts were seeded at a density of
70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture
medium the cells were allowed to attach for 24 hours in a humidified incubator at
37degC
Properties of the Light Emitting Diode
Prior to LED treatment all dishes were microscopically checked to guarantee that the
cells are adherent and to assure that there is no confluence nor contamination The
dishes were divided randomly into the treated or the control group Medium was then
removed by tipping the dishes and aspirating with a sterile pipette Following the
aspiration 2 ml fresh medium was added and treatment started
A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser
Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-
1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32
single LEDrsquos For the treatments in this study an average power of 160 mW at
continuous mode was applied The irradiation lasted 6 minutes resulting in an energy
density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result
of the divergence in function of this distance the surface of the LED (18 cm2) covered
the complete surface of the used petriplates (215 cm2)
After these manipulations 3 ml medium was added to each dish followed by 24 hours
incubation
One LED irradiation was performed daily during three consecutive days according
this procedure Control cultures underwent the same handling during these three days
but were sham-irradiated
Proliferation analysis
After the last treatment a trypsination was performed to detach the cells from the
culture dishes followed by centrifugation Once the cells were isolated from the used
trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts
Chapter 1
32
within this suspension as reflection for the proliferation was quantified by means of a
Buumlrker Chamber or hemocytometry
The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-
Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab
Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on
the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator
using an inverted light microscope counted the number of cells in 25 small squares
In order to calculate the number of cells one should multiply the amount of cells
counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and
the dilution factor (the amount of Trypan blue suspended with the cells 21=3)
Statistical methods
The data were analysed statistical in order to examine the hypothesis that LED
irradiation enhances fibroblast proliferation They were processed as absolute figures
for both trials separately In a second phase the counted cell numbers were converted
in relative figures so the data of both trials could be analysed as the data of one test
These relative figures were obtained by expressing each figure as a percentage from the
highest figure (=100) of that trial and this for each assay separately
A Kolmogorov-Smirnoff test of normality was performed on the data followed by a
Mann-Whitney-U test when the test of normality was significant and otherwise a T-
test Differences were accepted as significant when plt005 For this analysis SPSSreg
100 was used
RESULTS
The descriptive data for both trials are depicted in figure I The mean number of cells
in trial A is higher than in trial B for the controls as for the treated wells There is a
mean difference of 1252500 fibroblasts between the controls and 1223000 between
the irradiated wells of trial A and B The averages of both trials show that in control
cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no
From an in vitro trial to a patient treatment
33
statistically significant difference could be found between the two groups in either trial
nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not
significant for trial A (p=020) nor trial B (p=020) Only the combined data from both
trials were significant (plt001) for normality Further analysis respectively T-test for
the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the
combined data (p=0474) revealed no statistical significant differences
DESCRIPTIVE DATA
1730000181750029530003070000
00E+00
50E+05
10E+06
15E+06
20E+06
25E+06
30E+06
35E+06
40E+06
Trial A Trial A Trial B Trial B
Mea
n n
um
ber
of
cells
Control
Irradiated
Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B
DISCUSSION
Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities
(lt500 mW) in vitro have been analysed by evaluating various factors involving
(pro)collagen production25-27 cell viability2829 growth factor production28 and
myofibroblast formation30 Fibroblast proliferation also is an important factor to
consider In accordance with wound healing fibroblasts fulfil an essential role especially
in the late inflammatory phase and the early granulation phase31 Despite the failure of
some studies to demonstrate beneficial effects of LPL irradiation on fibroblast
proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very
Chapter 1
34
significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -
208 ) Atabey et al35 also revealed a significant increase in cell number two or more
irradiations resulted in an increased fibroblast proliferation Several other studies
confirmed these positive findings25263637
The results of this present in vitro study indicate that LED treatment does not
influence fibroblast proliferation Although the dosimetric parameters (in particular the
arbitrary energy density of 32 Jcm2) used in this study are well within the
recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous
studies about LPL therapy raising enhanced fibroblast proliferation252634-37
Van Breugel et al36 gave a possible explanation for these controversial results
According to them the fibroblast proliferation is not inherent at the energy density
They provide evidence that independent of the energy density the power density and
the exposure time determine the biostimulative effects of LPL irradiation LPL with a
power below 291 mW could enhance cell proliferation while a higher power had no
effect
Some authors also argued that the absorption spectrum of human fibroblasts show
several absorption peaks and pointed out that a wavelength of 950 nm is far above the
highest peak of about 730 nm3638 At longer wavelengths they determined a general
decrease in absorption Despite these results several investigators pose biostimulative
effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or
even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is
ranged from 800 nm to 830 nm principally because of the presence of cytochrome
oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts
at lower wavelengths is restricted by an inferior skin transmission than at higher
wavelengths38
Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting
respective green and red light The 950 nm beam of light was used for its high power
density but according to a range of remarks mentioned above the effects of the two
other probes must be as well evaluated
From an in vitro trial to a patient treatment
35
Another factor one can not ignore is that besides fibroblast proliferation other
processes or morphologic changes were not analysed although several authors have
posed that those changes and processes could be responsible for the biostimulative
effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et
al30 for example described a massive transformation of fibroblasts into myofibroblasts
after LPL treatment These modified fibroblasts play an important role in contraction
of granulation tissue30 A second example is an increased (pro)collagen production
after low power light therapy25-27 which is also considered as a responsible factor for
accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast
proliferation3640
It may be wondered if the light sources mostly LPL in the consulted literature are
representative for the LED used in this study although this LPL literature is often
used for that purpose As in the early days of LPL the stimulative effects upon
biological objects were explained by its coherence the beam emitted by the Bio-Dio on
the contrary produces incoherent light Nowadays contradictory research results are
responsible for a new discussion the clinical and biological significance of coherence
The findings of some authors172341-43 pose that the coherence of light is of no
importance of LPL and its effects although the opposite has also been stated4445
Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be
accounted for the non-enhanced fibroblast proliferation in this trial
Another possible explanation for the absence of biostimulative effect is related to the
moment of analysis of the proliferation The evaluation one day after the last
irradiation did not allow a delayed enhancement of proliferation while it is determined
in numerous investigations that the effects occur more than 24 hours after the last
treatment273746 and that they weaken after a further undefined period of time34
The fluctuation in cell numbers between both trials despite the use of an identical
protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor
reproducibility of their technique In this study the fluctuations are attributable to the
counting of the cells by Buumlrker hemocytometer before seeding According to some
authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large
Chapter 1
36
variability48 and it is often difficult to standardize48 Overestimation of the cell
concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity
was contradicted by Lin et al50 moreover satisfactory correlations with flow-
cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay
for cell counting (MTT)51 were determined
An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it
provides accurate cell counts in a short period of time with less intervention from the
investigator52
These remarks and controversies point out the possible deficiencies from the used
proliferation analyses and the relativity from the obtained results Other analyse
methods and analyses from different cell processes and morphologic changes could be
considered for further investigation
IN VIVO INVESTIGATION
MATERIALS AND METHODS
The effects of LED on wound healing in vivo were studied by treating a postsurgical
incision A male patient received chirurgical treatment for the removal of a cyst
situated approximately 15 cm posterior from the lateral malleolus of his right foot For
removal of the cyst an incision of 6 cm was made The incision was sutured and 12
days after the surgery the stitches were removed Visual inspection demonstrated that
the healing process of the wound proceeded well but not equally over the whole 6
centimetres (figure II) Epithelialization and wound contraction appeared to have
progressed better in the upper part (approximately 3 cm) of the cicatrice than at the
lower part (covered with eschar) No evidence of infection was noted in either part
LED treatment started the 13th day The incision was treated partially the lowest part
(26 cm) with the inferior epithelialization and wound contraction was irradiated the
remaining 34 cm served as control area This control area was screened from radiation
with cardboard and opaque black cling film
From an in vitro trial to a patient treatment
37
The light source destinated for the treatment was the same device used for the in vitro
irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED
output parameters were identical with those applicated in the preceding in vitro
investigation In particular a continuous wave at an average power of 160 mW and 6
minutes of treatment duration corresponding to an energy density of 32 Jcm2 An
equal distance from the probe to the target tissue as from the probe to the culture
medium was respected A plastic applicant of according height guaranteed constant
distance of 06 cm from the surface of the skin
Figure II Surgical incision before the first treatment 13 days after initial stitching
Therapy was performed once a day during five consecutive days repeatedly at the same
time resulting in an extension of the duration of the in vitro therapy with two days
Visual macroscopic observations were accomplished 6 52 and 175 days after the first
treatment
Comparison of the cutaneous sensitivity at the irradiated area and the control area was
accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175
days after the first treatment A control measurement also occurred at the same region
Chapter 1
38
on the left foot The aesthesiometer used in this study consisted of five hand-held
nylon monofilaments with a length of 38 mm and varying diameter
Sensitivity threshold is traced by presenting a monofilament of a certain diameter
vertically to the skin The monofilament bends when a specific pressure has been
reached with a velocity proportional to its diameter Measurements allow mapping
areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified
from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest
filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished
protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to
finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the
filaments
RESULTS
Visual estimation at any point of time after irradiation divulged no occurrence of
problems with dehiscence or infection in either part of the wound During the five
days of therapy the irradiated area looked dryer than the control area After the last
irradiation this was no longer recorded
Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area
From an in vitro trial to a patient treatment
39
Figure III representing the first evaluation six days after the initial treatment
illustrates that the wound healing has evolved slightly in both parts Though the lower
irradiated part remains of inferior quality as regards to epithelialization and wound
contraction In the course of the reparative process the influence of light exposures
were registered At 52 days after the first irradiation beneficial effects of LED
treatment are clearly present (Figure IV)
Figure IV Surgical incision 52 days after initiating LED treatment
The irradiated area (26 cm) showed a more appropriate contracture than the control
area (34 cm) characterized by less discoloration at scar level and a less hypertrophic
scar A similar trend was noticed at a third visual observation 175 days after the initial
treatment At that moment no impairments at cutaneous sensitivity level were stated
and the sensitivity showed no differences between left or right foot nor between the
two areas of the cicatrice
Chapter 1
40
DISCUSSION
The results of this case study indicate that LED had a positive influence on wound
healing in humans as determined by visual observations Many investigators
examining the effects of LPL on wound healing by means of a range of observation
and treatment methods reported accelerated and enhanced wound healing8-10 others
described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and
LED21 studies were unable to repeat these results
The late but beneficial findings in this study seem to be to the credit of LED-therapy
Though several authors establish positive results in an earlier stage of the wound
healing process8-1020 one should question why the differences did not occur at the first
evaluation on day 6 An explanation can be found in the start of the treatment Most
investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so
they influence a first cellular and vascular reaction with the production of chemical
mediators of inflammation resulting in an enhanced collagen production9 tremendous
proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the
time the first treatment in this study took place the traumatized tissue was in an
overlapping stage between an almost finished inflammatory phase and a scarcely
initiated re-epithelialization and wound contraction phase At that moment an infiltrate
of fibroblasts is present So fibroblast proliferation a possible mechanism of the
biostimulative effect had already occurred and could no longer be influenced Growth
factor production and collagen deposition have also decreased at that stage
Granulation tissue formation and fibroplasia in the contrary are initiating by that time
Those prolonged and slow processes with belated results are of significant importance
for the course of the final stage of wound healing and for the outlook of the future
scar31
The experimental findings revealed that the sensitivity of the skin according to the
threshold detection method of Semmes and Weinstein was normal at all the
investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a
good and objective predictor of normal skin sensitivity No other LPL nor LED
studies investigating this quality of the skin were found
From an in vitro trial to a patient treatment
41
CONCLUSION
This study demonstrates that although LED application at the applied energy density
and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to
have beneficial biostimulative effects on wound healing in human skin confirmed by
the favourable re-epithelialization and contracture
These results are discussed in the context of other experimental findings but no
reasonable explanation for this discrepancy could be found The literature on wound
healing after LED treatment in animal models or in humans is presently very limited
and contradictory The diversity in used radiation parameters and the absence of
references on how the wounds were measured or evaluated or what the end point was
for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro
investigations are better standardised nevertheless these results show a number of
conflicts One can conclude that until today the controversial findings are characteristic
for many results obtained with light photobiomodulation
However the postponed favourable results in the case study confirm some facts of the
discussion Namely the short period of incubation 24 hours in the in vitro part of the
study can be responsible for the lack of enhanced fibroblast proliferation It also
confirms that other cell processes and morphologic changes possibly are responsible
for biostimulative effects in vivo other observation methods should be considered for
future in vivo experiments
Despite these remarks we believe that LED application on cutaneous wounds of
human skin is useful with a single flash daily at the dose applied in this study for at
least three days
Furthermore future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Chapter 1
42
ACKNOWLEDGMENTS
The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the
material necessary for this study as well as Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
From an in vitro trial to a patient treatment
43
REFERENCES
1 G Baxter D Walsh J Allen A Lowe and A Bell Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79(2) 227-34 (1994)
2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)
3 J Stelian I Gil B Habot et al Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy J Am Geriatr Soc 40(1) 23-6 (1992)
4 A Honmura A Ishii M Yanase J Obata and E Haruki Analgesic effect of Ga-Al-As diode laser irradiation on hyperalgesia in carrageenin-induced inflammation Lasers Surg Med 13(4) 463-9 (1993)
5 D Cambier K Blom E Witvrouw G Ollevier M De Muynck and G Vanderstraeten The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15 195-200 (2000)
6 G Reddy L Stehno Bittel and C Enwemeka Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-7 (1998)
7 K Moore N Hira I Broome and J Cruikshank The effect of infrared diode laser irradiation on the duration and severity of postoperative pain a double blind trial Laser Therapy 4 145-148 (1992)
8 A Amir A Solomon S Giler M Cordoba and D Hauben The influence of helium-neon laser irradiation on the viability of skin flaps in the rat Br J Plast Surg 53(1) 58-62 (2000)
9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)
10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)
11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)
12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)
13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)
14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)
15 A Schlager P Kronberger F Petschke and H Ulmer Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1) 39-42 (2000)
16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)
17 GD Baxter and J Allen Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone (1994)
18 G Baxter A Bell J Allen and J Ravey Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77(3) 171-178 (1991)
19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)
20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)
Chapter 1
44
21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)
22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)
23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)
24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)
25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)
26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)
27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)
28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)
29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)
30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)
31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)
32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)
33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)
34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)
35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)
36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)
37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)
38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)
39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)
40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)
41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)
42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)
From an in vitro trial to a patient treatment
45
43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)
44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)
45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)
46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)
47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)
48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)
49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)
50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)
51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)
52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)
53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)
54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)
CHAPTER 2
INCREASED FIBROBLAST PROLIFERATION INDUCED BY
LIGHT EMITTING DIODE AND LOW LEVEL LASER
IRRADIATION
Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Lasers in Medical Science 2003 18(2) 95-99
Chapter 2
48
ABSTRACT
Background and Objective As Light Emitting Diode (LED) devices are
commercially introduced as an alternative for Low Level Laser (LLL) Therapy the
ability of LED in influencing wound healing processes at cellular level was examined
Study DesignMaterials and Methods Cultured fibroblasts were treated in a
controlled randomized manner during three consecutive days either with a infrared
LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and
570 nm) and respective power outputs Treatment duration varied in relation to
varying surface energy densities (radiant exposures)
Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all
irradiated cultures in comparison with the controls Green light yielded a significantly
higher number of cells than red (plt0001) and infrared LED light (plt0001) and than
the cultures irradiated with the LLL (plt0001) the red probe provided a higher
increase (plt0001) than the infrared LED probe and than the LLL source
Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation
in vitro This study therefore postulates possible stimulatory effects on wound healing
in vivo at the applied dosimetric parameters
Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low
Level Laser Tetrazolium salt
LED induced increase of fibroblast proliferation
49
INTRODUCTION
Since the introduction of photobiostimulation into medicine the effectiveness and
applicability of a variety of light sources in the treatment of a wide range of medical
conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results
of several investigations are remarkably contradictory This is at least in part a
consequence of the wide range of indications as well as the wide range of suitable
parameters for irradiation and even the inability to measure the possible effects after
irradiation with the necessary objectivity [457] A lack of theoretical understanding
can also be responsible for the existing controversies In fact theoretical understanding
of the mechanisms is not necessary to establish effects though it is necessary to
simplify the evaluation and interpretation of the obtained results As a consequence
the widespread acceptance of especially Low Level Laser (LLL) therapy in the early
seventies is faded nowadays and biostimulation by light is often viewed with scepticism
[8] According to Baxter [49] contemporary research and consumption in
physiotherapy is in particular focused on the stimulation of wound healing Tissue
repair and healing of injured skin are complex processes that involve a dynamic series
of events including coagulation inflammation granulation tissue formation wound
contraction and tissue remodelling [10] This complexity aggravates research within this
cardinal indication
Research in this domain mostly covers LLL studies but the current commercial
availability of other light sources appeals research to investigate as well the effects of
those alternative light sources eg Light Emitting Diode (LED) apparatus
The scarcity of literature on LED is responsible for consultation of literature
originating from LLL studies [11] but it may be wondered if this literature is
representative for that purpose As in the early days of LLL therapy the stimulating
effects upon biological objects were explained by its coherence [1213] while the beam
emitted by LEDrsquos on the contrary produces incoherent light Though the findings of
some scientists [914151617] pose nowadays that the coherence of the light beam is
not responsible for the effects of LLL therapy Given that the cardinal difference
between LED and LLL therapy coherence is not of remarkable importance in
Chapter 2
50
providing biological response in cellular monolayers [5] one may consult literature
from LLL studies to refer to in this LED studies
The purpose of this preliminary study is to examine the hypothesis that LED
irradiation at specific output parameters can influence fibroblast proliferation
Therefore irradiated fibroblasts cultures were compared with controls The article
reports the findings of this study in an attempt to promote further discussion and
establish the use of LED
MATERIALS AND METHODS
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos Isolation and
disaggregation of the cells was performed with warm trypsin according the protocol
described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in
Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1
L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants
reached confluence cells were detached with trypsine and subcultured during 24 hours
in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours
the cells were removed from the culture flasks by trypsinization and counted by Buumlrker
hemocytometry For the experiment cells from the third passage were plated in 96-well
plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a
density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C
during 24 hours
All supplies for cell culture were delivered by NV Life Technologies Belgium except
for Fetal Calf Serum (Invitrogen Corporation UK)
Irradiation sources
In this study two light sources a Light Emitting Diode (LED) device and a Low Level
Laser (LLL) device were used in comparison to control cultures
The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)
LED induced increase of fibroblast proliferation
51
with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400
mW and a frequency range from 0-1500 Hz
The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)
consisted of three wavelengths emitted by separate probes A first probe emitting
green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the
red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third
probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared
light The area of all three probes was 18 cm2 and their frequency was variable within
the range of 0-1500 Hz
Exposure regime
Prior to irradiation the 96-well plates were microscopically verified to guarantee that
the cells were adherent and to assure that there was no confluence nor contamination
Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining
25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation
The 96-well plates were randomly assigned in the treated (LLL or green red or infrared
LEDrsquos) or the control group
For the treatments in this study the continuous mode was applied as well for the LLL
as for the three LED-probes The distance from light source to fibroblasts was 06 cm
LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting
in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant
exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to
exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective
power output of 160 mW 80 mW or 10 mW
After these handlings the remaining medium was removed and new Hanksrsquoculture
medium was added followed by 24 hours of incubation
One irradiation (LLL or LED) was performed daily during three consecutive days
according to the aforementioned procedure Control cultures underwent the same
handling but were sham-irradiated
Chapter 2
52
Determination of cell proliferation
The number of cells within the 96-well plates as a measure for repair [19] was
quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The
colorimetric assay was performed at two different points of time to determine the
duration of the effect of the used light sources
This assay exists of a replacement of Hanksrsquoculture medium by fresh medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as
described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT
solution was substituted by lysing buffer isopropyl alcohol The plates were
temporarily shaken to allow dissolution of the produced formazan crystals After 30
minutes of exposure to the lysing buffer absorbance was measured The absorbance at
400 to 750 nm which was proportional to fibroblast proliferation was determined
using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)
The complete procedure from isolation to MTT assay was executed six times (Trial A
B C D E and F) while it was impossible to irradiate all the investigated number of
wells with the same LED apparatus on one day All the trials included as much control
as irradiated wells but the number of control and irradiated wells in each trial varied
depending on the number of available cells after the second subculturing A further
consequence of the available number of cells is the number of probes examined per
trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E
Incubation period before proliferation analyses numbered 24 hours To investigate if
the stimulatory effect tends to occur immediately after irradiation or after a longer
period of time incubation in trial F lasted 72 hours
An overview of the followed procedures regarding incubation time before proliferation
analysis number of analysed wells for each trial and the number of probes examined
per trial is given in table 1 As a consequence of the differences in procedures followed
and because each trial started from a new cell line the results of the five trials must be
discussed separately
LED induced increase of fibroblast proliferation
53
Statistical analysis
Depending on the amount of groups to be compared within each trial and depending
on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way
ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons Statistical significance for all tests was
accepted at the 005 level For this analysis Statistical Package for Social Sciences 100
(SPSS 100) was used
RESULTS
The results presented in table 1 show that cell counts by means of MTT assay
revealed a significant (plt0001) increase in the number of cells in comparison to their
respective sham-irradiated controls for all the irradiated cultures of trial A B C D
and E except the irradiated groups in trial F
Moreover the results of trial A showed that the effect of the green and red LED probe
was significantly (plt0001) higher than the effect of the LLL probe With regard to the
amount of proliferation the green probe yielded a significantly higher number of cells
than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe
provided a higher increase in cells (plt0001) than the infrared probe
The infrared LED source and the LLL provided a significant (plt0001) higher number
of cells than the control cultures but no statistical significant difference was recorded
between both light sources
The trials A B C D and E regardless of the number of probes used in each trial
were analysed after 24 hours of incubation after the last irradiation The incubation
period of trial F lasted 72 hours
The means of trial F illustrated that the effect was opposite after such a long
incubation The control cultures had significantly (plt0001) more fibroblasts than the
irradiated cultures with the exception of the LED-infrared group that showed a not
significant increase of cells Further analysis revealed that the green probe yielded a
significantly lower number of cells than the red (plt0001) and the infrared probe
(plt0001) and that the red probe provided a higher decrease (plt0001) than the
Chapter 2
54
infrared probe Laser irradiation induced a significant decrease of fibroblasts in
comparison to the infrared irradiated cultures (plt0001) and the control cultures
(p=0001) LED irradiation with the green and the red probe revealed no statistical
significant differences
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups
Absorbency (proportional to the number of fibroblasts)a
Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050
Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis
DISCUSSION
Despite the failure of some studies [223] to demonstrate beneficial effects of laser and
photodiode irradiation at relatively low intensities (lt500mW) on fibroblast
LED induced increase of fibroblast proliferation
55
proliferation this study provides experimental support for a significant increased cell
proliferation Therefore these results confirm previous studies that yielded beneficial
stimulating effect [1152425] Remarkably though is the higher increase noted after
irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general
decrease in absorption at longer wavelengths and concluded that several molecules in
fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445
470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the
closest to one of these peaks
Karu [5] also emphasises that the use of the appropriate wavelength namely within the
bandwidth of the absorption spectra of photoacceptor molecules is an important
factor to consider
In this particular context penetration depth can almost be ignored as virtually all
wavelengths in the visible and infrared spectrum will pass through a monolayer cell
culture [12] The irradiance (Wcm2) on the contrary could have had an important
influence on the outcome of this study The higher increased proliferation by the lower
wavelengths is possibly a result of the lower irradiance of these wavelengths Lower
irradiances are confirmed by other experiments to be more effective than higher
irradiances [111626]
The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as
described by Poumlntinen [17] but in the scope of these results it also needs to be noticed
that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)
the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)
Consequently the results of especially trial A and F must be interpreted with the
necessary caution It is possible that the determined distinction between the used light
sources and the used probes is a result from the various radiant exposures applied
during the treatments of the cultures
Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after
the last irradiation this study was unable to demonstrate a stimulating effect when
analysis was performed 72 hours after the last irradiation Moreover this longer
incubation period even yielded an adverse effect Although a weakening of the
Chapter 2
56
photostimulating influence over time is acceptable it can not explain a complete
inversion Especially in the knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period [2427] In an attempt to illuminate
this finding one can suppose that the circadian response of the cells triggered by the
LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark
The most obvious explanation is even though a decreased vitality and untimely cell
death in the irradiated cell cultures as a result of reaching confluence at an earlier point
of time than the control cultures The cells of a confluent monolayer have the tendency
to inhibit growth and finally die when they are not subcultured in time No other
reasonable explanations could be found for this discrepancy
Photo-modulated stimulation of wound healing is often viewed with scepticism The
real benefits of Light Emitting Diodes if any can only be established by histological
and clinical investigations performed under well controlled protocols Despite these
remarks this study suggests beneficial effects of LED and LLL irradiation at the
cellular level assuming potential beneficial clinical results LED application on
cutaneous wounds of human skin may be assumed useful at the applied dosimetric
parameters but future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Persons in good health rarely require treatment for wound healing as posed by Reddy
et al [13] light has a possible optimal effect under conditions of impaired healing
Postponed wound healing is a time-consuming and often expensive complication
Thus future prospects must remind to examine the therapeutic efficacy of LED on
healing-resistant wounds
LED induced increase of fibroblast proliferation
57
ACKNOWLEDGMENTS
The authors are grateful to Prof Deridder for supplying the laboratory as well as the
material necessary for this investigation and to Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
Chapter 2
58
REFERENCES
1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55
2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32
3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7
4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994
5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998
6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32
7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42
8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5
9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8
10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7
11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8
12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34
13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9
14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18
15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43
16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6
17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475
18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J
Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9
21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601
22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63
LED induced increase of fibroblast proliferation
59
23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9
24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301
25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are
more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37
27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8
28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26
CHAPTER 3
GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES
FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL
Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Photomedicine and Laser Surgery 2005 23(2) 167-171
Chapter 3
62
ABSTRACT
Background and Objective The chronic metabolic disorder diabetes mellitus is an
important cause of morbidity and mortality due to a series of common secondary
metabolic complications such as the development of severe often slow healing skin
lesions
In view of promoting the wound-healing process in diabetic patients this preliminary
in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on
fibroblast proliferation and viability under hyperglycemic circumstances
Materials and Methods To achieve hyperglycemic circumstances embryonic chicken
fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL
glucose LED irradiation was performed on 3 consecutive days with a probe emitting
green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min
resulting in a radiation exposure of 01 Jcm2
Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all
irradiated cultures in comparison with the controls
Conclusion According to these results the effectiveness of green LED irradiation on
fibroblasts in hyperglycemic circumstances is established Future in vivo investigation
would be worthwhile to investigate whether there are equivalent positive results in
diabetic patients
Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes
Fibroblast proliferation under hyperglycemic circumstances
63
INTRODUCTION
The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide
geographic variation in incidence and prevalence generally 11 of the world
population is affected and worldwide it is the twelfth leading cause of death1 Those
figures may be higher for urban regions as well as for industrialized countries Due to
multiple factors involving the aging process of the population and lifestyle changes
(such as reduced physical activity hypercaloric eating habits and concomitant obesity)
these figures may increase in the future2-6 Therefore diabetes mellitus could become
the most common chronic disease in certain regions as stated by Gale it ldquotargets the
rich in poor countries and the poor in rich countriesrdquo6
The harmful disruption of the metabolic equilibrium in diabetes mellitus results in
characteristic end-organ damage that occurs in various combinations and that follows
an unpredictable clinical pathway
Accordingly the major consequence of diabetes mellitus in terms of morbidity
mortality and economic burden principally concerns macroangiopathies or
arteriosclerosis and microangiopathies including nephropathy neuropathy and
retinopathy7-10
One of these devastating consequences which often appears in time is the
development of various skin defects that are frequently resistant to healing and that
tend to be more severe than similar lesions in nondiabetic individuals Diabetes
mellitus even increases the risk of infection by an increased susceptibility to bacteria
and an impaired ability of the body to eliminate bacteria1112
Skin problems are a severe complication in diabetic individuals and require a
comprehensive and appropriate multidisciplinary approach to prevention and
treatment12
Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to
play the most prominent role in the development of diabetic complications With the
development of insulin treatment for type I diabetes and various oral hypoglycemic
agents for type 2 diabetes a reduction in the development of skin defects due to
hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply
Chapter 3
64
waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in
diabetic patients often need special care in comparison to those persons in good
health who rarely require treatment for wound healing1617 Special care is directed
besides of course toward optimal diabetes regulation toward patient education
maximum pressure relief controlling infection recovery of circulation in case of
ischemia and different modalities of intensive wound treatment18
In the last few years various therapies have been introduced with varying success An
example of such a therapy is the photo-modulated stimulation of diabetic lesions In
vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been
subject of extensive investigation1920 Due to contradictory research results LLL-
photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of
light in the domain of wound healing is less time-consuming less expensive less
invasive than many of the other introduced treatment modalities and practical to use
however it seems worthwhile to investigate the value and benefits of a newly
introduced and alternative light source the light emitting diodes (LEDrsquos)
Preliminary research has proved that green LED with particular properties (an
exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01
Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These
results may be of great importance to the diabetic patient because as posed by Reddy et
al light has a possible beneficial effect in the case of impaired healing1617
To obtain insight into the ability of LED to stimulate fibroblast proliferation under
diabetic-specific conditions of impaired healing the proliferation was assessed in
irradiated and control cultures cultivated in medium with a high quantity of glucose
MATERIAL amp METHODS
Cell cultivation
Primary fibroblast cultures were established by outgrowth from 8-day-old chicken
embryos After isolation and disaggregating as described by Freshney (1994)24 the cells
were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10
Fibroblast proliferation under hyperglycemic circumstances
65
fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin
Secondary cultures were initiated by trypsinization followed by plating of the cells in
80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were
disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently
231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third
passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the
cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified
atmosphere at 37deg C to allow them to attach to the bottom of the wells
Light source specifications and illumination procedure
To control adherence of the cells and to assure that there was no confluence or
contamination the 96-well plates were microscopically examined before irradiation
Subsequently the tissue culture plates were randomly assigned for use in the treated
and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium
was aspirated The remaining 25 (50 microl) medium avoided dehydration of the
fibroblasts throughout irradiation
Irradiation was performed with a light emitting diode (LED) device The LED device
(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power
range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable
within the range of 0-1500 Hz
The investigation used the following illumination properties the continuous mode a
distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2
radiant exposure This procedure resulted in an exposure time of 3 min and a power
output of 10 mW Immediately after irradiation the remaining medium was aspirated
and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM
glucose (30 gL) and incubated at 37deg C
Irradiation and medium changes occurred at 1-day intervals so one irradiation was
implemented each 24 h for 3 days in a row and from the first irradiation onwards all
medium renewals occurred with glucose-supplemented Hanksrsquo culture medium
Control cultures were handled in the same manner but were sham-irradiated
Chapter 3
66
Proliferation assay
Fibroblast survival and proliferation were determined by a sensitive and reproducible
colorimetric assay the assay which detects merely living cells and the signal generated
bears a constant ratio to the degree of activation of the fibroblasts and the number of
fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than
only a small subsample26
Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented
Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution
was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during
30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous
solution The optical density of the final solution was measured on an ELtimes800 counter
(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test
wavelength varying from 400 to 750 nm
The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies
for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except
for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)
Data analysis
On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a
Mann-Whitney U test was performed for nonparametrical comparison of the results
Statistical significance for all tests was accepted at the 005 level For this analysis the
Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used
RESULTS
The MTT measurements from each of the 256 control wells and 256 irradiated wells
and the subsequent nonparametrical analysis from the optical densities obtained
disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic
Fibroblast proliferation under hyperglycemic circumstances
67
circumstances after irradiation than in the same circumstances without irradiation (Fig
1)
Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)
DISCUSSION
The outcome of these in vitro experiments based on the above-described light source
properties and the illumination procedure described clearly demonstrated the
stimulatory potential of LED on fibroblast proliferation and the cell viability of
fibroblasts cultured in hyperglycemic medium Preliminary research has already
demonstrated that under these conditions (an exposure time of 3 min a wavelength of
570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this
procedure allowed the highest number of living cells The nature of the light and the
usual questions concerning coherence wavelength power output and radiant
exposures have been discussed previously23
Although these findings confirm the results previously found one cannot ignore the
important methodological difference between previous investigations and the current
study as the cells in this experiment were cultured in hyperglycemic medium2328-30
Absorbency - Proportional to the number of fibroblasts
621 x 10-1 682 x 10-1
0010203040506070809
1
Control Irradiated
Groups
Ab
sorb
ency
Chapter 3
68
After a growth period with normal Hanksrsquo culture medium a necessary step to ensure
normal growth of these secondary subcultures and normal attachment to the bottom
of the wells the Hanksrsquo culture medium was supplemented with glucose
Several earlier studies have established that exposure to glucose concentrations (20-40
mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human
vascular endothelial cell proliferation1531-34 This restraint is more pronounced for
higher glucose15 concentrations and is expressed especially after protracted exposure to
high glucose levels31 A similar restraint was found for cultured fibroblasts by
Hehenberger et al3536 According to some authors however cultured fibroblasts
conversely have been shown to maintain responsiveness to ambient high glucose323738
As there are some ambiguities in literature regarding normal or inhibited growth of
fibroblasts in medium supplemented with glucose39 a pilot study was performed to
determine the amount of glucose necessary to inhibit normal growth after 72 h of
culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study
demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease
of cell viability and to bring about a decline in fibroblast proliferation
This concentration resulted in a remarkable reduction of cell viability and a noteworthy
decrease in the proliferation rate in comparison to control cultures grown in 55 mM
glucose although this concentration is too high to mimic severe diabetic
hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to
investigate the effect of LED in distinct destructive conditions in order to obtain an
incontrovertible result
In addition it is possible that the present investigation needed a higher amount of
glucose to result in a remarkable reduction of proliferation as exposure to glucose was
limited to 72 h and as previous studies revealed that the antiproliferative effect of high
glucose was more pronounced with prolonged exposure with a maximal inhibition
attained by 7-14 days1531
Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo
cell growth are too complex to compare A key question is whether fibroblast
senescence in tissue culture and in the intact organism are similar Cristofalo et al40
Fibroblast proliferation under hyperglycemic circumstances
69
reported that this is not the case as fibroblasts have a finite ability to divide and
replicate but apparently the pathway or the morphologic characteristics leading to the
replicative senescence is not identical in vivo compared to in vitro
Furthermore extrinsic aging related to environmental damage which in diabetic
patients is mainly due to a chronic exposure to high levels of glucose during life is
unachievable in vitro
Unless a number of questions regarding the mechanism according to which LED
stimulates fibroblast proliferation in this particular condition remain unanswered the
results ascertain the potential effects of LED on fibroblast proliferation and viability
CONCLUSION
The current results should be interpreted with caution However these results
demonstrate the effectiveness of green LED irradiation at the above-described light
source properties and the illumination procedure described on cells in hyperglycemic
circumstances
The findings of the present study using an experimental in vitro model indicate that the
use of LED irradiation to promote wound healing in diabetic patients may have
promising future results As the present study establishes the possibility of using LED
irradiation in experimental in vitro situations it would be a worthwhile extension to
perform in vivo investigations to determine whether these in vitro observations were
relevant to the physiological situation and to determine the effect of these LED
properties on human tissue response
ACKNOWLEDGMENTS
The authors are greatly indebted to P Coorevits for assistance with the statistical
analysis and to Professor L Deridder and Ms N Franccedilois of the department of
Human Anatomy Embryology Histology and Medical Physics for providing access to
the laboratory and for helpful discussions
Chapter 3
70
REFERENCES
1 World Health Organisation The world health report of 2002 Statistical Annex 2002 pp 179-201
2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418
3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957
4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538
5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308
6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J
Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot
wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-
glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A
Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood
glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13
14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309
15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501
16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287
17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255
18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412
19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192
20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137
21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675
22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246
23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99
Fibroblast proliferation under hyperglycemic circumstances
71
24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729
27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63
28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301
29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -
progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of
cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA
damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in
culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia
23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar
K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71
36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141
37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588
38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252
39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97
40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619
PART II ANALGESIA
CHAPTER 4
EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION
MEDIATED BY LIGHT EMITTING DIODE IRRADIATION
Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy
Vanderstraetenab and Dirk Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Lasers in Medical Science 2005 20(1) 35-40
Chapter 4
76
ABSTRACT
The introduction of light emitting diode (LED) devices as a novel treatment for pain
relief in place of low-level laser warrants fundamental research on the effect of LED
devices on one of the potential explanatory mechanisms peripheral neurophysiology in
vivo
A randomised controlled study was conducted by measuring antidromic nerve
conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline
measurement and five post-irradiation recordings (2 min interval each) were performed
of the nerve conduction velocity (NCV) and negative peak latency (NPL)
Interventional set-up was identical for all subjects but the experimental group (=32)
received an irradiation (2 min at a continuous power output of 160 mW resulting in a
radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype
MDB-Laser Belgium) while the placebo group was treated by sham irradiation
Statistical analysis (general regression model for repeated measures) of NCV and NPL
difference scores revealed a significant interactive effect for both NCV (p=0003) and
NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical
significant decreased NCV and an increased NPL in the experimental group and a
statistical significant difference between placebo and experimental group at various
points of time
Based on these results it can be concluded that LED irradiation applied to intact skin
at the described irradiation parameters produces an immediate and localized effect
upon conduction characteristics in underlying nerves Therefore the outcome of this in
vivo experiment yields a potential explanation for pain relief induced by LED
Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative
peak latency middot Analgesic effect
Nerve conduction characteristics
77
INTRODUCTION
Since the introduction of photobiostimulation into medicine the light sources used
have advanced technologically and varied in characteristics over the years
Advancement and variation of the sources implicate a concomitant necessity to revise
research results in the respective domains of application Research and clinical
applications in the past particularly focused on the effectiveness of low-level lasers
have shifted now to novel treatment units such as light emitting diode (LED) devices
The efficacy and applicability of LED irradiation within the field of wound healing has
already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED
is not only promoted for its beneficial effects on the wound-healing process it is also
suggested to be potentially effective in the treatment of pain of various aetiology
although this claim has not yet been investigated thoroughly either experimentally or
clinically The putative analgesic effects of LED remain to be further explored
As the basic vehicle of pain is the neuronal system [7] measuring the
neurophysiological effect of LED treatment would be an appropriate experimental
approach to investigate the efficacy of LED on pain inhibition Nerve conduction
studies have become a technique for investigating the neurophysiologic effects of light
therapy [8-9]
Review of literature regarding standard nerve conduction studies revealed that previous
human studies on the influence of various light sources on peripheral nerves have
utilized different methods which hampers a comprehensive comparison In general
this research was performed on the superficial radial nerve [10-13] described by Shin J
Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve
[891315-17] Following the method of Cambier et al [18] the authors of this study
decided to investigate the effect of the light source used on the conduction
characteristics of the sural nerve By investigating this solely sensory nerve interaction
of motor nerve fibres (motor response can easily be provoked by antidromic nerve
stimulation [19]) can be avoided and given the superficial nature of the nerve it should
be sufficiently amenable to the effects of percutaneous LED irradiation
Chapter 4
78
A second major difference between the trials and therefore also hindering an
appropriate comparison between the results is the wide range of used light sources
HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared
multisource treatment unit [15]
With respect to the potential importance of LED irradiation for the treatment of pain
the current investigation was designed to assess the putative neurophysiological effects
of LED on the sensory nerve conduction of the human superficial peripheral sural
nerve and to establish a time course of the supposed phenomenon
The experimental hypothesis postulates that LED generates an immediate decrease in
conduction velocity and increase in negative peak latency In addition it can be
postulated that this effect is most prominent immediately after the irradiation and will
weaken as time progresses
STUDY DESIGN
The study was approved by the Ethical Committee of the Ghent University Hospital
After explanation of the experimental procedure a written informed consent was
obtained from each subject
Subjects
After screening based on a brief medical history excluding subjects with
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever inflammation of the skin) or conditions
that might affect sensory nerve conduction (such as diabetes peripheral neuropathy
radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral
edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40
females (mean age 26plusmn6 years range 18-42 years) participated in this study The body
mass index (BMI) of each subject varied within the normal range (=185-249) [20]
(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo
Nerve conduction characteristics
79
or an experimental group Each group of 32 subjects was composed of 12 males and
20 females
Experimental Procedure and Data Acquisition
In order to be able to quantify the negative peak latency (NPL) (measured from the
start of the stimulus artefact to the peak of the negative portion of the nerve action
potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was
followed
With respect to the known relationship between nerve conduction characteristics and
temperature the ambient temperature was kept constant (23ordmC-26ordmC room
temperature) during the investigation In view of this temperature issue the
standardized protocol started with 10 min of accommodation during which the
subjects rested in prone position on a treatment table
Immediately before this adjustment period the skin over the dorsolateral aspect of the
left calf and foot was cleaned with alcohol to remove surface lipids This preparation of
the treatment area was followed by the placement of the electrodes (TECA
Accessories Oxford Instruments Medical Systems Division Old Woking UK) as
described by Delisa et al [21]
The two-posted (2 cm separation anode distal) surface caption electrode was placed
distal and posterior of the lateral malleolus on the skin covering the sural nerve The
fixation of the earth electrode (Medelec Oxford Instruments Medical Systems
Division Old Woking UK) occurred 12 cm above the caption electrode according to
the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm
above the caption electrode to map the ideal stimulation point To level off
intraindividual variations in the amount of sensory response attributable to the
successive placement of the bipolar stimulator in course of the investigation a two-
posted (2 cm separation cathode distal) bar stimulating electrode was attached at the
point where the maximal response was obtained
This placement of the electrodes allows antidromic stimulation of the sural nerve
Electrophysiological stimulation and recordings were obtained with a Medelec
Chapter 4
80
Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse
of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was
used to produce each evoked sensory response
Baseline measurements of NPL and NCV were immediately followed by treatment of
the subjects according the protocol detailed below Recordings were subsequently
repeated at 2-min intervals over an 8-min period resulting in five recordings (one
immediately after the completion of the treatment and one at 2 4 6 and 8 min after
irradiation) Skin temperature was recorded concomitantly throughout the procedure
at the time of baseline measurement immediately after LED irradiation at the time of
the first recording and consequently at 2-min intervals together with the four final
electrophysiological recordings For this a surface digital C9001 thermometer
(Comark UK) sensitive to temperature changes of 01degC was used at the same point
of LED administration namely at 7 cm above the caption electrode The procedure
was identical for both conditions but subjects in the placebo group received a sham
LED irradiation
Light Characteristics and Irradiation Procedure
Irradiation was administrated with a light emitting diode device (BIO-DIO
preprototype MDB-Laser Belgium) The probe used emitted infrared light with a
wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2
and the frequency was variable within the range of 0-1500 Hz
Preceding baseline measurement the treatment point was marked on the skin overlying
the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-
point between the stimulation and capture electrode The LED probe was held in
contact with the skin perpendicular to the skin surface during the complete irradiation
procedure LED treatment consisted for all subjects of the experimental group out of 2
minutes lasting irradiation The LED was set to deliver a continuous energy density of
107 Jcm2 at a power output of 160 mW These parameters were selected as they are
appropriate for the treatment of pain in a clinical setting First of all because the
Nerve conduction characteristics
81
duration of the treatment is clinically feasible and secondly because the parameters are
within the scope of previously described light source characteristics [1-36915]
Statistics
Although superficial skin temperature did not change significantly in course of the
investigation the influence of the measured skin temperature on NPL and NCV was
taken into account by using a correction factor of respectively 02 msdegC and 147
ms degC All corrections were calculated towards a reference skin temperature of 32degC
Difference scores ie the variation between baseline measurements and each post-
irradiation recording were used as the basis for statistical analysis A General
Regression Model for repeated measures with one within-subjects factor (time 0
min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and
one between-subjects factor (group placebo or LED irradiated) was performed
followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least
Significant Difference) to determine whether any differences between baseline
measurements and post-irradiation recordings were statistically significant
The Statistical package for social sciences (SPSS 110) was used for analysis and
statistical significance for all tests was accepted at the 005 level
RESULTS
Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated
group plotted against time in minutes The values of the irradiated subjects decrease
directly after the irradiation and reach a first low point 2 min after finishing LED
treatment This decrease is followed by a marginal increase at 4 and 6 min and again an
important decrease at 8 min Statistical analysis (general regression model for repeated
measures) of these data indicated a significant interactive effect (P=0003)
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82
Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)
Post hoc LSD further showed significant differences between baseline measurements
and all post-treatment recordings (Table 1) Mutual comparison of the values from the
post-treatment recordings did not reveal any significant difference In addition there
was no significant difference determined in the placebo group in course of time
Table 1 Summary of the influence of LED irradiation on nerve conduction velocity
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001
2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002
4 0111plusmn0377 0647 -0908plusmn1898 0021 0004
6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001
8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve Conduction Velocity
-14
-12
-1
-08
-06
-04
-02
0
02
04
Baseline 0 min 2 min 4 min 6 min 8 min
Time Course
Dif
fere
nce
Sco
re (
m
s)
PlaceboLED
Nerve conduction characteristics
83
A similar representation was used for the results of the NPL Figure 2 reproduces NPL
plotted against time in minutes revealing for the irradiated group an increased latency
with two important peaks one at 4 min and one at 8 min
Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)
Statistical analysis of the mean difference scores again indicated a significant interactive
effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2
showed significant differences between baseline measurements and all post-treatment
recordings of the experimental group The mean difference score of the first post-
treatment recording of this same group (LED irradiated) differed significantly with the
recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED
irradiation As well as the recording 2 min after irradiation which differed significantly
(P=0013) with the 8 min post-treatment recording As observed for the NCV the
NPL of the placebo group did not reveal any significant difference in time course
At the time of the final recording the NCV and NPL mean difference scores of the
irradiated group did not return to their respective baseline values
Negative Peak Latency
-001
0
001
002
003
004
005
006
007
Baseline 0 min 2 min 4 min 6 min 8 min
Time course
Dif
fere
nce
Sco
re (
ms)
PlaceboLED
Chapter 4
84
Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group
significance) revealed statistical differences between the experimental and the placebo
group for NCV as well as for NPL NCV and NPL were statistical significant between
both groups at all points of time except from the NPL recording immediately after
finishing irradiation
DISCUSSION
Notwithstanding the above-mentioned difficulties in comparing results between
different trials on nerve conduction we attempt to discuss the current findings in view
of the results of the previous studies
This investigation revealed that percutaneous LED irradiation at feasible and current
clinical parameters generates measurable and significant changes in human sural nerve
antidromic conduction latency and velocity These results thus support previous
findings of light-mediated nerve conduction latency shifts in vivo [8101218]
although there are several important issues to be discussed
Table 2 Summary of the influence of LED irradiation on negative peak latency
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0004 plusmn0053 0755 0029plusmn0080 0019 0145
2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021
4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004
6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034
8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve conduction characteristics
85
A first comment deals with the progress of the NCV and NPL in function of time As
postulated the NCV decreases significantly immediately after irradiation
corresponding with a significant increased NPL However this effect does not weaken
as time progresses both variables remain significant throughout the 8 min during
observation period
Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the
conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]
although this slight increase in NPL was not significant at any moment Two other
studies [822] with a GaAlAs laser even registered comparable effects over a period of
55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous
studies post-treatment conduction measurements should be extended in time At
present for all studies it remains unclear at what point of time the effect extinguishes
although the interval of time during which LED treatment remains effective is
clinically important when treating pain
Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least
45 min) mediated by a monochromatic multisource infrared diode device although it
needs to be mentioned that this study performed with a comparable light source as the
current investigation revealed a significant decrease of the NPL These inverse results
between the study of Noble et al [15] and the current investigation could be attributed
to the concomitant increase of the skin temperature [15] As it has been well
recognised that a variation in tissue temperature causes a corresponding alteration in
nerve conduction velocities and peak latencies [91523-27] the temperature changes
may indeed provide an explanation for the observed findings In an attempt to analyse
the influence of a direct photobiological effect on sural nerve conduction
characteristics rather than working out the effects based upon thermal mechanisms
the present study corrected the skin temperature towards a reference temperature of
32degC This correction was performed notwithstanding the fact that the superficial skin
temperature did not change significantly before and after LED irradiation as well as
despite the fact that influencing nerve temperature takes place long after affecting skin
temperature [23] and thus being (almost) impossible after 2 min of irradiation
Chapter 4
86
followed by 8 min of registration Introduction of the correction factor implies likewise
that eventual influence on nerve conduction by cooling of the limb due to inactivity as
described by Greathouse et al [11] can be excluded
These facts suggest that temperature changes did not contribute to the demonstrated
effects of LED on nerve conduction Nevertheless the underlying mechanism of the
observed effects remains indistinct
A following remark regarding the fluctuation of NCV and NPL in function of time
considers the fact that both the NCV and the NPL do not change in a constant way up
to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the
increase in NPL display a small though not significant inversion of the effect at 4 and
(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of
fluctuation is to be expected when measuring NCV and NPL besides there is a similar
variation in the placebo groups
Although investigating dose dependency was not intended an additional remark
considers the fact that the use of optimal irradiation parameters is essential to obtain
the observed neurophysiological effect Nevertheless it is impossible to determine
ideal light source characteristics for effective treatment as the range of used
wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency
(pulsed or continuous) are not sufficiently similar between the different studies It can
only be concluded that a pulsing light source [91028] does not provide the postulated
results Radiant exposure exposure time power range and wavelength are not yet
established but based on this study and previously described assays it can be
speculated that the ranges of these parameters are quite large
In comparison with other studies where the number of subjects is 10 or less [8-
1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-
Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number
of subjects (n=32) was investigated in each group In spite of the large investigated
population it should be noted that the magnitude of the described changes in NCV
and NPL can simply be replicated by lowering the temperature of the extremity as the
observed changes are within the expected physiological ranges making the clinical
Nerve conduction characteristics
87
significance of the change questionable (This fact does not implement that the
decrease and the significant changes were temperature mediated)
A key question and meanwhile the initial impetus for future investigation is whether
the measured effects can be extrapolated to the actual nociceptive afferents namely the
myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])
respectively conducting acute and chronic pain The functional testing of these
nociceptive pathways has recently been extensively evaluated The currently accepted
neurophysiological method of assessing nociceptive pathways relies on laser-evoked
potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]
As up till now LEP is not available in this or any surrounding research centre the
investigators of this study had to perform a standard nerve conduction study (assessing
the large myelinated Aβ afferents) Therefore the current and previous beneficial
results of low level light therapy on conduction characteristics of nerves in vivo should
initiate measurements of clinical effectiveness first of all in laboratory settings and
afterward at a clinical level
CONCLUSION
Despite these remarks and the limited knowledge regarding the underlying mechanism
the present findings enable the following conclusions to be drawn LED irradiation at
clinical applied energy densities produces an immediate and localized effect upon
conduction characteristics in underlying nerves More specifically it is proven that
LED treatment lowers the NCV and augments the NPL resulting in a reduced
number of impulses per unit of time Therefore the outcome of this in vivo experiment
assumes that LED possibly induces pain relief
In order to encourage a widespread acceptance for the use of this non-invasive pain-
reducing modality in clinical settings prospective research should establish the precise
relationship between LED and pain relief as well as determine the ideal irradiation
parameters and verify which painful conditions can be treated with this treatment unit
Chapter 4
88
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the
equipment and MDB-Laser Belgium for generously providing the Light Emitting
Diode equipment
Nerve conduction characteristics
89
REFERENCES
1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9
2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)
3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18
4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8
5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43
6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65
7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53
8 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-34
9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6
10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90
11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7
12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5
13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9
14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore
15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5
16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5
17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604
18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200
19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21
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90
20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083
21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press
22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9
24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9
25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203
26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13
27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12
28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9
29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445
30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8
31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56
CHAPTER 5
PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE
IRRADIATION A PILOT STUDY ON EXPERIMENTALLY
INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS
Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Accepted for publication in Lasers in Medical Science December 2005
Chapter 5
92
ABSTRACT
Objective The present pilot study investigated the analgesic efficacy of light emitting
diode (LED) In view of a standardised and controlled pain reduction study design this
in vivo trial was conducted on experimentally induced delayed-onset muscle soreness
(DOMS)
Design Thirty-two eligible human volunteers were randomly assigned to either an
experimental (n=16) or placebo group (n=16) Immediately following the induction of
muscle soreness perceived pain was measured by means of a visual analog scale (VAS)
followed by a more objective mechanical pain threshold (MPT) measurement and
finally an eccentricconcentric isokinetic peak torque (IPT) assessment The
experimental group was treated with infrared LED at one of both arms the other arm
served as control Irradiation lasted 6 min at a continuous power output of 160 mW
resulting in an energy density of 32 Jcm2 The subjects of the placebo group received
sham irradiation at both sides In post-treatment a second daily assessment of MPT
and VAS took place The treatment and assessment procedure (MPT VAS and IPT)
was performed during 4 consecutive days
Results Statistical analysis (a general linear model followed by post hoc least
significant difference) revealed no apparent significant analgesic effects of LED at the
above-described light parameters and treatment procedure for none of the three
outcome measures However as the means of all VAS and MPT variables disclose a
general analgesic effect of LED irradiation in favour of the experimental group
precaution should be taken in view of any clinical decision on LED
Conclusion Future research should therefore focus on the investigation of the
mechanisms of LED action and on the exploration of the analgesic effects of LED in a
larger randomised clinical trial and eventually in more clinical settings
Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset
muscle soreness middot Musculus biceps brachii
Delayed-onset muscle soreness
93
INTRODUCTION
The analgesic efficacy of light emitting diode (LED) irradiation is recently being
investigated by means of a nerve conduction study on the superficial peripheral sural
nerve [1] It was demonstrated that LED irradiation at clinical applied densities
produces an immediate and localized effect upon conduction characteristics in
underlying nerves More specific LED induces a decreased number of sensory
impulses per unit of time thus possibly inducing pain relief [1]
Given the established influence of this treatment modality on the nerve conduction
velocity and thereby its potential analgesic ability the current investigation was
designed
Studies investigating the efficacy of a therapeutic modality on pain often experience
difficulties regarding standardisation of the population as analysis or comparison of
pain with different aetiologies is almost impossible Therefore we opted to measure the
analgesic effects of LED in a laboratory setting on a sample with experimentally
induced delayed-onset of muscle soreness (DOMS)
Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the
induction exercise and then spreads throughout the muscle [2-4] The correlates of
DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10
[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force
decreased range of motion and in particular muscle pain which is more pronounced
during movement and palpation [8 11] Despite the large volume of research that has
been undertaken to identify the underlying pathophysiology of DOMS the precise
mechanism is not yet universally accepted Several theories such as the torn-tissue
theory the connective tissue damage theory the muscle spasm theory and the
inflammation theory still remain viable though the current opinion states that DOMS
arises from a sequence of events in which several theories occupy an important place
[2 6 12 13]
DOMS has been used as a representative model of musculoskeletal pain and stiffness
in a number of studies [4 7 11 14 15] as it has a number of advantages it can be
induced in a relatively easy and standardised manner in a group of healthy subjects the
Chapter 5
94
time-course is relatively predictable and the symptoms have the same aetiology and are
of transitory nature [14 16] Nevertheless it should be emphasised that the use of this
particular experimental model to test the effectiveness of LED does not mean that this
treatment modality is necessarily advocated for the treatment of DOMS but merely
that it may be helpful in documenting the efficacy of LED in a clinical model of
musculoskeletal pain and stiffness In addition studies based on the induction of
DOMS under carefully controlled laboratory conditions can not replace research
involving actual patients but offer the opportunity to assess the effectiveness of
particular therapeutic interventions and might help to define additional clinical research
[14]
The experimental hypothesis of the current study postulates that infrared LED reduces
pain and muscle sensitivity associated with DOMS
MATERIALS AND METHODS
The study was approved by the ethical committee of the Ghent University Hospital
After providing information regarding the study design and possible consequences
related to participation at the study written informed consent was obtained from each
subject
Subjects
Healthy human volunteers were recruited from the university population Individuals
with any upper limb pathology neurological deficit and recent injury to either upper
extremity or undiagnosed pain were excluded Other exclusion criteria were
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever and inflammation of the skin) or
conditions in which physical exertion is contraindicated (such as cardiovascular deficits
hypertension and respiratory problems)
Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age
23plusmn4 years) were enrolled All subjects were randomly assigned using a random table
Delayed-onset muscle soreness
95
of numbers to the experimental or placebo group Each group of 16 subjects
consisted by stratification of equal numbers of men and women Age height and
weight did not differ significantly between the three groups
All subjects were physically active however none performed on a regular basis any type
of upper body weight-training Subjects were requested to refrain from any form of
strenuous physical activity and they were asked to avoid any form of medication
including anti-inflammatory agents as well as alcohol for 2 days before testing and for
the duration of the study
Overview of experimental design
The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to
induce pain related to DOMS Immediately following induction exercise an initial
assessment of the outcome measures (visual analog scale or VAS mechanical pain
threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the
subjects were treated under blinded conditions according to the randomised group
allocation In post-treatment the MPT was re-recorded and perceived pain was
reassessed with a VAS Contrary to these outcome measures the muscle strength was
only measured in pre-treatment at the one hand because short-term effects of LED
on muscle strength were not postulated and on the other hand because post-
treatment muscle strength can be influenced by too many different physiological
factors related to the pre-treatment measurement On the succeeding days (day 2 3
and 4) the treatment and assessment procedure was similar with approximately 24 h
separating each treatment
In both of the groups the two arms of the participants were included in the study In
the experimental group an equal number of dominant and non-dominant arms were
treated The non-treated arm served as control arm In the placebo group also an equal
number of dominant and non-dominant arms were considered as treated arm and the
other arm was classified in the non-treated group The procedure was identical for
both conditions but the subjects in the placebo group received sham LED irradiation
on both arms
Chapter 5
96
Specific aspects of the experimental design and procedures are detailed below
Pain induction
Muscle soreness was induced in a standardised fashion via a daily calibrated computer-
operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY
USA) Induction occurred separately and in random order in the elbow flexors of both
arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex
Prior to induction of DOMS the subjects were allowed an initial familiarization session
to become comfortable performing maximum voluntary contractions at the required
angular velocities This was immediately followed by determination of the maximum
eccentric and concentric peak torque at an angular velocity of 60degs and 120degs
Subsequently four sessions of eccentricconcentric work were performed with each
arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs
first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg
and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-
range) followed by two sessions at an angular velocity of 120degs again the first time
along an arch of 120deg and followed by the mid-range performance The subjects were
asked to accomplish maximum voluntary contractions during all the sessions Each
session was performed until exhaustion which was defined as the point when the
subject lost 70 of the initial eccentric and concentric peak torque There was a 1-
minute rest between each session This procedure was based on a pilot study and
previously described induction protocols [17-21]
Outcome measures
Outcome measures of subjective pain measurements MPT and muscle strength were
measured in this order on days 1-4 Subjective pain measurements and MPT occurred
immediately prior to and following irradiation whereas muscle strength measurements
only took place before LED treatment
Measurement of subjective pain Perceived muscle soreness was measured
subjectively by means of a 100-mm VAS A series of scales were completed separately
Delayed-onset muscle soreness
97
for each arm pain at rest followed by pain perception associated with full extension of
the arms and finally with maximal flexion of the arms The subjects were not allowed
to compare one VAS result with another
This assessment tool commonly used in measuring experimentally induced pain [22
23] has been found to be a reliable and valid method [24-26]
MPT Tenderness MPT used as a more objective correlate of muscle tenderness
has been demonstrated to be a reliable method to measure experimental induced
muscle soreness [27] This outcome measure was assessed by using a handheld
pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries
South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a
line from the radial insertion of the musculus biceps brachii at the elbow to the
intertubercular groove of the humerus thus resulting in three measure points one at
the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A
pressure of 4Ns was delivered The subjects were instructed to say yes at the exact
moment the pressure perceived became painful Each point was recorded three times
in pre-treatment as well as in post-treatment The average MPT score for each point in
pre- and post-treatment was used for statistical analyses
Muscle strength assessment Eccentric and concentric IPT were measured on the
same computerised dynamometer as was used for the induction of pain and an
identical standardisation procedure regarding positioning was followed
A warm-up session of two maximum voluntary contractions at the required angular
velocities was followed by determination of the eccentric and concentric peak torque
The first session at 60degs consisted of three repetitions followed by a 1-min during
rest and for the second session at 120degs five repetitions were performed The
subjects were instructed to flex and extend the elbow through the entire range of
motion as forcefully and rapidly as possible for each repetition The maximum
eccentric and concentric torque produced during the respective repetitions was used
for statistical analysis
Chapter 5
98
Light source specifications and treatment procedure
Light treatment was applied daily according to group allocation Irradiation occurred
with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The
probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160
mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The
frequency was variable within the range of 0ndash1500 Hz
During the complete irradiation procedure the LED probe was held in contact with
the skin perpendicular to the skin surface and at the exact mid-point between the MPT
mark at 4 cm and the one at 8 cm Light source properties were identical for all
subjects of the experimental group and consisted out of irradiation of 6-min lasting
duration at a continuous power output of 160 mW resulting in an energy density of
32 Jcm2 To conceal the treated side and condition the subjects were blinded to the
treatment status For the experimental condition a probe was held in contact with each
arm but only one of the two probes was attached to the LED device The subjects of
the placebo group received sham irradiation at both sides
The selected parameters are within the scope of previously described light source
characteristics for pain reduction [1 28-30] and they are appropriate for the treatment
of pain in a clinical setting because the duration of the treatment is clinically feasible
Statistical analysis
The three outcome measures were analysed separately For the VAS and MPT
measurements the same procedure was followed a general linear model (GLM) for
repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-
post preceding and following LED irradiation) and one between-subject factor (group
placebo or infrared LED irradiated) was performed If necessary the GLM was
followed by appropriate pairwise comparisons (post hoc least significant difference or
LSD) to determine whether any differences between measurements were statistically
significant A similar model was carried out separately for both the treated and the
control arm
Delayed-onset muscle soreness
99
In contrast to MPT and VAS the muscle strength was analysed differently The peak
torque values recomputed towards body weight of the subjects were statistically
analysed using a GLM for repeated measures This model consisted of one within-
subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo
or infrared LED irradiated) The model was completed twice first for the treated arm
and consequently for the control arm
The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)
was used for analysis and statistical significance for all tests was accepted at the 005
level
RESULTS
Statistical analysis of all variables of the three outcome measures revealed no significant
interactive effects of the main interaction (time times group times pre-post) The means and
standard deviations of the variables for both the treated and the control arm are
outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The
means of all VAS and MPT variables disclose a non-statistical significant general
analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher
MPT values in the irradiated group than in the placebo group The lower VAS rates are
present from day 1 until the last day of the study but they are more clearly present
from day 3 pre-treatment The higher MPT values are present from day 1 post-
irradiation until the last day and they are more visible at 4 cm followed by 12 cm and
finally at 8 cm In addition to the analgesic influence of LED an increased
convalescence of muscle strength was noted It should be remarked that this outcome
is similar for the treated as well as for the control arm of the irradiated group The
findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms
of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively
Graphical presentation of the other variables shows a similar course
Chapter 5
100
Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42
Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36
Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Visual Analog Scale
0
02
04
06
08
1
12
14
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n V
AS
scor
e (a
t re
st)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
101
Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249
Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Mechanical Pain Threshold
0
5
10
15
20
25
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n M
PT
sco
re (
at 4
cm)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Chapter 5
102
Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group
Day 1 Day 2 Day 3 Day 4
Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04
Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04
Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Isokinetic Peak Torque
04
045
05
055
06
065
Day 1 Day 2 Day 3 Day 4
Time course
Mea
n I
PT
sco
re (
ecce
ntr
ic a
t 60
degse
c)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
103
Despite the absence of significant main interaction effects the remaining interactions
as well as the main effects were statistically significant for some variables Only the
significant interactions including the between-subject factor group as well as the main-
effect group will be discussed The other interactions and effects establish the successful
induction of DOMS but are not relevant in view of the postulated hypothesis
The interaction between group and time is significant (p=014) for the VAS in
association with full extension for the control arm Post hoc LSD reveals no difference
between both groups a significant effect over time for both groups is found
Consequently this will not be further evaluated
A second significant interaction (p=0002) is the one among the within-subject factor
pre-post and the between-subject factor group for the MPT at 12 cm for the control arm
Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more
pressure after than before the treatment whereas in the placebo group a not
significant decrease of supported pressure is noted
Finally GLM analysis revealed that at the treated arm the irradiated group tolerates
significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)
DISCUSSION
It has previously been demonstrated that the LED source used might assist in
accelerating wound healing [31] that it has a direct cellular effect [3233] and that it
changes nerve conduction characteristics [1] Nevertheless LED-treated experimental
induced DOMS failed to prove the analgesic efficacy of LED at the above-described
light parameters and treatment procedure The current outcome concurs with other
research that demonstrated a lack of effect of various forms of light therapy on DOMS
[8 11 15] However despite the absence of an apparent and overall definitive finding
the present results cannot exclude favourable effects of LED treatment on pain Since
first of all an isolated statistical significant pre-post difference between groups (control
arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)
revealed that subjects of the irradiated group tolerate more pressure than the subjects
of the placebo group Second of all the overall means identified generally lower VAS
Chapter 5
104
scores higher MPT values and higher peak torques in the irradiated group This
implied that the treated subjects experienced noticeable less pain supported more
pressure on the painful muscle and generated more force than the non-treated
participants However these results are not statistically significant consequently it is
possible that these differences were found by coincidence and that there is no
relationship between the treatment and the described results of the three outcome
measures though it should be mentioned that the absence of significant findings is
more probably attributable to the small sample size involved in this study This
assumption is based on a post hoc power analysis It was calculated that for the small
effect size measured after treatment and for the measured control group event rate a
sample size of 80 subjects in each group was required at α=005 and power=080
(two-sided) to reveal significant results
Another factor conceivably responsible for the lack of solid evidence of the beneficial
effects of LED treatment upon DOMS-associated pain is related to the size of the
treatment effect in relation to the severity of the induced DOMS It is possible that by
using multiple exhaustive sets of exercise severe DOMS were induced which masked
relatively small but apparent treatment effects [4 11] In this same context it is
possible that the results only become significantly different after a prolonged treatment
and follow-up period as previous research noticed that recuperation subsequent to
DOMS induction can last up to 10 days [8]
Although it needs to be stressed that these results are not statistically significant critical
analysis of the overall means leads up to three additional remarks A primary comment
relates to the pre- and post-treatment courses of the results Starting at day 2 a clear
reduction of pain and muscle sensitivity was observed immediately post-treatment
Still one cannot conclude that this is indicative for the analgesic effect of LED
irradiation as a similar decrease in VAS and increase in MPT values was noted in the
treated and the control arm of the placebo group Perhaps this was caused by placebo
effect as reported by Pollo et al [34] the expectation of the participant can easily result
in pain relief but it can only be elucidated by implementation of a control group
Delayed-onset muscle soreness
105
Nevertheless in the current study this particular finding can be most probably
attributed to the physiological effects of the peak torque measurement performed
between the pre- and post-treatment recordings of VAS and MPT on the painful
flexor muscle of the upper arm For the assessment of muscle strength two short
series of alternative concentric and eccentric efforts were performed in succession
involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist
in the removal of inflammatory markers and exudate consequently reducing local
tenderness [4] In addition the force assessment can be considered as a form of active
warming-up resulting in an increased muscle temperature which can reduce muscle
viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness
[3536] thus decreasing the sensitivity of the muscle and moderating pain during
movement In any case the beneficial influence of LED immediately after irradiation
can not be securely interpreted due to the sequential assessment of the outcome
measures
A second additional remark considers the fact that both arms of the irradiated subjects
demonstrated evidence of the beneficial effects of LED as a similar reduction of pain
and muscle sensitivity and higher peak torques were found in course of time at the
treated arm as well as at the control arm of the irradiated subjects This ascertainment
points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]
stated that in case LED works via systemic effects the use of the contralateral side as a
control arm might be ill-advised Thus reinforcing that future research should include a
control group to bring clarification [4 7 16]
Finally it needs to be mentioned that although the extent of DOMS was probably
relatively high for investigating the postulated hypothesis the time-course of the
present study corresponds to that reported by other investigators [2 3 5-10]
Significant time effects in many of the variables revealed that muscle damage was
evident diffuse muscle soreness became progressively worse 24-48 h after DOMS
induction followed by a small amelioration after 72 h [35910] After 72 h the follow-
Chapter 5
106
up was ceased consequently further regain of force and attenuation of pain and
muscle sensitivity could not be evaluated Extending the duration of the assessment
period could be useful in assessing any longer-term effects of LED treatment
particularly because as mentioned above differences between both groups are more
clearly present from day 3 pre-treatment and also because DOMS may last for up to 10
days when induced with the described protocol [715]
Lack of knowledge regarding both the precise mechanism of action of LED and the
specific pathophysiology of DOMS hampers the way to offer a definitive explanation
for the absence of more obvious statistically significant differences Still the small
number of significant findings and the mean values suggest that possible analgesic
effects of infrared LED may not be excluded yet but to be able to estimate the real
value of LED further research is necessary A large-scaled randomised clinical trial
which takes the above-mentioned remarks into consideration should be performed
CONCLUSION
Regardless of the reasons for the absence of statistical significant effects reported here
and although LED may have some potential in the management of pain and functional
impairment associated with DOMS its effectiveness at the applied densities has not
been established
Future research should focus on evaluation of the appropriateness of DOMS as an
experimental model of pain and muscle damage Validation of this model would
enhance the ability to study various modalities for their potential effects on pain and
muscle injuries Besides the mechanisms of LED action are not known thus further
fundamental investigations need to address the underlying mechanism and
physiological basis of pain modulation utilizing LED treatment
Once LED irradiation has finally proven its treatment value in an experimental model
the most important prospect considers establishing the effectiveness of LED to reduce
pain in clinical settings
Delayed-onset muscle soreness
107
ACKNOWLEDGMENTS
The authors would like to thank Mr T Barbe and Mr R Deridder for their technical
assistance in the collection of the data as well as for their valuable input into the
research design Sincere appreciation is extended to the volunteers that participated in
this study and to MDB-Laser (Belgium) for generously providing the light emitting
diode equipment The authors also gratefully recognize Prof Dr G Van Maele for
assistance with the statistical analysis and for helpful discussion
Chapter 5
108
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2 Cheung K Hume PA and Maxwell L (2003) Delayed Onset Muscle Soreness - Treatment Strategies and Performance Factors Sports Med 33(2)145-164
3 MacIntyre DL Reid WD and McKenzie DC (1995) Delayed muscle soreness The inflammatory response to muscle injury and its clinical implications Sports Med 20(1)24-40
4 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
5 Clarkson PM and Tremblay I (1988) Exercise-induced muscle damage repair and adaptation in humans J Appl Physiol 65(1)1-6
6 Cleak MJ and Eston RG (1992) Delayed onset muscle soreness mechanisms and management J Sports Sci 10(4)325-341
7 Craig JA Cunningham MB Walsh DM Baxter GD and Allen JM (1996) Lack of Effect of Transcutaneous Electrical Nerve Stimulation Upon Experimentally Induced Delayed Onset Muscle Soreness in Humans Pain 67(2-3)285-289
8 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
9 Ebbeling CB and Clarkson PM (1989) Exercise-Induced Muscle Damage and Adaptation Sports Med 7(4)207-234
10 Tiidus PM and Ianuzzo CD (1983) Effects of Intensity and Duration of Muscular Exercise on Delayed Soreness and Serum Enzyme-Activities Med Sci Sports Exerc 15(6)461-465
11 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
12 Armstrong RB (1984) Mechanisms of exercise-induced delayed onset muscular soreness a brief review Med Sci Sports Exerc 16(6)529-538
13 Rodenburg JB Steenbeek D Schiereck P and Bar PR (1994) Warm-up stretching and massage diminish harmful effects of eccentric exercise Int J Sports Med 15(7)414-419
14 Ciccone CD Leggin BG and Callamaro JJ (1991) Effects of ultrasound and trolamine salicylate phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-675 discussion 675-678
15 Craig J Barlas P Baxter D Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
16 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
17 Dvir Z (2003) Isokinetics muscle testing interpretation and clinical applications Churchill Livingstone Edinburgh
18 Lambert MI Marcus P Burgess T and Noakes TD (2002) Electro-Membrane Microcurrent Therapy Reduces Signs and Symptoms of Muscle Damage Med Sci Sports Exerc 34(4)602-607
19 Sumida K Greenberg M and Hill J (2003) Hot gel packs and reduction of delayed-onset muscle soreness 30 minutes after treatment J Sport Rehabil 12221-228
20 Tiidus PM and Shoemaker JK (1995) Effleurage massage muscle blood flow and long-term post-exercise strength recovery Int J Sports Med 16(7)478-483
21 Zhang J Clement D and Taunton J (2000) The efficacy of Farabloc an electromagnetic shield in attenuating delayed-onset muscle soreness Clin J Sport Med 10(1)15-21
22 Fitzgerald GK Rothstein JM Mayhew TP and Lamb RL (1991) Exercise-Induced Muscle Soreness After Concentric and Eccentric Isokinetic Contractions Phys Ther 71(7)505-513
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23 Nosaka K and Clarkson PM (1997) Influence of Previous Concentric Exercise on Eccentric Exercise-Induced Muscle Damage J Sports Sci 15(5)477-483
24 Jensen MP Karoly P and Braver S (1986) The Measurement of Clinical Pain Intensity - a Comparison of 6 Methods Pain 27(1)117-126
25 Price DD Mcgrath PA Rafii A and Buckingham B (1983) The Validation of Visual Analog Scales as Ratio Scale Measures for Chronic and Experimental Pain Pain 17(1)45-56
26 Revill SI Robinson JO Rosen M and Hogg MIJ (1976) Reliability of a Linear Analog for Evaluating Pain Anaesthesia 31(9)1191-1198
27 Nussbaum EL and Downes L (1998) Reliability of Clinical Pressure-Pain Algometric Measurements Obtained on Consecutive Days Phys Ther 78(2)160-169
28 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
29 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
30 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118
31 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in biomedical optics and imaging 3(28 Proceedings of SPIE 4903)156-165
32 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18(2)95-99
33 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2005) Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level J Photomed and Laser Surg 23(2)167-171
34 Pollo A Amanzio M Arslanian A Casadio C Maggi G and Benedetti F (2001) Response Expectancies in Placebo Analgesia and Their Clinical Relevance Pain 93(1)77-84
35 Shellock FG and Prentice WE (1985) Warming-up and Stretching for Improved Physical Performance and Prevention of Sports-Related Injuries Sports Med 2(4)267-278
36 Safran MR Seaber AV and Garrett WE (1989) Warm-up and Muscular Injury Prevention an Update Sports Med 8(4)239-249
37 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
38 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
GENERAL DISCUSSION
General discussion
113
SUMMARY
As outlined in the general introduction the overall objective of this doctoral thesis is to
develop the current knowledge about the mechanisms of LED action in view of the
eventual provision of evidence-based support for the clinical use of LED as a
biostimulatory and analgesic treatment modality especially in the field of
physiotherapy
Part I Wound healing
The investigations described in chapter 1 and 2 were conducted to gain insight into the
potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As
fibroblasts are principal cells for biostimulation (in view of growing and dividing in
healing wounds) the influence of LED irradiation on fibroblast proliferation was
assessed1
The first investigation consisted of a pilot study performed in order to evaluate the
appropriateness of the cell isolation technique cell culture protocol and proliferation
analysis as well as to appraise the feasibility of the light source properties and
illumination procedure
Data analysis revealed no statistically significant differences between the infrared LED
irradiated and control petri dishes for the used parameters (table 1) Considering this
outcome other experimental findings disclose that the absence of stimulatory effects of
LED irradiation on fibroblast proliferation can partly be attributed to the use of
inappropriate light source properties However the applied external dosimetric
parameters are well within the recommended spectrum described by previous studies
investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be
excluded that changes in the illumination procedure (such as the use of lower power
shorter exposure times wavelengths with finer coverage of the absorption spectrum of
the irradiated cells and a longer incubation period between the last irradiation and cell
counting) could still result in an increased fibroblast proliferation467 Of equal
importance in interpreting the lack of distinctive results are the imperfections of the
applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails
114
considerable intervention from the investigator compromising the reliability of the
method It is also a time-consuming technique with an insufficient sensitivity for some
purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11
To avoid contamination of the results by these modifiable remarks a similar
experiment (chapter 2) was performed in which wavelength power and output mode of
the infrared LED source were not modified (table 1) only the exposure time was
reduced resulting in a lower radiant exposure In addition the effect of two other
emission spectra was evaluated These probes emitting red and green light had a
shorter wavelength than the infrared LED source and the power was half or a
sixteenth of the power from the infrared probe Consequently the red LED irradiation
occurred with a different exposure time than the infrared one in order to attain the
same radiant exposure (053 Jcm2) With respect to the green LED it was not
endeavoured to achieve the same radiant exposure as 16 min of irradiation is not
feasible for in vitro or clinical application
Finally also an LLL light source was integrated Although it was not attempted to
analyse the effectiveness of LED in comparison to LLL enclosure of this modality was
interesting in order to join in with the available literature covering mostly LLL studies
To bypass the described problems regarding analysis of fibroblast proliferation
counting of the cells was carried out this time by means of a colorimetric MTT assay
This method provides more accurate cell counts in a short period of time and therefore
can be considered as a more reliable alternative to Buumlrker hemocytometer11
MTT assay 24 h after the last irradiation revealed a significantly increased number of
cells in the irradiated wells in comparison to their (respective sham-irradiated) controls
Although the study supplied experimental support for a significantly increased cell
proliferation by all external dosimetric properties based on the results of the
comparative trial with an incubation period of 24 hours irradiation with the green
LED source yielded the highest number of fibroblasts Thus it can be concluded that
the wavelength of the green LED is probably within the bandwidth of the absorption
spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that
General discussion
115
the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast
cultures in vitro612
Table 1 External dosimetric properties summarized for each chapter
Wavelength Power Exposure
time Output mode
Radiant exposure
PART I Chapter 1
In vitro part
LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2
LED-infrared 950 nm 160 mW 1 min continuous 053 Jcm2
LED-red 660 nm 80 mW 2 min continuous 053 Jcm2
LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2
Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2
PART II Chapter 4
LED-infrared 950 nm 160 mW 2 min continuous 107 Jcm2
Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
The next aim of the first part of this doctoral thesis was to explore whether LED
treatment could ameliorate in vitro cell proliferation under conditions of impaired
healing In the pursuit of this aim fibroblasts were cultured in medium supplemented
with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a
pilot study the amount of glucose necessary to inhibit normal growth was determined
In order to attain an important reduction of cell viability and decreased proliferation
rate a relatively high concentration of glucose (1667 mM) was necessary in
comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia
(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity
between both circumstances in vitro limited to 72 h whereas the human tissue of a
diabetic patient in vivo is chronically exposed to glucose
Treatment of the fibroblasts occurred in respect of the previously described results
with the same irradiation parameters and illumination procedure (chapter 2)
Accordingly green LED irradiation labelled as the most appropriate treatment for
116
irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in
the previous study (table 1)
Analysis of the cell proliferation by means of MTT measurements yielded a
significantly higher rate of proliferation in hyperglycaemic circumstances after
irradiation than in the control conditions (ie hyperglycaemic circumstances without
irradiation) Thus this outcome supported the stimulatory potential of green LED
irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a
considerable destructive hyperglycaemic medium
Finally although the results of the in vivo part of chapter 1 were persuasive and
encouraging they will not be further discussed in this summary of part I as it was not
aimed in this doctoral thesis to investigate the wound healing process in vivo However
the results of this case study can be a valuable hold for future in vivo research
The possible clinical implications of these results and future research directions in the
scope of wound healing will be discussed below
Part II Analgesia
In the second part two studies investigated the effects of LED irradiation as a
potential intervention mode in one of the most important fields in physiotherapy
practice analgesia Chapter 4 describes the influence of LED treatment on changing
sensory nerve conduction characteristics of a human superficial peripheral nerve
Altering nerve conduction characteristics may not be the sole beneficial purpose to
attain with LED irradiation in view of analgesia but the advantage of nerve conduction
characteristics is that they are objective measurable physical variables and changes in
these characteristics provide a potential explanatory mechanism of pain inhibition by
LED treatment17
The results showed that percutaneous LED irradiation at feasible clinical parameters
can generate a significant decrease in NCV and increase in NPL for all recordings post-
treatment in comparison to the baseline measurement The data in the placebo group
did not reveal any significant difference in the same course of time Statistical analysis
General discussion
117
revealed significant differences between the experimental and the placebo group for
NCV as well as for NPL at all time-points of observation with exception of the NPL
recording immediately after finishing irradiation
It was also observed that the noted effects did not weaken as time progressed It can
be concluded that post-treatment conduction measurements should be extended in
time which is in accordance with the findings of some previous studies18-21 Clarifying
the point of time at which the effect extinguishes is necessary and clinically relevant
when treating pain by means of LED irradiation Besides obtaining the desired
neurophysiological effects ideally the optimal irradiation parameters should be
applied The most favourable dosimetric properties are not yet determined but based
on this study and previously described assays it can be speculated that the dosimetric
window is quite large
Regardless of these clinically important remarks the present findings allow to draw the
following conclusion LED irradiation at clinically applied densities can generate an
immediate and localized effect upon conduction characteristics in underlying nerves as
LED treatment results in lowering the NCV and augmenting the NPL Therefore the
outcome of this in vivo experiment assumes a potential pain relief by means of LED
treatment and justifies further research regarding its clinical effectiveness in laboratory
settings and at a clinical level
The fourth and final aim was to determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5
illustrates a clinical study observing the effect of LED treatment on a model
comprising experimentally induced DOMS in a healthy population The progress of
pain perception and peak torque was evaluated during 4 consecutive days commencing
on the day of DOMS induction The effect of infrared LED treatment at the light
parameters described (table 1) was assessed with regard to three different factors time
(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures
(VAS MPT and IPT) revealed no significant interactive effects of the main interaction
118
(timegrouppre-post) For the remaining interactions and for the main effects only a
few significant findings were relevant in view of the postulated hypothesis
Notwithstanding the absence of an apparent and overall statistically significant finding
the present results indicate favourable trends of LED treatment on pain as the means
of all VAS and MPT variables show a statistically nonsignificant general analgesic
effect of infrared LED irradiation expressed by lower subjective pain rates and higher
MPT values in the irradiated group In addition to the analgesic influence of LED an
augmented restoration of muscle strength was noted The lack of solid statistically
significant evidence for these beneficial effects of LED treatment upon DOMS-
associated pain can possibly be attributed to the small sample size in this study or even
to the size of the treatment effect in relation to the severity of the induced DOMS as
induction of severe DOMS can mask relatively small but apparent treatment
effects2223 A final possibility is that the results only become significantly different after
a prolonged treatment and follow up period as previous research demonstrated that
recuperation subsequent to DOMS induction can last up to 10 days24
It should also be noted that the described general analgesic effect of LED irradiation
was identical for the treated as well as for the control arm in the irradiated group
proposing that infrared LED might induce systemic effects 2526 However it needs to
be stressed that these results were not statistically significant
Regardless of the absence of statistically significant findings the mean values suggest a
potential role for infrared LED irradiation in the management of pain and functional
impairment associated with DOMS Notwithstanding this postulation future research
is absolutely required to establish the effectiveness of LED treatment to reduce pain as
well at the applied densities as for other dosimetric parameters
CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS
In the course of the past years during the process of the genesis of this thesis
therapeutic physical agents in general and phototherapeutic modalities in particular
became less important as physiotherapeutic modes of treatment than during the
preceding two decades The diminished use of these treatment modalities in the
General discussion
119
physiotherapy practice is to a certain degree a consequence of the controversial
research findings regarding the use of these physical agents This issue of controversy
led to less support for the use of these treatment modalities and a growing scepticism
regarding the effectiveness of these physical agents within the scope of the growing
climate of evidence-based practice A second responsible protagonist for the loss of
popularity of physical agents is linked with the current tendency within physiotherapy
emphasising active remedial therapy The establishment of this development was based
on various experiments mainly performed during the last decade demonstrating that
active treatment modalities are for numerous impairments and disabilities preferable to
more passive forms of therapy In Belgium the prevailing nomenclature which came
into use on 1 May 2002 went along with this tendency In the appendix to the Royal
decree of 14 September 1984 towards settlement of the nomenclature of medicinal
treatments concerning compulsory insurance for medical care and allowances the
personal involvement of the physical therapist during the physiotherapeutic session
was emphasized and it was even defined that massage physical techniques within the
framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal
application can only be remunerated when they are applied supplementarily and not as a sole therapy
This implies that passive treatment modalities should not be used as sole method of
treatment and should always be considered as an adjunct to an active treatment
program This development needs to be applauded in many cases such as various
painful musculoskeletal problems functional instability rehabilitation of neurological
patients re-activation of the elderly population psychomotor rehabilitation
cardiovascular and respiratory convalescence Nevertheless it would be erroneous to
entirely reject physical agents including LED treatment Based on the findings of the
above described experiments it needs to be stressed that for some purposes especially
within the scope of impaired wound healing LED irradiation could be a suitable
therapeutic measure This statement is founded on the results of part I of the present
thesis they provided satisfactory fundamental evidence for the advantageous effects of
LED treatment on a crucial exponent of the wound healing process namely fibroblast
proliferation The beneficial findings are the result of basic in vitro research As it is
120
inaccurate to simply extrapolate these results to the clinical practice the clinical use of
LED irradiation for wound healing needs to be preceded by purposive and specific in
vivo investigations to substantiate these basic research findings27
The case study described in chapter 1 indicates a foundation for further in vivo research
Visual appraisal of the surgical incision revealed (from the 65th day in the course of the
reparative process onwards) that the irradiated area -which initially showed inferior
epithelialization and wound contraction- showed a more appropriate contracture than
the control area characterized by less discoloration at scar level and a less hypertrophic
scar These clear beneficial effects of LED treatment on a human cutaneous wound
can serve as preliminary impetus for further research into the clinical applicability of
LED therapy although this case study is insufficient in order to guarantee a safe
correct and effective use of LED as a therapeutic modality
Despite these remarks it tentatively can be concluded that based on a detailed analysis
of the available data of the present in vitro studies and the given case report in
combination with the small number of previously published human studies the
beneficial effects of LED irradiation at the cellular level are obvious and therefore a
potentially favourable outcome can be assumed in clinical practice28-30 LED-
modulated stimulation of wound healing can be gradually and vigilantly implemented
clinically Nevertheless the real benefits of LED irradiation within the scope of wound
healing can only be established by additional clinical trials as thus far clinical
application and stipulation of dosimetry still occurs on a trial-and-error basis which is
not conducive to a generally accepted clinical use of LED To lend more credibility to
the treatment of wounds by means of LED irradiation and to expel the existing
controversy and scepticism surrounding this topic in vivo investigations on wound
healing using a number of different animal models and adequately controlled human
studies are necessary In addition these studies should be performed preferably on a
population suffering from impaired healing as a consequence of diabetes mellitus or as
a result of any other debilitating reason because as posed by Reddy et al3132 and as
mentioned above light has possible optimal clinical effects in the treatment of healing-
resistant wounds
General discussion
121
Drawing general conclusions and formulating clinical implications for analgesia is
obviously less manifest first of all because only a limited number of possible
mechanisms of action in order to obtain analgesia were highlighted and secondly
because both studies did not come to a joint or complementary conclusion The
outcome of the first study revealed that LED treatment lowers the NCV and augments
the NPL resulting in a slower stimulus conduction and consequently a reduced number
of sensory pulses per unit of time Thus it could be assumed that LED induces pain
relief but the results of the study describing the effect of LED treatment on
experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy
In addition it needs to be emphasised that the first study (chapter 4) measured the effect
of LED irradiation on the large myelinated Aβ afferents A noteworthy question and
meanwhile a stimulus for future investigation is whether the measured effects can be
extrapolated from these sensory nerve fibres to the actual nociceptive afferents
notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of
these nociceptive pathways relies on laser-evoked potentials which selectively activate
Aδ-fibres and C-fibres3334 This technique was presently not available therefore a
standard sensory nerve conduction study was performed
Whereas stimulation of wound healing by means of LED irradiation can be cautiously
implemented in the clinical practice at this stage it is too early to promote LED
irradiation as a treatment modality for pain To make this possible it is essential to
conduct numerous studies with regards to the use of LED in the field of analgesia
Future research should focus on fundamental investigations in order to discover the
underlying mechanisms and physiological basis of pain modulation utilizing LED
treatment Furthermore the evaluation of the appropriateness of DOMS as an
experimental model of pain is an important prospect to consider as validation of this
model would enhance the ability to study various modalities for their potential effects
on pain Irrespective of the difficulties regarding standardisation of the research
population and evaluation of soreness inextricably linked with clinical pain studies the
122
ultimate objective of future research should be the establishment of the effectiveness
of LED irradiation to reduce pain of miscellaneous origin in a clinical setting
Regardless of the encouraging results of the described studies and besides the earlier
proposed specific directions for future research (directed towards wound healing or
pain relief) it is necessary in the interest of the patientrsquos well being and to the
advantage of the prospective clinical use of LED to highlight a few more issues for
future research Therefore one has to deal with some limitations of the performed
investigations A first limitation concerns the fact that only two mechanisms of LED
action were investigated (notably changed fibroblast proliferation and alteration of the
nerve conduction characteristics) So one can conclude that for further and better
understanding of the mechanisms of action it is necessary to perform more basic
research Answering the questions regarding the functioning of LED irradiation will
simplify the evaluation and reinforce the interpretation of the obtained results and
ultimately contribute to a more widespread and well definded acceptance of the use of
LED in clinical settings
A second general limitation of this doctoral thesis is the substantial difference in the
used external dosimetric parameters between the different chapters and even within
one and the same study (illustrated in table 1) this complicates the comparison
between the different trials In each trial the dosimetry was individually ascertained
based on previous studies within the given field As not for every application the same
dosimetry is suggested in literature a range of dosages were used Another important
factor in deciding on the dosimetry was the clinical applicability of the dosage as it is
useless to investigate the appropriateness of a treatment modality at a clinically
unrealistic dose As a result of this limitation the current findings do not fully
contribute to the explanation regarding the ideal parameters one should use although
this was not set as a principal purpose Based on this thesis and previously described
assays it can be speculated that the possible window for these parameters is quite large
the ideal irradiation parameters and proper timing or sequencing of LED irradiation
General discussion
123
for example to the various phases of wound healing and to different painful conditions
are therefore possibly unattainable
The establishment of an appropriate dosimetry should also consist of investigating the
absolute and relative penetration depth of LED irradiation into human tissue This is
less crucial within the scope of wound healing but it is of key importance while
treating deep-seated tissue (eg nerve fibres muscles circulatory components et
cetera)
Finally this thesis only investigated the efficiency of LED in a very limited number of
conditions notebly wound healing and pain Within the scope of physiotherapy and
medicine in general there are numerous other purposes for which LED irradiation is
promoted such as oedema arthritis miscellaneous orthodontic applications seasonal
affective disorder neonatal jaundice photodynamical therapy et cetera2835-41
In summary additional work on establishing proper dosimetry and identifying the
biochemical or photobiologic phenomena that are responsible for improving wound
healing and reducing pain or even other effects within a broader spectrum of
conditions remains to be done in order to answer unreciprocated questions Until that
time the potential clinical usefulness and actual value of LED irradiation for wound
healing and even to a larger extent for analgesia should always be approached with
appropriate professionalism and even caution
FINAL CONCLUSION
LED devices are promoted for clinical use but the currently available scientific
documentation regarding effectiveness of this physical agent is rather scarce Through
providing scientific support for the biostimulatory and analgesic effectiveness of LED
irradiation this doctoral thesis attempted to bridge in some degree this gap
The conducted studies revealed that LED irradiation undeniably has potential
beneficial effects on wound healing and to a lesser degree within the scope of
analgesia However based on the present results it can be corroborated that light
124
therapy in the guise of LED irradiation is not magic but these results can raise some
corrective doubts in fundamental disbelievers and antagonists
Nevertheless we have to join the queue of scientists who have found beneficial results
but cannot elucidate with certainty how this outcome was established Thus although
the present results are encouraging a continuing development and integration of new
knowledge based on further research is necessary in various domains of intervention
Therefore several directions for future investigations were proposed in order to cover
as many existing gaps and to answer the utmost number of remaining questions as
possible Still one ought to be aware not to carry future fundamental research at a
disproportional level and the inevitable quest for mechanisms of LED action should
not hypothecate the potential clinical value implying that at a certain point it should be
appropriate to make the transfer from science to the application of the available
knowledge in clinical practice
The described findings regarding LED irradiation are comparable to the results of
previously published studies performed with other light sources Consequently as
postulated by some LED providers it can be speculated that the biological response of
tissue to light irradiation can probably not be equated merely to a light source but
rather to a broad photo-energy window
General discussion
125
REFERENCES
1 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
2 Abergel R Lyons R Castel J Dwyer R and Uitto J (1987) Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2)127-133
3 Skinner S Gage J Wilce P and Shaw R (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-192
4 Webb C Dyson M and Lewis W (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5)294-301
5 Atabey A Karademir S Atabey N and Barutcu A (1995) The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 1899-102
6 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
7 Ben-Dov N Shefer G Irinitchev A Wernig A Oron U and Halevy O (1999) Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3)372-380
8 Rebulla P Porretti L Bertolini F et al (1993) White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2)128-133
9 Deneys V Mazzon A Robert A Duvillier H and De Bruyere M (1994) Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2)172-177
10 Mahmoud A Depoorter B Piens N and Comhaire F (1997) The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2)340-345
11 Haskard D and Revell P (1984) Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3)319-322
12 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers
13 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of cultured human endothelial cells Diabetes 36(11)1261-1267
14 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA damage in cultured human endothelial cells J Clin Invest 77(1)322-325
15 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7)621-627
16 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4)491-501
17 Kramer J and Sandrin M (1993) Effect of low-power laser and white light on sensory conduction rate of the superficial radial nerve Physiotherapy Canada 45(3)165-170
18 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
19 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
126
21 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
22 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
23 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
24 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
25 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
26 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer
G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
29 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
30 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255
32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287
33 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-28
34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856
35 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
36 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
37 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263
39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64
40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74
General discussion
127
41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press
NEDERLANDSTALIGE SAMENVATTING
Nederlandstalige samenvatting
131
NEDERLANDSTALIGE SAMENVATTING
Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de
tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te
vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie
van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen
Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest
enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is
ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als
op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang
van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve
interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken
Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van
ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen
moeilijk te verantwoorden
Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin
de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere
overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit
methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan
consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in
de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen
laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in
de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag
aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies
inclusief het gebruik van licht
Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat
de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde
doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en
dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht
komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt
geloosd
132
De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo
lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en
gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te
anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te
voorkomen lijkt een gerichte en rationele a priori aanpak conform de
wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen
Te meer daar grondige literatuurstudie leert dat men ten behoeve van de
werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie
zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser
De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de
extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is
geboden De introductie van alternatieve lichtbronnen in de huidige
kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk
weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus
eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood
naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based
gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder
binnen de domeinen van haar potentieel beloftevolle klinische toepassingen
wondheling en analgesie
Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de
wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op
prominente protagonisten van de wondheling de fibroblasten werd getracht het
fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten
zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de
proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd
respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand
waarbij de normale celgroei werd verstoord
In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het
effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische
Nederlandstalige samenvatting
133
data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden
verklaard door het gebruik van een inadequate LED dosering en anderzijds een
methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie
vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de
precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en
inter-tester variabiliteit tot gevolg
In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde
onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de
bestralingsparameters (dosering) De effecten van de drie verschillende LED
golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten
werden hierbij geanalyseerd door middel van een meer betrouwbare en minder
subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-
yl)-25-diphenyl tetrazolium bromide (MTT)
De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir
effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden
tevens een basis voor meer coherente en relevante inzichten aangaande de globale
bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)
Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het
wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend
karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp
rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde
vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De
fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met
extreem hoge concentraties glucose Deze modificatie van het medium staat model
voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische
praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan
vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de
gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en
proliferatie
134
Het tweede deel van dit proefschrift exploreert het domein van het potentieel
analgetisch effect van LED binnen de kinesitherapie aan de hand van twee
fundamentele onderzoeken
In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere
sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele
hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en
een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale
verklaring van een analgetisch effect van het medium zou kunnen belichamen
Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)
meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf
verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen
aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke
significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de
negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele
hypothese
Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend
fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale
pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp
van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische
krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED
behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier
keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED
op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd
(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde
isokinetische krachtmeting en een registratie van de waargenomen spierpijn De
spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met
behulp van een kwantitatieve hand-hold algometer
Analyse van de bekomen data bracht geen significante verschillen tussen de
controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden
Nederlandstalige samenvatting
135
kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED
behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van
de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de
proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene
afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine
proefgroep die werd onderzocht enof door de grootte van het behandeleffect in
verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers
een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up
kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel
absolute omzichtigheid geboden en moet deze visie louter als speculatief worden
beschouwd
Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de
kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de
laatste jaren aanzienlijk afgenomen
De positieve resultaten van de verschillende in vitro studies in het kader van wondheling
vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing
vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel
gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een
belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek
Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie
Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme
onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch
complementair resultaat Verder onderzoek ter exploratie van de mogelijke
onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom
onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op
termijn wetenschappelijk te rechtvaardigen
136
LED tovenarij trend of therapie
LED mag geen magische krachten worden toegemeten maar verdient het lot van een
kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een
wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen
zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke
indicaties
Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds
the darkness has always got there first and is waiting for it
(Terry Pratchett Reaper Man 1991)
Do not attempt to do a thing unless you are sure of yourself
but do not relinquish it simply because someone else is not sure of you
(Stewart E White)
IX
ACKNOWLEDGEMENTS
I wish to thank those people who supported me over the years and who helped me to
shape my life and work
First of all I would like to express my gratitude towards my promotor prof dr D
Cambier as without his encouraging words criticism inspiration and unremitting
support I would still be floundering about the contents of chapter 1 Thank you for
your good advice when I needed it the most
The members of the supervisory committee prof dr M Cornelissen prof dr M De
Muynck and prof dr G Vanderstraeten thank you for your assistance and helpful
feedback during the process of formation of this thesis
I also gratefully acknowledge the external members of the examination board prof dr
P Calders prof dr M Dyson prof dr P Lievens and prof dr K Peers for their
constructive reflections which contributed to the improvement of this thesis
I am greatly indebted to my special mentor prof dr J Anders of the Uniformed
Services University of Bethesda Maryland for the research suggestions she made as
well as for her unlimited belief in the value of my work
I wish to thank prof dr L Deridder for providing access to the laboratory of
Histology the Centre of Sports Medicine of the Ghent University Hospital for
allowing me to use their equipment as well as MDB-Laser Belgium for generously
providing the light emitting diode equipment
Sincere appreciation is extended to the volunteers that participated in this study and to
Tom and Roel for their valuable technical assistance in the collection of the data as
well as for their useful input into the research design of the investigation described in
chapter 5
X
Warm thanks go to the colleagues of the department of Human Anatomy
Embryology Histology and Medical Physics for providing the culture medium for the
technical support for the helpful discussions and principally for the amusing pastime
aseptic chats
In addition I also want to thank my colleagues of the associated institute Kinesitherapie
Gent and above all the colleagues of the department of Rehabilitation Sciences and
Physiotherapy Ann Axel Barbara Bart Bert Bihiyga Bruno Carine Damien Els2
Erik Frike Helga Inge James Karlien Kathy Kat(h)leen Koen Marc Martine
Mieke Paris (Nele) Patrick Roland Sophie Stefan Stijn (Veerle) Wiene Wim and
Youri thank you for the organisation and your attendance at many memorable
sidelines such as the survival-weekend the first department-day Fata Revaki our
legendary Thursday-sport activities and Friday-afternoon coffee breaks St Nicholas
visit skiing holiday New Yearrsquos lunch with quiz bowling spinning the introduction of
ldquosubetelyrdquo during our conspicuous presence at WCPT 2003 et cetera We shared many
treasured moments thanks to you a common working day often turned out to be very
pleasant I know that it will be impossible to find a comparable team of colleagues to
work with in the future
I especially want to thank Barbara to remind me on a regularly basis of my deadlines
to listen to my grieves and joy and to be willing to offer me a window-seat in our
office Kurt (although you abandoned at a certain moment) for solving my computer
problems Pascal for assistance with the statistical analyses Lieven for your motivating
interest and finally Fabienne Tine and Kim as loyal and appreciated friends who
worn-out several sports shoesbathing suits to supply in the weekly portion of sports I
needed to remain physically and mentally fit
I also extend my appreciation to my family and friends for their interest in my research
activities permanent mental support for the adoption of the surviving chickens but in
particular for looking after Louka and for the numerous relaxing moments Special
thanks are going to Katelijne and Frank for the delicious hot meals on lonely evenings
XI
Thomas Stijn and Johan thank you for the repeated (mostly futile) attempts to
convince me to do something together Sebastiaan each time during the past few years
when I doubted about the sense of my work it was your ridiculous story about a man
who wanted to invent superglue but instead invented the well-known yellow post-it
which stimulated me to continue my scientific quest
Of course I owe most gratitude to Luc my most devoted supporter Dearest I know
that since august 2004 you lived a solitary life in Dubai Although I think it was
possibly easier not to live under the same roof with me these last stressful months I
am aware that it was very difficult for you not to be able to play with Louka and to
miss some precious months of her life
Louka thank you for your radiant smile and daily baby speeches I am sorry that you
had to miss your daddy I promise that we will be reunited very soon
Elke Vinck
Ghent March 2006
GENERAL INTRODUCTION
General introduction
3
BACKGROUND
The use of light for therapeutic purposes reaches far back in time Current interest for
photomedicine with his its biological and medical effects relies fundamentally on two
major evolutions in the given field (1) the research results regarding the use of
ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in
the light amplification by stimulated emission of radiation (laser)-technology The production
of the first laser the ruby pulsed laser was rapidly succeeded by the development of
the helium-neon laser and other lasers like the argon the neodymium-glass and the
neodymium-yttrium-aluminium-garnet lasers1
As in the mid-1990s semiconductor and diode-based lasers gained popularity the
principally massive gas and dye lasers were rendered obsolete Therapeutic light
technology further continued to evolve and todayrsquos therapeutic light source is as likely
to be a light emitting diode (LED) or polarized light as a semiconductor or diode
laser1
Technological advancement and variation of the light sources necessitate a
concomitant update and revision of research in the respective domains of application
Unfortunately this logical and rational necessity has rarely been fulfilled From a
historical perspective this lack of appropriate research has led to disenchanting
evolutions in the use of light especially in physiotherapy The experience exists in this
medical field that light sources were promoted and commercialised for a vast regimen
of indications without foregoing scientific backup Consequently research developed
often after the commercial introduction in physiotherapy As these investigations
frequently gave rise to conflicting results for certain indications scepticism arose and
the use of the given modality knew a waning popularity for all its indications The final
result of such an inappropriate frame of promotion commercialisation and research is
a growing clinical disuse of a given modality even for motivated indications In view of
the actual increasing interest in LED treatment and based on former ascertainment
one has to state that a literature review for the given source reveals that research
mostly covers only low level laser (LLL) studies23 Although recently a number of
papers can be noted that report on the effects of LEDs and polarized light still
4
numerous source-specific-questions need to be answered as research concerning
mechanisms of action and efficacy of the current light sources remains limited in view
of a substantiated clinical application4-17
The reason for the contemporary light-oriented interest in physiotherapeutic practice
for LED devices is in essence based on several advantages of LED in comparison with
LLL For example the use of LEDs is esteemed to be safer as the delivered power
does not damage tissue LEDs can be made to produce multiple wavelengths thereby
stimulating outright a broader range of tissue types and probes that cover a large
treatment area are available18 In addition from a commercial point of view LEDs are
far more interesting as they are a good deal cheaper than laser diodes and they have a
long life span as these solid devices stand robust handling
As a result of the above-mentioned lack of literature on LED some providers of these
devices have taken for granted that the biological response of tissue to light irradiation
cannot be equated merely to a light source They declare that a given response solely
depends on the extent of absorption of radiated light by the tissue19 Consequently
these providers state that it is acceptable to extrapolate scientific findings of LLL
studies for explaining the mechanisms of action and detailing the efficacy of LED and
other alternative light sources Thus actually without appropriate scientific support
equal biological effects are attributed to LED as to LLL Nevertheless prudence is
called for such an extrapolation firstly because it is irrespective of the mentioned
dissimilarities and by simple projection one ignores a number of physical differences
between LLL and LED (eg coherence and degree of collimation or divergence)
Secondly LLL therapy is still not yet an established and evidence-based clinical tool20
Notwithstanding the historical efforts there still remains a considerable amount of
ignorance scepticism and controversy concerning the use and clinical efficacy of
LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed
parameters for irradiation as well as to the difficult objective measurement of possible
irradiation effects and even to the exceptional range of unsubstantiated indications for
General introduction
5
which light therapy was promoted27-29 A lack of theoretical understanding can also be
responsible for the existing controversies as the evaluation and interpretation of
research results would be simplified largely when the appropriate knowledge about the
mechanisms of light action would be available
LLL literature can undoubtedly be used as basis for research on LED and as a
comparative reference for these given investigations However to guarantee evidence-
based use of LED within physiotherapy the need for specific research in view of an
accurate consumption of LED is definite especially for potential promising clinical
applications in physiotherapy according to LLL literature mainly wound healing and
analgesia3031
Hitherto the most substantial research concerning the use of LED for improvement
of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded
under the influence of prolonged exposure to microgravity (eg during long-term space
flights) and in case of absence of exposure to sunlight such as in submarine
atmospheres they performed wound healing experiments for military application in the
given circumstances3233 In vitro experiments revealed that LED treatment increased
proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA
synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and
osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies
demonstrated therapeutic benefits of LED in speeding the early phase of wound
closure and in changing gene expression in a type 2 diabetic mouse model183234
Human studies noted 50 faster healing of lacerations a return of sensation and
increased tissue granulation as a result of LED irradiation1833
Associates of the Rehabilitation Sciences Research Group of the Ulster University in
Northern Ireland extensively investigated the effectiveness of light in the treatment of
pain The emphasis was laid primarily on the analysis of the effects of various low level
laser light sources35-44 However in the year 2001 two studies gave an account on the
efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46
6
noticed relatively long-lasting neurophysiological effects a significant change of the
nerve conduction characteristics (decrease of the negative peak latency) was mediated
by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a
comparable multisource diode device was ineffective in the management of delayed-
onset of muscle soreness (DOMS)
Despite the major value of these described trials a definitive answer regarding the
ability of LED in influencing wound healing or pain is not forthcoming cardinally
because a number of aspects are not yet investigated Consequently more research is
required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the
formerly mentioned scepticism regarding the effectiveness of a medium and possibly
to the undeserved fall into disuse of the treatment modality which happened in a way
with LLL therapy
PHYSICAL CHARACTERISTICS
This chapter supplies a short but comprehensive review of opto-physics A brief
description of the physical characteristics of the LED source used is essential as the
physical properties of light play an important part in the ultimate efficacy of treatment
According to the International Electrotechnical Commission (IEC 60825-1) an LED
can be defined as
Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by
radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process
of spontaneous emission1947
The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO
preprototype MDB-Laser Belgium) This illustration shows that a probe consists of
32 single LEDs disseminated over a surface of 18 cm2
General introduction
7
Figure 1 LED device and three available probes (infrared red and green)
Three highly monochromatic probes were available each emitting light of a different
wavelength within the above-defined range (table 1)2748 The wavelength of the light
emitted and thus its colour depends on the band gap energy of the materials forming
the p-n junctiona This light property is a key determinant to obtain maximum
photochemical or biological responses as light absorption by tissue molecules is
wavelength specific27 Only by absorbing radiation of the appropriate wavelength
(namely the wavelengths equal to the energy states of the valence electrons)
photoacceptor molecules will be stimulated resulting in a direct photochemical
reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750
8
absorption spectrum of a particular molecule is limited absorption often only occurs
over a waveband range of about 40-60 nm274851 Nevertheless the absorption
spectrum at cell or tissue level is broad because cells are composed of many different
molecules
Besides its influence on the absorption by means of tissue molecules there is a crucial
link between wavelength and penetration depth of the irradiated light Penetration into
tissue decreases as the wavelength shortens hence green light penetrates less than red
light which at his turn penetrates less into tissue than infrared light2748 Detailed
principles of light penetration will be discussed below
The LED device used emits non-coherent light In the 1980s the observed biological
responses after laser irradiation were generally thought to be attributable to the
coherenceb of the light485253 Though currently the clinical and biological significance
of coherence is seriously questioned54 According to several authors coherence does
not play an essential role in laser-tissue interactions firstly as it was proven that both
coherent and non-coherent light clinically show equal efficacy75556 Secondly as
according to some authors almost immediately after transmission of light through the
skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et
al1957 state that both findings are incorrect coherence is not lost in tissue due to the
phenomenon of scattering and non-coherent light is not as efficient as coherent light
This lack of consensus makes it necessary to mention whether or not light is
coherent2758
Further decisive characteristics to accomplish phototherapeutic efficacy are the power
exposure time output mode and beam area Based on these parameters both
irradiancec and radiant exposured can be calculated According to numerous authors
some of these parameters are more crucial than others to determine whether
b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48
c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48
d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248
General introduction
9
absorption of light will lead to a photobiological event192728485455 However the
literature yields several controversial findings as not all authors attribute an equal
importance to a given parameter For example according to Nussbaum et al59
irradiance was the determinant characteristic in the biomodulation of Pseudomonas
aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49
found that in order to stimulate tissue cell proliferation a specific combination of
irradiance and exposure time are more important than the actual radiant exposure Low
et al3940 on the contrary highlighted the critical importance of the radiant exposure in
observing neurophysiological effects Whereas Mendez et al60 reported that both
parameters influence the final results of light therapy
Koutna et al61 even suggested that the output mode of light applications plays a more
prominent role in the treatment outcome than the wavelength of the used light source
Nevertheless this finding could not be confirmed by other research results Besides
more controversial findings have been published regarding the output mode although
the repetition rate in a pulsed mode was considered as an important treatment
parameter several investigations failed to prove its value19272840414461-64
Based on these findings it was opted within the investigations of this doctoral thesis to
irradiate in a continuous mode The remaining dosimetric parameters (wavelength
exposure time and power) depended on the purpose of each investigation they are
described in the respective chapters The data necessary for the calculation of the
radiant exposure for the equipment used in the respective trials are summarized in
table 1
Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High
Infrared 950 80 120 160 Red 660 15 46 80
Green 570 02 42 10
10
The radiant exposure of the used LED can be calculated as follows65
RE =
Radiant Exposure [Jcm2]
T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]
PRE = α S T
α = 006 (continuous mode) or
003 (pulsed mode)
The parameters commented on so far can be considered as the external dosimetry
involving all parameters directly controlled by the operator limited by the apparatus
used Furthermore there is the so-called internal dosimetry referring to (1) several
physical phenomena (reflection transmission scattering and absorption) influencing
the light distribution within the tissue during energy transfer (2) the optical
characteristics of the irradiated tissue as well as (3) the relation between the external
dosimetry and these respective elements5466
This internal dosimetry determines to a considerable extend the penetration of light
into tissue Penetration can be defined as the tissue depth at which the radiant
exposure is reduced to 37 of its original value1948 However this definition only
accounts for the absolute penetration depth resulting in direct effects of light at that
depth In addition there is also a relative penetration depth leading up to effects
deeper in the irradiated tissue and even in certain degree throughout the entire
body1967 These so-called systemic effects can be caused by chemical processes initiated
at superficial levels at their turn mediating effects at a deeper tissue level57
Involvement of several forms of communication in the tissue such as blood circulation
and transport of transmitters or signal substances is possible1967 This means that light
sources with poor absolute penetration do not necessarily give inferior results than
those with a good absolute penetration19
In the same context it should be noted that calculation and even measurement of the
exact light distribution during irradiation is highly complicated principally as tissues
have complex structures and also because the optical properties of tissues vary largely
inter-individual2768
General introduction
11
Studies regarding actual penetration depth of LED light are scarce consequently the
knowledge on the topic of penetration depth of LED light is based on literature
originating from LLL research19 These findings established with various LLL sources
revealed that there is an obvious relation between penetration depth and
wavelength27486769-71
Three final remarks can be made on the dosimetry First of all it should be noted that
partly as a result of the above-mentioned contrasting findings on dosimetry ideal light
source characteristics for effective treatment of various medical applications are not yet
established and probably never really will be28 Therefore in the attempt to offer
sufficient guidelines for correct use of treatment parameters one should always try to
provide detailed description of light source properties used in any trial so the
practitioner can interpret the scientific results adequately and accordingly draw the
correct conclusions for his clinical practice
A second comment is based on the mentioned possible influence of the external and
internal dosimetric parameters on the photobiological effectiveness of light the
intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation
between radiated wavelength and penetration depth19546572 So it should be
emphasized that caution is recommended when comparing research results of light
sources with different wavelengths or other dissimilar dosimetric parameters
A third and final remark considers the extrapolation issue Comparison of the
therapeutic usefulness of the same light source used on different species should occur
cautiously So simply extrapolating the dosage used for one species to another is
inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo
clinical practice is inappropriate So purposive and specific research is the prerequisite
to produce safe and correct use of light as a therapeutic modality27
12
MECHANISMS OF ACTION
In the past decennia several mechanisms of action for biostimulation and pain
inhibition have been proposed and investigated73 Research was primarily based on
studies at the molecular and cellular levels and as a second resort investigations
occurred at the organism level resulting in numerous possible explanatory
mechanisms272858
It is the common view that light triggers a cascade of cellular and molecular reactions
resulting in various biological responses Thus different mechanisms of whom the
causal relationships are very difficult to establish- underlie the effects of light3448557475
To illustrate this complex matter the various mechanisms of action will be summarised
by means of a comprehensive model (fig 2) Detailed discussion about the different
individual components of the proposed model and other effects than those regarding
wound healing or analgesia were not provided as this was beyond the scope of this
general introduction
As depicted in figure 2 exposure to light leads to photon absorption by a
photoacceptor molecule causing excitation of the electronic state or increased
vibrational state of the given molecule275173 This process is followed by primary
photochemical reactions7475 Several key mechanisms have been discussed in the
literature Respiratory chain activation is the central point and can occur by an
alteration in redox properties acceleration of electron transfer generation of reactive
oxygen species (namely singlet oxygen formation and superoxide generation) as well as
by induction of local transient heating of absorbing chromophores192848515576-83 It is
supposed that each of these respective mechanisms plays a part in obtaining a
measurable biological effect It is yet not clear if one mechanism is more prominent
and decisive than another nevertheless recent experimental evidence has revealed that
mechanisms based on changes in redox properties of terminal enzymes of respiratory
chains might be of crucial importance2848517679
The primary mechanisms occurring during light exposure are followed by the dark
reactions (secondary mechanisms) occurring when the effective radiation is switched
General introduction
13
off2851 Activation of respiratory chain components is followed by the initiation of a
complicated cellular signalling cascade or a photosignal transduction and amplification
chain associated with eg changes in the cellular homeostasis alterations in ATP or
cAMP levels modulation of DNA and RNA synthesis membrane permeability
alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87
The sequence of events finally results in a range of physiological effects essential for
the promotion of the wound healing process for supplying analgesia or other
advantageous responses (acceleration of inflammatory processes oedema re-
absorption increased lymph vessel regeneration or increased nerve
regeneration)12181927486188-93
Photostimulation of the wound healing process can be mediated by increased
fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis
and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations
have been especially focussed on fibroblasts but other possible physiological effects
attributing to an accelerated wound healing were also observed suppression and
alteration of undesirable immune processes increased leukocyte activity new
formation of capillaries increased production of growth factors and enzymes while
monocytes and macrophages can provide an enlarged release of a variety of substances
related to immunity and wound healing1619277376
As pain and nociception are even less understood than wound healing the possible
mechanisms in obtaining pain relief by the use of light are less underpinned However
it is established that light therapy influences the synthesis release and metabolism of
numerous transmitter signal substances involved in analgesia such as endorphin nitric
oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these
neuropharmacological effects there is experimental evidence for diminished
inflammation decreased C-fibre activity increased blood circulation and reduced
excitability of the nervous system1927848899
One should be aware that a large amount of research regarding the possible
mechanisms of light action was conducted at the cellular level The described cascade
of reactions at the organism level is possibly even more complex as in contradiction to
14
the in vitro situation in vivo a range of supplementary interactions can influence the
sequence of effects and accordingly the final responses Besides it needs to be
mentioned that this summary did not take into account the origin of the light or the
external dosimetry thus the description is based on investigations performed with
various light sources and different dosages
Figure 2 Model summarizing the identified mechanisms of light action
Secondarymechanisms
Primary mechanisms
Final effects
Trigger
Stimulated wound healing Analgesia
Exposure to light
Photon absorption by photoacceptors
Respiratory chain activation
Accelerated electrontransfer
Reactive oxygen generation
Heating of absorbing chromophores
Altered redox properties
darr inflammation uarr oedema resorption
uarr lymph vessel regenerationuarr blood circulation
Photosignal transduction and amplification chain
uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation
darr C-fibre activity darr nervous excitability neuropharmacological effects
General introduction
15
Regardless of the large number of previous investigations identification of underlying
mechanisms of light action remains an important issue as these are not yet fully
understood and because probably not all mechanisms of action are currently
identified Convincing explanation of the mechanisms in normal as well as in
pathological tissue could banish the existing suspicion concerning the use of light as a
treatment modality2732547678
AIMS AND OUTLINE
The introduction of LED in medicine and in physiotherapy more specifically requires
particular scientific research especially within the fields of its clinical potential
application wound healing and analgesia The above described gaps in literature
regarding the use of LED laid the foundation of this doctoral thesis
Consequently the general purpose of this thesis is to explore a scientific approach for
the supposed biostimulatory and analgesic effect of LED and to formulate an answer
in view of an evidence-based clinical use of this treatment modality
The detailed objectives can be phrased as follows
Aim 1 To assess the biostimulatory effectiveness of LED
irradiation under normal in vitro conditions
Aim 2 To investigate the value of LED treatment to ameliorate
in vitro cell proliferation under conditions of impaired healing
Aim 3 To examine the effectiveness of LED in changing the
nerve conduction characteristics in view of analgesia
Aim 4 To determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting
Part I investigates the influence of LED on wound healing In pursuit of the first aim
chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial
16
measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these
cells needs to be considered as an exponent of the wound healing process as
fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase
and early remodelling100 Secondly an in vivo case study exploring the postulation that
LED irradiation could accelerate and ameliorate the healing of a surgical incision was
described
The results contrasted sharply with the findings of the in vitro part Two fundamental
causes were proposed in order to explain the different biological effect of LED
irradiation observed in vitro and in vivo the used irradiation parameters and evaluation
method
The experiment described in chapter 2 endeavoured to explore these considerations A
similar study was therefore performed but as distinctive characteristics different light
source properties an adapted irradiation procedure and the use of a colorimetric assay
based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the
counting of the cells were used
As stimulation of the wound healing process is virtually mainly indicated under
conditions of impaired healing (resulting in a situation which threatens to become
chronic and debilitating) proper attention for this matter is warranted192855 Besides
the medical consequences the costs involved with impaired healing yield also a socially
relevant problem to tackle Impaired healing will become even more common as the
world population continues to age After all senescence of systems and age-committed
comorbid conditions are commonly the culprits responsible for poor wound healing101
Thus finding cost-effective time-sparing non-invasive and practical treatment
modalities to cure wounds is a necessity
Aiming to assess the biostimulative effects by means of LED in these circumstances a
third study was conducted with respect of the previous results regarding irradiation
parameters and cell proliferation analysis The irradiation experiment described in
chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose
This medium modification serves as a pattern for cell proliferation in diabetic patients
General introduction
17
a population for whom stimulation of the wound healing process is a clinical relevant
feature
In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored
by means of two studies A first investigation (chapter 4) evaluated the influence of LED
on the sensory nerve conduction characteristics of a human superficial peripheral
nerve as a potential explanatory mechanism of pain inhibition by LED which is based
on the putative neurophysiological effects of this treatment modality The experimental
hypothesis postulated that LED generates an immediate decrease in conduction
velocity and increase in negative peak latency In addition it was postulated that this
effect is most prominent immediately after the irradiation and will weaken as time
progresses
The values of nerve conduction velocity and negative peak latency of a baseline
antidromic nerve conduction measurement were compared with the results of five
identical recordings performed at several points of time after LED irradiation
Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory
setting To guarantee an adequate standardized and controlled pain reduction study
there was opted to observe a healthy population with experimentally induced DOMS
Induction of DOMS has been described in a number of studies as a representative
model of musculoskeletal pain and stiffness because it can be induced in a relatively
easy and standardised manner the time course is quite predictable and the symptoms
have the same aetiology and are of transitory nature4445102-105
The treatment as well as the assessment procedure was performed during 4
consecutive days The first day isokinetic exercise was performed to induce pain
related to DOMS Subsequently the volunteers of the experimental group received an
infrared LED treatment and those of the placebo group received sham-irradiation
Evaluation of the effect of the treatment on perceived pain was registered by a visual
analog scale and by a mechanical pain threshold these observations occurred every day
18
prior to and following LED irradiation Eccentricconcentric isokinetic peak torque
assessment took place daily before each treatment
For the analysis of the results three different factors were taken into consideration
time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental)
In completion of this thesis the most prominent findings are summarized and the
clinical implications are discussed The general discussion also includes some future
research directions and a final conclusion
General introduction
19
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neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3)340-345
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20
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monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5)291-298
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science
Publishers 29 Basford J (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg
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Northern Ireland Physiotherapy 77(3)171-178 31 Cambier D and Vanderstraeten G (1997) Low-level laser therapy The experience in flanders
Eur J Phys Med Rehabil 7(4)102-105 32 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D
Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
33 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
34 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
35 Basford J Hallman H Matsumoto J Moyer S Buss J and Baxter G (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6)597-604
36 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol (Lond) 43563
37 Baxter G Allen J Walsh D Bell A and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol (Lond) 446445
38 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
39 Lowe A Baxter G Walsh D and Allen J (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
40 Lowe A Baxter G Walsh D and Allen J M (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Lasers Med Sci 10(4)253-259
41 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
General introduction
21
42 Baxter G Effect of combined phototherapylow intensity laser therapy upon experimental ischaemic pain Potential relevance of experimental design 14th World Congress Physical Therapy Barcelona Spain 2004 Proceedings CD
43 Craig J Barron J Walsh D and Baxter G (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
44 Craig J Barlas P Baxter G Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
45 Glasgow P Hill I McKevitt A Lowe A and Baxter G (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1)33-39
46 Noble J Lowe A Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
47 IEC 60825-1 (1993) International Electrotechnical Commission Safety of laser products Part 1 Equipment classification requirements and users guide
48 Nussbaum E Baxter G and Lilge L (2003) A review of laser technology and light-tissue interactions as a background to therapeutic applications of low intensity lasers and other light sources Phys Ther Rev 831-44
49 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
50 Nakamura S (1998) The roles of structural imperfections in InGaN-based blue light-emitting diodes and laser diodes Science 281956-961
51 Karu Tiina (consulted on 2005 august 22) Cellular mechanism of low-power laser therapy httpwwwtinnitusustiinakarupresentationhtml
52 Mester E Mester A and Mester A (1985) The biomedical effects of laser application Lasers Surg Med 5(1)31-39
53 Moore K Calderhead G (1991) The clinical application of low incident power density 830 nm GaAlAs diode laser radiation in the therapy of chronic intractable pain A historical and optoelectronic rationale and clinical review Int J Optoelectron 6503-520
54 King P (1989) Low level laser therapy A review Lasers Med Sci 4141-148 55 Karu T (1989) Photobiology of low-power laser effects Health Phys 56(5)691-704 56 Simunovic Z (2000) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical
Application of Low Energy-Laser Laser Therapy LLLT Rijeka Vitgraf 57 Hode L and Tuner J (2000) Low level laser therapy (LLLT) contra light emitting diode therapy
(LEDT) - What is the difference Proceedings of SPIE 416690-97 58 Kitchen S and Partridge C (1991) A review of low level laser therapy Part I Background
physiological effects and hazards Physiotherapy 77(3)161-163 59 Nussbaum E Lilge L and Mazzulli T (2003) Effects of low-level laser therapy (LLLT) of 810 nm
upon in vitro growth of bacteria Relevance of irradiance and radiant exposure J Clin Laser Med Sur 21(5)283-290
60 Mendez T Pinheiro A Pacheco M Nascimento P and Ramalho L (2004) Dose and wavelength of laser light have influence on the repair of cutaneous wounds J Clin Laser Med Sur 22(1)19-25
61 Koutna M Janisch R and Veselska R (2003) Effects of low-power laser irradiation on cell proliferation Scripta Medica 76(3)163-172
62 Al-Watban F and Zhang X (2004) The comparison of effects between pulsed and CW lasers on wound healing J Clin Laser Med Sur 22(1)15-18
63 Panjehpour M Overholt B DeNovo R Petersen M and Sneed R (1993) Comparative study between pulsed and continuous wave lasers for photofrin photodynamic therapy Lasers Surg Med 13(3)296-304
64 Dyson M (2005) The significance of pulsing in the stimulation of tissue repair by light A review Lasers Med Sci 20S21
22
65 MDB Laser (2000) Bio Dio (Preprotoype) User guide Belgium Ekeren 66 Fisher J (1992) Photons physiatrics and physicians A practical guide to understanding laser light
interaction with living tissue Part I J Clin Laser Med Sur 10(6)419-426 67 Tuner J and Hode L (2000) Depth of penetration of laser light in tissue Laser Partner
Clinixperience 151-3 68 Anderson R and Parrish J (1981) The optics of human skin J Invest Dermatol 77(1)13-19 69 De Cuyper H and Lambert H (1991) Penetration depth of infrared- and helium-neon-laserlight
An assessment in vivo Eur J Phys Med Rehabil 1(5)133-137 70 Oshiro T Ogata H Yoshida M Tanaka Y Sasaki K and Yoshimi K (1996) Penetration depths
of 830nm diode laser irradiation in the head and neck assessed using a radiographic phanom model and wavelength-specific imaging film Laser Ther 8197-204
71 Kolarova H Ditrichova D and Wagner J (1999) Penetration of the laser light into the skin in vitro Lasers Surg Med 24(3)231-235
72 Chen Q Huang Z Chen H Shapiro H Beckers J and Hetzel F (2002) Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy Photochem Photobiol 76(2)197ndash203
73 Conlan M Rapley J and Cobb C (1996) Biostimulation of wound healing by low-energy laser irradiation A review J Clin Periodontol 23(5)492-496
74 Parrish J (1981) New concepts in therapeutic photomedicine photochemistry optical targeting and the therapeutic window J Invest Dermatol 7745-50
75 Smith K (1981) Photobiology and photomedicine the future is bright J Invest Dermatol 772-7 76 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees D (1989) Systemic effects
of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
77 Polo L Presti F Schindl A Schindl L Jori G and Bertoloni G (1999) Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth Biochem Biophys Res Commun 257(3)753-758
78 Bertoloni G Sacchetto R Baro E Ceccherelli F and Jori G (1993) Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 6328 nm light J Photochem Photobiol B-Biol 18191-196
79 Lubart R Friedmann H Sinykov M and Grossman N (1995) Biostimulation of photosensitized fibroblasts by low incident levels of visible light energy Laser Ther 7101-106
80 Harris D (1991) Biomolecular mechanisms of laser biostimulation J Clin Laser Med Sur 9277-280
81 Grossman N Schneid N Reuveni H Halevy S and Lubart R (1998) 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures involvement of reactive oxygen species Lasers Surg Med 22212-218
82 Oren D Charney D Lavi R Sinyakov M and Lubart R (2001) Stimulation of reactive oxygen species production by an antidepressant visible light source Biol Psychiatry 49464-467
83 Lavi R Shainberg A Friedmann H Shneyvays V Rickover O Eichler M Kaplan D and Lubart R (2003) Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells 278(42)40917-40922
84 Reddy K (2004) Photobiological basis and clinical role of low-intensity lasers in biology and medicine J Clin Laser Med Sur 22(2)141-150
85 Greco M Guida G Perlino E Marra E and Quagliariello E (1989) Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser Biochem Biophys Res Commun 163(3)1428-1434
86 Vacca R Marra E Quagliariello E and Greco M (1993) Activation of mitochondrial DNA replication by He-Ne laser irradiation Biochem Biophys Res Commun 195(2)704-709
87 Vacca R Marra E Quagliariello E and Greco M (1994) Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser Biochem Biophys Res Commun 203(2)991-997
88 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral
General introduction
23
nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
89 Anders J Borke R Woolery S and Van de Merwe W (1993) Low power laser irradiation alters the rate of regeneration of the rat facial nerve Lasers Surg Med 13(1)72-82
90 Rochkind S Nissan M Barr-Nea L Razon N Schwartz M and Bartal A (1987) Response of peripheral nerve to He-Ne laser Experimental studies Lasers Surg Med 7441-443
91 Lievens P (1986) Lasertherapie (deel II) De invloed van laser-bestralingen op het lymfesysteem en de wondheling Ned T Fysiotherapie 96140-142
92 Lievens P (1991) The effect of a combined HeNe and IR laser treatment on the regeneration of the lymphatic sysytem during the process of wound healing Lasers Med Sci 6193-199
93 Lievens P (1991) The effect of IR laser irradiation on the vasomotricity of the lymphatic system Lasers Med Sci 6189-191
94 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin J and Martin P (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1)171-178
95 Bosatra M Jucci A Olliaro P Quacci D and Sacchi S (1984) In vitro fibroblast and dermis fibroblast activation by laser irradiation at low energy An electron microscopic study Dermatologica 168(4)157-162
96 Enwemeka C Rodriquez O Gall N and Walsh N (1990) Morphometrics of collagen fibril populations in HeNe laser photostimulated tendons J Clin Laser Med Sur 847-52
97 Abergel P Lam T Meeker C Castel C Dwyer R and Uitto J (1984) Biostimulation of procollagen production by low energy lasers in human skin fibroblast culturesJ Invest Dermatol 82(4)395
98 Abergel P Lam T Meeker C Dwyer R and Uitto J (1984) Low energy lasers stimulate collagen production in human skin fibroblast cultures Clin Res 32(1)16A
99 Shimoyama N Lijima K Shimoyama M and Mizuguchi T (1992) The effects of helium-neon laser on formalin-induced activity of dorsal horn neurons in the rat J Clin Laser Med Sur 1091-94
100 Kloth L McCulloch J and Feedar J (1990) Wound healing Alternatives in management USA FA Davis Company
101 Lanzafame R (2004) Revolutions and revelations J Clin Laser Med Surg 22(1)1-2 102 Ciccone C Leggin B and Callamaro J (1991) Effects of ultrasound and trolamine salicylate
phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-678 103 Craig J Cunningham M Walsh D Baxter G and Allen J (1996) Lack of effect of transcutaneous
electrical nerve stimulation upon experimentally induced delayed onset muscle soreness in humans Pain 67(2-3)285-289
104 Minder P Noble J Alves-Guerreiro J Hill I Lowe A Walsh D and Baxter G (2002) Interferential therapy Lack of effect upon experimentally induced delayed onset muscle soreness Clin Physiol Funct Imaging 22(5)339-347
105 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
PART I WOUND HEALING
CHAPTER 1
DO INFRARED LIGHT EMITTING DIODES HAVE A
STIMULATORY EFFECT ON WOUND HEALING FROM AN IN
VITRO TRIAL TO A PATIENT TREATMENT
Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Proceedings of SPIE 2002 4903 156-165
Chapter 1
28
ABSTRACT
Variable effects of different forms of light therapy on wound healing have been
reported This preliminary study covers the efficacy of infrared light emitting diodes
(LED) in this domain
Cultured embryonic chicken fibroblasts were treated in a controlled randomised
manner LED irradiation was performed three consecutive days with a wavelength of
950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each
treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2
The results indicated that LED treatment does not influence fibroblast proliferation at
the applied energy density and irradiation frequency (p=0474)
Meanwhile the effects of LED on wound healing in vivo were studied by treating a
surgical incision (6 cm) on the lateral side of the right foot of a male patient The
treatment started after 13 days when initial stitches were removed The same
parameters as the in vitro study were used but the treatment was performed five times
The healing could only be evaluated clinically the irradiated area (26 cm) showed a
more appropriate contraction less discoloration and a less hypertrophic scar than the
control area (34 cm)
The used parameters failed to demonstrate any biological effect of LED irradiation in
vitro although the case study on the other hand illustrated a beneficial effect
Keywords Light Emitting Diodes Fibroblasts Wound healing
From an in vitro trial to a patient treatment
29
INTRODUCTION
Various beneficial effects of lasers and photodiodes at relatively low intensities have
been reported involving treatment of neurological impairments12 treatment of pain3-5
treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the
enhancement of wound healing has been a focus of contemporary research11-16 It
seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing
remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the
basis of a questionnaire about low power laser (LPL) in the current clinical practice in
Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey
into clinical LPL experience in Flanders
Nevertheless there remains a considerable amount of ignorance scepticism and
controversial issues concerning the use and clinical efficacy of LPL even in the domain
of wound healing12152021 This is at least in part a consequence of the inability to
measure and control operating variables related to connective tissue repair and of the
wide range of suitable parameters for irradiation
Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent
characteristics which make their use in a clinical setting problematic including
limitations in wavelength capabilities and beam width The combined wavelength of
light optimal for wound healing cannot be efficiently produced and the size of
wounds which may be treated by LPLs is limited Some companies offer an
alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be
made to produce multiple wavelengths and can have probes with large surface area
allowing treatment of large wounds Still one can not accept this light source as an
alternative for LPL therapy based on the cited advantages without proper investigation
regarding its biostimulatory effects
The effectiveness of this possible alternative for LPLs must be studied in vitro and in
addition in animal models or in humans because the effects of LED at the cellular level
do not necessarily translate to a noticeable effect in vivo The small amount of previous
investigations demonstrate that LED effects are as difficult to isolate162223 as LPL
Chapter 1
30
effects and the results are conflicting just like the results in literature specific on the
use of LPL121520
The purpose of the first part of this study is to examine the hypothesis stating that
LED irradiation can influence fibroblast proliferation Therefore a comparison of the
proliferation from fibroblasts in irradiated and control wells was performed The in vitro
investigation was linked with an in vivo case study This part enquired the assumption if
LED irradiation could accelerate and ameliorate the healing of a surgical incision
IN VITRO INVESTIGATION
MATERIALS AND METHODS
The complete procedure from isolation to proliferation analysis was executed twice
(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of
control dishes The second trial consisted of 27 irradiated and 27 control dishes
Cell isolation and culture procedures
Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation
and disaggregating of the cells occurred with warm trypsin (NV Life Technologies
Belgium) according the protocol described by Ian Freshney (1994)24 The primary
explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies
Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1
Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life
Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies
Belgium) When cell growth from the explants reached confluence cells were detached
with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV
Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the
cells were removed from the culture flasks by trypsinization and counted by
hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc
From an in vitro trial to a patient treatment
31
NV Life Technologies Belgium) The fibroblasts were seeded at a density of
70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture
medium the cells were allowed to attach for 24 hours in a humidified incubator at
37degC
Properties of the Light Emitting Diode
Prior to LED treatment all dishes were microscopically checked to guarantee that the
cells are adherent and to assure that there is no confluence nor contamination The
dishes were divided randomly into the treated or the control group Medium was then
removed by tipping the dishes and aspirating with a sterile pipette Following the
aspiration 2 ml fresh medium was added and treatment started
A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser
Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-
1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32
single LEDrsquos For the treatments in this study an average power of 160 mW at
continuous mode was applied The irradiation lasted 6 minutes resulting in an energy
density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result
of the divergence in function of this distance the surface of the LED (18 cm2) covered
the complete surface of the used petriplates (215 cm2)
After these manipulations 3 ml medium was added to each dish followed by 24 hours
incubation
One LED irradiation was performed daily during three consecutive days according
this procedure Control cultures underwent the same handling during these three days
but were sham-irradiated
Proliferation analysis
After the last treatment a trypsination was performed to detach the cells from the
culture dishes followed by centrifugation Once the cells were isolated from the used
trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts
Chapter 1
32
within this suspension as reflection for the proliferation was quantified by means of a
Buumlrker Chamber or hemocytometry
The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-
Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab
Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on
the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator
using an inverted light microscope counted the number of cells in 25 small squares
In order to calculate the number of cells one should multiply the amount of cells
counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and
the dilution factor (the amount of Trypan blue suspended with the cells 21=3)
Statistical methods
The data were analysed statistical in order to examine the hypothesis that LED
irradiation enhances fibroblast proliferation They were processed as absolute figures
for both trials separately In a second phase the counted cell numbers were converted
in relative figures so the data of both trials could be analysed as the data of one test
These relative figures were obtained by expressing each figure as a percentage from the
highest figure (=100) of that trial and this for each assay separately
A Kolmogorov-Smirnoff test of normality was performed on the data followed by a
Mann-Whitney-U test when the test of normality was significant and otherwise a T-
test Differences were accepted as significant when plt005 For this analysis SPSSreg
100 was used
RESULTS
The descriptive data for both trials are depicted in figure I The mean number of cells
in trial A is higher than in trial B for the controls as for the treated wells There is a
mean difference of 1252500 fibroblasts between the controls and 1223000 between
the irradiated wells of trial A and B The averages of both trials show that in control
cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no
From an in vitro trial to a patient treatment
33
statistically significant difference could be found between the two groups in either trial
nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not
significant for trial A (p=020) nor trial B (p=020) Only the combined data from both
trials were significant (plt001) for normality Further analysis respectively T-test for
the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the
combined data (p=0474) revealed no statistical significant differences
DESCRIPTIVE DATA
1730000181750029530003070000
00E+00
50E+05
10E+06
15E+06
20E+06
25E+06
30E+06
35E+06
40E+06
Trial A Trial A Trial B Trial B
Mea
n n
um
ber
of
cells
Control
Irradiated
Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B
DISCUSSION
Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities
(lt500 mW) in vitro have been analysed by evaluating various factors involving
(pro)collagen production25-27 cell viability2829 growth factor production28 and
myofibroblast formation30 Fibroblast proliferation also is an important factor to
consider In accordance with wound healing fibroblasts fulfil an essential role especially
in the late inflammatory phase and the early granulation phase31 Despite the failure of
some studies to demonstrate beneficial effects of LPL irradiation on fibroblast
proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very
Chapter 1
34
significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -
208 ) Atabey et al35 also revealed a significant increase in cell number two or more
irradiations resulted in an increased fibroblast proliferation Several other studies
confirmed these positive findings25263637
The results of this present in vitro study indicate that LED treatment does not
influence fibroblast proliferation Although the dosimetric parameters (in particular the
arbitrary energy density of 32 Jcm2) used in this study are well within the
recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous
studies about LPL therapy raising enhanced fibroblast proliferation252634-37
Van Breugel et al36 gave a possible explanation for these controversial results
According to them the fibroblast proliferation is not inherent at the energy density
They provide evidence that independent of the energy density the power density and
the exposure time determine the biostimulative effects of LPL irradiation LPL with a
power below 291 mW could enhance cell proliferation while a higher power had no
effect
Some authors also argued that the absorption spectrum of human fibroblasts show
several absorption peaks and pointed out that a wavelength of 950 nm is far above the
highest peak of about 730 nm3638 At longer wavelengths they determined a general
decrease in absorption Despite these results several investigators pose biostimulative
effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or
even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is
ranged from 800 nm to 830 nm principally because of the presence of cytochrome
oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts
at lower wavelengths is restricted by an inferior skin transmission than at higher
wavelengths38
Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting
respective green and red light The 950 nm beam of light was used for its high power
density but according to a range of remarks mentioned above the effects of the two
other probes must be as well evaluated
From an in vitro trial to a patient treatment
35
Another factor one can not ignore is that besides fibroblast proliferation other
processes or morphologic changes were not analysed although several authors have
posed that those changes and processes could be responsible for the biostimulative
effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et
al30 for example described a massive transformation of fibroblasts into myofibroblasts
after LPL treatment These modified fibroblasts play an important role in contraction
of granulation tissue30 A second example is an increased (pro)collagen production
after low power light therapy25-27 which is also considered as a responsible factor for
accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast
proliferation3640
It may be wondered if the light sources mostly LPL in the consulted literature are
representative for the LED used in this study although this LPL literature is often
used for that purpose As in the early days of LPL the stimulative effects upon
biological objects were explained by its coherence the beam emitted by the Bio-Dio on
the contrary produces incoherent light Nowadays contradictory research results are
responsible for a new discussion the clinical and biological significance of coherence
The findings of some authors172341-43 pose that the coherence of light is of no
importance of LPL and its effects although the opposite has also been stated4445
Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be
accounted for the non-enhanced fibroblast proliferation in this trial
Another possible explanation for the absence of biostimulative effect is related to the
moment of analysis of the proliferation The evaluation one day after the last
irradiation did not allow a delayed enhancement of proliferation while it is determined
in numerous investigations that the effects occur more than 24 hours after the last
treatment273746 and that they weaken after a further undefined period of time34
The fluctuation in cell numbers between both trials despite the use of an identical
protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor
reproducibility of their technique In this study the fluctuations are attributable to the
counting of the cells by Buumlrker hemocytometer before seeding According to some
authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large
Chapter 1
36
variability48 and it is often difficult to standardize48 Overestimation of the cell
concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity
was contradicted by Lin et al50 moreover satisfactory correlations with flow-
cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay
for cell counting (MTT)51 were determined
An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it
provides accurate cell counts in a short period of time with less intervention from the
investigator52
These remarks and controversies point out the possible deficiencies from the used
proliferation analyses and the relativity from the obtained results Other analyse
methods and analyses from different cell processes and morphologic changes could be
considered for further investigation
IN VIVO INVESTIGATION
MATERIALS AND METHODS
The effects of LED on wound healing in vivo were studied by treating a postsurgical
incision A male patient received chirurgical treatment for the removal of a cyst
situated approximately 15 cm posterior from the lateral malleolus of his right foot For
removal of the cyst an incision of 6 cm was made The incision was sutured and 12
days after the surgery the stitches were removed Visual inspection demonstrated that
the healing process of the wound proceeded well but not equally over the whole 6
centimetres (figure II) Epithelialization and wound contraction appeared to have
progressed better in the upper part (approximately 3 cm) of the cicatrice than at the
lower part (covered with eschar) No evidence of infection was noted in either part
LED treatment started the 13th day The incision was treated partially the lowest part
(26 cm) with the inferior epithelialization and wound contraction was irradiated the
remaining 34 cm served as control area This control area was screened from radiation
with cardboard and opaque black cling film
From an in vitro trial to a patient treatment
37
The light source destinated for the treatment was the same device used for the in vitro
irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED
output parameters were identical with those applicated in the preceding in vitro
investigation In particular a continuous wave at an average power of 160 mW and 6
minutes of treatment duration corresponding to an energy density of 32 Jcm2 An
equal distance from the probe to the target tissue as from the probe to the culture
medium was respected A plastic applicant of according height guaranteed constant
distance of 06 cm from the surface of the skin
Figure II Surgical incision before the first treatment 13 days after initial stitching
Therapy was performed once a day during five consecutive days repeatedly at the same
time resulting in an extension of the duration of the in vitro therapy with two days
Visual macroscopic observations were accomplished 6 52 and 175 days after the first
treatment
Comparison of the cutaneous sensitivity at the irradiated area and the control area was
accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175
days after the first treatment A control measurement also occurred at the same region
Chapter 1
38
on the left foot The aesthesiometer used in this study consisted of five hand-held
nylon monofilaments with a length of 38 mm and varying diameter
Sensitivity threshold is traced by presenting a monofilament of a certain diameter
vertically to the skin The monofilament bends when a specific pressure has been
reached with a velocity proportional to its diameter Measurements allow mapping
areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified
from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest
filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished
protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to
finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the
filaments
RESULTS
Visual estimation at any point of time after irradiation divulged no occurrence of
problems with dehiscence or infection in either part of the wound During the five
days of therapy the irradiated area looked dryer than the control area After the last
irradiation this was no longer recorded
Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area
From an in vitro trial to a patient treatment
39
Figure III representing the first evaluation six days after the initial treatment
illustrates that the wound healing has evolved slightly in both parts Though the lower
irradiated part remains of inferior quality as regards to epithelialization and wound
contraction In the course of the reparative process the influence of light exposures
were registered At 52 days after the first irradiation beneficial effects of LED
treatment are clearly present (Figure IV)
Figure IV Surgical incision 52 days after initiating LED treatment
The irradiated area (26 cm) showed a more appropriate contracture than the control
area (34 cm) characterized by less discoloration at scar level and a less hypertrophic
scar A similar trend was noticed at a third visual observation 175 days after the initial
treatment At that moment no impairments at cutaneous sensitivity level were stated
and the sensitivity showed no differences between left or right foot nor between the
two areas of the cicatrice
Chapter 1
40
DISCUSSION
The results of this case study indicate that LED had a positive influence on wound
healing in humans as determined by visual observations Many investigators
examining the effects of LPL on wound healing by means of a range of observation
and treatment methods reported accelerated and enhanced wound healing8-10 others
described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and
LED21 studies were unable to repeat these results
The late but beneficial findings in this study seem to be to the credit of LED-therapy
Though several authors establish positive results in an earlier stage of the wound
healing process8-1020 one should question why the differences did not occur at the first
evaluation on day 6 An explanation can be found in the start of the treatment Most
investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so
they influence a first cellular and vascular reaction with the production of chemical
mediators of inflammation resulting in an enhanced collagen production9 tremendous
proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the
time the first treatment in this study took place the traumatized tissue was in an
overlapping stage between an almost finished inflammatory phase and a scarcely
initiated re-epithelialization and wound contraction phase At that moment an infiltrate
of fibroblasts is present So fibroblast proliferation a possible mechanism of the
biostimulative effect had already occurred and could no longer be influenced Growth
factor production and collagen deposition have also decreased at that stage
Granulation tissue formation and fibroplasia in the contrary are initiating by that time
Those prolonged and slow processes with belated results are of significant importance
for the course of the final stage of wound healing and for the outlook of the future
scar31
The experimental findings revealed that the sensitivity of the skin according to the
threshold detection method of Semmes and Weinstein was normal at all the
investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a
good and objective predictor of normal skin sensitivity No other LPL nor LED
studies investigating this quality of the skin were found
From an in vitro trial to a patient treatment
41
CONCLUSION
This study demonstrates that although LED application at the applied energy density
and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to
have beneficial biostimulative effects on wound healing in human skin confirmed by
the favourable re-epithelialization and contracture
These results are discussed in the context of other experimental findings but no
reasonable explanation for this discrepancy could be found The literature on wound
healing after LED treatment in animal models or in humans is presently very limited
and contradictory The diversity in used radiation parameters and the absence of
references on how the wounds were measured or evaluated or what the end point was
for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro
investigations are better standardised nevertheless these results show a number of
conflicts One can conclude that until today the controversial findings are characteristic
for many results obtained with light photobiomodulation
However the postponed favourable results in the case study confirm some facts of the
discussion Namely the short period of incubation 24 hours in the in vitro part of the
study can be responsible for the lack of enhanced fibroblast proliferation It also
confirms that other cell processes and morphologic changes possibly are responsible
for biostimulative effects in vivo other observation methods should be considered for
future in vivo experiments
Despite these remarks we believe that LED application on cutaneous wounds of
human skin is useful with a single flash daily at the dose applied in this study for at
least three days
Furthermore future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Chapter 1
42
ACKNOWLEDGMENTS
The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the
material necessary for this study as well as Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
From an in vitro trial to a patient treatment
43
REFERENCES
1 G Baxter D Walsh J Allen A Lowe and A Bell Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79(2) 227-34 (1994)
2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)
3 J Stelian I Gil B Habot et al Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy J Am Geriatr Soc 40(1) 23-6 (1992)
4 A Honmura A Ishii M Yanase J Obata and E Haruki Analgesic effect of Ga-Al-As diode laser irradiation on hyperalgesia in carrageenin-induced inflammation Lasers Surg Med 13(4) 463-9 (1993)
5 D Cambier K Blom E Witvrouw G Ollevier M De Muynck and G Vanderstraeten The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15 195-200 (2000)
6 G Reddy L Stehno Bittel and C Enwemeka Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-7 (1998)
7 K Moore N Hira I Broome and J Cruikshank The effect of infrared diode laser irradiation on the duration and severity of postoperative pain a double blind trial Laser Therapy 4 145-148 (1992)
8 A Amir A Solomon S Giler M Cordoba and D Hauben The influence of helium-neon laser irradiation on the viability of skin flaps in the rat Br J Plast Surg 53(1) 58-62 (2000)
9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)
10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)
11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)
12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)
13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)
14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)
15 A Schlager P Kronberger F Petschke and H Ulmer Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1) 39-42 (2000)
16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)
17 GD Baxter and J Allen Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone (1994)
18 G Baxter A Bell J Allen and J Ravey Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77(3) 171-178 (1991)
19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)
20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)
Chapter 1
44
21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)
22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)
23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)
24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)
25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)
26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)
27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)
28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)
29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)
30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)
31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)
32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)
33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)
34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)
35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)
36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)
37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)
38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)
39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)
40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)
41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)
42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)
From an in vitro trial to a patient treatment
45
43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)
44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)
45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)
46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)
47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)
48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)
49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)
50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)
51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)
52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)
53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)
54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)
CHAPTER 2
INCREASED FIBROBLAST PROLIFERATION INDUCED BY
LIGHT EMITTING DIODE AND LOW LEVEL LASER
IRRADIATION
Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Lasers in Medical Science 2003 18(2) 95-99
Chapter 2
48
ABSTRACT
Background and Objective As Light Emitting Diode (LED) devices are
commercially introduced as an alternative for Low Level Laser (LLL) Therapy the
ability of LED in influencing wound healing processes at cellular level was examined
Study DesignMaterials and Methods Cultured fibroblasts were treated in a
controlled randomized manner during three consecutive days either with a infrared
LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and
570 nm) and respective power outputs Treatment duration varied in relation to
varying surface energy densities (radiant exposures)
Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all
irradiated cultures in comparison with the controls Green light yielded a significantly
higher number of cells than red (plt0001) and infrared LED light (plt0001) and than
the cultures irradiated with the LLL (plt0001) the red probe provided a higher
increase (plt0001) than the infrared LED probe and than the LLL source
Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation
in vitro This study therefore postulates possible stimulatory effects on wound healing
in vivo at the applied dosimetric parameters
Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low
Level Laser Tetrazolium salt
LED induced increase of fibroblast proliferation
49
INTRODUCTION
Since the introduction of photobiostimulation into medicine the effectiveness and
applicability of a variety of light sources in the treatment of a wide range of medical
conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results
of several investigations are remarkably contradictory This is at least in part a
consequence of the wide range of indications as well as the wide range of suitable
parameters for irradiation and even the inability to measure the possible effects after
irradiation with the necessary objectivity [457] A lack of theoretical understanding
can also be responsible for the existing controversies In fact theoretical understanding
of the mechanisms is not necessary to establish effects though it is necessary to
simplify the evaluation and interpretation of the obtained results As a consequence
the widespread acceptance of especially Low Level Laser (LLL) therapy in the early
seventies is faded nowadays and biostimulation by light is often viewed with scepticism
[8] According to Baxter [49] contemporary research and consumption in
physiotherapy is in particular focused on the stimulation of wound healing Tissue
repair and healing of injured skin are complex processes that involve a dynamic series
of events including coagulation inflammation granulation tissue formation wound
contraction and tissue remodelling [10] This complexity aggravates research within this
cardinal indication
Research in this domain mostly covers LLL studies but the current commercial
availability of other light sources appeals research to investigate as well the effects of
those alternative light sources eg Light Emitting Diode (LED) apparatus
The scarcity of literature on LED is responsible for consultation of literature
originating from LLL studies [11] but it may be wondered if this literature is
representative for that purpose As in the early days of LLL therapy the stimulating
effects upon biological objects were explained by its coherence [1213] while the beam
emitted by LEDrsquos on the contrary produces incoherent light Though the findings of
some scientists [914151617] pose nowadays that the coherence of the light beam is
not responsible for the effects of LLL therapy Given that the cardinal difference
between LED and LLL therapy coherence is not of remarkable importance in
Chapter 2
50
providing biological response in cellular monolayers [5] one may consult literature
from LLL studies to refer to in this LED studies
The purpose of this preliminary study is to examine the hypothesis that LED
irradiation at specific output parameters can influence fibroblast proliferation
Therefore irradiated fibroblasts cultures were compared with controls The article
reports the findings of this study in an attempt to promote further discussion and
establish the use of LED
MATERIALS AND METHODS
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos Isolation and
disaggregation of the cells was performed with warm trypsin according the protocol
described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in
Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1
L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants
reached confluence cells were detached with trypsine and subcultured during 24 hours
in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours
the cells were removed from the culture flasks by trypsinization and counted by Buumlrker
hemocytometry For the experiment cells from the third passage were plated in 96-well
plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a
density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C
during 24 hours
All supplies for cell culture were delivered by NV Life Technologies Belgium except
for Fetal Calf Serum (Invitrogen Corporation UK)
Irradiation sources
In this study two light sources a Light Emitting Diode (LED) device and a Low Level
Laser (LLL) device were used in comparison to control cultures
The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)
LED induced increase of fibroblast proliferation
51
with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400
mW and a frequency range from 0-1500 Hz
The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)
consisted of three wavelengths emitted by separate probes A first probe emitting
green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the
red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third
probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared
light The area of all three probes was 18 cm2 and their frequency was variable within
the range of 0-1500 Hz
Exposure regime
Prior to irradiation the 96-well plates were microscopically verified to guarantee that
the cells were adherent and to assure that there was no confluence nor contamination
Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining
25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation
The 96-well plates were randomly assigned in the treated (LLL or green red or infrared
LEDrsquos) or the control group
For the treatments in this study the continuous mode was applied as well for the LLL
as for the three LED-probes The distance from light source to fibroblasts was 06 cm
LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting
in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant
exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to
exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective
power output of 160 mW 80 mW or 10 mW
After these handlings the remaining medium was removed and new Hanksrsquoculture
medium was added followed by 24 hours of incubation
One irradiation (LLL or LED) was performed daily during three consecutive days
according to the aforementioned procedure Control cultures underwent the same
handling but were sham-irradiated
Chapter 2
52
Determination of cell proliferation
The number of cells within the 96-well plates as a measure for repair [19] was
quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The
colorimetric assay was performed at two different points of time to determine the
duration of the effect of the used light sources
This assay exists of a replacement of Hanksrsquoculture medium by fresh medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as
described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT
solution was substituted by lysing buffer isopropyl alcohol The plates were
temporarily shaken to allow dissolution of the produced formazan crystals After 30
minutes of exposure to the lysing buffer absorbance was measured The absorbance at
400 to 750 nm which was proportional to fibroblast proliferation was determined
using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)
The complete procedure from isolation to MTT assay was executed six times (Trial A
B C D E and F) while it was impossible to irradiate all the investigated number of
wells with the same LED apparatus on one day All the trials included as much control
as irradiated wells but the number of control and irradiated wells in each trial varied
depending on the number of available cells after the second subculturing A further
consequence of the available number of cells is the number of probes examined per
trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E
Incubation period before proliferation analyses numbered 24 hours To investigate if
the stimulatory effect tends to occur immediately after irradiation or after a longer
period of time incubation in trial F lasted 72 hours
An overview of the followed procedures regarding incubation time before proliferation
analysis number of analysed wells for each trial and the number of probes examined
per trial is given in table 1 As a consequence of the differences in procedures followed
and because each trial started from a new cell line the results of the five trials must be
discussed separately
LED induced increase of fibroblast proliferation
53
Statistical analysis
Depending on the amount of groups to be compared within each trial and depending
on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way
ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons Statistical significance for all tests was
accepted at the 005 level For this analysis Statistical Package for Social Sciences 100
(SPSS 100) was used
RESULTS
The results presented in table 1 show that cell counts by means of MTT assay
revealed a significant (plt0001) increase in the number of cells in comparison to their
respective sham-irradiated controls for all the irradiated cultures of trial A B C D
and E except the irradiated groups in trial F
Moreover the results of trial A showed that the effect of the green and red LED probe
was significantly (plt0001) higher than the effect of the LLL probe With regard to the
amount of proliferation the green probe yielded a significantly higher number of cells
than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe
provided a higher increase in cells (plt0001) than the infrared probe
The infrared LED source and the LLL provided a significant (plt0001) higher number
of cells than the control cultures but no statistical significant difference was recorded
between both light sources
The trials A B C D and E regardless of the number of probes used in each trial
were analysed after 24 hours of incubation after the last irradiation The incubation
period of trial F lasted 72 hours
The means of trial F illustrated that the effect was opposite after such a long
incubation The control cultures had significantly (plt0001) more fibroblasts than the
irradiated cultures with the exception of the LED-infrared group that showed a not
significant increase of cells Further analysis revealed that the green probe yielded a
significantly lower number of cells than the red (plt0001) and the infrared probe
(plt0001) and that the red probe provided a higher decrease (plt0001) than the
Chapter 2
54
infrared probe Laser irradiation induced a significant decrease of fibroblasts in
comparison to the infrared irradiated cultures (plt0001) and the control cultures
(p=0001) LED irradiation with the green and the red probe revealed no statistical
significant differences
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups
Absorbency (proportional to the number of fibroblasts)a
Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050
Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis
DISCUSSION
Despite the failure of some studies [223] to demonstrate beneficial effects of laser and
photodiode irradiation at relatively low intensities (lt500mW) on fibroblast
LED induced increase of fibroblast proliferation
55
proliferation this study provides experimental support for a significant increased cell
proliferation Therefore these results confirm previous studies that yielded beneficial
stimulating effect [1152425] Remarkably though is the higher increase noted after
irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general
decrease in absorption at longer wavelengths and concluded that several molecules in
fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445
470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the
closest to one of these peaks
Karu [5] also emphasises that the use of the appropriate wavelength namely within the
bandwidth of the absorption spectra of photoacceptor molecules is an important
factor to consider
In this particular context penetration depth can almost be ignored as virtually all
wavelengths in the visible and infrared spectrum will pass through a monolayer cell
culture [12] The irradiance (Wcm2) on the contrary could have had an important
influence on the outcome of this study The higher increased proliferation by the lower
wavelengths is possibly a result of the lower irradiance of these wavelengths Lower
irradiances are confirmed by other experiments to be more effective than higher
irradiances [111626]
The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as
described by Poumlntinen [17] but in the scope of these results it also needs to be noticed
that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)
the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)
Consequently the results of especially trial A and F must be interpreted with the
necessary caution It is possible that the determined distinction between the used light
sources and the used probes is a result from the various radiant exposures applied
during the treatments of the cultures
Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after
the last irradiation this study was unable to demonstrate a stimulating effect when
analysis was performed 72 hours after the last irradiation Moreover this longer
incubation period even yielded an adverse effect Although a weakening of the
Chapter 2
56
photostimulating influence over time is acceptable it can not explain a complete
inversion Especially in the knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period [2427] In an attempt to illuminate
this finding one can suppose that the circadian response of the cells triggered by the
LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark
The most obvious explanation is even though a decreased vitality and untimely cell
death in the irradiated cell cultures as a result of reaching confluence at an earlier point
of time than the control cultures The cells of a confluent monolayer have the tendency
to inhibit growth and finally die when they are not subcultured in time No other
reasonable explanations could be found for this discrepancy
Photo-modulated stimulation of wound healing is often viewed with scepticism The
real benefits of Light Emitting Diodes if any can only be established by histological
and clinical investigations performed under well controlled protocols Despite these
remarks this study suggests beneficial effects of LED and LLL irradiation at the
cellular level assuming potential beneficial clinical results LED application on
cutaneous wounds of human skin may be assumed useful at the applied dosimetric
parameters but future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Persons in good health rarely require treatment for wound healing as posed by Reddy
et al [13] light has a possible optimal effect under conditions of impaired healing
Postponed wound healing is a time-consuming and often expensive complication
Thus future prospects must remind to examine the therapeutic efficacy of LED on
healing-resistant wounds
LED induced increase of fibroblast proliferation
57
ACKNOWLEDGMENTS
The authors are grateful to Prof Deridder for supplying the laboratory as well as the
material necessary for this investigation and to Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
Chapter 2
58
REFERENCES
1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55
2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32
3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7
4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994
5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998
6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32
7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42
8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5
9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8
10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7
11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8
12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34
13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9
14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18
15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43
16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6
17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475
18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J
Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9
21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601
22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63
LED induced increase of fibroblast proliferation
59
23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9
24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301
25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are
more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37
27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8
28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26
CHAPTER 3
GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES
FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL
Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Photomedicine and Laser Surgery 2005 23(2) 167-171
Chapter 3
62
ABSTRACT
Background and Objective The chronic metabolic disorder diabetes mellitus is an
important cause of morbidity and mortality due to a series of common secondary
metabolic complications such as the development of severe often slow healing skin
lesions
In view of promoting the wound-healing process in diabetic patients this preliminary
in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on
fibroblast proliferation and viability under hyperglycemic circumstances
Materials and Methods To achieve hyperglycemic circumstances embryonic chicken
fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL
glucose LED irradiation was performed on 3 consecutive days with a probe emitting
green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min
resulting in a radiation exposure of 01 Jcm2
Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all
irradiated cultures in comparison with the controls
Conclusion According to these results the effectiveness of green LED irradiation on
fibroblasts in hyperglycemic circumstances is established Future in vivo investigation
would be worthwhile to investigate whether there are equivalent positive results in
diabetic patients
Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes
Fibroblast proliferation under hyperglycemic circumstances
63
INTRODUCTION
The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide
geographic variation in incidence and prevalence generally 11 of the world
population is affected and worldwide it is the twelfth leading cause of death1 Those
figures may be higher for urban regions as well as for industrialized countries Due to
multiple factors involving the aging process of the population and lifestyle changes
(such as reduced physical activity hypercaloric eating habits and concomitant obesity)
these figures may increase in the future2-6 Therefore diabetes mellitus could become
the most common chronic disease in certain regions as stated by Gale it ldquotargets the
rich in poor countries and the poor in rich countriesrdquo6
The harmful disruption of the metabolic equilibrium in diabetes mellitus results in
characteristic end-organ damage that occurs in various combinations and that follows
an unpredictable clinical pathway
Accordingly the major consequence of diabetes mellitus in terms of morbidity
mortality and economic burden principally concerns macroangiopathies or
arteriosclerosis and microangiopathies including nephropathy neuropathy and
retinopathy7-10
One of these devastating consequences which often appears in time is the
development of various skin defects that are frequently resistant to healing and that
tend to be more severe than similar lesions in nondiabetic individuals Diabetes
mellitus even increases the risk of infection by an increased susceptibility to bacteria
and an impaired ability of the body to eliminate bacteria1112
Skin problems are a severe complication in diabetic individuals and require a
comprehensive and appropriate multidisciplinary approach to prevention and
treatment12
Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to
play the most prominent role in the development of diabetic complications With the
development of insulin treatment for type I diabetes and various oral hypoglycemic
agents for type 2 diabetes a reduction in the development of skin defects due to
hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply
Chapter 3
64
waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in
diabetic patients often need special care in comparison to those persons in good
health who rarely require treatment for wound healing1617 Special care is directed
besides of course toward optimal diabetes regulation toward patient education
maximum pressure relief controlling infection recovery of circulation in case of
ischemia and different modalities of intensive wound treatment18
In the last few years various therapies have been introduced with varying success An
example of such a therapy is the photo-modulated stimulation of diabetic lesions In
vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been
subject of extensive investigation1920 Due to contradictory research results LLL-
photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of
light in the domain of wound healing is less time-consuming less expensive less
invasive than many of the other introduced treatment modalities and practical to use
however it seems worthwhile to investigate the value and benefits of a newly
introduced and alternative light source the light emitting diodes (LEDrsquos)
Preliminary research has proved that green LED with particular properties (an
exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01
Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These
results may be of great importance to the diabetic patient because as posed by Reddy et
al light has a possible beneficial effect in the case of impaired healing1617
To obtain insight into the ability of LED to stimulate fibroblast proliferation under
diabetic-specific conditions of impaired healing the proliferation was assessed in
irradiated and control cultures cultivated in medium with a high quantity of glucose
MATERIAL amp METHODS
Cell cultivation
Primary fibroblast cultures were established by outgrowth from 8-day-old chicken
embryos After isolation and disaggregating as described by Freshney (1994)24 the cells
were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10
Fibroblast proliferation under hyperglycemic circumstances
65
fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin
Secondary cultures were initiated by trypsinization followed by plating of the cells in
80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were
disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently
231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third
passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the
cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified
atmosphere at 37deg C to allow them to attach to the bottom of the wells
Light source specifications and illumination procedure
To control adherence of the cells and to assure that there was no confluence or
contamination the 96-well plates were microscopically examined before irradiation
Subsequently the tissue culture plates were randomly assigned for use in the treated
and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium
was aspirated The remaining 25 (50 microl) medium avoided dehydration of the
fibroblasts throughout irradiation
Irradiation was performed with a light emitting diode (LED) device The LED device
(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power
range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable
within the range of 0-1500 Hz
The investigation used the following illumination properties the continuous mode a
distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2
radiant exposure This procedure resulted in an exposure time of 3 min and a power
output of 10 mW Immediately after irradiation the remaining medium was aspirated
and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM
glucose (30 gL) and incubated at 37deg C
Irradiation and medium changes occurred at 1-day intervals so one irradiation was
implemented each 24 h for 3 days in a row and from the first irradiation onwards all
medium renewals occurred with glucose-supplemented Hanksrsquo culture medium
Control cultures were handled in the same manner but were sham-irradiated
Chapter 3
66
Proliferation assay
Fibroblast survival and proliferation were determined by a sensitive and reproducible
colorimetric assay the assay which detects merely living cells and the signal generated
bears a constant ratio to the degree of activation of the fibroblasts and the number of
fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than
only a small subsample26
Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented
Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution
was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during
30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous
solution The optical density of the final solution was measured on an ELtimes800 counter
(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test
wavelength varying from 400 to 750 nm
The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies
for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except
for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)
Data analysis
On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a
Mann-Whitney U test was performed for nonparametrical comparison of the results
Statistical significance for all tests was accepted at the 005 level For this analysis the
Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used
RESULTS
The MTT measurements from each of the 256 control wells and 256 irradiated wells
and the subsequent nonparametrical analysis from the optical densities obtained
disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic
Fibroblast proliferation under hyperglycemic circumstances
67
circumstances after irradiation than in the same circumstances without irradiation (Fig
1)
Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)
DISCUSSION
The outcome of these in vitro experiments based on the above-described light source
properties and the illumination procedure described clearly demonstrated the
stimulatory potential of LED on fibroblast proliferation and the cell viability of
fibroblasts cultured in hyperglycemic medium Preliminary research has already
demonstrated that under these conditions (an exposure time of 3 min a wavelength of
570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this
procedure allowed the highest number of living cells The nature of the light and the
usual questions concerning coherence wavelength power output and radiant
exposures have been discussed previously23
Although these findings confirm the results previously found one cannot ignore the
important methodological difference between previous investigations and the current
study as the cells in this experiment were cultured in hyperglycemic medium2328-30
Absorbency - Proportional to the number of fibroblasts
621 x 10-1 682 x 10-1
0010203040506070809
1
Control Irradiated
Groups
Ab
sorb
ency
Chapter 3
68
After a growth period with normal Hanksrsquo culture medium a necessary step to ensure
normal growth of these secondary subcultures and normal attachment to the bottom
of the wells the Hanksrsquo culture medium was supplemented with glucose
Several earlier studies have established that exposure to glucose concentrations (20-40
mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human
vascular endothelial cell proliferation1531-34 This restraint is more pronounced for
higher glucose15 concentrations and is expressed especially after protracted exposure to
high glucose levels31 A similar restraint was found for cultured fibroblasts by
Hehenberger et al3536 According to some authors however cultured fibroblasts
conversely have been shown to maintain responsiveness to ambient high glucose323738
As there are some ambiguities in literature regarding normal or inhibited growth of
fibroblasts in medium supplemented with glucose39 a pilot study was performed to
determine the amount of glucose necessary to inhibit normal growth after 72 h of
culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study
demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease
of cell viability and to bring about a decline in fibroblast proliferation
This concentration resulted in a remarkable reduction of cell viability and a noteworthy
decrease in the proliferation rate in comparison to control cultures grown in 55 mM
glucose although this concentration is too high to mimic severe diabetic
hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to
investigate the effect of LED in distinct destructive conditions in order to obtain an
incontrovertible result
In addition it is possible that the present investigation needed a higher amount of
glucose to result in a remarkable reduction of proliferation as exposure to glucose was
limited to 72 h and as previous studies revealed that the antiproliferative effect of high
glucose was more pronounced with prolonged exposure with a maximal inhibition
attained by 7-14 days1531
Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo
cell growth are too complex to compare A key question is whether fibroblast
senescence in tissue culture and in the intact organism are similar Cristofalo et al40
Fibroblast proliferation under hyperglycemic circumstances
69
reported that this is not the case as fibroblasts have a finite ability to divide and
replicate but apparently the pathway or the morphologic characteristics leading to the
replicative senescence is not identical in vivo compared to in vitro
Furthermore extrinsic aging related to environmental damage which in diabetic
patients is mainly due to a chronic exposure to high levels of glucose during life is
unachievable in vitro
Unless a number of questions regarding the mechanism according to which LED
stimulates fibroblast proliferation in this particular condition remain unanswered the
results ascertain the potential effects of LED on fibroblast proliferation and viability
CONCLUSION
The current results should be interpreted with caution However these results
demonstrate the effectiveness of green LED irradiation at the above-described light
source properties and the illumination procedure described on cells in hyperglycemic
circumstances
The findings of the present study using an experimental in vitro model indicate that the
use of LED irradiation to promote wound healing in diabetic patients may have
promising future results As the present study establishes the possibility of using LED
irradiation in experimental in vitro situations it would be a worthwhile extension to
perform in vivo investigations to determine whether these in vitro observations were
relevant to the physiological situation and to determine the effect of these LED
properties on human tissue response
ACKNOWLEDGMENTS
The authors are greatly indebted to P Coorevits for assistance with the statistical
analysis and to Professor L Deridder and Ms N Franccedilois of the department of
Human Anatomy Embryology Histology and Medical Physics for providing access to
the laboratory and for helpful discussions
Chapter 3
70
REFERENCES
1 World Health Organisation The world health report of 2002 Statistical Annex 2002 pp 179-201
2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418
3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957
4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538
5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308
6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J
Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot
wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-
glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A
Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood
glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13
14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309
15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501
16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287
17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255
18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412
19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192
20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137
21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675
22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246
23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99
Fibroblast proliferation under hyperglycemic circumstances
71
24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729
27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63
28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301
29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -
progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of
cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA
damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in
culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia
23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar
K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71
36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141
37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588
38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252
39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97
40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619
PART II ANALGESIA
CHAPTER 4
EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION
MEDIATED BY LIGHT EMITTING DIODE IRRADIATION
Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy
Vanderstraetenab and Dirk Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Lasers in Medical Science 2005 20(1) 35-40
Chapter 4
76
ABSTRACT
The introduction of light emitting diode (LED) devices as a novel treatment for pain
relief in place of low-level laser warrants fundamental research on the effect of LED
devices on one of the potential explanatory mechanisms peripheral neurophysiology in
vivo
A randomised controlled study was conducted by measuring antidromic nerve
conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline
measurement and five post-irradiation recordings (2 min interval each) were performed
of the nerve conduction velocity (NCV) and negative peak latency (NPL)
Interventional set-up was identical for all subjects but the experimental group (=32)
received an irradiation (2 min at a continuous power output of 160 mW resulting in a
radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype
MDB-Laser Belgium) while the placebo group was treated by sham irradiation
Statistical analysis (general regression model for repeated measures) of NCV and NPL
difference scores revealed a significant interactive effect for both NCV (p=0003) and
NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical
significant decreased NCV and an increased NPL in the experimental group and a
statistical significant difference between placebo and experimental group at various
points of time
Based on these results it can be concluded that LED irradiation applied to intact skin
at the described irradiation parameters produces an immediate and localized effect
upon conduction characteristics in underlying nerves Therefore the outcome of this in
vivo experiment yields a potential explanation for pain relief induced by LED
Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative
peak latency middot Analgesic effect
Nerve conduction characteristics
77
INTRODUCTION
Since the introduction of photobiostimulation into medicine the light sources used
have advanced technologically and varied in characteristics over the years
Advancement and variation of the sources implicate a concomitant necessity to revise
research results in the respective domains of application Research and clinical
applications in the past particularly focused on the effectiveness of low-level lasers
have shifted now to novel treatment units such as light emitting diode (LED) devices
The efficacy and applicability of LED irradiation within the field of wound healing has
already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED
is not only promoted for its beneficial effects on the wound-healing process it is also
suggested to be potentially effective in the treatment of pain of various aetiology
although this claim has not yet been investigated thoroughly either experimentally or
clinically The putative analgesic effects of LED remain to be further explored
As the basic vehicle of pain is the neuronal system [7] measuring the
neurophysiological effect of LED treatment would be an appropriate experimental
approach to investigate the efficacy of LED on pain inhibition Nerve conduction
studies have become a technique for investigating the neurophysiologic effects of light
therapy [8-9]
Review of literature regarding standard nerve conduction studies revealed that previous
human studies on the influence of various light sources on peripheral nerves have
utilized different methods which hampers a comprehensive comparison In general
this research was performed on the superficial radial nerve [10-13] described by Shin J
Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve
[891315-17] Following the method of Cambier et al [18] the authors of this study
decided to investigate the effect of the light source used on the conduction
characteristics of the sural nerve By investigating this solely sensory nerve interaction
of motor nerve fibres (motor response can easily be provoked by antidromic nerve
stimulation [19]) can be avoided and given the superficial nature of the nerve it should
be sufficiently amenable to the effects of percutaneous LED irradiation
Chapter 4
78
A second major difference between the trials and therefore also hindering an
appropriate comparison between the results is the wide range of used light sources
HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared
multisource treatment unit [15]
With respect to the potential importance of LED irradiation for the treatment of pain
the current investigation was designed to assess the putative neurophysiological effects
of LED on the sensory nerve conduction of the human superficial peripheral sural
nerve and to establish a time course of the supposed phenomenon
The experimental hypothesis postulates that LED generates an immediate decrease in
conduction velocity and increase in negative peak latency In addition it can be
postulated that this effect is most prominent immediately after the irradiation and will
weaken as time progresses
STUDY DESIGN
The study was approved by the Ethical Committee of the Ghent University Hospital
After explanation of the experimental procedure a written informed consent was
obtained from each subject
Subjects
After screening based on a brief medical history excluding subjects with
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever inflammation of the skin) or conditions
that might affect sensory nerve conduction (such as diabetes peripheral neuropathy
radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral
edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40
females (mean age 26plusmn6 years range 18-42 years) participated in this study The body
mass index (BMI) of each subject varied within the normal range (=185-249) [20]
(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo
Nerve conduction characteristics
79
or an experimental group Each group of 32 subjects was composed of 12 males and
20 females
Experimental Procedure and Data Acquisition
In order to be able to quantify the negative peak latency (NPL) (measured from the
start of the stimulus artefact to the peak of the negative portion of the nerve action
potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was
followed
With respect to the known relationship between nerve conduction characteristics and
temperature the ambient temperature was kept constant (23ordmC-26ordmC room
temperature) during the investigation In view of this temperature issue the
standardized protocol started with 10 min of accommodation during which the
subjects rested in prone position on a treatment table
Immediately before this adjustment period the skin over the dorsolateral aspect of the
left calf and foot was cleaned with alcohol to remove surface lipids This preparation of
the treatment area was followed by the placement of the electrodes (TECA
Accessories Oxford Instruments Medical Systems Division Old Woking UK) as
described by Delisa et al [21]
The two-posted (2 cm separation anode distal) surface caption electrode was placed
distal and posterior of the lateral malleolus on the skin covering the sural nerve The
fixation of the earth electrode (Medelec Oxford Instruments Medical Systems
Division Old Woking UK) occurred 12 cm above the caption electrode according to
the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm
above the caption electrode to map the ideal stimulation point To level off
intraindividual variations in the amount of sensory response attributable to the
successive placement of the bipolar stimulator in course of the investigation a two-
posted (2 cm separation cathode distal) bar stimulating electrode was attached at the
point where the maximal response was obtained
This placement of the electrodes allows antidromic stimulation of the sural nerve
Electrophysiological stimulation and recordings were obtained with a Medelec
Chapter 4
80
Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse
of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was
used to produce each evoked sensory response
Baseline measurements of NPL and NCV were immediately followed by treatment of
the subjects according the protocol detailed below Recordings were subsequently
repeated at 2-min intervals over an 8-min period resulting in five recordings (one
immediately after the completion of the treatment and one at 2 4 6 and 8 min after
irradiation) Skin temperature was recorded concomitantly throughout the procedure
at the time of baseline measurement immediately after LED irradiation at the time of
the first recording and consequently at 2-min intervals together with the four final
electrophysiological recordings For this a surface digital C9001 thermometer
(Comark UK) sensitive to temperature changes of 01degC was used at the same point
of LED administration namely at 7 cm above the caption electrode The procedure
was identical for both conditions but subjects in the placebo group received a sham
LED irradiation
Light Characteristics and Irradiation Procedure
Irradiation was administrated with a light emitting diode device (BIO-DIO
preprototype MDB-Laser Belgium) The probe used emitted infrared light with a
wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2
and the frequency was variable within the range of 0-1500 Hz
Preceding baseline measurement the treatment point was marked on the skin overlying
the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-
point between the stimulation and capture electrode The LED probe was held in
contact with the skin perpendicular to the skin surface during the complete irradiation
procedure LED treatment consisted for all subjects of the experimental group out of 2
minutes lasting irradiation The LED was set to deliver a continuous energy density of
107 Jcm2 at a power output of 160 mW These parameters were selected as they are
appropriate for the treatment of pain in a clinical setting First of all because the
Nerve conduction characteristics
81
duration of the treatment is clinically feasible and secondly because the parameters are
within the scope of previously described light source characteristics [1-36915]
Statistics
Although superficial skin temperature did not change significantly in course of the
investigation the influence of the measured skin temperature on NPL and NCV was
taken into account by using a correction factor of respectively 02 msdegC and 147
ms degC All corrections were calculated towards a reference skin temperature of 32degC
Difference scores ie the variation between baseline measurements and each post-
irradiation recording were used as the basis for statistical analysis A General
Regression Model for repeated measures with one within-subjects factor (time 0
min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and
one between-subjects factor (group placebo or LED irradiated) was performed
followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least
Significant Difference) to determine whether any differences between baseline
measurements and post-irradiation recordings were statistically significant
The Statistical package for social sciences (SPSS 110) was used for analysis and
statistical significance for all tests was accepted at the 005 level
RESULTS
Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated
group plotted against time in minutes The values of the irradiated subjects decrease
directly after the irradiation and reach a first low point 2 min after finishing LED
treatment This decrease is followed by a marginal increase at 4 and 6 min and again an
important decrease at 8 min Statistical analysis (general regression model for repeated
measures) of these data indicated a significant interactive effect (P=0003)
Chapter 4
82
Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)
Post hoc LSD further showed significant differences between baseline measurements
and all post-treatment recordings (Table 1) Mutual comparison of the values from the
post-treatment recordings did not reveal any significant difference In addition there
was no significant difference determined in the placebo group in course of time
Table 1 Summary of the influence of LED irradiation on nerve conduction velocity
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001
2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002
4 0111plusmn0377 0647 -0908plusmn1898 0021 0004
6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001
8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve Conduction Velocity
-14
-12
-1
-08
-06
-04
-02
0
02
04
Baseline 0 min 2 min 4 min 6 min 8 min
Time Course
Dif
fere
nce
Sco
re (
m
s)
PlaceboLED
Nerve conduction characteristics
83
A similar representation was used for the results of the NPL Figure 2 reproduces NPL
plotted against time in minutes revealing for the irradiated group an increased latency
with two important peaks one at 4 min and one at 8 min
Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)
Statistical analysis of the mean difference scores again indicated a significant interactive
effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2
showed significant differences between baseline measurements and all post-treatment
recordings of the experimental group The mean difference score of the first post-
treatment recording of this same group (LED irradiated) differed significantly with the
recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED
irradiation As well as the recording 2 min after irradiation which differed significantly
(P=0013) with the 8 min post-treatment recording As observed for the NCV the
NPL of the placebo group did not reveal any significant difference in time course
At the time of the final recording the NCV and NPL mean difference scores of the
irradiated group did not return to their respective baseline values
Negative Peak Latency
-001
0
001
002
003
004
005
006
007
Baseline 0 min 2 min 4 min 6 min 8 min
Time course
Dif
fere
nce
Sco
re (
ms)
PlaceboLED
Chapter 4
84
Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group
significance) revealed statistical differences between the experimental and the placebo
group for NCV as well as for NPL NCV and NPL were statistical significant between
both groups at all points of time except from the NPL recording immediately after
finishing irradiation
DISCUSSION
Notwithstanding the above-mentioned difficulties in comparing results between
different trials on nerve conduction we attempt to discuss the current findings in view
of the results of the previous studies
This investigation revealed that percutaneous LED irradiation at feasible and current
clinical parameters generates measurable and significant changes in human sural nerve
antidromic conduction latency and velocity These results thus support previous
findings of light-mediated nerve conduction latency shifts in vivo [8101218]
although there are several important issues to be discussed
Table 2 Summary of the influence of LED irradiation on negative peak latency
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0004 plusmn0053 0755 0029plusmn0080 0019 0145
2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021
4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004
6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034
8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve conduction characteristics
85
A first comment deals with the progress of the NCV and NPL in function of time As
postulated the NCV decreases significantly immediately after irradiation
corresponding with a significant increased NPL However this effect does not weaken
as time progresses both variables remain significant throughout the 8 min during
observation period
Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the
conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]
although this slight increase in NPL was not significant at any moment Two other
studies [822] with a GaAlAs laser even registered comparable effects over a period of
55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous
studies post-treatment conduction measurements should be extended in time At
present for all studies it remains unclear at what point of time the effect extinguishes
although the interval of time during which LED treatment remains effective is
clinically important when treating pain
Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least
45 min) mediated by a monochromatic multisource infrared diode device although it
needs to be mentioned that this study performed with a comparable light source as the
current investigation revealed a significant decrease of the NPL These inverse results
between the study of Noble et al [15] and the current investigation could be attributed
to the concomitant increase of the skin temperature [15] As it has been well
recognised that a variation in tissue temperature causes a corresponding alteration in
nerve conduction velocities and peak latencies [91523-27] the temperature changes
may indeed provide an explanation for the observed findings In an attempt to analyse
the influence of a direct photobiological effect on sural nerve conduction
characteristics rather than working out the effects based upon thermal mechanisms
the present study corrected the skin temperature towards a reference temperature of
32degC This correction was performed notwithstanding the fact that the superficial skin
temperature did not change significantly before and after LED irradiation as well as
despite the fact that influencing nerve temperature takes place long after affecting skin
temperature [23] and thus being (almost) impossible after 2 min of irradiation
Chapter 4
86
followed by 8 min of registration Introduction of the correction factor implies likewise
that eventual influence on nerve conduction by cooling of the limb due to inactivity as
described by Greathouse et al [11] can be excluded
These facts suggest that temperature changes did not contribute to the demonstrated
effects of LED on nerve conduction Nevertheless the underlying mechanism of the
observed effects remains indistinct
A following remark regarding the fluctuation of NCV and NPL in function of time
considers the fact that both the NCV and the NPL do not change in a constant way up
to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the
increase in NPL display a small though not significant inversion of the effect at 4 and
(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of
fluctuation is to be expected when measuring NCV and NPL besides there is a similar
variation in the placebo groups
Although investigating dose dependency was not intended an additional remark
considers the fact that the use of optimal irradiation parameters is essential to obtain
the observed neurophysiological effect Nevertheless it is impossible to determine
ideal light source characteristics for effective treatment as the range of used
wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency
(pulsed or continuous) are not sufficiently similar between the different studies It can
only be concluded that a pulsing light source [91028] does not provide the postulated
results Radiant exposure exposure time power range and wavelength are not yet
established but based on this study and previously described assays it can be
speculated that the ranges of these parameters are quite large
In comparison with other studies where the number of subjects is 10 or less [8-
1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-
Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number
of subjects (n=32) was investigated in each group In spite of the large investigated
population it should be noted that the magnitude of the described changes in NCV
and NPL can simply be replicated by lowering the temperature of the extremity as the
observed changes are within the expected physiological ranges making the clinical
Nerve conduction characteristics
87
significance of the change questionable (This fact does not implement that the
decrease and the significant changes were temperature mediated)
A key question and meanwhile the initial impetus for future investigation is whether
the measured effects can be extrapolated to the actual nociceptive afferents namely the
myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])
respectively conducting acute and chronic pain The functional testing of these
nociceptive pathways has recently been extensively evaluated The currently accepted
neurophysiological method of assessing nociceptive pathways relies on laser-evoked
potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]
As up till now LEP is not available in this or any surrounding research centre the
investigators of this study had to perform a standard nerve conduction study (assessing
the large myelinated Aβ afferents) Therefore the current and previous beneficial
results of low level light therapy on conduction characteristics of nerves in vivo should
initiate measurements of clinical effectiveness first of all in laboratory settings and
afterward at a clinical level
CONCLUSION
Despite these remarks and the limited knowledge regarding the underlying mechanism
the present findings enable the following conclusions to be drawn LED irradiation at
clinical applied energy densities produces an immediate and localized effect upon
conduction characteristics in underlying nerves More specifically it is proven that
LED treatment lowers the NCV and augments the NPL resulting in a reduced
number of impulses per unit of time Therefore the outcome of this in vivo experiment
assumes that LED possibly induces pain relief
In order to encourage a widespread acceptance for the use of this non-invasive pain-
reducing modality in clinical settings prospective research should establish the precise
relationship between LED and pain relief as well as determine the ideal irradiation
parameters and verify which painful conditions can be treated with this treatment unit
Chapter 4
88
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the
equipment and MDB-Laser Belgium for generously providing the Light Emitting
Diode equipment
Nerve conduction characteristics
89
REFERENCES
1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9
2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)
3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18
4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8
5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43
6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65
7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53
8 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-34
9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6
10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90
11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7
12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5
13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9
14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore
15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5
16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5
17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604
18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200
19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21
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20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083
21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press
22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9
24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9
25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203
26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13
27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12
28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9
29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445
30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8
31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56
CHAPTER 5
PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE
IRRADIATION A PILOT STUDY ON EXPERIMENTALLY
INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS
Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Accepted for publication in Lasers in Medical Science December 2005
Chapter 5
92
ABSTRACT
Objective The present pilot study investigated the analgesic efficacy of light emitting
diode (LED) In view of a standardised and controlled pain reduction study design this
in vivo trial was conducted on experimentally induced delayed-onset muscle soreness
(DOMS)
Design Thirty-two eligible human volunteers were randomly assigned to either an
experimental (n=16) or placebo group (n=16) Immediately following the induction of
muscle soreness perceived pain was measured by means of a visual analog scale (VAS)
followed by a more objective mechanical pain threshold (MPT) measurement and
finally an eccentricconcentric isokinetic peak torque (IPT) assessment The
experimental group was treated with infrared LED at one of both arms the other arm
served as control Irradiation lasted 6 min at a continuous power output of 160 mW
resulting in an energy density of 32 Jcm2 The subjects of the placebo group received
sham irradiation at both sides In post-treatment a second daily assessment of MPT
and VAS took place The treatment and assessment procedure (MPT VAS and IPT)
was performed during 4 consecutive days
Results Statistical analysis (a general linear model followed by post hoc least
significant difference) revealed no apparent significant analgesic effects of LED at the
above-described light parameters and treatment procedure for none of the three
outcome measures However as the means of all VAS and MPT variables disclose a
general analgesic effect of LED irradiation in favour of the experimental group
precaution should be taken in view of any clinical decision on LED
Conclusion Future research should therefore focus on the investigation of the
mechanisms of LED action and on the exploration of the analgesic effects of LED in a
larger randomised clinical trial and eventually in more clinical settings
Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset
muscle soreness middot Musculus biceps brachii
Delayed-onset muscle soreness
93
INTRODUCTION
The analgesic efficacy of light emitting diode (LED) irradiation is recently being
investigated by means of a nerve conduction study on the superficial peripheral sural
nerve [1] It was demonstrated that LED irradiation at clinical applied densities
produces an immediate and localized effect upon conduction characteristics in
underlying nerves More specific LED induces a decreased number of sensory
impulses per unit of time thus possibly inducing pain relief [1]
Given the established influence of this treatment modality on the nerve conduction
velocity and thereby its potential analgesic ability the current investigation was
designed
Studies investigating the efficacy of a therapeutic modality on pain often experience
difficulties regarding standardisation of the population as analysis or comparison of
pain with different aetiologies is almost impossible Therefore we opted to measure the
analgesic effects of LED in a laboratory setting on a sample with experimentally
induced delayed-onset of muscle soreness (DOMS)
Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the
induction exercise and then spreads throughout the muscle [2-4] The correlates of
DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10
[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force
decreased range of motion and in particular muscle pain which is more pronounced
during movement and palpation [8 11] Despite the large volume of research that has
been undertaken to identify the underlying pathophysiology of DOMS the precise
mechanism is not yet universally accepted Several theories such as the torn-tissue
theory the connective tissue damage theory the muscle spasm theory and the
inflammation theory still remain viable though the current opinion states that DOMS
arises from a sequence of events in which several theories occupy an important place
[2 6 12 13]
DOMS has been used as a representative model of musculoskeletal pain and stiffness
in a number of studies [4 7 11 14 15] as it has a number of advantages it can be
induced in a relatively easy and standardised manner in a group of healthy subjects the
Chapter 5
94
time-course is relatively predictable and the symptoms have the same aetiology and are
of transitory nature [14 16] Nevertheless it should be emphasised that the use of this
particular experimental model to test the effectiveness of LED does not mean that this
treatment modality is necessarily advocated for the treatment of DOMS but merely
that it may be helpful in documenting the efficacy of LED in a clinical model of
musculoskeletal pain and stiffness In addition studies based on the induction of
DOMS under carefully controlled laboratory conditions can not replace research
involving actual patients but offer the opportunity to assess the effectiveness of
particular therapeutic interventions and might help to define additional clinical research
[14]
The experimental hypothesis of the current study postulates that infrared LED reduces
pain and muscle sensitivity associated with DOMS
MATERIALS AND METHODS
The study was approved by the ethical committee of the Ghent University Hospital
After providing information regarding the study design and possible consequences
related to participation at the study written informed consent was obtained from each
subject
Subjects
Healthy human volunteers were recruited from the university population Individuals
with any upper limb pathology neurological deficit and recent injury to either upper
extremity or undiagnosed pain were excluded Other exclusion criteria were
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever and inflammation of the skin) or
conditions in which physical exertion is contraindicated (such as cardiovascular deficits
hypertension and respiratory problems)
Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age
23plusmn4 years) were enrolled All subjects were randomly assigned using a random table
Delayed-onset muscle soreness
95
of numbers to the experimental or placebo group Each group of 16 subjects
consisted by stratification of equal numbers of men and women Age height and
weight did not differ significantly between the three groups
All subjects were physically active however none performed on a regular basis any type
of upper body weight-training Subjects were requested to refrain from any form of
strenuous physical activity and they were asked to avoid any form of medication
including anti-inflammatory agents as well as alcohol for 2 days before testing and for
the duration of the study
Overview of experimental design
The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to
induce pain related to DOMS Immediately following induction exercise an initial
assessment of the outcome measures (visual analog scale or VAS mechanical pain
threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the
subjects were treated under blinded conditions according to the randomised group
allocation In post-treatment the MPT was re-recorded and perceived pain was
reassessed with a VAS Contrary to these outcome measures the muscle strength was
only measured in pre-treatment at the one hand because short-term effects of LED
on muscle strength were not postulated and on the other hand because post-
treatment muscle strength can be influenced by too many different physiological
factors related to the pre-treatment measurement On the succeeding days (day 2 3
and 4) the treatment and assessment procedure was similar with approximately 24 h
separating each treatment
In both of the groups the two arms of the participants were included in the study In
the experimental group an equal number of dominant and non-dominant arms were
treated The non-treated arm served as control arm In the placebo group also an equal
number of dominant and non-dominant arms were considered as treated arm and the
other arm was classified in the non-treated group The procedure was identical for
both conditions but the subjects in the placebo group received sham LED irradiation
on both arms
Chapter 5
96
Specific aspects of the experimental design and procedures are detailed below
Pain induction
Muscle soreness was induced in a standardised fashion via a daily calibrated computer-
operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY
USA) Induction occurred separately and in random order in the elbow flexors of both
arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex
Prior to induction of DOMS the subjects were allowed an initial familiarization session
to become comfortable performing maximum voluntary contractions at the required
angular velocities This was immediately followed by determination of the maximum
eccentric and concentric peak torque at an angular velocity of 60degs and 120degs
Subsequently four sessions of eccentricconcentric work were performed with each
arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs
first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg
and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-
range) followed by two sessions at an angular velocity of 120degs again the first time
along an arch of 120deg and followed by the mid-range performance The subjects were
asked to accomplish maximum voluntary contractions during all the sessions Each
session was performed until exhaustion which was defined as the point when the
subject lost 70 of the initial eccentric and concentric peak torque There was a 1-
minute rest between each session This procedure was based on a pilot study and
previously described induction protocols [17-21]
Outcome measures
Outcome measures of subjective pain measurements MPT and muscle strength were
measured in this order on days 1-4 Subjective pain measurements and MPT occurred
immediately prior to and following irradiation whereas muscle strength measurements
only took place before LED treatment
Measurement of subjective pain Perceived muscle soreness was measured
subjectively by means of a 100-mm VAS A series of scales were completed separately
Delayed-onset muscle soreness
97
for each arm pain at rest followed by pain perception associated with full extension of
the arms and finally with maximal flexion of the arms The subjects were not allowed
to compare one VAS result with another
This assessment tool commonly used in measuring experimentally induced pain [22
23] has been found to be a reliable and valid method [24-26]
MPT Tenderness MPT used as a more objective correlate of muscle tenderness
has been demonstrated to be a reliable method to measure experimental induced
muscle soreness [27] This outcome measure was assessed by using a handheld
pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries
South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a
line from the radial insertion of the musculus biceps brachii at the elbow to the
intertubercular groove of the humerus thus resulting in three measure points one at
the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A
pressure of 4Ns was delivered The subjects were instructed to say yes at the exact
moment the pressure perceived became painful Each point was recorded three times
in pre-treatment as well as in post-treatment The average MPT score for each point in
pre- and post-treatment was used for statistical analyses
Muscle strength assessment Eccentric and concentric IPT were measured on the
same computerised dynamometer as was used for the induction of pain and an
identical standardisation procedure regarding positioning was followed
A warm-up session of two maximum voluntary contractions at the required angular
velocities was followed by determination of the eccentric and concentric peak torque
The first session at 60degs consisted of three repetitions followed by a 1-min during
rest and for the second session at 120degs five repetitions were performed The
subjects were instructed to flex and extend the elbow through the entire range of
motion as forcefully and rapidly as possible for each repetition The maximum
eccentric and concentric torque produced during the respective repetitions was used
for statistical analysis
Chapter 5
98
Light source specifications and treatment procedure
Light treatment was applied daily according to group allocation Irradiation occurred
with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The
probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160
mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The
frequency was variable within the range of 0ndash1500 Hz
During the complete irradiation procedure the LED probe was held in contact with
the skin perpendicular to the skin surface and at the exact mid-point between the MPT
mark at 4 cm and the one at 8 cm Light source properties were identical for all
subjects of the experimental group and consisted out of irradiation of 6-min lasting
duration at a continuous power output of 160 mW resulting in an energy density of
32 Jcm2 To conceal the treated side and condition the subjects were blinded to the
treatment status For the experimental condition a probe was held in contact with each
arm but only one of the two probes was attached to the LED device The subjects of
the placebo group received sham irradiation at both sides
The selected parameters are within the scope of previously described light source
characteristics for pain reduction [1 28-30] and they are appropriate for the treatment
of pain in a clinical setting because the duration of the treatment is clinically feasible
Statistical analysis
The three outcome measures were analysed separately For the VAS and MPT
measurements the same procedure was followed a general linear model (GLM) for
repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-
post preceding and following LED irradiation) and one between-subject factor (group
placebo or infrared LED irradiated) was performed If necessary the GLM was
followed by appropriate pairwise comparisons (post hoc least significant difference or
LSD) to determine whether any differences between measurements were statistically
significant A similar model was carried out separately for both the treated and the
control arm
Delayed-onset muscle soreness
99
In contrast to MPT and VAS the muscle strength was analysed differently The peak
torque values recomputed towards body weight of the subjects were statistically
analysed using a GLM for repeated measures This model consisted of one within-
subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo
or infrared LED irradiated) The model was completed twice first for the treated arm
and consequently for the control arm
The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)
was used for analysis and statistical significance for all tests was accepted at the 005
level
RESULTS
Statistical analysis of all variables of the three outcome measures revealed no significant
interactive effects of the main interaction (time times group times pre-post) The means and
standard deviations of the variables for both the treated and the control arm are
outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The
means of all VAS and MPT variables disclose a non-statistical significant general
analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher
MPT values in the irradiated group than in the placebo group The lower VAS rates are
present from day 1 until the last day of the study but they are more clearly present
from day 3 pre-treatment The higher MPT values are present from day 1 post-
irradiation until the last day and they are more visible at 4 cm followed by 12 cm and
finally at 8 cm In addition to the analgesic influence of LED an increased
convalescence of muscle strength was noted It should be remarked that this outcome
is similar for the treated as well as for the control arm of the irradiated group The
findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms
of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively
Graphical presentation of the other variables shows a similar course
Chapter 5
100
Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42
Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36
Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Visual Analog Scale
0
02
04
06
08
1
12
14
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n V
AS
scor
e (a
t re
st)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
101
Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249
Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Mechanical Pain Threshold
0
5
10
15
20
25
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n M
PT
sco
re (
at 4
cm)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Chapter 5
102
Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group
Day 1 Day 2 Day 3 Day 4
Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04
Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04
Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Isokinetic Peak Torque
04
045
05
055
06
065
Day 1 Day 2 Day 3 Day 4
Time course
Mea
n I
PT
sco
re (
ecce
ntr
ic a
t 60
degse
c)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
103
Despite the absence of significant main interaction effects the remaining interactions
as well as the main effects were statistically significant for some variables Only the
significant interactions including the between-subject factor group as well as the main-
effect group will be discussed The other interactions and effects establish the successful
induction of DOMS but are not relevant in view of the postulated hypothesis
The interaction between group and time is significant (p=014) for the VAS in
association with full extension for the control arm Post hoc LSD reveals no difference
between both groups a significant effect over time for both groups is found
Consequently this will not be further evaluated
A second significant interaction (p=0002) is the one among the within-subject factor
pre-post and the between-subject factor group for the MPT at 12 cm for the control arm
Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more
pressure after than before the treatment whereas in the placebo group a not
significant decrease of supported pressure is noted
Finally GLM analysis revealed that at the treated arm the irradiated group tolerates
significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)
DISCUSSION
It has previously been demonstrated that the LED source used might assist in
accelerating wound healing [31] that it has a direct cellular effect [3233] and that it
changes nerve conduction characteristics [1] Nevertheless LED-treated experimental
induced DOMS failed to prove the analgesic efficacy of LED at the above-described
light parameters and treatment procedure The current outcome concurs with other
research that demonstrated a lack of effect of various forms of light therapy on DOMS
[8 11 15] However despite the absence of an apparent and overall definitive finding
the present results cannot exclude favourable effects of LED treatment on pain Since
first of all an isolated statistical significant pre-post difference between groups (control
arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)
revealed that subjects of the irradiated group tolerate more pressure than the subjects
of the placebo group Second of all the overall means identified generally lower VAS
Chapter 5
104
scores higher MPT values and higher peak torques in the irradiated group This
implied that the treated subjects experienced noticeable less pain supported more
pressure on the painful muscle and generated more force than the non-treated
participants However these results are not statistically significant consequently it is
possible that these differences were found by coincidence and that there is no
relationship between the treatment and the described results of the three outcome
measures though it should be mentioned that the absence of significant findings is
more probably attributable to the small sample size involved in this study This
assumption is based on a post hoc power analysis It was calculated that for the small
effect size measured after treatment and for the measured control group event rate a
sample size of 80 subjects in each group was required at α=005 and power=080
(two-sided) to reveal significant results
Another factor conceivably responsible for the lack of solid evidence of the beneficial
effects of LED treatment upon DOMS-associated pain is related to the size of the
treatment effect in relation to the severity of the induced DOMS It is possible that by
using multiple exhaustive sets of exercise severe DOMS were induced which masked
relatively small but apparent treatment effects [4 11] In this same context it is
possible that the results only become significantly different after a prolonged treatment
and follow-up period as previous research noticed that recuperation subsequent to
DOMS induction can last up to 10 days [8]
Although it needs to be stressed that these results are not statistically significant critical
analysis of the overall means leads up to three additional remarks A primary comment
relates to the pre- and post-treatment courses of the results Starting at day 2 a clear
reduction of pain and muscle sensitivity was observed immediately post-treatment
Still one cannot conclude that this is indicative for the analgesic effect of LED
irradiation as a similar decrease in VAS and increase in MPT values was noted in the
treated and the control arm of the placebo group Perhaps this was caused by placebo
effect as reported by Pollo et al [34] the expectation of the participant can easily result
in pain relief but it can only be elucidated by implementation of a control group
Delayed-onset muscle soreness
105
Nevertheless in the current study this particular finding can be most probably
attributed to the physiological effects of the peak torque measurement performed
between the pre- and post-treatment recordings of VAS and MPT on the painful
flexor muscle of the upper arm For the assessment of muscle strength two short
series of alternative concentric and eccentric efforts were performed in succession
involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist
in the removal of inflammatory markers and exudate consequently reducing local
tenderness [4] In addition the force assessment can be considered as a form of active
warming-up resulting in an increased muscle temperature which can reduce muscle
viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness
[3536] thus decreasing the sensitivity of the muscle and moderating pain during
movement In any case the beneficial influence of LED immediately after irradiation
can not be securely interpreted due to the sequential assessment of the outcome
measures
A second additional remark considers the fact that both arms of the irradiated subjects
demonstrated evidence of the beneficial effects of LED as a similar reduction of pain
and muscle sensitivity and higher peak torques were found in course of time at the
treated arm as well as at the control arm of the irradiated subjects This ascertainment
points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]
stated that in case LED works via systemic effects the use of the contralateral side as a
control arm might be ill-advised Thus reinforcing that future research should include a
control group to bring clarification [4 7 16]
Finally it needs to be mentioned that although the extent of DOMS was probably
relatively high for investigating the postulated hypothesis the time-course of the
present study corresponds to that reported by other investigators [2 3 5-10]
Significant time effects in many of the variables revealed that muscle damage was
evident diffuse muscle soreness became progressively worse 24-48 h after DOMS
induction followed by a small amelioration after 72 h [35910] After 72 h the follow-
Chapter 5
106
up was ceased consequently further regain of force and attenuation of pain and
muscle sensitivity could not be evaluated Extending the duration of the assessment
period could be useful in assessing any longer-term effects of LED treatment
particularly because as mentioned above differences between both groups are more
clearly present from day 3 pre-treatment and also because DOMS may last for up to 10
days when induced with the described protocol [715]
Lack of knowledge regarding both the precise mechanism of action of LED and the
specific pathophysiology of DOMS hampers the way to offer a definitive explanation
for the absence of more obvious statistically significant differences Still the small
number of significant findings and the mean values suggest that possible analgesic
effects of infrared LED may not be excluded yet but to be able to estimate the real
value of LED further research is necessary A large-scaled randomised clinical trial
which takes the above-mentioned remarks into consideration should be performed
CONCLUSION
Regardless of the reasons for the absence of statistical significant effects reported here
and although LED may have some potential in the management of pain and functional
impairment associated with DOMS its effectiveness at the applied densities has not
been established
Future research should focus on evaluation of the appropriateness of DOMS as an
experimental model of pain and muscle damage Validation of this model would
enhance the ability to study various modalities for their potential effects on pain and
muscle injuries Besides the mechanisms of LED action are not known thus further
fundamental investigations need to address the underlying mechanism and
physiological basis of pain modulation utilizing LED treatment
Once LED irradiation has finally proven its treatment value in an experimental model
the most important prospect considers establishing the effectiveness of LED to reduce
pain in clinical settings
Delayed-onset muscle soreness
107
ACKNOWLEDGMENTS
The authors would like to thank Mr T Barbe and Mr R Deridder for their technical
assistance in the collection of the data as well as for their valuable input into the
research design Sincere appreciation is extended to the volunteers that participated in
this study and to MDB-Laser (Belgium) for generously providing the light emitting
diode equipment The authors also gratefully recognize Prof Dr G Van Maele for
assistance with the statistical analysis and for helpful discussion
Chapter 5
108
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18 Lambert MI Marcus P Burgess T and Noakes TD (2002) Electro-Membrane Microcurrent Therapy Reduces Signs and Symptoms of Muscle Damage Med Sci Sports Exerc 34(4)602-607
19 Sumida K Greenberg M and Hill J (2003) Hot gel packs and reduction of delayed-onset muscle soreness 30 minutes after treatment J Sport Rehabil 12221-228
20 Tiidus PM and Shoemaker JK (1995) Effleurage massage muscle blood flow and long-term post-exercise strength recovery Int J Sports Med 16(7)478-483
21 Zhang J Clement D and Taunton J (2000) The efficacy of Farabloc an electromagnetic shield in attenuating delayed-onset muscle soreness Clin J Sport Med 10(1)15-21
22 Fitzgerald GK Rothstein JM Mayhew TP and Lamb RL (1991) Exercise-Induced Muscle Soreness After Concentric and Eccentric Isokinetic Contractions Phys Ther 71(7)505-513
Delayed-onset muscle soreness
109
23 Nosaka K and Clarkson PM (1997) Influence of Previous Concentric Exercise on Eccentric Exercise-Induced Muscle Damage J Sports Sci 15(5)477-483
24 Jensen MP Karoly P and Braver S (1986) The Measurement of Clinical Pain Intensity - a Comparison of 6 Methods Pain 27(1)117-126
25 Price DD Mcgrath PA Rafii A and Buckingham B (1983) The Validation of Visual Analog Scales as Ratio Scale Measures for Chronic and Experimental Pain Pain 17(1)45-56
26 Revill SI Robinson JO Rosen M and Hogg MIJ (1976) Reliability of a Linear Analog for Evaluating Pain Anaesthesia 31(9)1191-1198
27 Nussbaum EL and Downes L (1998) Reliability of Clinical Pressure-Pain Algometric Measurements Obtained on Consecutive Days Phys Ther 78(2)160-169
28 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
29 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
30 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118
31 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in biomedical optics and imaging 3(28 Proceedings of SPIE 4903)156-165
32 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18(2)95-99
33 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2005) Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level J Photomed and Laser Surg 23(2)167-171
34 Pollo A Amanzio M Arslanian A Casadio C Maggi G and Benedetti F (2001) Response Expectancies in Placebo Analgesia and Their Clinical Relevance Pain 93(1)77-84
35 Shellock FG and Prentice WE (1985) Warming-up and Stretching for Improved Physical Performance and Prevention of Sports-Related Injuries Sports Med 2(4)267-278
36 Safran MR Seaber AV and Garrett WE (1989) Warm-up and Muscular Injury Prevention an Update Sports Med 8(4)239-249
37 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
38 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
GENERAL DISCUSSION
General discussion
113
SUMMARY
As outlined in the general introduction the overall objective of this doctoral thesis is to
develop the current knowledge about the mechanisms of LED action in view of the
eventual provision of evidence-based support for the clinical use of LED as a
biostimulatory and analgesic treatment modality especially in the field of
physiotherapy
Part I Wound healing
The investigations described in chapter 1 and 2 were conducted to gain insight into the
potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As
fibroblasts are principal cells for biostimulation (in view of growing and dividing in
healing wounds) the influence of LED irradiation on fibroblast proliferation was
assessed1
The first investigation consisted of a pilot study performed in order to evaluate the
appropriateness of the cell isolation technique cell culture protocol and proliferation
analysis as well as to appraise the feasibility of the light source properties and
illumination procedure
Data analysis revealed no statistically significant differences between the infrared LED
irradiated and control petri dishes for the used parameters (table 1) Considering this
outcome other experimental findings disclose that the absence of stimulatory effects of
LED irradiation on fibroblast proliferation can partly be attributed to the use of
inappropriate light source properties However the applied external dosimetric
parameters are well within the recommended spectrum described by previous studies
investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be
excluded that changes in the illumination procedure (such as the use of lower power
shorter exposure times wavelengths with finer coverage of the absorption spectrum of
the irradiated cells and a longer incubation period between the last irradiation and cell
counting) could still result in an increased fibroblast proliferation467 Of equal
importance in interpreting the lack of distinctive results are the imperfections of the
applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails
114
considerable intervention from the investigator compromising the reliability of the
method It is also a time-consuming technique with an insufficient sensitivity for some
purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11
To avoid contamination of the results by these modifiable remarks a similar
experiment (chapter 2) was performed in which wavelength power and output mode of
the infrared LED source were not modified (table 1) only the exposure time was
reduced resulting in a lower radiant exposure In addition the effect of two other
emission spectra was evaluated These probes emitting red and green light had a
shorter wavelength than the infrared LED source and the power was half or a
sixteenth of the power from the infrared probe Consequently the red LED irradiation
occurred with a different exposure time than the infrared one in order to attain the
same radiant exposure (053 Jcm2) With respect to the green LED it was not
endeavoured to achieve the same radiant exposure as 16 min of irradiation is not
feasible for in vitro or clinical application
Finally also an LLL light source was integrated Although it was not attempted to
analyse the effectiveness of LED in comparison to LLL enclosure of this modality was
interesting in order to join in with the available literature covering mostly LLL studies
To bypass the described problems regarding analysis of fibroblast proliferation
counting of the cells was carried out this time by means of a colorimetric MTT assay
This method provides more accurate cell counts in a short period of time and therefore
can be considered as a more reliable alternative to Buumlrker hemocytometer11
MTT assay 24 h after the last irradiation revealed a significantly increased number of
cells in the irradiated wells in comparison to their (respective sham-irradiated) controls
Although the study supplied experimental support for a significantly increased cell
proliferation by all external dosimetric properties based on the results of the
comparative trial with an incubation period of 24 hours irradiation with the green
LED source yielded the highest number of fibroblasts Thus it can be concluded that
the wavelength of the green LED is probably within the bandwidth of the absorption
spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that
General discussion
115
the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast
cultures in vitro612
Table 1 External dosimetric properties summarized for each chapter
Wavelength Power Exposure
time Output mode
Radiant exposure
PART I Chapter 1
In vitro part
LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2
LED-infrared 950 nm 160 mW 1 min continuous 053 Jcm2
LED-red 660 nm 80 mW 2 min continuous 053 Jcm2
LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2
Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2
PART II Chapter 4
LED-infrared 950 nm 160 mW 2 min continuous 107 Jcm2
Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
The next aim of the first part of this doctoral thesis was to explore whether LED
treatment could ameliorate in vitro cell proliferation under conditions of impaired
healing In the pursuit of this aim fibroblasts were cultured in medium supplemented
with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a
pilot study the amount of glucose necessary to inhibit normal growth was determined
In order to attain an important reduction of cell viability and decreased proliferation
rate a relatively high concentration of glucose (1667 mM) was necessary in
comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia
(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity
between both circumstances in vitro limited to 72 h whereas the human tissue of a
diabetic patient in vivo is chronically exposed to glucose
Treatment of the fibroblasts occurred in respect of the previously described results
with the same irradiation parameters and illumination procedure (chapter 2)
Accordingly green LED irradiation labelled as the most appropriate treatment for
116
irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in
the previous study (table 1)
Analysis of the cell proliferation by means of MTT measurements yielded a
significantly higher rate of proliferation in hyperglycaemic circumstances after
irradiation than in the control conditions (ie hyperglycaemic circumstances without
irradiation) Thus this outcome supported the stimulatory potential of green LED
irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a
considerable destructive hyperglycaemic medium
Finally although the results of the in vivo part of chapter 1 were persuasive and
encouraging they will not be further discussed in this summary of part I as it was not
aimed in this doctoral thesis to investigate the wound healing process in vivo However
the results of this case study can be a valuable hold for future in vivo research
The possible clinical implications of these results and future research directions in the
scope of wound healing will be discussed below
Part II Analgesia
In the second part two studies investigated the effects of LED irradiation as a
potential intervention mode in one of the most important fields in physiotherapy
practice analgesia Chapter 4 describes the influence of LED treatment on changing
sensory nerve conduction characteristics of a human superficial peripheral nerve
Altering nerve conduction characteristics may not be the sole beneficial purpose to
attain with LED irradiation in view of analgesia but the advantage of nerve conduction
characteristics is that they are objective measurable physical variables and changes in
these characteristics provide a potential explanatory mechanism of pain inhibition by
LED treatment17
The results showed that percutaneous LED irradiation at feasible clinical parameters
can generate a significant decrease in NCV and increase in NPL for all recordings post-
treatment in comparison to the baseline measurement The data in the placebo group
did not reveal any significant difference in the same course of time Statistical analysis
General discussion
117
revealed significant differences between the experimental and the placebo group for
NCV as well as for NPL at all time-points of observation with exception of the NPL
recording immediately after finishing irradiation
It was also observed that the noted effects did not weaken as time progressed It can
be concluded that post-treatment conduction measurements should be extended in
time which is in accordance with the findings of some previous studies18-21 Clarifying
the point of time at which the effect extinguishes is necessary and clinically relevant
when treating pain by means of LED irradiation Besides obtaining the desired
neurophysiological effects ideally the optimal irradiation parameters should be
applied The most favourable dosimetric properties are not yet determined but based
on this study and previously described assays it can be speculated that the dosimetric
window is quite large
Regardless of these clinically important remarks the present findings allow to draw the
following conclusion LED irradiation at clinically applied densities can generate an
immediate and localized effect upon conduction characteristics in underlying nerves as
LED treatment results in lowering the NCV and augmenting the NPL Therefore the
outcome of this in vivo experiment assumes a potential pain relief by means of LED
treatment and justifies further research regarding its clinical effectiveness in laboratory
settings and at a clinical level
The fourth and final aim was to determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5
illustrates a clinical study observing the effect of LED treatment on a model
comprising experimentally induced DOMS in a healthy population The progress of
pain perception and peak torque was evaluated during 4 consecutive days commencing
on the day of DOMS induction The effect of infrared LED treatment at the light
parameters described (table 1) was assessed with regard to three different factors time
(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures
(VAS MPT and IPT) revealed no significant interactive effects of the main interaction
118
(timegrouppre-post) For the remaining interactions and for the main effects only a
few significant findings were relevant in view of the postulated hypothesis
Notwithstanding the absence of an apparent and overall statistically significant finding
the present results indicate favourable trends of LED treatment on pain as the means
of all VAS and MPT variables show a statistically nonsignificant general analgesic
effect of infrared LED irradiation expressed by lower subjective pain rates and higher
MPT values in the irradiated group In addition to the analgesic influence of LED an
augmented restoration of muscle strength was noted The lack of solid statistically
significant evidence for these beneficial effects of LED treatment upon DOMS-
associated pain can possibly be attributed to the small sample size in this study or even
to the size of the treatment effect in relation to the severity of the induced DOMS as
induction of severe DOMS can mask relatively small but apparent treatment
effects2223 A final possibility is that the results only become significantly different after
a prolonged treatment and follow up period as previous research demonstrated that
recuperation subsequent to DOMS induction can last up to 10 days24
It should also be noted that the described general analgesic effect of LED irradiation
was identical for the treated as well as for the control arm in the irradiated group
proposing that infrared LED might induce systemic effects 2526 However it needs to
be stressed that these results were not statistically significant
Regardless of the absence of statistically significant findings the mean values suggest a
potential role for infrared LED irradiation in the management of pain and functional
impairment associated with DOMS Notwithstanding this postulation future research
is absolutely required to establish the effectiveness of LED treatment to reduce pain as
well at the applied densities as for other dosimetric parameters
CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS
In the course of the past years during the process of the genesis of this thesis
therapeutic physical agents in general and phototherapeutic modalities in particular
became less important as physiotherapeutic modes of treatment than during the
preceding two decades The diminished use of these treatment modalities in the
General discussion
119
physiotherapy practice is to a certain degree a consequence of the controversial
research findings regarding the use of these physical agents This issue of controversy
led to less support for the use of these treatment modalities and a growing scepticism
regarding the effectiveness of these physical agents within the scope of the growing
climate of evidence-based practice A second responsible protagonist for the loss of
popularity of physical agents is linked with the current tendency within physiotherapy
emphasising active remedial therapy The establishment of this development was based
on various experiments mainly performed during the last decade demonstrating that
active treatment modalities are for numerous impairments and disabilities preferable to
more passive forms of therapy In Belgium the prevailing nomenclature which came
into use on 1 May 2002 went along with this tendency In the appendix to the Royal
decree of 14 September 1984 towards settlement of the nomenclature of medicinal
treatments concerning compulsory insurance for medical care and allowances the
personal involvement of the physical therapist during the physiotherapeutic session
was emphasized and it was even defined that massage physical techniques within the
framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal
application can only be remunerated when they are applied supplementarily and not as a sole therapy
This implies that passive treatment modalities should not be used as sole method of
treatment and should always be considered as an adjunct to an active treatment
program This development needs to be applauded in many cases such as various
painful musculoskeletal problems functional instability rehabilitation of neurological
patients re-activation of the elderly population psychomotor rehabilitation
cardiovascular and respiratory convalescence Nevertheless it would be erroneous to
entirely reject physical agents including LED treatment Based on the findings of the
above described experiments it needs to be stressed that for some purposes especially
within the scope of impaired wound healing LED irradiation could be a suitable
therapeutic measure This statement is founded on the results of part I of the present
thesis they provided satisfactory fundamental evidence for the advantageous effects of
LED treatment on a crucial exponent of the wound healing process namely fibroblast
proliferation The beneficial findings are the result of basic in vitro research As it is
120
inaccurate to simply extrapolate these results to the clinical practice the clinical use of
LED irradiation for wound healing needs to be preceded by purposive and specific in
vivo investigations to substantiate these basic research findings27
The case study described in chapter 1 indicates a foundation for further in vivo research
Visual appraisal of the surgical incision revealed (from the 65th day in the course of the
reparative process onwards) that the irradiated area -which initially showed inferior
epithelialization and wound contraction- showed a more appropriate contracture than
the control area characterized by less discoloration at scar level and a less hypertrophic
scar These clear beneficial effects of LED treatment on a human cutaneous wound
can serve as preliminary impetus for further research into the clinical applicability of
LED therapy although this case study is insufficient in order to guarantee a safe
correct and effective use of LED as a therapeutic modality
Despite these remarks it tentatively can be concluded that based on a detailed analysis
of the available data of the present in vitro studies and the given case report in
combination with the small number of previously published human studies the
beneficial effects of LED irradiation at the cellular level are obvious and therefore a
potentially favourable outcome can be assumed in clinical practice28-30 LED-
modulated stimulation of wound healing can be gradually and vigilantly implemented
clinically Nevertheless the real benefits of LED irradiation within the scope of wound
healing can only be established by additional clinical trials as thus far clinical
application and stipulation of dosimetry still occurs on a trial-and-error basis which is
not conducive to a generally accepted clinical use of LED To lend more credibility to
the treatment of wounds by means of LED irradiation and to expel the existing
controversy and scepticism surrounding this topic in vivo investigations on wound
healing using a number of different animal models and adequately controlled human
studies are necessary In addition these studies should be performed preferably on a
population suffering from impaired healing as a consequence of diabetes mellitus or as
a result of any other debilitating reason because as posed by Reddy et al3132 and as
mentioned above light has possible optimal clinical effects in the treatment of healing-
resistant wounds
General discussion
121
Drawing general conclusions and formulating clinical implications for analgesia is
obviously less manifest first of all because only a limited number of possible
mechanisms of action in order to obtain analgesia were highlighted and secondly
because both studies did not come to a joint or complementary conclusion The
outcome of the first study revealed that LED treatment lowers the NCV and augments
the NPL resulting in a slower stimulus conduction and consequently a reduced number
of sensory pulses per unit of time Thus it could be assumed that LED induces pain
relief but the results of the study describing the effect of LED treatment on
experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy
In addition it needs to be emphasised that the first study (chapter 4) measured the effect
of LED irradiation on the large myelinated Aβ afferents A noteworthy question and
meanwhile a stimulus for future investigation is whether the measured effects can be
extrapolated from these sensory nerve fibres to the actual nociceptive afferents
notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of
these nociceptive pathways relies on laser-evoked potentials which selectively activate
Aδ-fibres and C-fibres3334 This technique was presently not available therefore a
standard sensory nerve conduction study was performed
Whereas stimulation of wound healing by means of LED irradiation can be cautiously
implemented in the clinical practice at this stage it is too early to promote LED
irradiation as a treatment modality for pain To make this possible it is essential to
conduct numerous studies with regards to the use of LED in the field of analgesia
Future research should focus on fundamental investigations in order to discover the
underlying mechanisms and physiological basis of pain modulation utilizing LED
treatment Furthermore the evaluation of the appropriateness of DOMS as an
experimental model of pain is an important prospect to consider as validation of this
model would enhance the ability to study various modalities for their potential effects
on pain Irrespective of the difficulties regarding standardisation of the research
population and evaluation of soreness inextricably linked with clinical pain studies the
122
ultimate objective of future research should be the establishment of the effectiveness
of LED irradiation to reduce pain of miscellaneous origin in a clinical setting
Regardless of the encouraging results of the described studies and besides the earlier
proposed specific directions for future research (directed towards wound healing or
pain relief) it is necessary in the interest of the patientrsquos well being and to the
advantage of the prospective clinical use of LED to highlight a few more issues for
future research Therefore one has to deal with some limitations of the performed
investigations A first limitation concerns the fact that only two mechanisms of LED
action were investigated (notably changed fibroblast proliferation and alteration of the
nerve conduction characteristics) So one can conclude that for further and better
understanding of the mechanisms of action it is necessary to perform more basic
research Answering the questions regarding the functioning of LED irradiation will
simplify the evaluation and reinforce the interpretation of the obtained results and
ultimately contribute to a more widespread and well definded acceptance of the use of
LED in clinical settings
A second general limitation of this doctoral thesis is the substantial difference in the
used external dosimetric parameters between the different chapters and even within
one and the same study (illustrated in table 1) this complicates the comparison
between the different trials In each trial the dosimetry was individually ascertained
based on previous studies within the given field As not for every application the same
dosimetry is suggested in literature a range of dosages were used Another important
factor in deciding on the dosimetry was the clinical applicability of the dosage as it is
useless to investigate the appropriateness of a treatment modality at a clinically
unrealistic dose As a result of this limitation the current findings do not fully
contribute to the explanation regarding the ideal parameters one should use although
this was not set as a principal purpose Based on this thesis and previously described
assays it can be speculated that the possible window for these parameters is quite large
the ideal irradiation parameters and proper timing or sequencing of LED irradiation
General discussion
123
for example to the various phases of wound healing and to different painful conditions
are therefore possibly unattainable
The establishment of an appropriate dosimetry should also consist of investigating the
absolute and relative penetration depth of LED irradiation into human tissue This is
less crucial within the scope of wound healing but it is of key importance while
treating deep-seated tissue (eg nerve fibres muscles circulatory components et
cetera)
Finally this thesis only investigated the efficiency of LED in a very limited number of
conditions notebly wound healing and pain Within the scope of physiotherapy and
medicine in general there are numerous other purposes for which LED irradiation is
promoted such as oedema arthritis miscellaneous orthodontic applications seasonal
affective disorder neonatal jaundice photodynamical therapy et cetera2835-41
In summary additional work on establishing proper dosimetry and identifying the
biochemical or photobiologic phenomena that are responsible for improving wound
healing and reducing pain or even other effects within a broader spectrum of
conditions remains to be done in order to answer unreciprocated questions Until that
time the potential clinical usefulness and actual value of LED irradiation for wound
healing and even to a larger extent for analgesia should always be approached with
appropriate professionalism and even caution
FINAL CONCLUSION
LED devices are promoted for clinical use but the currently available scientific
documentation regarding effectiveness of this physical agent is rather scarce Through
providing scientific support for the biostimulatory and analgesic effectiveness of LED
irradiation this doctoral thesis attempted to bridge in some degree this gap
The conducted studies revealed that LED irradiation undeniably has potential
beneficial effects on wound healing and to a lesser degree within the scope of
analgesia However based on the present results it can be corroborated that light
124
therapy in the guise of LED irradiation is not magic but these results can raise some
corrective doubts in fundamental disbelievers and antagonists
Nevertheless we have to join the queue of scientists who have found beneficial results
but cannot elucidate with certainty how this outcome was established Thus although
the present results are encouraging a continuing development and integration of new
knowledge based on further research is necessary in various domains of intervention
Therefore several directions for future investigations were proposed in order to cover
as many existing gaps and to answer the utmost number of remaining questions as
possible Still one ought to be aware not to carry future fundamental research at a
disproportional level and the inevitable quest for mechanisms of LED action should
not hypothecate the potential clinical value implying that at a certain point it should be
appropriate to make the transfer from science to the application of the available
knowledge in clinical practice
The described findings regarding LED irradiation are comparable to the results of
previously published studies performed with other light sources Consequently as
postulated by some LED providers it can be speculated that the biological response of
tissue to light irradiation can probably not be equated merely to a light source but
rather to a broad photo-energy window
General discussion
125
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20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
126
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25 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
26 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
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30 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255
32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287
33 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-28
34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856
35 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
36 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
37 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263
39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64
40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74
General discussion
127
41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press
NEDERLANDSTALIGE SAMENVATTING
Nederlandstalige samenvatting
131
NEDERLANDSTALIGE SAMENVATTING
Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de
tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te
vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie
van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen
Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest
enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is
ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als
op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang
van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve
interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken
Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van
ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen
moeilijk te verantwoorden
Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin
de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere
overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit
methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan
consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in
de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen
laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in
de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag
aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies
inclusief het gebruik van licht
Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat
de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde
doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en
dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht
komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt
geloosd
132
De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo
lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en
gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te
anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te
voorkomen lijkt een gerichte en rationele a priori aanpak conform de
wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen
Te meer daar grondige literatuurstudie leert dat men ten behoeve van de
werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie
zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser
De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de
extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is
geboden De introductie van alternatieve lichtbronnen in de huidige
kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk
weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus
eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood
naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based
gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder
binnen de domeinen van haar potentieel beloftevolle klinische toepassingen
wondheling en analgesie
Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de
wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op
prominente protagonisten van de wondheling de fibroblasten werd getracht het
fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten
zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de
proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd
respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand
waarbij de normale celgroei werd verstoord
In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het
effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische
Nederlandstalige samenvatting
133
data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden
verklaard door het gebruik van een inadequate LED dosering en anderzijds een
methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie
vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de
precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en
inter-tester variabiliteit tot gevolg
In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde
onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de
bestralingsparameters (dosering) De effecten van de drie verschillende LED
golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten
werden hierbij geanalyseerd door middel van een meer betrouwbare en minder
subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-
yl)-25-diphenyl tetrazolium bromide (MTT)
De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir
effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden
tevens een basis voor meer coherente en relevante inzichten aangaande de globale
bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)
Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het
wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend
karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp
rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde
vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De
fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met
extreem hoge concentraties glucose Deze modificatie van het medium staat model
voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische
praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan
vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de
gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en
proliferatie
134
Het tweede deel van dit proefschrift exploreert het domein van het potentieel
analgetisch effect van LED binnen de kinesitherapie aan de hand van twee
fundamentele onderzoeken
In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere
sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele
hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en
een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale
verklaring van een analgetisch effect van het medium zou kunnen belichamen
Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)
meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf
verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen
aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke
significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de
negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele
hypothese
Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend
fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale
pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp
van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische
krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED
behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier
keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED
op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd
(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde
isokinetische krachtmeting en een registratie van de waargenomen spierpijn De
spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met
behulp van een kwantitatieve hand-hold algometer
Analyse van de bekomen data bracht geen significante verschillen tussen de
controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden
Nederlandstalige samenvatting
135
kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED
behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van
de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de
proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene
afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine
proefgroep die werd onderzocht enof door de grootte van het behandeleffect in
verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers
een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up
kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel
absolute omzichtigheid geboden en moet deze visie louter als speculatief worden
beschouwd
Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de
kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de
laatste jaren aanzienlijk afgenomen
De positieve resultaten van de verschillende in vitro studies in het kader van wondheling
vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing
vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel
gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een
belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek
Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie
Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme
onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch
complementair resultaat Verder onderzoek ter exploratie van de mogelijke
onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom
onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op
termijn wetenschappelijk te rechtvaardigen
136
LED tovenarij trend of therapie
LED mag geen magische krachten worden toegemeten maar verdient het lot van een
kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een
wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen
zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke
indicaties
Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds
the darkness has always got there first and is waiting for it
(Terry Pratchett Reaper Man 1991)
IX
ACKNOWLEDGEMENTS
I wish to thank those people who supported me over the years and who helped me to
shape my life and work
First of all I would like to express my gratitude towards my promotor prof dr D
Cambier as without his encouraging words criticism inspiration and unremitting
support I would still be floundering about the contents of chapter 1 Thank you for
your good advice when I needed it the most
The members of the supervisory committee prof dr M Cornelissen prof dr M De
Muynck and prof dr G Vanderstraeten thank you for your assistance and helpful
feedback during the process of formation of this thesis
I also gratefully acknowledge the external members of the examination board prof dr
P Calders prof dr M Dyson prof dr P Lievens and prof dr K Peers for their
constructive reflections which contributed to the improvement of this thesis
I am greatly indebted to my special mentor prof dr J Anders of the Uniformed
Services University of Bethesda Maryland for the research suggestions she made as
well as for her unlimited belief in the value of my work
I wish to thank prof dr L Deridder for providing access to the laboratory of
Histology the Centre of Sports Medicine of the Ghent University Hospital for
allowing me to use their equipment as well as MDB-Laser Belgium for generously
providing the light emitting diode equipment
Sincere appreciation is extended to the volunteers that participated in this study and to
Tom and Roel for their valuable technical assistance in the collection of the data as
well as for their useful input into the research design of the investigation described in
chapter 5
X
Warm thanks go to the colleagues of the department of Human Anatomy
Embryology Histology and Medical Physics for providing the culture medium for the
technical support for the helpful discussions and principally for the amusing pastime
aseptic chats
In addition I also want to thank my colleagues of the associated institute Kinesitherapie
Gent and above all the colleagues of the department of Rehabilitation Sciences and
Physiotherapy Ann Axel Barbara Bart Bert Bihiyga Bruno Carine Damien Els2
Erik Frike Helga Inge James Karlien Kathy Kat(h)leen Koen Marc Martine
Mieke Paris (Nele) Patrick Roland Sophie Stefan Stijn (Veerle) Wiene Wim and
Youri thank you for the organisation and your attendance at many memorable
sidelines such as the survival-weekend the first department-day Fata Revaki our
legendary Thursday-sport activities and Friday-afternoon coffee breaks St Nicholas
visit skiing holiday New Yearrsquos lunch with quiz bowling spinning the introduction of
ldquosubetelyrdquo during our conspicuous presence at WCPT 2003 et cetera We shared many
treasured moments thanks to you a common working day often turned out to be very
pleasant I know that it will be impossible to find a comparable team of colleagues to
work with in the future
I especially want to thank Barbara to remind me on a regularly basis of my deadlines
to listen to my grieves and joy and to be willing to offer me a window-seat in our
office Kurt (although you abandoned at a certain moment) for solving my computer
problems Pascal for assistance with the statistical analyses Lieven for your motivating
interest and finally Fabienne Tine and Kim as loyal and appreciated friends who
worn-out several sports shoesbathing suits to supply in the weekly portion of sports I
needed to remain physically and mentally fit
I also extend my appreciation to my family and friends for their interest in my research
activities permanent mental support for the adoption of the surviving chickens but in
particular for looking after Louka and for the numerous relaxing moments Special
thanks are going to Katelijne and Frank for the delicious hot meals on lonely evenings
XI
Thomas Stijn and Johan thank you for the repeated (mostly futile) attempts to
convince me to do something together Sebastiaan each time during the past few years
when I doubted about the sense of my work it was your ridiculous story about a man
who wanted to invent superglue but instead invented the well-known yellow post-it
which stimulated me to continue my scientific quest
Of course I owe most gratitude to Luc my most devoted supporter Dearest I know
that since august 2004 you lived a solitary life in Dubai Although I think it was
possibly easier not to live under the same roof with me these last stressful months I
am aware that it was very difficult for you not to be able to play with Louka and to
miss some precious months of her life
Louka thank you for your radiant smile and daily baby speeches I am sorry that you
had to miss your daddy I promise that we will be reunited very soon
Elke Vinck
Ghent March 2006
GENERAL INTRODUCTION
General introduction
3
BACKGROUND
The use of light for therapeutic purposes reaches far back in time Current interest for
photomedicine with his its biological and medical effects relies fundamentally on two
major evolutions in the given field (1) the research results regarding the use of
ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in
the light amplification by stimulated emission of radiation (laser)-technology The production
of the first laser the ruby pulsed laser was rapidly succeeded by the development of
the helium-neon laser and other lasers like the argon the neodymium-glass and the
neodymium-yttrium-aluminium-garnet lasers1
As in the mid-1990s semiconductor and diode-based lasers gained popularity the
principally massive gas and dye lasers were rendered obsolete Therapeutic light
technology further continued to evolve and todayrsquos therapeutic light source is as likely
to be a light emitting diode (LED) or polarized light as a semiconductor or diode
laser1
Technological advancement and variation of the light sources necessitate a
concomitant update and revision of research in the respective domains of application
Unfortunately this logical and rational necessity has rarely been fulfilled From a
historical perspective this lack of appropriate research has led to disenchanting
evolutions in the use of light especially in physiotherapy The experience exists in this
medical field that light sources were promoted and commercialised for a vast regimen
of indications without foregoing scientific backup Consequently research developed
often after the commercial introduction in physiotherapy As these investigations
frequently gave rise to conflicting results for certain indications scepticism arose and
the use of the given modality knew a waning popularity for all its indications The final
result of such an inappropriate frame of promotion commercialisation and research is
a growing clinical disuse of a given modality even for motivated indications In view of
the actual increasing interest in LED treatment and based on former ascertainment
one has to state that a literature review for the given source reveals that research
mostly covers only low level laser (LLL) studies23 Although recently a number of
papers can be noted that report on the effects of LEDs and polarized light still
4
numerous source-specific-questions need to be answered as research concerning
mechanisms of action and efficacy of the current light sources remains limited in view
of a substantiated clinical application4-17
The reason for the contemporary light-oriented interest in physiotherapeutic practice
for LED devices is in essence based on several advantages of LED in comparison with
LLL For example the use of LEDs is esteemed to be safer as the delivered power
does not damage tissue LEDs can be made to produce multiple wavelengths thereby
stimulating outright a broader range of tissue types and probes that cover a large
treatment area are available18 In addition from a commercial point of view LEDs are
far more interesting as they are a good deal cheaper than laser diodes and they have a
long life span as these solid devices stand robust handling
As a result of the above-mentioned lack of literature on LED some providers of these
devices have taken for granted that the biological response of tissue to light irradiation
cannot be equated merely to a light source They declare that a given response solely
depends on the extent of absorption of radiated light by the tissue19 Consequently
these providers state that it is acceptable to extrapolate scientific findings of LLL
studies for explaining the mechanisms of action and detailing the efficacy of LED and
other alternative light sources Thus actually without appropriate scientific support
equal biological effects are attributed to LED as to LLL Nevertheless prudence is
called for such an extrapolation firstly because it is irrespective of the mentioned
dissimilarities and by simple projection one ignores a number of physical differences
between LLL and LED (eg coherence and degree of collimation or divergence)
Secondly LLL therapy is still not yet an established and evidence-based clinical tool20
Notwithstanding the historical efforts there still remains a considerable amount of
ignorance scepticism and controversy concerning the use and clinical efficacy of
LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed
parameters for irradiation as well as to the difficult objective measurement of possible
irradiation effects and even to the exceptional range of unsubstantiated indications for
General introduction
5
which light therapy was promoted27-29 A lack of theoretical understanding can also be
responsible for the existing controversies as the evaluation and interpretation of
research results would be simplified largely when the appropriate knowledge about the
mechanisms of light action would be available
LLL literature can undoubtedly be used as basis for research on LED and as a
comparative reference for these given investigations However to guarantee evidence-
based use of LED within physiotherapy the need for specific research in view of an
accurate consumption of LED is definite especially for potential promising clinical
applications in physiotherapy according to LLL literature mainly wound healing and
analgesia3031
Hitherto the most substantial research concerning the use of LED for improvement
of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded
under the influence of prolonged exposure to microgravity (eg during long-term space
flights) and in case of absence of exposure to sunlight such as in submarine
atmospheres they performed wound healing experiments for military application in the
given circumstances3233 In vitro experiments revealed that LED treatment increased
proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA
synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and
osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies
demonstrated therapeutic benefits of LED in speeding the early phase of wound
closure and in changing gene expression in a type 2 diabetic mouse model183234
Human studies noted 50 faster healing of lacerations a return of sensation and
increased tissue granulation as a result of LED irradiation1833
Associates of the Rehabilitation Sciences Research Group of the Ulster University in
Northern Ireland extensively investigated the effectiveness of light in the treatment of
pain The emphasis was laid primarily on the analysis of the effects of various low level
laser light sources35-44 However in the year 2001 two studies gave an account on the
efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46
6
noticed relatively long-lasting neurophysiological effects a significant change of the
nerve conduction characteristics (decrease of the negative peak latency) was mediated
by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a
comparable multisource diode device was ineffective in the management of delayed-
onset of muscle soreness (DOMS)
Despite the major value of these described trials a definitive answer regarding the
ability of LED in influencing wound healing or pain is not forthcoming cardinally
because a number of aspects are not yet investigated Consequently more research is
required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the
formerly mentioned scepticism regarding the effectiveness of a medium and possibly
to the undeserved fall into disuse of the treatment modality which happened in a way
with LLL therapy
PHYSICAL CHARACTERISTICS
This chapter supplies a short but comprehensive review of opto-physics A brief
description of the physical characteristics of the LED source used is essential as the
physical properties of light play an important part in the ultimate efficacy of treatment
According to the International Electrotechnical Commission (IEC 60825-1) an LED
can be defined as
Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by
radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process
of spontaneous emission1947
The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO
preprototype MDB-Laser Belgium) This illustration shows that a probe consists of
32 single LEDs disseminated over a surface of 18 cm2
General introduction
7
Figure 1 LED device and three available probes (infrared red and green)
Three highly monochromatic probes were available each emitting light of a different
wavelength within the above-defined range (table 1)2748 The wavelength of the light
emitted and thus its colour depends on the band gap energy of the materials forming
the p-n junctiona This light property is a key determinant to obtain maximum
photochemical or biological responses as light absorption by tissue molecules is
wavelength specific27 Only by absorbing radiation of the appropriate wavelength
(namely the wavelengths equal to the energy states of the valence electrons)
photoacceptor molecules will be stimulated resulting in a direct photochemical
reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750
8
absorption spectrum of a particular molecule is limited absorption often only occurs
over a waveband range of about 40-60 nm274851 Nevertheless the absorption
spectrum at cell or tissue level is broad because cells are composed of many different
molecules
Besides its influence on the absorption by means of tissue molecules there is a crucial
link between wavelength and penetration depth of the irradiated light Penetration into
tissue decreases as the wavelength shortens hence green light penetrates less than red
light which at his turn penetrates less into tissue than infrared light2748 Detailed
principles of light penetration will be discussed below
The LED device used emits non-coherent light In the 1980s the observed biological
responses after laser irradiation were generally thought to be attributable to the
coherenceb of the light485253 Though currently the clinical and biological significance
of coherence is seriously questioned54 According to several authors coherence does
not play an essential role in laser-tissue interactions firstly as it was proven that both
coherent and non-coherent light clinically show equal efficacy75556 Secondly as
according to some authors almost immediately after transmission of light through the
skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et
al1957 state that both findings are incorrect coherence is not lost in tissue due to the
phenomenon of scattering and non-coherent light is not as efficient as coherent light
This lack of consensus makes it necessary to mention whether or not light is
coherent2758
Further decisive characteristics to accomplish phototherapeutic efficacy are the power
exposure time output mode and beam area Based on these parameters both
irradiancec and radiant exposured can be calculated According to numerous authors
some of these parameters are more crucial than others to determine whether
b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48
c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48
d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248
General introduction
9
absorption of light will lead to a photobiological event192728485455 However the
literature yields several controversial findings as not all authors attribute an equal
importance to a given parameter For example according to Nussbaum et al59
irradiance was the determinant characteristic in the biomodulation of Pseudomonas
aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49
found that in order to stimulate tissue cell proliferation a specific combination of
irradiance and exposure time are more important than the actual radiant exposure Low
et al3940 on the contrary highlighted the critical importance of the radiant exposure in
observing neurophysiological effects Whereas Mendez et al60 reported that both
parameters influence the final results of light therapy
Koutna et al61 even suggested that the output mode of light applications plays a more
prominent role in the treatment outcome than the wavelength of the used light source
Nevertheless this finding could not be confirmed by other research results Besides
more controversial findings have been published regarding the output mode although
the repetition rate in a pulsed mode was considered as an important treatment
parameter several investigations failed to prove its value19272840414461-64
Based on these findings it was opted within the investigations of this doctoral thesis to
irradiate in a continuous mode The remaining dosimetric parameters (wavelength
exposure time and power) depended on the purpose of each investigation they are
described in the respective chapters The data necessary for the calculation of the
radiant exposure for the equipment used in the respective trials are summarized in
table 1
Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High
Infrared 950 80 120 160 Red 660 15 46 80
Green 570 02 42 10
10
The radiant exposure of the used LED can be calculated as follows65
RE =
Radiant Exposure [Jcm2]
T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]
PRE = α S T
α = 006 (continuous mode) or
003 (pulsed mode)
The parameters commented on so far can be considered as the external dosimetry
involving all parameters directly controlled by the operator limited by the apparatus
used Furthermore there is the so-called internal dosimetry referring to (1) several
physical phenomena (reflection transmission scattering and absorption) influencing
the light distribution within the tissue during energy transfer (2) the optical
characteristics of the irradiated tissue as well as (3) the relation between the external
dosimetry and these respective elements5466
This internal dosimetry determines to a considerable extend the penetration of light
into tissue Penetration can be defined as the tissue depth at which the radiant
exposure is reduced to 37 of its original value1948 However this definition only
accounts for the absolute penetration depth resulting in direct effects of light at that
depth In addition there is also a relative penetration depth leading up to effects
deeper in the irradiated tissue and even in certain degree throughout the entire
body1967 These so-called systemic effects can be caused by chemical processes initiated
at superficial levels at their turn mediating effects at a deeper tissue level57
Involvement of several forms of communication in the tissue such as blood circulation
and transport of transmitters or signal substances is possible1967 This means that light
sources with poor absolute penetration do not necessarily give inferior results than
those with a good absolute penetration19
In the same context it should be noted that calculation and even measurement of the
exact light distribution during irradiation is highly complicated principally as tissues
have complex structures and also because the optical properties of tissues vary largely
inter-individual2768
General introduction
11
Studies regarding actual penetration depth of LED light are scarce consequently the
knowledge on the topic of penetration depth of LED light is based on literature
originating from LLL research19 These findings established with various LLL sources
revealed that there is an obvious relation between penetration depth and
wavelength27486769-71
Three final remarks can be made on the dosimetry First of all it should be noted that
partly as a result of the above-mentioned contrasting findings on dosimetry ideal light
source characteristics for effective treatment of various medical applications are not yet
established and probably never really will be28 Therefore in the attempt to offer
sufficient guidelines for correct use of treatment parameters one should always try to
provide detailed description of light source properties used in any trial so the
practitioner can interpret the scientific results adequately and accordingly draw the
correct conclusions for his clinical practice
A second comment is based on the mentioned possible influence of the external and
internal dosimetric parameters on the photobiological effectiveness of light the
intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation
between radiated wavelength and penetration depth19546572 So it should be
emphasized that caution is recommended when comparing research results of light
sources with different wavelengths or other dissimilar dosimetric parameters
A third and final remark considers the extrapolation issue Comparison of the
therapeutic usefulness of the same light source used on different species should occur
cautiously So simply extrapolating the dosage used for one species to another is
inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo
clinical practice is inappropriate So purposive and specific research is the prerequisite
to produce safe and correct use of light as a therapeutic modality27
12
MECHANISMS OF ACTION
In the past decennia several mechanisms of action for biostimulation and pain
inhibition have been proposed and investigated73 Research was primarily based on
studies at the molecular and cellular levels and as a second resort investigations
occurred at the organism level resulting in numerous possible explanatory
mechanisms272858
It is the common view that light triggers a cascade of cellular and molecular reactions
resulting in various biological responses Thus different mechanisms of whom the
causal relationships are very difficult to establish- underlie the effects of light3448557475
To illustrate this complex matter the various mechanisms of action will be summarised
by means of a comprehensive model (fig 2) Detailed discussion about the different
individual components of the proposed model and other effects than those regarding
wound healing or analgesia were not provided as this was beyond the scope of this
general introduction
As depicted in figure 2 exposure to light leads to photon absorption by a
photoacceptor molecule causing excitation of the electronic state or increased
vibrational state of the given molecule275173 This process is followed by primary
photochemical reactions7475 Several key mechanisms have been discussed in the
literature Respiratory chain activation is the central point and can occur by an
alteration in redox properties acceleration of electron transfer generation of reactive
oxygen species (namely singlet oxygen formation and superoxide generation) as well as
by induction of local transient heating of absorbing chromophores192848515576-83 It is
supposed that each of these respective mechanisms plays a part in obtaining a
measurable biological effect It is yet not clear if one mechanism is more prominent
and decisive than another nevertheless recent experimental evidence has revealed that
mechanisms based on changes in redox properties of terminal enzymes of respiratory
chains might be of crucial importance2848517679
The primary mechanisms occurring during light exposure are followed by the dark
reactions (secondary mechanisms) occurring when the effective radiation is switched
General introduction
13
off2851 Activation of respiratory chain components is followed by the initiation of a
complicated cellular signalling cascade or a photosignal transduction and amplification
chain associated with eg changes in the cellular homeostasis alterations in ATP or
cAMP levels modulation of DNA and RNA synthesis membrane permeability
alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87
The sequence of events finally results in a range of physiological effects essential for
the promotion of the wound healing process for supplying analgesia or other
advantageous responses (acceleration of inflammatory processes oedema re-
absorption increased lymph vessel regeneration or increased nerve
regeneration)12181927486188-93
Photostimulation of the wound healing process can be mediated by increased
fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis
and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations
have been especially focussed on fibroblasts but other possible physiological effects
attributing to an accelerated wound healing were also observed suppression and
alteration of undesirable immune processes increased leukocyte activity new
formation of capillaries increased production of growth factors and enzymes while
monocytes and macrophages can provide an enlarged release of a variety of substances
related to immunity and wound healing1619277376
As pain and nociception are even less understood than wound healing the possible
mechanisms in obtaining pain relief by the use of light are less underpinned However
it is established that light therapy influences the synthesis release and metabolism of
numerous transmitter signal substances involved in analgesia such as endorphin nitric
oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these
neuropharmacological effects there is experimental evidence for diminished
inflammation decreased C-fibre activity increased blood circulation and reduced
excitability of the nervous system1927848899
One should be aware that a large amount of research regarding the possible
mechanisms of light action was conducted at the cellular level The described cascade
of reactions at the organism level is possibly even more complex as in contradiction to
14
the in vitro situation in vivo a range of supplementary interactions can influence the
sequence of effects and accordingly the final responses Besides it needs to be
mentioned that this summary did not take into account the origin of the light or the
external dosimetry thus the description is based on investigations performed with
various light sources and different dosages
Figure 2 Model summarizing the identified mechanisms of light action
Secondarymechanisms
Primary mechanisms
Final effects
Trigger
Stimulated wound healing Analgesia
Exposure to light
Photon absorption by photoacceptors
Respiratory chain activation
Accelerated electrontransfer
Reactive oxygen generation
Heating of absorbing chromophores
Altered redox properties
darr inflammation uarr oedema resorption
uarr lymph vessel regenerationuarr blood circulation
Photosignal transduction and amplification chain
uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation
darr C-fibre activity darr nervous excitability neuropharmacological effects
General introduction
15
Regardless of the large number of previous investigations identification of underlying
mechanisms of light action remains an important issue as these are not yet fully
understood and because probably not all mechanisms of action are currently
identified Convincing explanation of the mechanisms in normal as well as in
pathological tissue could banish the existing suspicion concerning the use of light as a
treatment modality2732547678
AIMS AND OUTLINE
The introduction of LED in medicine and in physiotherapy more specifically requires
particular scientific research especially within the fields of its clinical potential
application wound healing and analgesia The above described gaps in literature
regarding the use of LED laid the foundation of this doctoral thesis
Consequently the general purpose of this thesis is to explore a scientific approach for
the supposed biostimulatory and analgesic effect of LED and to formulate an answer
in view of an evidence-based clinical use of this treatment modality
The detailed objectives can be phrased as follows
Aim 1 To assess the biostimulatory effectiveness of LED
irradiation under normal in vitro conditions
Aim 2 To investigate the value of LED treatment to ameliorate
in vitro cell proliferation under conditions of impaired healing
Aim 3 To examine the effectiveness of LED in changing the
nerve conduction characteristics in view of analgesia
Aim 4 To determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting
Part I investigates the influence of LED on wound healing In pursuit of the first aim
chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial
16
measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these
cells needs to be considered as an exponent of the wound healing process as
fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase
and early remodelling100 Secondly an in vivo case study exploring the postulation that
LED irradiation could accelerate and ameliorate the healing of a surgical incision was
described
The results contrasted sharply with the findings of the in vitro part Two fundamental
causes were proposed in order to explain the different biological effect of LED
irradiation observed in vitro and in vivo the used irradiation parameters and evaluation
method
The experiment described in chapter 2 endeavoured to explore these considerations A
similar study was therefore performed but as distinctive characteristics different light
source properties an adapted irradiation procedure and the use of a colorimetric assay
based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the
counting of the cells were used
As stimulation of the wound healing process is virtually mainly indicated under
conditions of impaired healing (resulting in a situation which threatens to become
chronic and debilitating) proper attention for this matter is warranted192855 Besides
the medical consequences the costs involved with impaired healing yield also a socially
relevant problem to tackle Impaired healing will become even more common as the
world population continues to age After all senescence of systems and age-committed
comorbid conditions are commonly the culprits responsible for poor wound healing101
Thus finding cost-effective time-sparing non-invasive and practical treatment
modalities to cure wounds is a necessity
Aiming to assess the biostimulative effects by means of LED in these circumstances a
third study was conducted with respect of the previous results regarding irradiation
parameters and cell proliferation analysis The irradiation experiment described in
chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose
This medium modification serves as a pattern for cell proliferation in diabetic patients
General introduction
17
a population for whom stimulation of the wound healing process is a clinical relevant
feature
In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored
by means of two studies A first investigation (chapter 4) evaluated the influence of LED
on the sensory nerve conduction characteristics of a human superficial peripheral
nerve as a potential explanatory mechanism of pain inhibition by LED which is based
on the putative neurophysiological effects of this treatment modality The experimental
hypothesis postulated that LED generates an immediate decrease in conduction
velocity and increase in negative peak latency In addition it was postulated that this
effect is most prominent immediately after the irradiation and will weaken as time
progresses
The values of nerve conduction velocity and negative peak latency of a baseline
antidromic nerve conduction measurement were compared with the results of five
identical recordings performed at several points of time after LED irradiation
Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory
setting To guarantee an adequate standardized and controlled pain reduction study
there was opted to observe a healthy population with experimentally induced DOMS
Induction of DOMS has been described in a number of studies as a representative
model of musculoskeletal pain and stiffness because it can be induced in a relatively
easy and standardised manner the time course is quite predictable and the symptoms
have the same aetiology and are of transitory nature4445102-105
The treatment as well as the assessment procedure was performed during 4
consecutive days The first day isokinetic exercise was performed to induce pain
related to DOMS Subsequently the volunteers of the experimental group received an
infrared LED treatment and those of the placebo group received sham-irradiation
Evaluation of the effect of the treatment on perceived pain was registered by a visual
analog scale and by a mechanical pain threshold these observations occurred every day
18
prior to and following LED irradiation Eccentricconcentric isokinetic peak torque
assessment took place daily before each treatment
For the analysis of the results three different factors were taken into consideration
time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental)
In completion of this thesis the most prominent findings are summarized and the
clinical implications are discussed The general discussion also includes some future
research directions and a final conclusion
General introduction
19
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General introduction
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97 Abergel P Lam T Meeker C Castel C Dwyer R and Uitto J (1984) Biostimulation of procollagen production by low energy lasers in human skin fibroblast culturesJ Invest Dermatol 82(4)395
98 Abergel P Lam T Meeker C Dwyer R and Uitto J (1984) Low energy lasers stimulate collagen production in human skin fibroblast cultures Clin Res 32(1)16A
99 Shimoyama N Lijima K Shimoyama M and Mizuguchi T (1992) The effects of helium-neon laser on formalin-induced activity of dorsal horn neurons in the rat J Clin Laser Med Sur 1091-94
100 Kloth L McCulloch J and Feedar J (1990) Wound healing Alternatives in management USA FA Davis Company
101 Lanzafame R (2004) Revolutions and revelations J Clin Laser Med Surg 22(1)1-2 102 Ciccone C Leggin B and Callamaro J (1991) Effects of ultrasound and trolamine salicylate
phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-678 103 Craig J Cunningham M Walsh D Baxter G and Allen J (1996) Lack of effect of transcutaneous
electrical nerve stimulation upon experimentally induced delayed onset muscle soreness in humans Pain 67(2-3)285-289
104 Minder P Noble J Alves-Guerreiro J Hill I Lowe A Walsh D and Baxter G (2002) Interferential therapy Lack of effect upon experimentally induced delayed onset muscle soreness Clin Physiol Funct Imaging 22(5)339-347
105 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
PART I WOUND HEALING
CHAPTER 1
DO INFRARED LIGHT EMITTING DIODES HAVE A
STIMULATORY EFFECT ON WOUND HEALING FROM AN IN
VITRO TRIAL TO A PATIENT TREATMENT
Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Proceedings of SPIE 2002 4903 156-165
Chapter 1
28
ABSTRACT
Variable effects of different forms of light therapy on wound healing have been
reported This preliminary study covers the efficacy of infrared light emitting diodes
(LED) in this domain
Cultured embryonic chicken fibroblasts were treated in a controlled randomised
manner LED irradiation was performed three consecutive days with a wavelength of
950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each
treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2
The results indicated that LED treatment does not influence fibroblast proliferation at
the applied energy density and irradiation frequency (p=0474)
Meanwhile the effects of LED on wound healing in vivo were studied by treating a
surgical incision (6 cm) on the lateral side of the right foot of a male patient The
treatment started after 13 days when initial stitches were removed The same
parameters as the in vitro study were used but the treatment was performed five times
The healing could only be evaluated clinically the irradiated area (26 cm) showed a
more appropriate contraction less discoloration and a less hypertrophic scar than the
control area (34 cm)
The used parameters failed to demonstrate any biological effect of LED irradiation in
vitro although the case study on the other hand illustrated a beneficial effect
Keywords Light Emitting Diodes Fibroblasts Wound healing
From an in vitro trial to a patient treatment
29
INTRODUCTION
Various beneficial effects of lasers and photodiodes at relatively low intensities have
been reported involving treatment of neurological impairments12 treatment of pain3-5
treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the
enhancement of wound healing has been a focus of contemporary research11-16 It
seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing
remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the
basis of a questionnaire about low power laser (LPL) in the current clinical practice in
Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey
into clinical LPL experience in Flanders
Nevertheless there remains a considerable amount of ignorance scepticism and
controversial issues concerning the use and clinical efficacy of LPL even in the domain
of wound healing12152021 This is at least in part a consequence of the inability to
measure and control operating variables related to connective tissue repair and of the
wide range of suitable parameters for irradiation
Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent
characteristics which make their use in a clinical setting problematic including
limitations in wavelength capabilities and beam width The combined wavelength of
light optimal for wound healing cannot be efficiently produced and the size of
wounds which may be treated by LPLs is limited Some companies offer an
alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be
made to produce multiple wavelengths and can have probes with large surface area
allowing treatment of large wounds Still one can not accept this light source as an
alternative for LPL therapy based on the cited advantages without proper investigation
regarding its biostimulatory effects
The effectiveness of this possible alternative for LPLs must be studied in vitro and in
addition in animal models or in humans because the effects of LED at the cellular level
do not necessarily translate to a noticeable effect in vivo The small amount of previous
investigations demonstrate that LED effects are as difficult to isolate162223 as LPL
Chapter 1
30
effects and the results are conflicting just like the results in literature specific on the
use of LPL121520
The purpose of the first part of this study is to examine the hypothesis stating that
LED irradiation can influence fibroblast proliferation Therefore a comparison of the
proliferation from fibroblasts in irradiated and control wells was performed The in vitro
investigation was linked with an in vivo case study This part enquired the assumption if
LED irradiation could accelerate and ameliorate the healing of a surgical incision
IN VITRO INVESTIGATION
MATERIALS AND METHODS
The complete procedure from isolation to proliferation analysis was executed twice
(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of
control dishes The second trial consisted of 27 irradiated and 27 control dishes
Cell isolation and culture procedures
Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation
and disaggregating of the cells occurred with warm trypsin (NV Life Technologies
Belgium) according the protocol described by Ian Freshney (1994)24 The primary
explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies
Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1
Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life
Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies
Belgium) When cell growth from the explants reached confluence cells were detached
with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV
Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the
cells were removed from the culture flasks by trypsinization and counted by
hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc
From an in vitro trial to a patient treatment
31
NV Life Technologies Belgium) The fibroblasts were seeded at a density of
70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture
medium the cells were allowed to attach for 24 hours in a humidified incubator at
37degC
Properties of the Light Emitting Diode
Prior to LED treatment all dishes were microscopically checked to guarantee that the
cells are adherent and to assure that there is no confluence nor contamination The
dishes were divided randomly into the treated or the control group Medium was then
removed by tipping the dishes and aspirating with a sterile pipette Following the
aspiration 2 ml fresh medium was added and treatment started
A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser
Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-
1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32
single LEDrsquos For the treatments in this study an average power of 160 mW at
continuous mode was applied The irradiation lasted 6 minutes resulting in an energy
density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result
of the divergence in function of this distance the surface of the LED (18 cm2) covered
the complete surface of the used petriplates (215 cm2)
After these manipulations 3 ml medium was added to each dish followed by 24 hours
incubation
One LED irradiation was performed daily during three consecutive days according
this procedure Control cultures underwent the same handling during these three days
but were sham-irradiated
Proliferation analysis
After the last treatment a trypsination was performed to detach the cells from the
culture dishes followed by centrifugation Once the cells were isolated from the used
trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts
Chapter 1
32
within this suspension as reflection for the proliferation was quantified by means of a
Buumlrker Chamber or hemocytometry
The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-
Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab
Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on
the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator
using an inverted light microscope counted the number of cells in 25 small squares
In order to calculate the number of cells one should multiply the amount of cells
counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and
the dilution factor (the amount of Trypan blue suspended with the cells 21=3)
Statistical methods
The data were analysed statistical in order to examine the hypothesis that LED
irradiation enhances fibroblast proliferation They were processed as absolute figures
for both trials separately In a second phase the counted cell numbers were converted
in relative figures so the data of both trials could be analysed as the data of one test
These relative figures were obtained by expressing each figure as a percentage from the
highest figure (=100) of that trial and this for each assay separately
A Kolmogorov-Smirnoff test of normality was performed on the data followed by a
Mann-Whitney-U test when the test of normality was significant and otherwise a T-
test Differences were accepted as significant when plt005 For this analysis SPSSreg
100 was used
RESULTS
The descriptive data for both trials are depicted in figure I The mean number of cells
in trial A is higher than in trial B for the controls as for the treated wells There is a
mean difference of 1252500 fibroblasts between the controls and 1223000 between
the irradiated wells of trial A and B The averages of both trials show that in control
cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no
From an in vitro trial to a patient treatment
33
statistically significant difference could be found between the two groups in either trial
nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not
significant for trial A (p=020) nor trial B (p=020) Only the combined data from both
trials were significant (plt001) for normality Further analysis respectively T-test for
the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the
combined data (p=0474) revealed no statistical significant differences
DESCRIPTIVE DATA
1730000181750029530003070000
00E+00
50E+05
10E+06
15E+06
20E+06
25E+06
30E+06
35E+06
40E+06
Trial A Trial A Trial B Trial B
Mea
n n
um
ber
of
cells
Control
Irradiated
Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B
DISCUSSION
Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities
(lt500 mW) in vitro have been analysed by evaluating various factors involving
(pro)collagen production25-27 cell viability2829 growth factor production28 and
myofibroblast formation30 Fibroblast proliferation also is an important factor to
consider In accordance with wound healing fibroblasts fulfil an essential role especially
in the late inflammatory phase and the early granulation phase31 Despite the failure of
some studies to demonstrate beneficial effects of LPL irradiation on fibroblast
proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very
Chapter 1
34
significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -
208 ) Atabey et al35 also revealed a significant increase in cell number two or more
irradiations resulted in an increased fibroblast proliferation Several other studies
confirmed these positive findings25263637
The results of this present in vitro study indicate that LED treatment does not
influence fibroblast proliferation Although the dosimetric parameters (in particular the
arbitrary energy density of 32 Jcm2) used in this study are well within the
recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous
studies about LPL therapy raising enhanced fibroblast proliferation252634-37
Van Breugel et al36 gave a possible explanation for these controversial results
According to them the fibroblast proliferation is not inherent at the energy density
They provide evidence that independent of the energy density the power density and
the exposure time determine the biostimulative effects of LPL irradiation LPL with a
power below 291 mW could enhance cell proliferation while a higher power had no
effect
Some authors also argued that the absorption spectrum of human fibroblasts show
several absorption peaks and pointed out that a wavelength of 950 nm is far above the
highest peak of about 730 nm3638 At longer wavelengths they determined a general
decrease in absorption Despite these results several investigators pose biostimulative
effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or
even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is
ranged from 800 nm to 830 nm principally because of the presence of cytochrome
oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts
at lower wavelengths is restricted by an inferior skin transmission than at higher
wavelengths38
Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting
respective green and red light The 950 nm beam of light was used for its high power
density but according to a range of remarks mentioned above the effects of the two
other probes must be as well evaluated
From an in vitro trial to a patient treatment
35
Another factor one can not ignore is that besides fibroblast proliferation other
processes or morphologic changes were not analysed although several authors have
posed that those changes and processes could be responsible for the biostimulative
effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et
al30 for example described a massive transformation of fibroblasts into myofibroblasts
after LPL treatment These modified fibroblasts play an important role in contraction
of granulation tissue30 A second example is an increased (pro)collagen production
after low power light therapy25-27 which is also considered as a responsible factor for
accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast
proliferation3640
It may be wondered if the light sources mostly LPL in the consulted literature are
representative for the LED used in this study although this LPL literature is often
used for that purpose As in the early days of LPL the stimulative effects upon
biological objects were explained by its coherence the beam emitted by the Bio-Dio on
the contrary produces incoherent light Nowadays contradictory research results are
responsible for a new discussion the clinical and biological significance of coherence
The findings of some authors172341-43 pose that the coherence of light is of no
importance of LPL and its effects although the opposite has also been stated4445
Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be
accounted for the non-enhanced fibroblast proliferation in this trial
Another possible explanation for the absence of biostimulative effect is related to the
moment of analysis of the proliferation The evaluation one day after the last
irradiation did not allow a delayed enhancement of proliferation while it is determined
in numerous investigations that the effects occur more than 24 hours after the last
treatment273746 and that they weaken after a further undefined period of time34
The fluctuation in cell numbers between both trials despite the use of an identical
protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor
reproducibility of their technique In this study the fluctuations are attributable to the
counting of the cells by Buumlrker hemocytometer before seeding According to some
authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large
Chapter 1
36
variability48 and it is often difficult to standardize48 Overestimation of the cell
concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity
was contradicted by Lin et al50 moreover satisfactory correlations with flow-
cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay
for cell counting (MTT)51 were determined
An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it
provides accurate cell counts in a short period of time with less intervention from the
investigator52
These remarks and controversies point out the possible deficiencies from the used
proliferation analyses and the relativity from the obtained results Other analyse
methods and analyses from different cell processes and morphologic changes could be
considered for further investigation
IN VIVO INVESTIGATION
MATERIALS AND METHODS
The effects of LED on wound healing in vivo were studied by treating a postsurgical
incision A male patient received chirurgical treatment for the removal of a cyst
situated approximately 15 cm posterior from the lateral malleolus of his right foot For
removal of the cyst an incision of 6 cm was made The incision was sutured and 12
days after the surgery the stitches were removed Visual inspection demonstrated that
the healing process of the wound proceeded well but not equally over the whole 6
centimetres (figure II) Epithelialization and wound contraction appeared to have
progressed better in the upper part (approximately 3 cm) of the cicatrice than at the
lower part (covered with eschar) No evidence of infection was noted in either part
LED treatment started the 13th day The incision was treated partially the lowest part
(26 cm) with the inferior epithelialization and wound contraction was irradiated the
remaining 34 cm served as control area This control area was screened from radiation
with cardboard and opaque black cling film
From an in vitro trial to a patient treatment
37
The light source destinated for the treatment was the same device used for the in vitro
irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED
output parameters were identical with those applicated in the preceding in vitro
investigation In particular a continuous wave at an average power of 160 mW and 6
minutes of treatment duration corresponding to an energy density of 32 Jcm2 An
equal distance from the probe to the target tissue as from the probe to the culture
medium was respected A plastic applicant of according height guaranteed constant
distance of 06 cm from the surface of the skin
Figure II Surgical incision before the first treatment 13 days after initial stitching
Therapy was performed once a day during five consecutive days repeatedly at the same
time resulting in an extension of the duration of the in vitro therapy with two days
Visual macroscopic observations were accomplished 6 52 and 175 days after the first
treatment
Comparison of the cutaneous sensitivity at the irradiated area and the control area was
accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175
days after the first treatment A control measurement also occurred at the same region
Chapter 1
38
on the left foot The aesthesiometer used in this study consisted of five hand-held
nylon monofilaments with a length of 38 mm and varying diameter
Sensitivity threshold is traced by presenting a monofilament of a certain diameter
vertically to the skin The monofilament bends when a specific pressure has been
reached with a velocity proportional to its diameter Measurements allow mapping
areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified
from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest
filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished
protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to
finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the
filaments
RESULTS
Visual estimation at any point of time after irradiation divulged no occurrence of
problems with dehiscence or infection in either part of the wound During the five
days of therapy the irradiated area looked dryer than the control area After the last
irradiation this was no longer recorded
Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area
From an in vitro trial to a patient treatment
39
Figure III representing the first evaluation six days after the initial treatment
illustrates that the wound healing has evolved slightly in both parts Though the lower
irradiated part remains of inferior quality as regards to epithelialization and wound
contraction In the course of the reparative process the influence of light exposures
were registered At 52 days after the first irradiation beneficial effects of LED
treatment are clearly present (Figure IV)
Figure IV Surgical incision 52 days after initiating LED treatment
The irradiated area (26 cm) showed a more appropriate contracture than the control
area (34 cm) characterized by less discoloration at scar level and a less hypertrophic
scar A similar trend was noticed at a third visual observation 175 days after the initial
treatment At that moment no impairments at cutaneous sensitivity level were stated
and the sensitivity showed no differences between left or right foot nor between the
two areas of the cicatrice
Chapter 1
40
DISCUSSION
The results of this case study indicate that LED had a positive influence on wound
healing in humans as determined by visual observations Many investigators
examining the effects of LPL on wound healing by means of a range of observation
and treatment methods reported accelerated and enhanced wound healing8-10 others
described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and
LED21 studies were unable to repeat these results
The late but beneficial findings in this study seem to be to the credit of LED-therapy
Though several authors establish positive results in an earlier stage of the wound
healing process8-1020 one should question why the differences did not occur at the first
evaluation on day 6 An explanation can be found in the start of the treatment Most
investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so
they influence a first cellular and vascular reaction with the production of chemical
mediators of inflammation resulting in an enhanced collagen production9 tremendous
proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the
time the first treatment in this study took place the traumatized tissue was in an
overlapping stage between an almost finished inflammatory phase and a scarcely
initiated re-epithelialization and wound contraction phase At that moment an infiltrate
of fibroblasts is present So fibroblast proliferation a possible mechanism of the
biostimulative effect had already occurred and could no longer be influenced Growth
factor production and collagen deposition have also decreased at that stage
Granulation tissue formation and fibroplasia in the contrary are initiating by that time
Those prolonged and slow processes with belated results are of significant importance
for the course of the final stage of wound healing and for the outlook of the future
scar31
The experimental findings revealed that the sensitivity of the skin according to the
threshold detection method of Semmes and Weinstein was normal at all the
investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a
good and objective predictor of normal skin sensitivity No other LPL nor LED
studies investigating this quality of the skin were found
From an in vitro trial to a patient treatment
41
CONCLUSION
This study demonstrates that although LED application at the applied energy density
and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to
have beneficial biostimulative effects on wound healing in human skin confirmed by
the favourable re-epithelialization and contracture
These results are discussed in the context of other experimental findings but no
reasonable explanation for this discrepancy could be found The literature on wound
healing after LED treatment in animal models or in humans is presently very limited
and contradictory The diversity in used radiation parameters and the absence of
references on how the wounds were measured or evaluated or what the end point was
for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro
investigations are better standardised nevertheless these results show a number of
conflicts One can conclude that until today the controversial findings are characteristic
for many results obtained with light photobiomodulation
However the postponed favourable results in the case study confirm some facts of the
discussion Namely the short period of incubation 24 hours in the in vitro part of the
study can be responsible for the lack of enhanced fibroblast proliferation It also
confirms that other cell processes and morphologic changes possibly are responsible
for biostimulative effects in vivo other observation methods should be considered for
future in vivo experiments
Despite these remarks we believe that LED application on cutaneous wounds of
human skin is useful with a single flash daily at the dose applied in this study for at
least three days
Furthermore future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Chapter 1
42
ACKNOWLEDGMENTS
The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the
material necessary for this study as well as Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
From an in vitro trial to a patient treatment
43
REFERENCES
1 G Baxter D Walsh J Allen A Lowe and A Bell Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79(2) 227-34 (1994)
2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)
3 J Stelian I Gil B Habot et al Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy J Am Geriatr Soc 40(1) 23-6 (1992)
4 A Honmura A Ishii M Yanase J Obata and E Haruki Analgesic effect of Ga-Al-As diode laser irradiation on hyperalgesia in carrageenin-induced inflammation Lasers Surg Med 13(4) 463-9 (1993)
5 D Cambier K Blom E Witvrouw G Ollevier M De Muynck and G Vanderstraeten The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15 195-200 (2000)
6 G Reddy L Stehno Bittel and C Enwemeka Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-7 (1998)
7 K Moore N Hira I Broome and J Cruikshank The effect of infrared diode laser irradiation on the duration and severity of postoperative pain a double blind trial Laser Therapy 4 145-148 (1992)
8 A Amir A Solomon S Giler M Cordoba and D Hauben The influence of helium-neon laser irradiation on the viability of skin flaps in the rat Br J Plast Surg 53(1) 58-62 (2000)
9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)
10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)
11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)
12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)
13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)
14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)
15 A Schlager P Kronberger F Petschke and H Ulmer Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1) 39-42 (2000)
16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)
17 GD Baxter and J Allen Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone (1994)
18 G Baxter A Bell J Allen and J Ravey Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77(3) 171-178 (1991)
19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)
20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)
Chapter 1
44
21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)
22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)
23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)
24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)
25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)
26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)
27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)
28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)
29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)
30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)
31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)
32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)
33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)
34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)
35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)
36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)
37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)
38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)
39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)
40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)
41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)
42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)
From an in vitro trial to a patient treatment
45
43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)
44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)
45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)
46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)
47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)
48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)
49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)
50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)
51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)
52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)
53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)
54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)
CHAPTER 2
INCREASED FIBROBLAST PROLIFERATION INDUCED BY
LIGHT EMITTING DIODE AND LOW LEVEL LASER
IRRADIATION
Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Lasers in Medical Science 2003 18(2) 95-99
Chapter 2
48
ABSTRACT
Background and Objective As Light Emitting Diode (LED) devices are
commercially introduced as an alternative for Low Level Laser (LLL) Therapy the
ability of LED in influencing wound healing processes at cellular level was examined
Study DesignMaterials and Methods Cultured fibroblasts were treated in a
controlled randomized manner during three consecutive days either with a infrared
LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and
570 nm) and respective power outputs Treatment duration varied in relation to
varying surface energy densities (radiant exposures)
Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all
irradiated cultures in comparison with the controls Green light yielded a significantly
higher number of cells than red (plt0001) and infrared LED light (plt0001) and than
the cultures irradiated with the LLL (plt0001) the red probe provided a higher
increase (plt0001) than the infrared LED probe and than the LLL source
Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation
in vitro This study therefore postulates possible stimulatory effects on wound healing
in vivo at the applied dosimetric parameters
Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low
Level Laser Tetrazolium salt
LED induced increase of fibroblast proliferation
49
INTRODUCTION
Since the introduction of photobiostimulation into medicine the effectiveness and
applicability of a variety of light sources in the treatment of a wide range of medical
conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results
of several investigations are remarkably contradictory This is at least in part a
consequence of the wide range of indications as well as the wide range of suitable
parameters for irradiation and even the inability to measure the possible effects after
irradiation with the necessary objectivity [457] A lack of theoretical understanding
can also be responsible for the existing controversies In fact theoretical understanding
of the mechanisms is not necessary to establish effects though it is necessary to
simplify the evaluation and interpretation of the obtained results As a consequence
the widespread acceptance of especially Low Level Laser (LLL) therapy in the early
seventies is faded nowadays and biostimulation by light is often viewed with scepticism
[8] According to Baxter [49] contemporary research and consumption in
physiotherapy is in particular focused on the stimulation of wound healing Tissue
repair and healing of injured skin are complex processes that involve a dynamic series
of events including coagulation inflammation granulation tissue formation wound
contraction and tissue remodelling [10] This complexity aggravates research within this
cardinal indication
Research in this domain mostly covers LLL studies but the current commercial
availability of other light sources appeals research to investigate as well the effects of
those alternative light sources eg Light Emitting Diode (LED) apparatus
The scarcity of literature on LED is responsible for consultation of literature
originating from LLL studies [11] but it may be wondered if this literature is
representative for that purpose As in the early days of LLL therapy the stimulating
effects upon biological objects were explained by its coherence [1213] while the beam
emitted by LEDrsquos on the contrary produces incoherent light Though the findings of
some scientists [914151617] pose nowadays that the coherence of the light beam is
not responsible for the effects of LLL therapy Given that the cardinal difference
between LED and LLL therapy coherence is not of remarkable importance in
Chapter 2
50
providing biological response in cellular monolayers [5] one may consult literature
from LLL studies to refer to in this LED studies
The purpose of this preliminary study is to examine the hypothesis that LED
irradiation at specific output parameters can influence fibroblast proliferation
Therefore irradiated fibroblasts cultures were compared with controls The article
reports the findings of this study in an attempt to promote further discussion and
establish the use of LED
MATERIALS AND METHODS
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos Isolation and
disaggregation of the cells was performed with warm trypsin according the protocol
described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in
Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1
L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants
reached confluence cells were detached with trypsine and subcultured during 24 hours
in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours
the cells were removed from the culture flasks by trypsinization and counted by Buumlrker
hemocytometry For the experiment cells from the third passage were plated in 96-well
plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a
density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C
during 24 hours
All supplies for cell culture were delivered by NV Life Technologies Belgium except
for Fetal Calf Serum (Invitrogen Corporation UK)
Irradiation sources
In this study two light sources a Light Emitting Diode (LED) device and a Low Level
Laser (LLL) device were used in comparison to control cultures
The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)
LED induced increase of fibroblast proliferation
51
with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400
mW and a frequency range from 0-1500 Hz
The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)
consisted of three wavelengths emitted by separate probes A first probe emitting
green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the
red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third
probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared
light The area of all three probes was 18 cm2 and their frequency was variable within
the range of 0-1500 Hz
Exposure regime
Prior to irradiation the 96-well plates were microscopically verified to guarantee that
the cells were adherent and to assure that there was no confluence nor contamination
Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining
25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation
The 96-well plates were randomly assigned in the treated (LLL or green red or infrared
LEDrsquos) or the control group
For the treatments in this study the continuous mode was applied as well for the LLL
as for the three LED-probes The distance from light source to fibroblasts was 06 cm
LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting
in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant
exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to
exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective
power output of 160 mW 80 mW or 10 mW
After these handlings the remaining medium was removed and new Hanksrsquoculture
medium was added followed by 24 hours of incubation
One irradiation (LLL or LED) was performed daily during three consecutive days
according to the aforementioned procedure Control cultures underwent the same
handling but were sham-irradiated
Chapter 2
52
Determination of cell proliferation
The number of cells within the 96-well plates as a measure for repair [19] was
quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The
colorimetric assay was performed at two different points of time to determine the
duration of the effect of the used light sources
This assay exists of a replacement of Hanksrsquoculture medium by fresh medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as
described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT
solution was substituted by lysing buffer isopropyl alcohol The plates were
temporarily shaken to allow dissolution of the produced formazan crystals After 30
minutes of exposure to the lysing buffer absorbance was measured The absorbance at
400 to 750 nm which was proportional to fibroblast proliferation was determined
using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)
The complete procedure from isolation to MTT assay was executed six times (Trial A
B C D E and F) while it was impossible to irradiate all the investigated number of
wells with the same LED apparatus on one day All the trials included as much control
as irradiated wells but the number of control and irradiated wells in each trial varied
depending on the number of available cells after the second subculturing A further
consequence of the available number of cells is the number of probes examined per
trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E
Incubation period before proliferation analyses numbered 24 hours To investigate if
the stimulatory effect tends to occur immediately after irradiation or after a longer
period of time incubation in trial F lasted 72 hours
An overview of the followed procedures regarding incubation time before proliferation
analysis number of analysed wells for each trial and the number of probes examined
per trial is given in table 1 As a consequence of the differences in procedures followed
and because each trial started from a new cell line the results of the five trials must be
discussed separately
LED induced increase of fibroblast proliferation
53
Statistical analysis
Depending on the amount of groups to be compared within each trial and depending
on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way
ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons Statistical significance for all tests was
accepted at the 005 level For this analysis Statistical Package for Social Sciences 100
(SPSS 100) was used
RESULTS
The results presented in table 1 show that cell counts by means of MTT assay
revealed a significant (plt0001) increase in the number of cells in comparison to their
respective sham-irradiated controls for all the irradiated cultures of trial A B C D
and E except the irradiated groups in trial F
Moreover the results of trial A showed that the effect of the green and red LED probe
was significantly (plt0001) higher than the effect of the LLL probe With regard to the
amount of proliferation the green probe yielded a significantly higher number of cells
than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe
provided a higher increase in cells (plt0001) than the infrared probe
The infrared LED source and the LLL provided a significant (plt0001) higher number
of cells than the control cultures but no statistical significant difference was recorded
between both light sources
The trials A B C D and E regardless of the number of probes used in each trial
were analysed after 24 hours of incubation after the last irradiation The incubation
period of trial F lasted 72 hours
The means of trial F illustrated that the effect was opposite after such a long
incubation The control cultures had significantly (plt0001) more fibroblasts than the
irradiated cultures with the exception of the LED-infrared group that showed a not
significant increase of cells Further analysis revealed that the green probe yielded a
significantly lower number of cells than the red (plt0001) and the infrared probe
(plt0001) and that the red probe provided a higher decrease (plt0001) than the
Chapter 2
54
infrared probe Laser irradiation induced a significant decrease of fibroblasts in
comparison to the infrared irradiated cultures (plt0001) and the control cultures
(p=0001) LED irradiation with the green and the red probe revealed no statistical
significant differences
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups
Absorbency (proportional to the number of fibroblasts)a
Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050
Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis
DISCUSSION
Despite the failure of some studies [223] to demonstrate beneficial effects of laser and
photodiode irradiation at relatively low intensities (lt500mW) on fibroblast
LED induced increase of fibroblast proliferation
55
proliferation this study provides experimental support for a significant increased cell
proliferation Therefore these results confirm previous studies that yielded beneficial
stimulating effect [1152425] Remarkably though is the higher increase noted after
irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general
decrease in absorption at longer wavelengths and concluded that several molecules in
fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445
470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the
closest to one of these peaks
Karu [5] also emphasises that the use of the appropriate wavelength namely within the
bandwidth of the absorption spectra of photoacceptor molecules is an important
factor to consider
In this particular context penetration depth can almost be ignored as virtually all
wavelengths in the visible and infrared spectrum will pass through a monolayer cell
culture [12] The irradiance (Wcm2) on the contrary could have had an important
influence on the outcome of this study The higher increased proliferation by the lower
wavelengths is possibly a result of the lower irradiance of these wavelengths Lower
irradiances are confirmed by other experiments to be more effective than higher
irradiances [111626]
The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as
described by Poumlntinen [17] but in the scope of these results it also needs to be noticed
that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)
the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)
Consequently the results of especially trial A and F must be interpreted with the
necessary caution It is possible that the determined distinction between the used light
sources and the used probes is a result from the various radiant exposures applied
during the treatments of the cultures
Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after
the last irradiation this study was unable to demonstrate a stimulating effect when
analysis was performed 72 hours after the last irradiation Moreover this longer
incubation period even yielded an adverse effect Although a weakening of the
Chapter 2
56
photostimulating influence over time is acceptable it can not explain a complete
inversion Especially in the knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period [2427] In an attempt to illuminate
this finding one can suppose that the circadian response of the cells triggered by the
LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark
The most obvious explanation is even though a decreased vitality and untimely cell
death in the irradiated cell cultures as a result of reaching confluence at an earlier point
of time than the control cultures The cells of a confluent monolayer have the tendency
to inhibit growth and finally die when they are not subcultured in time No other
reasonable explanations could be found for this discrepancy
Photo-modulated stimulation of wound healing is often viewed with scepticism The
real benefits of Light Emitting Diodes if any can only be established by histological
and clinical investigations performed under well controlled protocols Despite these
remarks this study suggests beneficial effects of LED and LLL irradiation at the
cellular level assuming potential beneficial clinical results LED application on
cutaneous wounds of human skin may be assumed useful at the applied dosimetric
parameters but future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Persons in good health rarely require treatment for wound healing as posed by Reddy
et al [13] light has a possible optimal effect under conditions of impaired healing
Postponed wound healing is a time-consuming and often expensive complication
Thus future prospects must remind to examine the therapeutic efficacy of LED on
healing-resistant wounds
LED induced increase of fibroblast proliferation
57
ACKNOWLEDGMENTS
The authors are grateful to Prof Deridder for supplying the laboratory as well as the
material necessary for this investigation and to Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
Chapter 2
58
REFERENCES
1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55
2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32
3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7
4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994
5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998
6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32
7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42
8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5
9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8
10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7
11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8
12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34
13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9
14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18
15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43
16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6
17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475
18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J
Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9
21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601
22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63
LED induced increase of fibroblast proliferation
59
23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9
24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301
25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are
more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37
27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8
28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26
CHAPTER 3
GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES
FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL
Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Photomedicine and Laser Surgery 2005 23(2) 167-171
Chapter 3
62
ABSTRACT
Background and Objective The chronic metabolic disorder diabetes mellitus is an
important cause of morbidity and mortality due to a series of common secondary
metabolic complications such as the development of severe often slow healing skin
lesions
In view of promoting the wound-healing process in diabetic patients this preliminary
in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on
fibroblast proliferation and viability under hyperglycemic circumstances
Materials and Methods To achieve hyperglycemic circumstances embryonic chicken
fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL
glucose LED irradiation was performed on 3 consecutive days with a probe emitting
green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min
resulting in a radiation exposure of 01 Jcm2
Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all
irradiated cultures in comparison with the controls
Conclusion According to these results the effectiveness of green LED irradiation on
fibroblasts in hyperglycemic circumstances is established Future in vivo investigation
would be worthwhile to investigate whether there are equivalent positive results in
diabetic patients
Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes
Fibroblast proliferation under hyperglycemic circumstances
63
INTRODUCTION
The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide
geographic variation in incidence and prevalence generally 11 of the world
population is affected and worldwide it is the twelfth leading cause of death1 Those
figures may be higher for urban regions as well as for industrialized countries Due to
multiple factors involving the aging process of the population and lifestyle changes
(such as reduced physical activity hypercaloric eating habits and concomitant obesity)
these figures may increase in the future2-6 Therefore diabetes mellitus could become
the most common chronic disease in certain regions as stated by Gale it ldquotargets the
rich in poor countries and the poor in rich countriesrdquo6
The harmful disruption of the metabolic equilibrium in diabetes mellitus results in
characteristic end-organ damage that occurs in various combinations and that follows
an unpredictable clinical pathway
Accordingly the major consequence of diabetes mellitus in terms of morbidity
mortality and economic burden principally concerns macroangiopathies or
arteriosclerosis and microangiopathies including nephropathy neuropathy and
retinopathy7-10
One of these devastating consequences which often appears in time is the
development of various skin defects that are frequently resistant to healing and that
tend to be more severe than similar lesions in nondiabetic individuals Diabetes
mellitus even increases the risk of infection by an increased susceptibility to bacteria
and an impaired ability of the body to eliminate bacteria1112
Skin problems are a severe complication in diabetic individuals and require a
comprehensive and appropriate multidisciplinary approach to prevention and
treatment12
Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to
play the most prominent role in the development of diabetic complications With the
development of insulin treatment for type I diabetes and various oral hypoglycemic
agents for type 2 diabetes a reduction in the development of skin defects due to
hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply
Chapter 3
64
waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in
diabetic patients often need special care in comparison to those persons in good
health who rarely require treatment for wound healing1617 Special care is directed
besides of course toward optimal diabetes regulation toward patient education
maximum pressure relief controlling infection recovery of circulation in case of
ischemia and different modalities of intensive wound treatment18
In the last few years various therapies have been introduced with varying success An
example of such a therapy is the photo-modulated stimulation of diabetic lesions In
vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been
subject of extensive investigation1920 Due to contradictory research results LLL-
photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of
light in the domain of wound healing is less time-consuming less expensive less
invasive than many of the other introduced treatment modalities and practical to use
however it seems worthwhile to investigate the value and benefits of a newly
introduced and alternative light source the light emitting diodes (LEDrsquos)
Preliminary research has proved that green LED with particular properties (an
exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01
Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These
results may be of great importance to the diabetic patient because as posed by Reddy et
al light has a possible beneficial effect in the case of impaired healing1617
To obtain insight into the ability of LED to stimulate fibroblast proliferation under
diabetic-specific conditions of impaired healing the proliferation was assessed in
irradiated and control cultures cultivated in medium with a high quantity of glucose
MATERIAL amp METHODS
Cell cultivation
Primary fibroblast cultures were established by outgrowth from 8-day-old chicken
embryos After isolation and disaggregating as described by Freshney (1994)24 the cells
were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10
Fibroblast proliferation under hyperglycemic circumstances
65
fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin
Secondary cultures were initiated by trypsinization followed by plating of the cells in
80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were
disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently
231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third
passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the
cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified
atmosphere at 37deg C to allow them to attach to the bottom of the wells
Light source specifications and illumination procedure
To control adherence of the cells and to assure that there was no confluence or
contamination the 96-well plates were microscopically examined before irradiation
Subsequently the tissue culture plates were randomly assigned for use in the treated
and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium
was aspirated The remaining 25 (50 microl) medium avoided dehydration of the
fibroblasts throughout irradiation
Irradiation was performed with a light emitting diode (LED) device The LED device
(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power
range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable
within the range of 0-1500 Hz
The investigation used the following illumination properties the continuous mode a
distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2
radiant exposure This procedure resulted in an exposure time of 3 min and a power
output of 10 mW Immediately after irradiation the remaining medium was aspirated
and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM
glucose (30 gL) and incubated at 37deg C
Irradiation and medium changes occurred at 1-day intervals so one irradiation was
implemented each 24 h for 3 days in a row and from the first irradiation onwards all
medium renewals occurred with glucose-supplemented Hanksrsquo culture medium
Control cultures were handled in the same manner but were sham-irradiated
Chapter 3
66
Proliferation assay
Fibroblast survival and proliferation were determined by a sensitive and reproducible
colorimetric assay the assay which detects merely living cells and the signal generated
bears a constant ratio to the degree of activation of the fibroblasts and the number of
fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than
only a small subsample26
Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented
Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution
was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during
30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous
solution The optical density of the final solution was measured on an ELtimes800 counter
(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test
wavelength varying from 400 to 750 nm
The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies
for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except
for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)
Data analysis
On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a
Mann-Whitney U test was performed for nonparametrical comparison of the results
Statistical significance for all tests was accepted at the 005 level For this analysis the
Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used
RESULTS
The MTT measurements from each of the 256 control wells and 256 irradiated wells
and the subsequent nonparametrical analysis from the optical densities obtained
disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic
Fibroblast proliferation under hyperglycemic circumstances
67
circumstances after irradiation than in the same circumstances without irradiation (Fig
1)
Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)
DISCUSSION
The outcome of these in vitro experiments based on the above-described light source
properties and the illumination procedure described clearly demonstrated the
stimulatory potential of LED on fibroblast proliferation and the cell viability of
fibroblasts cultured in hyperglycemic medium Preliminary research has already
demonstrated that under these conditions (an exposure time of 3 min a wavelength of
570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this
procedure allowed the highest number of living cells The nature of the light and the
usual questions concerning coherence wavelength power output and radiant
exposures have been discussed previously23
Although these findings confirm the results previously found one cannot ignore the
important methodological difference between previous investigations and the current
study as the cells in this experiment were cultured in hyperglycemic medium2328-30
Absorbency - Proportional to the number of fibroblasts
621 x 10-1 682 x 10-1
0010203040506070809
1
Control Irradiated
Groups
Ab
sorb
ency
Chapter 3
68
After a growth period with normal Hanksrsquo culture medium a necessary step to ensure
normal growth of these secondary subcultures and normal attachment to the bottom
of the wells the Hanksrsquo culture medium was supplemented with glucose
Several earlier studies have established that exposure to glucose concentrations (20-40
mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human
vascular endothelial cell proliferation1531-34 This restraint is more pronounced for
higher glucose15 concentrations and is expressed especially after protracted exposure to
high glucose levels31 A similar restraint was found for cultured fibroblasts by
Hehenberger et al3536 According to some authors however cultured fibroblasts
conversely have been shown to maintain responsiveness to ambient high glucose323738
As there are some ambiguities in literature regarding normal or inhibited growth of
fibroblasts in medium supplemented with glucose39 a pilot study was performed to
determine the amount of glucose necessary to inhibit normal growth after 72 h of
culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study
demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease
of cell viability and to bring about a decline in fibroblast proliferation
This concentration resulted in a remarkable reduction of cell viability and a noteworthy
decrease in the proliferation rate in comparison to control cultures grown in 55 mM
glucose although this concentration is too high to mimic severe diabetic
hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to
investigate the effect of LED in distinct destructive conditions in order to obtain an
incontrovertible result
In addition it is possible that the present investigation needed a higher amount of
glucose to result in a remarkable reduction of proliferation as exposure to glucose was
limited to 72 h and as previous studies revealed that the antiproliferative effect of high
glucose was more pronounced with prolonged exposure with a maximal inhibition
attained by 7-14 days1531
Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo
cell growth are too complex to compare A key question is whether fibroblast
senescence in tissue culture and in the intact organism are similar Cristofalo et al40
Fibroblast proliferation under hyperglycemic circumstances
69
reported that this is not the case as fibroblasts have a finite ability to divide and
replicate but apparently the pathway or the morphologic characteristics leading to the
replicative senescence is not identical in vivo compared to in vitro
Furthermore extrinsic aging related to environmental damage which in diabetic
patients is mainly due to a chronic exposure to high levels of glucose during life is
unachievable in vitro
Unless a number of questions regarding the mechanism according to which LED
stimulates fibroblast proliferation in this particular condition remain unanswered the
results ascertain the potential effects of LED on fibroblast proliferation and viability
CONCLUSION
The current results should be interpreted with caution However these results
demonstrate the effectiveness of green LED irradiation at the above-described light
source properties and the illumination procedure described on cells in hyperglycemic
circumstances
The findings of the present study using an experimental in vitro model indicate that the
use of LED irradiation to promote wound healing in diabetic patients may have
promising future results As the present study establishes the possibility of using LED
irradiation in experimental in vitro situations it would be a worthwhile extension to
perform in vivo investigations to determine whether these in vitro observations were
relevant to the physiological situation and to determine the effect of these LED
properties on human tissue response
ACKNOWLEDGMENTS
The authors are greatly indebted to P Coorevits for assistance with the statistical
analysis and to Professor L Deridder and Ms N Franccedilois of the department of
Human Anatomy Embryology Histology and Medical Physics for providing access to
the laboratory and for helpful discussions
Chapter 3
70
REFERENCES
1 World Health Organisation The world health report of 2002 Statistical Annex 2002 pp 179-201
2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418
3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957
4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538
5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308
6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J
Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot
wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-
glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A
Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood
glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13
14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309
15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501
16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287
17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255
18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412
19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192
20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137
21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675
22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246
23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99
Fibroblast proliferation under hyperglycemic circumstances
71
24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729
27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63
28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301
29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -
progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of
cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA
damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in
culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia
23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar
K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71
36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141
37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588
38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252
39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97
40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619
PART II ANALGESIA
CHAPTER 4
EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION
MEDIATED BY LIGHT EMITTING DIODE IRRADIATION
Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy
Vanderstraetenab and Dirk Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Lasers in Medical Science 2005 20(1) 35-40
Chapter 4
76
ABSTRACT
The introduction of light emitting diode (LED) devices as a novel treatment for pain
relief in place of low-level laser warrants fundamental research on the effect of LED
devices on one of the potential explanatory mechanisms peripheral neurophysiology in
vivo
A randomised controlled study was conducted by measuring antidromic nerve
conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline
measurement and five post-irradiation recordings (2 min interval each) were performed
of the nerve conduction velocity (NCV) and negative peak latency (NPL)
Interventional set-up was identical for all subjects but the experimental group (=32)
received an irradiation (2 min at a continuous power output of 160 mW resulting in a
radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype
MDB-Laser Belgium) while the placebo group was treated by sham irradiation
Statistical analysis (general regression model for repeated measures) of NCV and NPL
difference scores revealed a significant interactive effect for both NCV (p=0003) and
NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical
significant decreased NCV and an increased NPL in the experimental group and a
statistical significant difference between placebo and experimental group at various
points of time
Based on these results it can be concluded that LED irradiation applied to intact skin
at the described irradiation parameters produces an immediate and localized effect
upon conduction characteristics in underlying nerves Therefore the outcome of this in
vivo experiment yields a potential explanation for pain relief induced by LED
Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative
peak latency middot Analgesic effect
Nerve conduction characteristics
77
INTRODUCTION
Since the introduction of photobiostimulation into medicine the light sources used
have advanced technologically and varied in characteristics over the years
Advancement and variation of the sources implicate a concomitant necessity to revise
research results in the respective domains of application Research and clinical
applications in the past particularly focused on the effectiveness of low-level lasers
have shifted now to novel treatment units such as light emitting diode (LED) devices
The efficacy and applicability of LED irradiation within the field of wound healing has
already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED
is not only promoted for its beneficial effects on the wound-healing process it is also
suggested to be potentially effective in the treatment of pain of various aetiology
although this claim has not yet been investigated thoroughly either experimentally or
clinically The putative analgesic effects of LED remain to be further explored
As the basic vehicle of pain is the neuronal system [7] measuring the
neurophysiological effect of LED treatment would be an appropriate experimental
approach to investigate the efficacy of LED on pain inhibition Nerve conduction
studies have become a technique for investigating the neurophysiologic effects of light
therapy [8-9]
Review of literature regarding standard nerve conduction studies revealed that previous
human studies on the influence of various light sources on peripheral nerves have
utilized different methods which hampers a comprehensive comparison In general
this research was performed on the superficial radial nerve [10-13] described by Shin J
Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve
[891315-17] Following the method of Cambier et al [18] the authors of this study
decided to investigate the effect of the light source used on the conduction
characteristics of the sural nerve By investigating this solely sensory nerve interaction
of motor nerve fibres (motor response can easily be provoked by antidromic nerve
stimulation [19]) can be avoided and given the superficial nature of the nerve it should
be sufficiently amenable to the effects of percutaneous LED irradiation
Chapter 4
78
A second major difference between the trials and therefore also hindering an
appropriate comparison between the results is the wide range of used light sources
HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared
multisource treatment unit [15]
With respect to the potential importance of LED irradiation for the treatment of pain
the current investigation was designed to assess the putative neurophysiological effects
of LED on the sensory nerve conduction of the human superficial peripheral sural
nerve and to establish a time course of the supposed phenomenon
The experimental hypothesis postulates that LED generates an immediate decrease in
conduction velocity and increase in negative peak latency In addition it can be
postulated that this effect is most prominent immediately after the irradiation and will
weaken as time progresses
STUDY DESIGN
The study was approved by the Ethical Committee of the Ghent University Hospital
After explanation of the experimental procedure a written informed consent was
obtained from each subject
Subjects
After screening based on a brief medical history excluding subjects with
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever inflammation of the skin) or conditions
that might affect sensory nerve conduction (such as diabetes peripheral neuropathy
radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral
edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40
females (mean age 26plusmn6 years range 18-42 years) participated in this study The body
mass index (BMI) of each subject varied within the normal range (=185-249) [20]
(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo
Nerve conduction characteristics
79
or an experimental group Each group of 32 subjects was composed of 12 males and
20 females
Experimental Procedure and Data Acquisition
In order to be able to quantify the negative peak latency (NPL) (measured from the
start of the stimulus artefact to the peak of the negative portion of the nerve action
potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was
followed
With respect to the known relationship between nerve conduction characteristics and
temperature the ambient temperature was kept constant (23ordmC-26ordmC room
temperature) during the investigation In view of this temperature issue the
standardized protocol started with 10 min of accommodation during which the
subjects rested in prone position on a treatment table
Immediately before this adjustment period the skin over the dorsolateral aspect of the
left calf and foot was cleaned with alcohol to remove surface lipids This preparation of
the treatment area was followed by the placement of the electrodes (TECA
Accessories Oxford Instruments Medical Systems Division Old Woking UK) as
described by Delisa et al [21]
The two-posted (2 cm separation anode distal) surface caption electrode was placed
distal and posterior of the lateral malleolus on the skin covering the sural nerve The
fixation of the earth electrode (Medelec Oxford Instruments Medical Systems
Division Old Woking UK) occurred 12 cm above the caption electrode according to
the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm
above the caption electrode to map the ideal stimulation point To level off
intraindividual variations in the amount of sensory response attributable to the
successive placement of the bipolar stimulator in course of the investigation a two-
posted (2 cm separation cathode distal) bar stimulating electrode was attached at the
point where the maximal response was obtained
This placement of the electrodes allows antidromic stimulation of the sural nerve
Electrophysiological stimulation and recordings were obtained with a Medelec
Chapter 4
80
Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse
of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was
used to produce each evoked sensory response
Baseline measurements of NPL and NCV were immediately followed by treatment of
the subjects according the protocol detailed below Recordings were subsequently
repeated at 2-min intervals over an 8-min period resulting in five recordings (one
immediately after the completion of the treatment and one at 2 4 6 and 8 min after
irradiation) Skin temperature was recorded concomitantly throughout the procedure
at the time of baseline measurement immediately after LED irradiation at the time of
the first recording and consequently at 2-min intervals together with the four final
electrophysiological recordings For this a surface digital C9001 thermometer
(Comark UK) sensitive to temperature changes of 01degC was used at the same point
of LED administration namely at 7 cm above the caption electrode The procedure
was identical for both conditions but subjects in the placebo group received a sham
LED irradiation
Light Characteristics and Irradiation Procedure
Irradiation was administrated with a light emitting diode device (BIO-DIO
preprototype MDB-Laser Belgium) The probe used emitted infrared light with a
wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2
and the frequency was variable within the range of 0-1500 Hz
Preceding baseline measurement the treatment point was marked on the skin overlying
the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-
point between the stimulation and capture electrode The LED probe was held in
contact with the skin perpendicular to the skin surface during the complete irradiation
procedure LED treatment consisted for all subjects of the experimental group out of 2
minutes lasting irradiation The LED was set to deliver a continuous energy density of
107 Jcm2 at a power output of 160 mW These parameters were selected as they are
appropriate for the treatment of pain in a clinical setting First of all because the
Nerve conduction characteristics
81
duration of the treatment is clinically feasible and secondly because the parameters are
within the scope of previously described light source characteristics [1-36915]
Statistics
Although superficial skin temperature did not change significantly in course of the
investigation the influence of the measured skin temperature on NPL and NCV was
taken into account by using a correction factor of respectively 02 msdegC and 147
ms degC All corrections were calculated towards a reference skin temperature of 32degC
Difference scores ie the variation between baseline measurements and each post-
irradiation recording were used as the basis for statistical analysis A General
Regression Model for repeated measures with one within-subjects factor (time 0
min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and
one between-subjects factor (group placebo or LED irradiated) was performed
followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least
Significant Difference) to determine whether any differences between baseline
measurements and post-irradiation recordings were statistically significant
The Statistical package for social sciences (SPSS 110) was used for analysis and
statistical significance for all tests was accepted at the 005 level
RESULTS
Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated
group plotted against time in minutes The values of the irradiated subjects decrease
directly after the irradiation and reach a first low point 2 min after finishing LED
treatment This decrease is followed by a marginal increase at 4 and 6 min and again an
important decrease at 8 min Statistical analysis (general regression model for repeated
measures) of these data indicated a significant interactive effect (P=0003)
Chapter 4
82
Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)
Post hoc LSD further showed significant differences between baseline measurements
and all post-treatment recordings (Table 1) Mutual comparison of the values from the
post-treatment recordings did not reveal any significant difference In addition there
was no significant difference determined in the placebo group in course of time
Table 1 Summary of the influence of LED irradiation on nerve conduction velocity
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001
2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002
4 0111plusmn0377 0647 -0908plusmn1898 0021 0004
6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001
8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve Conduction Velocity
-14
-12
-1
-08
-06
-04
-02
0
02
04
Baseline 0 min 2 min 4 min 6 min 8 min
Time Course
Dif
fere
nce
Sco
re (
m
s)
PlaceboLED
Nerve conduction characteristics
83
A similar representation was used for the results of the NPL Figure 2 reproduces NPL
plotted against time in minutes revealing for the irradiated group an increased latency
with two important peaks one at 4 min and one at 8 min
Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)
Statistical analysis of the mean difference scores again indicated a significant interactive
effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2
showed significant differences between baseline measurements and all post-treatment
recordings of the experimental group The mean difference score of the first post-
treatment recording of this same group (LED irradiated) differed significantly with the
recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED
irradiation As well as the recording 2 min after irradiation which differed significantly
(P=0013) with the 8 min post-treatment recording As observed for the NCV the
NPL of the placebo group did not reveal any significant difference in time course
At the time of the final recording the NCV and NPL mean difference scores of the
irradiated group did not return to their respective baseline values
Negative Peak Latency
-001
0
001
002
003
004
005
006
007
Baseline 0 min 2 min 4 min 6 min 8 min
Time course
Dif
fere
nce
Sco
re (
ms)
PlaceboLED
Chapter 4
84
Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group
significance) revealed statistical differences between the experimental and the placebo
group for NCV as well as for NPL NCV and NPL were statistical significant between
both groups at all points of time except from the NPL recording immediately after
finishing irradiation
DISCUSSION
Notwithstanding the above-mentioned difficulties in comparing results between
different trials on nerve conduction we attempt to discuss the current findings in view
of the results of the previous studies
This investigation revealed that percutaneous LED irradiation at feasible and current
clinical parameters generates measurable and significant changes in human sural nerve
antidromic conduction latency and velocity These results thus support previous
findings of light-mediated nerve conduction latency shifts in vivo [8101218]
although there are several important issues to be discussed
Table 2 Summary of the influence of LED irradiation on negative peak latency
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0004 plusmn0053 0755 0029plusmn0080 0019 0145
2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021
4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004
6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034
8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve conduction characteristics
85
A first comment deals with the progress of the NCV and NPL in function of time As
postulated the NCV decreases significantly immediately after irradiation
corresponding with a significant increased NPL However this effect does not weaken
as time progresses both variables remain significant throughout the 8 min during
observation period
Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the
conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]
although this slight increase in NPL was not significant at any moment Two other
studies [822] with a GaAlAs laser even registered comparable effects over a period of
55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous
studies post-treatment conduction measurements should be extended in time At
present for all studies it remains unclear at what point of time the effect extinguishes
although the interval of time during which LED treatment remains effective is
clinically important when treating pain
Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least
45 min) mediated by a monochromatic multisource infrared diode device although it
needs to be mentioned that this study performed with a comparable light source as the
current investigation revealed a significant decrease of the NPL These inverse results
between the study of Noble et al [15] and the current investigation could be attributed
to the concomitant increase of the skin temperature [15] As it has been well
recognised that a variation in tissue temperature causes a corresponding alteration in
nerve conduction velocities and peak latencies [91523-27] the temperature changes
may indeed provide an explanation for the observed findings In an attempt to analyse
the influence of a direct photobiological effect on sural nerve conduction
characteristics rather than working out the effects based upon thermal mechanisms
the present study corrected the skin temperature towards a reference temperature of
32degC This correction was performed notwithstanding the fact that the superficial skin
temperature did not change significantly before and after LED irradiation as well as
despite the fact that influencing nerve temperature takes place long after affecting skin
temperature [23] and thus being (almost) impossible after 2 min of irradiation
Chapter 4
86
followed by 8 min of registration Introduction of the correction factor implies likewise
that eventual influence on nerve conduction by cooling of the limb due to inactivity as
described by Greathouse et al [11] can be excluded
These facts suggest that temperature changes did not contribute to the demonstrated
effects of LED on nerve conduction Nevertheless the underlying mechanism of the
observed effects remains indistinct
A following remark regarding the fluctuation of NCV and NPL in function of time
considers the fact that both the NCV and the NPL do not change in a constant way up
to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the
increase in NPL display a small though not significant inversion of the effect at 4 and
(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of
fluctuation is to be expected when measuring NCV and NPL besides there is a similar
variation in the placebo groups
Although investigating dose dependency was not intended an additional remark
considers the fact that the use of optimal irradiation parameters is essential to obtain
the observed neurophysiological effect Nevertheless it is impossible to determine
ideal light source characteristics for effective treatment as the range of used
wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency
(pulsed or continuous) are not sufficiently similar between the different studies It can
only be concluded that a pulsing light source [91028] does not provide the postulated
results Radiant exposure exposure time power range and wavelength are not yet
established but based on this study and previously described assays it can be
speculated that the ranges of these parameters are quite large
In comparison with other studies where the number of subjects is 10 or less [8-
1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-
Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number
of subjects (n=32) was investigated in each group In spite of the large investigated
population it should be noted that the magnitude of the described changes in NCV
and NPL can simply be replicated by lowering the temperature of the extremity as the
observed changes are within the expected physiological ranges making the clinical
Nerve conduction characteristics
87
significance of the change questionable (This fact does not implement that the
decrease and the significant changes were temperature mediated)
A key question and meanwhile the initial impetus for future investigation is whether
the measured effects can be extrapolated to the actual nociceptive afferents namely the
myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])
respectively conducting acute and chronic pain The functional testing of these
nociceptive pathways has recently been extensively evaluated The currently accepted
neurophysiological method of assessing nociceptive pathways relies on laser-evoked
potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]
As up till now LEP is not available in this or any surrounding research centre the
investigators of this study had to perform a standard nerve conduction study (assessing
the large myelinated Aβ afferents) Therefore the current and previous beneficial
results of low level light therapy on conduction characteristics of nerves in vivo should
initiate measurements of clinical effectiveness first of all in laboratory settings and
afterward at a clinical level
CONCLUSION
Despite these remarks and the limited knowledge regarding the underlying mechanism
the present findings enable the following conclusions to be drawn LED irradiation at
clinical applied energy densities produces an immediate and localized effect upon
conduction characteristics in underlying nerves More specifically it is proven that
LED treatment lowers the NCV and augments the NPL resulting in a reduced
number of impulses per unit of time Therefore the outcome of this in vivo experiment
assumes that LED possibly induces pain relief
In order to encourage a widespread acceptance for the use of this non-invasive pain-
reducing modality in clinical settings prospective research should establish the precise
relationship between LED and pain relief as well as determine the ideal irradiation
parameters and verify which painful conditions can be treated with this treatment unit
Chapter 4
88
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the
equipment and MDB-Laser Belgium for generously providing the Light Emitting
Diode equipment
Nerve conduction characteristics
89
REFERENCES
1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9
2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)
3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18
4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8
5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43
6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65
7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53
8 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-34
9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6
10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90
11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7
12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5
13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9
14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore
15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5
16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5
17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604
18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200
19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21
Chapter 4
90
20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083
21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press
22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9
24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9
25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203
26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13
27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12
28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9
29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445
30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8
31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56
CHAPTER 5
PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE
IRRADIATION A PILOT STUDY ON EXPERIMENTALLY
INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS
Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Accepted for publication in Lasers in Medical Science December 2005
Chapter 5
92
ABSTRACT
Objective The present pilot study investigated the analgesic efficacy of light emitting
diode (LED) In view of a standardised and controlled pain reduction study design this
in vivo trial was conducted on experimentally induced delayed-onset muscle soreness
(DOMS)
Design Thirty-two eligible human volunteers were randomly assigned to either an
experimental (n=16) or placebo group (n=16) Immediately following the induction of
muscle soreness perceived pain was measured by means of a visual analog scale (VAS)
followed by a more objective mechanical pain threshold (MPT) measurement and
finally an eccentricconcentric isokinetic peak torque (IPT) assessment The
experimental group was treated with infrared LED at one of both arms the other arm
served as control Irradiation lasted 6 min at a continuous power output of 160 mW
resulting in an energy density of 32 Jcm2 The subjects of the placebo group received
sham irradiation at both sides In post-treatment a second daily assessment of MPT
and VAS took place The treatment and assessment procedure (MPT VAS and IPT)
was performed during 4 consecutive days
Results Statistical analysis (a general linear model followed by post hoc least
significant difference) revealed no apparent significant analgesic effects of LED at the
above-described light parameters and treatment procedure for none of the three
outcome measures However as the means of all VAS and MPT variables disclose a
general analgesic effect of LED irradiation in favour of the experimental group
precaution should be taken in view of any clinical decision on LED
Conclusion Future research should therefore focus on the investigation of the
mechanisms of LED action and on the exploration of the analgesic effects of LED in a
larger randomised clinical trial and eventually in more clinical settings
Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset
muscle soreness middot Musculus biceps brachii
Delayed-onset muscle soreness
93
INTRODUCTION
The analgesic efficacy of light emitting diode (LED) irradiation is recently being
investigated by means of a nerve conduction study on the superficial peripheral sural
nerve [1] It was demonstrated that LED irradiation at clinical applied densities
produces an immediate and localized effect upon conduction characteristics in
underlying nerves More specific LED induces a decreased number of sensory
impulses per unit of time thus possibly inducing pain relief [1]
Given the established influence of this treatment modality on the nerve conduction
velocity and thereby its potential analgesic ability the current investigation was
designed
Studies investigating the efficacy of a therapeutic modality on pain often experience
difficulties regarding standardisation of the population as analysis or comparison of
pain with different aetiologies is almost impossible Therefore we opted to measure the
analgesic effects of LED in a laboratory setting on a sample with experimentally
induced delayed-onset of muscle soreness (DOMS)
Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the
induction exercise and then spreads throughout the muscle [2-4] The correlates of
DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10
[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force
decreased range of motion and in particular muscle pain which is more pronounced
during movement and palpation [8 11] Despite the large volume of research that has
been undertaken to identify the underlying pathophysiology of DOMS the precise
mechanism is not yet universally accepted Several theories such as the torn-tissue
theory the connective tissue damage theory the muscle spasm theory and the
inflammation theory still remain viable though the current opinion states that DOMS
arises from a sequence of events in which several theories occupy an important place
[2 6 12 13]
DOMS has been used as a representative model of musculoskeletal pain and stiffness
in a number of studies [4 7 11 14 15] as it has a number of advantages it can be
induced in a relatively easy and standardised manner in a group of healthy subjects the
Chapter 5
94
time-course is relatively predictable and the symptoms have the same aetiology and are
of transitory nature [14 16] Nevertheless it should be emphasised that the use of this
particular experimental model to test the effectiveness of LED does not mean that this
treatment modality is necessarily advocated for the treatment of DOMS but merely
that it may be helpful in documenting the efficacy of LED in a clinical model of
musculoskeletal pain and stiffness In addition studies based on the induction of
DOMS under carefully controlled laboratory conditions can not replace research
involving actual patients but offer the opportunity to assess the effectiveness of
particular therapeutic interventions and might help to define additional clinical research
[14]
The experimental hypothesis of the current study postulates that infrared LED reduces
pain and muscle sensitivity associated with DOMS
MATERIALS AND METHODS
The study was approved by the ethical committee of the Ghent University Hospital
After providing information regarding the study design and possible consequences
related to participation at the study written informed consent was obtained from each
subject
Subjects
Healthy human volunteers were recruited from the university population Individuals
with any upper limb pathology neurological deficit and recent injury to either upper
extremity or undiagnosed pain were excluded Other exclusion criteria were
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever and inflammation of the skin) or
conditions in which physical exertion is contraindicated (such as cardiovascular deficits
hypertension and respiratory problems)
Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age
23plusmn4 years) were enrolled All subjects were randomly assigned using a random table
Delayed-onset muscle soreness
95
of numbers to the experimental or placebo group Each group of 16 subjects
consisted by stratification of equal numbers of men and women Age height and
weight did not differ significantly between the three groups
All subjects were physically active however none performed on a regular basis any type
of upper body weight-training Subjects were requested to refrain from any form of
strenuous physical activity and they were asked to avoid any form of medication
including anti-inflammatory agents as well as alcohol for 2 days before testing and for
the duration of the study
Overview of experimental design
The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to
induce pain related to DOMS Immediately following induction exercise an initial
assessment of the outcome measures (visual analog scale or VAS mechanical pain
threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the
subjects were treated under blinded conditions according to the randomised group
allocation In post-treatment the MPT was re-recorded and perceived pain was
reassessed with a VAS Contrary to these outcome measures the muscle strength was
only measured in pre-treatment at the one hand because short-term effects of LED
on muscle strength were not postulated and on the other hand because post-
treatment muscle strength can be influenced by too many different physiological
factors related to the pre-treatment measurement On the succeeding days (day 2 3
and 4) the treatment and assessment procedure was similar with approximately 24 h
separating each treatment
In both of the groups the two arms of the participants were included in the study In
the experimental group an equal number of dominant and non-dominant arms were
treated The non-treated arm served as control arm In the placebo group also an equal
number of dominant and non-dominant arms were considered as treated arm and the
other arm was classified in the non-treated group The procedure was identical for
both conditions but the subjects in the placebo group received sham LED irradiation
on both arms
Chapter 5
96
Specific aspects of the experimental design and procedures are detailed below
Pain induction
Muscle soreness was induced in a standardised fashion via a daily calibrated computer-
operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY
USA) Induction occurred separately and in random order in the elbow flexors of both
arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex
Prior to induction of DOMS the subjects were allowed an initial familiarization session
to become comfortable performing maximum voluntary contractions at the required
angular velocities This was immediately followed by determination of the maximum
eccentric and concentric peak torque at an angular velocity of 60degs and 120degs
Subsequently four sessions of eccentricconcentric work were performed with each
arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs
first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg
and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-
range) followed by two sessions at an angular velocity of 120degs again the first time
along an arch of 120deg and followed by the mid-range performance The subjects were
asked to accomplish maximum voluntary contractions during all the sessions Each
session was performed until exhaustion which was defined as the point when the
subject lost 70 of the initial eccentric and concentric peak torque There was a 1-
minute rest between each session This procedure was based on a pilot study and
previously described induction protocols [17-21]
Outcome measures
Outcome measures of subjective pain measurements MPT and muscle strength were
measured in this order on days 1-4 Subjective pain measurements and MPT occurred
immediately prior to and following irradiation whereas muscle strength measurements
only took place before LED treatment
Measurement of subjective pain Perceived muscle soreness was measured
subjectively by means of a 100-mm VAS A series of scales were completed separately
Delayed-onset muscle soreness
97
for each arm pain at rest followed by pain perception associated with full extension of
the arms and finally with maximal flexion of the arms The subjects were not allowed
to compare one VAS result with another
This assessment tool commonly used in measuring experimentally induced pain [22
23] has been found to be a reliable and valid method [24-26]
MPT Tenderness MPT used as a more objective correlate of muscle tenderness
has been demonstrated to be a reliable method to measure experimental induced
muscle soreness [27] This outcome measure was assessed by using a handheld
pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries
South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a
line from the radial insertion of the musculus biceps brachii at the elbow to the
intertubercular groove of the humerus thus resulting in three measure points one at
the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A
pressure of 4Ns was delivered The subjects were instructed to say yes at the exact
moment the pressure perceived became painful Each point was recorded three times
in pre-treatment as well as in post-treatment The average MPT score for each point in
pre- and post-treatment was used for statistical analyses
Muscle strength assessment Eccentric and concentric IPT were measured on the
same computerised dynamometer as was used for the induction of pain and an
identical standardisation procedure regarding positioning was followed
A warm-up session of two maximum voluntary contractions at the required angular
velocities was followed by determination of the eccentric and concentric peak torque
The first session at 60degs consisted of three repetitions followed by a 1-min during
rest and for the second session at 120degs five repetitions were performed The
subjects were instructed to flex and extend the elbow through the entire range of
motion as forcefully and rapidly as possible for each repetition The maximum
eccentric and concentric torque produced during the respective repetitions was used
for statistical analysis
Chapter 5
98
Light source specifications and treatment procedure
Light treatment was applied daily according to group allocation Irradiation occurred
with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The
probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160
mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The
frequency was variable within the range of 0ndash1500 Hz
During the complete irradiation procedure the LED probe was held in contact with
the skin perpendicular to the skin surface and at the exact mid-point between the MPT
mark at 4 cm and the one at 8 cm Light source properties were identical for all
subjects of the experimental group and consisted out of irradiation of 6-min lasting
duration at a continuous power output of 160 mW resulting in an energy density of
32 Jcm2 To conceal the treated side and condition the subjects were blinded to the
treatment status For the experimental condition a probe was held in contact with each
arm but only one of the two probes was attached to the LED device The subjects of
the placebo group received sham irradiation at both sides
The selected parameters are within the scope of previously described light source
characteristics for pain reduction [1 28-30] and they are appropriate for the treatment
of pain in a clinical setting because the duration of the treatment is clinically feasible
Statistical analysis
The three outcome measures were analysed separately For the VAS and MPT
measurements the same procedure was followed a general linear model (GLM) for
repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-
post preceding and following LED irradiation) and one between-subject factor (group
placebo or infrared LED irradiated) was performed If necessary the GLM was
followed by appropriate pairwise comparisons (post hoc least significant difference or
LSD) to determine whether any differences between measurements were statistically
significant A similar model was carried out separately for both the treated and the
control arm
Delayed-onset muscle soreness
99
In contrast to MPT and VAS the muscle strength was analysed differently The peak
torque values recomputed towards body weight of the subjects were statistically
analysed using a GLM for repeated measures This model consisted of one within-
subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo
or infrared LED irradiated) The model was completed twice first for the treated arm
and consequently for the control arm
The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)
was used for analysis and statistical significance for all tests was accepted at the 005
level
RESULTS
Statistical analysis of all variables of the three outcome measures revealed no significant
interactive effects of the main interaction (time times group times pre-post) The means and
standard deviations of the variables for both the treated and the control arm are
outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The
means of all VAS and MPT variables disclose a non-statistical significant general
analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher
MPT values in the irradiated group than in the placebo group The lower VAS rates are
present from day 1 until the last day of the study but they are more clearly present
from day 3 pre-treatment The higher MPT values are present from day 1 post-
irradiation until the last day and they are more visible at 4 cm followed by 12 cm and
finally at 8 cm In addition to the analgesic influence of LED an increased
convalescence of muscle strength was noted It should be remarked that this outcome
is similar for the treated as well as for the control arm of the irradiated group The
findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms
of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively
Graphical presentation of the other variables shows a similar course
Chapter 5
100
Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42
Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36
Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Visual Analog Scale
0
02
04
06
08
1
12
14
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n V
AS
scor
e (a
t re
st)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
101
Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249
Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Mechanical Pain Threshold
0
5
10
15
20
25
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n M
PT
sco
re (
at 4
cm)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Chapter 5
102
Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group
Day 1 Day 2 Day 3 Day 4
Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04
Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04
Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Isokinetic Peak Torque
04
045
05
055
06
065
Day 1 Day 2 Day 3 Day 4
Time course
Mea
n I
PT
sco
re (
ecce
ntr
ic a
t 60
degse
c)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
103
Despite the absence of significant main interaction effects the remaining interactions
as well as the main effects were statistically significant for some variables Only the
significant interactions including the between-subject factor group as well as the main-
effect group will be discussed The other interactions and effects establish the successful
induction of DOMS but are not relevant in view of the postulated hypothesis
The interaction between group and time is significant (p=014) for the VAS in
association with full extension for the control arm Post hoc LSD reveals no difference
between both groups a significant effect over time for both groups is found
Consequently this will not be further evaluated
A second significant interaction (p=0002) is the one among the within-subject factor
pre-post and the between-subject factor group for the MPT at 12 cm for the control arm
Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more
pressure after than before the treatment whereas in the placebo group a not
significant decrease of supported pressure is noted
Finally GLM analysis revealed that at the treated arm the irradiated group tolerates
significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)
DISCUSSION
It has previously been demonstrated that the LED source used might assist in
accelerating wound healing [31] that it has a direct cellular effect [3233] and that it
changes nerve conduction characteristics [1] Nevertheless LED-treated experimental
induced DOMS failed to prove the analgesic efficacy of LED at the above-described
light parameters and treatment procedure The current outcome concurs with other
research that demonstrated a lack of effect of various forms of light therapy on DOMS
[8 11 15] However despite the absence of an apparent and overall definitive finding
the present results cannot exclude favourable effects of LED treatment on pain Since
first of all an isolated statistical significant pre-post difference between groups (control
arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)
revealed that subjects of the irradiated group tolerate more pressure than the subjects
of the placebo group Second of all the overall means identified generally lower VAS
Chapter 5
104
scores higher MPT values and higher peak torques in the irradiated group This
implied that the treated subjects experienced noticeable less pain supported more
pressure on the painful muscle and generated more force than the non-treated
participants However these results are not statistically significant consequently it is
possible that these differences were found by coincidence and that there is no
relationship between the treatment and the described results of the three outcome
measures though it should be mentioned that the absence of significant findings is
more probably attributable to the small sample size involved in this study This
assumption is based on a post hoc power analysis It was calculated that for the small
effect size measured after treatment and for the measured control group event rate a
sample size of 80 subjects in each group was required at α=005 and power=080
(two-sided) to reveal significant results
Another factor conceivably responsible for the lack of solid evidence of the beneficial
effects of LED treatment upon DOMS-associated pain is related to the size of the
treatment effect in relation to the severity of the induced DOMS It is possible that by
using multiple exhaustive sets of exercise severe DOMS were induced which masked
relatively small but apparent treatment effects [4 11] In this same context it is
possible that the results only become significantly different after a prolonged treatment
and follow-up period as previous research noticed that recuperation subsequent to
DOMS induction can last up to 10 days [8]
Although it needs to be stressed that these results are not statistically significant critical
analysis of the overall means leads up to three additional remarks A primary comment
relates to the pre- and post-treatment courses of the results Starting at day 2 a clear
reduction of pain and muscle sensitivity was observed immediately post-treatment
Still one cannot conclude that this is indicative for the analgesic effect of LED
irradiation as a similar decrease in VAS and increase in MPT values was noted in the
treated and the control arm of the placebo group Perhaps this was caused by placebo
effect as reported by Pollo et al [34] the expectation of the participant can easily result
in pain relief but it can only be elucidated by implementation of a control group
Delayed-onset muscle soreness
105
Nevertheless in the current study this particular finding can be most probably
attributed to the physiological effects of the peak torque measurement performed
between the pre- and post-treatment recordings of VAS and MPT on the painful
flexor muscle of the upper arm For the assessment of muscle strength two short
series of alternative concentric and eccentric efforts were performed in succession
involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist
in the removal of inflammatory markers and exudate consequently reducing local
tenderness [4] In addition the force assessment can be considered as a form of active
warming-up resulting in an increased muscle temperature which can reduce muscle
viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness
[3536] thus decreasing the sensitivity of the muscle and moderating pain during
movement In any case the beneficial influence of LED immediately after irradiation
can not be securely interpreted due to the sequential assessment of the outcome
measures
A second additional remark considers the fact that both arms of the irradiated subjects
demonstrated evidence of the beneficial effects of LED as a similar reduction of pain
and muscle sensitivity and higher peak torques were found in course of time at the
treated arm as well as at the control arm of the irradiated subjects This ascertainment
points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]
stated that in case LED works via systemic effects the use of the contralateral side as a
control arm might be ill-advised Thus reinforcing that future research should include a
control group to bring clarification [4 7 16]
Finally it needs to be mentioned that although the extent of DOMS was probably
relatively high for investigating the postulated hypothesis the time-course of the
present study corresponds to that reported by other investigators [2 3 5-10]
Significant time effects in many of the variables revealed that muscle damage was
evident diffuse muscle soreness became progressively worse 24-48 h after DOMS
induction followed by a small amelioration after 72 h [35910] After 72 h the follow-
Chapter 5
106
up was ceased consequently further regain of force and attenuation of pain and
muscle sensitivity could not be evaluated Extending the duration of the assessment
period could be useful in assessing any longer-term effects of LED treatment
particularly because as mentioned above differences between both groups are more
clearly present from day 3 pre-treatment and also because DOMS may last for up to 10
days when induced with the described protocol [715]
Lack of knowledge regarding both the precise mechanism of action of LED and the
specific pathophysiology of DOMS hampers the way to offer a definitive explanation
for the absence of more obvious statistically significant differences Still the small
number of significant findings and the mean values suggest that possible analgesic
effects of infrared LED may not be excluded yet but to be able to estimate the real
value of LED further research is necessary A large-scaled randomised clinical trial
which takes the above-mentioned remarks into consideration should be performed
CONCLUSION
Regardless of the reasons for the absence of statistical significant effects reported here
and although LED may have some potential in the management of pain and functional
impairment associated with DOMS its effectiveness at the applied densities has not
been established
Future research should focus on evaluation of the appropriateness of DOMS as an
experimental model of pain and muscle damage Validation of this model would
enhance the ability to study various modalities for their potential effects on pain and
muscle injuries Besides the mechanisms of LED action are not known thus further
fundamental investigations need to address the underlying mechanism and
physiological basis of pain modulation utilizing LED treatment
Once LED irradiation has finally proven its treatment value in an experimental model
the most important prospect considers establishing the effectiveness of LED to reduce
pain in clinical settings
Delayed-onset muscle soreness
107
ACKNOWLEDGMENTS
The authors would like to thank Mr T Barbe and Mr R Deridder for their technical
assistance in the collection of the data as well as for their valuable input into the
research design Sincere appreciation is extended to the volunteers that participated in
this study and to MDB-Laser (Belgium) for generously providing the light emitting
diode equipment The authors also gratefully recognize Prof Dr G Van Maele for
assistance with the statistical analysis and for helpful discussion
Chapter 5
108
REFERENCES
1 Vinck E Coorevits P Cagnie B Muynck MD Vanderstraeten G and Cambier D (2005) Evidence of changes in sural nerve conduction mediated by light emitting diode irradiation Lasers Med Sci DOI101007s10103-005-0333-2
2 Cheung K Hume PA and Maxwell L (2003) Delayed Onset Muscle Soreness - Treatment Strategies and Performance Factors Sports Med 33(2)145-164
3 MacIntyre DL Reid WD and McKenzie DC (1995) Delayed muscle soreness The inflammatory response to muscle injury and its clinical implications Sports Med 20(1)24-40
4 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
5 Clarkson PM and Tremblay I (1988) Exercise-induced muscle damage repair and adaptation in humans J Appl Physiol 65(1)1-6
6 Cleak MJ and Eston RG (1992) Delayed onset muscle soreness mechanisms and management J Sports Sci 10(4)325-341
7 Craig JA Cunningham MB Walsh DM Baxter GD and Allen JM (1996) Lack of Effect of Transcutaneous Electrical Nerve Stimulation Upon Experimentally Induced Delayed Onset Muscle Soreness in Humans Pain 67(2-3)285-289
8 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
9 Ebbeling CB and Clarkson PM (1989) Exercise-Induced Muscle Damage and Adaptation Sports Med 7(4)207-234
10 Tiidus PM and Ianuzzo CD (1983) Effects of Intensity and Duration of Muscular Exercise on Delayed Soreness and Serum Enzyme-Activities Med Sci Sports Exerc 15(6)461-465
11 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
12 Armstrong RB (1984) Mechanisms of exercise-induced delayed onset muscular soreness a brief review Med Sci Sports Exerc 16(6)529-538
13 Rodenburg JB Steenbeek D Schiereck P and Bar PR (1994) Warm-up stretching and massage diminish harmful effects of eccentric exercise Int J Sports Med 15(7)414-419
14 Ciccone CD Leggin BG and Callamaro JJ (1991) Effects of ultrasound and trolamine salicylate phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-675 discussion 675-678
15 Craig J Barlas P Baxter D Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
16 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
17 Dvir Z (2003) Isokinetics muscle testing interpretation and clinical applications Churchill Livingstone Edinburgh
18 Lambert MI Marcus P Burgess T and Noakes TD (2002) Electro-Membrane Microcurrent Therapy Reduces Signs and Symptoms of Muscle Damage Med Sci Sports Exerc 34(4)602-607
19 Sumida K Greenberg M and Hill J (2003) Hot gel packs and reduction of delayed-onset muscle soreness 30 minutes after treatment J Sport Rehabil 12221-228
20 Tiidus PM and Shoemaker JK (1995) Effleurage massage muscle blood flow and long-term post-exercise strength recovery Int J Sports Med 16(7)478-483
21 Zhang J Clement D and Taunton J (2000) The efficacy of Farabloc an electromagnetic shield in attenuating delayed-onset muscle soreness Clin J Sport Med 10(1)15-21
22 Fitzgerald GK Rothstein JM Mayhew TP and Lamb RL (1991) Exercise-Induced Muscle Soreness After Concentric and Eccentric Isokinetic Contractions Phys Ther 71(7)505-513
Delayed-onset muscle soreness
109
23 Nosaka K and Clarkson PM (1997) Influence of Previous Concentric Exercise on Eccentric Exercise-Induced Muscle Damage J Sports Sci 15(5)477-483
24 Jensen MP Karoly P and Braver S (1986) The Measurement of Clinical Pain Intensity - a Comparison of 6 Methods Pain 27(1)117-126
25 Price DD Mcgrath PA Rafii A and Buckingham B (1983) The Validation of Visual Analog Scales as Ratio Scale Measures for Chronic and Experimental Pain Pain 17(1)45-56
26 Revill SI Robinson JO Rosen M and Hogg MIJ (1976) Reliability of a Linear Analog for Evaluating Pain Anaesthesia 31(9)1191-1198
27 Nussbaum EL and Downes L (1998) Reliability of Clinical Pressure-Pain Algometric Measurements Obtained on Consecutive Days Phys Ther 78(2)160-169
28 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
29 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
30 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118
31 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in biomedical optics and imaging 3(28 Proceedings of SPIE 4903)156-165
32 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18(2)95-99
33 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2005) Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level J Photomed and Laser Surg 23(2)167-171
34 Pollo A Amanzio M Arslanian A Casadio C Maggi G and Benedetti F (2001) Response Expectancies in Placebo Analgesia and Their Clinical Relevance Pain 93(1)77-84
35 Shellock FG and Prentice WE (1985) Warming-up and Stretching for Improved Physical Performance and Prevention of Sports-Related Injuries Sports Med 2(4)267-278
36 Safran MR Seaber AV and Garrett WE (1989) Warm-up and Muscular Injury Prevention an Update Sports Med 8(4)239-249
37 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
38 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
GENERAL DISCUSSION
General discussion
113
SUMMARY
As outlined in the general introduction the overall objective of this doctoral thesis is to
develop the current knowledge about the mechanisms of LED action in view of the
eventual provision of evidence-based support for the clinical use of LED as a
biostimulatory and analgesic treatment modality especially in the field of
physiotherapy
Part I Wound healing
The investigations described in chapter 1 and 2 were conducted to gain insight into the
potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As
fibroblasts are principal cells for biostimulation (in view of growing and dividing in
healing wounds) the influence of LED irradiation on fibroblast proliferation was
assessed1
The first investigation consisted of a pilot study performed in order to evaluate the
appropriateness of the cell isolation technique cell culture protocol and proliferation
analysis as well as to appraise the feasibility of the light source properties and
illumination procedure
Data analysis revealed no statistically significant differences between the infrared LED
irradiated and control petri dishes for the used parameters (table 1) Considering this
outcome other experimental findings disclose that the absence of stimulatory effects of
LED irradiation on fibroblast proliferation can partly be attributed to the use of
inappropriate light source properties However the applied external dosimetric
parameters are well within the recommended spectrum described by previous studies
investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be
excluded that changes in the illumination procedure (such as the use of lower power
shorter exposure times wavelengths with finer coverage of the absorption spectrum of
the irradiated cells and a longer incubation period between the last irradiation and cell
counting) could still result in an increased fibroblast proliferation467 Of equal
importance in interpreting the lack of distinctive results are the imperfections of the
applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails
114
considerable intervention from the investigator compromising the reliability of the
method It is also a time-consuming technique with an insufficient sensitivity for some
purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11
To avoid contamination of the results by these modifiable remarks a similar
experiment (chapter 2) was performed in which wavelength power and output mode of
the infrared LED source were not modified (table 1) only the exposure time was
reduced resulting in a lower radiant exposure In addition the effect of two other
emission spectra was evaluated These probes emitting red and green light had a
shorter wavelength than the infrared LED source and the power was half or a
sixteenth of the power from the infrared probe Consequently the red LED irradiation
occurred with a different exposure time than the infrared one in order to attain the
same radiant exposure (053 Jcm2) With respect to the green LED it was not
endeavoured to achieve the same radiant exposure as 16 min of irradiation is not
feasible for in vitro or clinical application
Finally also an LLL light source was integrated Although it was not attempted to
analyse the effectiveness of LED in comparison to LLL enclosure of this modality was
interesting in order to join in with the available literature covering mostly LLL studies
To bypass the described problems regarding analysis of fibroblast proliferation
counting of the cells was carried out this time by means of a colorimetric MTT assay
This method provides more accurate cell counts in a short period of time and therefore
can be considered as a more reliable alternative to Buumlrker hemocytometer11
MTT assay 24 h after the last irradiation revealed a significantly increased number of
cells in the irradiated wells in comparison to their (respective sham-irradiated) controls
Although the study supplied experimental support for a significantly increased cell
proliferation by all external dosimetric properties based on the results of the
comparative trial with an incubation period of 24 hours irradiation with the green
LED source yielded the highest number of fibroblasts Thus it can be concluded that
the wavelength of the green LED is probably within the bandwidth of the absorption
spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that
General discussion
115
the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast
cultures in vitro612
Table 1 External dosimetric properties summarized for each chapter
Wavelength Power Exposure
time Output mode
Radiant exposure
PART I Chapter 1
In vitro part
LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2
LED-infrared 950 nm 160 mW 1 min continuous 053 Jcm2
LED-red 660 nm 80 mW 2 min continuous 053 Jcm2
LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2
Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2
PART II Chapter 4
LED-infrared 950 nm 160 mW 2 min continuous 107 Jcm2
Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
The next aim of the first part of this doctoral thesis was to explore whether LED
treatment could ameliorate in vitro cell proliferation under conditions of impaired
healing In the pursuit of this aim fibroblasts were cultured in medium supplemented
with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a
pilot study the amount of glucose necessary to inhibit normal growth was determined
In order to attain an important reduction of cell viability and decreased proliferation
rate a relatively high concentration of glucose (1667 mM) was necessary in
comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia
(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity
between both circumstances in vitro limited to 72 h whereas the human tissue of a
diabetic patient in vivo is chronically exposed to glucose
Treatment of the fibroblasts occurred in respect of the previously described results
with the same irradiation parameters and illumination procedure (chapter 2)
Accordingly green LED irradiation labelled as the most appropriate treatment for
116
irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in
the previous study (table 1)
Analysis of the cell proliferation by means of MTT measurements yielded a
significantly higher rate of proliferation in hyperglycaemic circumstances after
irradiation than in the control conditions (ie hyperglycaemic circumstances without
irradiation) Thus this outcome supported the stimulatory potential of green LED
irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a
considerable destructive hyperglycaemic medium
Finally although the results of the in vivo part of chapter 1 were persuasive and
encouraging they will not be further discussed in this summary of part I as it was not
aimed in this doctoral thesis to investigate the wound healing process in vivo However
the results of this case study can be a valuable hold for future in vivo research
The possible clinical implications of these results and future research directions in the
scope of wound healing will be discussed below
Part II Analgesia
In the second part two studies investigated the effects of LED irradiation as a
potential intervention mode in one of the most important fields in physiotherapy
practice analgesia Chapter 4 describes the influence of LED treatment on changing
sensory nerve conduction characteristics of a human superficial peripheral nerve
Altering nerve conduction characteristics may not be the sole beneficial purpose to
attain with LED irradiation in view of analgesia but the advantage of nerve conduction
characteristics is that they are objective measurable physical variables and changes in
these characteristics provide a potential explanatory mechanism of pain inhibition by
LED treatment17
The results showed that percutaneous LED irradiation at feasible clinical parameters
can generate a significant decrease in NCV and increase in NPL for all recordings post-
treatment in comparison to the baseline measurement The data in the placebo group
did not reveal any significant difference in the same course of time Statistical analysis
General discussion
117
revealed significant differences between the experimental and the placebo group for
NCV as well as for NPL at all time-points of observation with exception of the NPL
recording immediately after finishing irradiation
It was also observed that the noted effects did not weaken as time progressed It can
be concluded that post-treatment conduction measurements should be extended in
time which is in accordance with the findings of some previous studies18-21 Clarifying
the point of time at which the effect extinguishes is necessary and clinically relevant
when treating pain by means of LED irradiation Besides obtaining the desired
neurophysiological effects ideally the optimal irradiation parameters should be
applied The most favourable dosimetric properties are not yet determined but based
on this study and previously described assays it can be speculated that the dosimetric
window is quite large
Regardless of these clinically important remarks the present findings allow to draw the
following conclusion LED irradiation at clinically applied densities can generate an
immediate and localized effect upon conduction characteristics in underlying nerves as
LED treatment results in lowering the NCV and augmenting the NPL Therefore the
outcome of this in vivo experiment assumes a potential pain relief by means of LED
treatment and justifies further research regarding its clinical effectiveness in laboratory
settings and at a clinical level
The fourth and final aim was to determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5
illustrates a clinical study observing the effect of LED treatment on a model
comprising experimentally induced DOMS in a healthy population The progress of
pain perception and peak torque was evaluated during 4 consecutive days commencing
on the day of DOMS induction The effect of infrared LED treatment at the light
parameters described (table 1) was assessed with regard to three different factors time
(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures
(VAS MPT and IPT) revealed no significant interactive effects of the main interaction
118
(timegrouppre-post) For the remaining interactions and for the main effects only a
few significant findings were relevant in view of the postulated hypothesis
Notwithstanding the absence of an apparent and overall statistically significant finding
the present results indicate favourable trends of LED treatment on pain as the means
of all VAS and MPT variables show a statistically nonsignificant general analgesic
effect of infrared LED irradiation expressed by lower subjective pain rates and higher
MPT values in the irradiated group In addition to the analgesic influence of LED an
augmented restoration of muscle strength was noted The lack of solid statistically
significant evidence for these beneficial effects of LED treatment upon DOMS-
associated pain can possibly be attributed to the small sample size in this study or even
to the size of the treatment effect in relation to the severity of the induced DOMS as
induction of severe DOMS can mask relatively small but apparent treatment
effects2223 A final possibility is that the results only become significantly different after
a prolonged treatment and follow up period as previous research demonstrated that
recuperation subsequent to DOMS induction can last up to 10 days24
It should also be noted that the described general analgesic effect of LED irradiation
was identical for the treated as well as for the control arm in the irradiated group
proposing that infrared LED might induce systemic effects 2526 However it needs to
be stressed that these results were not statistically significant
Regardless of the absence of statistically significant findings the mean values suggest a
potential role for infrared LED irradiation in the management of pain and functional
impairment associated with DOMS Notwithstanding this postulation future research
is absolutely required to establish the effectiveness of LED treatment to reduce pain as
well at the applied densities as for other dosimetric parameters
CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS
In the course of the past years during the process of the genesis of this thesis
therapeutic physical agents in general and phototherapeutic modalities in particular
became less important as physiotherapeutic modes of treatment than during the
preceding two decades The diminished use of these treatment modalities in the
General discussion
119
physiotherapy practice is to a certain degree a consequence of the controversial
research findings regarding the use of these physical agents This issue of controversy
led to less support for the use of these treatment modalities and a growing scepticism
regarding the effectiveness of these physical agents within the scope of the growing
climate of evidence-based practice A second responsible protagonist for the loss of
popularity of physical agents is linked with the current tendency within physiotherapy
emphasising active remedial therapy The establishment of this development was based
on various experiments mainly performed during the last decade demonstrating that
active treatment modalities are for numerous impairments and disabilities preferable to
more passive forms of therapy In Belgium the prevailing nomenclature which came
into use on 1 May 2002 went along with this tendency In the appendix to the Royal
decree of 14 September 1984 towards settlement of the nomenclature of medicinal
treatments concerning compulsory insurance for medical care and allowances the
personal involvement of the physical therapist during the physiotherapeutic session
was emphasized and it was even defined that massage physical techniques within the
framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal
application can only be remunerated when they are applied supplementarily and not as a sole therapy
This implies that passive treatment modalities should not be used as sole method of
treatment and should always be considered as an adjunct to an active treatment
program This development needs to be applauded in many cases such as various
painful musculoskeletal problems functional instability rehabilitation of neurological
patients re-activation of the elderly population psychomotor rehabilitation
cardiovascular and respiratory convalescence Nevertheless it would be erroneous to
entirely reject physical agents including LED treatment Based on the findings of the
above described experiments it needs to be stressed that for some purposes especially
within the scope of impaired wound healing LED irradiation could be a suitable
therapeutic measure This statement is founded on the results of part I of the present
thesis they provided satisfactory fundamental evidence for the advantageous effects of
LED treatment on a crucial exponent of the wound healing process namely fibroblast
proliferation The beneficial findings are the result of basic in vitro research As it is
120
inaccurate to simply extrapolate these results to the clinical practice the clinical use of
LED irradiation for wound healing needs to be preceded by purposive and specific in
vivo investigations to substantiate these basic research findings27
The case study described in chapter 1 indicates a foundation for further in vivo research
Visual appraisal of the surgical incision revealed (from the 65th day in the course of the
reparative process onwards) that the irradiated area -which initially showed inferior
epithelialization and wound contraction- showed a more appropriate contracture than
the control area characterized by less discoloration at scar level and a less hypertrophic
scar These clear beneficial effects of LED treatment on a human cutaneous wound
can serve as preliminary impetus for further research into the clinical applicability of
LED therapy although this case study is insufficient in order to guarantee a safe
correct and effective use of LED as a therapeutic modality
Despite these remarks it tentatively can be concluded that based on a detailed analysis
of the available data of the present in vitro studies and the given case report in
combination with the small number of previously published human studies the
beneficial effects of LED irradiation at the cellular level are obvious and therefore a
potentially favourable outcome can be assumed in clinical practice28-30 LED-
modulated stimulation of wound healing can be gradually and vigilantly implemented
clinically Nevertheless the real benefits of LED irradiation within the scope of wound
healing can only be established by additional clinical trials as thus far clinical
application and stipulation of dosimetry still occurs on a trial-and-error basis which is
not conducive to a generally accepted clinical use of LED To lend more credibility to
the treatment of wounds by means of LED irradiation and to expel the existing
controversy and scepticism surrounding this topic in vivo investigations on wound
healing using a number of different animal models and adequately controlled human
studies are necessary In addition these studies should be performed preferably on a
population suffering from impaired healing as a consequence of diabetes mellitus or as
a result of any other debilitating reason because as posed by Reddy et al3132 and as
mentioned above light has possible optimal clinical effects in the treatment of healing-
resistant wounds
General discussion
121
Drawing general conclusions and formulating clinical implications for analgesia is
obviously less manifest first of all because only a limited number of possible
mechanisms of action in order to obtain analgesia were highlighted and secondly
because both studies did not come to a joint or complementary conclusion The
outcome of the first study revealed that LED treatment lowers the NCV and augments
the NPL resulting in a slower stimulus conduction and consequently a reduced number
of sensory pulses per unit of time Thus it could be assumed that LED induces pain
relief but the results of the study describing the effect of LED treatment on
experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy
In addition it needs to be emphasised that the first study (chapter 4) measured the effect
of LED irradiation on the large myelinated Aβ afferents A noteworthy question and
meanwhile a stimulus for future investigation is whether the measured effects can be
extrapolated from these sensory nerve fibres to the actual nociceptive afferents
notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of
these nociceptive pathways relies on laser-evoked potentials which selectively activate
Aδ-fibres and C-fibres3334 This technique was presently not available therefore a
standard sensory nerve conduction study was performed
Whereas stimulation of wound healing by means of LED irradiation can be cautiously
implemented in the clinical practice at this stage it is too early to promote LED
irradiation as a treatment modality for pain To make this possible it is essential to
conduct numerous studies with regards to the use of LED in the field of analgesia
Future research should focus on fundamental investigations in order to discover the
underlying mechanisms and physiological basis of pain modulation utilizing LED
treatment Furthermore the evaluation of the appropriateness of DOMS as an
experimental model of pain is an important prospect to consider as validation of this
model would enhance the ability to study various modalities for their potential effects
on pain Irrespective of the difficulties regarding standardisation of the research
population and evaluation of soreness inextricably linked with clinical pain studies the
122
ultimate objective of future research should be the establishment of the effectiveness
of LED irradiation to reduce pain of miscellaneous origin in a clinical setting
Regardless of the encouraging results of the described studies and besides the earlier
proposed specific directions for future research (directed towards wound healing or
pain relief) it is necessary in the interest of the patientrsquos well being and to the
advantage of the prospective clinical use of LED to highlight a few more issues for
future research Therefore one has to deal with some limitations of the performed
investigations A first limitation concerns the fact that only two mechanisms of LED
action were investigated (notably changed fibroblast proliferation and alteration of the
nerve conduction characteristics) So one can conclude that for further and better
understanding of the mechanisms of action it is necessary to perform more basic
research Answering the questions regarding the functioning of LED irradiation will
simplify the evaluation and reinforce the interpretation of the obtained results and
ultimately contribute to a more widespread and well definded acceptance of the use of
LED in clinical settings
A second general limitation of this doctoral thesis is the substantial difference in the
used external dosimetric parameters between the different chapters and even within
one and the same study (illustrated in table 1) this complicates the comparison
between the different trials In each trial the dosimetry was individually ascertained
based on previous studies within the given field As not for every application the same
dosimetry is suggested in literature a range of dosages were used Another important
factor in deciding on the dosimetry was the clinical applicability of the dosage as it is
useless to investigate the appropriateness of a treatment modality at a clinically
unrealistic dose As a result of this limitation the current findings do not fully
contribute to the explanation regarding the ideal parameters one should use although
this was not set as a principal purpose Based on this thesis and previously described
assays it can be speculated that the possible window for these parameters is quite large
the ideal irradiation parameters and proper timing or sequencing of LED irradiation
General discussion
123
for example to the various phases of wound healing and to different painful conditions
are therefore possibly unattainable
The establishment of an appropriate dosimetry should also consist of investigating the
absolute and relative penetration depth of LED irradiation into human tissue This is
less crucial within the scope of wound healing but it is of key importance while
treating deep-seated tissue (eg nerve fibres muscles circulatory components et
cetera)
Finally this thesis only investigated the efficiency of LED in a very limited number of
conditions notebly wound healing and pain Within the scope of physiotherapy and
medicine in general there are numerous other purposes for which LED irradiation is
promoted such as oedema arthritis miscellaneous orthodontic applications seasonal
affective disorder neonatal jaundice photodynamical therapy et cetera2835-41
In summary additional work on establishing proper dosimetry and identifying the
biochemical or photobiologic phenomena that are responsible for improving wound
healing and reducing pain or even other effects within a broader spectrum of
conditions remains to be done in order to answer unreciprocated questions Until that
time the potential clinical usefulness and actual value of LED irradiation for wound
healing and even to a larger extent for analgesia should always be approached with
appropriate professionalism and even caution
FINAL CONCLUSION
LED devices are promoted for clinical use but the currently available scientific
documentation regarding effectiveness of this physical agent is rather scarce Through
providing scientific support for the biostimulatory and analgesic effectiveness of LED
irradiation this doctoral thesis attempted to bridge in some degree this gap
The conducted studies revealed that LED irradiation undeniably has potential
beneficial effects on wound healing and to a lesser degree within the scope of
analgesia However based on the present results it can be corroborated that light
124
therapy in the guise of LED irradiation is not magic but these results can raise some
corrective doubts in fundamental disbelievers and antagonists
Nevertheless we have to join the queue of scientists who have found beneficial results
but cannot elucidate with certainty how this outcome was established Thus although
the present results are encouraging a continuing development and integration of new
knowledge based on further research is necessary in various domains of intervention
Therefore several directions for future investigations were proposed in order to cover
as many existing gaps and to answer the utmost number of remaining questions as
possible Still one ought to be aware not to carry future fundamental research at a
disproportional level and the inevitable quest for mechanisms of LED action should
not hypothecate the potential clinical value implying that at a certain point it should be
appropriate to make the transfer from science to the application of the available
knowledge in clinical practice
The described findings regarding LED irradiation are comparable to the results of
previously published studies performed with other light sources Consequently as
postulated by some LED providers it can be speculated that the biological response of
tissue to light irradiation can probably not be equated merely to a light source but
rather to a broad photo-energy window
General discussion
125
REFERENCES
1 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
2 Abergel R Lyons R Castel J Dwyer R and Uitto J (1987) Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2)127-133
3 Skinner S Gage J Wilce P and Shaw R (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-192
4 Webb C Dyson M and Lewis W (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5)294-301
5 Atabey A Karademir S Atabey N and Barutcu A (1995) The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 1899-102
6 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
7 Ben-Dov N Shefer G Irinitchev A Wernig A Oron U and Halevy O (1999) Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3)372-380
8 Rebulla P Porretti L Bertolini F et al (1993) White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2)128-133
9 Deneys V Mazzon A Robert A Duvillier H and De Bruyere M (1994) Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2)172-177
10 Mahmoud A Depoorter B Piens N and Comhaire F (1997) The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2)340-345
11 Haskard D and Revell P (1984) Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3)319-322
12 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers
13 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of cultured human endothelial cells Diabetes 36(11)1261-1267
14 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA damage in cultured human endothelial cells J Clin Invest 77(1)322-325
15 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7)621-627
16 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4)491-501
17 Kramer J and Sandrin M (1993) Effect of low-power laser and white light on sensory conduction rate of the superficial radial nerve Physiotherapy Canada 45(3)165-170
18 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
19 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
126
21 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
22 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
23 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
24 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
25 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
26 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer
G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
29 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
30 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255
32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287
33 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-28
34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856
35 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
36 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
37 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263
39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64
40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74
General discussion
127
41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press
NEDERLANDSTALIGE SAMENVATTING
Nederlandstalige samenvatting
131
NEDERLANDSTALIGE SAMENVATTING
Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de
tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te
vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie
van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen
Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest
enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is
ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als
op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang
van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve
interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken
Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van
ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen
moeilijk te verantwoorden
Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin
de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere
overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit
methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan
consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in
de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen
laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in
de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag
aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies
inclusief het gebruik van licht
Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat
de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde
doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en
dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht
komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt
geloosd
132
De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo
lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en
gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te
anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te
voorkomen lijkt een gerichte en rationele a priori aanpak conform de
wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen
Te meer daar grondige literatuurstudie leert dat men ten behoeve van de
werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie
zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser
De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de
extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is
geboden De introductie van alternatieve lichtbronnen in de huidige
kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk
weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus
eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood
naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based
gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder
binnen de domeinen van haar potentieel beloftevolle klinische toepassingen
wondheling en analgesie
Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de
wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op
prominente protagonisten van de wondheling de fibroblasten werd getracht het
fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten
zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de
proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd
respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand
waarbij de normale celgroei werd verstoord
In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het
effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische
Nederlandstalige samenvatting
133
data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden
verklaard door het gebruik van een inadequate LED dosering en anderzijds een
methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie
vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de
precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en
inter-tester variabiliteit tot gevolg
In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde
onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de
bestralingsparameters (dosering) De effecten van de drie verschillende LED
golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten
werden hierbij geanalyseerd door middel van een meer betrouwbare en minder
subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-
yl)-25-diphenyl tetrazolium bromide (MTT)
De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir
effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden
tevens een basis voor meer coherente en relevante inzichten aangaande de globale
bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)
Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het
wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend
karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp
rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde
vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De
fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met
extreem hoge concentraties glucose Deze modificatie van het medium staat model
voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische
praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan
vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de
gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en
proliferatie
134
Het tweede deel van dit proefschrift exploreert het domein van het potentieel
analgetisch effect van LED binnen de kinesitherapie aan de hand van twee
fundamentele onderzoeken
In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere
sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele
hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en
een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale
verklaring van een analgetisch effect van het medium zou kunnen belichamen
Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)
meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf
verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen
aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke
significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de
negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele
hypothese
Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend
fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale
pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp
van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische
krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED
behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier
keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED
op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd
(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde
isokinetische krachtmeting en een registratie van de waargenomen spierpijn De
spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met
behulp van een kwantitatieve hand-hold algometer
Analyse van de bekomen data bracht geen significante verschillen tussen de
controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden
Nederlandstalige samenvatting
135
kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED
behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van
de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de
proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene
afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine
proefgroep die werd onderzocht enof door de grootte van het behandeleffect in
verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers
een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up
kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel
absolute omzichtigheid geboden en moet deze visie louter als speculatief worden
beschouwd
Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de
kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de
laatste jaren aanzienlijk afgenomen
De positieve resultaten van de verschillende in vitro studies in het kader van wondheling
vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing
vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel
gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een
belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek
Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie
Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme
onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch
complementair resultaat Verder onderzoek ter exploratie van de mogelijke
onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom
onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op
termijn wetenschappelijk te rechtvaardigen
136
LED tovenarij trend of therapie
LED mag geen magische krachten worden toegemeten maar verdient het lot van een
kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een
wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen
zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke
indicaties
Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds
the darkness has always got there first and is waiting for it
(Terry Pratchett Reaper Man 1991)
X
Warm thanks go to the colleagues of the department of Human Anatomy
Embryology Histology and Medical Physics for providing the culture medium for the
technical support for the helpful discussions and principally for the amusing pastime
aseptic chats
In addition I also want to thank my colleagues of the associated institute Kinesitherapie
Gent and above all the colleagues of the department of Rehabilitation Sciences and
Physiotherapy Ann Axel Barbara Bart Bert Bihiyga Bruno Carine Damien Els2
Erik Frike Helga Inge James Karlien Kathy Kat(h)leen Koen Marc Martine
Mieke Paris (Nele) Patrick Roland Sophie Stefan Stijn (Veerle) Wiene Wim and
Youri thank you for the organisation and your attendance at many memorable
sidelines such as the survival-weekend the first department-day Fata Revaki our
legendary Thursday-sport activities and Friday-afternoon coffee breaks St Nicholas
visit skiing holiday New Yearrsquos lunch with quiz bowling spinning the introduction of
ldquosubetelyrdquo during our conspicuous presence at WCPT 2003 et cetera We shared many
treasured moments thanks to you a common working day often turned out to be very
pleasant I know that it will be impossible to find a comparable team of colleagues to
work with in the future
I especially want to thank Barbara to remind me on a regularly basis of my deadlines
to listen to my grieves and joy and to be willing to offer me a window-seat in our
office Kurt (although you abandoned at a certain moment) for solving my computer
problems Pascal for assistance with the statistical analyses Lieven for your motivating
interest and finally Fabienne Tine and Kim as loyal and appreciated friends who
worn-out several sports shoesbathing suits to supply in the weekly portion of sports I
needed to remain physically and mentally fit
I also extend my appreciation to my family and friends for their interest in my research
activities permanent mental support for the adoption of the surviving chickens but in
particular for looking after Louka and for the numerous relaxing moments Special
thanks are going to Katelijne and Frank for the delicious hot meals on lonely evenings
XI
Thomas Stijn and Johan thank you for the repeated (mostly futile) attempts to
convince me to do something together Sebastiaan each time during the past few years
when I doubted about the sense of my work it was your ridiculous story about a man
who wanted to invent superglue but instead invented the well-known yellow post-it
which stimulated me to continue my scientific quest
Of course I owe most gratitude to Luc my most devoted supporter Dearest I know
that since august 2004 you lived a solitary life in Dubai Although I think it was
possibly easier not to live under the same roof with me these last stressful months I
am aware that it was very difficult for you not to be able to play with Louka and to
miss some precious months of her life
Louka thank you for your radiant smile and daily baby speeches I am sorry that you
had to miss your daddy I promise that we will be reunited very soon
Elke Vinck
Ghent March 2006
GENERAL INTRODUCTION
General introduction
3
BACKGROUND
The use of light for therapeutic purposes reaches far back in time Current interest for
photomedicine with his its biological and medical effects relies fundamentally on two
major evolutions in the given field (1) the research results regarding the use of
ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in
the light amplification by stimulated emission of radiation (laser)-technology The production
of the first laser the ruby pulsed laser was rapidly succeeded by the development of
the helium-neon laser and other lasers like the argon the neodymium-glass and the
neodymium-yttrium-aluminium-garnet lasers1
As in the mid-1990s semiconductor and diode-based lasers gained popularity the
principally massive gas and dye lasers were rendered obsolete Therapeutic light
technology further continued to evolve and todayrsquos therapeutic light source is as likely
to be a light emitting diode (LED) or polarized light as a semiconductor or diode
laser1
Technological advancement and variation of the light sources necessitate a
concomitant update and revision of research in the respective domains of application
Unfortunately this logical and rational necessity has rarely been fulfilled From a
historical perspective this lack of appropriate research has led to disenchanting
evolutions in the use of light especially in physiotherapy The experience exists in this
medical field that light sources were promoted and commercialised for a vast regimen
of indications without foregoing scientific backup Consequently research developed
often after the commercial introduction in physiotherapy As these investigations
frequently gave rise to conflicting results for certain indications scepticism arose and
the use of the given modality knew a waning popularity for all its indications The final
result of such an inappropriate frame of promotion commercialisation and research is
a growing clinical disuse of a given modality even for motivated indications In view of
the actual increasing interest in LED treatment and based on former ascertainment
one has to state that a literature review for the given source reveals that research
mostly covers only low level laser (LLL) studies23 Although recently a number of
papers can be noted that report on the effects of LEDs and polarized light still
4
numerous source-specific-questions need to be answered as research concerning
mechanisms of action and efficacy of the current light sources remains limited in view
of a substantiated clinical application4-17
The reason for the contemporary light-oriented interest in physiotherapeutic practice
for LED devices is in essence based on several advantages of LED in comparison with
LLL For example the use of LEDs is esteemed to be safer as the delivered power
does not damage tissue LEDs can be made to produce multiple wavelengths thereby
stimulating outright a broader range of tissue types and probes that cover a large
treatment area are available18 In addition from a commercial point of view LEDs are
far more interesting as they are a good deal cheaper than laser diodes and they have a
long life span as these solid devices stand robust handling
As a result of the above-mentioned lack of literature on LED some providers of these
devices have taken for granted that the biological response of tissue to light irradiation
cannot be equated merely to a light source They declare that a given response solely
depends on the extent of absorption of radiated light by the tissue19 Consequently
these providers state that it is acceptable to extrapolate scientific findings of LLL
studies for explaining the mechanisms of action and detailing the efficacy of LED and
other alternative light sources Thus actually without appropriate scientific support
equal biological effects are attributed to LED as to LLL Nevertheless prudence is
called for such an extrapolation firstly because it is irrespective of the mentioned
dissimilarities and by simple projection one ignores a number of physical differences
between LLL and LED (eg coherence and degree of collimation or divergence)
Secondly LLL therapy is still not yet an established and evidence-based clinical tool20
Notwithstanding the historical efforts there still remains a considerable amount of
ignorance scepticism and controversy concerning the use and clinical efficacy of
LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed
parameters for irradiation as well as to the difficult objective measurement of possible
irradiation effects and even to the exceptional range of unsubstantiated indications for
General introduction
5
which light therapy was promoted27-29 A lack of theoretical understanding can also be
responsible for the existing controversies as the evaluation and interpretation of
research results would be simplified largely when the appropriate knowledge about the
mechanisms of light action would be available
LLL literature can undoubtedly be used as basis for research on LED and as a
comparative reference for these given investigations However to guarantee evidence-
based use of LED within physiotherapy the need for specific research in view of an
accurate consumption of LED is definite especially for potential promising clinical
applications in physiotherapy according to LLL literature mainly wound healing and
analgesia3031
Hitherto the most substantial research concerning the use of LED for improvement
of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded
under the influence of prolonged exposure to microgravity (eg during long-term space
flights) and in case of absence of exposure to sunlight such as in submarine
atmospheres they performed wound healing experiments for military application in the
given circumstances3233 In vitro experiments revealed that LED treatment increased
proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA
synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and
osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies
demonstrated therapeutic benefits of LED in speeding the early phase of wound
closure and in changing gene expression in a type 2 diabetic mouse model183234
Human studies noted 50 faster healing of lacerations a return of sensation and
increased tissue granulation as a result of LED irradiation1833
Associates of the Rehabilitation Sciences Research Group of the Ulster University in
Northern Ireland extensively investigated the effectiveness of light in the treatment of
pain The emphasis was laid primarily on the analysis of the effects of various low level
laser light sources35-44 However in the year 2001 two studies gave an account on the
efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46
6
noticed relatively long-lasting neurophysiological effects a significant change of the
nerve conduction characteristics (decrease of the negative peak latency) was mediated
by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a
comparable multisource diode device was ineffective in the management of delayed-
onset of muscle soreness (DOMS)
Despite the major value of these described trials a definitive answer regarding the
ability of LED in influencing wound healing or pain is not forthcoming cardinally
because a number of aspects are not yet investigated Consequently more research is
required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the
formerly mentioned scepticism regarding the effectiveness of a medium and possibly
to the undeserved fall into disuse of the treatment modality which happened in a way
with LLL therapy
PHYSICAL CHARACTERISTICS
This chapter supplies a short but comprehensive review of opto-physics A brief
description of the physical characteristics of the LED source used is essential as the
physical properties of light play an important part in the ultimate efficacy of treatment
According to the International Electrotechnical Commission (IEC 60825-1) an LED
can be defined as
Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by
radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process
of spontaneous emission1947
The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO
preprototype MDB-Laser Belgium) This illustration shows that a probe consists of
32 single LEDs disseminated over a surface of 18 cm2
General introduction
7
Figure 1 LED device and three available probes (infrared red and green)
Three highly monochromatic probes were available each emitting light of a different
wavelength within the above-defined range (table 1)2748 The wavelength of the light
emitted and thus its colour depends on the band gap energy of the materials forming
the p-n junctiona This light property is a key determinant to obtain maximum
photochemical or biological responses as light absorption by tissue molecules is
wavelength specific27 Only by absorbing radiation of the appropriate wavelength
(namely the wavelengths equal to the energy states of the valence electrons)
photoacceptor molecules will be stimulated resulting in a direct photochemical
reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750
8
absorption spectrum of a particular molecule is limited absorption often only occurs
over a waveband range of about 40-60 nm274851 Nevertheless the absorption
spectrum at cell or tissue level is broad because cells are composed of many different
molecules
Besides its influence on the absorption by means of tissue molecules there is a crucial
link between wavelength and penetration depth of the irradiated light Penetration into
tissue decreases as the wavelength shortens hence green light penetrates less than red
light which at his turn penetrates less into tissue than infrared light2748 Detailed
principles of light penetration will be discussed below
The LED device used emits non-coherent light In the 1980s the observed biological
responses after laser irradiation were generally thought to be attributable to the
coherenceb of the light485253 Though currently the clinical and biological significance
of coherence is seriously questioned54 According to several authors coherence does
not play an essential role in laser-tissue interactions firstly as it was proven that both
coherent and non-coherent light clinically show equal efficacy75556 Secondly as
according to some authors almost immediately after transmission of light through the
skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et
al1957 state that both findings are incorrect coherence is not lost in tissue due to the
phenomenon of scattering and non-coherent light is not as efficient as coherent light
This lack of consensus makes it necessary to mention whether or not light is
coherent2758
Further decisive characteristics to accomplish phototherapeutic efficacy are the power
exposure time output mode and beam area Based on these parameters both
irradiancec and radiant exposured can be calculated According to numerous authors
some of these parameters are more crucial than others to determine whether
b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48
c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48
d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248
General introduction
9
absorption of light will lead to a photobiological event192728485455 However the
literature yields several controversial findings as not all authors attribute an equal
importance to a given parameter For example according to Nussbaum et al59
irradiance was the determinant characteristic in the biomodulation of Pseudomonas
aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49
found that in order to stimulate tissue cell proliferation a specific combination of
irradiance and exposure time are more important than the actual radiant exposure Low
et al3940 on the contrary highlighted the critical importance of the radiant exposure in
observing neurophysiological effects Whereas Mendez et al60 reported that both
parameters influence the final results of light therapy
Koutna et al61 even suggested that the output mode of light applications plays a more
prominent role in the treatment outcome than the wavelength of the used light source
Nevertheless this finding could not be confirmed by other research results Besides
more controversial findings have been published regarding the output mode although
the repetition rate in a pulsed mode was considered as an important treatment
parameter several investigations failed to prove its value19272840414461-64
Based on these findings it was opted within the investigations of this doctoral thesis to
irradiate in a continuous mode The remaining dosimetric parameters (wavelength
exposure time and power) depended on the purpose of each investigation they are
described in the respective chapters The data necessary for the calculation of the
radiant exposure for the equipment used in the respective trials are summarized in
table 1
Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High
Infrared 950 80 120 160 Red 660 15 46 80
Green 570 02 42 10
10
The radiant exposure of the used LED can be calculated as follows65
RE =
Radiant Exposure [Jcm2]
T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]
PRE = α S T
α = 006 (continuous mode) or
003 (pulsed mode)
The parameters commented on so far can be considered as the external dosimetry
involving all parameters directly controlled by the operator limited by the apparatus
used Furthermore there is the so-called internal dosimetry referring to (1) several
physical phenomena (reflection transmission scattering and absorption) influencing
the light distribution within the tissue during energy transfer (2) the optical
characteristics of the irradiated tissue as well as (3) the relation between the external
dosimetry and these respective elements5466
This internal dosimetry determines to a considerable extend the penetration of light
into tissue Penetration can be defined as the tissue depth at which the radiant
exposure is reduced to 37 of its original value1948 However this definition only
accounts for the absolute penetration depth resulting in direct effects of light at that
depth In addition there is also a relative penetration depth leading up to effects
deeper in the irradiated tissue and even in certain degree throughout the entire
body1967 These so-called systemic effects can be caused by chemical processes initiated
at superficial levels at their turn mediating effects at a deeper tissue level57
Involvement of several forms of communication in the tissue such as blood circulation
and transport of transmitters or signal substances is possible1967 This means that light
sources with poor absolute penetration do not necessarily give inferior results than
those with a good absolute penetration19
In the same context it should be noted that calculation and even measurement of the
exact light distribution during irradiation is highly complicated principally as tissues
have complex structures and also because the optical properties of tissues vary largely
inter-individual2768
General introduction
11
Studies regarding actual penetration depth of LED light are scarce consequently the
knowledge on the topic of penetration depth of LED light is based on literature
originating from LLL research19 These findings established with various LLL sources
revealed that there is an obvious relation between penetration depth and
wavelength27486769-71
Three final remarks can be made on the dosimetry First of all it should be noted that
partly as a result of the above-mentioned contrasting findings on dosimetry ideal light
source characteristics for effective treatment of various medical applications are not yet
established and probably never really will be28 Therefore in the attempt to offer
sufficient guidelines for correct use of treatment parameters one should always try to
provide detailed description of light source properties used in any trial so the
practitioner can interpret the scientific results adequately and accordingly draw the
correct conclusions for his clinical practice
A second comment is based on the mentioned possible influence of the external and
internal dosimetric parameters on the photobiological effectiveness of light the
intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation
between radiated wavelength and penetration depth19546572 So it should be
emphasized that caution is recommended when comparing research results of light
sources with different wavelengths or other dissimilar dosimetric parameters
A third and final remark considers the extrapolation issue Comparison of the
therapeutic usefulness of the same light source used on different species should occur
cautiously So simply extrapolating the dosage used for one species to another is
inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo
clinical practice is inappropriate So purposive and specific research is the prerequisite
to produce safe and correct use of light as a therapeutic modality27
12
MECHANISMS OF ACTION
In the past decennia several mechanisms of action for biostimulation and pain
inhibition have been proposed and investigated73 Research was primarily based on
studies at the molecular and cellular levels and as a second resort investigations
occurred at the organism level resulting in numerous possible explanatory
mechanisms272858
It is the common view that light triggers a cascade of cellular and molecular reactions
resulting in various biological responses Thus different mechanisms of whom the
causal relationships are very difficult to establish- underlie the effects of light3448557475
To illustrate this complex matter the various mechanisms of action will be summarised
by means of a comprehensive model (fig 2) Detailed discussion about the different
individual components of the proposed model and other effects than those regarding
wound healing or analgesia were not provided as this was beyond the scope of this
general introduction
As depicted in figure 2 exposure to light leads to photon absorption by a
photoacceptor molecule causing excitation of the electronic state or increased
vibrational state of the given molecule275173 This process is followed by primary
photochemical reactions7475 Several key mechanisms have been discussed in the
literature Respiratory chain activation is the central point and can occur by an
alteration in redox properties acceleration of electron transfer generation of reactive
oxygen species (namely singlet oxygen formation and superoxide generation) as well as
by induction of local transient heating of absorbing chromophores192848515576-83 It is
supposed that each of these respective mechanisms plays a part in obtaining a
measurable biological effect It is yet not clear if one mechanism is more prominent
and decisive than another nevertheless recent experimental evidence has revealed that
mechanisms based on changes in redox properties of terminal enzymes of respiratory
chains might be of crucial importance2848517679
The primary mechanisms occurring during light exposure are followed by the dark
reactions (secondary mechanisms) occurring when the effective radiation is switched
General introduction
13
off2851 Activation of respiratory chain components is followed by the initiation of a
complicated cellular signalling cascade or a photosignal transduction and amplification
chain associated with eg changes in the cellular homeostasis alterations in ATP or
cAMP levels modulation of DNA and RNA synthesis membrane permeability
alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87
The sequence of events finally results in a range of physiological effects essential for
the promotion of the wound healing process for supplying analgesia or other
advantageous responses (acceleration of inflammatory processes oedema re-
absorption increased lymph vessel regeneration or increased nerve
regeneration)12181927486188-93
Photostimulation of the wound healing process can be mediated by increased
fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis
and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations
have been especially focussed on fibroblasts but other possible physiological effects
attributing to an accelerated wound healing were also observed suppression and
alteration of undesirable immune processes increased leukocyte activity new
formation of capillaries increased production of growth factors and enzymes while
monocytes and macrophages can provide an enlarged release of a variety of substances
related to immunity and wound healing1619277376
As pain and nociception are even less understood than wound healing the possible
mechanisms in obtaining pain relief by the use of light are less underpinned However
it is established that light therapy influences the synthesis release and metabolism of
numerous transmitter signal substances involved in analgesia such as endorphin nitric
oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these
neuropharmacological effects there is experimental evidence for diminished
inflammation decreased C-fibre activity increased blood circulation and reduced
excitability of the nervous system1927848899
One should be aware that a large amount of research regarding the possible
mechanisms of light action was conducted at the cellular level The described cascade
of reactions at the organism level is possibly even more complex as in contradiction to
14
the in vitro situation in vivo a range of supplementary interactions can influence the
sequence of effects and accordingly the final responses Besides it needs to be
mentioned that this summary did not take into account the origin of the light or the
external dosimetry thus the description is based on investigations performed with
various light sources and different dosages
Figure 2 Model summarizing the identified mechanisms of light action
Secondarymechanisms
Primary mechanisms
Final effects
Trigger
Stimulated wound healing Analgesia
Exposure to light
Photon absorption by photoacceptors
Respiratory chain activation
Accelerated electrontransfer
Reactive oxygen generation
Heating of absorbing chromophores
Altered redox properties
darr inflammation uarr oedema resorption
uarr lymph vessel regenerationuarr blood circulation
Photosignal transduction and amplification chain
uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation
darr C-fibre activity darr nervous excitability neuropharmacological effects
General introduction
15
Regardless of the large number of previous investigations identification of underlying
mechanisms of light action remains an important issue as these are not yet fully
understood and because probably not all mechanisms of action are currently
identified Convincing explanation of the mechanisms in normal as well as in
pathological tissue could banish the existing suspicion concerning the use of light as a
treatment modality2732547678
AIMS AND OUTLINE
The introduction of LED in medicine and in physiotherapy more specifically requires
particular scientific research especially within the fields of its clinical potential
application wound healing and analgesia The above described gaps in literature
regarding the use of LED laid the foundation of this doctoral thesis
Consequently the general purpose of this thesis is to explore a scientific approach for
the supposed biostimulatory and analgesic effect of LED and to formulate an answer
in view of an evidence-based clinical use of this treatment modality
The detailed objectives can be phrased as follows
Aim 1 To assess the biostimulatory effectiveness of LED
irradiation under normal in vitro conditions
Aim 2 To investigate the value of LED treatment to ameliorate
in vitro cell proliferation under conditions of impaired healing
Aim 3 To examine the effectiveness of LED in changing the
nerve conduction characteristics in view of analgesia
Aim 4 To determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting
Part I investigates the influence of LED on wound healing In pursuit of the first aim
chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial
16
measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these
cells needs to be considered as an exponent of the wound healing process as
fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase
and early remodelling100 Secondly an in vivo case study exploring the postulation that
LED irradiation could accelerate and ameliorate the healing of a surgical incision was
described
The results contrasted sharply with the findings of the in vitro part Two fundamental
causes were proposed in order to explain the different biological effect of LED
irradiation observed in vitro and in vivo the used irradiation parameters and evaluation
method
The experiment described in chapter 2 endeavoured to explore these considerations A
similar study was therefore performed but as distinctive characteristics different light
source properties an adapted irradiation procedure and the use of a colorimetric assay
based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the
counting of the cells were used
As stimulation of the wound healing process is virtually mainly indicated under
conditions of impaired healing (resulting in a situation which threatens to become
chronic and debilitating) proper attention for this matter is warranted192855 Besides
the medical consequences the costs involved with impaired healing yield also a socially
relevant problem to tackle Impaired healing will become even more common as the
world population continues to age After all senescence of systems and age-committed
comorbid conditions are commonly the culprits responsible for poor wound healing101
Thus finding cost-effective time-sparing non-invasive and practical treatment
modalities to cure wounds is a necessity
Aiming to assess the biostimulative effects by means of LED in these circumstances a
third study was conducted with respect of the previous results regarding irradiation
parameters and cell proliferation analysis The irradiation experiment described in
chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose
This medium modification serves as a pattern for cell proliferation in diabetic patients
General introduction
17
a population for whom stimulation of the wound healing process is a clinical relevant
feature
In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored
by means of two studies A first investigation (chapter 4) evaluated the influence of LED
on the sensory nerve conduction characteristics of a human superficial peripheral
nerve as a potential explanatory mechanism of pain inhibition by LED which is based
on the putative neurophysiological effects of this treatment modality The experimental
hypothesis postulated that LED generates an immediate decrease in conduction
velocity and increase in negative peak latency In addition it was postulated that this
effect is most prominent immediately after the irradiation and will weaken as time
progresses
The values of nerve conduction velocity and negative peak latency of a baseline
antidromic nerve conduction measurement were compared with the results of five
identical recordings performed at several points of time after LED irradiation
Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory
setting To guarantee an adequate standardized and controlled pain reduction study
there was opted to observe a healthy population with experimentally induced DOMS
Induction of DOMS has been described in a number of studies as a representative
model of musculoskeletal pain and stiffness because it can be induced in a relatively
easy and standardised manner the time course is quite predictable and the symptoms
have the same aetiology and are of transitory nature4445102-105
The treatment as well as the assessment procedure was performed during 4
consecutive days The first day isokinetic exercise was performed to induce pain
related to DOMS Subsequently the volunteers of the experimental group received an
infrared LED treatment and those of the placebo group received sham-irradiation
Evaluation of the effect of the treatment on perceived pain was registered by a visual
analog scale and by a mechanical pain threshold these observations occurred every day
18
prior to and following LED irradiation Eccentricconcentric isokinetic peak torque
assessment took place daily before each treatment
For the analysis of the results three different factors were taken into consideration
time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental)
In completion of this thesis the most prominent findings are summarized and the
clinical implications are discussed The general discussion also includes some future
research directions and a final conclusion
General introduction
19
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General introduction
21
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45 Glasgow P Hill I McKevitt A Lowe A and Baxter G (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1)33-39
46 Noble J Lowe A Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
47 IEC 60825-1 (1993) International Electrotechnical Commission Safety of laser products Part 1 Equipment classification requirements and users guide
48 Nussbaum E Baxter G and Lilge L (2003) A review of laser technology and light-tissue interactions as a background to therapeutic applications of low intensity lasers and other light sources Phys Ther Rev 831-44
49 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
50 Nakamura S (1998) The roles of structural imperfections in InGaN-based blue light-emitting diodes and laser diodes Science 281956-961
51 Karu Tiina (consulted on 2005 august 22) Cellular mechanism of low-power laser therapy httpwwwtinnitusustiinakarupresentationhtml
52 Mester E Mester A and Mester A (1985) The biomedical effects of laser application Lasers Surg Med 5(1)31-39
53 Moore K Calderhead G (1991) The clinical application of low incident power density 830 nm GaAlAs diode laser radiation in the therapy of chronic intractable pain A historical and optoelectronic rationale and clinical review Int J Optoelectron 6503-520
54 King P (1989) Low level laser therapy A review Lasers Med Sci 4141-148 55 Karu T (1989) Photobiology of low-power laser effects Health Phys 56(5)691-704 56 Simunovic Z (2000) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical
Application of Low Energy-Laser Laser Therapy LLLT Rijeka Vitgraf 57 Hode L and Tuner J (2000) Low level laser therapy (LLLT) contra light emitting diode therapy
(LEDT) - What is the difference Proceedings of SPIE 416690-97 58 Kitchen S and Partridge C (1991) A review of low level laser therapy Part I Background
physiological effects and hazards Physiotherapy 77(3)161-163 59 Nussbaum E Lilge L and Mazzulli T (2003) Effects of low-level laser therapy (LLLT) of 810 nm
upon in vitro growth of bacteria Relevance of irradiance and radiant exposure J Clin Laser Med Sur 21(5)283-290
60 Mendez T Pinheiro A Pacheco M Nascimento P and Ramalho L (2004) Dose and wavelength of laser light have influence on the repair of cutaneous wounds J Clin Laser Med Sur 22(1)19-25
61 Koutna M Janisch R and Veselska R (2003) Effects of low-power laser irradiation on cell proliferation Scripta Medica 76(3)163-172
62 Al-Watban F and Zhang X (2004) The comparison of effects between pulsed and CW lasers on wound healing J Clin Laser Med Sur 22(1)15-18
63 Panjehpour M Overholt B DeNovo R Petersen M and Sneed R (1993) Comparative study between pulsed and continuous wave lasers for photofrin photodynamic therapy Lasers Surg Med 13(3)296-304
64 Dyson M (2005) The significance of pulsing in the stimulation of tissue repair by light A review Lasers Med Sci 20S21
22
65 MDB Laser (2000) Bio Dio (Preprotoype) User guide Belgium Ekeren 66 Fisher J (1992) Photons physiatrics and physicians A practical guide to understanding laser light
interaction with living tissue Part I J Clin Laser Med Sur 10(6)419-426 67 Tuner J and Hode L (2000) Depth of penetration of laser light in tissue Laser Partner
Clinixperience 151-3 68 Anderson R and Parrish J (1981) The optics of human skin J Invest Dermatol 77(1)13-19 69 De Cuyper H and Lambert H (1991) Penetration depth of infrared- and helium-neon-laserlight
An assessment in vivo Eur J Phys Med Rehabil 1(5)133-137 70 Oshiro T Ogata H Yoshida M Tanaka Y Sasaki K and Yoshimi K (1996) Penetration depths
of 830nm diode laser irradiation in the head and neck assessed using a radiographic phanom model and wavelength-specific imaging film Laser Ther 8197-204
71 Kolarova H Ditrichova D and Wagner J (1999) Penetration of the laser light into the skin in vitro Lasers Surg Med 24(3)231-235
72 Chen Q Huang Z Chen H Shapiro H Beckers J and Hetzel F (2002) Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy Photochem Photobiol 76(2)197ndash203
73 Conlan M Rapley J and Cobb C (1996) Biostimulation of wound healing by low-energy laser irradiation A review J Clin Periodontol 23(5)492-496
74 Parrish J (1981) New concepts in therapeutic photomedicine photochemistry optical targeting and the therapeutic window J Invest Dermatol 7745-50
75 Smith K (1981) Photobiology and photomedicine the future is bright J Invest Dermatol 772-7 76 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees D (1989) Systemic effects
of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
77 Polo L Presti F Schindl A Schindl L Jori G and Bertoloni G (1999) Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth Biochem Biophys Res Commun 257(3)753-758
78 Bertoloni G Sacchetto R Baro E Ceccherelli F and Jori G (1993) Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 6328 nm light J Photochem Photobiol B-Biol 18191-196
79 Lubart R Friedmann H Sinykov M and Grossman N (1995) Biostimulation of photosensitized fibroblasts by low incident levels of visible light energy Laser Ther 7101-106
80 Harris D (1991) Biomolecular mechanisms of laser biostimulation J Clin Laser Med Sur 9277-280
81 Grossman N Schneid N Reuveni H Halevy S and Lubart R (1998) 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures involvement of reactive oxygen species Lasers Surg Med 22212-218
82 Oren D Charney D Lavi R Sinyakov M and Lubart R (2001) Stimulation of reactive oxygen species production by an antidepressant visible light source Biol Psychiatry 49464-467
83 Lavi R Shainberg A Friedmann H Shneyvays V Rickover O Eichler M Kaplan D and Lubart R (2003) Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells 278(42)40917-40922
84 Reddy K (2004) Photobiological basis and clinical role of low-intensity lasers in biology and medicine J Clin Laser Med Sur 22(2)141-150
85 Greco M Guida G Perlino E Marra E and Quagliariello E (1989) Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser Biochem Biophys Res Commun 163(3)1428-1434
86 Vacca R Marra E Quagliariello E and Greco M (1993) Activation of mitochondrial DNA replication by He-Ne laser irradiation Biochem Biophys Res Commun 195(2)704-709
87 Vacca R Marra E Quagliariello E and Greco M (1994) Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser Biochem Biophys Res Commun 203(2)991-997
88 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral
General introduction
23
nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
89 Anders J Borke R Woolery S and Van de Merwe W (1993) Low power laser irradiation alters the rate of regeneration of the rat facial nerve Lasers Surg Med 13(1)72-82
90 Rochkind S Nissan M Barr-Nea L Razon N Schwartz M and Bartal A (1987) Response of peripheral nerve to He-Ne laser Experimental studies Lasers Surg Med 7441-443
91 Lievens P (1986) Lasertherapie (deel II) De invloed van laser-bestralingen op het lymfesysteem en de wondheling Ned T Fysiotherapie 96140-142
92 Lievens P (1991) The effect of a combined HeNe and IR laser treatment on the regeneration of the lymphatic sysytem during the process of wound healing Lasers Med Sci 6193-199
93 Lievens P (1991) The effect of IR laser irradiation on the vasomotricity of the lymphatic system Lasers Med Sci 6189-191
94 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin J and Martin P (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1)171-178
95 Bosatra M Jucci A Olliaro P Quacci D and Sacchi S (1984) In vitro fibroblast and dermis fibroblast activation by laser irradiation at low energy An electron microscopic study Dermatologica 168(4)157-162
96 Enwemeka C Rodriquez O Gall N and Walsh N (1990) Morphometrics of collagen fibril populations in HeNe laser photostimulated tendons J Clin Laser Med Sur 847-52
97 Abergel P Lam T Meeker C Castel C Dwyer R and Uitto J (1984) Biostimulation of procollagen production by low energy lasers in human skin fibroblast culturesJ Invest Dermatol 82(4)395
98 Abergel P Lam T Meeker C Dwyer R and Uitto J (1984) Low energy lasers stimulate collagen production in human skin fibroblast cultures Clin Res 32(1)16A
99 Shimoyama N Lijima K Shimoyama M and Mizuguchi T (1992) The effects of helium-neon laser on formalin-induced activity of dorsal horn neurons in the rat J Clin Laser Med Sur 1091-94
100 Kloth L McCulloch J and Feedar J (1990) Wound healing Alternatives in management USA FA Davis Company
101 Lanzafame R (2004) Revolutions and revelations J Clin Laser Med Surg 22(1)1-2 102 Ciccone C Leggin B and Callamaro J (1991) Effects of ultrasound and trolamine salicylate
phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-678 103 Craig J Cunningham M Walsh D Baxter G and Allen J (1996) Lack of effect of transcutaneous
electrical nerve stimulation upon experimentally induced delayed onset muscle soreness in humans Pain 67(2-3)285-289
104 Minder P Noble J Alves-Guerreiro J Hill I Lowe A Walsh D and Baxter G (2002) Interferential therapy Lack of effect upon experimentally induced delayed onset muscle soreness Clin Physiol Funct Imaging 22(5)339-347
105 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
PART I WOUND HEALING
CHAPTER 1
DO INFRARED LIGHT EMITTING DIODES HAVE A
STIMULATORY EFFECT ON WOUND HEALING FROM AN IN
VITRO TRIAL TO A PATIENT TREATMENT
Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Proceedings of SPIE 2002 4903 156-165
Chapter 1
28
ABSTRACT
Variable effects of different forms of light therapy on wound healing have been
reported This preliminary study covers the efficacy of infrared light emitting diodes
(LED) in this domain
Cultured embryonic chicken fibroblasts were treated in a controlled randomised
manner LED irradiation was performed three consecutive days with a wavelength of
950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each
treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2
The results indicated that LED treatment does not influence fibroblast proliferation at
the applied energy density and irradiation frequency (p=0474)
Meanwhile the effects of LED on wound healing in vivo were studied by treating a
surgical incision (6 cm) on the lateral side of the right foot of a male patient The
treatment started after 13 days when initial stitches were removed The same
parameters as the in vitro study were used but the treatment was performed five times
The healing could only be evaluated clinically the irradiated area (26 cm) showed a
more appropriate contraction less discoloration and a less hypertrophic scar than the
control area (34 cm)
The used parameters failed to demonstrate any biological effect of LED irradiation in
vitro although the case study on the other hand illustrated a beneficial effect
Keywords Light Emitting Diodes Fibroblasts Wound healing
From an in vitro trial to a patient treatment
29
INTRODUCTION
Various beneficial effects of lasers and photodiodes at relatively low intensities have
been reported involving treatment of neurological impairments12 treatment of pain3-5
treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the
enhancement of wound healing has been a focus of contemporary research11-16 It
seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing
remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the
basis of a questionnaire about low power laser (LPL) in the current clinical practice in
Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey
into clinical LPL experience in Flanders
Nevertheless there remains a considerable amount of ignorance scepticism and
controversial issues concerning the use and clinical efficacy of LPL even in the domain
of wound healing12152021 This is at least in part a consequence of the inability to
measure and control operating variables related to connective tissue repair and of the
wide range of suitable parameters for irradiation
Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent
characteristics which make their use in a clinical setting problematic including
limitations in wavelength capabilities and beam width The combined wavelength of
light optimal for wound healing cannot be efficiently produced and the size of
wounds which may be treated by LPLs is limited Some companies offer an
alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be
made to produce multiple wavelengths and can have probes with large surface area
allowing treatment of large wounds Still one can not accept this light source as an
alternative for LPL therapy based on the cited advantages without proper investigation
regarding its biostimulatory effects
The effectiveness of this possible alternative for LPLs must be studied in vitro and in
addition in animal models or in humans because the effects of LED at the cellular level
do not necessarily translate to a noticeable effect in vivo The small amount of previous
investigations demonstrate that LED effects are as difficult to isolate162223 as LPL
Chapter 1
30
effects and the results are conflicting just like the results in literature specific on the
use of LPL121520
The purpose of the first part of this study is to examine the hypothesis stating that
LED irradiation can influence fibroblast proliferation Therefore a comparison of the
proliferation from fibroblasts in irradiated and control wells was performed The in vitro
investigation was linked with an in vivo case study This part enquired the assumption if
LED irradiation could accelerate and ameliorate the healing of a surgical incision
IN VITRO INVESTIGATION
MATERIALS AND METHODS
The complete procedure from isolation to proliferation analysis was executed twice
(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of
control dishes The second trial consisted of 27 irradiated and 27 control dishes
Cell isolation and culture procedures
Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation
and disaggregating of the cells occurred with warm trypsin (NV Life Technologies
Belgium) according the protocol described by Ian Freshney (1994)24 The primary
explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies
Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1
Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life
Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies
Belgium) When cell growth from the explants reached confluence cells were detached
with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV
Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the
cells were removed from the culture flasks by trypsinization and counted by
hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc
From an in vitro trial to a patient treatment
31
NV Life Technologies Belgium) The fibroblasts were seeded at a density of
70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture
medium the cells were allowed to attach for 24 hours in a humidified incubator at
37degC
Properties of the Light Emitting Diode
Prior to LED treatment all dishes were microscopically checked to guarantee that the
cells are adherent and to assure that there is no confluence nor contamination The
dishes were divided randomly into the treated or the control group Medium was then
removed by tipping the dishes and aspirating with a sterile pipette Following the
aspiration 2 ml fresh medium was added and treatment started
A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser
Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-
1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32
single LEDrsquos For the treatments in this study an average power of 160 mW at
continuous mode was applied The irradiation lasted 6 minutes resulting in an energy
density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result
of the divergence in function of this distance the surface of the LED (18 cm2) covered
the complete surface of the used petriplates (215 cm2)
After these manipulations 3 ml medium was added to each dish followed by 24 hours
incubation
One LED irradiation was performed daily during three consecutive days according
this procedure Control cultures underwent the same handling during these three days
but were sham-irradiated
Proliferation analysis
After the last treatment a trypsination was performed to detach the cells from the
culture dishes followed by centrifugation Once the cells were isolated from the used
trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts
Chapter 1
32
within this suspension as reflection for the proliferation was quantified by means of a
Buumlrker Chamber or hemocytometry
The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-
Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab
Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on
the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator
using an inverted light microscope counted the number of cells in 25 small squares
In order to calculate the number of cells one should multiply the amount of cells
counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and
the dilution factor (the amount of Trypan blue suspended with the cells 21=3)
Statistical methods
The data were analysed statistical in order to examine the hypothesis that LED
irradiation enhances fibroblast proliferation They were processed as absolute figures
for both trials separately In a second phase the counted cell numbers were converted
in relative figures so the data of both trials could be analysed as the data of one test
These relative figures were obtained by expressing each figure as a percentage from the
highest figure (=100) of that trial and this for each assay separately
A Kolmogorov-Smirnoff test of normality was performed on the data followed by a
Mann-Whitney-U test when the test of normality was significant and otherwise a T-
test Differences were accepted as significant when plt005 For this analysis SPSSreg
100 was used
RESULTS
The descriptive data for both trials are depicted in figure I The mean number of cells
in trial A is higher than in trial B for the controls as for the treated wells There is a
mean difference of 1252500 fibroblasts between the controls and 1223000 between
the irradiated wells of trial A and B The averages of both trials show that in control
cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no
From an in vitro trial to a patient treatment
33
statistically significant difference could be found between the two groups in either trial
nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not
significant for trial A (p=020) nor trial B (p=020) Only the combined data from both
trials were significant (plt001) for normality Further analysis respectively T-test for
the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the
combined data (p=0474) revealed no statistical significant differences
DESCRIPTIVE DATA
1730000181750029530003070000
00E+00
50E+05
10E+06
15E+06
20E+06
25E+06
30E+06
35E+06
40E+06
Trial A Trial A Trial B Trial B
Mea
n n
um
ber
of
cells
Control
Irradiated
Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B
DISCUSSION
Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities
(lt500 mW) in vitro have been analysed by evaluating various factors involving
(pro)collagen production25-27 cell viability2829 growth factor production28 and
myofibroblast formation30 Fibroblast proliferation also is an important factor to
consider In accordance with wound healing fibroblasts fulfil an essential role especially
in the late inflammatory phase and the early granulation phase31 Despite the failure of
some studies to demonstrate beneficial effects of LPL irradiation on fibroblast
proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very
Chapter 1
34
significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -
208 ) Atabey et al35 also revealed a significant increase in cell number two or more
irradiations resulted in an increased fibroblast proliferation Several other studies
confirmed these positive findings25263637
The results of this present in vitro study indicate that LED treatment does not
influence fibroblast proliferation Although the dosimetric parameters (in particular the
arbitrary energy density of 32 Jcm2) used in this study are well within the
recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous
studies about LPL therapy raising enhanced fibroblast proliferation252634-37
Van Breugel et al36 gave a possible explanation for these controversial results
According to them the fibroblast proliferation is not inherent at the energy density
They provide evidence that independent of the energy density the power density and
the exposure time determine the biostimulative effects of LPL irradiation LPL with a
power below 291 mW could enhance cell proliferation while a higher power had no
effect
Some authors also argued that the absorption spectrum of human fibroblasts show
several absorption peaks and pointed out that a wavelength of 950 nm is far above the
highest peak of about 730 nm3638 At longer wavelengths they determined a general
decrease in absorption Despite these results several investigators pose biostimulative
effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or
even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is
ranged from 800 nm to 830 nm principally because of the presence of cytochrome
oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts
at lower wavelengths is restricted by an inferior skin transmission than at higher
wavelengths38
Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting
respective green and red light The 950 nm beam of light was used for its high power
density but according to a range of remarks mentioned above the effects of the two
other probes must be as well evaluated
From an in vitro trial to a patient treatment
35
Another factor one can not ignore is that besides fibroblast proliferation other
processes or morphologic changes were not analysed although several authors have
posed that those changes and processes could be responsible for the biostimulative
effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et
al30 for example described a massive transformation of fibroblasts into myofibroblasts
after LPL treatment These modified fibroblasts play an important role in contraction
of granulation tissue30 A second example is an increased (pro)collagen production
after low power light therapy25-27 which is also considered as a responsible factor for
accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast
proliferation3640
It may be wondered if the light sources mostly LPL in the consulted literature are
representative for the LED used in this study although this LPL literature is often
used for that purpose As in the early days of LPL the stimulative effects upon
biological objects were explained by its coherence the beam emitted by the Bio-Dio on
the contrary produces incoherent light Nowadays contradictory research results are
responsible for a new discussion the clinical and biological significance of coherence
The findings of some authors172341-43 pose that the coherence of light is of no
importance of LPL and its effects although the opposite has also been stated4445
Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be
accounted for the non-enhanced fibroblast proliferation in this trial
Another possible explanation for the absence of biostimulative effect is related to the
moment of analysis of the proliferation The evaluation one day after the last
irradiation did not allow a delayed enhancement of proliferation while it is determined
in numerous investigations that the effects occur more than 24 hours after the last
treatment273746 and that they weaken after a further undefined period of time34
The fluctuation in cell numbers between both trials despite the use of an identical
protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor
reproducibility of their technique In this study the fluctuations are attributable to the
counting of the cells by Buumlrker hemocytometer before seeding According to some
authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large
Chapter 1
36
variability48 and it is often difficult to standardize48 Overestimation of the cell
concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity
was contradicted by Lin et al50 moreover satisfactory correlations with flow-
cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay
for cell counting (MTT)51 were determined
An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it
provides accurate cell counts in a short period of time with less intervention from the
investigator52
These remarks and controversies point out the possible deficiencies from the used
proliferation analyses and the relativity from the obtained results Other analyse
methods and analyses from different cell processes and morphologic changes could be
considered for further investigation
IN VIVO INVESTIGATION
MATERIALS AND METHODS
The effects of LED on wound healing in vivo were studied by treating a postsurgical
incision A male patient received chirurgical treatment for the removal of a cyst
situated approximately 15 cm posterior from the lateral malleolus of his right foot For
removal of the cyst an incision of 6 cm was made The incision was sutured and 12
days after the surgery the stitches were removed Visual inspection demonstrated that
the healing process of the wound proceeded well but not equally over the whole 6
centimetres (figure II) Epithelialization and wound contraction appeared to have
progressed better in the upper part (approximately 3 cm) of the cicatrice than at the
lower part (covered with eschar) No evidence of infection was noted in either part
LED treatment started the 13th day The incision was treated partially the lowest part
(26 cm) with the inferior epithelialization and wound contraction was irradiated the
remaining 34 cm served as control area This control area was screened from radiation
with cardboard and opaque black cling film
From an in vitro trial to a patient treatment
37
The light source destinated for the treatment was the same device used for the in vitro
irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED
output parameters were identical with those applicated in the preceding in vitro
investigation In particular a continuous wave at an average power of 160 mW and 6
minutes of treatment duration corresponding to an energy density of 32 Jcm2 An
equal distance from the probe to the target tissue as from the probe to the culture
medium was respected A plastic applicant of according height guaranteed constant
distance of 06 cm from the surface of the skin
Figure II Surgical incision before the first treatment 13 days after initial stitching
Therapy was performed once a day during five consecutive days repeatedly at the same
time resulting in an extension of the duration of the in vitro therapy with two days
Visual macroscopic observations were accomplished 6 52 and 175 days after the first
treatment
Comparison of the cutaneous sensitivity at the irradiated area and the control area was
accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175
days after the first treatment A control measurement also occurred at the same region
Chapter 1
38
on the left foot The aesthesiometer used in this study consisted of five hand-held
nylon monofilaments with a length of 38 mm and varying diameter
Sensitivity threshold is traced by presenting a monofilament of a certain diameter
vertically to the skin The monofilament bends when a specific pressure has been
reached with a velocity proportional to its diameter Measurements allow mapping
areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified
from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest
filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished
protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to
finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the
filaments
RESULTS
Visual estimation at any point of time after irradiation divulged no occurrence of
problems with dehiscence or infection in either part of the wound During the five
days of therapy the irradiated area looked dryer than the control area After the last
irradiation this was no longer recorded
Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area
From an in vitro trial to a patient treatment
39
Figure III representing the first evaluation six days after the initial treatment
illustrates that the wound healing has evolved slightly in both parts Though the lower
irradiated part remains of inferior quality as regards to epithelialization and wound
contraction In the course of the reparative process the influence of light exposures
were registered At 52 days after the first irradiation beneficial effects of LED
treatment are clearly present (Figure IV)
Figure IV Surgical incision 52 days after initiating LED treatment
The irradiated area (26 cm) showed a more appropriate contracture than the control
area (34 cm) characterized by less discoloration at scar level and a less hypertrophic
scar A similar trend was noticed at a third visual observation 175 days after the initial
treatment At that moment no impairments at cutaneous sensitivity level were stated
and the sensitivity showed no differences between left or right foot nor between the
two areas of the cicatrice
Chapter 1
40
DISCUSSION
The results of this case study indicate that LED had a positive influence on wound
healing in humans as determined by visual observations Many investigators
examining the effects of LPL on wound healing by means of a range of observation
and treatment methods reported accelerated and enhanced wound healing8-10 others
described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and
LED21 studies were unable to repeat these results
The late but beneficial findings in this study seem to be to the credit of LED-therapy
Though several authors establish positive results in an earlier stage of the wound
healing process8-1020 one should question why the differences did not occur at the first
evaluation on day 6 An explanation can be found in the start of the treatment Most
investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so
they influence a first cellular and vascular reaction with the production of chemical
mediators of inflammation resulting in an enhanced collagen production9 tremendous
proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the
time the first treatment in this study took place the traumatized tissue was in an
overlapping stage between an almost finished inflammatory phase and a scarcely
initiated re-epithelialization and wound contraction phase At that moment an infiltrate
of fibroblasts is present So fibroblast proliferation a possible mechanism of the
biostimulative effect had already occurred and could no longer be influenced Growth
factor production and collagen deposition have also decreased at that stage
Granulation tissue formation and fibroplasia in the contrary are initiating by that time
Those prolonged and slow processes with belated results are of significant importance
for the course of the final stage of wound healing and for the outlook of the future
scar31
The experimental findings revealed that the sensitivity of the skin according to the
threshold detection method of Semmes and Weinstein was normal at all the
investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a
good and objective predictor of normal skin sensitivity No other LPL nor LED
studies investigating this quality of the skin were found
From an in vitro trial to a patient treatment
41
CONCLUSION
This study demonstrates that although LED application at the applied energy density
and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to
have beneficial biostimulative effects on wound healing in human skin confirmed by
the favourable re-epithelialization and contracture
These results are discussed in the context of other experimental findings but no
reasonable explanation for this discrepancy could be found The literature on wound
healing after LED treatment in animal models or in humans is presently very limited
and contradictory The diversity in used radiation parameters and the absence of
references on how the wounds were measured or evaluated or what the end point was
for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro
investigations are better standardised nevertheless these results show a number of
conflicts One can conclude that until today the controversial findings are characteristic
for many results obtained with light photobiomodulation
However the postponed favourable results in the case study confirm some facts of the
discussion Namely the short period of incubation 24 hours in the in vitro part of the
study can be responsible for the lack of enhanced fibroblast proliferation It also
confirms that other cell processes and morphologic changes possibly are responsible
for biostimulative effects in vivo other observation methods should be considered for
future in vivo experiments
Despite these remarks we believe that LED application on cutaneous wounds of
human skin is useful with a single flash daily at the dose applied in this study for at
least three days
Furthermore future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Chapter 1
42
ACKNOWLEDGMENTS
The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the
material necessary for this study as well as Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
From an in vitro trial to a patient treatment
43
REFERENCES
1 G Baxter D Walsh J Allen A Lowe and A Bell Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79(2) 227-34 (1994)
2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)
3 J Stelian I Gil B Habot et al Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy J Am Geriatr Soc 40(1) 23-6 (1992)
4 A Honmura A Ishii M Yanase J Obata and E Haruki Analgesic effect of Ga-Al-As diode laser irradiation on hyperalgesia in carrageenin-induced inflammation Lasers Surg Med 13(4) 463-9 (1993)
5 D Cambier K Blom E Witvrouw G Ollevier M De Muynck and G Vanderstraeten The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15 195-200 (2000)
6 G Reddy L Stehno Bittel and C Enwemeka Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-7 (1998)
7 K Moore N Hira I Broome and J Cruikshank The effect of infrared diode laser irradiation on the duration and severity of postoperative pain a double blind trial Laser Therapy 4 145-148 (1992)
8 A Amir A Solomon S Giler M Cordoba and D Hauben The influence of helium-neon laser irradiation on the viability of skin flaps in the rat Br J Plast Surg 53(1) 58-62 (2000)
9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)
10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)
11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)
12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)
13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)
14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)
15 A Schlager P Kronberger F Petschke and H Ulmer Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1) 39-42 (2000)
16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)
17 GD Baxter and J Allen Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone (1994)
18 G Baxter A Bell J Allen and J Ravey Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77(3) 171-178 (1991)
19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)
20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)
Chapter 1
44
21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)
22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)
23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)
24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)
25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)
26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)
27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)
28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)
29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)
30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)
31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)
32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)
33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)
34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)
35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)
36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)
37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)
38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)
39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)
40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)
41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)
42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)
From an in vitro trial to a patient treatment
45
43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)
44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)
45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)
46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)
47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)
48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)
49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)
50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)
51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)
52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)
53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)
54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)
CHAPTER 2
INCREASED FIBROBLAST PROLIFERATION INDUCED BY
LIGHT EMITTING DIODE AND LOW LEVEL LASER
IRRADIATION
Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Lasers in Medical Science 2003 18(2) 95-99
Chapter 2
48
ABSTRACT
Background and Objective As Light Emitting Diode (LED) devices are
commercially introduced as an alternative for Low Level Laser (LLL) Therapy the
ability of LED in influencing wound healing processes at cellular level was examined
Study DesignMaterials and Methods Cultured fibroblasts were treated in a
controlled randomized manner during three consecutive days either with a infrared
LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and
570 nm) and respective power outputs Treatment duration varied in relation to
varying surface energy densities (radiant exposures)
Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all
irradiated cultures in comparison with the controls Green light yielded a significantly
higher number of cells than red (plt0001) and infrared LED light (plt0001) and than
the cultures irradiated with the LLL (plt0001) the red probe provided a higher
increase (plt0001) than the infrared LED probe and than the LLL source
Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation
in vitro This study therefore postulates possible stimulatory effects on wound healing
in vivo at the applied dosimetric parameters
Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low
Level Laser Tetrazolium salt
LED induced increase of fibroblast proliferation
49
INTRODUCTION
Since the introduction of photobiostimulation into medicine the effectiveness and
applicability of a variety of light sources in the treatment of a wide range of medical
conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results
of several investigations are remarkably contradictory This is at least in part a
consequence of the wide range of indications as well as the wide range of suitable
parameters for irradiation and even the inability to measure the possible effects after
irradiation with the necessary objectivity [457] A lack of theoretical understanding
can also be responsible for the existing controversies In fact theoretical understanding
of the mechanisms is not necessary to establish effects though it is necessary to
simplify the evaluation and interpretation of the obtained results As a consequence
the widespread acceptance of especially Low Level Laser (LLL) therapy in the early
seventies is faded nowadays and biostimulation by light is often viewed with scepticism
[8] According to Baxter [49] contemporary research and consumption in
physiotherapy is in particular focused on the stimulation of wound healing Tissue
repair and healing of injured skin are complex processes that involve a dynamic series
of events including coagulation inflammation granulation tissue formation wound
contraction and tissue remodelling [10] This complexity aggravates research within this
cardinal indication
Research in this domain mostly covers LLL studies but the current commercial
availability of other light sources appeals research to investigate as well the effects of
those alternative light sources eg Light Emitting Diode (LED) apparatus
The scarcity of literature on LED is responsible for consultation of literature
originating from LLL studies [11] but it may be wondered if this literature is
representative for that purpose As in the early days of LLL therapy the stimulating
effects upon biological objects were explained by its coherence [1213] while the beam
emitted by LEDrsquos on the contrary produces incoherent light Though the findings of
some scientists [914151617] pose nowadays that the coherence of the light beam is
not responsible for the effects of LLL therapy Given that the cardinal difference
between LED and LLL therapy coherence is not of remarkable importance in
Chapter 2
50
providing biological response in cellular monolayers [5] one may consult literature
from LLL studies to refer to in this LED studies
The purpose of this preliminary study is to examine the hypothesis that LED
irradiation at specific output parameters can influence fibroblast proliferation
Therefore irradiated fibroblasts cultures were compared with controls The article
reports the findings of this study in an attempt to promote further discussion and
establish the use of LED
MATERIALS AND METHODS
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos Isolation and
disaggregation of the cells was performed with warm trypsin according the protocol
described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in
Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1
L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants
reached confluence cells were detached with trypsine and subcultured during 24 hours
in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours
the cells were removed from the culture flasks by trypsinization and counted by Buumlrker
hemocytometry For the experiment cells from the third passage were plated in 96-well
plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a
density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C
during 24 hours
All supplies for cell culture were delivered by NV Life Technologies Belgium except
for Fetal Calf Serum (Invitrogen Corporation UK)
Irradiation sources
In this study two light sources a Light Emitting Diode (LED) device and a Low Level
Laser (LLL) device were used in comparison to control cultures
The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)
LED induced increase of fibroblast proliferation
51
with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400
mW and a frequency range from 0-1500 Hz
The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)
consisted of three wavelengths emitted by separate probes A first probe emitting
green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the
red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third
probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared
light The area of all three probes was 18 cm2 and their frequency was variable within
the range of 0-1500 Hz
Exposure regime
Prior to irradiation the 96-well plates were microscopically verified to guarantee that
the cells were adherent and to assure that there was no confluence nor contamination
Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining
25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation
The 96-well plates were randomly assigned in the treated (LLL or green red or infrared
LEDrsquos) or the control group
For the treatments in this study the continuous mode was applied as well for the LLL
as for the three LED-probes The distance from light source to fibroblasts was 06 cm
LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting
in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant
exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to
exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective
power output of 160 mW 80 mW or 10 mW
After these handlings the remaining medium was removed and new Hanksrsquoculture
medium was added followed by 24 hours of incubation
One irradiation (LLL or LED) was performed daily during three consecutive days
according to the aforementioned procedure Control cultures underwent the same
handling but were sham-irradiated
Chapter 2
52
Determination of cell proliferation
The number of cells within the 96-well plates as a measure for repair [19] was
quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The
colorimetric assay was performed at two different points of time to determine the
duration of the effect of the used light sources
This assay exists of a replacement of Hanksrsquoculture medium by fresh medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as
described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT
solution was substituted by lysing buffer isopropyl alcohol The plates were
temporarily shaken to allow dissolution of the produced formazan crystals After 30
minutes of exposure to the lysing buffer absorbance was measured The absorbance at
400 to 750 nm which was proportional to fibroblast proliferation was determined
using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)
The complete procedure from isolation to MTT assay was executed six times (Trial A
B C D E and F) while it was impossible to irradiate all the investigated number of
wells with the same LED apparatus on one day All the trials included as much control
as irradiated wells but the number of control and irradiated wells in each trial varied
depending on the number of available cells after the second subculturing A further
consequence of the available number of cells is the number of probes examined per
trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E
Incubation period before proliferation analyses numbered 24 hours To investigate if
the stimulatory effect tends to occur immediately after irradiation or after a longer
period of time incubation in trial F lasted 72 hours
An overview of the followed procedures regarding incubation time before proliferation
analysis number of analysed wells for each trial and the number of probes examined
per trial is given in table 1 As a consequence of the differences in procedures followed
and because each trial started from a new cell line the results of the five trials must be
discussed separately
LED induced increase of fibroblast proliferation
53
Statistical analysis
Depending on the amount of groups to be compared within each trial and depending
on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way
ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons Statistical significance for all tests was
accepted at the 005 level For this analysis Statistical Package for Social Sciences 100
(SPSS 100) was used
RESULTS
The results presented in table 1 show that cell counts by means of MTT assay
revealed a significant (plt0001) increase in the number of cells in comparison to their
respective sham-irradiated controls for all the irradiated cultures of trial A B C D
and E except the irradiated groups in trial F
Moreover the results of trial A showed that the effect of the green and red LED probe
was significantly (plt0001) higher than the effect of the LLL probe With regard to the
amount of proliferation the green probe yielded a significantly higher number of cells
than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe
provided a higher increase in cells (plt0001) than the infrared probe
The infrared LED source and the LLL provided a significant (plt0001) higher number
of cells than the control cultures but no statistical significant difference was recorded
between both light sources
The trials A B C D and E regardless of the number of probes used in each trial
were analysed after 24 hours of incubation after the last irradiation The incubation
period of trial F lasted 72 hours
The means of trial F illustrated that the effect was opposite after such a long
incubation The control cultures had significantly (plt0001) more fibroblasts than the
irradiated cultures with the exception of the LED-infrared group that showed a not
significant increase of cells Further analysis revealed that the green probe yielded a
significantly lower number of cells than the red (plt0001) and the infrared probe
(plt0001) and that the red probe provided a higher decrease (plt0001) than the
Chapter 2
54
infrared probe Laser irradiation induced a significant decrease of fibroblasts in
comparison to the infrared irradiated cultures (plt0001) and the control cultures
(p=0001) LED irradiation with the green and the red probe revealed no statistical
significant differences
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups
Absorbency (proportional to the number of fibroblasts)a
Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050
Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis
DISCUSSION
Despite the failure of some studies [223] to demonstrate beneficial effects of laser and
photodiode irradiation at relatively low intensities (lt500mW) on fibroblast
LED induced increase of fibroblast proliferation
55
proliferation this study provides experimental support for a significant increased cell
proliferation Therefore these results confirm previous studies that yielded beneficial
stimulating effect [1152425] Remarkably though is the higher increase noted after
irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general
decrease in absorption at longer wavelengths and concluded that several molecules in
fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445
470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the
closest to one of these peaks
Karu [5] also emphasises that the use of the appropriate wavelength namely within the
bandwidth of the absorption spectra of photoacceptor molecules is an important
factor to consider
In this particular context penetration depth can almost be ignored as virtually all
wavelengths in the visible and infrared spectrum will pass through a monolayer cell
culture [12] The irradiance (Wcm2) on the contrary could have had an important
influence on the outcome of this study The higher increased proliferation by the lower
wavelengths is possibly a result of the lower irradiance of these wavelengths Lower
irradiances are confirmed by other experiments to be more effective than higher
irradiances [111626]
The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as
described by Poumlntinen [17] but in the scope of these results it also needs to be noticed
that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)
the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)
Consequently the results of especially trial A and F must be interpreted with the
necessary caution It is possible that the determined distinction between the used light
sources and the used probes is a result from the various radiant exposures applied
during the treatments of the cultures
Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after
the last irradiation this study was unable to demonstrate a stimulating effect when
analysis was performed 72 hours after the last irradiation Moreover this longer
incubation period even yielded an adverse effect Although a weakening of the
Chapter 2
56
photostimulating influence over time is acceptable it can not explain a complete
inversion Especially in the knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period [2427] In an attempt to illuminate
this finding one can suppose that the circadian response of the cells triggered by the
LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark
The most obvious explanation is even though a decreased vitality and untimely cell
death in the irradiated cell cultures as a result of reaching confluence at an earlier point
of time than the control cultures The cells of a confluent monolayer have the tendency
to inhibit growth and finally die when they are not subcultured in time No other
reasonable explanations could be found for this discrepancy
Photo-modulated stimulation of wound healing is often viewed with scepticism The
real benefits of Light Emitting Diodes if any can only be established by histological
and clinical investigations performed under well controlled protocols Despite these
remarks this study suggests beneficial effects of LED and LLL irradiation at the
cellular level assuming potential beneficial clinical results LED application on
cutaneous wounds of human skin may be assumed useful at the applied dosimetric
parameters but future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Persons in good health rarely require treatment for wound healing as posed by Reddy
et al [13] light has a possible optimal effect under conditions of impaired healing
Postponed wound healing is a time-consuming and often expensive complication
Thus future prospects must remind to examine the therapeutic efficacy of LED on
healing-resistant wounds
LED induced increase of fibroblast proliferation
57
ACKNOWLEDGMENTS
The authors are grateful to Prof Deridder for supplying the laboratory as well as the
material necessary for this investigation and to Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
Chapter 2
58
REFERENCES
1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55
2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32
3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7
4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994
5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998
6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32
7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42
8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5
9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8
10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7
11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8
12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34
13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9
14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18
15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43
16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6
17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475
18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J
Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9
21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601
22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63
LED induced increase of fibroblast proliferation
59
23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9
24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301
25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are
more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37
27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8
28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26
CHAPTER 3
GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES
FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL
Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Photomedicine and Laser Surgery 2005 23(2) 167-171
Chapter 3
62
ABSTRACT
Background and Objective The chronic metabolic disorder diabetes mellitus is an
important cause of morbidity and mortality due to a series of common secondary
metabolic complications such as the development of severe often slow healing skin
lesions
In view of promoting the wound-healing process in diabetic patients this preliminary
in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on
fibroblast proliferation and viability under hyperglycemic circumstances
Materials and Methods To achieve hyperglycemic circumstances embryonic chicken
fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL
glucose LED irradiation was performed on 3 consecutive days with a probe emitting
green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min
resulting in a radiation exposure of 01 Jcm2
Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all
irradiated cultures in comparison with the controls
Conclusion According to these results the effectiveness of green LED irradiation on
fibroblasts in hyperglycemic circumstances is established Future in vivo investigation
would be worthwhile to investigate whether there are equivalent positive results in
diabetic patients
Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes
Fibroblast proliferation under hyperglycemic circumstances
63
INTRODUCTION
The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide
geographic variation in incidence and prevalence generally 11 of the world
population is affected and worldwide it is the twelfth leading cause of death1 Those
figures may be higher for urban regions as well as for industrialized countries Due to
multiple factors involving the aging process of the population and lifestyle changes
(such as reduced physical activity hypercaloric eating habits and concomitant obesity)
these figures may increase in the future2-6 Therefore diabetes mellitus could become
the most common chronic disease in certain regions as stated by Gale it ldquotargets the
rich in poor countries and the poor in rich countriesrdquo6
The harmful disruption of the metabolic equilibrium in diabetes mellitus results in
characteristic end-organ damage that occurs in various combinations and that follows
an unpredictable clinical pathway
Accordingly the major consequence of diabetes mellitus in terms of morbidity
mortality and economic burden principally concerns macroangiopathies or
arteriosclerosis and microangiopathies including nephropathy neuropathy and
retinopathy7-10
One of these devastating consequences which often appears in time is the
development of various skin defects that are frequently resistant to healing and that
tend to be more severe than similar lesions in nondiabetic individuals Diabetes
mellitus even increases the risk of infection by an increased susceptibility to bacteria
and an impaired ability of the body to eliminate bacteria1112
Skin problems are a severe complication in diabetic individuals and require a
comprehensive and appropriate multidisciplinary approach to prevention and
treatment12
Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to
play the most prominent role in the development of diabetic complications With the
development of insulin treatment for type I diabetes and various oral hypoglycemic
agents for type 2 diabetes a reduction in the development of skin defects due to
hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply
Chapter 3
64
waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in
diabetic patients often need special care in comparison to those persons in good
health who rarely require treatment for wound healing1617 Special care is directed
besides of course toward optimal diabetes regulation toward patient education
maximum pressure relief controlling infection recovery of circulation in case of
ischemia and different modalities of intensive wound treatment18
In the last few years various therapies have been introduced with varying success An
example of such a therapy is the photo-modulated stimulation of diabetic lesions In
vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been
subject of extensive investigation1920 Due to contradictory research results LLL-
photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of
light in the domain of wound healing is less time-consuming less expensive less
invasive than many of the other introduced treatment modalities and practical to use
however it seems worthwhile to investigate the value and benefits of a newly
introduced and alternative light source the light emitting diodes (LEDrsquos)
Preliminary research has proved that green LED with particular properties (an
exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01
Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These
results may be of great importance to the diabetic patient because as posed by Reddy et
al light has a possible beneficial effect in the case of impaired healing1617
To obtain insight into the ability of LED to stimulate fibroblast proliferation under
diabetic-specific conditions of impaired healing the proliferation was assessed in
irradiated and control cultures cultivated in medium with a high quantity of glucose
MATERIAL amp METHODS
Cell cultivation
Primary fibroblast cultures were established by outgrowth from 8-day-old chicken
embryos After isolation and disaggregating as described by Freshney (1994)24 the cells
were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10
Fibroblast proliferation under hyperglycemic circumstances
65
fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin
Secondary cultures were initiated by trypsinization followed by plating of the cells in
80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were
disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently
231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third
passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the
cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified
atmosphere at 37deg C to allow them to attach to the bottom of the wells
Light source specifications and illumination procedure
To control adherence of the cells and to assure that there was no confluence or
contamination the 96-well plates were microscopically examined before irradiation
Subsequently the tissue culture plates were randomly assigned for use in the treated
and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium
was aspirated The remaining 25 (50 microl) medium avoided dehydration of the
fibroblasts throughout irradiation
Irradiation was performed with a light emitting diode (LED) device The LED device
(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power
range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable
within the range of 0-1500 Hz
The investigation used the following illumination properties the continuous mode a
distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2
radiant exposure This procedure resulted in an exposure time of 3 min and a power
output of 10 mW Immediately after irradiation the remaining medium was aspirated
and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM
glucose (30 gL) and incubated at 37deg C
Irradiation and medium changes occurred at 1-day intervals so one irradiation was
implemented each 24 h for 3 days in a row and from the first irradiation onwards all
medium renewals occurred with glucose-supplemented Hanksrsquo culture medium
Control cultures were handled in the same manner but were sham-irradiated
Chapter 3
66
Proliferation assay
Fibroblast survival and proliferation were determined by a sensitive and reproducible
colorimetric assay the assay which detects merely living cells and the signal generated
bears a constant ratio to the degree of activation of the fibroblasts and the number of
fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than
only a small subsample26
Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented
Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution
was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during
30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous
solution The optical density of the final solution was measured on an ELtimes800 counter
(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test
wavelength varying from 400 to 750 nm
The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies
for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except
for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)
Data analysis
On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a
Mann-Whitney U test was performed for nonparametrical comparison of the results
Statistical significance for all tests was accepted at the 005 level For this analysis the
Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used
RESULTS
The MTT measurements from each of the 256 control wells and 256 irradiated wells
and the subsequent nonparametrical analysis from the optical densities obtained
disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic
Fibroblast proliferation under hyperglycemic circumstances
67
circumstances after irradiation than in the same circumstances without irradiation (Fig
1)
Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)
DISCUSSION
The outcome of these in vitro experiments based on the above-described light source
properties and the illumination procedure described clearly demonstrated the
stimulatory potential of LED on fibroblast proliferation and the cell viability of
fibroblasts cultured in hyperglycemic medium Preliminary research has already
demonstrated that under these conditions (an exposure time of 3 min a wavelength of
570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this
procedure allowed the highest number of living cells The nature of the light and the
usual questions concerning coherence wavelength power output and radiant
exposures have been discussed previously23
Although these findings confirm the results previously found one cannot ignore the
important methodological difference between previous investigations and the current
study as the cells in this experiment were cultured in hyperglycemic medium2328-30
Absorbency - Proportional to the number of fibroblasts
621 x 10-1 682 x 10-1
0010203040506070809
1
Control Irradiated
Groups
Ab
sorb
ency
Chapter 3
68
After a growth period with normal Hanksrsquo culture medium a necessary step to ensure
normal growth of these secondary subcultures and normal attachment to the bottom
of the wells the Hanksrsquo culture medium was supplemented with glucose
Several earlier studies have established that exposure to glucose concentrations (20-40
mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human
vascular endothelial cell proliferation1531-34 This restraint is more pronounced for
higher glucose15 concentrations and is expressed especially after protracted exposure to
high glucose levels31 A similar restraint was found for cultured fibroblasts by
Hehenberger et al3536 According to some authors however cultured fibroblasts
conversely have been shown to maintain responsiveness to ambient high glucose323738
As there are some ambiguities in literature regarding normal or inhibited growth of
fibroblasts in medium supplemented with glucose39 a pilot study was performed to
determine the amount of glucose necessary to inhibit normal growth after 72 h of
culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study
demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease
of cell viability and to bring about a decline in fibroblast proliferation
This concentration resulted in a remarkable reduction of cell viability and a noteworthy
decrease in the proliferation rate in comparison to control cultures grown in 55 mM
glucose although this concentration is too high to mimic severe diabetic
hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to
investigate the effect of LED in distinct destructive conditions in order to obtain an
incontrovertible result
In addition it is possible that the present investigation needed a higher amount of
glucose to result in a remarkable reduction of proliferation as exposure to glucose was
limited to 72 h and as previous studies revealed that the antiproliferative effect of high
glucose was more pronounced with prolonged exposure with a maximal inhibition
attained by 7-14 days1531
Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo
cell growth are too complex to compare A key question is whether fibroblast
senescence in tissue culture and in the intact organism are similar Cristofalo et al40
Fibroblast proliferation under hyperglycemic circumstances
69
reported that this is not the case as fibroblasts have a finite ability to divide and
replicate but apparently the pathway or the morphologic characteristics leading to the
replicative senescence is not identical in vivo compared to in vitro
Furthermore extrinsic aging related to environmental damage which in diabetic
patients is mainly due to a chronic exposure to high levels of glucose during life is
unachievable in vitro
Unless a number of questions regarding the mechanism according to which LED
stimulates fibroblast proliferation in this particular condition remain unanswered the
results ascertain the potential effects of LED on fibroblast proliferation and viability
CONCLUSION
The current results should be interpreted with caution However these results
demonstrate the effectiveness of green LED irradiation at the above-described light
source properties and the illumination procedure described on cells in hyperglycemic
circumstances
The findings of the present study using an experimental in vitro model indicate that the
use of LED irradiation to promote wound healing in diabetic patients may have
promising future results As the present study establishes the possibility of using LED
irradiation in experimental in vitro situations it would be a worthwhile extension to
perform in vivo investigations to determine whether these in vitro observations were
relevant to the physiological situation and to determine the effect of these LED
properties on human tissue response
ACKNOWLEDGMENTS
The authors are greatly indebted to P Coorevits for assistance with the statistical
analysis and to Professor L Deridder and Ms N Franccedilois of the department of
Human Anatomy Embryology Histology and Medical Physics for providing access to
the laboratory and for helpful discussions
Chapter 3
70
REFERENCES
1 World Health Organisation The world health report of 2002 Statistical Annex 2002 pp 179-201
2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418
3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957
4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538
5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308
6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J
Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot
wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-
glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A
Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood
glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13
14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309
15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501
16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287
17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255
18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412
19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192
20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137
21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675
22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246
23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99
Fibroblast proliferation under hyperglycemic circumstances
71
24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729
27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63
28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301
29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -
progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of
cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA
damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in
culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia
23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar
K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71
36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141
37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588
38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252
39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97
40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619
PART II ANALGESIA
CHAPTER 4
EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION
MEDIATED BY LIGHT EMITTING DIODE IRRADIATION
Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy
Vanderstraetenab and Dirk Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Lasers in Medical Science 2005 20(1) 35-40
Chapter 4
76
ABSTRACT
The introduction of light emitting diode (LED) devices as a novel treatment for pain
relief in place of low-level laser warrants fundamental research on the effect of LED
devices on one of the potential explanatory mechanisms peripheral neurophysiology in
vivo
A randomised controlled study was conducted by measuring antidromic nerve
conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline
measurement and five post-irradiation recordings (2 min interval each) were performed
of the nerve conduction velocity (NCV) and negative peak latency (NPL)
Interventional set-up was identical for all subjects but the experimental group (=32)
received an irradiation (2 min at a continuous power output of 160 mW resulting in a
radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype
MDB-Laser Belgium) while the placebo group was treated by sham irradiation
Statistical analysis (general regression model for repeated measures) of NCV and NPL
difference scores revealed a significant interactive effect for both NCV (p=0003) and
NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical
significant decreased NCV and an increased NPL in the experimental group and a
statistical significant difference between placebo and experimental group at various
points of time
Based on these results it can be concluded that LED irradiation applied to intact skin
at the described irradiation parameters produces an immediate and localized effect
upon conduction characteristics in underlying nerves Therefore the outcome of this in
vivo experiment yields a potential explanation for pain relief induced by LED
Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative
peak latency middot Analgesic effect
Nerve conduction characteristics
77
INTRODUCTION
Since the introduction of photobiostimulation into medicine the light sources used
have advanced technologically and varied in characteristics over the years
Advancement and variation of the sources implicate a concomitant necessity to revise
research results in the respective domains of application Research and clinical
applications in the past particularly focused on the effectiveness of low-level lasers
have shifted now to novel treatment units such as light emitting diode (LED) devices
The efficacy and applicability of LED irradiation within the field of wound healing has
already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED
is not only promoted for its beneficial effects on the wound-healing process it is also
suggested to be potentially effective in the treatment of pain of various aetiology
although this claim has not yet been investigated thoroughly either experimentally or
clinically The putative analgesic effects of LED remain to be further explored
As the basic vehicle of pain is the neuronal system [7] measuring the
neurophysiological effect of LED treatment would be an appropriate experimental
approach to investigate the efficacy of LED on pain inhibition Nerve conduction
studies have become a technique for investigating the neurophysiologic effects of light
therapy [8-9]
Review of literature regarding standard nerve conduction studies revealed that previous
human studies on the influence of various light sources on peripheral nerves have
utilized different methods which hampers a comprehensive comparison In general
this research was performed on the superficial radial nerve [10-13] described by Shin J
Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve
[891315-17] Following the method of Cambier et al [18] the authors of this study
decided to investigate the effect of the light source used on the conduction
characteristics of the sural nerve By investigating this solely sensory nerve interaction
of motor nerve fibres (motor response can easily be provoked by antidromic nerve
stimulation [19]) can be avoided and given the superficial nature of the nerve it should
be sufficiently amenable to the effects of percutaneous LED irradiation
Chapter 4
78
A second major difference between the trials and therefore also hindering an
appropriate comparison between the results is the wide range of used light sources
HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared
multisource treatment unit [15]
With respect to the potential importance of LED irradiation for the treatment of pain
the current investigation was designed to assess the putative neurophysiological effects
of LED on the sensory nerve conduction of the human superficial peripheral sural
nerve and to establish a time course of the supposed phenomenon
The experimental hypothesis postulates that LED generates an immediate decrease in
conduction velocity and increase in negative peak latency In addition it can be
postulated that this effect is most prominent immediately after the irradiation and will
weaken as time progresses
STUDY DESIGN
The study was approved by the Ethical Committee of the Ghent University Hospital
After explanation of the experimental procedure a written informed consent was
obtained from each subject
Subjects
After screening based on a brief medical history excluding subjects with
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever inflammation of the skin) or conditions
that might affect sensory nerve conduction (such as diabetes peripheral neuropathy
radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral
edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40
females (mean age 26plusmn6 years range 18-42 years) participated in this study The body
mass index (BMI) of each subject varied within the normal range (=185-249) [20]
(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo
Nerve conduction characteristics
79
or an experimental group Each group of 32 subjects was composed of 12 males and
20 females
Experimental Procedure and Data Acquisition
In order to be able to quantify the negative peak latency (NPL) (measured from the
start of the stimulus artefact to the peak of the negative portion of the nerve action
potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was
followed
With respect to the known relationship between nerve conduction characteristics and
temperature the ambient temperature was kept constant (23ordmC-26ordmC room
temperature) during the investigation In view of this temperature issue the
standardized protocol started with 10 min of accommodation during which the
subjects rested in prone position on a treatment table
Immediately before this adjustment period the skin over the dorsolateral aspect of the
left calf and foot was cleaned with alcohol to remove surface lipids This preparation of
the treatment area was followed by the placement of the electrodes (TECA
Accessories Oxford Instruments Medical Systems Division Old Woking UK) as
described by Delisa et al [21]
The two-posted (2 cm separation anode distal) surface caption electrode was placed
distal and posterior of the lateral malleolus on the skin covering the sural nerve The
fixation of the earth electrode (Medelec Oxford Instruments Medical Systems
Division Old Woking UK) occurred 12 cm above the caption electrode according to
the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm
above the caption electrode to map the ideal stimulation point To level off
intraindividual variations in the amount of sensory response attributable to the
successive placement of the bipolar stimulator in course of the investigation a two-
posted (2 cm separation cathode distal) bar stimulating electrode was attached at the
point where the maximal response was obtained
This placement of the electrodes allows antidromic stimulation of the sural nerve
Electrophysiological stimulation and recordings were obtained with a Medelec
Chapter 4
80
Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse
of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was
used to produce each evoked sensory response
Baseline measurements of NPL and NCV were immediately followed by treatment of
the subjects according the protocol detailed below Recordings were subsequently
repeated at 2-min intervals over an 8-min period resulting in five recordings (one
immediately after the completion of the treatment and one at 2 4 6 and 8 min after
irradiation) Skin temperature was recorded concomitantly throughout the procedure
at the time of baseline measurement immediately after LED irradiation at the time of
the first recording and consequently at 2-min intervals together with the four final
electrophysiological recordings For this a surface digital C9001 thermometer
(Comark UK) sensitive to temperature changes of 01degC was used at the same point
of LED administration namely at 7 cm above the caption electrode The procedure
was identical for both conditions but subjects in the placebo group received a sham
LED irradiation
Light Characteristics and Irradiation Procedure
Irradiation was administrated with a light emitting diode device (BIO-DIO
preprototype MDB-Laser Belgium) The probe used emitted infrared light with a
wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2
and the frequency was variable within the range of 0-1500 Hz
Preceding baseline measurement the treatment point was marked on the skin overlying
the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-
point between the stimulation and capture electrode The LED probe was held in
contact with the skin perpendicular to the skin surface during the complete irradiation
procedure LED treatment consisted for all subjects of the experimental group out of 2
minutes lasting irradiation The LED was set to deliver a continuous energy density of
107 Jcm2 at a power output of 160 mW These parameters were selected as they are
appropriate for the treatment of pain in a clinical setting First of all because the
Nerve conduction characteristics
81
duration of the treatment is clinically feasible and secondly because the parameters are
within the scope of previously described light source characteristics [1-36915]
Statistics
Although superficial skin temperature did not change significantly in course of the
investigation the influence of the measured skin temperature on NPL and NCV was
taken into account by using a correction factor of respectively 02 msdegC and 147
ms degC All corrections were calculated towards a reference skin temperature of 32degC
Difference scores ie the variation between baseline measurements and each post-
irradiation recording were used as the basis for statistical analysis A General
Regression Model for repeated measures with one within-subjects factor (time 0
min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and
one between-subjects factor (group placebo or LED irradiated) was performed
followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least
Significant Difference) to determine whether any differences between baseline
measurements and post-irradiation recordings were statistically significant
The Statistical package for social sciences (SPSS 110) was used for analysis and
statistical significance for all tests was accepted at the 005 level
RESULTS
Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated
group plotted against time in minutes The values of the irradiated subjects decrease
directly after the irradiation and reach a first low point 2 min after finishing LED
treatment This decrease is followed by a marginal increase at 4 and 6 min and again an
important decrease at 8 min Statistical analysis (general regression model for repeated
measures) of these data indicated a significant interactive effect (P=0003)
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82
Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)
Post hoc LSD further showed significant differences between baseline measurements
and all post-treatment recordings (Table 1) Mutual comparison of the values from the
post-treatment recordings did not reveal any significant difference In addition there
was no significant difference determined in the placebo group in course of time
Table 1 Summary of the influence of LED irradiation on nerve conduction velocity
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001
2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002
4 0111plusmn0377 0647 -0908plusmn1898 0021 0004
6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001
8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve Conduction Velocity
-14
-12
-1
-08
-06
-04
-02
0
02
04
Baseline 0 min 2 min 4 min 6 min 8 min
Time Course
Dif
fere
nce
Sco
re (
m
s)
PlaceboLED
Nerve conduction characteristics
83
A similar representation was used for the results of the NPL Figure 2 reproduces NPL
plotted against time in minutes revealing for the irradiated group an increased latency
with two important peaks one at 4 min and one at 8 min
Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)
Statistical analysis of the mean difference scores again indicated a significant interactive
effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2
showed significant differences between baseline measurements and all post-treatment
recordings of the experimental group The mean difference score of the first post-
treatment recording of this same group (LED irradiated) differed significantly with the
recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED
irradiation As well as the recording 2 min after irradiation which differed significantly
(P=0013) with the 8 min post-treatment recording As observed for the NCV the
NPL of the placebo group did not reveal any significant difference in time course
At the time of the final recording the NCV and NPL mean difference scores of the
irradiated group did not return to their respective baseline values
Negative Peak Latency
-001
0
001
002
003
004
005
006
007
Baseline 0 min 2 min 4 min 6 min 8 min
Time course
Dif
fere
nce
Sco
re (
ms)
PlaceboLED
Chapter 4
84
Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group
significance) revealed statistical differences between the experimental and the placebo
group for NCV as well as for NPL NCV and NPL were statistical significant between
both groups at all points of time except from the NPL recording immediately after
finishing irradiation
DISCUSSION
Notwithstanding the above-mentioned difficulties in comparing results between
different trials on nerve conduction we attempt to discuss the current findings in view
of the results of the previous studies
This investigation revealed that percutaneous LED irradiation at feasible and current
clinical parameters generates measurable and significant changes in human sural nerve
antidromic conduction latency and velocity These results thus support previous
findings of light-mediated nerve conduction latency shifts in vivo [8101218]
although there are several important issues to be discussed
Table 2 Summary of the influence of LED irradiation on negative peak latency
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0004 plusmn0053 0755 0029plusmn0080 0019 0145
2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021
4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004
6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034
8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve conduction characteristics
85
A first comment deals with the progress of the NCV and NPL in function of time As
postulated the NCV decreases significantly immediately after irradiation
corresponding with a significant increased NPL However this effect does not weaken
as time progresses both variables remain significant throughout the 8 min during
observation period
Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the
conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]
although this slight increase in NPL was not significant at any moment Two other
studies [822] with a GaAlAs laser even registered comparable effects over a period of
55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous
studies post-treatment conduction measurements should be extended in time At
present for all studies it remains unclear at what point of time the effect extinguishes
although the interval of time during which LED treatment remains effective is
clinically important when treating pain
Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least
45 min) mediated by a monochromatic multisource infrared diode device although it
needs to be mentioned that this study performed with a comparable light source as the
current investigation revealed a significant decrease of the NPL These inverse results
between the study of Noble et al [15] and the current investigation could be attributed
to the concomitant increase of the skin temperature [15] As it has been well
recognised that a variation in tissue temperature causes a corresponding alteration in
nerve conduction velocities and peak latencies [91523-27] the temperature changes
may indeed provide an explanation for the observed findings In an attempt to analyse
the influence of a direct photobiological effect on sural nerve conduction
characteristics rather than working out the effects based upon thermal mechanisms
the present study corrected the skin temperature towards a reference temperature of
32degC This correction was performed notwithstanding the fact that the superficial skin
temperature did not change significantly before and after LED irradiation as well as
despite the fact that influencing nerve temperature takes place long after affecting skin
temperature [23] and thus being (almost) impossible after 2 min of irradiation
Chapter 4
86
followed by 8 min of registration Introduction of the correction factor implies likewise
that eventual influence on nerve conduction by cooling of the limb due to inactivity as
described by Greathouse et al [11] can be excluded
These facts suggest that temperature changes did not contribute to the demonstrated
effects of LED on nerve conduction Nevertheless the underlying mechanism of the
observed effects remains indistinct
A following remark regarding the fluctuation of NCV and NPL in function of time
considers the fact that both the NCV and the NPL do not change in a constant way up
to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the
increase in NPL display a small though not significant inversion of the effect at 4 and
(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of
fluctuation is to be expected when measuring NCV and NPL besides there is a similar
variation in the placebo groups
Although investigating dose dependency was not intended an additional remark
considers the fact that the use of optimal irradiation parameters is essential to obtain
the observed neurophysiological effect Nevertheless it is impossible to determine
ideal light source characteristics for effective treatment as the range of used
wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency
(pulsed or continuous) are not sufficiently similar between the different studies It can
only be concluded that a pulsing light source [91028] does not provide the postulated
results Radiant exposure exposure time power range and wavelength are not yet
established but based on this study and previously described assays it can be
speculated that the ranges of these parameters are quite large
In comparison with other studies where the number of subjects is 10 or less [8-
1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-
Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number
of subjects (n=32) was investigated in each group In spite of the large investigated
population it should be noted that the magnitude of the described changes in NCV
and NPL can simply be replicated by lowering the temperature of the extremity as the
observed changes are within the expected physiological ranges making the clinical
Nerve conduction characteristics
87
significance of the change questionable (This fact does not implement that the
decrease and the significant changes were temperature mediated)
A key question and meanwhile the initial impetus for future investigation is whether
the measured effects can be extrapolated to the actual nociceptive afferents namely the
myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])
respectively conducting acute and chronic pain The functional testing of these
nociceptive pathways has recently been extensively evaluated The currently accepted
neurophysiological method of assessing nociceptive pathways relies on laser-evoked
potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]
As up till now LEP is not available in this or any surrounding research centre the
investigators of this study had to perform a standard nerve conduction study (assessing
the large myelinated Aβ afferents) Therefore the current and previous beneficial
results of low level light therapy on conduction characteristics of nerves in vivo should
initiate measurements of clinical effectiveness first of all in laboratory settings and
afterward at a clinical level
CONCLUSION
Despite these remarks and the limited knowledge regarding the underlying mechanism
the present findings enable the following conclusions to be drawn LED irradiation at
clinical applied energy densities produces an immediate and localized effect upon
conduction characteristics in underlying nerves More specifically it is proven that
LED treatment lowers the NCV and augments the NPL resulting in a reduced
number of impulses per unit of time Therefore the outcome of this in vivo experiment
assumes that LED possibly induces pain relief
In order to encourage a widespread acceptance for the use of this non-invasive pain-
reducing modality in clinical settings prospective research should establish the precise
relationship between LED and pain relief as well as determine the ideal irradiation
parameters and verify which painful conditions can be treated with this treatment unit
Chapter 4
88
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the
equipment and MDB-Laser Belgium for generously providing the Light Emitting
Diode equipment
Nerve conduction characteristics
89
REFERENCES
1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9
2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)
3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18
4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8
5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43
6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65
7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53
8 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-34
9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6
10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90
11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7
12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5
13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9
14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore
15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5
16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5
17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604
18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200
19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21
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90
20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083
21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press
22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9
24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9
25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203
26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13
27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12
28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9
29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445
30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8
31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56
CHAPTER 5
PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE
IRRADIATION A PILOT STUDY ON EXPERIMENTALLY
INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS
Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Accepted for publication in Lasers in Medical Science December 2005
Chapter 5
92
ABSTRACT
Objective The present pilot study investigated the analgesic efficacy of light emitting
diode (LED) In view of a standardised and controlled pain reduction study design this
in vivo trial was conducted on experimentally induced delayed-onset muscle soreness
(DOMS)
Design Thirty-two eligible human volunteers were randomly assigned to either an
experimental (n=16) or placebo group (n=16) Immediately following the induction of
muscle soreness perceived pain was measured by means of a visual analog scale (VAS)
followed by a more objective mechanical pain threshold (MPT) measurement and
finally an eccentricconcentric isokinetic peak torque (IPT) assessment The
experimental group was treated with infrared LED at one of both arms the other arm
served as control Irradiation lasted 6 min at a continuous power output of 160 mW
resulting in an energy density of 32 Jcm2 The subjects of the placebo group received
sham irradiation at both sides In post-treatment a second daily assessment of MPT
and VAS took place The treatment and assessment procedure (MPT VAS and IPT)
was performed during 4 consecutive days
Results Statistical analysis (a general linear model followed by post hoc least
significant difference) revealed no apparent significant analgesic effects of LED at the
above-described light parameters and treatment procedure for none of the three
outcome measures However as the means of all VAS and MPT variables disclose a
general analgesic effect of LED irradiation in favour of the experimental group
precaution should be taken in view of any clinical decision on LED
Conclusion Future research should therefore focus on the investigation of the
mechanisms of LED action and on the exploration of the analgesic effects of LED in a
larger randomised clinical trial and eventually in more clinical settings
Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset
muscle soreness middot Musculus biceps brachii
Delayed-onset muscle soreness
93
INTRODUCTION
The analgesic efficacy of light emitting diode (LED) irradiation is recently being
investigated by means of a nerve conduction study on the superficial peripheral sural
nerve [1] It was demonstrated that LED irradiation at clinical applied densities
produces an immediate and localized effect upon conduction characteristics in
underlying nerves More specific LED induces a decreased number of sensory
impulses per unit of time thus possibly inducing pain relief [1]
Given the established influence of this treatment modality on the nerve conduction
velocity and thereby its potential analgesic ability the current investigation was
designed
Studies investigating the efficacy of a therapeutic modality on pain often experience
difficulties regarding standardisation of the population as analysis or comparison of
pain with different aetiologies is almost impossible Therefore we opted to measure the
analgesic effects of LED in a laboratory setting on a sample with experimentally
induced delayed-onset of muscle soreness (DOMS)
Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the
induction exercise and then spreads throughout the muscle [2-4] The correlates of
DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10
[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force
decreased range of motion and in particular muscle pain which is more pronounced
during movement and palpation [8 11] Despite the large volume of research that has
been undertaken to identify the underlying pathophysiology of DOMS the precise
mechanism is not yet universally accepted Several theories such as the torn-tissue
theory the connective tissue damage theory the muscle spasm theory and the
inflammation theory still remain viable though the current opinion states that DOMS
arises from a sequence of events in which several theories occupy an important place
[2 6 12 13]
DOMS has been used as a representative model of musculoskeletal pain and stiffness
in a number of studies [4 7 11 14 15] as it has a number of advantages it can be
induced in a relatively easy and standardised manner in a group of healthy subjects the
Chapter 5
94
time-course is relatively predictable and the symptoms have the same aetiology and are
of transitory nature [14 16] Nevertheless it should be emphasised that the use of this
particular experimental model to test the effectiveness of LED does not mean that this
treatment modality is necessarily advocated for the treatment of DOMS but merely
that it may be helpful in documenting the efficacy of LED in a clinical model of
musculoskeletal pain and stiffness In addition studies based on the induction of
DOMS under carefully controlled laboratory conditions can not replace research
involving actual patients but offer the opportunity to assess the effectiveness of
particular therapeutic interventions and might help to define additional clinical research
[14]
The experimental hypothesis of the current study postulates that infrared LED reduces
pain and muscle sensitivity associated with DOMS
MATERIALS AND METHODS
The study was approved by the ethical committee of the Ghent University Hospital
After providing information regarding the study design and possible consequences
related to participation at the study written informed consent was obtained from each
subject
Subjects
Healthy human volunteers were recruited from the university population Individuals
with any upper limb pathology neurological deficit and recent injury to either upper
extremity or undiagnosed pain were excluded Other exclusion criteria were
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever and inflammation of the skin) or
conditions in which physical exertion is contraindicated (such as cardiovascular deficits
hypertension and respiratory problems)
Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age
23plusmn4 years) were enrolled All subjects were randomly assigned using a random table
Delayed-onset muscle soreness
95
of numbers to the experimental or placebo group Each group of 16 subjects
consisted by stratification of equal numbers of men and women Age height and
weight did not differ significantly between the three groups
All subjects were physically active however none performed on a regular basis any type
of upper body weight-training Subjects were requested to refrain from any form of
strenuous physical activity and they were asked to avoid any form of medication
including anti-inflammatory agents as well as alcohol for 2 days before testing and for
the duration of the study
Overview of experimental design
The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to
induce pain related to DOMS Immediately following induction exercise an initial
assessment of the outcome measures (visual analog scale or VAS mechanical pain
threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the
subjects were treated under blinded conditions according to the randomised group
allocation In post-treatment the MPT was re-recorded and perceived pain was
reassessed with a VAS Contrary to these outcome measures the muscle strength was
only measured in pre-treatment at the one hand because short-term effects of LED
on muscle strength were not postulated and on the other hand because post-
treatment muscle strength can be influenced by too many different physiological
factors related to the pre-treatment measurement On the succeeding days (day 2 3
and 4) the treatment and assessment procedure was similar with approximately 24 h
separating each treatment
In both of the groups the two arms of the participants were included in the study In
the experimental group an equal number of dominant and non-dominant arms were
treated The non-treated arm served as control arm In the placebo group also an equal
number of dominant and non-dominant arms were considered as treated arm and the
other arm was classified in the non-treated group The procedure was identical for
both conditions but the subjects in the placebo group received sham LED irradiation
on both arms
Chapter 5
96
Specific aspects of the experimental design and procedures are detailed below
Pain induction
Muscle soreness was induced in a standardised fashion via a daily calibrated computer-
operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY
USA) Induction occurred separately and in random order in the elbow flexors of both
arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex
Prior to induction of DOMS the subjects were allowed an initial familiarization session
to become comfortable performing maximum voluntary contractions at the required
angular velocities This was immediately followed by determination of the maximum
eccentric and concentric peak torque at an angular velocity of 60degs and 120degs
Subsequently four sessions of eccentricconcentric work were performed with each
arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs
first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg
and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-
range) followed by two sessions at an angular velocity of 120degs again the first time
along an arch of 120deg and followed by the mid-range performance The subjects were
asked to accomplish maximum voluntary contractions during all the sessions Each
session was performed until exhaustion which was defined as the point when the
subject lost 70 of the initial eccentric and concentric peak torque There was a 1-
minute rest between each session This procedure was based on a pilot study and
previously described induction protocols [17-21]
Outcome measures
Outcome measures of subjective pain measurements MPT and muscle strength were
measured in this order on days 1-4 Subjective pain measurements and MPT occurred
immediately prior to and following irradiation whereas muscle strength measurements
only took place before LED treatment
Measurement of subjective pain Perceived muscle soreness was measured
subjectively by means of a 100-mm VAS A series of scales were completed separately
Delayed-onset muscle soreness
97
for each arm pain at rest followed by pain perception associated with full extension of
the arms and finally with maximal flexion of the arms The subjects were not allowed
to compare one VAS result with another
This assessment tool commonly used in measuring experimentally induced pain [22
23] has been found to be a reliable and valid method [24-26]
MPT Tenderness MPT used as a more objective correlate of muscle tenderness
has been demonstrated to be a reliable method to measure experimental induced
muscle soreness [27] This outcome measure was assessed by using a handheld
pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries
South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a
line from the radial insertion of the musculus biceps brachii at the elbow to the
intertubercular groove of the humerus thus resulting in three measure points one at
the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A
pressure of 4Ns was delivered The subjects were instructed to say yes at the exact
moment the pressure perceived became painful Each point was recorded three times
in pre-treatment as well as in post-treatment The average MPT score for each point in
pre- and post-treatment was used for statistical analyses
Muscle strength assessment Eccentric and concentric IPT were measured on the
same computerised dynamometer as was used for the induction of pain and an
identical standardisation procedure regarding positioning was followed
A warm-up session of two maximum voluntary contractions at the required angular
velocities was followed by determination of the eccentric and concentric peak torque
The first session at 60degs consisted of three repetitions followed by a 1-min during
rest and for the second session at 120degs five repetitions were performed The
subjects were instructed to flex and extend the elbow through the entire range of
motion as forcefully and rapidly as possible for each repetition The maximum
eccentric and concentric torque produced during the respective repetitions was used
for statistical analysis
Chapter 5
98
Light source specifications and treatment procedure
Light treatment was applied daily according to group allocation Irradiation occurred
with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The
probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160
mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The
frequency was variable within the range of 0ndash1500 Hz
During the complete irradiation procedure the LED probe was held in contact with
the skin perpendicular to the skin surface and at the exact mid-point between the MPT
mark at 4 cm and the one at 8 cm Light source properties were identical for all
subjects of the experimental group and consisted out of irradiation of 6-min lasting
duration at a continuous power output of 160 mW resulting in an energy density of
32 Jcm2 To conceal the treated side and condition the subjects were blinded to the
treatment status For the experimental condition a probe was held in contact with each
arm but only one of the two probes was attached to the LED device The subjects of
the placebo group received sham irradiation at both sides
The selected parameters are within the scope of previously described light source
characteristics for pain reduction [1 28-30] and they are appropriate for the treatment
of pain in a clinical setting because the duration of the treatment is clinically feasible
Statistical analysis
The three outcome measures were analysed separately For the VAS and MPT
measurements the same procedure was followed a general linear model (GLM) for
repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-
post preceding and following LED irradiation) and one between-subject factor (group
placebo or infrared LED irradiated) was performed If necessary the GLM was
followed by appropriate pairwise comparisons (post hoc least significant difference or
LSD) to determine whether any differences between measurements were statistically
significant A similar model was carried out separately for both the treated and the
control arm
Delayed-onset muscle soreness
99
In contrast to MPT and VAS the muscle strength was analysed differently The peak
torque values recomputed towards body weight of the subjects were statistically
analysed using a GLM for repeated measures This model consisted of one within-
subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo
or infrared LED irradiated) The model was completed twice first for the treated arm
and consequently for the control arm
The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)
was used for analysis and statistical significance for all tests was accepted at the 005
level
RESULTS
Statistical analysis of all variables of the three outcome measures revealed no significant
interactive effects of the main interaction (time times group times pre-post) The means and
standard deviations of the variables for both the treated and the control arm are
outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The
means of all VAS and MPT variables disclose a non-statistical significant general
analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher
MPT values in the irradiated group than in the placebo group The lower VAS rates are
present from day 1 until the last day of the study but they are more clearly present
from day 3 pre-treatment The higher MPT values are present from day 1 post-
irradiation until the last day and they are more visible at 4 cm followed by 12 cm and
finally at 8 cm In addition to the analgesic influence of LED an increased
convalescence of muscle strength was noted It should be remarked that this outcome
is similar for the treated as well as for the control arm of the irradiated group The
findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms
of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively
Graphical presentation of the other variables shows a similar course
Chapter 5
100
Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42
Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36
Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Visual Analog Scale
0
02
04
06
08
1
12
14
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n V
AS
scor
e (a
t re
st)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
101
Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249
Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Mechanical Pain Threshold
0
5
10
15
20
25
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n M
PT
sco
re (
at 4
cm)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Chapter 5
102
Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group
Day 1 Day 2 Day 3 Day 4
Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04
Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04
Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Isokinetic Peak Torque
04
045
05
055
06
065
Day 1 Day 2 Day 3 Day 4
Time course
Mea
n I
PT
sco
re (
ecce
ntr
ic a
t 60
degse
c)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
103
Despite the absence of significant main interaction effects the remaining interactions
as well as the main effects were statistically significant for some variables Only the
significant interactions including the between-subject factor group as well as the main-
effect group will be discussed The other interactions and effects establish the successful
induction of DOMS but are not relevant in view of the postulated hypothesis
The interaction between group and time is significant (p=014) for the VAS in
association with full extension for the control arm Post hoc LSD reveals no difference
between both groups a significant effect over time for both groups is found
Consequently this will not be further evaluated
A second significant interaction (p=0002) is the one among the within-subject factor
pre-post and the between-subject factor group for the MPT at 12 cm for the control arm
Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more
pressure after than before the treatment whereas in the placebo group a not
significant decrease of supported pressure is noted
Finally GLM analysis revealed that at the treated arm the irradiated group tolerates
significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)
DISCUSSION
It has previously been demonstrated that the LED source used might assist in
accelerating wound healing [31] that it has a direct cellular effect [3233] and that it
changes nerve conduction characteristics [1] Nevertheless LED-treated experimental
induced DOMS failed to prove the analgesic efficacy of LED at the above-described
light parameters and treatment procedure The current outcome concurs with other
research that demonstrated a lack of effect of various forms of light therapy on DOMS
[8 11 15] However despite the absence of an apparent and overall definitive finding
the present results cannot exclude favourable effects of LED treatment on pain Since
first of all an isolated statistical significant pre-post difference between groups (control
arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)
revealed that subjects of the irradiated group tolerate more pressure than the subjects
of the placebo group Second of all the overall means identified generally lower VAS
Chapter 5
104
scores higher MPT values and higher peak torques in the irradiated group This
implied that the treated subjects experienced noticeable less pain supported more
pressure on the painful muscle and generated more force than the non-treated
participants However these results are not statistically significant consequently it is
possible that these differences were found by coincidence and that there is no
relationship between the treatment and the described results of the three outcome
measures though it should be mentioned that the absence of significant findings is
more probably attributable to the small sample size involved in this study This
assumption is based on a post hoc power analysis It was calculated that for the small
effect size measured after treatment and for the measured control group event rate a
sample size of 80 subjects in each group was required at α=005 and power=080
(two-sided) to reveal significant results
Another factor conceivably responsible for the lack of solid evidence of the beneficial
effects of LED treatment upon DOMS-associated pain is related to the size of the
treatment effect in relation to the severity of the induced DOMS It is possible that by
using multiple exhaustive sets of exercise severe DOMS were induced which masked
relatively small but apparent treatment effects [4 11] In this same context it is
possible that the results only become significantly different after a prolonged treatment
and follow-up period as previous research noticed that recuperation subsequent to
DOMS induction can last up to 10 days [8]
Although it needs to be stressed that these results are not statistically significant critical
analysis of the overall means leads up to three additional remarks A primary comment
relates to the pre- and post-treatment courses of the results Starting at day 2 a clear
reduction of pain and muscle sensitivity was observed immediately post-treatment
Still one cannot conclude that this is indicative for the analgesic effect of LED
irradiation as a similar decrease in VAS and increase in MPT values was noted in the
treated and the control arm of the placebo group Perhaps this was caused by placebo
effect as reported by Pollo et al [34] the expectation of the participant can easily result
in pain relief but it can only be elucidated by implementation of a control group
Delayed-onset muscle soreness
105
Nevertheless in the current study this particular finding can be most probably
attributed to the physiological effects of the peak torque measurement performed
between the pre- and post-treatment recordings of VAS and MPT on the painful
flexor muscle of the upper arm For the assessment of muscle strength two short
series of alternative concentric and eccentric efforts were performed in succession
involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist
in the removal of inflammatory markers and exudate consequently reducing local
tenderness [4] In addition the force assessment can be considered as a form of active
warming-up resulting in an increased muscle temperature which can reduce muscle
viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness
[3536] thus decreasing the sensitivity of the muscle and moderating pain during
movement In any case the beneficial influence of LED immediately after irradiation
can not be securely interpreted due to the sequential assessment of the outcome
measures
A second additional remark considers the fact that both arms of the irradiated subjects
demonstrated evidence of the beneficial effects of LED as a similar reduction of pain
and muscle sensitivity and higher peak torques were found in course of time at the
treated arm as well as at the control arm of the irradiated subjects This ascertainment
points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]
stated that in case LED works via systemic effects the use of the contralateral side as a
control arm might be ill-advised Thus reinforcing that future research should include a
control group to bring clarification [4 7 16]
Finally it needs to be mentioned that although the extent of DOMS was probably
relatively high for investigating the postulated hypothesis the time-course of the
present study corresponds to that reported by other investigators [2 3 5-10]
Significant time effects in many of the variables revealed that muscle damage was
evident diffuse muscle soreness became progressively worse 24-48 h after DOMS
induction followed by a small amelioration after 72 h [35910] After 72 h the follow-
Chapter 5
106
up was ceased consequently further regain of force and attenuation of pain and
muscle sensitivity could not be evaluated Extending the duration of the assessment
period could be useful in assessing any longer-term effects of LED treatment
particularly because as mentioned above differences between both groups are more
clearly present from day 3 pre-treatment and also because DOMS may last for up to 10
days when induced with the described protocol [715]
Lack of knowledge regarding both the precise mechanism of action of LED and the
specific pathophysiology of DOMS hampers the way to offer a definitive explanation
for the absence of more obvious statistically significant differences Still the small
number of significant findings and the mean values suggest that possible analgesic
effects of infrared LED may not be excluded yet but to be able to estimate the real
value of LED further research is necessary A large-scaled randomised clinical trial
which takes the above-mentioned remarks into consideration should be performed
CONCLUSION
Regardless of the reasons for the absence of statistical significant effects reported here
and although LED may have some potential in the management of pain and functional
impairment associated with DOMS its effectiveness at the applied densities has not
been established
Future research should focus on evaluation of the appropriateness of DOMS as an
experimental model of pain and muscle damage Validation of this model would
enhance the ability to study various modalities for their potential effects on pain and
muscle injuries Besides the mechanisms of LED action are not known thus further
fundamental investigations need to address the underlying mechanism and
physiological basis of pain modulation utilizing LED treatment
Once LED irradiation has finally proven its treatment value in an experimental model
the most important prospect considers establishing the effectiveness of LED to reduce
pain in clinical settings
Delayed-onset muscle soreness
107
ACKNOWLEDGMENTS
The authors would like to thank Mr T Barbe and Mr R Deridder for their technical
assistance in the collection of the data as well as for their valuable input into the
research design Sincere appreciation is extended to the volunteers that participated in
this study and to MDB-Laser (Belgium) for generously providing the light emitting
diode equipment The authors also gratefully recognize Prof Dr G Van Maele for
assistance with the statistical analysis and for helpful discussion
Chapter 5
108
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1 Vinck E Coorevits P Cagnie B Muynck MD Vanderstraeten G and Cambier D (2005) Evidence of changes in sural nerve conduction mediated by light emitting diode irradiation Lasers Med Sci DOI101007s10103-005-0333-2
2 Cheung K Hume PA and Maxwell L (2003) Delayed Onset Muscle Soreness - Treatment Strategies and Performance Factors Sports Med 33(2)145-164
3 MacIntyre DL Reid WD and McKenzie DC (1995) Delayed muscle soreness The inflammatory response to muscle injury and its clinical implications Sports Med 20(1)24-40
4 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
5 Clarkson PM and Tremblay I (1988) Exercise-induced muscle damage repair and adaptation in humans J Appl Physiol 65(1)1-6
6 Cleak MJ and Eston RG (1992) Delayed onset muscle soreness mechanisms and management J Sports Sci 10(4)325-341
7 Craig JA Cunningham MB Walsh DM Baxter GD and Allen JM (1996) Lack of Effect of Transcutaneous Electrical Nerve Stimulation Upon Experimentally Induced Delayed Onset Muscle Soreness in Humans Pain 67(2-3)285-289
8 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
9 Ebbeling CB and Clarkson PM (1989) Exercise-Induced Muscle Damage and Adaptation Sports Med 7(4)207-234
10 Tiidus PM and Ianuzzo CD (1983) Effects of Intensity and Duration of Muscular Exercise on Delayed Soreness and Serum Enzyme-Activities Med Sci Sports Exerc 15(6)461-465
11 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
12 Armstrong RB (1984) Mechanisms of exercise-induced delayed onset muscular soreness a brief review Med Sci Sports Exerc 16(6)529-538
13 Rodenburg JB Steenbeek D Schiereck P and Bar PR (1994) Warm-up stretching and massage diminish harmful effects of eccentric exercise Int J Sports Med 15(7)414-419
14 Ciccone CD Leggin BG and Callamaro JJ (1991) Effects of ultrasound and trolamine salicylate phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-675 discussion 675-678
15 Craig J Barlas P Baxter D Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
16 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
17 Dvir Z (2003) Isokinetics muscle testing interpretation and clinical applications Churchill Livingstone Edinburgh
18 Lambert MI Marcus P Burgess T and Noakes TD (2002) Electro-Membrane Microcurrent Therapy Reduces Signs and Symptoms of Muscle Damage Med Sci Sports Exerc 34(4)602-607
19 Sumida K Greenberg M and Hill J (2003) Hot gel packs and reduction of delayed-onset muscle soreness 30 minutes after treatment J Sport Rehabil 12221-228
20 Tiidus PM and Shoemaker JK (1995) Effleurage massage muscle blood flow and long-term post-exercise strength recovery Int J Sports Med 16(7)478-483
21 Zhang J Clement D and Taunton J (2000) The efficacy of Farabloc an electromagnetic shield in attenuating delayed-onset muscle soreness Clin J Sport Med 10(1)15-21
22 Fitzgerald GK Rothstein JM Mayhew TP and Lamb RL (1991) Exercise-Induced Muscle Soreness After Concentric and Eccentric Isokinetic Contractions Phys Ther 71(7)505-513
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23 Nosaka K and Clarkson PM (1997) Influence of Previous Concentric Exercise on Eccentric Exercise-Induced Muscle Damage J Sports Sci 15(5)477-483
24 Jensen MP Karoly P and Braver S (1986) The Measurement of Clinical Pain Intensity - a Comparison of 6 Methods Pain 27(1)117-126
25 Price DD Mcgrath PA Rafii A and Buckingham B (1983) The Validation of Visual Analog Scales as Ratio Scale Measures for Chronic and Experimental Pain Pain 17(1)45-56
26 Revill SI Robinson JO Rosen M and Hogg MIJ (1976) Reliability of a Linear Analog for Evaluating Pain Anaesthesia 31(9)1191-1198
27 Nussbaum EL and Downes L (1998) Reliability of Clinical Pressure-Pain Algometric Measurements Obtained on Consecutive Days Phys Ther 78(2)160-169
28 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
29 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
30 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118
31 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in biomedical optics and imaging 3(28 Proceedings of SPIE 4903)156-165
32 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18(2)95-99
33 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2005) Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level J Photomed and Laser Surg 23(2)167-171
34 Pollo A Amanzio M Arslanian A Casadio C Maggi G and Benedetti F (2001) Response Expectancies in Placebo Analgesia and Their Clinical Relevance Pain 93(1)77-84
35 Shellock FG and Prentice WE (1985) Warming-up and Stretching for Improved Physical Performance and Prevention of Sports-Related Injuries Sports Med 2(4)267-278
36 Safran MR Seaber AV and Garrett WE (1989) Warm-up and Muscular Injury Prevention an Update Sports Med 8(4)239-249
37 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
38 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
GENERAL DISCUSSION
General discussion
113
SUMMARY
As outlined in the general introduction the overall objective of this doctoral thesis is to
develop the current knowledge about the mechanisms of LED action in view of the
eventual provision of evidence-based support for the clinical use of LED as a
biostimulatory and analgesic treatment modality especially in the field of
physiotherapy
Part I Wound healing
The investigations described in chapter 1 and 2 were conducted to gain insight into the
potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As
fibroblasts are principal cells for biostimulation (in view of growing and dividing in
healing wounds) the influence of LED irradiation on fibroblast proliferation was
assessed1
The first investigation consisted of a pilot study performed in order to evaluate the
appropriateness of the cell isolation technique cell culture protocol and proliferation
analysis as well as to appraise the feasibility of the light source properties and
illumination procedure
Data analysis revealed no statistically significant differences between the infrared LED
irradiated and control petri dishes for the used parameters (table 1) Considering this
outcome other experimental findings disclose that the absence of stimulatory effects of
LED irradiation on fibroblast proliferation can partly be attributed to the use of
inappropriate light source properties However the applied external dosimetric
parameters are well within the recommended spectrum described by previous studies
investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be
excluded that changes in the illumination procedure (such as the use of lower power
shorter exposure times wavelengths with finer coverage of the absorption spectrum of
the irradiated cells and a longer incubation period between the last irradiation and cell
counting) could still result in an increased fibroblast proliferation467 Of equal
importance in interpreting the lack of distinctive results are the imperfections of the
applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails
114
considerable intervention from the investigator compromising the reliability of the
method It is also a time-consuming technique with an insufficient sensitivity for some
purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11
To avoid contamination of the results by these modifiable remarks a similar
experiment (chapter 2) was performed in which wavelength power and output mode of
the infrared LED source were not modified (table 1) only the exposure time was
reduced resulting in a lower radiant exposure In addition the effect of two other
emission spectra was evaluated These probes emitting red and green light had a
shorter wavelength than the infrared LED source and the power was half or a
sixteenth of the power from the infrared probe Consequently the red LED irradiation
occurred with a different exposure time than the infrared one in order to attain the
same radiant exposure (053 Jcm2) With respect to the green LED it was not
endeavoured to achieve the same radiant exposure as 16 min of irradiation is not
feasible for in vitro or clinical application
Finally also an LLL light source was integrated Although it was not attempted to
analyse the effectiveness of LED in comparison to LLL enclosure of this modality was
interesting in order to join in with the available literature covering mostly LLL studies
To bypass the described problems regarding analysis of fibroblast proliferation
counting of the cells was carried out this time by means of a colorimetric MTT assay
This method provides more accurate cell counts in a short period of time and therefore
can be considered as a more reliable alternative to Buumlrker hemocytometer11
MTT assay 24 h after the last irradiation revealed a significantly increased number of
cells in the irradiated wells in comparison to their (respective sham-irradiated) controls
Although the study supplied experimental support for a significantly increased cell
proliferation by all external dosimetric properties based on the results of the
comparative trial with an incubation period of 24 hours irradiation with the green
LED source yielded the highest number of fibroblasts Thus it can be concluded that
the wavelength of the green LED is probably within the bandwidth of the absorption
spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that
General discussion
115
the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast
cultures in vitro612
Table 1 External dosimetric properties summarized for each chapter
Wavelength Power Exposure
time Output mode
Radiant exposure
PART I Chapter 1
In vitro part
LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2
LED-infrared 950 nm 160 mW 1 min continuous 053 Jcm2
LED-red 660 nm 80 mW 2 min continuous 053 Jcm2
LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2
Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2
PART II Chapter 4
LED-infrared 950 nm 160 mW 2 min continuous 107 Jcm2
Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
The next aim of the first part of this doctoral thesis was to explore whether LED
treatment could ameliorate in vitro cell proliferation under conditions of impaired
healing In the pursuit of this aim fibroblasts were cultured in medium supplemented
with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a
pilot study the amount of glucose necessary to inhibit normal growth was determined
In order to attain an important reduction of cell viability and decreased proliferation
rate a relatively high concentration of glucose (1667 mM) was necessary in
comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia
(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity
between both circumstances in vitro limited to 72 h whereas the human tissue of a
diabetic patient in vivo is chronically exposed to glucose
Treatment of the fibroblasts occurred in respect of the previously described results
with the same irradiation parameters and illumination procedure (chapter 2)
Accordingly green LED irradiation labelled as the most appropriate treatment for
116
irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in
the previous study (table 1)
Analysis of the cell proliferation by means of MTT measurements yielded a
significantly higher rate of proliferation in hyperglycaemic circumstances after
irradiation than in the control conditions (ie hyperglycaemic circumstances without
irradiation) Thus this outcome supported the stimulatory potential of green LED
irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a
considerable destructive hyperglycaemic medium
Finally although the results of the in vivo part of chapter 1 were persuasive and
encouraging they will not be further discussed in this summary of part I as it was not
aimed in this doctoral thesis to investigate the wound healing process in vivo However
the results of this case study can be a valuable hold for future in vivo research
The possible clinical implications of these results and future research directions in the
scope of wound healing will be discussed below
Part II Analgesia
In the second part two studies investigated the effects of LED irradiation as a
potential intervention mode in one of the most important fields in physiotherapy
practice analgesia Chapter 4 describes the influence of LED treatment on changing
sensory nerve conduction characteristics of a human superficial peripheral nerve
Altering nerve conduction characteristics may not be the sole beneficial purpose to
attain with LED irradiation in view of analgesia but the advantage of nerve conduction
characteristics is that they are objective measurable physical variables and changes in
these characteristics provide a potential explanatory mechanism of pain inhibition by
LED treatment17
The results showed that percutaneous LED irradiation at feasible clinical parameters
can generate a significant decrease in NCV and increase in NPL for all recordings post-
treatment in comparison to the baseline measurement The data in the placebo group
did not reveal any significant difference in the same course of time Statistical analysis
General discussion
117
revealed significant differences between the experimental and the placebo group for
NCV as well as for NPL at all time-points of observation with exception of the NPL
recording immediately after finishing irradiation
It was also observed that the noted effects did not weaken as time progressed It can
be concluded that post-treatment conduction measurements should be extended in
time which is in accordance with the findings of some previous studies18-21 Clarifying
the point of time at which the effect extinguishes is necessary and clinically relevant
when treating pain by means of LED irradiation Besides obtaining the desired
neurophysiological effects ideally the optimal irradiation parameters should be
applied The most favourable dosimetric properties are not yet determined but based
on this study and previously described assays it can be speculated that the dosimetric
window is quite large
Regardless of these clinically important remarks the present findings allow to draw the
following conclusion LED irradiation at clinically applied densities can generate an
immediate and localized effect upon conduction characteristics in underlying nerves as
LED treatment results in lowering the NCV and augmenting the NPL Therefore the
outcome of this in vivo experiment assumes a potential pain relief by means of LED
treatment and justifies further research regarding its clinical effectiveness in laboratory
settings and at a clinical level
The fourth and final aim was to determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5
illustrates a clinical study observing the effect of LED treatment on a model
comprising experimentally induced DOMS in a healthy population The progress of
pain perception and peak torque was evaluated during 4 consecutive days commencing
on the day of DOMS induction The effect of infrared LED treatment at the light
parameters described (table 1) was assessed with regard to three different factors time
(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures
(VAS MPT and IPT) revealed no significant interactive effects of the main interaction
118
(timegrouppre-post) For the remaining interactions and for the main effects only a
few significant findings were relevant in view of the postulated hypothesis
Notwithstanding the absence of an apparent and overall statistically significant finding
the present results indicate favourable trends of LED treatment on pain as the means
of all VAS and MPT variables show a statistically nonsignificant general analgesic
effect of infrared LED irradiation expressed by lower subjective pain rates and higher
MPT values in the irradiated group In addition to the analgesic influence of LED an
augmented restoration of muscle strength was noted The lack of solid statistically
significant evidence for these beneficial effects of LED treatment upon DOMS-
associated pain can possibly be attributed to the small sample size in this study or even
to the size of the treatment effect in relation to the severity of the induced DOMS as
induction of severe DOMS can mask relatively small but apparent treatment
effects2223 A final possibility is that the results only become significantly different after
a prolonged treatment and follow up period as previous research demonstrated that
recuperation subsequent to DOMS induction can last up to 10 days24
It should also be noted that the described general analgesic effect of LED irradiation
was identical for the treated as well as for the control arm in the irradiated group
proposing that infrared LED might induce systemic effects 2526 However it needs to
be stressed that these results were not statistically significant
Regardless of the absence of statistically significant findings the mean values suggest a
potential role for infrared LED irradiation in the management of pain and functional
impairment associated with DOMS Notwithstanding this postulation future research
is absolutely required to establish the effectiveness of LED treatment to reduce pain as
well at the applied densities as for other dosimetric parameters
CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS
In the course of the past years during the process of the genesis of this thesis
therapeutic physical agents in general and phototherapeutic modalities in particular
became less important as physiotherapeutic modes of treatment than during the
preceding two decades The diminished use of these treatment modalities in the
General discussion
119
physiotherapy practice is to a certain degree a consequence of the controversial
research findings regarding the use of these physical agents This issue of controversy
led to less support for the use of these treatment modalities and a growing scepticism
regarding the effectiveness of these physical agents within the scope of the growing
climate of evidence-based practice A second responsible protagonist for the loss of
popularity of physical agents is linked with the current tendency within physiotherapy
emphasising active remedial therapy The establishment of this development was based
on various experiments mainly performed during the last decade demonstrating that
active treatment modalities are for numerous impairments and disabilities preferable to
more passive forms of therapy In Belgium the prevailing nomenclature which came
into use on 1 May 2002 went along with this tendency In the appendix to the Royal
decree of 14 September 1984 towards settlement of the nomenclature of medicinal
treatments concerning compulsory insurance for medical care and allowances the
personal involvement of the physical therapist during the physiotherapeutic session
was emphasized and it was even defined that massage physical techniques within the
framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal
application can only be remunerated when they are applied supplementarily and not as a sole therapy
This implies that passive treatment modalities should not be used as sole method of
treatment and should always be considered as an adjunct to an active treatment
program This development needs to be applauded in many cases such as various
painful musculoskeletal problems functional instability rehabilitation of neurological
patients re-activation of the elderly population psychomotor rehabilitation
cardiovascular and respiratory convalescence Nevertheless it would be erroneous to
entirely reject physical agents including LED treatment Based on the findings of the
above described experiments it needs to be stressed that for some purposes especially
within the scope of impaired wound healing LED irradiation could be a suitable
therapeutic measure This statement is founded on the results of part I of the present
thesis they provided satisfactory fundamental evidence for the advantageous effects of
LED treatment on a crucial exponent of the wound healing process namely fibroblast
proliferation The beneficial findings are the result of basic in vitro research As it is
120
inaccurate to simply extrapolate these results to the clinical practice the clinical use of
LED irradiation for wound healing needs to be preceded by purposive and specific in
vivo investigations to substantiate these basic research findings27
The case study described in chapter 1 indicates a foundation for further in vivo research
Visual appraisal of the surgical incision revealed (from the 65th day in the course of the
reparative process onwards) that the irradiated area -which initially showed inferior
epithelialization and wound contraction- showed a more appropriate contracture than
the control area characterized by less discoloration at scar level and a less hypertrophic
scar These clear beneficial effects of LED treatment on a human cutaneous wound
can serve as preliminary impetus for further research into the clinical applicability of
LED therapy although this case study is insufficient in order to guarantee a safe
correct and effective use of LED as a therapeutic modality
Despite these remarks it tentatively can be concluded that based on a detailed analysis
of the available data of the present in vitro studies and the given case report in
combination with the small number of previously published human studies the
beneficial effects of LED irradiation at the cellular level are obvious and therefore a
potentially favourable outcome can be assumed in clinical practice28-30 LED-
modulated stimulation of wound healing can be gradually and vigilantly implemented
clinically Nevertheless the real benefits of LED irradiation within the scope of wound
healing can only be established by additional clinical trials as thus far clinical
application and stipulation of dosimetry still occurs on a trial-and-error basis which is
not conducive to a generally accepted clinical use of LED To lend more credibility to
the treatment of wounds by means of LED irradiation and to expel the existing
controversy and scepticism surrounding this topic in vivo investigations on wound
healing using a number of different animal models and adequately controlled human
studies are necessary In addition these studies should be performed preferably on a
population suffering from impaired healing as a consequence of diabetes mellitus or as
a result of any other debilitating reason because as posed by Reddy et al3132 and as
mentioned above light has possible optimal clinical effects in the treatment of healing-
resistant wounds
General discussion
121
Drawing general conclusions and formulating clinical implications for analgesia is
obviously less manifest first of all because only a limited number of possible
mechanisms of action in order to obtain analgesia were highlighted and secondly
because both studies did not come to a joint or complementary conclusion The
outcome of the first study revealed that LED treatment lowers the NCV and augments
the NPL resulting in a slower stimulus conduction and consequently a reduced number
of sensory pulses per unit of time Thus it could be assumed that LED induces pain
relief but the results of the study describing the effect of LED treatment on
experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy
In addition it needs to be emphasised that the first study (chapter 4) measured the effect
of LED irradiation on the large myelinated Aβ afferents A noteworthy question and
meanwhile a stimulus for future investigation is whether the measured effects can be
extrapolated from these sensory nerve fibres to the actual nociceptive afferents
notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of
these nociceptive pathways relies on laser-evoked potentials which selectively activate
Aδ-fibres and C-fibres3334 This technique was presently not available therefore a
standard sensory nerve conduction study was performed
Whereas stimulation of wound healing by means of LED irradiation can be cautiously
implemented in the clinical practice at this stage it is too early to promote LED
irradiation as a treatment modality for pain To make this possible it is essential to
conduct numerous studies with regards to the use of LED in the field of analgesia
Future research should focus on fundamental investigations in order to discover the
underlying mechanisms and physiological basis of pain modulation utilizing LED
treatment Furthermore the evaluation of the appropriateness of DOMS as an
experimental model of pain is an important prospect to consider as validation of this
model would enhance the ability to study various modalities for their potential effects
on pain Irrespective of the difficulties regarding standardisation of the research
population and evaluation of soreness inextricably linked with clinical pain studies the
122
ultimate objective of future research should be the establishment of the effectiveness
of LED irradiation to reduce pain of miscellaneous origin in a clinical setting
Regardless of the encouraging results of the described studies and besides the earlier
proposed specific directions for future research (directed towards wound healing or
pain relief) it is necessary in the interest of the patientrsquos well being and to the
advantage of the prospective clinical use of LED to highlight a few more issues for
future research Therefore one has to deal with some limitations of the performed
investigations A first limitation concerns the fact that only two mechanisms of LED
action were investigated (notably changed fibroblast proliferation and alteration of the
nerve conduction characteristics) So one can conclude that for further and better
understanding of the mechanisms of action it is necessary to perform more basic
research Answering the questions regarding the functioning of LED irradiation will
simplify the evaluation and reinforce the interpretation of the obtained results and
ultimately contribute to a more widespread and well definded acceptance of the use of
LED in clinical settings
A second general limitation of this doctoral thesis is the substantial difference in the
used external dosimetric parameters between the different chapters and even within
one and the same study (illustrated in table 1) this complicates the comparison
between the different trials In each trial the dosimetry was individually ascertained
based on previous studies within the given field As not for every application the same
dosimetry is suggested in literature a range of dosages were used Another important
factor in deciding on the dosimetry was the clinical applicability of the dosage as it is
useless to investigate the appropriateness of a treatment modality at a clinically
unrealistic dose As a result of this limitation the current findings do not fully
contribute to the explanation regarding the ideal parameters one should use although
this was not set as a principal purpose Based on this thesis and previously described
assays it can be speculated that the possible window for these parameters is quite large
the ideal irradiation parameters and proper timing or sequencing of LED irradiation
General discussion
123
for example to the various phases of wound healing and to different painful conditions
are therefore possibly unattainable
The establishment of an appropriate dosimetry should also consist of investigating the
absolute and relative penetration depth of LED irradiation into human tissue This is
less crucial within the scope of wound healing but it is of key importance while
treating deep-seated tissue (eg nerve fibres muscles circulatory components et
cetera)
Finally this thesis only investigated the efficiency of LED in a very limited number of
conditions notebly wound healing and pain Within the scope of physiotherapy and
medicine in general there are numerous other purposes for which LED irradiation is
promoted such as oedema arthritis miscellaneous orthodontic applications seasonal
affective disorder neonatal jaundice photodynamical therapy et cetera2835-41
In summary additional work on establishing proper dosimetry and identifying the
biochemical or photobiologic phenomena that are responsible for improving wound
healing and reducing pain or even other effects within a broader spectrum of
conditions remains to be done in order to answer unreciprocated questions Until that
time the potential clinical usefulness and actual value of LED irradiation for wound
healing and even to a larger extent for analgesia should always be approached with
appropriate professionalism and even caution
FINAL CONCLUSION
LED devices are promoted for clinical use but the currently available scientific
documentation regarding effectiveness of this physical agent is rather scarce Through
providing scientific support for the biostimulatory and analgesic effectiveness of LED
irradiation this doctoral thesis attempted to bridge in some degree this gap
The conducted studies revealed that LED irradiation undeniably has potential
beneficial effects on wound healing and to a lesser degree within the scope of
analgesia However based on the present results it can be corroborated that light
124
therapy in the guise of LED irradiation is not magic but these results can raise some
corrective doubts in fundamental disbelievers and antagonists
Nevertheless we have to join the queue of scientists who have found beneficial results
but cannot elucidate with certainty how this outcome was established Thus although
the present results are encouraging a continuing development and integration of new
knowledge based on further research is necessary in various domains of intervention
Therefore several directions for future investigations were proposed in order to cover
as many existing gaps and to answer the utmost number of remaining questions as
possible Still one ought to be aware not to carry future fundamental research at a
disproportional level and the inevitable quest for mechanisms of LED action should
not hypothecate the potential clinical value implying that at a certain point it should be
appropriate to make the transfer from science to the application of the available
knowledge in clinical practice
The described findings regarding LED irradiation are comparable to the results of
previously published studies performed with other light sources Consequently as
postulated by some LED providers it can be speculated that the biological response of
tissue to light irradiation can probably not be equated merely to a light source but
rather to a broad photo-energy window
General discussion
125
REFERENCES
1 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
2 Abergel R Lyons R Castel J Dwyer R and Uitto J (1987) Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2)127-133
3 Skinner S Gage J Wilce P and Shaw R (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-192
4 Webb C Dyson M and Lewis W (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5)294-301
5 Atabey A Karademir S Atabey N and Barutcu A (1995) The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 1899-102
6 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
7 Ben-Dov N Shefer G Irinitchev A Wernig A Oron U and Halevy O (1999) Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3)372-380
8 Rebulla P Porretti L Bertolini F et al (1993) White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2)128-133
9 Deneys V Mazzon A Robert A Duvillier H and De Bruyere M (1994) Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2)172-177
10 Mahmoud A Depoorter B Piens N and Comhaire F (1997) The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2)340-345
11 Haskard D and Revell P (1984) Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3)319-322
12 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers
13 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of cultured human endothelial cells Diabetes 36(11)1261-1267
14 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA damage in cultured human endothelial cells J Clin Invest 77(1)322-325
15 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7)621-627
16 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4)491-501
17 Kramer J and Sandrin M (1993) Effect of low-power laser and white light on sensory conduction rate of the superficial radial nerve Physiotherapy Canada 45(3)165-170
18 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
19 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
126
21 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
22 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
23 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
24 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
25 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
26 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer
G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
29 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
30 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255
32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287
33 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-28
34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856
35 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
36 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
37 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263
39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64
40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74
General discussion
127
41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press
NEDERLANDSTALIGE SAMENVATTING
Nederlandstalige samenvatting
131
NEDERLANDSTALIGE SAMENVATTING
Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de
tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te
vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie
van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen
Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest
enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is
ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als
op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang
van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve
interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken
Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van
ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen
moeilijk te verantwoorden
Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin
de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere
overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit
methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan
consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in
de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen
laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in
de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag
aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies
inclusief het gebruik van licht
Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat
de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde
doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en
dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht
komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt
geloosd
132
De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo
lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en
gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te
anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te
voorkomen lijkt een gerichte en rationele a priori aanpak conform de
wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen
Te meer daar grondige literatuurstudie leert dat men ten behoeve van de
werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie
zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser
De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de
extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is
geboden De introductie van alternatieve lichtbronnen in de huidige
kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk
weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus
eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood
naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based
gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder
binnen de domeinen van haar potentieel beloftevolle klinische toepassingen
wondheling en analgesie
Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de
wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op
prominente protagonisten van de wondheling de fibroblasten werd getracht het
fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten
zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de
proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd
respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand
waarbij de normale celgroei werd verstoord
In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het
effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische
Nederlandstalige samenvatting
133
data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden
verklaard door het gebruik van een inadequate LED dosering en anderzijds een
methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie
vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de
precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en
inter-tester variabiliteit tot gevolg
In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde
onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de
bestralingsparameters (dosering) De effecten van de drie verschillende LED
golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten
werden hierbij geanalyseerd door middel van een meer betrouwbare en minder
subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-
yl)-25-diphenyl tetrazolium bromide (MTT)
De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir
effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden
tevens een basis voor meer coherente en relevante inzichten aangaande de globale
bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)
Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het
wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend
karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp
rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde
vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De
fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met
extreem hoge concentraties glucose Deze modificatie van het medium staat model
voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische
praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan
vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de
gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en
proliferatie
134
Het tweede deel van dit proefschrift exploreert het domein van het potentieel
analgetisch effect van LED binnen de kinesitherapie aan de hand van twee
fundamentele onderzoeken
In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere
sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele
hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en
een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale
verklaring van een analgetisch effect van het medium zou kunnen belichamen
Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)
meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf
verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen
aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke
significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de
negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele
hypothese
Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend
fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale
pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp
van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische
krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED
behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier
keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED
op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd
(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde
isokinetische krachtmeting en een registratie van de waargenomen spierpijn De
spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met
behulp van een kwantitatieve hand-hold algometer
Analyse van de bekomen data bracht geen significante verschillen tussen de
controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden
Nederlandstalige samenvatting
135
kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED
behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van
de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de
proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene
afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine
proefgroep die werd onderzocht enof door de grootte van het behandeleffect in
verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers
een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up
kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel
absolute omzichtigheid geboden en moet deze visie louter als speculatief worden
beschouwd
Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de
kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de
laatste jaren aanzienlijk afgenomen
De positieve resultaten van de verschillende in vitro studies in het kader van wondheling
vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing
vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel
gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een
belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek
Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie
Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme
onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch
complementair resultaat Verder onderzoek ter exploratie van de mogelijke
onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom
onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op
termijn wetenschappelijk te rechtvaardigen
136
LED tovenarij trend of therapie
LED mag geen magische krachten worden toegemeten maar verdient het lot van een
kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een
wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen
zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke
indicaties
Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds
the darkness has always got there first and is waiting for it
(Terry Pratchett Reaper Man 1991)
XI
Thomas Stijn and Johan thank you for the repeated (mostly futile) attempts to
convince me to do something together Sebastiaan each time during the past few years
when I doubted about the sense of my work it was your ridiculous story about a man
who wanted to invent superglue but instead invented the well-known yellow post-it
which stimulated me to continue my scientific quest
Of course I owe most gratitude to Luc my most devoted supporter Dearest I know
that since august 2004 you lived a solitary life in Dubai Although I think it was
possibly easier not to live under the same roof with me these last stressful months I
am aware that it was very difficult for you not to be able to play with Louka and to
miss some precious months of her life
Louka thank you for your radiant smile and daily baby speeches I am sorry that you
had to miss your daddy I promise that we will be reunited very soon
Elke Vinck
Ghent March 2006
GENERAL INTRODUCTION
General introduction
3
BACKGROUND
The use of light for therapeutic purposes reaches far back in time Current interest for
photomedicine with his its biological and medical effects relies fundamentally on two
major evolutions in the given field (1) the research results regarding the use of
ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in
the light amplification by stimulated emission of radiation (laser)-technology The production
of the first laser the ruby pulsed laser was rapidly succeeded by the development of
the helium-neon laser and other lasers like the argon the neodymium-glass and the
neodymium-yttrium-aluminium-garnet lasers1
As in the mid-1990s semiconductor and diode-based lasers gained popularity the
principally massive gas and dye lasers were rendered obsolete Therapeutic light
technology further continued to evolve and todayrsquos therapeutic light source is as likely
to be a light emitting diode (LED) or polarized light as a semiconductor or diode
laser1
Technological advancement and variation of the light sources necessitate a
concomitant update and revision of research in the respective domains of application
Unfortunately this logical and rational necessity has rarely been fulfilled From a
historical perspective this lack of appropriate research has led to disenchanting
evolutions in the use of light especially in physiotherapy The experience exists in this
medical field that light sources were promoted and commercialised for a vast regimen
of indications without foregoing scientific backup Consequently research developed
often after the commercial introduction in physiotherapy As these investigations
frequently gave rise to conflicting results for certain indications scepticism arose and
the use of the given modality knew a waning popularity for all its indications The final
result of such an inappropriate frame of promotion commercialisation and research is
a growing clinical disuse of a given modality even for motivated indications In view of
the actual increasing interest in LED treatment and based on former ascertainment
one has to state that a literature review for the given source reveals that research
mostly covers only low level laser (LLL) studies23 Although recently a number of
papers can be noted that report on the effects of LEDs and polarized light still
4
numerous source-specific-questions need to be answered as research concerning
mechanisms of action and efficacy of the current light sources remains limited in view
of a substantiated clinical application4-17
The reason for the contemporary light-oriented interest in physiotherapeutic practice
for LED devices is in essence based on several advantages of LED in comparison with
LLL For example the use of LEDs is esteemed to be safer as the delivered power
does not damage tissue LEDs can be made to produce multiple wavelengths thereby
stimulating outright a broader range of tissue types and probes that cover a large
treatment area are available18 In addition from a commercial point of view LEDs are
far more interesting as they are a good deal cheaper than laser diodes and they have a
long life span as these solid devices stand robust handling
As a result of the above-mentioned lack of literature on LED some providers of these
devices have taken for granted that the biological response of tissue to light irradiation
cannot be equated merely to a light source They declare that a given response solely
depends on the extent of absorption of radiated light by the tissue19 Consequently
these providers state that it is acceptable to extrapolate scientific findings of LLL
studies for explaining the mechanisms of action and detailing the efficacy of LED and
other alternative light sources Thus actually without appropriate scientific support
equal biological effects are attributed to LED as to LLL Nevertheless prudence is
called for such an extrapolation firstly because it is irrespective of the mentioned
dissimilarities and by simple projection one ignores a number of physical differences
between LLL and LED (eg coherence and degree of collimation or divergence)
Secondly LLL therapy is still not yet an established and evidence-based clinical tool20
Notwithstanding the historical efforts there still remains a considerable amount of
ignorance scepticism and controversy concerning the use and clinical efficacy of
LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed
parameters for irradiation as well as to the difficult objective measurement of possible
irradiation effects and even to the exceptional range of unsubstantiated indications for
General introduction
5
which light therapy was promoted27-29 A lack of theoretical understanding can also be
responsible for the existing controversies as the evaluation and interpretation of
research results would be simplified largely when the appropriate knowledge about the
mechanisms of light action would be available
LLL literature can undoubtedly be used as basis for research on LED and as a
comparative reference for these given investigations However to guarantee evidence-
based use of LED within physiotherapy the need for specific research in view of an
accurate consumption of LED is definite especially for potential promising clinical
applications in physiotherapy according to LLL literature mainly wound healing and
analgesia3031
Hitherto the most substantial research concerning the use of LED for improvement
of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded
under the influence of prolonged exposure to microgravity (eg during long-term space
flights) and in case of absence of exposure to sunlight such as in submarine
atmospheres they performed wound healing experiments for military application in the
given circumstances3233 In vitro experiments revealed that LED treatment increased
proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA
synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and
osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies
demonstrated therapeutic benefits of LED in speeding the early phase of wound
closure and in changing gene expression in a type 2 diabetic mouse model183234
Human studies noted 50 faster healing of lacerations a return of sensation and
increased tissue granulation as a result of LED irradiation1833
Associates of the Rehabilitation Sciences Research Group of the Ulster University in
Northern Ireland extensively investigated the effectiveness of light in the treatment of
pain The emphasis was laid primarily on the analysis of the effects of various low level
laser light sources35-44 However in the year 2001 two studies gave an account on the
efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46
6
noticed relatively long-lasting neurophysiological effects a significant change of the
nerve conduction characteristics (decrease of the negative peak latency) was mediated
by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a
comparable multisource diode device was ineffective in the management of delayed-
onset of muscle soreness (DOMS)
Despite the major value of these described trials a definitive answer regarding the
ability of LED in influencing wound healing or pain is not forthcoming cardinally
because a number of aspects are not yet investigated Consequently more research is
required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the
formerly mentioned scepticism regarding the effectiveness of a medium and possibly
to the undeserved fall into disuse of the treatment modality which happened in a way
with LLL therapy
PHYSICAL CHARACTERISTICS
This chapter supplies a short but comprehensive review of opto-physics A brief
description of the physical characteristics of the LED source used is essential as the
physical properties of light play an important part in the ultimate efficacy of treatment
According to the International Electrotechnical Commission (IEC 60825-1) an LED
can be defined as
Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by
radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process
of spontaneous emission1947
The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO
preprototype MDB-Laser Belgium) This illustration shows that a probe consists of
32 single LEDs disseminated over a surface of 18 cm2
General introduction
7
Figure 1 LED device and three available probes (infrared red and green)
Three highly monochromatic probes were available each emitting light of a different
wavelength within the above-defined range (table 1)2748 The wavelength of the light
emitted and thus its colour depends on the band gap energy of the materials forming
the p-n junctiona This light property is a key determinant to obtain maximum
photochemical or biological responses as light absorption by tissue molecules is
wavelength specific27 Only by absorbing radiation of the appropriate wavelength
(namely the wavelengths equal to the energy states of the valence electrons)
photoacceptor molecules will be stimulated resulting in a direct photochemical
reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750
8
absorption spectrum of a particular molecule is limited absorption often only occurs
over a waveband range of about 40-60 nm274851 Nevertheless the absorption
spectrum at cell or tissue level is broad because cells are composed of many different
molecules
Besides its influence on the absorption by means of tissue molecules there is a crucial
link between wavelength and penetration depth of the irradiated light Penetration into
tissue decreases as the wavelength shortens hence green light penetrates less than red
light which at his turn penetrates less into tissue than infrared light2748 Detailed
principles of light penetration will be discussed below
The LED device used emits non-coherent light In the 1980s the observed biological
responses after laser irradiation were generally thought to be attributable to the
coherenceb of the light485253 Though currently the clinical and biological significance
of coherence is seriously questioned54 According to several authors coherence does
not play an essential role in laser-tissue interactions firstly as it was proven that both
coherent and non-coherent light clinically show equal efficacy75556 Secondly as
according to some authors almost immediately after transmission of light through the
skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et
al1957 state that both findings are incorrect coherence is not lost in tissue due to the
phenomenon of scattering and non-coherent light is not as efficient as coherent light
This lack of consensus makes it necessary to mention whether or not light is
coherent2758
Further decisive characteristics to accomplish phototherapeutic efficacy are the power
exposure time output mode and beam area Based on these parameters both
irradiancec and radiant exposured can be calculated According to numerous authors
some of these parameters are more crucial than others to determine whether
b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48
c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48
d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248
General introduction
9
absorption of light will lead to a photobiological event192728485455 However the
literature yields several controversial findings as not all authors attribute an equal
importance to a given parameter For example according to Nussbaum et al59
irradiance was the determinant characteristic in the biomodulation of Pseudomonas
aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49
found that in order to stimulate tissue cell proliferation a specific combination of
irradiance and exposure time are more important than the actual radiant exposure Low
et al3940 on the contrary highlighted the critical importance of the radiant exposure in
observing neurophysiological effects Whereas Mendez et al60 reported that both
parameters influence the final results of light therapy
Koutna et al61 even suggested that the output mode of light applications plays a more
prominent role in the treatment outcome than the wavelength of the used light source
Nevertheless this finding could not be confirmed by other research results Besides
more controversial findings have been published regarding the output mode although
the repetition rate in a pulsed mode was considered as an important treatment
parameter several investigations failed to prove its value19272840414461-64
Based on these findings it was opted within the investigations of this doctoral thesis to
irradiate in a continuous mode The remaining dosimetric parameters (wavelength
exposure time and power) depended on the purpose of each investigation they are
described in the respective chapters The data necessary for the calculation of the
radiant exposure for the equipment used in the respective trials are summarized in
table 1
Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High
Infrared 950 80 120 160 Red 660 15 46 80
Green 570 02 42 10
10
The radiant exposure of the used LED can be calculated as follows65
RE =
Radiant Exposure [Jcm2]
T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]
PRE = α S T
α = 006 (continuous mode) or
003 (pulsed mode)
The parameters commented on so far can be considered as the external dosimetry
involving all parameters directly controlled by the operator limited by the apparatus
used Furthermore there is the so-called internal dosimetry referring to (1) several
physical phenomena (reflection transmission scattering and absorption) influencing
the light distribution within the tissue during energy transfer (2) the optical
characteristics of the irradiated tissue as well as (3) the relation between the external
dosimetry and these respective elements5466
This internal dosimetry determines to a considerable extend the penetration of light
into tissue Penetration can be defined as the tissue depth at which the radiant
exposure is reduced to 37 of its original value1948 However this definition only
accounts for the absolute penetration depth resulting in direct effects of light at that
depth In addition there is also a relative penetration depth leading up to effects
deeper in the irradiated tissue and even in certain degree throughout the entire
body1967 These so-called systemic effects can be caused by chemical processes initiated
at superficial levels at their turn mediating effects at a deeper tissue level57
Involvement of several forms of communication in the tissue such as blood circulation
and transport of transmitters or signal substances is possible1967 This means that light
sources with poor absolute penetration do not necessarily give inferior results than
those with a good absolute penetration19
In the same context it should be noted that calculation and even measurement of the
exact light distribution during irradiation is highly complicated principally as tissues
have complex structures and also because the optical properties of tissues vary largely
inter-individual2768
General introduction
11
Studies regarding actual penetration depth of LED light are scarce consequently the
knowledge on the topic of penetration depth of LED light is based on literature
originating from LLL research19 These findings established with various LLL sources
revealed that there is an obvious relation between penetration depth and
wavelength27486769-71
Three final remarks can be made on the dosimetry First of all it should be noted that
partly as a result of the above-mentioned contrasting findings on dosimetry ideal light
source characteristics for effective treatment of various medical applications are not yet
established and probably never really will be28 Therefore in the attempt to offer
sufficient guidelines for correct use of treatment parameters one should always try to
provide detailed description of light source properties used in any trial so the
practitioner can interpret the scientific results adequately and accordingly draw the
correct conclusions for his clinical practice
A second comment is based on the mentioned possible influence of the external and
internal dosimetric parameters on the photobiological effectiveness of light the
intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation
between radiated wavelength and penetration depth19546572 So it should be
emphasized that caution is recommended when comparing research results of light
sources with different wavelengths or other dissimilar dosimetric parameters
A third and final remark considers the extrapolation issue Comparison of the
therapeutic usefulness of the same light source used on different species should occur
cautiously So simply extrapolating the dosage used for one species to another is
inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo
clinical practice is inappropriate So purposive and specific research is the prerequisite
to produce safe and correct use of light as a therapeutic modality27
12
MECHANISMS OF ACTION
In the past decennia several mechanisms of action for biostimulation and pain
inhibition have been proposed and investigated73 Research was primarily based on
studies at the molecular and cellular levels and as a second resort investigations
occurred at the organism level resulting in numerous possible explanatory
mechanisms272858
It is the common view that light triggers a cascade of cellular and molecular reactions
resulting in various biological responses Thus different mechanisms of whom the
causal relationships are very difficult to establish- underlie the effects of light3448557475
To illustrate this complex matter the various mechanisms of action will be summarised
by means of a comprehensive model (fig 2) Detailed discussion about the different
individual components of the proposed model and other effects than those regarding
wound healing or analgesia were not provided as this was beyond the scope of this
general introduction
As depicted in figure 2 exposure to light leads to photon absorption by a
photoacceptor molecule causing excitation of the electronic state or increased
vibrational state of the given molecule275173 This process is followed by primary
photochemical reactions7475 Several key mechanisms have been discussed in the
literature Respiratory chain activation is the central point and can occur by an
alteration in redox properties acceleration of electron transfer generation of reactive
oxygen species (namely singlet oxygen formation and superoxide generation) as well as
by induction of local transient heating of absorbing chromophores192848515576-83 It is
supposed that each of these respective mechanisms plays a part in obtaining a
measurable biological effect It is yet not clear if one mechanism is more prominent
and decisive than another nevertheless recent experimental evidence has revealed that
mechanisms based on changes in redox properties of terminal enzymes of respiratory
chains might be of crucial importance2848517679
The primary mechanisms occurring during light exposure are followed by the dark
reactions (secondary mechanisms) occurring when the effective radiation is switched
General introduction
13
off2851 Activation of respiratory chain components is followed by the initiation of a
complicated cellular signalling cascade or a photosignal transduction and amplification
chain associated with eg changes in the cellular homeostasis alterations in ATP or
cAMP levels modulation of DNA and RNA synthesis membrane permeability
alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87
The sequence of events finally results in a range of physiological effects essential for
the promotion of the wound healing process for supplying analgesia or other
advantageous responses (acceleration of inflammatory processes oedema re-
absorption increased lymph vessel regeneration or increased nerve
regeneration)12181927486188-93
Photostimulation of the wound healing process can be mediated by increased
fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis
and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations
have been especially focussed on fibroblasts but other possible physiological effects
attributing to an accelerated wound healing were also observed suppression and
alteration of undesirable immune processes increased leukocyte activity new
formation of capillaries increased production of growth factors and enzymes while
monocytes and macrophages can provide an enlarged release of a variety of substances
related to immunity and wound healing1619277376
As pain and nociception are even less understood than wound healing the possible
mechanisms in obtaining pain relief by the use of light are less underpinned However
it is established that light therapy influences the synthesis release and metabolism of
numerous transmitter signal substances involved in analgesia such as endorphin nitric
oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these
neuropharmacological effects there is experimental evidence for diminished
inflammation decreased C-fibre activity increased blood circulation and reduced
excitability of the nervous system1927848899
One should be aware that a large amount of research regarding the possible
mechanisms of light action was conducted at the cellular level The described cascade
of reactions at the organism level is possibly even more complex as in contradiction to
14
the in vitro situation in vivo a range of supplementary interactions can influence the
sequence of effects and accordingly the final responses Besides it needs to be
mentioned that this summary did not take into account the origin of the light or the
external dosimetry thus the description is based on investigations performed with
various light sources and different dosages
Figure 2 Model summarizing the identified mechanisms of light action
Secondarymechanisms
Primary mechanisms
Final effects
Trigger
Stimulated wound healing Analgesia
Exposure to light
Photon absorption by photoacceptors
Respiratory chain activation
Accelerated electrontransfer
Reactive oxygen generation
Heating of absorbing chromophores
Altered redox properties
darr inflammation uarr oedema resorption
uarr lymph vessel regenerationuarr blood circulation
Photosignal transduction and amplification chain
uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation
darr C-fibre activity darr nervous excitability neuropharmacological effects
General introduction
15
Regardless of the large number of previous investigations identification of underlying
mechanisms of light action remains an important issue as these are not yet fully
understood and because probably not all mechanisms of action are currently
identified Convincing explanation of the mechanisms in normal as well as in
pathological tissue could banish the existing suspicion concerning the use of light as a
treatment modality2732547678
AIMS AND OUTLINE
The introduction of LED in medicine and in physiotherapy more specifically requires
particular scientific research especially within the fields of its clinical potential
application wound healing and analgesia The above described gaps in literature
regarding the use of LED laid the foundation of this doctoral thesis
Consequently the general purpose of this thesis is to explore a scientific approach for
the supposed biostimulatory and analgesic effect of LED and to formulate an answer
in view of an evidence-based clinical use of this treatment modality
The detailed objectives can be phrased as follows
Aim 1 To assess the biostimulatory effectiveness of LED
irradiation under normal in vitro conditions
Aim 2 To investigate the value of LED treatment to ameliorate
in vitro cell proliferation under conditions of impaired healing
Aim 3 To examine the effectiveness of LED in changing the
nerve conduction characteristics in view of analgesia
Aim 4 To determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting
Part I investigates the influence of LED on wound healing In pursuit of the first aim
chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial
16
measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these
cells needs to be considered as an exponent of the wound healing process as
fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase
and early remodelling100 Secondly an in vivo case study exploring the postulation that
LED irradiation could accelerate and ameliorate the healing of a surgical incision was
described
The results contrasted sharply with the findings of the in vitro part Two fundamental
causes were proposed in order to explain the different biological effect of LED
irradiation observed in vitro and in vivo the used irradiation parameters and evaluation
method
The experiment described in chapter 2 endeavoured to explore these considerations A
similar study was therefore performed but as distinctive characteristics different light
source properties an adapted irradiation procedure and the use of a colorimetric assay
based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the
counting of the cells were used
As stimulation of the wound healing process is virtually mainly indicated under
conditions of impaired healing (resulting in a situation which threatens to become
chronic and debilitating) proper attention for this matter is warranted192855 Besides
the medical consequences the costs involved with impaired healing yield also a socially
relevant problem to tackle Impaired healing will become even more common as the
world population continues to age After all senescence of systems and age-committed
comorbid conditions are commonly the culprits responsible for poor wound healing101
Thus finding cost-effective time-sparing non-invasive and practical treatment
modalities to cure wounds is a necessity
Aiming to assess the biostimulative effects by means of LED in these circumstances a
third study was conducted with respect of the previous results regarding irradiation
parameters and cell proliferation analysis The irradiation experiment described in
chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose
This medium modification serves as a pattern for cell proliferation in diabetic patients
General introduction
17
a population for whom stimulation of the wound healing process is a clinical relevant
feature
In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored
by means of two studies A first investigation (chapter 4) evaluated the influence of LED
on the sensory nerve conduction characteristics of a human superficial peripheral
nerve as a potential explanatory mechanism of pain inhibition by LED which is based
on the putative neurophysiological effects of this treatment modality The experimental
hypothesis postulated that LED generates an immediate decrease in conduction
velocity and increase in negative peak latency In addition it was postulated that this
effect is most prominent immediately after the irradiation and will weaken as time
progresses
The values of nerve conduction velocity and negative peak latency of a baseline
antidromic nerve conduction measurement were compared with the results of five
identical recordings performed at several points of time after LED irradiation
Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory
setting To guarantee an adequate standardized and controlled pain reduction study
there was opted to observe a healthy population with experimentally induced DOMS
Induction of DOMS has been described in a number of studies as a representative
model of musculoskeletal pain and stiffness because it can be induced in a relatively
easy and standardised manner the time course is quite predictable and the symptoms
have the same aetiology and are of transitory nature4445102-105
The treatment as well as the assessment procedure was performed during 4
consecutive days The first day isokinetic exercise was performed to induce pain
related to DOMS Subsequently the volunteers of the experimental group received an
infrared LED treatment and those of the placebo group received sham-irradiation
Evaluation of the effect of the treatment on perceived pain was registered by a visual
analog scale and by a mechanical pain threshold these observations occurred every day
18
prior to and following LED irradiation Eccentricconcentric isokinetic peak torque
assessment took place daily before each treatment
For the analysis of the results three different factors were taken into consideration
time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental)
In completion of this thesis the most prominent findings are summarized and the
clinical implications are discussed The general discussion also includes some future
research directions and a final conclusion
General introduction
19
REFERENCES
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radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-92 3 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy
in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austriaca 33(3)132-137
4 Stahl F Ashworth S Jandt K and Mills R (2000) Light-emitting diode (LED) polymerisation of dental composites flexural properties and polymerisation potential Biomaterials 21(13)1379-1385
5 Mills R Jandt K and Ashworth S (1999) Dental composite depth of cure with halogen and blue light emitting diode technology Br Dent J 186(8)388-391
6 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
7 Pontinen P Aaltokallio T and Kolari P (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118
8 Schmidt M Reichert K Ozker K Meyer G Donohoe D Bajic D Whelan N and Whelan H (1999) Preclinical evaluation of benzoporphyrin derivative combined with a light-emitting diode array for photodynamic therapy of brain tumors Pediatr Neurosurg 30(5)225-231
9 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
10 Sommer A Pinheiro A Mester A Franke RP and Whelan H (2001) Biostimulatory windows in low-intensity laser activation lasers scanners and NASAs light-emitting diode array systems J Clin Laser Med Sur 19(1)29-33
11 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
12 Whelan H Smits R Buchman E Whelan N Turner S Margolis D Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Philippi A Graf W Hodgson B Gould L Kane M Chen G and Caviness J (2001) Effect of NASA light-emitting diode irradiation on wound healing J Clin Laser Med Sur 19(6)305-314
13 Ojeda A Redondo E Gonzalez Diaz G and Martil I (1997) Analysis of light-emission processes in light-emitting diodes and semiconductor lasers Eur J Phys 18(2)63-67
14 Monstrey S Hoeksema H Saelens H Depuydt K Hamdi M Van Landuyt K and Blondeel P (2002) Conservative approach for deep dermal burn wounds using polarised-light therapy Br J Plast Surg 55(5)420-426
15 Monstrey S Hoeksema H Depuydt K Van Maele G Van Landuyt K and Blondeel P (2002) The effect of polarized light on wound healing Eur J Plast Surg 24377-382
16 Bolton P Dyson M and Young S (1992) The effect of polarized light on the release of growth factors from the U-937 macrophage-like cell line Laser Ther 233-42
17 Stasinopoulos D (2005) The use of polarized polychromatic non-coherent light as therapy for acute tennis elbowlateral epicondylalgia A pilot study Photomed Laser Surg 23(1)66-69
18 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
19 Tuner J Hode L (2004) The laser therapy handbook Tallinn Prima Books AB 20 Allendorf J Bessler M Huang J Kayton M Laird D Nowygrod R and Treat M (1997) Helium-
neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3)340-345
21 Basford J (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8)671-675
20
22 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
23 Lagan K Clements B McDonough S and Baxter G (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1)27-32
24 Schlager A Kronberger P Petschke F and Ulmer H (2000) Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1)39-42
25 Nemeth A J (1993) Lasers and wound healing Dermatol Clin 11(4)783-789 26 Lowe A Walker M OByrne M Baxter G and Hirst D (1998) Effect of low intensity
monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5)291-298
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science
Publishers 29 Basford J (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg
Med 16(4)331-342 30 Baxter G Bell A Allen J and Ravey J (1991) Low level laser therapy Current clinical practice in
Northern Ireland Physiotherapy 77(3)171-178 31 Cambier D and Vanderstraeten G (1997) Low-level laser therapy The experience in flanders
Eur J Phys Med Rehabil 7(4)102-105 32 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D
Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
33 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
34 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
35 Basford J Hallman H Matsumoto J Moyer S Buss J and Baxter G (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6)597-604
36 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol (Lond) 43563
37 Baxter G Allen J Walsh D Bell A and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol (Lond) 446445
38 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
39 Lowe A Baxter G Walsh D and Allen J (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
40 Lowe A Baxter G Walsh D and Allen J M (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Lasers Med Sci 10(4)253-259
41 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
General introduction
21
42 Baxter G Effect of combined phototherapylow intensity laser therapy upon experimental ischaemic pain Potential relevance of experimental design 14th World Congress Physical Therapy Barcelona Spain 2004 Proceedings CD
43 Craig J Barron J Walsh D and Baxter G (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
44 Craig J Barlas P Baxter G Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
45 Glasgow P Hill I McKevitt A Lowe A and Baxter G (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1)33-39
46 Noble J Lowe A Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
47 IEC 60825-1 (1993) International Electrotechnical Commission Safety of laser products Part 1 Equipment classification requirements and users guide
48 Nussbaum E Baxter G and Lilge L (2003) A review of laser technology and light-tissue interactions as a background to therapeutic applications of low intensity lasers and other light sources Phys Ther Rev 831-44
49 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
50 Nakamura S (1998) The roles of structural imperfections in InGaN-based blue light-emitting diodes and laser diodes Science 281956-961
51 Karu Tiina (consulted on 2005 august 22) Cellular mechanism of low-power laser therapy httpwwwtinnitusustiinakarupresentationhtml
52 Mester E Mester A and Mester A (1985) The biomedical effects of laser application Lasers Surg Med 5(1)31-39
53 Moore K Calderhead G (1991) The clinical application of low incident power density 830 nm GaAlAs diode laser radiation in the therapy of chronic intractable pain A historical and optoelectronic rationale and clinical review Int J Optoelectron 6503-520
54 King P (1989) Low level laser therapy A review Lasers Med Sci 4141-148 55 Karu T (1989) Photobiology of low-power laser effects Health Phys 56(5)691-704 56 Simunovic Z (2000) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical
Application of Low Energy-Laser Laser Therapy LLLT Rijeka Vitgraf 57 Hode L and Tuner J (2000) Low level laser therapy (LLLT) contra light emitting diode therapy
(LEDT) - What is the difference Proceedings of SPIE 416690-97 58 Kitchen S and Partridge C (1991) A review of low level laser therapy Part I Background
physiological effects and hazards Physiotherapy 77(3)161-163 59 Nussbaum E Lilge L and Mazzulli T (2003) Effects of low-level laser therapy (LLLT) of 810 nm
upon in vitro growth of bacteria Relevance of irradiance and radiant exposure J Clin Laser Med Sur 21(5)283-290
60 Mendez T Pinheiro A Pacheco M Nascimento P and Ramalho L (2004) Dose and wavelength of laser light have influence on the repair of cutaneous wounds J Clin Laser Med Sur 22(1)19-25
61 Koutna M Janisch R and Veselska R (2003) Effects of low-power laser irradiation on cell proliferation Scripta Medica 76(3)163-172
62 Al-Watban F and Zhang X (2004) The comparison of effects between pulsed and CW lasers on wound healing J Clin Laser Med Sur 22(1)15-18
63 Panjehpour M Overholt B DeNovo R Petersen M and Sneed R (1993) Comparative study between pulsed and continuous wave lasers for photofrin photodynamic therapy Lasers Surg Med 13(3)296-304
64 Dyson M (2005) The significance of pulsing in the stimulation of tissue repair by light A review Lasers Med Sci 20S21
22
65 MDB Laser (2000) Bio Dio (Preprotoype) User guide Belgium Ekeren 66 Fisher J (1992) Photons physiatrics and physicians A practical guide to understanding laser light
interaction with living tissue Part I J Clin Laser Med Sur 10(6)419-426 67 Tuner J and Hode L (2000) Depth of penetration of laser light in tissue Laser Partner
Clinixperience 151-3 68 Anderson R and Parrish J (1981) The optics of human skin J Invest Dermatol 77(1)13-19 69 De Cuyper H and Lambert H (1991) Penetration depth of infrared- and helium-neon-laserlight
An assessment in vivo Eur J Phys Med Rehabil 1(5)133-137 70 Oshiro T Ogata H Yoshida M Tanaka Y Sasaki K and Yoshimi K (1996) Penetration depths
of 830nm diode laser irradiation in the head and neck assessed using a radiographic phanom model and wavelength-specific imaging film Laser Ther 8197-204
71 Kolarova H Ditrichova D and Wagner J (1999) Penetration of the laser light into the skin in vitro Lasers Surg Med 24(3)231-235
72 Chen Q Huang Z Chen H Shapiro H Beckers J and Hetzel F (2002) Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy Photochem Photobiol 76(2)197ndash203
73 Conlan M Rapley J and Cobb C (1996) Biostimulation of wound healing by low-energy laser irradiation A review J Clin Periodontol 23(5)492-496
74 Parrish J (1981) New concepts in therapeutic photomedicine photochemistry optical targeting and the therapeutic window J Invest Dermatol 7745-50
75 Smith K (1981) Photobiology and photomedicine the future is bright J Invest Dermatol 772-7 76 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees D (1989) Systemic effects
of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
77 Polo L Presti F Schindl A Schindl L Jori G and Bertoloni G (1999) Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth Biochem Biophys Res Commun 257(3)753-758
78 Bertoloni G Sacchetto R Baro E Ceccherelli F and Jori G (1993) Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 6328 nm light J Photochem Photobiol B-Biol 18191-196
79 Lubart R Friedmann H Sinykov M and Grossman N (1995) Biostimulation of photosensitized fibroblasts by low incident levels of visible light energy Laser Ther 7101-106
80 Harris D (1991) Biomolecular mechanisms of laser biostimulation J Clin Laser Med Sur 9277-280
81 Grossman N Schneid N Reuveni H Halevy S and Lubart R (1998) 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures involvement of reactive oxygen species Lasers Surg Med 22212-218
82 Oren D Charney D Lavi R Sinyakov M and Lubart R (2001) Stimulation of reactive oxygen species production by an antidepressant visible light source Biol Psychiatry 49464-467
83 Lavi R Shainberg A Friedmann H Shneyvays V Rickover O Eichler M Kaplan D and Lubart R (2003) Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells 278(42)40917-40922
84 Reddy K (2004) Photobiological basis and clinical role of low-intensity lasers in biology and medicine J Clin Laser Med Sur 22(2)141-150
85 Greco M Guida G Perlino E Marra E and Quagliariello E (1989) Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser Biochem Biophys Res Commun 163(3)1428-1434
86 Vacca R Marra E Quagliariello E and Greco M (1993) Activation of mitochondrial DNA replication by He-Ne laser irradiation Biochem Biophys Res Commun 195(2)704-709
87 Vacca R Marra E Quagliariello E and Greco M (1994) Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser Biochem Biophys Res Commun 203(2)991-997
88 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral
General introduction
23
nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
89 Anders J Borke R Woolery S and Van de Merwe W (1993) Low power laser irradiation alters the rate of regeneration of the rat facial nerve Lasers Surg Med 13(1)72-82
90 Rochkind S Nissan M Barr-Nea L Razon N Schwartz M and Bartal A (1987) Response of peripheral nerve to He-Ne laser Experimental studies Lasers Surg Med 7441-443
91 Lievens P (1986) Lasertherapie (deel II) De invloed van laser-bestralingen op het lymfesysteem en de wondheling Ned T Fysiotherapie 96140-142
92 Lievens P (1991) The effect of a combined HeNe and IR laser treatment on the regeneration of the lymphatic sysytem during the process of wound healing Lasers Med Sci 6193-199
93 Lievens P (1991) The effect of IR laser irradiation on the vasomotricity of the lymphatic system Lasers Med Sci 6189-191
94 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin J and Martin P (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1)171-178
95 Bosatra M Jucci A Olliaro P Quacci D and Sacchi S (1984) In vitro fibroblast and dermis fibroblast activation by laser irradiation at low energy An electron microscopic study Dermatologica 168(4)157-162
96 Enwemeka C Rodriquez O Gall N and Walsh N (1990) Morphometrics of collagen fibril populations in HeNe laser photostimulated tendons J Clin Laser Med Sur 847-52
97 Abergel P Lam T Meeker C Castel C Dwyer R and Uitto J (1984) Biostimulation of procollagen production by low energy lasers in human skin fibroblast culturesJ Invest Dermatol 82(4)395
98 Abergel P Lam T Meeker C Dwyer R and Uitto J (1984) Low energy lasers stimulate collagen production in human skin fibroblast cultures Clin Res 32(1)16A
99 Shimoyama N Lijima K Shimoyama M and Mizuguchi T (1992) The effects of helium-neon laser on formalin-induced activity of dorsal horn neurons in the rat J Clin Laser Med Sur 1091-94
100 Kloth L McCulloch J and Feedar J (1990) Wound healing Alternatives in management USA FA Davis Company
101 Lanzafame R (2004) Revolutions and revelations J Clin Laser Med Surg 22(1)1-2 102 Ciccone C Leggin B and Callamaro J (1991) Effects of ultrasound and trolamine salicylate
phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-678 103 Craig J Cunningham M Walsh D Baxter G and Allen J (1996) Lack of effect of transcutaneous
electrical nerve stimulation upon experimentally induced delayed onset muscle soreness in humans Pain 67(2-3)285-289
104 Minder P Noble J Alves-Guerreiro J Hill I Lowe A Walsh D and Baxter G (2002) Interferential therapy Lack of effect upon experimentally induced delayed onset muscle soreness Clin Physiol Funct Imaging 22(5)339-347
105 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
PART I WOUND HEALING
CHAPTER 1
DO INFRARED LIGHT EMITTING DIODES HAVE A
STIMULATORY EFFECT ON WOUND HEALING FROM AN IN
VITRO TRIAL TO A PATIENT TREATMENT
Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Proceedings of SPIE 2002 4903 156-165
Chapter 1
28
ABSTRACT
Variable effects of different forms of light therapy on wound healing have been
reported This preliminary study covers the efficacy of infrared light emitting diodes
(LED) in this domain
Cultured embryonic chicken fibroblasts were treated in a controlled randomised
manner LED irradiation was performed three consecutive days with a wavelength of
950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each
treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2
The results indicated that LED treatment does not influence fibroblast proliferation at
the applied energy density and irradiation frequency (p=0474)
Meanwhile the effects of LED on wound healing in vivo were studied by treating a
surgical incision (6 cm) on the lateral side of the right foot of a male patient The
treatment started after 13 days when initial stitches were removed The same
parameters as the in vitro study were used but the treatment was performed five times
The healing could only be evaluated clinically the irradiated area (26 cm) showed a
more appropriate contraction less discoloration and a less hypertrophic scar than the
control area (34 cm)
The used parameters failed to demonstrate any biological effect of LED irradiation in
vitro although the case study on the other hand illustrated a beneficial effect
Keywords Light Emitting Diodes Fibroblasts Wound healing
From an in vitro trial to a patient treatment
29
INTRODUCTION
Various beneficial effects of lasers and photodiodes at relatively low intensities have
been reported involving treatment of neurological impairments12 treatment of pain3-5
treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the
enhancement of wound healing has been a focus of contemporary research11-16 It
seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing
remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the
basis of a questionnaire about low power laser (LPL) in the current clinical practice in
Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey
into clinical LPL experience in Flanders
Nevertheless there remains a considerable amount of ignorance scepticism and
controversial issues concerning the use and clinical efficacy of LPL even in the domain
of wound healing12152021 This is at least in part a consequence of the inability to
measure and control operating variables related to connective tissue repair and of the
wide range of suitable parameters for irradiation
Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent
characteristics which make their use in a clinical setting problematic including
limitations in wavelength capabilities and beam width The combined wavelength of
light optimal for wound healing cannot be efficiently produced and the size of
wounds which may be treated by LPLs is limited Some companies offer an
alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be
made to produce multiple wavelengths and can have probes with large surface area
allowing treatment of large wounds Still one can not accept this light source as an
alternative for LPL therapy based on the cited advantages without proper investigation
regarding its biostimulatory effects
The effectiveness of this possible alternative for LPLs must be studied in vitro and in
addition in animal models or in humans because the effects of LED at the cellular level
do not necessarily translate to a noticeable effect in vivo The small amount of previous
investigations demonstrate that LED effects are as difficult to isolate162223 as LPL
Chapter 1
30
effects and the results are conflicting just like the results in literature specific on the
use of LPL121520
The purpose of the first part of this study is to examine the hypothesis stating that
LED irradiation can influence fibroblast proliferation Therefore a comparison of the
proliferation from fibroblasts in irradiated and control wells was performed The in vitro
investigation was linked with an in vivo case study This part enquired the assumption if
LED irradiation could accelerate and ameliorate the healing of a surgical incision
IN VITRO INVESTIGATION
MATERIALS AND METHODS
The complete procedure from isolation to proliferation analysis was executed twice
(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of
control dishes The second trial consisted of 27 irradiated and 27 control dishes
Cell isolation and culture procedures
Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation
and disaggregating of the cells occurred with warm trypsin (NV Life Technologies
Belgium) according the protocol described by Ian Freshney (1994)24 The primary
explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies
Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1
Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life
Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies
Belgium) When cell growth from the explants reached confluence cells were detached
with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV
Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the
cells were removed from the culture flasks by trypsinization and counted by
hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc
From an in vitro trial to a patient treatment
31
NV Life Technologies Belgium) The fibroblasts were seeded at a density of
70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture
medium the cells were allowed to attach for 24 hours in a humidified incubator at
37degC
Properties of the Light Emitting Diode
Prior to LED treatment all dishes were microscopically checked to guarantee that the
cells are adherent and to assure that there is no confluence nor contamination The
dishes were divided randomly into the treated or the control group Medium was then
removed by tipping the dishes and aspirating with a sterile pipette Following the
aspiration 2 ml fresh medium was added and treatment started
A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser
Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-
1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32
single LEDrsquos For the treatments in this study an average power of 160 mW at
continuous mode was applied The irradiation lasted 6 minutes resulting in an energy
density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result
of the divergence in function of this distance the surface of the LED (18 cm2) covered
the complete surface of the used petriplates (215 cm2)
After these manipulations 3 ml medium was added to each dish followed by 24 hours
incubation
One LED irradiation was performed daily during three consecutive days according
this procedure Control cultures underwent the same handling during these three days
but were sham-irradiated
Proliferation analysis
After the last treatment a trypsination was performed to detach the cells from the
culture dishes followed by centrifugation Once the cells were isolated from the used
trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts
Chapter 1
32
within this suspension as reflection for the proliferation was quantified by means of a
Buumlrker Chamber or hemocytometry
The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-
Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab
Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on
the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator
using an inverted light microscope counted the number of cells in 25 small squares
In order to calculate the number of cells one should multiply the amount of cells
counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and
the dilution factor (the amount of Trypan blue suspended with the cells 21=3)
Statistical methods
The data were analysed statistical in order to examine the hypothesis that LED
irradiation enhances fibroblast proliferation They were processed as absolute figures
for both trials separately In a second phase the counted cell numbers were converted
in relative figures so the data of both trials could be analysed as the data of one test
These relative figures were obtained by expressing each figure as a percentage from the
highest figure (=100) of that trial and this for each assay separately
A Kolmogorov-Smirnoff test of normality was performed on the data followed by a
Mann-Whitney-U test when the test of normality was significant and otherwise a T-
test Differences were accepted as significant when plt005 For this analysis SPSSreg
100 was used
RESULTS
The descriptive data for both trials are depicted in figure I The mean number of cells
in trial A is higher than in trial B for the controls as for the treated wells There is a
mean difference of 1252500 fibroblasts between the controls and 1223000 between
the irradiated wells of trial A and B The averages of both trials show that in control
cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no
From an in vitro trial to a patient treatment
33
statistically significant difference could be found between the two groups in either trial
nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not
significant for trial A (p=020) nor trial B (p=020) Only the combined data from both
trials were significant (plt001) for normality Further analysis respectively T-test for
the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the
combined data (p=0474) revealed no statistical significant differences
DESCRIPTIVE DATA
1730000181750029530003070000
00E+00
50E+05
10E+06
15E+06
20E+06
25E+06
30E+06
35E+06
40E+06
Trial A Trial A Trial B Trial B
Mea
n n
um
ber
of
cells
Control
Irradiated
Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B
DISCUSSION
Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities
(lt500 mW) in vitro have been analysed by evaluating various factors involving
(pro)collagen production25-27 cell viability2829 growth factor production28 and
myofibroblast formation30 Fibroblast proliferation also is an important factor to
consider In accordance with wound healing fibroblasts fulfil an essential role especially
in the late inflammatory phase and the early granulation phase31 Despite the failure of
some studies to demonstrate beneficial effects of LPL irradiation on fibroblast
proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very
Chapter 1
34
significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -
208 ) Atabey et al35 also revealed a significant increase in cell number two or more
irradiations resulted in an increased fibroblast proliferation Several other studies
confirmed these positive findings25263637
The results of this present in vitro study indicate that LED treatment does not
influence fibroblast proliferation Although the dosimetric parameters (in particular the
arbitrary energy density of 32 Jcm2) used in this study are well within the
recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous
studies about LPL therapy raising enhanced fibroblast proliferation252634-37
Van Breugel et al36 gave a possible explanation for these controversial results
According to them the fibroblast proliferation is not inherent at the energy density
They provide evidence that independent of the energy density the power density and
the exposure time determine the biostimulative effects of LPL irradiation LPL with a
power below 291 mW could enhance cell proliferation while a higher power had no
effect
Some authors also argued that the absorption spectrum of human fibroblasts show
several absorption peaks and pointed out that a wavelength of 950 nm is far above the
highest peak of about 730 nm3638 At longer wavelengths they determined a general
decrease in absorption Despite these results several investigators pose biostimulative
effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or
even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is
ranged from 800 nm to 830 nm principally because of the presence of cytochrome
oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts
at lower wavelengths is restricted by an inferior skin transmission than at higher
wavelengths38
Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting
respective green and red light The 950 nm beam of light was used for its high power
density but according to a range of remarks mentioned above the effects of the two
other probes must be as well evaluated
From an in vitro trial to a patient treatment
35
Another factor one can not ignore is that besides fibroblast proliferation other
processes or morphologic changes were not analysed although several authors have
posed that those changes and processes could be responsible for the biostimulative
effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et
al30 for example described a massive transformation of fibroblasts into myofibroblasts
after LPL treatment These modified fibroblasts play an important role in contraction
of granulation tissue30 A second example is an increased (pro)collagen production
after low power light therapy25-27 which is also considered as a responsible factor for
accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast
proliferation3640
It may be wondered if the light sources mostly LPL in the consulted literature are
representative for the LED used in this study although this LPL literature is often
used for that purpose As in the early days of LPL the stimulative effects upon
biological objects were explained by its coherence the beam emitted by the Bio-Dio on
the contrary produces incoherent light Nowadays contradictory research results are
responsible for a new discussion the clinical and biological significance of coherence
The findings of some authors172341-43 pose that the coherence of light is of no
importance of LPL and its effects although the opposite has also been stated4445
Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be
accounted for the non-enhanced fibroblast proliferation in this trial
Another possible explanation for the absence of biostimulative effect is related to the
moment of analysis of the proliferation The evaluation one day after the last
irradiation did not allow a delayed enhancement of proliferation while it is determined
in numerous investigations that the effects occur more than 24 hours after the last
treatment273746 and that they weaken after a further undefined period of time34
The fluctuation in cell numbers between both trials despite the use of an identical
protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor
reproducibility of their technique In this study the fluctuations are attributable to the
counting of the cells by Buumlrker hemocytometer before seeding According to some
authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large
Chapter 1
36
variability48 and it is often difficult to standardize48 Overestimation of the cell
concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity
was contradicted by Lin et al50 moreover satisfactory correlations with flow-
cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay
for cell counting (MTT)51 were determined
An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it
provides accurate cell counts in a short period of time with less intervention from the
investigator52
These remarks and controversies point out the possible deficiencies from the used
proliferation analyses and the relativity from the obtained results Other analyse
methods and analyses from different cell processes and morphologic changes could be
considered for further investigation
IN VIVO INVESTIGATION
MATERIALS AND METHODS
The effects of LED on wound healing in vivo were studied by treating a postsurgical
incision A male patient received chirurgical treatment for the removal of a cyst
situated approximately 15 cm posterior from the lateral malleolus of his right foot For
removal of the cyst an incision of 6 cm was made The incision was sutured and 12
days after the surgery the stitches were removed Visual inspection demonstrated that
the healing process of the wound proceeded well but not equally over the whole 6
centimetres (figure II) Epithelialization and wound contraction appeared to have
progressed better in the upper part (approximately 3 cm) of the cicatrice than at the
lower part (covered with eschar) No evidence of infection was noted in either part
LED treatment started the 13th day The incision was treated partially the lowest part
(26 cm) with the inferior epithelialization and wound contraction was irradiated the
remaining 34 cm served as control area This control area was screened from radiation
with cardboard and opaque black cling film
From an in vitro trial to a patient treatment
37
The light source destinated for the treatment was the same device used for the in vitro
irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED
output parameters were identical with those applicated in the preceding in vitro
investigation In particular a continuous wave at an average power of 160 mW and 6
minutes of treatment duration corresponding to an energy density of 32 Jcm2 An
equal distance from the probe to the target tissue as from the probe to the culture
medium was respected A plastic applicant of according height guaranteed constant
distance of 06 cm from the surface of the skin
Figure II Surgical incision before the first treatment 13 days after initial stitching
Therapy was performed once a day during five consecutive days repeatedly at the same
time resulting in an extension of the duration of the in vitro therapy with two days
Visual macroscopic observations were accomplished 6 52 and 175 days after the first
treatment
Comparison of the cutaneous sensitivity at the irradiated area and the control area was
accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175
days after the first treatment A control measurement also occurred at the same region
Chapter 1
38
on the left foot The aesthesiometer used in this study consisted of five hand-held
nylon monofilaments with a length of 38 mm and varying diameter
Sensitivity threshold is traced by presenting a monofilament of a certain diameter
vertically to the skin The monofilament bends when a specific pressure has been
reached with a velocity proportional to its diameter Measurements allow mapping
areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified
from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest
filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished
protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to
finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the
filaments
RESULTS
Visual estimation at any point of time after irradiation divulged no occurrence of
problems with dehiscence or infection in either part of the wound During the five
days of therapy the irradiated area looked dryer than the control area After the last
irradiation this was no longer recorded
Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area
From an in vitro trial to a patient treatment
39
Figure III representing the first evaluation six days after the initial treatment
illustrates that the wound healing has evolved slightly in both parts Though the lower
irradiated part remains of inferior quality as regards to epithelialization and wound
contraction In the course of the reparative process the influence of light exposures
were registered At 52 days after the first irradiation beneficial effects of LED
treatment are clearly present (Figure IV)
Figure IV Surgical incision 52 days after initiating LED treatment
The irradiated area (26 cm) showed a more appropriate contracture than the control
area (34 cm) characterized by less discoloration at scar level and a less hypertrophic
scar A similar trend was noticed at a third visual observation 175 days after the initial
treatment At that moment no impairments at cutaneous sensitivity level were stated
and the sensitivity showed no differences between left or right foot nor between the
two areas of the cicatrice
Chapter 1
40
DISCUSSION
The results of this case study indicate that LED had a positive influence on wound
healing in humans as determined by visual observations Many investigators
examining the effects of LPL on wound healing by means of a range of observation
and treatment methods reported accelerated and enhanced wound healing8-10 others
described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and
LED21 studies were unable to repeat these results
The late but beneficial findings in this study seem to be to the credit of LED-therapy
Though several authors establish positive results in an earlier stage of the wound
healing process8-1020 one should question why the differences did not occur at the first
evaluation on day 6 An explanation can be found in the start of the treatment Most
investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so
they influence a first cellular and vascular reaction with the production of chemical
mediators of inflammation resulting in an enhanced collagen production9 tremendous
proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the
time the first treatment in this study took place the traumatized tissue was in an
overlapping stage between an almost finished inflammatory phase and a scarcely
initiated re-epithelialization and wound contraction phase At that moment an infiltrate
of fibroblasts is present So fibroblast proliferation a possible mechanism of the
biostimulative effect had already occurred and could no longer be influenced Growth
factor production and collagen deposition have also decreased at that stage
Granulation tissue formation and fibroplasia in the contrary are initiating by that time
Those prolonged and slow processes with belated results are of significant importance
for the course of the final stage of wound healing and for the outlook of the future
scar31
The experimental findings revealed that the sensitivity of the skin according to the
threshold detection method of Semmes and Weinstein was normal at all the
investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a
good and objective predictor of normal skin sensitivity No other LPL nor LED
studies investigating this quality of the skin were found
From an in vitro trial to a patient treatment
41
CONCLUSION
This study demonstrates that although LED application at the applied energy density
and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to
have beneficial biostimulative effects on wound healing in human skin confirmed by
the favourable re-epithelialization and contracture
These results are discussed in the context of other experimental findings but no
reasonable explanation for this discrepancy could be found The literature on wound
healing after LED treatment in animal models or in humans is presently very limited
and contradictory The diversity in used radiation parameters and the absence of
references on how the wounds were measured or evaluated or what the end point was
for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro
investigations are better standardised nevertheless these results show a number of
conflicts One can conclude that until today the controversial findings are characteristic
for many results obtained with light photobiomodulation
However the postponed favourable results in the case study confirm some facts of the
discussion Namely the short period of incubation 24 hours in the in vitro part of the
study can be responsible for the lack of enhanced fibroblast proliferation It also
confirms that other cell processes and morphologic changes possibly are responsible
for biostimulative effects in vivo other observation methods should be considered for
future in vivo experiments
Despite these remarks we believe that LED application on cutaneous wounds of
human skin is useful with a single flash daily at the dose applied in this study for at
least three days
Furthermore future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Chapter 1
42
ACKNOWLEDGMENTS
The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the
material necessary for this study as well as Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
From an in vitro trial to a patient treatment
43
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1 G Baxter D Walsh J Allen A Lowe and A Bell Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79(2) 227-34 (1994)
2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)
3 J Stelian I Gil B Habot et al Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy J Am Geriatr Soc 40(1) 23-6 (1992)
4 A Honmura A Ishii M Yanase J Obata and E Haruki Analgesic effect of Ga-Al-As diode laser irradiation on hyperalgesia in carrageenin-induced inflammation Lasers Surg Med 13(4) 463-9 (1993)
5 D Cambier K Blom E Witvrouw G Ollevier M De Muynck and G Vanderstraeten The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15 195-200 (2000)
6 G Reddy L Stehno Bittel and C Enwemeka Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-7 (1998)
7 K Moore N Hira I Broome and J Cruikshank The effect of infrared diode laser irradiation on the duration and severity of postoperative pain a double blind trial Laser Therapy 4 145-148 (1992)
8 A Amir A Solomon S Giler M Cordoba and D Hauben The influence of helium-neon laser irradiation on the viability of skin flaps in the rat Br J Plast Surg 53(1) 58-62 (2000)
9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)
10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)
11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)
12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)
13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)
14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)
15 A Schlager P Kronberger F Petschke and H Ulmer Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1) 39-42 (2000)
16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)
17 GD Baxter and J Allen Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone (1994)
18 G Baxter A Bell J Allen and J Ravey Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77(3) 171-178 (1991)
19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)
20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)
Chapter 1
44
21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)
22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)
23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)
24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)
25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)
26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)
27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)
28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)
29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)
30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)
31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)
32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)
33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)
34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)
35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)
36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)
37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)
38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)
39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)
40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)
41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)
42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)
From an in vitro trial to a patient treatment
45
43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)
44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)
45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)
46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)
47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)
48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)
49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)
50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)
51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)
52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)
53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)
54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)
CHAPTER 2
INCREASED FIBROBLAST PROLIFERATION INDUCED BY
LIGHT EMITTING DIODE AND LOW LEVEL LASER
IRRADIATION
Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Lasers in Medical Science 2003 18(2) 95-99
Chapter 2
48
ABSTRACT
Background and Objective As Light Emitting Diode (LED) devices are
commercially introduced as an alternative for Low Level Laser (LLL) Therapy the
ability of LED in influencing wound healing processes at cellular level was examined
Study DesignMaterials and Methods Cultured fibroblasts were treated in a
controlled randomized manner during three consecutive days either with a infrared
LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and
570 nm) and respective power outputs Treatment duration varied in relation to
varying surface energy densities (radiant exposures)
Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all
irradiated cultures in comparison with the controls Green light yielded a significantly
higher number of cells than red (plt0001) and infrared LED light (plt0001) and than
the cultures irradiated with the LLL (plt0001) the red probe provided a higher
increase (plt0001) than the infrared LED probe and than the LLL source
Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation
in vitro This study therefore postulates possible stimulatory effects on wound healing
in vivo at the applied dosimetric parameters
Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low
Level Laser Tetrazolium salt
LED induced increase of fibroblast proliferation
49
INTRODUCTION
Since the introduction of photobiostimulation into medicine the effectiveness and
applicability of a variety of light sources in the treatment of a wide range of medical
conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results
of several investigations are remarkably contradictory This is at least in part a
consequence of the wide range of indications as well as the wide range of suitable
parameters for irradiation and even the inability to measure the possible effects after
irradiation with the necessary objectivity [457] A lack of theoretical understanding
can also be responsible for the existing controversies In fact theoretical understanding
of the mechanisms is not necessary to establish effects though it is necessary to
simplify the evaluation and interpretation of the obtained results As a consequence
the widespread acceptance of especially Low Level Laser (LLL) therapy in the early
seventies is faded nowadays and biostimulation by light is often viewed with scepticism
[8] According to Baxter [49] contemporary research and consumption in
physiotherapy is in particular focused on the stimulation of wound healing Tissue
repair and healing of injured skin are complex processes that involve a dynamic series
of events including coagulation inflammation granulation tissue formation wound
contraction and tissue remodelling [10] This complexity aggravates research within this
cardinal indication
Research in this domain mostly covers LLL studies but the current commercial
availability of other light sources appeals research to investigate as well the effects of
those alternative light sources eg Light Emitting Diode (LED) apparatus
The scarcity of literature on LED is responsible for consultation of literature
originating from LLL studies [11] but it may be wondered if this literature is
representative for that purpose As in the early days of LLL therapy the stimulating
effects upon biological objects were explained by its coherence [1213] while the beam
emitted by LEDrsquos on the contrary produces incoherent light Though the findings of
some scientists [914151617] pose nowadays that the coherence of the light beam is
not responsible for the effects of LLL therapy Given that the cardinal difference
between LED and LLL therapy coherence is not of remarkable importance in
Chapter 2
50
providing biological response in cellular monolayers [5] one may consult literature
from LLL studies to refer to in this LED studies
The purpose of this preliminary study is to examine the hypothesis that LED
irradiation at specific output parameters can influence fibroblast proliferation
Therefore irradiated fibroblasts cultures were compared with controls The article
reports the findings of this study in an attempt to promote further discussion and
establish the use of LED
MATERIALS AND METHODS
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos Isolation and
disaggregation of the cells was performed with warm trypsin according the protocol
described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in
Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1
L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants
reached confluence cells were detached with trypsine and subcultured during 24 hours
in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours
the cells were removed from the culture flasks by trypsinization and counted by Buumlrker
hemocytometry For the experiment cells from the third passage were plated in 96-well
plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a
density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C
during 24 hours
All supplies for cell culture were delivered by NV Life Technologies Belgium except
for Fetal Calf Serum (Invitrogen Corporation UK)
Irradiation sources
In this study two light sources a Light Emitting Diode (LED) device and a Low Level
Laser (LLL) device were used in comparison to control cultures
The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)
LED induced increase of fibroblast proliferation
51
with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400
mW and a frequency range from 0-1500 Hz
The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)
consisted of three wavelengths emitted by separate probes A first probe emitting
green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the
red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third
probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared
light The area of all three probes was 18 cm2 and their frequency was variable within
the range of 0-1500 Hz
Exposure regime
Prior to irradiation the 96-well plates were microscopically verified to guarantee that
the cells were adherent and to assure that there was no confluence nor contamination
Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining
25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation
The 96-well plates were randomly assigned in the treated (LLL or green red or infrared
LEDrsquos) or the control group
For the treatments in this study the continuous mode was applied as well for the LLL
as for the three LED-probes The distance from light source to fibroblasts was 06 cm
LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting
in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant
exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to
exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective
power output of 160 mW 80 mW or 10 mW
After these handlings the remaining medium was removed and new Hanksrsquoculture
medium was added followed by 24 hours of incubation
One irradiation (LLL or LED) was performed daily during three consecutive days
according to the aforementioned procedure Control cultures underwent the same
handling but were sham-irradiated
Chapter 2
52
Determination of cell proliferation
The number of cells within the 96-well plates as a measure for repair [19] was
quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The
colorimetric assay was performed at two different points of time to determine the
duration of the effect of the used light sources
This assay exists of a replacement of Hanksrsquoculture medium by fresh medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as
described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT
solution was substituted by lysing buffer isopropyl alcohol The plates were
temporarily shaken to allow dissolution of the produced formazan crystals After 30
minutes of exposure to the lysing buffer absorbance was measured The absorbance at
400 to 750 nm which was proportional to fibroblast proliferation was determined
using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)
The complete procedure from isolation to MTT assay was executed six times (Trial A
B C D E and F) while it was impossible to irradiate all the investigated number of
wells with the same LED apparatus on one day All the trials included as much control
as irradiated wells but the number of control and irradiated wells in each trial varied
depending on the number of available cells after the second subculturing A further
consequence of the available number of cells is the number of probes examined per
trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E
Incubation period before proliferation analyses numbered 24 hours To investigate if
the stimulatory effect tends to occur immediately after irradiation or after a longer
period of time incubation in trial F lasted 72 hours
An overview of the followed procedures regarding incubation time before proliferation
analysis number of analysed wells for each trial and the number of probes examined
per trial is given in table 1 As a consequence of the differences in procedures followed
and because each trial started from a new cell line the results of the five trials must be
discussed separately
LED induced increase of fibroblast proliferation
53
Statistical analysis
Depending on the amount of groups to be compared within each trial and depending
on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way
ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons Statistical significance for all tests was
accepted at the 005 level For this analysis Statistical Package for Social Sciences 100
(SPSS 100) was used
RESULTS
The results presented in table 1 show that cell counts by means of MTT assay
revealed a significant (plt0001) increase in the number of cells in comparison to their
respective sham-irradiated controls for all the irradiated cultures of trial A B C D
and E except the irradiated groups in trial F
Moreover the results of trial A showed that the effect of the green and red LED probe
was significantly (plt0001) higher than the effect of the LLL probe With regard to the
amount of proliferation the green probe yielded a significantly higher number of cells
than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe
provided a higher increase in cells (plt0001) than the infrared probe
The infrared LED source and the LLL provided a significant (plt0001) higher number
of cells than the control cultures but no statistical significant difference was recorded
between both light sources
The trials A B C D and E regardless of the number of probes used in each trial
were analysed after 24 hours of incubation after the last irradiation The incubation
period of trial F lasted 72 hours
The means of trial F illustrated that the effect was opposite after such a long
incubation The control cultures had significantly (plt0001) more fibroblasts than the
irradiated cultures with the exception of the LED-infrared group that showed a not
significant increase of cells Further analysis revealed that the green probe yielded a
significantly lower number of cells than the red (plt0001) and the infrared probe
(plt0001) and that the red probe provided a higher decrease (plt0001) than the
Chapter 2
54
infrared probe Laser irradiation induced a significant decrease of fibroblasts in
comparison to the infrared irradiated cultures (plt0001) and the control cultures
(p=0001) LED irradiation with the green and the red probe revealed no statistical
significant differences
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups
Absorbency (proportional to the number of fibroblasts)a
Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050
Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis
DISCUSSION
Despite the failure of some studies [223] to demonstrate beneficial effects of laser and
photodiode irradiation at relatively low intensities (lt500mW) on fibroblast
LED induced increase of fibroblast proliferation
55
proliferation this study provides experimental support for a significant increased cell
proliferation Therefore these results confirm previous studies that yielded beneficial
stimulating effect [1152425] Remarkably though is the higher increase noted after
irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general
decrease in absorption at longer wavelengths and concluded that several molecules in
fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445
470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the
closest to one of these peaks
Karu [5] also emphasises that the use of the appropriate wavelength namely within the
bandwidth of the absorption spectra of photoacceptor molecules is an important
factor to consider
In this particular context penetration depth can almost be ignored as virtually all
wavelengths in the visible and infrared spectrum will pass through a monolayer cell
culture [12] The irradiance (Wcm2) on the contrary could have had an important
influence on the outcome of this study The higher increased proliferation by the lower
wavelengths is possibly a result of the lower irradiance of these wavelengths Lower
irradiances are confirmed by other experiments to be more effective than higher
irradiances [111626]
The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as
described by Poumlntinen [17] but in the scope of these results it also needs to be noticed
that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)
the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)
Consequently the results of especially trial A and F must be interpreted with the
necessary caution It is possible that the determined distinction between the used light
sources and the used probes is a result from the various radiant exposures applied
during the treatments of the cultures
Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after
the last irradiation this study was unable to demonstrate a stimulating effect when
analysis was performed 72 hours after the last irradiation Moreover this longer
incubation period even yielded an adverse effect Although a weakening of the
Chapter 2
56
photostimulating influence over time is acceptable it can not explain a complete
inversion Especially in the knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period [2427] In an attempt to illuminate
this finding one can suppose that the circadian response of the cells triggered by the
LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark
The most obvious explanation is even though a decreased vitality and untimely cell
death in the irradiated cell cultures as a result of reaching confluence at an earlier point
of time than the control cultures The cells of a confluent monolayer have the tendency
to inhibit growth and finally die when they are not subcultured in time No other
reasonable explanations could be found for this discrepancy
Photo-modulated stimulation of wound healing is often viewed with scepticism The
real benefits of Light Emitting Diodes if any can only be established by histological
and clinical investigations performed under well controlled protocols Despite these
remarks this study suggests beneficial effects of LED and LLL irradiation at the
cellular level assuming potential beneficial clinical results LED application on
cutaneous wounds of human skin may be assumed useful at the applied dosimetric
parameters but future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Persons in good health rarely require treatment for wound healing as posed by Reddy
et al [13] light has a possible optimal effect under conditions of impaired healing
Postponed wound healing is a time-consuming and often expensive complication
Thus future prospects must remind to examine the therapeutic efficacy of LED on
healing-resistant wounds
LED induced increase of fibroblast proliferation
57
ACKNOWLEDGMENTS
The authors are grateful to Prof Deridder for supplying the laboratory as well as the
material necessary for this investigation and to Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
Chapter 2
58
REFERENCES
1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55
2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32
3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7
4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994
5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998
6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32
7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42
8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5
9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8
10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7
11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8
12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34
13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9
14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18
15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43
16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6
17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475
18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J
Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9
21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601
22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63
LED induced increase of fibroblast proliferation
59
23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9
24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301
25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are
more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37
27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8
28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26
CHAPTER 3
GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES
FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL
Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Photomedicine and Laser Surgery 2005 23(2) 167-171
Chapter 3
62
ABSTRACT
Background and Objective The chronic metabolic disorder diabetes mellitus is an
important cause of morbidity and mortality due to a series of common secondary
metabolic complications such as the development of severe often slow healing skin
lesions
In view of promoting the wound-healing process in diabetic patients this preliminary
in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on
fibroblast proliferation and viability under hyperglycemic circumstances
Materials and Methods To achieve hyperglycemic circumstances embryonic chicken
fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL
glucose LED irradiation was performed on 3 consecutive days with a probe emitting
green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min
resulting in a radiation exposure of 01 Jcm2
Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all
irradiated cultures in comparison with the controls
Conclusion According to these results the effectiveness of green LED irradiation on
fibroblasts in hyperglycemic circumstances is established Future in vivo investigation
would be worthwhile to investigate whether there are equivalent positive results in
diabetic patients
Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes
Fibroblast proliferation under hyperglycemic circumstances
63
INTRODUCTION
The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide
geographic variation in incidence and prevalence generally 11 of the world
population is affected and worldwide it is the twelfth leading cause of death1 Those
figures may be higher for urban regions as well as for industrialized countries Due to
multiple factors involving the aging process of the population and lifestyle changes
(such as reduced physical activity hypercaloric eating habits and concomitant obesity)
these figures may increase in the future2-6 Therefore diabetes mellitus could become
the most common chronic disease in certain regions as stated by Gale it ldquotargets the
rich in poor countries and the poor in rich countriesrdquo6
The harmful disruption of the metabolic equilibrium in diabetes mellitus results in
characteristic end-organ damage that occurs in various combinations and that follows
an unpredictable clinical pathway
Accordingly the major consequence of diabetes mellitus in terms of morbidity
mortality and economic burden principally concerns macroangiopathies or
arteriosclerosis and microangiopathies including nephropathy neuropathy and
retinopathy7-10
One of these devastating consequences which often appears in time is the
development of various skin defects that are frequently resistant to healing and that
tend to be more severe than similar lesions in nondiabetic individuals Diabetes
mellitus even increases the risk of infection by an increased susceptibility to bacteria
and an impaired ability of the body to eliminate bacteria1112
Skin problems are a severe complication in diabetic individuals and require a
comprehensive and appropriate multidisciplinary approach to prevention and
treatment12
Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to
play the most prominent role in the development of diabetic complications With the
development of insulin treatment for type I diabetes and various oral hypoglycemic
agents for type 2 diabetes a reduction in the development of skin defects due to
hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply
Chapter 3
64
waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in
diabetic patients often need special care in comparison to those persons in good
health who rarely require treatment for wound healing1617 Special care is directed
besides of course toward optimal diabetes regulation toward patient education
maximum pressure relief controlling infection recovery of circulation in case of
ischemia and different modalities of intensive wound treatment18
In the last few years various therapies have been introduced with varying success An
example of such a therapy is the photo-modulated stimulation of diabetic lesions In
vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been
subject of extensive investigation1920 Due to contradictory research results LLL-
photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of
light in the domain of wound healing is less time-consuming less expensive less
invasive than many of the other introduced treatment modalities and practical to use
however it seems worthwhile to investigate the value and benefits of a newly
introduced and alternative light source the light emitting diodes (LEDrsquos)
Preliminary research has proved that green LED with particular properties (an
exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01
Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These
results may be of great importance to the diabetic patient because as posed by Reddy et
al light has a possible beneficial effect in the case of impaired healing1617
To obtain insight into the ability of LED to stimulate fibroblast proliferation under
diabetic-specific conditions of impaired healing the proliferation was assessed in
irradiated and control cultures cultivated in medium with a high quantity of glucose
MATERIAL amp METHODS
Cell cultivation
Primary fibroblast cultures were established by outgrowth from 8-day-old chicken
embryos After isolation and disaggregating as described by Freshney (1994)24 the cells
were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10
Fibroblast proliferation under hyperglycemic circumstances
65
fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin
Secondary cultures were initiated by trypsinization followed by plating of the cells in
80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were
disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently
231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third
passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the
cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified
atmosphere at 37deg C to allow them to attach to the bottom of the wells
Light source specifications and illumination procedure
To control adherence of the cells and to assure that there was no confluence or
contamination the 96-well plates were microscopically examined before irradiation
Subsequently the tissue culture plates were randomly assigned for use in the treated
and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium
was aspirated The remaining 25 (50 microl) medium avoided dehydration of the
fibroblasts throughout irradiation
Irradiation was performed with a light emitting diode (LED) device The LED device
(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power
range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable
within the range of 0-1500 Hz
The investigation used the following illumination properties the continuous mode a
distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2
radiant exposure This procedure resulted in an exposure time of 3 min and a power
output of 10 mW Immediately after irradiation the remaining medium was aspirated
and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM
glucose (30 gL) and incubated at 37deg C
Irradiation and medium changes occurred at 1-day intervals so one irradiation was
implemented each 24 h for 3 days in a row and from the first irradiation onwards all
medium renewals occurred with glucose-supplemented Hanksrsquo culture medium
Control cultures were handled in the same manner but were sham-irradiated
Chapter 3
66
Proliferation assay
Fibroblast survival and proliferation were determined by a sensitive and reproducible
colorimetric assay the assay which detects merely living cells and the signal generated
bears a constant ratio to the degree of activation of the fibroblasts and the number of
fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than
only a small subsample26
Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented
Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution
was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during
30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous
solution The optical density of the final solution was measured on an ELtimes800 counter
(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test
wavelength varying from 400 to 750 nm
The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies
for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except
for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)
Data analysis
On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a
Mann-Whitney U test was performed for nonparametrical comparison of the results
Statistical significance for all tests was accepted at the 005 level For this analysis the
Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used
RESULTS
The MTT measurements from each of the 256 control wells and 256 irradiated wells
and the subsequent nonparametrical analysis from the optical densities obtained
disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic
Fibroblast proliferation under hyperglycemic circumstances
67
circumstances after irradiation than in the same circumstances without irradiation (Fig
1)
Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)
DISCUSSION
The outcome of these in vitro experiments based on the above-described light source
properties and the illumination procedure described clearly demonstrated the
stimulatory potential of LED on fibroblast proliferation and the cell viability of
fibroblasts cultured in hyperglycemic medium Preliminary research has already
demonstrated that under these conditions (an exposure time of 3 min a wavelength of
570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this
procedure allowed the highest number of living cells The nature of the light and the
usual questions concerning coherence wavelength power output and radiant
exposures have been discussed previously23
Although these findings confirm the results previously found one cannot ignore the
important methodological difference between previous investigations and the current
study as the cells in this experiment were cultured in hyperglycemic medium2328-30
Absorbency - Proportional to the number of fibroblasts
621 x 10-1 682 x 10-1
0010203040506070809
1
Control Irradiated
Groups
Ab
sorb
ency
Chapter 3
68
After a growth period with normal Hanksrsquo culture medium a necessary step to ensure
normal growth of these secondary subcultures and normal attachment to the bottom
of the wells the Hanksrsquo culture medium was supplemented with glucose
Several earlier studies have established that exposure to glucose concentrations (20-40
mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human
vascular endothelial cell proliferation1531-34 This restraint is more pronounced for
higher glucose15 concentrations and is expressed especially after protracted exposure to
high glucose levels31 A similar restraint was found for cultured fibroblasts by
Hehenberger et al3536 According to some authors however cultured fibroblasts
conversely have been shown to maintain responsiveness to ambient high glucose323738
As there are some ambiguities in literature regarding normal or inhibited growth of
fibroblasts in medium supplemented with glucose39 a pilot study was performed to
determine the amount of glucose necessary to inhibit normal growth after 72 h of
culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study
demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease
of cell viability and to bring about a decline in fibroblast proliferation
This concentration resulted in a remarkable reduction of cell viability and a noteworthy
decrease in the proliferation rate in comparison to control cultures grown in 55 mM
glucose although this concentration is too high to mimic severe diabetic
hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to
investigate the effect of LED in distinct destructive conditions in order to obtain an
incontrovertible result
In addition it is possible that the present investigation needed a higher amount of
glucose to result in a remarkable reduction of proliferation as exposure to glucose was
limited to 72 h and as previous studies revealed that the antiproliferative effect of high
glucose was more pronounced with prolonged exposure with a maximal inhibition
attained by 7-14 days1531
Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo
cell growth are too complex to compare A key question is whether fibroblast
senescence in tissue culture and in the intact organism are similar Cristofalo et al40
Fibroblast proliferation under hyperglycemic circumstances
69
reported that this is not the case as fibroblasts have a finite ability to divide and
replicate but apparently the pathway or the morphologic characteristics leading to the
replicative senescence is not identical in vivo compared to in vitro
Furthermore extrinsic aging related to environmental damage which in diabetic
patients is mainly due to a chronic exposure to high levels of glucose during life is
unachievable in vitro
Unless a number of questions regarding the mechanism according to which LED
stimulates fibroblast proliferation in this particular condition remain unanswered the
results ascertain the potential effects of LED on fibroblast proliferation and viability
CONCLUSION
The current results should be interpreted with caution However these results
demonstrate the effectiveness of green LED irradiation at the above-described light
source properties and the illumination procedure described on cells in hyperglycemic
circumstances
The findings of the present study using an experimental in vitro model indicate that the
use of LED irradiation to promote wound healing in diabetic patients may have
promising future results As the present study establishes the possibility of using LED
irradiation in experimental in vitro situations it would be a worthwhile extension to
perform in vivo investigations to determine whether these in vitro observations were
relevant to the physiological situation and to determine the effect of these LED
properties on human tissue response
ACKNOWLEDGMENTS
The authors are greatly indebted to P Coorevits for assistance with the statistical
analysis and to Professor L Deridder and Ms N Franccedilois of the department of
Human Anatomy Embryology Histology and Medical Physics for providing access to
the laboratory and for helpful discussions
Chapter 3
70
REFERENCES
1 World Health Organisation The world health report of 2002 Statistical Annex 2002 pp 179-201
2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418
3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957
4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538
5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308
6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J
Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot
wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-
glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A
Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood
glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13
14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309
15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501
16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287
17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255
18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412
19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192
20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137
21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675
22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246
23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99
Fibroblast proliferation under hyperglycemic circumstances
71
24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729
27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63
28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301
29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -
progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of
cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA
damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in
culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia
23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar
K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71
36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141
37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588
38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252
39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97
40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619
PART II ANALGESIA
CHAPTER 4
EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION
MEDIATED BY LIGHT EMITTING DIODE IRRADIATION
Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy
Vanderstraetenab and Dirk Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Lasers in Medical Science 2005 20(1) 35-40
Chapter 4
76
ABSTRACT
The introduction of light emitting diode (LED) devices as a novel treatment for pain
relief in place of low-level laser warrants fundamental research on the effect of LED
devices on one of the potential explanatory mechanisms peripheral neurophysiology in
vivo
A randomised controlled study was conducted by measuring antidromic nerve
conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline
measurement and five post-irradiation recordings (2 min interval each) were performed
of the nerve conduction velocity (NCV) and negative peak latency (NPL)
Interventional set-up was identical for all subjects but the experimental group (=32)
received an irradiation (2 min at a continuous power output of 160 mW resulting in a
radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype
MDB-Laser Belgium) while the placebo group was treated by sham irradiation
Statistical analysis (general regression model for repeated measures) of NCV and NPL
difference scores revealed a significant interactive effect for both NCV (p=0003) and
NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical
significant decreased NCV and an increased NPL in the experimental group and a
statistical significant difference between placebo and experimental group at various
points of time
Based on these results it can be concluded that LED irradiation applied to intact skin
at the described irradiation parameters produces an immediate and localized effect
upon conduction characteristics in underlying nerves Therefore the outcome of this in
vivo experiment yields a potential explanation for pain relief induced by LED
Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative
peak latency middot Analgesic effect
Nerve conduction characteristics
77
INTRODUCTION
Since the introduction of photobiostimulation into medicine the light sources used
have advanced technologically and varied in characteristics over the years
Advancement and variation of the sources implicate a concomitant necessity to revise
research results in the respective domains of application Research and clinical
applications in the past particularly focused on the effectiveness of low-level lasers
have shifted now to novel treatment units such as light emitting diode (LED) devices
The efficacy and applicability of LED irradiation within the field of wound healing has
already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED
is not only promoted for its beneficial effects on the wound-healing process it is also
suggested to be potentially effective in the treatment of pain of various aetiology
although this claim has not yet been investigated thoroughly either experimentally or
clinically The putative analgesic effects of LED remain to be further explored
As the basic vehicle of pain is the neuronal system [7] measuring the
neurophysiological effect of LED treatment would be an appropriate experimental
approach to investigate the efficacy of LED on pain inhibition Nerve conduction
studies have become a technique for investigating the neurophysiologic effects of light
therapy [8-9]
Review of literature regarding standard nerve conduction studies revealed that previous
human studies on the influence of various light sources on peripheral nerves have
utilized different methods which hampers a comprehensive comparison In general
this research was performed on the superficial radial nerve [10-13] described by Shin J
Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve
[891315-17] Following the method of Cambier et al [18] the authors of this study
decided to investigate the effect of the light source used on the conduction
characteristics of the sural nerve By investigating this solely sensory nerve interaction
of motor nerve fibres (motor response can easily be provoked by antidromic nerve
stimulation [19]) can be avoided and given the superficial nature of the nerve it should
be sufficiently amenable to the effects of percutaneous LED irradiation
Chapter 4
78
A second major difference between the trials and therefore also hindering an
appropriate comparison between the results is the wide range of used light sources
HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared
multisource treatment unit [15]
With respect to the potential importance of LED irradiation for the treatment of pain
the current investigation was designed to assess the putative neurophysiological effects
of LED on the sensory nerve conduction of the human superficial peripheral sural
nerve and to establish a time course of the supposed phenomenon
The experimental hypothesis postulates that LED generates an immediate decrease in
conduction velocity and increase in negative peak latency In addition it can be
postulated that this effect is most prominent immediately after the irradiation and will
weaken as time progresses
STUDY DESIGN
The study was approved by the Ethical Committee of the Ghent University Hospital
After explanation of the experimental procedure a written informed consent was
obtained from each subject
Subjects
After screening based on a brief medical history excluding subjects with
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever inflammation of the skin) or conditions
that might affect sensory nerve conduction (such as diabetes peripheral neuropathy
radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral
edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40
females (mean age 26plusmn6 years range 18-42 years) participated in this study The body
mass index (BMI) of each subject varied within the normal range (=185-249) [20]
(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo
Nerve conduction characteristics
79
or an experimental group Each group of 32 subjects was composed of 12 males and
20 females
Experimental Procedure and Data Acquisition
In order to be able to quantify the negative peak latency (NPL) (measured from the
start of the stimulus artefact to the peak of the negative portion of the nerve action
potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was
followed
With respect to the known relationship between nerve conduction characteristics and
temperature the ambient temperature was kept constant (23ordmC-26ordmC room
temperature) during the investigation In view of this temperature issue the
standardized protocol started with 10 min of accommodation during which the
subjects rested in prone position on a treatment table
Immediately before this adjustment period the skin over the dorsolateral aspect of the
left calf and foot was cleaned with alcohol to remove surface lipids This preparation of
the treatment area was followed by the placement of the electrodes (TECA
Accessories Oxford Instruments Medical Systems Division Old Woking UK) as
described by Delisa et al [21]
The two-posted (2 cm separation anode distal) surface caption electrode was placed
distal and posterior of the lateral malleolus on the skin covering the sural nerve The
fixation of the earth electrode (Medelec Oxford Instruments Medical Systems
Division Old Woking UK) occurred 12 cm above the caption electrode according to
the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm
above the caption electrode to map the ideal stimulation point To level off
intraindividual variations in the amount of sensory response attributable to the
successive placement of the bipolar stimulator in course of the investigation a two-
posted (2 cm separation cathode distal) bar stimulating electrode was attached at the
point where the maximal response was obtained
This placement of the electrodes allows antidromic stimulation of the sural nerve
Electrophysiological stimulation and recordings were obtained with a Medelec
Chapter 4
80
Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse
of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was
used to produce each evoked sensory response
Baseline measurements of NPL and NCV were immediately followed by treatment of
the subjects according the protocol detailed below Recordings were subsequently
repeated at 2-min intervals over an 8-min period resulting in five recordings (one
immediately after the completion of the treatment and one at 2 4 6 and 8 min after
irradiation) Skin temperature was recorded concomitantly throughout the procedure
at the time of baseline measurement immediately after LED irradiation at the time of
the first recording and consequently at 2-min intervals together with the four final
electrophysiological recordings For this a surface digital C9001 thermometer
(Comark UK) sensitive to temperature changes of 01degC was used at the same point
of LED administration namely at 7 cm above the caption electrode The procedure
was identical for both conditions but subjects in the placebo group received a sham
LED irradiation
Light Characteristics and Irradiation Procedure
Irradiation was administrated with a light emitting diode device (BIO-DIO
preprototype MDB-Laser Belgium) The probe used emitted infrared light with a
wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2
and the frequency was variable within the range of 0-1500 Hz
Preceding baseline measurement the treatment point was marked on the skin overlying
the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-
point between the stimulation and capture electrode The LED probe was held in
contact with the skin perpendicular to the skin surface during the complete irradiation
procedure LED treatment consisted for all subjects of the experimental group out of 2
minutes lasting irradiation The LED was set to deliver a continuous energy density of
107 Jcm2 at a power output of 160 mW These parameters were selected as they are
appropriate for the treatment of pain in a clinical setting First of all because the
Nerve conduction characteristics
81
duration of the treatment is clinically feasible and secondly because the parameters are
within the scope of previously described light source characteristics [1-36915]
Statistics
Although superficial skin temperature did not change significantly in course of the
investigation the influence of the measured skin temperature on NPL and NCV was
taken into account by using a correction factor of respectively 02 msdegC and 147
ms degC All corrections were calculated towards a reference skin temperature of 32degC
Difference scores ie the variation between baseline measurements and each post-
irradiation recording were used as the basis for statistical analysis A General
Regression Model for repeated measures with one within-subjects factor (time 0
min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and
one between-subjects factor (group placebo or LED irradiated) was performed
followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least
Significant Difference) to determine whether any differences between baseline
measurements and post-irradiation recordings were statistically significant
The Statistical package for social sciences (SPSS 110) was used for analysis and
statistical significance for all tests was accepted at the 005 level
RESULTS
Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated
group plotted against time in minutes The values of the irradiated subjects decrease
directly after the irradiation and reach a first low point 2 min after finishing LED
treatment This decrease is followed by a marginal increase at 4 and 6 min and again an
important decrease at 8 min Statistical analysis (general regression model for repeated
measures) of these data indicated a significant interactive effect (P=0003)
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82
Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)
Post hoc LSD further showed significant differences between baseline measurements
and all post-treatment recordings (Table 1) Mutual comparison of the values from the
post-treatment recordings did not reveal any significant difference In addition there
was no significant difference determined in the placebo group in course of time
Table 1 Summary of the influence of LED irradiation on nerve conduction velocity
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001
2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002
4 0111plusmn0377 0647 -0908plusmn1898 0021 0004
6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001
8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve Conduction Velocity
-14
-12
-1
-08
-06
-04
-02
0
02
04
Baseline 0 min 2 min 4 min 6 min 8 min
Time Course
Dif
fere
nce
Sco
re (
m
s)
PlaceboLED
Nerve conduction characteristics
83
A similar representation was used for the results of the NPL Figure 2 reproduces NPL
plotted against time in minutes revealing for the irradiated group an increased latency
with two important peaks one at 4 min and one at 8 min
Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)
Statistical analysis of the mean difference scores again indicated a significant interactive
effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2
showed significant differences between baseline measurements and all post-treatment
recordings of the experimental group The mean difference score of the first post-
treatment recording of this same group (LED irradiated) differed significantly with the
recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED
irradiation As well as the recording 2 min after irradiation which differed significantly
(P=0013) with the 8 min post-treatment recording As observed for the NCV the
NPL of the placebo group did not reveal any significant difference in time course
At the time of the final recording the NCV and NPL mean difference scores of the
irradiated group did not return to their respective baseline values
Negative Peak Latency
-001
0
001
002
003
004
005
006
007
Baseline 0 min 2 min 4 min 6 min 8 min
Time course
Dif
fere
nce
Sco
re (
ms)
PlaceboLED
Chapter 4
84
Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group
significance) revealed statistical differences between the experimental and the placebo
group for NCV as well as for NPL NCV and NPL were statistical significant between
both groups at all points of time except from the NPL recording immediately after
finishing irradiation
DISCUSSION
Notwithstanding the above-mentioned difficulties in comparing results between
different trials on nerve conduction we attempt to discuss the current findings in view
of the results of the previous studies
This investigation revealed that percutaneous LED irradiation at feasible and current
clinical parameters generates measurable and significant changes in human sural nerve
antidromic conduction latency and velocity These results thus support previous
findings of light-mediated nerve conduction latency shifts in vivo [8101218]
although there are several important issues to be discussed
Table 2 Summary of the influence of LED irradiation on negative peak latency
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0004 plusmn0053 0755 0029plusmn0080 0019 0145
2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021
4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004
6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034
8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve conduction characteristics
85
A first comment deals with the progress of the NCV and NPL in function of time As
postulated the NCV decreases significantly immediately after irradiation
corresponding with a significant increased NPL However this effect does not weaken
as time progresses both variables remain significant throughout the 8 min during
observation period
Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the
conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]
although this slight increase in NPL was not significant at any moment Two other
studies [822] with a GaAlAs laser even registered comparable effects over a period of
55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous
studies post-treatment conduction measurements should be extended in time At
present for all studies it remains unclear at what point of time the effect extinguishes
although the interval of time during which LED treatment remains effective is
clinically important when treating pain
Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least
45 min) mediated by a monochromatic multisource infrared diode device although it
needs to be mentioned that this study performed with a comparable light source as the
current investigation revealed a significant decrease of the NPL These inverse results
between the study of Noble et al [15] and the current investigation could be attributed
to the concomitant increase of the skin temperature [15] As it has been well
recognised that a variation in tissue temperature causes a corresponding alteration in
nerve conduction velocities and peak latencies [91523-27] the temperature changes
may indeed provide an explanation for the observed findings In an attempt to analyse
the influence of a direct photobiological effect on sural nerve conduction
characteristics rather than working out the effects based upon thermal mechanisms
the present study corrected the skin temperature towards a reference temperature of
32degC This correction was performed notwithstanding the fact that the superficial skin
temperature did not change significantly before and after LED irradiation as well as
despite the fact that influencing nerve temperature takes place long after affecting skin
temperature [23] and thus being (almost) impossible after 2 min of irradiation
Chapter 4
86
followed by 8 min of registration Introduction of the correction factor implies likewise
that eventual influence on nerve conduction by cooling of the limb due to inactivity as
described by Greathouse et al [11] can be excluded
These facts suggest that temperature changes did not contribute to the demonstrated
effects of LED on nerve conduction Nevertheless the underlying mechanism of the
observed effects remains indistinct
A following remark regarding the fluctuation of NCV and NPL in function of time
considers the fact that both the NCV and the NPL do not change in a constant way up
to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the
increase in NPL display a small though not significant inversion of the effect at 4 and
(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of
fluctuation is to be expected when measuring NCV and NPL besides there is a similar
variation in the placebo groups
Although investigating dose dependency was not intended an additional remark
considers the fact that the use of optimal irradiation parameters is essential to obtain
the observed neurophysiological effect Nevertheless it is impossible to determine
ideal light source characteristics for effective treatment as the range of used
wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency
(pulsed or continuous) are not sufficiently similar between the different studies It can
only be concluded that a pulsing light source [91028] does not provide the postulated
results Radiant exposure exposure time power range and wavelength are not yet
established but based on this study and previously described assays it can be
speculated that the ranges of these parameters are quite large
In comparison with other studies where the number of subjects is 10 or less [8-
1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-
Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number
of subjects (n=32) was investigated in each group In spite of the large investigated
population it should be noted that the magnitude of the described changes in NCV
and NPL can simply be replicated by lowering the temperature of the extremity as the
observed changes are within the expected physiological ranges making the clinical
Nerve conduction characteristics
87
significance of the change questionable (This fact does not implement that the
decrease and the significant changes were temperature mediated)
A key question and meanwhile the initial impetus for future investigation is whether
the measured effects can be extrapolated to the actual nociceptive afferents namely the
myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])
respectively conducting acute and chronic pain The functional testing of these
nociceptive pathways has recently been extensively evaluated The currently accepted
neurophysiological method of assessing nociceptive pathways relies on laser-evoked
potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]
As up till now LEP is not available in this or any surrounding research centre the
investigators of this study had to perform a standard nerve conduction study (assessing
the large myelinated Aβ afferents) Therefore the current and previous beneficial
results of low level light therapy on conduction characteristics of nerves in vivo should
initiate measurements of clinical effectiveness first of all in laboratory settings and
afterward at a clinical level
CONCLUSION
Despite these remarks and the limited knowledge regarding the underlying mechanism
the present findings enable the following conclusions to be drawn LED irradiation at
clinical applied energy densities produces an immediate and localized effect upon
conduction characteristics in underlying nerves More specifically it is proven that
LED treatment lowers the NCV and augments the NPL resulting in a reduced
number of impulses per unit of time Therefore the outcome of this in vivo experiment
assumes that LED possibly induces pain relief
In order to encourage a widespread acceptance for the use of this non-invasive pain-
reducing modality in clinical settings prospective research should establish the precise
relationship between LED and pain relief as well as determine the ideal irradiation
parameters and verify which painful conditions can be treated with this treatment unit
Chapter 4
88
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the
equipment and MDB-Laser Belgium for generously providing the Light Emitting
Diode equipment
Nerve conduction characteristics
89
REFERENCES
1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9
2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)
3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18
4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8
5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43
6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65
7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53
8 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-34
9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6
10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90
11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7
12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5
13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9
14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore
15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5
16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5
17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604
18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200
19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21
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20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083
21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press
22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9
24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9
25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203
26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13
27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12
28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9
29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445
30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8
31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56
CHAPTER 5
PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE
IRRADIATION A PILOT STUDY ON EXPERIMENTALLY
INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS
Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Accepted for publication in Lasers in Medical Science December 2005
Chapter 5
92
ABSTRACT
Objective The present pilot study investigated the analgesic efficacy of light emitting
diode (LED) In view of a standardised and controlled pain reduction study design this
in vivo trial was conducted on experimentally induced delayed-onset muscle soreness
(DOMS)
Design Thirty-two eligible human volunteers were randomly assigned to either an
experimental (n=16) or placebo group (n=16) Immediately following the induction of
muscle soreness perceived pain was measured by means of a visual analog scale (VAS)
followed by a more objective mechanical pain threshold (MPT) measurement and
finally an eccentricconcentric isokinetic peak torque (IPT) assessment The
experimental group was treated with infrared LED at one of both arms the other arm
served as control Irradiation lasted 6 min at a continuous power output of 160 mW
resulting in an energy density of 32 Jcm2 The subjects of the placebo group received
sham irradiation at both sides In post-treatment a second daily assessment of MPT
and VAS took place The treatment and assessment procedure (MPT VAS and IPT)
was performed during 4 consecutive days
Results Statistical analysis (a general linear model followed by post hoc least
significant difference) revealed no apparent significant analgesic effects of LED at the
above-described light parameters and treatment procedure for none of the three
outcome measures However as the means of all VAS and MPT variables disclose a
general analgesic effect of LED irradiation in favour of the experimental group
precaution should be taken in view of any clinical decision on LED
Conclusion Future research should therefore focus on the investigation of the
mechanisms of LED action and on the exploration of the analgesic effects of LED in a
larger randomised clinical trial and eventually in more clinical settings
Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset
muscle soreness middot Musculus biceps brachii
Delayed-onset muscle soreness
93
INTRODUCTION
The analgesic efficacy of light emitting diode (LED) irradiation is recently being
investigated by means of a nerve conduction study on the superficial peripheral sural
nerve [1] It was demonstrated that LED irradiation at clinical applied densities
produces an immediate and localized effect upon conduction characteristics in
underlying nerves More specific LED induces a decreased number of sensory
impulses per unit of time thus possibly inducing pain relief [1]
Given the established influence of this treatment modality on the nerve conduction
velocity and thereby its potential analgesic ability the current investigation was
designed
Studies investigating the efficacy of a therapeutic modality on pain often experience
difficulties regarding standardisation of the population as analysis or comparison of
pain with different aetiologies is almost impossible Therefore we opted to measure the
analgesic effects of LED in a laboratory setting on a sample with experimentally
induced delayed-onset of muscle soreness (DOMS)
Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the
induction exercise and then spreads throughout the muscle [2-4] The correlates of
DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10
[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force
decreased range of motion and in particular muscle pain which is more pronounced
during movement and palpation [8 11] Despite the large volume of research that has
been undertaken to identify the underlying pathophysiology of DOMS the precise
mechanism is not yet universally accepted Several theories such as the torn-tissue
theory the connective tissue damage theory the muscle spasm theory and the
inflammation theory still remain viable though the current opinion states that DOMS
arises from a sequence of events in which several theories occupy an important place
[2 6 12 13]
DOMS has been used as a representative model of musculoskeletal pain and stiffness
in a number of studies [4 7 11 14 15] as it has a number of advantages it can be
induced in a relatively easy and standardised manner in a group of healthy subjects the
Chapter 5
94
time-course is relatively predictable and the symptoms have the same aetiology and are
of transitory nature [14 16] Nevertheless it should be emphasised that the use of this
particular experimental model to test the effectiveness of LED does not mean that this
treatment modality is necessarily advocated for the treatment of DOMS but merely
that it may be helpful in documenting the efficacy of LED in a clinical model of
musculoskeletal pain and stiffness In addition studies based on the induction of
DOMS under carefully controlled laboratory conditions can not replace research
involving actual patients but offer the opportunity to assess the effectiveness of
particular therapeutic interventions and might help to define additional clinical research
[14]
The experimental hypothesis of the current study postulates that infrared LED reduces
pain and muscle sensitivity associated with DOMS
MATERIALS AND METHODS
The study was approved by the ethical committee of the Ghent University Hospital
After providing information regarding the study design and possible consequences
related to participation at the study written informed consent was obtained from each
subject
Subjects
Healthy human volunteers were recruited from the university population Individuals
with any upper limb pathology neurological deficit and recent injury to either upper
extremity or undiagnosed pain were excluded Other exclusion criteria were
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever and inflammation of the skin) or
conditions in which physical exertion is contraindicated (such as cardiovascular deficits
hypertension and respiratory problems)
Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age
23plusmn4 years) were enrolled All subjects were randomly assigned using a random table
Delayed-onset muscle soreness
95
of numbers to the experimental or placebo group Each group of 16 subjects
consisted by stratification of equal numbers of men and women Age height and
weight did not differ significantly between the three groups
All subjects were physically active however none performed on a regular basis any type
of upper body weight-training Subjects were requested to refrain from any form of
strenuous physical activity and they were asked to avoid any form of medication
including anti-inflammatory agents as well as alcohol for 2 days before testing and for
the duration of the study
Overview of experimental design
The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to
induce pain related to DOMS Immediately following induction exercise an initial
assessment of the outcome measures (visual analog scale or VAS mechanical pain
threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the
subjects were treated under blinded conditions according to the randomised group
allocation In post-treatment the MPT was re-recorded and perceived pain was
reassessed with a VAS Contrary to these outcome measures the muscle strength was
only measured in pre-treatment at the one hand because short-term effects of LED
on muscle strength were not postulated and on the other hand because post-
treatment muscle strength can be influenced by too many different physiological
factors related to the pre-treatment measurement On the succeeding days (day 2 3
and 4) the treatment and assessment procedure was similar with approximately 24 h
separating each treatment
In both of the groups the two arms of the participants were included in the study In
the experimental group an equal number of dominant and non-dominant arms were
treated The non-treated arm served as control arm In the placebo group also an equal
number of dominant and non-dominant arms were considered as treated arm and the
other arm was classified in the non-treated group The procedure was identical for
both conditions but the subjects in the placebo group received sham LED irradiation
on both arms
Chapter 5
96
Specific aspects of the experimental design and procedures are detailed below
Pain induction
Muscle soreness was induced in a standardised fashion via a daily calibrated computer-
operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY
USA) Induction occurred separately and in random order in the elbow flexors of both
arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex
Prior to induction of DOMS the subjects were allowed an initial familiarization session
to become comfortable performing maximum voluntary contractions at the required
angular velocities This was immediately followed by determination of the maximum
eccentric and concentric peak torque at an angular velocity of 60degs and 120degs
Subsequently four sessions of eccentricconcentric work were performed with each
arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs
first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg
and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-
range) followed by two sessions at an angular velocity of 120degs again the first time
along an arch of 120deg and followed by the mid-range performance The subjects were
asked to accomplish maximum voluntary contractions during all the sessions Each
session was performed until exhaustion which was defined as the point when the
subject lost 70 of the initial eccentric and concentric peak torque There was a 1-
minute rest between each session This procedure was based on a pilot study and
previously described induction protocols [17-21]
Outcome measures
Outcome measures of subjective pain measurements MPT and muscle strength were
measured in this order on days 1-4 Subjective pain measurements and MPT occurred
immediately prior to and following irradiation whereas muscle strength measurements
only took place before LED treatment
Measurement of subjective pain Perceived muscle soreness was measured
subjectively by means of a 100-mm VAS A series of scales were completed separately
Delayed-onset muscle soreness
97
for each arm pain at rest followed by pain perception associated with full extension of
the arms and finally with maximal flexion of the arms The subjects were not allowed
to compare one VAS result with another
This assessment tool commonly used in measuring experimentally induced pain [22
23] has been found to be a reliable and valid method [24-26]
MPT Tenderness MPT used as a more objective correlate of muscle tenderness
has been demonstrated to be a reliable method to measure experimental induced
muscle soreness [27] This outcome measure was assessed by using a handheld
pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries
South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a
line from the radial insertion of the musculus biceps brachii at the elbow to the
intertubercular groove of the humerus thus resulting in three measure points one at
the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A
pressure of 4Ns was delivered The subjects were instructed to say yes at the exact
moment the pressure perceived became painful Each point was recorded three times
in pre-treatment as well as in post-treatment The average MPT score for each point in
pre- and post-treatment was used for statistical analyses
Muscle strength assessment Eccentric and concentric IPT were measured on the
same computerised dynamometer as was used for the induction of pain and an
identical standardisation procedure regarding positioning was followed
A warm-up session of two maximum voluntary contractions at the required angular
velocities was followed by determination of the eccentric and concentric peak torque
The first session at 60degs consisted of three repetitions followed by a 1-min during
rest and for the second session at 120degs five repetitions were performed The
subjects were instructed to flex and extend the elbow through the entire range of
motion as forcefully and rapidly as possible for each repetition The maximum
eccentric and concentric torque produced during the respective repetitions was used
for statistical analysis
Chapter 5
98
Light source specifications and treatment procedure
Light treatment was applied daily according to group allocation Irradiation occurred
with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The
probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160
mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The
frequency was variable within the range of 0ndash1500 Hz
During the complete irradiation procedure the LED probe was held in contact with
the skin perpendicular to the skin surface and at the exact mid-point between the MPT
mark at 4 cm and the one at 8 cm Light source properties were identical for all
subjects of the experimental group and consisted out of irradiation of 6-min lasting
duration at a continuous power output of 160 mW resulting in an energy density of
32 Jcm2 To conceal the treated side and condition the subjects were blinded to the
treatment status For the experimental condition a probe was held in contact with each
arm but only one of the two probes was attached to the LED device The subjects of
the placebo group received sham irradiation at both sides
The selected parameters are within the scope of previously described light source
characteristics for pain reduction [1 28-30] and they are appropriate for the treatment
of pain in a clinical setting because the duration of the treatment is clinically feasible
Statistical analysis
The three outcome measures were analysed separately For the VAS and MPT
measurements the same procedure was followed a general linear model (GLM) for
repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-
post preceding and following LED irradiation) and one between-subject factor (group
placebo or infrared LED irradiated) was performed If necessary the GLM was
followed by appropriate pairwise comparisons (post hoc least significant difference or
LSD) to determine whether any differences between measurements were statistically
significant A similar model was carried out separately for both the treated and the
control arm
Delayed-onset muscle soreness
99
In contrast to MPT and VAS the muscle strength was analysed differently The peak
torque values recomputed towards body weight of the subjects were statistically
analysed using a GLM for repeated measures This model consisted of one within-
subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo
or infrared LED irradiated) The model was completed twice first for the treated arm
and consequently for the control arm
The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)
was used for analysis and statistical significance for all tests was accepted at the 005
level
RESULTS
Statistical analysis of all variables of the three outcome measures revealed no significant
interactive effects of the main interaction (time times group times pre-post) The means and
standard deviations of the variables for both the treated and the control arm are
outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The
means of all VAS and MPT variables disclose a non-statistical significant general
analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher
MPT values in the irradiated group than in the placebo group The lower VAS rates are
present from day 1 until the last day of the study but they are more clearly present
from day 3 pre-treatment The higher MPT values are present from day 1 post-
irradiation until the last day and they are more visible at 4 cm followed by 12 cm and
finally at 8 cm In addition to the analgesic influence of LED an increased
convalescence of muscle strength was noted It should be remarked that this outcome
is similar for the treated as well as for the control arm of the irradiated group The
findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms
of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively
Graphical presentation of the other variables shows a similar course
Chapter 5
100
Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42
Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36
Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Visual Analog Scale
0
02
04
06
08
1
12
14
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n V
AS
scor
e (a
t re
st)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
101
Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249
Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Mechanical Pain Threshold
0
5
10
15
20
25
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n M
PT
sco
re (
at 4
cm)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Chapter 5
102
Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group
Day 1 Day 2 Day 3 Day 4
Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04
Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04
Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Isokinetic Peak Torque
04
045
05
055
06
065
Day 1 Day 2 Day 3 Day 4
Time course
Mea
n I
PT
sco
re (
ecce
ntr
ic a
t 60
degse
c)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
103
Despite the absence of significant main interaction effects the remaining interactions
as well as the main effects were statistically significant for some variables Only the
significant interactions including the between-subject factor group as well as the main-
effect group will be discussed The other interactions and effects establish the successful
induction of DOMS but are not relevant in view of the postulated hypothesis
The interaction between group and time is significant (p=014) for the VAS in
association with full extension for the control arm Post hoc LSD reveals no difference
between both groups a significant effect over time for both groups is found
Consequently this will not be further evaluated
A second significant interaction (p=0002) is the one among the within-subject factor
pre-post and the between-subject factor group for the MPT at 12 cm for the control arm
Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more
pressure after than before the treatment whereas in the placebo group a not
significant decrease of supported pressure is noted
Finally GLM analysis revealed that at the treated arm the irradiated group tolerates
significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)
DISCUSSION
It has previously been demonstrated that the LED source used might assist in
accelerating wound healing [31] that it has a direct cellular effect [3233] and that it
changes nerve conduction characteristics [1] Nevertheless LED-treated experimental
induced DOMS failed to prove the analgesic efficacy of LED at the above-described
light parameters and treatment procedure The current outcome concurs with other
research that demonstrated a lack of effect of various forms of light therapy on DOMS
[8 11 15] However despite the absence of an apparent and overall definitive finding
the present results cannot exclude favourable effects of LED treatment on pain Since
first of all an isolated statistical significant pre-post difference between groups (control
arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)
revealed that subjects of the irradiated group tolerate more pressure than the subjects
of the placebo group Second of all the overall means identified generally lower VAS
Chapter 5
104
scores higher MPT values and higher peak torques in the irradiated group This
implied that the treated subjects experienced noticeable less pain supported more
pressure on the painful muscle and generated more force than the non-treated
participants However these results are not statistically significant consequently it is
possible that these differences were found by coincidence and that there is no
relationship between the treatment and the described results of the three outcome
measures though it should be mentioned that the absence of significant findings is
more probably attributable to the small sample size involved in this study This
assumption is based on a post hoc power analysis It was calculated that for the small
effect size measured after treatment and for the measured control group event rate a
sample size of 80 subjects in each group was required at α=005 and power=080
(two-sided) to reveal significant results
Another factor conceivably responsible for the lack of solid evidence of the beneficial
effects of LED treatment upon DOMS-associated pain is related to the size of the
treatment effect in relation to the severity of the induced DOMS It is possible that by
using multiple exhaustive sets of exercise severe DOMS were induced which masked
relatively small but apparent treatment effects [4 11] In this same context it is
possible that the results only become significantly different after a prolonged treatment
and follow-up period as previous research noticed that recuperation subsequent to
DOMS induction can last up to 10 days [8]
Although it needs to be stressed that these results are not statistically significant critical
analysis of the overall means leads up to three additional remarks A primary comment
relates to the pre- and post-treatment courses of the results Starting at day 2 a clear
reduction of pain and muscle sensitivity was observed immediately post-treatment
Still one cannot conclude that this is indicative for the analgesic effect of LED
irradiation as a similar decrease in VAS and increase in MPT values was noted in the
treated and the control arm of the placebo group Perhaps this was caused by placebo
effect as reported by Pollo et al [34] the expectation of the participant can easily result
in pain relief but it can only be elucidated by implementation of a control group
Delayed-onset muscle soreness
105
Nevertheless in the current study this particular finding can be most probably
attributed to the physiological effects of the peak torque measurement performed
between the pre- and post-treatment recordings of VAS and MPT on the painful
flexor muscle of the upper arm For the assessment of muscle strength two short
series of alternative concentric and eccentric efforts were performed in succession
involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist
in the removal of inflammatory markers and exudate consequently reducing local
tenderness [4] In addition the force assessment can be considered as a form of active
warming-up resulting in an increased muscle temperature which can reduce muscle
viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness
[3536] thus decreasing the sensitivity of the muscle and moderating pain during
movement In any case the beneficial influence of LED immediately after irradiation
can not be securely interpreted due to the sequential assessment of the outcome
measures
A second additional remark considers the fact that both arms of the irradiated subjects
demonstrated evidence of the beneficial effects of LED as a similar reduction of pain
and muscle sensitivity and higher peak torques were found in course of time at the
treated arm as well as at the control arm of the irradiated subjects This ascertainment
points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]
stated that in case LED works via systemic effects the use of the contralateral side as a
control arm might be ill-advised Thus reinforcing that future research should include a
control group to bring clarification [4 7 16]
Finally it needs to be mentioned that although the extent of DOMS was probably
relatively high for investigating the postulated hypothesis the time-course of the
present study corresponds to that reported by other investigators [2 3 5-10]
Significant time effects in many of the variables revealed that muscle damage was
evident diffuse muscle soreness became progressively worse 24-48 h after DOMS
induction followed by a small amelioration after 72 h [35910] After 72 h the follow-
Chapter 5
106
up was ceased consequently further regain of force and attenuation of pain and
muscle sensitivity could not be evaluated Extending the duration of the assessment
period could be useful in assessing any longer-term effects of LED treatment
particularly because as mentioned above differences between both groups are more
clearly present from day 3 pre-treatment and also because DOMS may last for up to 10
days when induced with the described protocol [715]
Lack of knowledge regarding both the precise mechanism of action of LED and the
specific pathophysiology of DOMS hampers the way to offer a definitive explanation
for the absence of more obvious statistically significant differences Still the small
number of significant findings and the mean values suggest that possible analgesic
effects of infrared LED may not be excluded yet but to be able to estimate the real
value of LED further research is necessary A large-scaled randomised clinical trial
which takes the above-mentioned remarks into consideration should be performed
CONCLUSION
Regardless of the reasons for the absence of statistical significant effects reported here
and although LED may have some potential in the management of pain and functional
impairment associated with DOMS its effectiveness at the applied densities has not
been established
Future research should focus on evaluation of the appropriateness of DOMS as an
experimental model of pain and muscle damage Validation of this model would
enhance the ability to study various modalities for their potential effects on pain and
muscle injuries Besides the mechanisms of LED action are not known thus further
fundamental investigations need to address the underlying mechanism and
physiological basis of pain modulation utilizing LED treatment
Once LED irradiation has finally proven its treatment value in an experimental model
the most important prospect considers establishing the effectiveness of LED to reduce
pain in clinical settings
Delayed-onset muscle soreness
107
ACKNOWLEDGMENTS
The authors would like to thank Mr T Barbe and Mr R Deridder for their technical
assistance in the collection of the data as well as for their valuable input into the
research design Sincere appreciation is extended to the volunteers that participated in
this study and to MDB-Laser (Belgium) for generously providing the light emitting
diode equipment The authors also gratefully recognize Prof Dr G Van Maele for
assistance with the statistical analysis and for helpful discussion
Chapter 5
108
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11 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
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23 Nosaka K and Clarkson PM (1997) Influence of Previous Concentric Exercise on Eccentric Exercise-Induced Muscle Damage J Sports Sci 15(5)477-483
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GENERAL DISCUSSION
General discussion
113
SUMMARY
As outlined in the general introduction the overall objective of this doctoral thesis is to
develop the current knowledge about the mechanisms of LED action in view of the
eventual provision of evidence-based support for the clinical use of LED as a
biostimulatory and analgesic treatment modality especially in the field of
physiotherapy
Part I Wound healing
The investigations described in chapter 1 and 2 were conducted to gain insight into the
potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As
fibroblasts are principal cells for biostimulation (in view of growing and dividing in
healing wounds) the influence of LED irradiation on fibroblast proliferation was
assessed1
The first investigation consisted of a pilot study performed in order to evaluate the
appropriateness of the cell isolation technique cell culture protocol and proliferation
analysis as well as to appraise the feasibility of the light source properties and
illumination procedure
Data analysis revealed no statistically significant differences between the infrared LED
irradiated and control petri dishes for the used parameters (table 1) Considering this
outcome other experimental findings disclose that the absence of stimulatory effects of
LED irradiation on fibroblast proliferation can partly be attributed to the use of
inappropriate light source properties However the applied external dosimetric
parameters are well within the recommended spectrum described by previous studies
investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be
excluded that changes in the illumination procedure (such as the use of lower power
shorter exposure times wavelengths with finer coverage of the absorption spectrum of
the irradiated cells and a longer incubation period between the last irradiation and cell
counting) could still result in an increased fibroblast proliferation467 Of equal
importance in interpreting the lack of distinctive results are the imperfections of the
applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails
114
considerable intervention from the investigator compromising the reliability of the
method It is also a time-consuming technique with an insufficient sensitivity for some
purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11
To avoid contamination of the results by these modifiable remarks a similar
experiment (chapter 2) was performed in which wavelength power and output mode of
the infrared LED source were not modified (table 1) only the exposure time was
reduced resulting in a lower radiant exposure In addition the effect of two other
emission spectra was evaluated These probes emitting red and green light had a
shorter wavelength than the infrared LED source and the power was half or a
sixteenth of the power from the infrared probe Consequently the red LED irradiation
occurred with a different exposure time than the infrared one in order to attain the
same radiant exposure (053 Jcm2) With respect to the green LED it was not
endeavoured to achieve the same radiant exposure as 16 min of irradiation is not
feasible for in vitro or clinical application
Finally also an LLL light source was integrated Although it was not attempted to
analyse the effectiveness of LED in comparison to LLL enclosure of this modality was
interesting in order to join in with the available literature covering mostly LLL studies
To bypass the described problems regarding analysis of fibroblast proliferation
counting of the cells was carried out this time by means of a colorimetric MTT assay
This method provides more accurate cell counts in a short period of time and therefore
can be considered as a more reliable alternative to Buumlrker hemocytometer11
MTT assay 24 h after the last irradiation revealed a significantly increased number of
cells in the irradiated wells in comparison to their (respective sham-irradiated) controls
Although the study supplied experimental support for a significantly increased cell
proliferation by all external dosimetric properties based on the results of the
comparative trial with an incubation period of 24 hours irradiation with the green
LED source yielded the highest number of fibroblasts Thus it can be concluded that
the wavelength of the green LED is probably within the bandwidth of the absorption
spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that
General discussion
115
the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast
cultures in vitro612
Table 1 External dosimetric properties summarized for each chapter
Wavelength Power Exposure
time Output mode
Radiant exposure
PART I Chapter 1
In vitro part
LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2
LED-infrared 950 nm 160 mW 1 min continuous 053 Jcm2
LED-red 660 nm 80 mW 2 min continuous 053 Jcm2
LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2
Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2
PART II Chapter 4
LED-infrared 950 nm 160 mW 2 min continuous 107 Jcm2
Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
The next aim of the first part of this doctoral thesis was to explore whether LED
treatment could ameliorate in vitro cell proliferation under conditions of impaired
healing In the pursuit of this aim fibroblasts were cultured in medium supplemented
with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a
pilot study the amount of glucose necessary to inhibit normal growth was determined
In order to attain an important reduction of cell viability and decreased proliferation
rate a relatively high concentration of glucose (1667 mM) was necessary in
comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia
(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity
between both circumstances in vitro limited to 72 h whereas the human tissue of a
diabetic patient in vivo is chronically exposed to glucose
Treatment of the fibroblasts occurred in respect of the previously described results
with the same irradiation parameters and illumination procedure (chapter 2)
Accordingly green LED irradiation labelled as the most appropriate treatment for
116
irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in
the previous study (table 1)
Analysis of the cell proliferation by means of MTT measurements yielded a
significantly higher rate of proliferation in hyperglycaemic circumstances after
irradiation than in the control conditions (ie hyperglycaemic circumstances without
irradiation) Thus this outcome supported the stimulatory potential of green LED
irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a
considerable destructive hyperglycaemic medium
Finally although the results of the in vivo part of chapter 1 were persuasive and
encouraging they will not be further discussed in this summary of part I as it was not
aimed in this doctoral thesis to investigate the wound healing process in vivo However
the results of this case study can be a valuable hold for future in vivo research
The possible clinical implications of these results and future research directions in the
scope of wound healing will be discussed below
Part II Analgesia
In the second part two studies investigated the effects of LED irradiation as a
potential intervention mode in one of the most important fields in physiotherapy
practice analgesia Chapter 4 describes the influence of LED treatment on changing
sensory nerve conduction characteristics of a human superficial peripheral nerve
Altering nerve conduction characteristics may not be the sole beneficial purpose to
attain with LED irradiation in view of analgesia but the advantage of nerve conduction
characteristics is that they are objective measurable physical variables and changes in
these characteristics provide a potential explanatory mechanism of pain inhibition by
LED treatment17
The results showed that percutaneous LED irradiation at feasible clinical parameters
can generate a significant decrease in NCV and increase in NPL for all recordings post-
treatment in comparison to the baseline measurement The data in the placebo group
did not reveal any significant difference in the same course of time Statistical analysis
General discussion
117
revealed significant differences between the experimental and the placebo group for
NCV as well as for NPL at all time-points of observation with exception of the NPL
recording immediately after finishing irradiation
It was also observed that the noted effects did not weaken as time progressed It can
be concluded that post-treatment conduction measurements should be extended in
time which is in accordance with the findings of some previous studies18-21 Clarifying
the point of time at which the effect extinguishes is necessary and clinically relevant
when treating pain by means of LED irradiation Besides obtaining the desired
neurophysiological effects ideally the optimal irradiation parameters should be
applied The most favourable dosimetric properties are not yet determined but based
on this study and previously described assays it can be speculated that the dosimetric
window is quite large
Regardless of these clinically important remarks the present findings allow to draw the
following conclusion LED irradiation at clinically applied densities can generate an
immediate and localized effect upon conduction characteristics in underlying nerves as
LED treatment results in lowering the NCV and augmenting the NPL Therefore the
outcome of this in vivo experiment assumes a potential pain relief by means of LED
treatment and justifies further research regarding its clinical effectiveness in laboratory
settings and at a clinical level
The fourth and final aim was to determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5
illustrates a clinical study observing the effect of LED treatment on a model
comprising experimentally induced DOMS in a healthy population The progress of
pain perception and peak torque was evaluated during 4 consecutive days commencing
on the day of DOMS induction The effect of infrared LED treatment at the light
parameters described (table 1) was assessed with regard to three different factors time
(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures
(VAS MPT and IPT) revealed no significant interactive effects of the main interaction
118
(timegrouppre-post) For the remaining interactions and for the main effects only a
few significant findings were relevant in view of the postulated hypothesis
Notwithstanding the absence of an apparent and overall statistically significant finding
the present results indicate favourable trends of LED treatment on pain as the means
of all VAS and MPT variables show a statistically nonsignificant general analgesic
effect of infrared LED irradiation expressed by lower subjective pain rates and higher
MPT values in the irradiated group In addition to the analgesic influence of LED an
augmented restoration of muscle strength was noted The lack of solid statistically
significant evidence for these beneficial effects of LED treatment upon DOMS-
associated pain can possibly be attributed to the small sample size in this study or even
to the size of the treatment effect in relation to the severity of the induced DOMS as
induction of severe DOMS can mask relatively small but apparent treatment
effects2223 A final possibility is that the results only become significantly different after
a prolonged treatment and follow up period as previous research demonstrated that
recuperation subsequent to DOMS induction can last up to 10 days24
It should also be noted that the described general analgesic effect of LED irradiation
was identical for the treated as well as for the control arm in the irradiated group
proposing that infrared LED might induce systemic effects 2526 However it needs to
be stressed that these results were not statistically significant
Regardless of the absence of statistically significant findings the mean values suggest a
potential role for infrared LED irradiation in the management of pain and functional
impairment associated with DOMS Notwithstanding this postulation future research
is absolutely required to establish the effectiveness of LED treatment to reduce pain as
well at the applied densities as for other dosimetric parameters
CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS
In the course of the past years during the process of the genesis of this thesis
therapeutic physical agents in general and phototherapeutic modalities in particular
became less important as physiotherapeutic modes of treatment than during the
preceding two decades The diminished use of these treatment modalities in the
General discussion
119
physiotherapy practice is to a certain degree a consequence of the controversial
research findings regarding the use of these physical agents This issue of controversy
led to less support for the use of these treatment modalities and a growing scepticism
regarding the effectiveness of these physical agents within the scope of the growing
climate of evidence-based practice A second responsible protagonist for the loss of
popularity of physical agents is linked with the current tendency within physiotherapy
emphasising active remedial therapy The establishment of this development was based
on various experiments mainly performed during the last decade demonstrating that
active treatment modalities are for numerous impairments and disabilities preferable to
more passive forms of therapy In Belgium the prevailing nomenclature which came
into use on 1 May 2002 went along with this tendency In the appendix to the Royal
decree of 14 September 1984 towards settlement of the nomenclature of medicinal
treatments concerning compulsory insurance for medical care and allowances the
personal involvement of the physical therapist during the physiotherapeutic session
was emphasized and it was even defined that massage physical techniques within the
framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal
application can only be remunerated when they are applied supplementarily and not as a sole therapy
This implies that passive treatment modalities should not be used as sole method of
treatment and should always be considered as an adjunct to an active treatment
program This development needs to be applauded in many cases such as various
painful musculoskeletal problems functional instability rehabilitation of neurological
patients re-activation of the elderly population psychomotor rehabilitation
cardiovascular and respiratory convalescence Nevertheless it would be erroneous to
entirely reject physical agents including LED treatment Based on the findings of the
above described experiments it needs to be stressed that for some purposes especially
within the scope of impaired wound healing LED irradiation could be a suitable
therapeutic measure This statement is founded on the results of part I of the present
thesis they provided satisfactory fundamental evidence for the advantageous effects of
LED treatment on a crucial exponent of the wound healing process namely fibroblast
proliferation The beneficial findings are the result of basic in vitro research As it is
120
inaccurate to simply extrapolate these results to the clinical practice the clinical use of
LED irradiation for wound healing needs to be preceded by purposive and specific in
vivo investigations to substantiate these basic research findings27
The case study described in chapter 1 indicates a foundation for further in vivo research
Visual appraisal of the surgical incision revealed (from the 65th day in the course of the
reparative process onwards) that the irradiated area -which initially showed inferior
epithelialization and wound contraction- showed a more appropriate contracture than
the control area characterized by less discoloration at scar level and a less hypertrophic
scar These clear beneficial effects of LED treatment on a human cutaneous wound
can serve as preliminary impetus for further research into the clinical applicability of
LED therapy although this case study is insufficient in order to guarantee a safe
correct and effective use of LED as a therapeutic modality
Despite these remarks it tentatively can be concluded that based on a detailed analysis
of the available data of the present in vitro studies and the given case report in
combination with the small number of previously published human studies the
beneficial effects of LED irradiation at the cellular level are obvious and therefore a
potentially favourable outcome can be assumed in clinical practice28-30 LED-
modulated stimulation of wound healing can be gradually and vigilantly implemented
clinically Nevertheless the real benefits of LED irradiation within the scope of wound
healing can only be established by additional clinical trials as thus far clinical
application and stipulation of dosimetry still occurs on a trial-and-error basis which is
not conducive to a generally accepted clinical use of LED To lend more credibility to
the treatment of wounds by means of LED irradiation and to expel the existing
controversy and scepticism surrounding this topic in vivo investigations on wound
healing using a number of different animal models and adequately controlled human
studies are necessary In addition these studies should be performed preferably on a
population suffering from impaired healing as a consequence of diabetes mellitus or as
a result of any other debilitating reason because as posed by Reddy et al3132 and as
mentioned above light has possible optimal clinical effects in the treatment of healing-
resistant wounds
General discussion
121
Drawing general conclusions and formulating clinical implications for analgesia is
obviously less manifest first of all because only a limited number of possible
mechanisms of action in order to obtain analgesia were highlighted and secondly
because both studies did not come to a joint or complementary conclusion The
outcome of the first study revealed that LED treatment lowers the NCV and augments
the NPL resulting in a slower stimulus conduction and consequently a reduced number
of sensory pulses per unit of time Thus it could be assumed that LED induces pain
relief but the results of the study describing the effect of LED treatment on
experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy
In addition it needs to be emphasised that the first study (chapter 4) measured the effect
of LED irradiation on the large myelinated Aβ afferents A noteworthy question and
meanwhile a stimulus for future investigation is whether the measured effects can be
extrapolated from these sensory nerve fibres to the actual nociceptive afferents
notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of
these nociceptive pathways relies on laser-evoked potentials which selectively activate
Aδ-fibres and C-fibres3334 This technique was presently not available therefore a
standard sensory nerve conduction study was performed
Whereas stimulation of wound healing by means of LED irradiation can be cautiously
implemented in the clinical practice at this stage it is too early to promote LED
irradiation as a treatment modality for pain To make this possible it is essential to
conduct numerous studies with regards to the use of LED in the field of analgesia
Future research should focus on fundamental investigations in order to discover the
underlying mechanisms and physiological basis of pain modulation utilizing LED
treatment Furthermore the evaluation of the appropriateness of DOMS as an
experimental model of pain is an important prospect to consider as validation of this
model would enhance the ability to study various modalities for their potential effects
on pain Irrespective of the difficulties regarding standardisation of the research
population and evaluation of soreness inextricably linked with clinical pain studies the
122
ultimate objective of future research should be the establishment of the effectiveness
of LED irradiation to reduce pain of miscellaneous origin in a clinical setting
Regardless of the encouraging results of the described studies and besides the earlier
proposed specific directions for future research (directed towards wound healing or
pain relief) it is necessary in the interest of the patientrsquos well being and to the
advantage of the prospective clinical use of LED to highlight a few more issues for
future research Therefore one has to deal with some limitations of the performed
investigations A first limitation concerns the fact that only two mechanisms of LED
action were investigated (notably changed fibroblast proliferation and alteration of the
nerve conduction characteristics) So one can conclude that for further and better
understanding of the mechanisms of action it is necessary to perform more basic
research Answering the questions regarding the functioning of LED irradiation will
simplify the evaluation and reinforce the interpretation of the obtained results and
ultimately contribute to a more widespread and well definded acceptance of the use of
LED in clinical settings
A second general limitation of this doctoral thesis is the substantial difference in the
used external dosimetric parameters between the different chapters and even within
one and the same study (illustrated in table 1) this complicates the comparison
between the different trials In each trial the dosimetry was individually ascertained
based on previous studies within the given field As not for every application the same
dosimetry is suggested in literature a range of dosages were used Another important
factor in deciding on the dosimetry was the clinical applicability of the dosage as it is
useless to investigate the appropriateness of a treatment modality at a clinically
unrealistic dose As a result of this limitation the current findings do not fully
contribute to the explanation regarding the ideal parameters one should use although
this was not set as a principal purpose Based on this thesis and previously described
assays it can be speculated that the possible window for these parameters is quite large
the ideal irradiation parameters and proper timing or sequencing of LED irradiation
General discussion
123
for example to the various phases of wound healing and to different painful conditions
are therefore possibly unattainable
The establishment of an appropriate dosimetry should also consist of investigating the
absolute and relative penetration depth of LED irradiation into human tissue This is
less crucial within the scope of wound healing but it is of key importance while
treating deep-seated tissue (eg nerve fibres muscles circulatory components et
cetera)
Finally this thesis only investigated the efficiency of LED in a very limited number of
conditions notebly wound healing and pain Within the scope of physiotherapy and
medicine in general there are numerous other purposes for which LED irradiation is
promoted such as oedema arthritis miscellaneous orthodontic applications seasonal
affective disorder neonatal jaundice photodynamical therapy et cetera2835-41
In summary additional work on establishing proper dosimetry and identifying the
biochemical or photobiologic phenomena that are responsible for improving wound
healing and reducing pain or even other effects within a broader spectrum of
conditions remains to be done in order to answer unreciprocated questions Until that
time the potential clinical usefulness and actual value of LED irradiation for wound
healing and even to a larger extent for analgesia should always be approached with
appropriate professionalism and even caution
FINAL CONCLUSION
LED devices are promoted for clinical use but the currently available scientific
documentation regarding effectiveness of this physical agent is rather scarce Through
providing scientific support for the biostimulatory and analgesic effectiveness of LED
irradiation this doctoral thesis attempted to bridge in some degree this gap
The conducted studies revealed that LED irradiation undeniably has potential
beneficial effects on wound healing and to a lesser degree within the scope of
analgesia However based on the present results it can be corroborated that light
124
therapy in the guise of LED irradiation is not magic but these results can raise some
corrective doubts in fundamental disbelievers and antagonists
Nevertheless we have to join the queue of scientists who have found beneficial results
but cannot elucidate with certainty how this outcome was established Thus although
the present results are encouraging a continuing development and integration of new
knowledge based on further research is necessary in various domains of intervention
Therefore several directions for future investigations were proposed in order to cover
as many existing gaps and to answer the utmost number of remaining questions as
possible Still one ought to be aware not to carry future fundamental research at a
disproportional level and the inevitable quest for mechanisms of LED action should
not hypothecate the potential clinical value implying that at a certain point it should be
appropriate to make the transfer from science to the application of the available
knowledge in clinical practice
The described findings regarding LED irradiation are comparable to the results of
previously published studies performed with other light sources Consequently as
postulated by some LED providers it can be speculated that the biological response of
tissue to light irradiation can probably not be equated merely to a light source but
rather to a broad photo-energy window
General discussion
125
REFERENCES
1 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
2 Abergel R Lyons R Castel J Dwyer R and Uitto J (1987) Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2)127-133
3 Skinner S Gage J Wilce P and Shaw R (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-192
4 Webb C Dyson M and Lewis W (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5)294-301
5 Atabey A Karademir S Atabey N and Barutcu A (1995) The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 1899-102
6 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
7 Ben-Dov N Shefer G Irinitchev A Wernig A Oron U and Halevy O (1999) Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3)372-380
8 Rebulla P Porretti L Bertolini F et al (1993) White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2)128-133
9 Deneys V Mazzon A Robert A Duvillier H and De Bruyere M (1994) Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2)172-177
10 Mahmoud A Depoorter B Piens N and Comhaire F (1997) The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2)340-345
11 Haskard D and Revell P (1984) Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3)319-322
12 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers
13 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of cultured human endothelial cells Diabetes 36(11)1261-1267
14 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA damage in cultured human endothelial cells J Clin Invest 77(1)322-325
15 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7)621-627
16 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4)491-501
17 Kramer J and Sandrin M (1993) Effect of low-power laser and white light on sensory conduction rate of the superficial radial nerve Physiotherapy Canada 45(3)165-170
18 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
19 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234
20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
126
21 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
22 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
23 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
24 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
25 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
26 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer
G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
29 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
30 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255
32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287
33 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-28
34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856
35 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
36 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
37 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263
39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64
40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74
General discussion
127
41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press
NEDERLANDSTALIGE SAMENVATTING
Nederlandstalige samenvatting
131
NEDERLANDSTALIGE SAMENVATTING
Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de
tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te
vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie
van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen
Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest
enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is
ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als
op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang
van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve
interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken
Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van
ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen
moeilijk te verantwoorden
Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin
de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere
overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit
methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan
consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in
de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen
laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in
de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag
aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies
inclusief het gebruik van licht
Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat
de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde
doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en
dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht
komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt
geloosd
132
De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo
lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en
gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te
anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te
voorkomen lijkt een gerichte en rationele a priori aanpak conform de
wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen
Te meer daar grondige literatuurstudie leert dat men ten behoeve van de
werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie
zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser
De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de
extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is
geboden De introductie van alternatieve lichtbronnen in de huidige
kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk
weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus
eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood
naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based
gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder
binnen de domeinen van haar potentieel beloftevolle klinische toepassingen
wondheling en analgesie
Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de
wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op
prominente protagonisten van de wondheling de fibroblasten werd getracht het
fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten
zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de
proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd
respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand
waarbij de normale celgroei werd verstoord
In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het
effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische
Nederlandstalige samenvatting
133
data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden
verklaard door het gebruik van een inadequate LED dosering en anderzijds een
methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie
vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de
precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en
inter-tester variabiliteit tot gevolg
In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde
onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de
bestralingsparameters (dosering) De effecten van de drie verschillende LED
golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten
werden hierbij geanalyseerd door middel van een meer betrouwbare en minder
subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-
yl)-25-diphenyl tetrazolium bromide (MTT)
De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir
effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden
tevens een basis voor meer coherente en relevante inzichten aangaande de globale
bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)
Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het
wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend
karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp
rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde
vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De
fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met
extreem hoge concentraties glucose Deze modificatie van het medium staat model
voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische
praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan
vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de
gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en
proliferatie
134
Het tweede deel van dit proefschrift exploreert het domein van het potentieel
analgetisch effect van LED binnen de kinesitherapie aan de hand van twee
fundamentele onderzoeken
In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere
sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele
hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en
een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale
verklaring van een analgetisch effect van het medium zou kunnen belichamen
Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)
meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf
verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen
aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke
significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de
negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele
hypothese
Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend
fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale
pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp
van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische
krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED
behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier
keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED
op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd
(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde
isokinetische krachtmeting en een registratie van de waargenomen spierpijn De
spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met
behulp van een kwantitatieve hand-hold algometer
Analyse van de bekomen data bracht geen significante verschillen tussen de
controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden
Nederlandstalige samenvatting
135
kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED
behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van
de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de
proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene
afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine
proefgroep die werd onderzocht enof door de grootte van het behandeleffect in
verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers
een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up
kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel
absolute omzichtigheid geboden en moet deze visie louter als speculatief worden
beschouwd
Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de
kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de
laatste jaren aanzienlijk afgenomen
De positieve resultaten van de verschillende in vitro studies in het kader van wondheling
vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing
vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel
gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een
belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek
Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie
Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme
onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch
complementair resultaat Verder onderzoek ter exploratie van de mogelijke
onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom
onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op
termijn wetenschappelijk te rechtvaardigen
136
LED tovenarij trend of therapie
LED mag geen magische krachten worden toegemeten maar verdient het lot van een
kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een
wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen
zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke
indicaties
Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds
the darkness has always got there first and is waiting for it
(Terry Pratchett Reaper Man 1991)
GENERAL INTRODUCTION
General introduction
3
BACKGROUND
The use of light for therapeutic purposes reaches far back in time Current interest for
photomedicine with his its biological and medical effects relies fundamentally on two
major evolutions in the given field (1) the research results regarding the use of
ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in
the light amplification by stimulated emission of radiation (laser)-technology The production
of the first laser the ruby pulsed laser was rapidly succeeded by the development of
the helium-neon laser and other lasers like the argon the neodymium-glass and the
neodymium-yttrium-aluminium-garnet lasers1
As in the mid-1990s semiconductor and diode-based lasers gained popularity the
principally massive gas and dye lasers were rendered obsolete Therapeutic light
technology further continued to evolve and todayrsquos therapeutic light source is as likely
to be a light emitting diode (LED) or polarized light as a semiconductor or diode
laser1
Technological advancement and variation of the light sources necessitate a
concomitant update and revision of research in the respective domains of application
Unfortunately this logical and rational necessity has rarely been fulfilled From a
historical perspective this lack of appropriate research has led to disenchanting
evolutions in the use of light especially in physiotherapy The experience exists in this
medical field that light sources were promoted and commercialised for a vast regimen
of indications without foregoing scientific backup Consequently research developed
often after the commercial introduction in physiotherapy As these investigations
frequently gave rise to conflicting results for certain indications scepticism arose and
the use of the given modality knew a waning popularity for all its indications The final
result of such an inappropriate frame of promotion commercialisation and research is
a growing clinical disuse of a given modality even for motivated indications In view of
the actual increasing interest in LED treatment and based on former ascertainment
one has to state that a literature review for the given source reveals that research
mostly covers only low level laser (LLL) studies23 Although recently a number of
papers can be noted that report on the effects of LEDs and polarized light still
4
numerous source-specific-questions need to be answered as research concerning
mechanisms of action and efficacy of the current light sources remains limited in view
of a substantiated clinical application4-17
The reason for the contemporary light-oriented interest in physiotherapeutic practice
for LED devices is in essence based on several advantages of LED in comparison with
LLL For example the use of LEDs is esteemed to be safer as the delivered power
does not damage tissue LEDs can be made to produce multiple wavelengths thereby
stimulating outright a broader range of tissue types and probes that cover a large
treatment area are available18 In addition from a commercial point of view LEDs are
far more interesting as they are a good deal cheaper than laser diodes and they have a
long life span as these solid devices stand robust handling
As a result of the above-mentioned lack of literature on LED some providers of these
devices have taken for granted that the biological response of tissue to light irradiation
cannot be equated merely to a light source They declare that a given response solely
depends on the extent of absorption of radiated light by the tissue19 Consequently
these providers state that it is acceptable to extrapolate scientific findings of LLL
studies for explaining the mechanisms of action and detailing the efficacy of LED and
other alternative light sources Thus actually without appropriate scientific support
equal biological effects are attributed to LED as to LLL Nevertheless prudence is
called for such an extrapolation firstly because it is irrespective of the mentioned
dissimilarities and by simple projection one ignores a number of physical differences
between LLL and LED (eg coherence and degree of collimation or divergence)
Secondly LLL therapy is still not yet an established and evidence-based clinical tool20
Notwithstanding the historical efforts there still remains a considerable amount of
ignorance scepticism and controversy concerning the use and clinical efficacy of
LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed
parameters for irradiation as well as to the difficult objective measurement of possible
irradiation effects and even to the exceptional range of unsubstantiated indications for
General introduction
5
which light therapy was promoted27-29 A lack of theoretical understanding can also be
responsible for the existing controversies as the evaluation and interpretation of
research results would be simplified largely when the appropriate knowledge about the
mechanisms of light action would be available
LLL literature can undoubtedly be used as basis for research on LED and as a
comparative reference for these given investigations However to guarantee evidence-
based use of LED within physiotherapy the need for specific research in view of an
accurate consumption of LED is definite especially for potential promising clinical
applications in physiotherapy according to LLL literature mainly wound healing and
analgesia3031
Hitherto the most substantial research concerning the use of LED for improvement
of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded
under the influence of prolonged exposure to microgravity (eg during long-term space
flights) and in case of absence of exposure to sunlight such as in submarine
atmospheres they performed wound healing experiments for military application in the
given circumstances3233 In vitro experiments revealed that LED treatment increased
proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA
synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and
osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies
demonstrated therapeutic benefits of LED in speeding the early phase of wound
closure and in changing gene expression in a type 2 diabetic mouse model183234
Human studies noted 50 faster healing of lacerations a return of sensation and
increased tissue granulation as a result of LED irradiation1833
Associates of the Rehabilitation Sciences Research Group of the Ulster University in
Northern Ireland extensively investigated the effectiveness of light in the treatment of
pain The emphasis was laid primarily on the analysis of the effects of various low level
laser light sources35-44 However in the year 2001 two studies gave an account on the
efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46
6
noticed relatively long-lasting neurophysiological effects a significant change of the
nerve conduction characteristics (decrease of the negative peak latency) was mediated
by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a
comparable multisource diode device was ineffective in the management of delayed-
onset of muscle soreness (DOMS)
Despite the major value of these described trials a definitive answer regarding the
ability of LED in influencing wound healing or pain is not forthcoming cardinally
because a number of aspects are not yet investigated Consequently more research is
required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the
formerly mentioned scepticism regarding the effectiveness of a medium and possibly
to the undeserved fall into disuse of the treatment modality which happened in a way
with LLL therapy
PHYSICAL CHARACTERISTICS
This chapter supplies a short but comprehensive review of opto-physics A brief
description of the physical characteristics of the LED source used is essential as the
physical properties of light play an important part in the ultimate efficacy of treatment
According to the International Electrotechnical Commission (IEC 60825-1) an LED
can be defined as
Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by
radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process
of spontaneous emission1947
The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO
preprototype MDB-Laser Belgium) This illustration shows that a probe consists of
32 single LEDs disseminated over a surface of 18 cm2
General introduction
7
Figure 1 LED device and three available probes (infrared red and green)
Three highly monochromatic probes were available each emitting light of a different
wavelength within the above-defined range (table 1)2748 The wavelength of the light
emitted and thus its colour depends on the band gap energy of the materials forming
the p-n junctiona This light property is a key determinant to obtain maximum
photochemical or biological responses as light absorption by tissue molecules is
wavelength specific27 Only by absorbing radiation of the appropriate wavelength
(namely the wavelengths equal to the energy states of the valence electrons)
photoacceptor molecules will be stimulated resulting in a direct photochemical
reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750
8
absorption spectrum of a particular molecule is limited absorption often only occurs
over a waveband range of about 40-60 nm274851 Nevertheless the absorption
spectrum at cell or tissue level is broad because cells are composed of many different
molecules
Besides its influence on the absorption by means of tissue molecules there is a crucial
link between wavelength and penetration depth of the irradiated light Penetration into
tissue decreases as the wavelength shortens hence green light penetrates less than red
light which at his turn penetrates less into tissue than infrared light2748 Detailed
principles of light penetration will be discussed below
The LED device used emits non-coherent light In the 1980s the observed biological
responses after laser irradiation were generally thought to be attributable to the
coherenceb of the light485253 Though currently the clinical and biological significance
of coherence is seriously questioned54 According to several authors coherence does
not play an essential role in laser-tissue interactions firstly as it was proven that both
coherent and non-coherent light clinically show equal efficacy75556 Secondly as
according to some authors almost immediately after transmission of light through the
skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et
al1957 state that both findings are incorrect coherence is not lost in tissue due to the
phenomenon of scattering and non-coherent light is not as efficient as coherent light
This lack of consensus makes it necessary to mention whether or not light is
coherent2758
Further decisive characteristics to accomplish phototherapeutic efficacy are the power
exposure time output mode and beam area Based on these parameters both
irradiancec and radiant exposured can be calculated According to numerous authors
some of these parameters are more crucial than others to determine whether
b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48
c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48
d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248
General introduction
9
absorption of light will lead to a photobiological event192728485455 However the
literature yields several controversial findings as not all authors attribute an equal
importance to a given parameter For example according to Nussbaum et al59
irradiance was the determinant characteristic in the biomodulation of Pseudomonas
aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49
found that in order to stimulate tissue cell proliferation a specific combination of
irradiance and exposure time are more important than the actual radiant exposure Low
et al3940 on the contrary highlighted the critical importance of the radiant exposure in
observing neurophysiological effects Whereas Mendez et al60 reported that both
parameters influence the final results of light therapy
Koutna et al61 even suggested that the output mode of light applications plays a more
prominent role in the treatment outcome than the wavelength of the used light source
Nevertheless this finding could not be confirmed by other research results Besides
more controversial findings have been published regarding the output mode although
the repetition rate in a pulsed mode was considered as an important treatment
parameter several investigations failed to prove its value19272840414461-64
Based on these findings it was opted within the investigations of this doctoral thesis to
irradiate in a continuous mode The remaining dosimetric parameters (wavelength
exposure time and power) depended on the purpose of each investigation they are
described in the respective chapters The data necessary for the calculation of the
radiant exposure for the equipment used in the respective trials are summarized in
table 1
Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High
Infrared 950 80 120 160 Red 660 15 46 80
Green 570 02 42 10
10
The radiant exposure of the used LED can be calculated as follows65
RE =
Radiant Exposure [Jcm2]
T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]
PRE = α S T
α = 006 (continuous mode) or
003 (pulsed mode)
The parameters commented on so far can be considered as the external dosimetry
involving all parameters directly controlled by the operator limited by the apparatus
used Furthermore there is the so-called internal dosimetry referring to (1) several
physical phenomena (reflection transmission scattering and absorption) influencing
the light distribution within the tissue during energy transfer (2) the optical
characteristics of the irradiated tissue as well as (3) the relation between the external
dosimetry and these respective elements5466
This internal dosimetry determines to a considerable extend the penetration of light
into tissue Penetration can be defined as the tissue depth at which the radiant
exposure is reduced to 37 of its original value1948 However this definition only
accounts for the absolute penetration depth resulting in direct effects of light at that
depth In addition there is also a relative penetration depth leading up to effects
deeper in the irradiated tissue and even in certain degree throughout the entire
body1967 These so-called systemic effects can be caused by chemical processes initiated
at superficial levels at their turn mediating effects at a deeper tissue level57
Involvement of several forms of communication in the tissue such as blood circulation
and transport of transmitters or signal substances is possible1967 This means that light
sources with poor absolute penetration do not necessarily give inferior results than
those with a good absolute penetration19
In the same context it should be noted that calculation and even measurement of the
exact light distribution during irradiation is highly complicated principally as tissues
have complex structures and also because the optical properties of tissues vary largely
inter-individual2768
General introduction
11
Studies regarding actual penetration depth of LED light are scarce consequently the
knowledge on the topic of penetration depth of LED light is based on literature
originating from LLL research19 These findings established with various LLL sources
revealed that there is an obvious relation between penetration depth and
wavelength27486769-71
Three final remarks can be made on the dosimetry First of all it should be noted that
partly as a result of the above-mentioned contrasting findings on dosimetry ideal light
source characteristics for effective treatment of various medical applications are not yet
established and probably never really will be28 Therefore in the attempt to offer
sufficient guidelines for correct use of treatment parameters one should always try to
provide detailed description of light source properties used in any trial so the
practitioner can interpret the scientific results adequately and accordingly draw the
correct conclusions for his clinical practice
A second comment is based on the mentioned possible influence of the external and
internal dosimetric parameters on the photobiological effectiveness of light the
intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation
between radiated wavelength and penetration depth19546572 So it should be
emphasized that caution is recommended when comparing research results of light
sources with different wavelengths or other dissimilar dosimetric parameters
A third and final remark considers the extrapolation issue Comparison of the
therapeutic usefulness of the same light source used on different species should occur
cautiously So simply extrapolating the dosage used for one species to another is
inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo
clinical practice is inappropriate So purposive and specific research is the prerequisite
to produce safe and correct use of light as a therapeutic modality27
12
MECHANISMS OF ACTION
In the past decennia several mechanisms of action for biostimulation and pain
inhibition have been proposed and investigated73 Research was primarily based on
studies at the molecular and cellular levels and as a second resort investigations
occurred at the organism level resulting in numerous possible explanatory
mechanisms272858
It is the common view that light triggers a cascade of cellular and molecular reactions
resulting in various biological responses Thus different mechanisms of whom the
causal relationships are very difficult to establish- underlie the effects of light3448557475
To illustrate this complex matter the various mechanisms of action will be summarised
by means of a comprehensive model (fig 2) Detailed discussion about the different
individual components of the proposed model and other effects than those regarding
wound healing or analgesia were not provided as this was beyond the scope of this
general introduction
As depicted in figure 2 exposure to light leads to photon absorption by a
photoacceptor molecule causing excitation of the electronic state or increased
vibrational state of the given molecule275173 This process is followed by primary
photochemical reactions7475 Several key mechanisms have been discussed in the
literature Respiratory chain activation is the central point and can occur by an
alteration in redox properties acceleration of electron transfer generation of reactive
oxygen species (namely singlet oxygen formation and superoxide generation) as well as
by induction of local transient heating of absorbing chromophores192848515576-83 It is
supposed that each of these respective mechanisms plays a part in obtaining a
measurable biological effect It is yet not clear if one mechanism is more prominent
and decisive than another nevertheless recent experimental evidence has revealed that
mechanisms based on changes in redox properties of terminal enzymes of respiratory
chains might be of crucial importance2848517679
The primary mechanisms occurring during light exposure are followed by the dark
reactions (secondary mechanisms) occurring when the effective radiation is switched
General introduction
13
off2851 Activation of respiratory chain components is followed by the initiation of a
complicated cellular signalling cascade or a photosignal transduction and amplification
chain associated with eg changes in the cellular homeostasis alterations in ATP or
cAMP levels modulation of DNA and RNA synthesis membrane permeability
alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87
The sequence of events finally results in a range of physiological effects essential for
the promotion of the wound healing process for supplying analgesia or other
advantageous responses (acceleration of inflammatory processes oedema re-
absorption increased lymph vessel regeneration or increased nerve
regeneration)12181927486188-93
Photostimulation of the wound healing process can be mediated by increased
fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis
and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations
have been especially focussed on fibroblasts but other possible physiological effects
attributing to an accelerated wound healing were also observed suppression and
alteration of undesirable immune processes increased leukocyte activity new
formation of capillaries increased production of growth factors and enzymes while
monocytes and macrophages can provide an enlarged release of a variety of substances
related to immunity and wound healing1619277376
As pain and nociception are even less understood than wound healing the possible
mechanisms in obtaining pain relief by the use of light are less underpinned However
it is established that light therapy influences the synthesis release and metabolism of
numerous transmitter signal substances involved in analgesia such as endorphin nitric
oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these
neuropharmacological effects there is experimental evidence for diminished
inflammation decreased C-fibre activity increased blood circulation and reduced
excitability of the nervous system1927848899
One should be aware that a large amount of research regarding the possible
mechanisms of light action was conducted at the cellular level The described cascade
of reactions at the organism level is possibly even more complex as in contradiction to
14
the in vitro situation in vivo a range of supplementary interactions can influence the
sequence of effects and accordingly the final responses Besides it needs to be
mentioned that this summary did not take into account the origin of the light or the
external dosimetry thus the description is based on investigations performed with
various light sources and different dosages
Figure 2 Model summarizing the identified mechanisms of light action
Secondarymechanisms
Primary mechanisms
Final effects
Trigger
Stimulated wound healing Analgesia
Exposure to light
Photon absorption by photoacceptors
Respiratory chain activation
Accelerated electrontransfer
Reactive oxygen generation
Heating of absorbing chromophores
Altered redox properties
darr inflammation uarr oedema resorption
uarr lymph vessel regenerationuarr blood circulation
Photosignal transduction and amplification chain
uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation
darr C-fibre activity darr nervous excitability neuropharmacological effects
General introduction
15
Regardless of the large number of previous investigations identification of underlying
mechanisms of light action remains an important issue as these are not yet fully
understood and because probably not all mechanisms of action are currently
identified Convincing explanation of the mechanisms in normal as well as in
pathological tissue could banish the existing suspicion concerning the use of light as a
treatment modality2732547678
AIMS AND OUTLINE
The introduction of LED in medicine and in physiotherapy more specifically requires
particular scientific research especially within the fields of its clinical potential
application wound healing and analgesia The above described gaps in literature
regarding the use of LED laid the foundation of this doctoral thesis
Consequently the general purpose of this thesis is to explore a scientific approach for
the supposed biostimulatory and analgesic effect of LED and to formulate an answer
in view of an evidence-based clinical use of this treatment modality
The detailed objectives can be phrased as follows
Aim 1 To assess the biostimulatory effectiveness of LED
irradiation under normal in vitro conditions
Aim 2 To investigate the value of LED treatment to ameliorate
in vitro cell proliferation under conditions of impaired healing
Aim 3 To examine the effectiveness of LED in changing the
nerve conduction characteristics in view of analgesia
Aim 4 To determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting
Part I investigates the influence of LED on wound healing In pursuit of the first aim
chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial
16
measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these
cells needs to be considered as an exponent of the wound healing process as
fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase
and early remodelling100 Secondly an in vivo case study exploring the postulation that
LED irradiation could accelerate and ameliorate the healing of a surgical incision was
described
The results contrasted sharply with the findings of the in vitro part Two fundamental
causes were proposed in order to explain the different biological effect of LED
irradiation observed in vitro and in vivo the used irradiation parameters and evaluation
method
The experiment described in chapter 2 endeavoured to explore these considerations A
similar study was therefore performed but as distinctive characteristics different light
source properties an adapted irradiation procedure and the use of a colorimetric assay
based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the
counting of the cells were used
As stimulation of the wound healing process is virtually mainly indicated under
conditions of impaired healing (resulting in a situation which threatens to become
chronic and debilitating) proper attention for this matter is warranted192855 Besides
the medical consequences the costs involved with impaired healing yield also a socially
relevant problem to tackle Impaired healing will become even more common as the
world population continues to age After all senescence of systems and age-committed
comorbid conditions are commonly the culprits responsible for poor wound healing101
Thus finding cost-effective time-sparing non-invasive and practical treatment
modalities to cure wounds is a necessity
Aiming to assess the biostimulative effects by means of LED in these circumstances a
third study was conducted with respect of the previous results regarding irradiation
parameters and cell proliferation analysis The irradiation experiment described in
chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose
This medium modification serves as a pattern for cell proliferation in diabetic patients
General introduction
17
a population for whom stimulation of the wound healing process is a clinical relevant
feature
In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored
by means of two studies A first investigation (chapter 4) evaluated the influence of LED
on the sensory nerve conduction characteristics of a human superficial peripheral
nerve as a potential explanatory mechanism of pain inhibition by LED which is based
on the putative neurophysiological effects of this treatment modality The experimental
hypothesis postulated that LED generates an immediate decrease in conduction
velocity and increase in negative peak latency In addition it was postulated that this
effect is most prominent immediately after the irradiation and will weaken as time
progresses
The values of nerve conduction velocity and negative peak latency of a baseline
antidromic nerve conduction measurement were compared with the results of five
identical recordings performed at several points of time after LED irradiation
Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory
setting To guarantee an adequate standardized and controlled pain reduction study
there was opted to observe a healthy population with experimentally induced DOMS
Induction of DOMS has been described in a number of studies as a representative
model of musculoskeletal pain and stiffness because it can be induced in a relatively
easy and standardised manner the time course is quite predictable and the symptoms
have the same aetiology and are of transitory nature4445102-105
The treatment as well as the assessment procedure was performed during 4
consecutive days The first day isokinetic exercise was performed to induce pain
related to DOMS Subsequently the volunteers of the experimental group received an
infrared LED treatment and those of the placebo group received sham-irradiation
Evaluation of the effect of the treatment on perceived pain was registered by a visual
analog scale and by a mechanical pain threshold these observations occurred every day
18
prior to and following LED irradiation Eccentricconcentric isokinetic peak torque
assessment took place daily before each treatment
For the analysis of the results three different factors were taken into consideration
time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental)
In completion of this thesis the most prominent findings are summarized and the
clinical implications are discussed The general discussion also includes some future
research directions and a final conclusion
General introduction
19
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neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3)340-345
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20
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Eur J Phys Med Rehabil 7(4)102-105 32 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D
Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15
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34 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
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41 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490
General introduction
21
42 Baxter G Effect of combined phototherapylow intensity laser therapy upon experimental ischaemic pain Potential relevance of experimental design 14th World Congress Physical Therapy Barcelona Spain 2004 Proceedings CD
43 Craig J Barron J Walsh D and Baxter G (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
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45 Glasgow P Hill I McKevitt A Lowe A and Baxter G (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1)33-39
46 Noble J Lowe A Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
47 IEC 60825-1 (1993) International Electrotechnical Commission Safety of laser products Part 1 Equipment classification requirements and users guide
48 Nussbaum E Baxter G and Lilge L (2003) A review of laser technology and light-tissue interactions as a background to therapeutic applications of low intensity lasers and other light sources Phys Ther Rev 831-44
49 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537
50 Nakamura S (1998) The roles of structural imperfections in InGaN-based blue light-emitting diodes and laser diodes Science 281956-961
51 Karu Tiina (consulted on 2005 august 22) Cellular mechanism of low-power laser therapy httpwwwtinnitusustiinakarupresentationhtml
52 Mester E Mester A and Mester A (1985) The biomedical effects of laser application Lasers Surg Med 5(1)31-39
53 Moore K Calderhead G (1991) The clinical application of low incident power density 830 nm GaAlAs diode laser radiation in the therapy of chronic intractable pain A historical and optoelectronic rationale and clinical review Int J Optoelectron 6503-520
54 King P (1989) Low level laser therapy A review Lasers Med Sci 4141-148 55 Karu T (1989) Photobiology of low-power laser effects Health Phys 56(5)691-704 56 Simunovic Z (2000) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical
Application of Low Energy-Laser Laser Therapy LLLT Rijeka Vitgraf 57 Hode L and Tuner J (2000) Low level laser therapy (LLLT) contra light emitting diode therapy
(LEDT) - What is the difference Proceedings of SPIE 416690-97 58 Kitchen S and Partridge C (1991) A review of low level laser therapy Part I Background
physiological effects and hazards Physiotherapy 77(3)161-163 59 Nussbaum E Lilge L and Mazzulli T (2003) Effects of low-level laser therapy (LLLT) of 810 nm
upon in vitro growth of bacteria Relevance of irradiance and radiant exposure J Clin Laser Med Sur 21(5)283-290
60 Mendez T Pinheiro A Pacheco M Nascimento P and Ramalho L (2004) Dose and wavelength of laser light have influence on the repair of cutaneous wounds J Clin Laser Med Sur 22(1)19-25
61 Koutna M Janisch R and Veselska R (2003) Effects of low-power laser irradiation on cell proliferation Scripta Medica 76(3)163-172
62 Al-Watban F and Zhang X (2004) The comparison of effects between pulsed and CW lasers on wound healing J Clin Laser Med Sur 22(1)15-18
63 Panjehpour M Overholt B DeNovo R Petersen M and Sneed R (1993) Comparative study between pulsed and continuous wave lasers for photofrin photodynamic therapy Lasers Surg Med 13(3)296-304
64 Dyson M (2005) The significance of pulsing in the stimulation of tissue repair by light A review Lasers Med Sci 20S21
22
65 MDB Laser (2000) Bio Dio (Preprotoype) User guide Belgium Ekeren 66 Fisher J (1992) Photons physiatrics and physicians A practical guide to understanding laser light
interaction with living tissue Part I J Clin Laser Med Sur 10(6)419-426 67 Tuner J and Hode L (2000) Depth of penetration of laser light in tissue Laser Partner
Clinixperience 151-3 68 Anderson R and Parrish J (1981) The optics of human skin J Invest Dermatol 77(1)13-19 69 De Cuyper H and Lambert H (1991) Penetration depth of infrared- and helium-neon-laserlight
An assessment in vivo Eur J Phys Med Rehabil 1(5)133-137 70 Oshiro T Ogata H Yoshida M Tanaka Y Sasaki K and Yoshimi K (1996) Penetration depths
of 830nm diode laser irradiation in the head and neck assessed using a radiographic phanom model and wavelength-specific imaging film Laser Ther 8197-204
71 Kolarova H Ditrichova D and Wagner J (1999) Penetration of the laser light into the skin in vitro Lasers Surg Med 24(3)231-235
72 Chen Q Huang Z Chen H Shapiro H Beckers J and Hetzel F (2002) Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy Photochem Photobiol 76(2)197ndash203
73 Conlan M Rapley J and Cobb C (1996) Biostimulation of wound healing by low-energy laser irradiation A review J Clin Periodontol 23(5)492-496
74 Parrish J (1981) New concepts in therapeutic photomedicine photochemistry optical targeting and the therapeutic window J Invest Dermatol 7745-50
75 Smith K (1981) Photobiology and photomedicine the future is bright J Invest Dermatol 772-7 76 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees D (1989) Systemic effects
of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
77 Polo L Presti F Schindl A Schindl L Jori G and Bertoloni G (1999) Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth Biochem Biophys Res Commun 257(3)753-758
78 Bertoloni G Sacchetto R Baro E Ceccherelli F and Jori G (1993) Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 6328 nm light J Photochem Photobiol B-Biol 18191-196
79 Lubart R Friedmann H Sinykov M and Grossman N (1995) Biostimulation of photosensitized fibroblasts by low incident levels of visible light energy Laser Ther 7101-106
80 Harris D (1991) Biomolecular mechanisms of laser biostimulation J Clin Laser Med Sur 9277-280
81 Grossman N Schneid N Reuveni H Halevy S and Lubart R (1998) 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures involvement of reactive oxygen species Lasers Surg Med 22212-218
82 Oren D Charney D Lavi R Sinyakov M and Lubart R (2001) Stimulation of reactive oxygen species production by an antidepressant visible light source Biol Psychiatry 49464-467
83 Lavi R Shainberg A Friedmann H Shneyvays V Rickover O Eichler M Kaplan D and Lubart R (2003) Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells 278(42)40917-40922
84 Reddy K (2004) Photobiological basis and clinical role of low-intensity lasers in biology and medicine J Clin Laser Med Sur 22(2)141-150
85 Greco M Guida G Perlino E Marra E and Quagliariello E (1989) Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser Biochem Biophys Res Commun 163(3)1428-1434
86 Vacca R Marra E Quagliariello E and Greco M (1993) Activation of mitochondrial DNA replication by He-Ne laser irradiation Biochem Biophys Res Commun 195(2)704-709
87 Vacca R Marra E Quagliariello E and Greco M (1994) Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser Biochem Biophys Res Commun 203(2)991-997
88 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral
General introduction
23
nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
89 Anders J Borke R Woolery S and Van de Merwe W (1993) Low power laser irradiation alters the rate of regeneration of the rat facial nerve Lasers Surg Med 13(1)72-82
90 Rochkind S Nissan M Barr-Nea L Razon N Schwartz M and Bartal A (1987) Response of peripheral nerve to He-Ne laser Experimental studies Lasers Surg Med 7441-443
91 Lievens P (1986) Lasertherapie (deel II) De invloed van laser-bestralingen op het lymfesysteem en de wondheling Ned T Fysiotherapie 96140-142
92 Lievens P (1991) The effect of a combined HeNe and IR laser treatment on the regeneration of the lymphatic sysytem during the process of wound healing Lasers Med Sci 6193-199
93 Lievens P (1991) The effect of IR laser irradiation on the vasomotricity of the lymphatic system Lasers Med Sci 6189-191
94 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin J and Martin P (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1)171-178
95 Bosatra M Jucci A Olliaro P Quacci D and Sacchi S (1984) In vitro fibroblast and dermis fibroblast activation by laser irradiation at low energy An electron microscopic study Dermatologica 168(4)157-162
96 Enwemeka C Rodriquez O Gall N and Walsh N (1990) Morphometrics of collagen fibril populations in HeNe laser photostimulated tendons J Clin Laser Med Sur 847-52
97 Abergel P Lam T Meeker C Castel C Dwyer R and Uitto J (1984) Biostimulation of procollagen production by low energy lasers in human skin fibroblast culturesJ Invest Dermatol 82(4)395
98 Abergel P Lam T Meeker C Dwyer R and Uitto J (1984) Low energy lasers stimulate collagen production in human skin fibroblast cultures Clin Res 32(1)16A
99 Shimoyama N Lijima K Shimoyama M and Mizuguchi T (1992) The effects of helium-neon laser on formalin-induced activity of dorsal horn neurons in the rat J Clin Laser Med Sur 1091-94
100 Kloth L McCulloch J and Feedar J (1990) Wound healing Alternatives in management USA FA Davis Company
101 Lanzafame R (2004) Revolutions and revelations J Clin Laser Med Surg 22(1)1-2 102 Ciccone C Leggin B and Callamaro J (1991) Effects of ultrasound and trolamine salicylate
phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-678 103 Craig J Cunningham M Walsh D Baxter G and Allen J (1996) Lack of effect of transcutaneous
electrical nerve stimulation upon experimentally induced delayed onset muscle soreness in humans Pain 67(2-3)285-289
104 Minder P Noble J Alves-Guerreiro J Hill I Lowe A Walsh D and Baxter G (2002) Interferential therapy Lack of effect upon experimentally induced delayed onset muscle soreness Clin Physiol Funct Imaging 22(5)339-347
105 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
PART I WOUND HEALING
CHAPTER 1
DO INFRARED LIGHT EMITTING DIODES HAVE A
STIMULATORY EFFECT ON WOUND HEALING FROM AN IN
VITRO TRIAL TO A PATIENT TREATMENT
Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Proceedings of SPIE 2002 4903 156-165
Chapter 1
28
ABSTRACT
Variable effects of different forms of light therapy on wound healing have been
reported This preliminary study covers the efficacy of infrared light emitting diodes
(LED) in this domain
Cultured embryonic chicken fibroblasts were treated in a controlled randomised
manner LED irradiation was performed three consecutive days with a wavelength of
950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each
treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2
The results indicated that LED treatment does not influence fibroblast proliferation at
the applied energy density and irradiation frequency (p=0474)
Meanwhile the effects of LED on wound healing in vivo were studied by treating a
surgical incision (6 cm) on the lateral side of the right foot of a male patient The
treatment started after 13 days when initial stitches were removed The same
parameters as the in vitro study were used but the treatment was performed five times
The healing could only be evaluated clinically the irradiated area (26 cm) showed a
more appropriate contraction less discoloration and a less hypertrophic scar than the
control area (34 cm)
The used parameters failed to demonstrate any biological effect of LED irradiation in
vitro although the case study on the other hand illustrated a beneficial effect
Keywords Light Emitting Diodes Fibroblasts Wound healing
From an in vitro trial to a patient treatment
29
INTRODUCTION
Various beneficial effects of lasers and photodiodes at relatively low intensities have
been reported involving treatment of neurological impairments12 treatment of pain3-5
treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the
enhancement of wound healing has been a focus of contemporary research11-16 It
seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing
remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the
basis of a questionnaire about low power laser (LPL) in the current clinical practice in
Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey
into clinical LPL experience in Flanders
Nevertheless there remains a considerable amount of ignorance scepticism and
controversial issues concerning the use and clinical efficacy of LPL even in the domain
of wound healing12152021 This is at least in part a consequence of the inability to
measure and control operating variables related to connective tissue repair and of the
wide range of suitable parameters for irradiation
Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent
characteristics which make their use in a clinical setting problematic including
limitations in wavelength capabilities and beam width The combined wavelength of
light optimal for wound healing cannot be efficiently produced and the size of
wounds which may be treated by LPLs is limited Some companies offer an
alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be
made to produce multiple wavelengths and can have probes with large surface area
allowing treatment of large wounds Still one can not accept this light source as an
alternative for LPL therapy based on the cited advantages without proper investigation
regarding its biostimulatory effects
The effectiveness of this possible alternative for LPLs must be studied in vitro and in
addition in animal models or in humans because the effects of LED at the cellular level
do not necessarily translate to a noticeable effect in vivo The small amount of previous
investigations demonstrate that LED effects are as difficult to isolate162223 as LPL
Chapter 1
30
effects and the results are conflicting just like the results in literature specific on the
use of LPL121520
The purpose of the first part of this study is to examine the hypothesis stating that
LED irradiation can influence fibroblast proliferation Therefore a comparison of the
proliferation from fibroblasts in irradiated and control wells was performed The in vitro
investigation was linked with an in vivo case study This part enquired the assumption if
LED irradiation could accelerate and ameliorate the healing of a surgical incision
IN VITRO INVESTIGATION
MATERIALS AND METHODS
The complete procedure from isolation to proliferation analysis was executed twice
(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of
control dishes The second trial consisted of 27 irradiated and 27 control dishes
Cell isolation and culture procedures
Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation
and disaggregating of the cells occurred with warm trypsin (NV Life Technologies
Belgium) according the protocol described by Ian Freshney (1994)24 The primary
explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies
Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1
Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life
Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies
Belgium) When cell growth from the explants reached confluence cells were detached
with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV
Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the
cells were removed from the culture flasks by trypsinization and counted by
hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc
From an in vitro trial to a patient treatment
31
NV Life Technologies Belgium) The fibroblasts were seeded at a density of
70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture
medium the cells were allowed to attach for 24 hours in a humidified incubator at
37degC
Properties of the Light Emitting Diode
Prior to LED treatment all dishes were microscopically checked to guarantee that the
cells are adherent and to assure that there is no confluence nor contamination The
dishes were divided randomly into the treated or the control group Medium was then
removed by tipping the dishes and aspirating with a sterile pipette Following the
aspiration 2 ml fresh medium was added and treatment started
A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser
Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-
1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32
single LEDrsquos For the treatments in this study an average power of 160 mW at
continuous mode was applied The irradiation lasted 6 minutes resulting in an energy
density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result
of the divergence in function of this distance the surface of the LED (18 cm2) covered
the complete surface of the used petriplates (215 cm2)
After these manipulations 3 ml medium was added to each dish followed by 24 hours
incubation
One LED irradiation was performed daily during three consecutive days according
this procedure Control cultures underwent the same handling during these three days
but were sham-irradiated
Proliferation analysis
After the last treatment a trypsination was performed to detach the cells from the
culture dishes followed by centrifugation Once the cells were isolated from the used
trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts
Chapter 1
32
within this suspension as reflection for the proliferation was quantified by means of a
Buumlrker Chamber or hemocytometry
The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-
Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab
Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on
the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator
using an inverted light microscope counted the number of cells in 25 small squares
In order to calculate the number of cells one should multiply the amount of cells
counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and
the dilution factor (the amount of Trypan blue suspended with the cells 21=3)
Statistical methods
The data were analysed statistical in order to examine the hypothesis that LED
irradiation enhances fibroblast proliferation They were processed as absolute figures
for both trials separately In a second phase the counted cell numbers were converted
in relative figures so the data of both trials could be analysed as the data of one test
These relative figures were obtained by expressing each figure as a percentage from the
highest figure (=100) of that trial and this for each assay separately
A Kolmogorov-Smirnoff test of normality was performed on the data followed by a
Mann-Whitney-U test when the test of normality was significant and otherwise a T-
test Differences were accepted as significant when plt005 For this analysis SPSSreg
100 was used
RESULTS
The descriptive data for both trials are depicted in figure I The mean number of cells
in trial A is higher than in trial B for the controls as for the treated wells There is a
mean difference of 1252500 fibroblasts between the controls and 1223000 between
the irradiated wells of trial A and B The averages of both trials show that in control
cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no
From an in vitro trial to a patient treatment
33
statistically significant difference could be found between the two groups in either trial
nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not
significant for trial A (p=020) nor trial B (p=020) Only the combined data from both
trials were significant (plt001) for normality Further analysis respectively T-test for
the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the
combined data (p=0474) revealed no statistical significant differences
DESCRIPTIVE DATA
1730000181750029530003070000
00E+00
50E+05
10E+06
15E+06
20E+06
25E+06
30E+06
35E+06
40E+06
Trial A Trial A Trial B Trial B
Mea
n n
um
ber
of
cells
Control
Irradiated
Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B
DISCUSSION
Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities
(lt500 mW) in vitro have been analysed by evaluating various factors involving
(pro)collagen production25-27 cell viability2829 growth factor production28 and
myofibroblast formation30 Fibroblast proliferation also is an important factor to
consider In accordance with wound healing fibroblasts fulfil an essential role especially
in the late inflammatory phase and the early granulation phase31 Despite the failure of
some studies to demonstrate beneficial effects of LPL irradiation on fibroblast
proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very
Chapter 1
34
significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -
208 ) Atabey et al35 also revealed a significant increase in cell number two or more
irradiations resulted in an increased fibroblast proliferation Several other studies
confirmed these positive findings25263637
The results of this present in vitro study indicate that LED treatment does not
influence fibroblast proliferation Although the dosimetric parameters (in particular the
arbitrary energy density of 32 Jcm2) used in this study are well within the
recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous
studies about LPL therapy raising enhanced fibroblast proliferation252634-37
Van Breugel et al36 gave a possible explanation for these controversial results
According to them the fibroblast proliferation is not inherent at the energy density
They provide evidence that independent of the energy density the power density and
the exposure time determine the biostimulative effects of LPL irradiation LPL with a
power below 291 mW could enhance cell proliferation while a higher power had no
effect
Some authors also argued that the absorption spectrum of human fibroblasts show
several absorption peaks and pointed out that a wavelength of 950 nm is far above the
highest peak of about 730 nm3638 At longer wavelengths they determined a general
decrease in absorption Despite these results several investigators pose biostimulative
effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or
even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is
ranged from 800 nm to 830 nm principally because of the presence of cytochrome
oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts
at lower wavelengths is restricted by an inferior skin transmission than at higher
wavelengths38
Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting
respective green and red light The 950 nm beam of light was used for its high power
density but according to a range of remarks mentioned above the effects of the two
other probes must be as well evaluated
From an in vitro trial to a patient treatment
35
Another factor one can not ignore is that besides fibroblast proliferation other
processes or morphologic changes were not analysed although several authors have
posed that those changes and processes could be responsible for the biostimulative
effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et
al30 for example described a massive transformation of fibroblasts into myofibroblasts
after LPL treatment These modified fibroblasts play an important role in contraction
of granulation tissue30 A second example is an increased (pro)collagen production
after low power light therapy25-27 which is also considered as a responsible factor for
accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast
proliferation3640
It may be wondered if the light sources mostly LPL in the consulted literature are
representative for the LED used in this study although this LPL literature is often
used for that purpose As in the early days of LPL the stimulative effects upon
biological objects were explained by its coherence the beam emitted by the Bio-Dio on
the contrary produces incoherent light Nowadays contradictory research results are
responsible for a new discussion the clinical and biological significance of coherence
The findings of some authors172341-43 pose that the coherence of light is of no
importance of LPL and its effects although the opposite has also been stated4445
Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be
accounted for the non-enhanced fibroblast proliferation in this trial
Another possible explanation for the absence of biostimulative effect is related to the
moment of analysis of the proliferation The evaluation one day after the last
irradiation did not allow a delayed enhancement of proliferation while it is determined
in numerous investigations that the effects occur more than 24 hours after the last
treatment273746 and that they weaken after a further undefined period of time34
The fluctuation in cell numbers between both trials despite the use of an identical
protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor
reproducibility of their technique In this study the fluctuations are attributable to the
counting of the cells by Buumlrker hemocytometer before seeding According to some
authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large
Chapter 1
36
variability48 and it is often difficult to standardize48 Overestimation of the cell
concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity
was contradicted by Lin et al50 moreover satisfactory correlations with flow-
cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay
for cell counting (MTT)51 were determined
An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it
provides accurate cell counts in a short period of time with less intervention from the
investigator52
These remarks and controversies point out the possible deficiencies from the used
proliferation analyses and the relativity from the obtained results Other analyse
methods and analyses from different cell processes and morphologic changes could be
considered for further investigation
IN VIVO INVESTIGATION
MATERIALS AND METHODS
The effects of LED on wound healing in vivo were studied by treating a postsurgical
incision A male patient received chirurgical treatment for the removal of a cyst
situated approximately 15 cm posterior from the lateral malleolus of his right foot For
removal of the cyst an incision of 6 cm was made The incision was sutured and 12
days after the surgery the stitches were removed Visual inspection demonstrated that
the healing process of the wound proceeded well but not equally over the whole 6
centimetres (figure II) Epithelialization and wound contraction appeared to have
progressed better in the upper part (approximately 3 cm) of the cicatrice than at the
lower part (covered with eschar) No evidence of infection was noted in either part
LED treatment started the 13th day The incision was treated partially the lowest part
(26 cm) with the inferior epithelialization and wound contraction was irradiated the
remaining 34 cm served as control area This control area was screened from radiation
with cardboard and opaque black cling film
From an in vitro trial to a patient treatment
37
The light source destinated for the treatment was the same device used for the in vitro
irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED
output parameters were identical with those applicated in the preceding in vitro
investigation In particular a continuous wave at an average power of 160 mW and 6
minutes of treatment duration corresponding to an energy density of 32 Jcm2 An
equal distance from the probe to the target tissue as from the probe to the culture
medium was respected A plastic applicant of according height guaranteed constant
distance of 06 cm from the surface of the skin
Figure II Surgical incision before the first treatment 13 days after initial stitching
Therapy was performed once a day during five consecutive days repeatedly at the same
time resulting in an extension of the duration of the in vitro therapy with two days
Visual macroscopic observations were accomplished 6 52 and 175 days after the first
treatment
Comparison of the cutaneous sensitivity at the irradiated area and the control area was
accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175
days after the first treatment A control measurement also occurred at the same region
Chapter 1
38
on the left foot The aesthesiometer used in this study consisted of five hand-held
nylon monofilaments with a length of 38 mm and varying diameter
Sensitivity threshold is traced by presenting a monofilament of a certain diameter
vertically to the skin The monofilament bends when a specific pressure has been
reached with a velocity proportional to its diameter Measurements allow mapping
areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified
from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest
filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished
protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to
finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the
filaments
RESULTS
Visual estimation at any point of time after irradiation divulged no occurrence of
problems with dehiscence or infection in either part of the wound During the five
days of therapy the irradiated area looked dryer than the control area After the last
irradiation this was no longer recorded
Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area
From an in vitro trial to a patient treatment
39
Figure III representing the first evaluation six days after the initial treatment
illustrates that the wound healing has evolved slightly in both parts Though the lower
irradiated part remains of inferior quality as regards to epithelialization and wound
contraction In the course of the reparative process the influence of light exposures
were registered At 52 days after the first irradiation beneficial effects of LED
treatment are clearly present (Figure IV)
Figure IV Surgical incision 52 days after initiating LED treatment
The irradiated area (26 cm) showed a more appropriate contracture than the control
area (34 cm) characterized by less discoloration at scar level and a less hypertrophic
scar A similar trend was noticed at a third visual observation 175 days after the initial
treatment At that moment no impairments at cutaneous sensitivity level were stated
and the sensitivity showed no differences between left or right foot nor between the
two areas of the cicatrice
Chapter 1
40
DISCUSSION
The results of this case study indicate that LED had a positive influence on wound
healing in humans as determined by visual observations Many investigators
examining the effects of LPL on wound healing by means of a range of observation
and treatment methods reported accelerated and enhanced wound healing8-10 others
described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and
LED21 studies were unable to repeat these results
The late but beneficial findings in this study seem to be to the credit of LED-therapy
Though several authors establish positive results in an earlier stage of the wound
healing process8-1020 one should question why the differences did not occur at the first
evaluation on day 6 An explanation can be found in the start of the treatment Most
investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so
they influence a first cellular and vascular reaction with the production of chemical
mediators of inflammation resulting in an enhanced collagen production9 tremendous
proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the
time the first treatment in this study took place the traumatized tissue was in an
overlapping stage between an almost finished inflammatory phase and a scarcely
initiated re-epithelialization and wound contraction phase At that moment an infiltrate
of fibroblasts is present So fibroblast proliferation a possible mechanism of the
biostimulative effect had already occurred and could no longer be influenced Growth
factor production and collagen deposition have also decreased at that stage
Granulation tissue formation and fibroplasia in the contrary are initiating by that time
Those prolonged and slow processes with belated results are of significant importance
for the course of the final stage of wound healing and for the outlook of the future
scar31
The experimental findings revealed that the sensitivity of the skin according to the
threshold detection method of Semmes and Weinstein was normal at all the
investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a
good and objective predictor of normal skin sensitivity No other LPL nor LED
studies investigating this quality of the skin were found
From an in vitro trial to a patient treatment
41
CONCLUSION
This study demonstrates that although LED application at the applied energy density
and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to
have beneficial biostimulative effects on wound healing in human skin confirmed by
the favourable re-epithelialization and contracture
These results are discussed in the context of other experimental findings but no
reasonable explanation for this discrepancy could be found The literature on wound
healing after LED treatment in animal models or in humans is presently very limited
and contradictory The diversity in used radiation parameters and the absence of
references on how the wounds were measured or evaluated or what the end point was
for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro
investigations are better standardised nevertheless these results show a number of
conflicts One can conclude that until today the controversial findings are characteristic
for many results obtained with light photobiomodulation
However the postponed favourable results in the case study confirm some facts of the
discussion Namely the short period of incubation 24 hours in the in vitro part of the
study can be responsible for the lack of enhanced fibroblast proliferation It also
confirms that other cell processes and morphologic changes possibly are responsible
for biostimulative effects in vivo other observation methods should be considered for
future in vivo experiments
Despite these remarks we believe that LED application on cutaneous wounds of
human skin is useful with a single flash daily at the dose applied in this study for at
least three days
Furthermore future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Chapter 1
42
ACKNOWLEDGMENTS
The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the
material necessary for this study as well as Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
From an in vitro trial to a patient treatment
43
REFERENCES
1 G Baxter D Walsh J Allen A Lowe and A Bell Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79(2) 227-34 (1994)
2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)
3 J Stelian I Gil B Habot et al Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy J Am Geriatr Soc 40(1) 23-6 (1992)
4 A Honmura A Ishii M Yanase J Obata and E Haruki Analgesic effect of Ga-Al-As diode laser irradiation on hyperalgesia in carrageenin-induced inflammation Lasers Surg Med 13(4) 463-9 (1993)
5 D Cambier K Blom E Witvrouw G Ollevier M De Muynck and G Vanderstraeten The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15 195-200 (2000)
6 G Reddy L Stehno Bittel and C Enwemeka Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-7 (1998)
7 K Moore N Hira I Broome and J Cruikshank The effect of infrared diode laser irradiation on the duration and severity of postoperative pain a double blind trial Laser Therapy 4 145-148 (1992)
8 A Amir A Solomon S Giler M Cordoba and D Hauben The influence of helium-neon laser irradiation on the viability of skin flaps in the rat Br J Plast Surg 53(1) 58-62 (2000)
9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)
10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)
11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)
12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)
13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)
14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)
15 A Schlager P Kronberger F Petschke and H Ulmer Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1) 39-42 (2000)
16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)
17 GD Baxter and J Allen Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone (1994)
18 G Baxter A Bell J Allen and J Ravey Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77(3) 171-178 (1991)
19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)
20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)
Chapter 1
44
21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)
22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)
23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)
24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)
25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)
26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)
27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)
28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)
29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)
30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)
31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)
32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)
33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)
34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)
35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)
36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)
37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)
38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)
39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)
40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)
41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)
42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)
From an in vitro trial to a patient treatment
45
43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)
44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)
45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)
46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)
47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)
48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)
49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)
50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)
51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)
52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)
53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)
54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)
CHAPTER 2
INCREASED FIBROBLAST PROLIFERATION INDUCED BY
LIGHT EMITTING DIODE AND LOW LEVEL LASER
IRRADIATION
Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Lasers in Medical Science 2003 18(2) 95-99
Chapter 2
48
ABSTRACT
Background and Objective As Light Emitting Diode (LED) devices are
commercially introduced as an alternative for Low Level Laser (LLL) Therapy the
ability of LED in influencing wound healing processes at cellular level was examined
Study DesignMaterials and Methods Cultured fibroblasts were treated in a
controlled randomized manner during three consecutive days either with a infrared
LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and
570 nm) and respective power outputs Treatment duration varied in relation to
varying surface energy densities (radiant exposures)
Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all
irradiated cultures in comparison with the controls Green light yielded a significantly
higher number of cells than red (plt0001) and infrared LED light (plt0001) and than
the cultures irradiated with the LLL (plt0001) the red probe provided a higher
increase (plt0001) than the infrared LED probe and than the LLL source
Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation
in vitro This study therefore postulates possible stimulatory effects on wound healing
in vivo at the applied dosimetric parameters
Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low
Level Laser Tetrazolium salt
LED induced increase of fibroblast proliferation
49
INTRODUCTION
Since the introduction of photobiostimulation into medicine the effectiveness and
applicability of a variety of light sources in the treatment of a wide range of medical
conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results
of several investigations are remarkably contradictory This is at least in part a
consequence of the wide range of indications as well as the wide range of suitable
parameters for irradiation and even the inability to measure the possible effects after
irradiation with the necessary objectivity [457] A lack of theoretical understanding
can also be responsible for the existing controversies In fact theoretical understanding
of the mechanisms is not necessary to establish effects though it is necessary to
simplify the evaluation and interpretation of the obtained results As a consequence
the widespread acceptance of especially Low Level Laser (LLL) therapy in the early
seventies is faded nowadays and biostimulation by light is often viewed with scepticism
[8] According to Baxter [49] contemporary research and consumption in
physiotherapy is in particular focused on the stimulation of wound healing Tissue
repair and healing of injured skin are complex processes that involve a dynamic series
of events including coagulation inflammation granulation tissue formation wound
contraction and tissue remodelling [10] This complexity aggravates research within this
cardinal indication
Research in this domain mostly covers LLL studies but the current commercial
availability of other light sources appeals research to investigate as well the effects of
those alternative light sources eg Light Emitting Diode (LED) apparatus
The scarcity of literature on LED is responsible for consultation of literature
originating from LLL studies [11] but it may be wondered if this literature is
representative for that purpose As in the early days of LLL therapy the stimulating
effects upon biological objects were explained by its coherence [1213] while the beam
emitted by LEDrsquos on the contrary produces incoherent light Though the findings of
some scientists [914151617] pose nowadays that the coherence of the light beam is
not responsible for the effects of LLL therapy Given that the cardinal difference
between LED and LLL therapy coherence is not of remarkable importance in
Chapter 2
50
providing biological response in cellular monolayers [5] one may consult literature
from LLL studies to refer to in this LED studies
The purpose of this preliminary study is to examine the hypothesis that LED
irradiation at specific output parameters can influence fibroblast proliferation
Therefore irradiated fibroblasts cultures were compared with controls The article
reports the findings of this study in an attempt to promote further discussion and
establish the use of LED
MATERIALS AND METHODS
Cell isolation and culture procedures
Fibroblasts were obtained from 8-days old chicken embryos Isolation and
disaggregation of the cells was performed with warm trypsin according the protocol
described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in
Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1
L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants
reached confluence cells were detached with trypsine and subcultured during 24 hours
in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours
the cells were removed from the culture flasks by trypsinization and counted by Buumlrker
hemocytometry For the experiment cells from the third passage were plated in 96-well
plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a
density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C
during 24 hours
All supplies for cell culture were delivered by NV Life Technologies Belgium except
for Fetal Calf Serum (Invitrogen Corporation UK)
Irradiation sources
In this study two light sources a Light Emitting Diode (LED) device and a Low Level
Laser (LLL) device were used in comparison to control cultures
The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)
LED induced increase of fibroblast proliferation
51
with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400
mW and a frequency range from 0-1500 Hz
The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)
consisted of three wavelengths emitted by separate probes A first probe emitting
green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the
red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third
probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared
light The area of all three probes was 18 cm2 and their frequency was variable within
the range of 0-1500 Hz
Exposure regime
Prior to irradiation the 96-well plates were microscopically verified to guarantee that
the cells were adherent and to assure that there was no confluence nor contamination
Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining
25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation
The 96-well plates were randomly assigned in the treated (LLL or green red or infrared
LEDrsquos) or the control group
For the treatments in this study the continuous mode was applied as well for the LLL
as for the three LED-probes The distance from light source to fibroblasts was 06 cm
LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting
in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant
exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to
exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective
power output of 160 mW 80 mW or 10 mW
After these handlings the remaining medium was removed and new Hanksrsquoculture
medium was added followed by 24 hours of incubation
One irradiation (LLL or LED) was performed daily during three consecutive days
according to the aforementioned procedure Control cultures underwent the same
handling but were sham-irradiated
Chapter 2
52
Determination of cell proliferation
The number of cells within the 96-well plates as a measure for repair [19] was
quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The
colorimetric assay was performed at two different points of time to determine the
duration of the effect of the used light sources
This assay exists of a replacement of Hanksrsquoculture medium by fresh medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as
described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT
solution was substituted by lysing buffer isopropyl alcohol The plates were
temporarily shaken to allow dissolution of the produced formazan crystals After 30
minutes of exposure to the lysing buffer absorbance was measured The absorbance at
400 to 750 nm which was proportional to fibroblast proliferation was determined
using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)
The complete procedure from isolation to MTT assay was executed six times (Trial A
B C D E and F) while it was impossible to irradiate all the investigated number of
wells with the same LED apparatus on one day All the trials included as much control
as irradiated wells but the number of control and irradiated wells in each trial varied
depending on the number of available cells after the second subculturing A further
consequence of the available number of cells is the number of probes examined per
trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E
Incubation period before proliferation analyses numbered 24 hours To investigate if
the stimulatory effect tends to occur immediately after irradiation or after a longer
period of time incubation in trial F lasted 72 hours
An overview of the followed procedures regarding incubation time before proliferation
analysis number of analysed wells for each trial and the number of probes examined
per trial is given in table 1 As a consequence of the differences in procedures followed
and because each trial started from a new cell line the results of the five trials must be
discussed separately
LED induced increase of fibroblast proliferation
53
Statistical analysis
Depending on the amount of groups to be compared within each trial and depending
on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way
ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U
test was used for nonparametrical comparisons Statistical significance for all tests was
accepted at the 005 level For this analysis Statistical Package for Social Sciences 100
(SPSS 100) was used
RESULTS
The results presented in table 1 show that cell counts by means of MTT assay
revealed a significant (plt0001) increase in the number of cells in comparison to their
respective sham-irradiated controls for all the irradiated cultures of trial A B C D
and E except the irradiated groups in trial F
Moreover the results of trial A showed that the effect of the green and red LED probe
was significantly (plt0001) higher than the effect of the LLL probe With regard to the
amount of proliferation the green probe yielded a significantly higher number of cells
than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe
provided a higher increase in cells (plt0001) than the infrared probe
The infrared LED source and the LLL provided a significant (plt0001) higher number
of cells than the control cultures but no statistical significant difference was recorded
between both light sources
The trials A B C D and E regardless of the number of probes used in each trial
were analysed after 24 hours of incubation after the last irradiation The incubation
period of trial F lasted 72 hours
The means of trial F illustrated that the effect was opposite after such a long
incubation The control cultures had significantly (plt0001) more fibroblasts than the
irradiated cultures with the exception of the LED-infrared group that showed a not
significant increase of cells Further analysis revealed that the green probe yielded a
significantly lower number of cells than the red (plt0001) and the infrared probe
(plt0001) and that the red probe provided a higher decrease (plt0001) than the
Chapter 2
54
infrared probe Laser irradiation induced a significant decrease of fibroblasts in
comparison to the infrared irradiated cultures (plt0001) and the control cultures
(p=0001) LED irradiation with the green and the red probe revealed no statistical
significant differences
Table 1 Fibroblast proliferation after LED and LLL irradiation
Groups
Absorbency (proportional to the number of fibroblasts)a
Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050
Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis
DISCUSSION
Despite the failure of some studies [223] to demonstrate beneficial effects of laser and
photodiode irradiation at relatively low intensities (lt500mW) on fibroblast
LED induced increase of fibroblast proliferation
55
proliferation this study provides experimental support for a significant increased cell
proliferation Therefore these results confirm previous studies that yielded beneficial
stimulating effect [1152425] Remarkably though is the higher increase noted after
irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general
decrease in absorption at longer wavelengths and concluded that several molecules in
fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445
470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the
closest to one of these peaks
Karu [5] also emphasises that the use of the appropriate wavelength namely within the
bandwidth of the absorption spectra of photoacceptor molecules is an important
factor to consider
In this particular context penetration depth can almost be ignored as virtually all
wavelengths in the visible and infrared spectrum will pass through a monolayer cell
culture [12] The irradiance (Wcm2) on the contrary could have had an important
influence on the outcome of this study The higher increased proliferation by the lower
wavelengths is possibly a result of the lower irradiance of these wavelengths Lower
irradiances are confirmed by other experiments to be more effective than higher
irradiances [111626]
The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as
described by Poumlntinen [17] but in the scope of these results it also needs to be noticed
that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)
the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)
Consequently the results of especially trial A and F must be interpreted with the
necessary caution It is possible that the determined distinction between the used light
sources and the used probes is a result from the various radiant exposures applied
during the treatments of the cultures
Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after
the last irradiation this study was unable to demonstrate a stimulating effect when
analysis was performed 72 hours after the last irradiation Moreover this longer
incubation period even yielded an adverse effect Although a weakening of the
Chapter 2
56
photostimulating influence over time is acceptable it can not explain a complete
inversion Especially in the knowledge that a considerable amount of authors still
ascertain an effect after a longer incubation period [2427] In an attempt to illuminate
this finding one can suppose that the circadian response of the cells triggered by the
LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark
The most obvious explanation is even though a decreased vitality and untimely cell
death in the irradiated cell cultures as a result of reaching confluence at an earlier point
of time than the control cultures The cells of a confluent monolayer have the tendency
to inhibit growth and finally die when they are not subcultured in time No other
reasonable explanations could be found for this discrepancy
Photo-modulated stimulation of wound healing is often viewed with scepticism The
real benefits of Light Emitting Diodes if any can only be established by histological
and clinical investigations performed under well controlled protocols Despite these
remarks this study suggests beneficial effects of LED and LLL irradiation at the
cellular level assuming potential beneficial clinical results LED application on
cutaneous wounds of human skin may be assumed useful at the applied dosimetric
parameters but future investigation is necessary to explain the mechanisms of LED
biomodulation and to provide sufficient guidelines in the use of the most effective
parameters for LED treatment Subsequently resolving the lack of scientific evidence
and nullifying the controversial acknowledgements of the effect of LED can bring
about a widespread acceptance for the use of LED in clinical settings
Persons in good health rarely require treatment for wound healing as posed by Reddy
et al [13] light has a possible optimal effect under conditions of impaired healing
Postponed wound healing is a time-consuming and often expensive complication
Thus future prospects must remind to examine the therapeutic efficacy of LED on
healing-resistant wounds
LED induced increase of fibroblast proliferation
57
ACKNOWLEDGMENTS
The authors are grateful to Prof Deridder for supplying the laboratory as well as the
material necessary for this investigation and to Ms Franccedilois laboratory worker for
providing the culture medium and for the technical support
Chapter 2
58
REFERENCES
1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55
2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32
3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7
4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994
5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998
6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32
7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42
8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5
9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8
10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7
11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8
12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34
13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9
14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18
15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43
16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6
17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475
18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J
Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9
21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601
22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63
LED induced increase of fibroblast proliferation
59
23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9
24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301
25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are
more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37
27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8
28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26
CHAPTER 3
GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES
FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL
Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium
Photomedicine and Laser Surgery 2005 23(2) 167-171
Chapter 3
62
ABSTRACT
Background and Objective The chronic metabolic disorder diabetes mellitus is an
important cause of morbidity and mortality due to a series of common secondary
metabolic complications such as the development of severe often slow healing skin
lesions
In view of promoting the wound-healing process in diabetic patients this preliminary
in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on
fibroblast proliferation and viability under hyperglycemic circumstances
Materials and Methods To achieve hyperglycemic circumstances embryonic chicken
fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL
glucose LED irradiation was performed on 3 consecutive days with a probe emitting
green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min
resulting in a radiation exposure of 01 Jcm2
Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all
irradiated cultures in comparison with the controls
Conclusion According to these results the effectiveness of green LED irradiation on
fibroblasts in hyperglycemic circumstances is established Future in vivo investigation
would be worthwhile to investigate whether there are equivalent positive results in
diabetic patients
Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes
Fibroblast proliferation under hyperglycemic circumstances
63
INTRODUCTION
The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide
geographic variation in incidence and prevalence generally 11 of the world
population is affected and worldwide it is the twelfth leading cause of death1 Those
figures may be higher for urban regions as well as for industrialized countries Due to
multiple factors involving the aging process of the population and lifestyle changes
(such as reduced physical activity hypercaloric eating habits and concomitant obesity)
these figures may increase in the future2-6 Therefore diabetes mellitus could become
the most common chronic disease in certain regions as stated by Gale it ldquotargets the
rich in poor countries and the poor in rich countriesrdquo6
The harmful disruption of the metabolic equilibrium in diabetes mellitus results in
characteristic end-organ damage that occurs in various combinations and that follows
an unpredictable clinical pathway
Accordingly the major consequence of diabetes mellitus in terms of morbidity
mortality and economic burden principally concerns macroangiopathies or
arteriosclerosis and microangiopathies including nephropathy neuropathy and
retinopathy7-10
One of these devastating consequences which often appears in time is the
development of various skin defects that are frequently resistant to healing and that
tend to be more severe than similar lesions in nondiabetic individuals Diabetes
mellitus even increases the risk of infection by an increased susceptibility to bacteria
and an impaired ability of the body to eliminate bacteria1112
Skin problems are a severe complication in diabetic individuals and require a
comprehensive and appropriate multidisciplinary approach to prevention and
treatment12
Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to
play the most prominent role in the development of diabetic complications With the
development of insulin treatment for type I diabetes and various oral hypoglycemic
agents for type 2 diabetes a reduction in the development of skin defects due to
hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply
Chapter 3
64
waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in
diabetic patients often need special care in comparison to those persons in good
health who rarely require treatment for wound healing1617 Special care is directed
besides of course toward optimal diabetes regulation toward patient education
maximum pressure relief controlling infection recovery of circulation in case of
ischemia and different modalities of intensive wound treatment18
In the last few years various therapies have been introduced with varying success An
example of such a therapy is the photo-modulated stimulation of diabetic lesions In
vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been
subject of extensive investigation1920 Due to contradictory research results LLL-
photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of
light in the domain of wound healing is less time-consuming less expensive less
invasive than many of the other introduced treatment modalities and practical to use
however it seems worthwhile to investigate the value and benefits of a newly
introduced and alternative light source the light emitting diodes (LEDrsquos)
Preliminary research has proved that green LED with particular properties (an
exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01
Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These
results may be of great importance to the diabetic patient because as posed by Reddy et
al light has a possible beneficial effect in the case of impaired healing1617
To obtain insight into the ability of LED to stimulate fibroblast proliferation under
diabetic-specific conditions of impaired healing the proliferation was assessed in
irradiated and control cultures cultivated in medium with a high quantity of glucose
MATERIAL amp METHODS
Cell cultivation
Primary fibroblast cultures were established by outgrowth from 8-day-old chicken
embryos After isolation and disaggregating as described by Freshney (1994)24 the cells
were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10
Fibroblast proliferation under hyperglycemic circumstances
65
fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin
Secondary cultures were initiated by trypsinization followed by plating of the cells in
80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were
disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently
231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third
passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the
cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified
atmosphere at 37deg C to allow them to attach to the bottom of the wells
Light source specifications and illumination procedure
To control adherence of the cells and to assure that there was no confluence or
contamination the 96-well plates were microscopically examined before irradiation
Subsequently the tissue culture plates were randomly assigned for use in the treated
and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium
was aspirated The remaining 25 (50 microl) medium avoided dehydration of the
fibroblasts throughout irradiation
Irradiation was performed with a light emitting diode (LED) device The LED device
(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power
range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable
within the range of 0-1500 Hz
The investigation used the following illumination properties the continuous mode a
distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2
radiant exposure This procedure resulted in an exposure time of 3 min and a power
output of 10 mW Immediately after irradiation the remaining medium was aspirated
and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM
glucose (30 gL) and incubated at 37deg C
Irradiation and medium changes occurred at 1-day intervals so one irradiation was
implemented each 24 h for 3 days in a row and from the first irradiation onwards all
medium renewals occurred with glucose-supplemented Hanksrsquo culture medium
Control cultures were handled in the same manner but were sham-irradiated
Chapter 3
66
Proliferation assay
Fibroblast survival and proliferation were determined by a sensitive and reproducible
colorimetric assay the assay which detects merely living cells and the signal generated
bears a constant ratio to the degree of activation of the fibroblasts and the number of
fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than
only a small subsample26
Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented
Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium
containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium
bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution
was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during
30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous
solution The optical density of the final solution was measured on an ELtimes800 counter
(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test
wavelength varying from 400 to 750 nm
The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies
for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except
for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)
Data analysis
On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a
Mann-Whitney U test was performed for nonparametrical comparison of the results
Statistical significance for all tests was accepted at the 005 level For this analysis the
Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used
RESULTS
The MTT measurements from each of the 256 control wells and 256 irradiated wells
and the subsequent nonparametrical analysis from the optical densities obtained
disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic
Fibroblast proliferation under hyperglycemic circumstances
67
circumstances after irradiation than in the same circumstances without irradiation (Fig
1)
Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)
DISCUSSION
The outcome of these in vitro experiments based on the above-described light source
properties and the illumination procedure described clearly demonstrated the
stimulatory potential of LED on fibroblast proliferation and the cell viability of
fibroblasts cultured in hyperglycemic medium Preliminary research has already
demonstrated that under these conditions (an exposure time of 3 min a wavelength of
570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this
procedure allowed the highest number of living cells The nature of the light and the
usual questions concerning coherence wavelength power output and radiant
exposures have been discussed previously23
Although these findings confirm the results previously found one cannot ignore the
important methodological difference between previous investigations and the current
study as the cells in this experiment were cultured in hyperglycemic medium2328-30
Absorbency - Proportional to the number of fibroblasts
621 x 10-1 682 x 10-1
0010203040506070809
1
Control Irradiated
Groups
Ab
sorb
ency
Chapter 3
68
After a growth period with normal Hanksrsquo culture medium a necessary step to ensure
normal growth of these secondary subcultures and normal attachment to the bottom
of the wells the Hanksrsquo culture medium was supplemented with glucose
Several earlier studies have established that exposure to glucose concentrations (20-40
mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human
vascular endothelial cell proliferation1531-34 This restraint is more pronounced for
higher glucose15 concentrations and is expressed especially after protracted exposure to
high glucose levels31 A similar restraint was found for cultured fibroblasts by
Hehenberger et al3536 According to some authors however cultured fibroblasts
conversely have been shown to maintain responsiveness to ambient high glucose323738
As there are some ambiguities in literature regarding normal or inhibited growth of
fibroblasts in medium supplemented with glucose39 a pilot study was performed to
determine the amount of glucose necessary to inhibit normal growth after 72 h of
culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study
demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease
of cell viability and to bring about a decline in fibroblast proliferation
This concentration resulted in a remarkable reduction of cell viability and a noteworthy
decrease in the proliferation rate in comparison to control cultures grown in 55 mM
glucose although this concentration is too high to mimic severe diabetic
hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to
investigate the effect of LED in distinct destructive conditions in order to obtain an
incontrovertible result
In addition it is possible that the present investigation needed a higher amount of
glucose to result in a remarkable reduction of proliferation as exposure to glucose was
limited to 72 h and as previous studies revealed that the antiproliferative effect of high
glucose was more pronounced with prolonged exposure with a maximal inhibition
attained by 7-14 days1531
Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo
cell growth are too complex to compare A key question is whether fibroblast
senescence in tissue culture and in the intact organism are similar Cristofalo et al40
Fibroblast proliferation under hyperglycemic circumstances
69
reported that this is not the case as fibroblasts have a finite ability to divide and
replicate but apparently the pathway or the morphologic characteristics leading to the
replicative senescence is not identical in vivo compared to in vitro
Furthermore extrinsic aging related to environmental damage which in diabetic
patients is mainly due to a chronic exposure to high levels of glucose during life is
unachievable in vitro
Unless a number of questions regarding the mechanism according to which LED
stimulates fibroblast proliferation in this particular condition remain unanswered the
results ascertain the potential effects of LED on fibroblast proliferation and viability
CONCLUSION
The current results should be interpreted with caution However these results
demonstrate the effectiveness of green LED irradiation at the above-described light
source properties and the illumination procedure described on cells in hyperglycemic
circumstances
The findings of the present study using an experimental in vitro model indicate that the
use of LED irradiation to promote wound healing in diabetic patients may have
promising future results As the present study establishes the possibility of using LED
irradiation in experimental in vitro situations it would be a worthwhile extension to
perform in vivo investigations to determine whether these in vitro observations were
relevant to the physiological situation and to determine the effect of these LED
properties on human tissue response
ACKNOWLEDGMENTS
The authors are greatly indebted to P Coorevits for assistance with the statistical
analysis and to Professor L Deridder and Ms N Franccedilois of the department of
Human Anatomy Embryology Histology and Medical Physics for providing access to
the laboratory and for helpful discussions
Chapter 3
70
REFERENCES
1 World Health Organisation The world health report of 2002 Statistical Annex 2002 pp 179-201
2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418
3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957
4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538
5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308
6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J
Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot
wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-
glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A
Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood
glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13
14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309
15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501
16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287
17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255
18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412
19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192
20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137
21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675
22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246
23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99
Fibroblast proliferation under hyperglycemic circumstances
71
24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-
25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729
27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63
28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301
29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -
progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of
cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA
damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in
culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia
23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar
K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71
36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141
37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588
38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252
39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97
40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619
PART II ANALGESIA
CHAPTER 4
EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION
MEDIATED BY LIGHT EMITTING DIODE IRRADIATION
Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy
Vanderstraetenab and Dirk Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Lasers in Medical Science 2005 20(1) 35-40
Chapter 4
76
ABSTRACT
The introduction of light emitting diode (LED) devices as a novel treatment for pain
relief in place of low-level laser warrants fundamental research on the effect of LED
devices on one of the potential explanatory mechanisms peripheral neurophysiology in
vivo
A randomised controlled study was conducted by measuring antidromic nerve
conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline
measurement and five post-irradiation recordings (2 min interval each) were performed
of the nerve conduction velocity (NCV) and negative peak latency (NPL)
Interventional set-up was identical for all subjects but the experimental group (=32)
received an irradiation (2 min at a continuous power output of 160 mW resulting in a
radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype
MDB-Laser Belgium) while the placebo group was treated by sham irradiation
Statistical analysis (general regression model for repeated measures) of NCV and NPL
difference scores revealed a significant interactive effect for both NCV (p=0003) and
NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical
significant decreased NCV and an increased NPL in the experimental group and a
statistical significant difference between placebo and experimental group at various
points of time
Based on these results it can be concluded that LED irradiation applied to intact skin
at the described irradiation parameters produces an immediate and localized effect
upon conduction characteristics in underlying nerves Therefore the outcome of this in
vivo experiment yields a potential explanation for pain relief induced by LED
Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative
peak latency middot Analgesic effect
Nerve conduction characteristics
77
INTRODUCTION
Since the introduction of photobiostimulation into medicine the light sources used
have advanced technologically and varied in characteristics over the years
Advancement and variation of the sources implicate a concomitant necessity to revise
research results in the respective domains of application Research and clinical
applications in the past particularly focused on the effectiveness of low-level lasers
have shifted now to novel treatment units such as light emitting diode (LED) devices
The efficacy and applicability of LED irradiation within the field of wound healing has
already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED
is not only promoted for its beneficial effects on the wound-healing process it is also
suggested to be potentially effective in the treatment of pain of various aetiology
although this claim has not yet been investigated thoroughly either experimentally or
clinically The putative analgesic effects of LED remain to be further explored
As the basic vehicle of pain is the neuronal system [7] measuring the
neurophysiological effect of LED treatment would be an appropriate experimental
approach to investigate the efficacy of LED on pain inhibition Nerve conduction
studies have become a technique for investigating the neurophysiologic effects of light
therapy [8-9]
Review of literature regarding standard nerve conduction studies revealed that previous
human studies on the influence of various light sources on peripheral nerves have
utilized different methods which hampers a comprehensive comparison In general
this research was performed on the superficial radial nerve [10-13] described by Shin J
Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve
[891315-17] Following the method of Cambier et al [18] the authors of this study
decided to investigate the effect of the light source used on the conduction
characteristics of the sural nerve By investigating this solely sensory nerve interaction
of motor nerve fibres (motor response can easily be provoked by antidromic nerve
stimulation [19]) can be avoided and given the superficial nature of the nerve it should
be sufficiently amenable to the effects of percutaneous LED irradiation
Chapter 4
78
A second major difference between the trials and therefore also hindering an
appropriate comparison between the results is the wide range of used light sources
HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared
multisource treatment unit [15]
With respect to the potential importance of LED irradiation for the treatment of pain
the current investigation was designed to assess the putative neurophysiological effects
of LED on the sensory nerve conduction of the human superficial peripheral sural
nerve and to establish a time course of the supposed phenomenon
The experimental hypothesis postulates that LED generates an immediate decrease in
conduction velocity and increase in negative peak latency In addition it can be
postulated that this effect is most prominent immediately after the irradiation and will
weaken as time progresses
STUDY DESIGN
The study was approved by the Ethical Committee of the Ghent University Hospital
After explanation of the experimental procedure a written informed consent was
obtained from each subject
Subjects
After screening based on a brief medical history excluding subjects with
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever inflammation of the skin) or conditions
that might affect sensory nerve conduction (such as diabetes peripheral neuropathy
radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral
edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40
females (mean age 26plusmn6 years range 18-42 years) participated in this study The body
mass index (BMI) of each subject varied within the normal range (=185-249) [20]
(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo
Nerve conduction characteristics
79
or an experimental group Each group of 32 subjects was composed of 12 males and
20 females
Experimental Procedure and Data Acquisition
In order to be able to quantify the negative peak latency (NPL) (measured from the
start of the stimulus artefact to the peak of the negative portion of the nerve action
potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was
followed
With respect to the known relationship between nerve conduction characteristics and
temperature the ambient temperature was kept constant (23ordmC-26ordmC room
temperature) during the investigation In view of this temperature issue the
standardized protocol started with 10 min of accommodation during which the
subjects rested in prone position on a treatment table
Immediately before this adjustment period the skin over the dorsolateral aspect of the
left calf and foot was cleaned with alcohol to remove surface lipids This preparation of
the treatment area was followed by the placement of the electrodes (TECA
Accessories Oxford Instruments Medical Systems Division Old Woking UK) as
described by Delisa et al [21]
The two-posted (2 cm separation anode distal) surface caption electrode was placed
distal and posterior of the lateral malleolus on the skin covering the sural nerve The
fixation of the earth electrode (Medelec Oxford Instruments Medical Systems
Division Old Woking UK) occurred 12 cm above the caption electrode according to
the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm
above the caption electrode to map the ideal stimulation point To level off
intraindividual variations in the amount of sensory response attributable to the
successive placement of the bipolar stimulator in course of the investigation a two-
posted (2 cm separation cathode distal) bar stimulating electrode was attached at the
point where the maximal response was obtained
This placement of the electrodes allows antidromic stimulation of the sural nerve
Electrophysiological stimulation and recordings were obtained with a Medelec
Chapter 4
80
Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse
of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was
used to produce each evoked sensory response
Baseline measurements of NPL and NCV were immediately followed by treatment of
the subjects according the protocol detailed below Recordings were subsequently
repeated at 2-min intervals over an 8-min period resulting in five recordings (one
immediately after the completion of the treatment and one at 2 4 6 and 8 min after
irradiation) Skin temperature was recorded concomitantly throughout the procedure
at the time of baseline measurement immediately after LED irradiation at the time of
the first recording and consequently at 2-min intervals together with the four final
electrophysiological recordings For this a surface digital C9001 thermometer
(Comark UK) sensitive to temperature changes of 01degC was used at the same point
of LED administration namely at 7 cm above the caption electrode The procedure
was identical for both conditions but subjects in the placebo group received a sham
LED irradiation
Light Characteristics and Irradiation Procedure
Irradiation was administrated with a light emitting diode device (BIO-DIO
preprototype MDB-Laser Belgium) The probe used emitted infrared light with a
wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2
and the frequency was variable within the range of 0-1500 Hz
Preceding baseline measurement the treatment point was marked on the skin overlying
the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-
point between the stimulation and capture electrode The LED probe was held in
contact with the skin perpendicular to the skin surface during the complete irradiation
procedure LED treatment consisted for all subjects of the experimental group out of 2
minutes lasting irradiation The LED was set to deliver a continuous energy density of
107 Jcm2 at a power output of 160 mW These parameters were selected as they are
appropriate for the treatment of pain in a clinical setting First of all because the
Nerve conduction characteristics
81
duration of the treatment is clinically feasible and secondly because the parameters are
within the scope of previously described light source characteristics [1-36915]
Statistics
Although superficial skin temperature did not change significantly in course of the
investigation the influence of the measured skin temperature on NPL and NCV was
taken into account by using a correction factor of respectively 02 msdegC and 147
ms degC All corrections were calculated towards a reference skin temperature of 32degC
Difference scores ie the variation between baseline measurements and each post-
irradiation recording were used as the basis for statistical analysis A General
Regression Model for repeated measures with one within-subjects factor (time 0
min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and
one between-subjects factor (group placebo or LED irradiated) was performed
followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least
Significant Difference) to determine whether any differences between baseline
measurements and post-irradiation recordings were statistically significant
The Statistical package for social sciences (SPSS 110) was used for analysis and
statistical significance for all tests was accepted at the 005 level
RESULTS
Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated
group plotted against time in minutes The values of the irradiated subjects decrease
directly after the irradiation and reach a first low point 2 min after finishing LED
treatment This decrease is followed by a marginal increase at 4 and 6 min and again an
important decrease at 8 min Statistical analysis (general regression model for repeated
measures) of these data indicated a significant interactive effect (P=0003)
Chapter 4
82
Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)
Post hoc LSD further showed significant differences between baseline measurements
and all post-treatment recordings (Table 1) Mutual comparison of the values from the
post-treatment recordings did not reveal any significant difference In addition there
was no significant difference determined in the placebo group in course of time
Table 1 Summary of the influence of LED irradiation on nerve conduction velocity
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001
2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002
4 0111plusmn0377 0647 -0908plusmn1898 0021 0004
6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001
8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve Conduction Velocity
-14
-12
-1
-08
-06
-04
-02
0
02
04
Baseline 0 min 2 min 4 min 6 min 8 min
Time Course
Dif
fere
nce
Sco
re (
m
s)
PlaceboLED
Nerve conduction characteristics
83
A similar representation was used for the results of the NPL Figure 2 reproduces NPL
plotted against time in minutes revealing for the irradiated group an increased latency
with two important peaks one at 4 min and one at 8 min
Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)
Statistical analysis of the mean difference scores again indicated a significant interactive
effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2
showed significant differences between baseline measurements and all post-treatment
recordings of the experimental group The mean difference score of the first post-
treatment recording of this same group (LED irradiated) differed significantly with the
recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED
irradiation As well as the recording 2 min after irradiation which differed significantly
(P=0013) with the 8 min post-treatment recording As observed for the NCV the
NPL of the placebo group did not reveal any significant difference in time course
At the time of the final recording the NCV and NPL mean difference scores of the
irradiated group did not return to their respective baseline values
Negative Peak Latency
-001
0
001
002
003
004
005
006
007
Baseline 0 min 2 min 4 min 6 min 8 min
Time course
Dif
fere
nce
Sco
re (
ms)
PlaceboLED
Chapter 4
84
Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group
significance) revealed statistical differences between the experimental and the placebo
group for NCV as well as for NPL NCV and NPL were statistical significant between
both groups at all points of time except from the NPL recording immediately after
finishing irradiation
DISCUSSION
Notwithstanding the above-mentioned difficulties in comparing results between
different trials on nerve conduction we attempt to discuss the current findings in view
of the results of the previous studies
This investigation revealed that percutaneous LED irradiation at feasible and current
clinical parameters generates measurable and significant changes in human sural nerve
antidromic conduction latency and velocity These results thus support previous
findings of light-mediated nerve conduction latency shifts in vivo [8101218]
although there are several important issues to be discussed
Table 2 Summary of the influence of LED irradiation on negative peak latency
Minutes Placeboa Time-Related Significance b
LEDa Time-Related Significance b
Group Significance c
0 0004 plusmn0053 0755 0029plusmn0080 0019 0145
2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021
4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004
6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034
8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)
Nerve conduction characteristics
85
A first comment deals with the progress of the NCV and NPL in function of time As
postulated the NCV decreases significantly immediately after irradiation
corresponding with a significant increased NPL However this effect does not weaken
as time progresses both variables remain significant throughout the 8 min during
observation period
Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the
conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]
although this slight increase in NPL was not significant at any moment Two other
studies [822] with a GaAlAs laser even registered comparable effects over a period of
55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous
studies post-treatment conduction measurements should be extended in time At
present for all studies it remains unclear at what point of time the effect extinguishes
although the interval of time during which LED treatment remains effective is
clinically important when treating pain
Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least
45 min) mediated by a monochromatic multisource infrared diode device although it
needs to be mentioned that this study performed with a comparable light source as the
current investigation revealed a significant decrease of the NPL These inverse results
between the study of Noble et al [15] and the current investigation could be attributed
to the concomitant increase of the skin temperature [15] As it has been well
recognised that a variation in tissue temperature causes a corresponding alteration in
nerve conduction velocities and peak latencies [91523-27] the temperature changes
may indeed provide an explanation for the observed findings In an attempt to analyse
the influence of a direct photobiological effect on sural nerve conduction
characteristics rather than working out the effects based upon thermal mechanisms
the present study corrected the skin temperature towards a reference temperature of
32degC This correction was performed notwithstanding the fact that the superficial skin
temperature did not change significantly before and after LED irradiation as well as
despite the fact that influencing nerve temperature takes place long after affecting skin
temperature [23] and thus being (almost) impossible after 2 min of irradiation
Chapter 4
86
followed by 8 min of registration Introduction of the correction factor implies likewise
that eventual influence on nerve conduction by cooling of the limb due to inactivity as
described by Greathouse et al [11] can be excluded
These facts suggest that temperature changes did not contribute to the demonstrated
effects of LED on nerve conduction Nevertheless the underlying mechanism of the
observed effects remains indistinct
A following remark regarding the fluctuation of NCV and NPL in function of time
considers the fact that both the NCV and the NPL do not change in a constant way up
to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the
increase in NPL display a small though not significant inversion of the effect at 4 and
(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of
fluctuation is to be expected when measuring NCV and NPL besides there is a similar
variation in the placebo groups
Although investigating dose dependency was not intended an additional remark
considers the fact that the use of optimal irradiation parameters is essential to obtain
the observed neurophysiological effect Nevertheless it is impossible to determine
ideal light source characteristics for effective treatment as the range of used
wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency
(pulsed or continuous) are not sufficiently similar between the different studies It can
only be concluded that a pulsing light source [91028] does not provide the postulated
results Radiant exposure exposure time power range and wavelength are not yet
established but based on this study and previously described assays it can be
speculated that the ranges of these parameters are quite large
In comparison with other studies where the number of subjects is 10 or less [8-
1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-
Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number
of subjects (n=32) was investigated in each group In spite of the large investigated
population it should be noted that the magnitude of the described changes in NCV
and NPL can simply be replicated by lowering the temperature of the extremity as the
observed changes are within the expected physiological ranges making the clinical
Nerve conduction characteristics
87
significance of the change questionable (This fact does not implement that the
decrease and the significant changes were temperature mediated)
A key question and meanwhile the initial impetus for future investigation is whether
the measured effects can be extrapolated to the actual nociceptive afferents namely the
myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])
respectively conducting acute and chronic pain The functional testing of these
nociceptive pathways has recently been extensively evaluated The currently accepted
neurophysiological method of assessing nociceptive pathways relies on laser-evoked
potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]
As up till now LEP is not available in this or any surrounding research centre the
investigators of this study had to perform a standard nerve conduction study (assessing
the large myelinated Aβ afferents) Therefore the current and previous beneficial
results of low level light therapy on conduction characteristics of nerves in vivo should
initiate measurements of clinical effectiveness first of all in laboratory settings and
afterward at a clinical level
CONCLUSION
Despite these remarks and the limited knowledge regarding the underlying mechanism
the present findings enable the following conclusions to be drawn LED irradiation at
clinical applied energy densities produces an immediate and localized effect upon
conduction characteristics in underlying nerves More specifically it is proven that
LED treatment lowers the NCV and augments the NPL resulting in a reduced
number of impulses per unit of time Therefore the outcome of this in vivo experiment
assumes that LED possibly induces pain relief
In order to encourage a widespread acceptance for the use of this non-invasive pain-
reducing modality in clinical settings prospective research should establish the precise
relationship between LED and pain relief as well as determine the ideal irradiation
parameters and verify which painful conditions can be treated with this treatment unit
Chapter 4
88
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the
equipment and MDB-Laser Belgium for generously providing the Light Emitting
Diode equipment
Nerve conduction characteristics
89
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1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9
2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)
3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18
4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8
5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43
6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65
7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53
8 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-34
9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6
10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90
11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7
12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5
13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9
14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore
15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5
16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5
17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604
18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200
19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21
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20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083
21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press
22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63
23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9
24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9
25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203
26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13
27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12
28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9
29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445
30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8
31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56
CHAPTER 5
PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE
IRRADIATION A PILOT STUDY ON EXPERIMENTALLY
INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS
Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk
Cambiera
a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium
b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium
Accepted for publication in Lasers in Medical Science December 2005
Chapter 5
92
ABSTRACT
Objective The present pilot study investigated the analgesic efficacy of light emitting
diode (LED) In view of a standardised and controlled pain reduction study design this
in vivo trial was conducted on experimentally induced delayed-onset muscle soreness
(DOMS)
Design Thirty-two eligible human volunteers were randomly assigned to either an
experimental (n=16) or placebo group (n=16) Immediately following the induction of
muscle soreness perceived pain was measured by means of a visual analog scale (VAS)
followed by a more objective mechanical pain threshold (MPT) measurement and
finally an eccentricconcentric isokinetic peak torque (IPT) assessment The
experimental group was treated with infrared LED at one of both arms the other arm
served as control Irradiation lasted 6 min at a continuous power output of 160 mW
resulting in an energy density of 32 Jcm2 The subjects of the placebo group received
sham irradiation at both sides In post-treatment a second daily assessment of MPT
and VAS took place The treatment and assessment procedure (MPT VAS and IPT)
was performed during 4 consecutive days
Results Statistical analysis (a general linear model followed by post hoc least
significant difference) revealed no apparent significant analgesic effects of LED at the
above-described light parameters and treatment procedure for none of the three
outcome measures However as the means of all VAS and MPT variables disclose a
general analgesic effect of LED irradiation in favour of the experimental group
precaution should be taken in view of any clinical decision on LED
Conclusion Future research should therefore focus on the investigation of the
mechanisms of LED action and on the exploration of the analgesic effects of LED in a
larger randomised clinical trial and eventually in more clinical settings
Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset
muscle soreness middot Musculus biceps brachii
Delayed-onset muscle soreness
93
INTRODUCTION
The analgesic efficacy of light emitting diode (LED) irradiation is recently being
investigated by means of a nerve conduction study on the superficial peripheral sural
nerve [1] It was demonstrated that LED irradiation at clinical applied densities
produces an immediate and localized effect upon conduction characteristics in
underlying nerves More specific LED induces a decreased number of sensory
impulses per unit of time thus possibly inducing pain relief [1]
Given the established influence of this treatment modality on the nerve conduction
velocity and thereby its potential analgesic ability the current investigation was
designed
Studies investigating the efficacy of a therapeutic modality on pain often experience
difficulties regarding standardisation of the population as analysis or comparison of
pain with different aetiologies is almost impossible Therefore we opted to measure the
analgesic effects of LED in a laboratory setting on a sample with experimentally
induced delayed-onset of muscle soreness (DOMS)
Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the
induction exercise and then spreads throughout the muscle [2-4] The correlates of
DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10
[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force
decreased range of motion and in particular muscle pain which is more pronounced
during movement and palpation [8 11] Despite the large volume of research that has
been undertaken to identify the underlying pathophysiology of DOMS the precise
mechanism is not yet universally accepted Several theories such as the torn-tissue
theory the connective tissue damage theory the muscle spasm theory and the
inflammation theory still remain viable though the current opinion states that DOMS
arises from a sequence of events in which several theories occupy an important place
[2 6 12 13]
DOMS has been used as a representative model of musculoskeletal pain and stiffness
in a number of studies [4 7 11 14 15] as it has a number of advantages it can be
induced in a relatively easy and standardised manner in a group of healthy subjects the
Chapter 5
94
time-course is relatively predictable and the symptoms have the same aetiology and are
of transitory nature [14 16] Nevertheless it should be emphasised that the use of this
particular experimental model to test the effectiveness of LED does not mean that this
treatment modality is necessarily advocated for the treatment of DOMS but merely
that it may be helpful in documenting the efficacy of LED in a clinical model of
musculoskeletal pain and stiffness In addition studies based on the induction of
DOMS under carefully controlled laboratory conditions can not replace research
involving actual patients but offer the opportunity to assess the effectiveness of
particular therapeutic interventions and might help to define additional clinical research
[14]
The experimental hypothesis of the current study postulates that infrared LED reduces
pain and muscle sensitivity associated with DOMS
MATERIALS AND METHODS
The study was approved by the ethical committee of the Ghent University Hospital
After providing information regarding the study design and possible consequences
related to participation at the study written informed consent was obtained from each
subject
Subjects
Healthy human volunteers were recruited from the university population Individuals
with any upper limb pathology neurological deficit and recent injury to either upper
extremity or undiagnosed pain were excluded Other exclusion criteria were
contraindications to LED irradiation (such as light hypersensitivity fluctuating blood
pressure insufficient blood circulation fever and inflammation of the skin) or
conditions in which physical exertion is contraindicated (such as cardiovascular deficits
hypertension and respiratory problems)
Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age
23plusmn4 years) were enrolled All subjects were randomly assigned using a random table
Delayed-onset muscle soreness
95
of numbers to the experimental or placebo group Each group of 16 subjects
consisted by stratification of equal numbers of men and women Age height and
weight did not differ significantly between the three groups
All subjects were physically active however none performed on a regular basis any type
of upper body weight-training Subjects were requested to refrain from any form of
strenuous physical activity and they were asked to avoid any form of medication
including anti-inflammatory agents as well as alcohol for 2 days before testing and for
the duration of the study
Overview of experimental design
The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to
induce pain related to DOMS Immediately following induction exercise an initial
assessment of the outcome measures (visual analog scale or VAS mechanical pain
threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the
subjects were treated under blinded conditions according to the randomised group
allocation In post-treatment the MPT was re-recorded and perceived pain was
reassessed with a VAS Contrary to these outcome measures the muscle strength was
only measured in pre-treatment at the one hand because short-term effects of LED
on muscle strength were not postulated and on the other hand because post-
treatment muscle strength can be influenced by too many different physiological
factors related to the pre-treatment measurement On the succeeding days (day 2 3
and 4) the treatment and assessment procedure was similar with approximately 24 h
separating each treatment
In both of the groups the two arms of the participants were included in the study In
the experimental group an equal number of dominant and non-dominant arms were
treated The non-treated arm served as control arm In the placebo group also an equal
number of dominant and non-dominant arms were considered as treated arm and the
other arm was classified in the non-treated group The procedure was identical for
both conditions but the subjects in the placebo group received sham LED irradiation
on both arms
Chapter 5
96
Specific aspects of the experimental design and procedures are detailed below
Pain induction
Muscle soreness was induced in a standardised fashion via a daily calibrated computer-
operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY
USA) Induction occurred separately and in random order in the elbow flexors of both
arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex
Prior to induction of DOMS the subjects were allowed an initial familiarization session
to become comfortable performing maximum voluntary contractions at the required
angular velocities This was immediately followed by determination of the maximum
eccentric and concentric peak torque at an angular velocity of 60degs and 120degs
Subsequently four sessions of eccentricconcentric work were performed with each
arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs
first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg
and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-
range) followed by two sessions at an angular velocity of 120degs again the first time
along an arch of 120deg and followed by the mid-range performance The subjects were
asked to accomplish maximum voluntary contractions during all the sessions Each
session was performed until exhaustion which was defined as the point when the
subject lost 70 of the initial eccentric and concentric peak torque There was a 1-
minute rest between each session This procedure was based on a pilot study and
previously described induction protocols [17-21]
Outcome measures
Outcome measures of subjective pain measurements MPT and muscle strength were
measured in this order on days 1-4 Subjective pain measurements and MPT occurred
immediately prior to and following irradiation whereas muscle strength measurements
only took place before LED treatment
Measurement of subjective pain Perceived muscle soreness was measured
subjectively by means of a 100-mm VAS A series of scales were completed separately
Delayed-onset muscle soreness
97
for each arm pain at rest followed by pain perception associated with full extension of
the arms and finally with maximal flexion of the arms The subjects were not allowed
to compare one VAS result with another
This assessment tool commonly used in measuring experimentally induced pain [22
23] has been found to be a reliable and valid method [24-26]
MPT Tenderness MPT used as a more objective correlate of muscle tenderness
has been demonstrated to be a reliable method to measure experimental induced
muscle soreness [27] This outcome measure was assessed by using a handheld
pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries
South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a
line from the radial insertion of the musculus biceps brachii at the elbow to the
intertubercular groove of the humerus thus resulting in three measure points one at
the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A
pressure of 4Ns was delivered The subjects were instructed to say yes at the exact
moment the pressure perceived became painful Each point was recorded three times
in pre-treatment as well as in post-treatment The average MPT score for each point in
pre- and post-treatment was used for statistical analyses
Muscle strength assessment Eccentric and concentric IPT were measured on the
same computerised dynamometer as was used for the induction of pain and an
identical standardisation procedure regarding positioning was followed
A warm-up session of two maximum voluntary contractions at the required angular
velocities was followed by determination of the eccentric and concentric peak torque
The first session at 60degs consisted of three repetitions followed by a 1-min during
rest and for the second session at 120degs five repetitions were performed The
subjects were instructed to flex and extend the elbow through the entire range of
motion as forcefully and rapidly as possible for each repetition The maximum
eccentric and concentric torque produced during the respective repetitions was used
for statistical analysis
Chapter 5
98
Light source specifications and treatment procedure
Light treatment was applied daily according to group allocation Irradiation occurred
with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The
probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160
mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The
frequency was variable within the range of 0ndash1500 Hz
During the complete irradiation procedure the LED probe was held in contact with
the skin perpendicular to the skin surface and at the exact mid-point between the MPT
mark at 4 cm and the one at 8 cm Light source properties were identical for all
subjects of the experimental group and consisted out of irradiation of 6-min lasting
duration at a continuous power output of 160 mW resulting in an energy density of
32 Jcm2 To conceal the treated side and condition the subjects were blinded to the
treatment status For the experimental condition a probe was held in contact with each
arm but only one of the two probes was attached to the LED device The subjects of
the placebo group received sham irradiation at both sides
The selected parameters are within the scope of previously described light source
characteristics for pain reduction [1 28-30] and they are appropriate for the treatment
of pain in a clinical setting because the duration of the treatment is clinically feasible
Statistical analysis
The three outcome measures were analysed separately For the VAS and MPT
measurements the same procedure was followed a general linear model (GLM) for
repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-
post preceding and following LED irradiation) and one between-subject factor (group
placebo or infrared LED irradiated) was performed If necessary the GLM was
followed by appropriate pairwise comparisons (post hoc least significant difference or
LSD) to determine whether any differences between measurements were statistically
significant A similar model was carried out separately for both the treated and the
control arm
Delayed-onset muscle soreness
99
In contrast to MPT and VAS the muscle strength was analysed differently The peak
torque values recomputed towards body weight of the subjects were statistically
analysed using a GLM for repeated measures This model consisted of one within-
subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo
or infrared LED irradiated) The model was completed twice first for the treated arm
and consequently for the control arm
The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)
was used for analysis and statistical significance for all tests was accepted at the 005
level
RESULTS
Statistical analysis of all variables of the three outcome measures revealed no significant
interactive effects of the main interaction (time times group times pre-post) The means and
standard deviations of the variables for both the treated and the control arm are
outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The
means of all VAS and MPT variables disclose a non-statistical significant general
analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher
MPT values in the irradiated group than in the placebo group The lower VAS rates are
present from day 1 until the last day of the study but they are more clearly present
from day 3 pre-treatment The higher MPT values are present from day 1 post-
irradiation until the last day and they are more visible at 4 cm followed by 12 cm and
finally at 8 cm In addition to the analgesic influence of LED an increased
convalescence of muscle strength was noted It should be remarked that this outcome
is similar for the treated as well as for the control arm of the irradiated group The
findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms
of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively
Graphical presentation of the other variables shows a similar course
Chapter 5
100
Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42
Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36
Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Visual Analog Scale
0
02
04
06
08
1
12
14
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n V
AS
scor
e (a
t re
st)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
101
Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group
Day 1 pre
Day 1 post
Day 2 pre
Day 2 post
Day 3 pre
Day 3 post
Day 4 pre
Day 4 post
Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249
Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Mechanical Pain Threshold
0
5
10
15
20
25
Day
1 -
Pre
Day
1 -
Post
Day
2 -
Pre
Day
2 -
Post
Day
3 -
Pre
Day
3 -
Post
Day
4 -
Pre
Day
4 -
Post
Time course
Mea
n M
PT
sco
re (
at 4
cm)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Chapter 5
102
Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group
Day 1 Day 2 Day 3 Day 4
Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04
Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04
Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)
Isokinetic Peak Torque
04
045
05
055
06
065
Day 1 Day 2 Day 3 Day 4
Time course
Mea
n I
PT
sco
re (
ecce
ntr
ic a
t 60
degse
c)
Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm
Delayed-onset muscle soreness
103
Despite the absence of significant main interaction effects the remaining interactions
as well as the main effects were statistically significant for some variables Only the
significant interactions including the between-subject factor group as well as the main-
effect group will be discussed The other interactions and effects establish the successful
induction of DOMS but are not relevant in view of the postulated hypothesis
The interaction between group and time is significant (p=014) for the VAS in
association with full extension for the control arm Post hoc LSD reveals no difference
between both groups a significant effect over time for both groups is found
Consequently this will not be further evaluated
A second significant interaction (p=0002) is the one among the within-subject factor
pre-post and the between-subject factor group for the MPT at 12 cm for the control arm
Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more
pressure after than before the treatment whereas in the placebo group a not
significant decrease of supported pressure is noted
Finally GLM analysis revealed that at the treated arm the irradiated group tolerates
significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)
DISCUSSION
It has previously been demonstrated that the LED source used might assist in
accelerating wound healing [31] that it has a direct cellular effect [3233] and that it
changes nerve conduction characteristics [1] Nevertheless LED-treated experimental
induced DOMS failed to prove the analgesic efficacy of LED at the above-described
light parameters and treatment procedure The current outcome concurs with other
research that demonstrated a lack of effect of various forms of light therapy on DOMS
[8 11 15] However despite the absence of an apparent and overall definitive finding
the present results cannot exclude favourable effects of LED treatment on pain Since
first of all an isolated statistical significant pre-post difference between groups (control
arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)
revealed that subjects of the irradiated group tolerate more pressure than the subjects
of the placebo group Second of all the overall means identified generally lower VAS
Chapter 5
104
scores higher MPT values and higher peak torques in the irradiated group This
implied that the treated subjects experienced noticeable less pain supported more
pressure on the painful muscle and generated more force than the non-treated
participants However these results are not statistically significant consequently it is
possible that these differences were found by coincidence and that there is no
relationship between the treatment and the described results of the three outcome
measures though it should be mentioned that the absence of significant findings is
more probably attributable to the small sample size involved in this study This
assumption is based on a post hoc power analysis It was calculated that for the small
effect size measured after treatment and for the measured control group event rate a
sample size of 80 subjects in each group was required at α=005 and power=080
(two-sided) to reveal significant results
Another factor conceivably responsible for the lack of solid evidence of the beneficial
effects of LED treatment upon DOMS-associated pain is related to the size of the
treatment effect in relation to the severity of the induced DOMS It is possible that by
using multiple exhaustive sets of exercise severe DOMS were induced which masked
relatively small but apparent treatment effects [4 11] In this same context it is
possible that the results only become significantly different after a prolonged treatment
and follow-up period as previous research noticed that recuperation subsequent to
DOMS induction can last up to 10 days [8]
Although it needs to be stressed that these results are not statistically significant critical
analysis of the overall means leads up to three additional remarks A primary comment
relates to the pre- and post-treatment courses of the results Starting at day 2 a clear
reduction of pain and muscle sensitivity was observed immediately post-treatment
Still one cannot conclude that this is indicative for the analgesic effect of LED
irradiation as a similar decrease in VAS and increase in MPT values was noted in the
treated and the control arm of the placebo group Perhaps this was caused by placebo
effect as reported by Pollo et al [34] the expectation of the participant can easily result
in pain relief but it can only be elucidated by implementation of a control group
Delayed-onset muscle soreness
105
Nevertheless in the current study this particular finding can be most probably
attributed to the physiological effects of the peak torque measurement performed
between the pre- and post-treatment recordings of VAS and MPT on the painful
flexor muscle of the upper arm For the assessment of muscle strength two short
series of alternative concentric and eccentric efforts were performed in succession
involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist
in the removal of inflammatory markers and exudate consequently reducing local
tenderness [4] In addition the force assessment can be considered as a form of active
warming-up resulting in an increased muscle temperature which can reduce muscle
viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness
[3536] thus decreasing the sensitivity of the muscle and moderating pain during
movement In any case the beneficial influence of LED immediately after irradiation
can not be securely interpreted due to the sequential assessment of the outcome
measures
A second additional remark considers the fact that both arms of the irradiated subjects
demonstrated evidence of the beneficial effects of LED as a similar reduction of pain
and muscle sensitivity and higher peak torques were found in course of time at the
treated arm as well as at the control arm of the irradiated subjects This ascertainment
points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]
stated that in case LED works via systemic effects the use of the contralateral side as a
control arm might be ill-advised Thus reinforcing that future research should include a
control group to bring clarification [4 7 16]
Finally it needs to be mentioned that although the extent of DOMS was probably
relatively high for investigating the postulated hypothesis the time-course of the
present study corresponds to that reported by other investigators [2 3 5-10]
Significant time effects in many of the variables revealed that muscle damage was
evident diffuse muscle soreness became progressively worse 24-48 h after DOMS
induction followed by a small amelioration after 72 h [35910] After 72 h the follow-
Chapter 5
106
up was ceased consequently further regain of force and attenuation of pain and
muscle sensitivity could not be evaluated Extending the duration of the assessment
period could be useful in assessing any longer-term effects of LED treatment
particularly because as mentioned above differences between both groups are more
clearly present from day 3 pre-treatment and also because DOMS may last for up to 10
days when induced with the described protocol [715]
Lack of knowledge regarding both the precise mechanism of action of LED and the
specific pathophysiology of DOMS hampers the way to offer a definitive explanation
for the absence of more obvious statistically significant differences Still the small
number of significant findings and the mean values suggest that possible analgesic
effects of infrared LED may not be excluded yet but to be able to estimate the real
value of LED further research is necessary A large-scaled randomised clinical trial
which takes the above-mentioned remarks into consideration should be performed
CONCLUSION
Regardless of the reasons for the absence of statistical significant effects reported here
and although LED may have some potential in the management of pain and functional
impairment associated with DOMS its effectiveness at the applied densities has not
been established
Future research should focus on evaluation of the appropriateness of DOMS as an
experimental model of pain and muscle damage Validation of this model would
enhance the ability to study various modalities for their potential effects on pain and
muscle injuries Besides the mechanisms of LED action are not known thus further
fundamental investigations need to address the underlying mechanism and
physiological basis of pain modulation utilizing LED treatment
Once LED irradiation has finally proven its treatment value in an experimental model
the most important prospect considers establishing the effectiveness of LED to reduce
pain in clinical settings
Delayed-onset muscle soreness
107
ACKNOWLEDGMENTS
The authors would like to thank Mr T Barbe and Mr R Deridder for their technical
assistance in the collection of the data as well as for their valuable input into the
research design Sincere appreciation is extended to the volunteers that participated in
this study and to MDB-Laser (Belgium) for generously providing the light emitting
diode equipment The authors also gratefully recognize Prof Dr G Van Maele for
assistance with the statistical analysis and for helpful discussion
Chapter 5
108
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11 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
12 Armstrong RB (1984) Mechanisms of exercise-induced delayed onset muscular soreness a brief review Med Sci Sports Exerc 16(6)529-538
13 Rodenburg JB Steenbeek D Schiereck P and Bar PR (1994) Warm-up stretching and massage diminish harmful effects of eccentric exercise Int J Sports Med 15(7)414-419
14 Ciccone CD Leggin BG and Callamaro JJ (1991) Effects of ultrasound and trolamine salicylate phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-675 discussion 675-678
15 Craig J Barlas P Baxter D Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380
16 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214
17 Dvir Z (2003) Isokinetics muscle testing interpretation and clinical applications Churchill Livingstone Edinburgh
18 Lambert MI Marcus P Burgess T and Noakes TD (2002) Electro-Membrane Microcurrent Therapy Reduces Signs and Symptoms of Muscle Damage Med Sci Sports Exerc 34(4)602-607
19 Sumida K Greenberg M and Hill J (2003) Hot gel packs and reduction of delayed-onset muscle soreness 30 minutes after treatment J Sport Rehabil 12221-228
20 Tiidus PM and Shoemaker JK (1995) Effleurage massage muscle blood flow and long-term post-exercise strength recovery Int J Sports Med 16(7)478-483
21 Zhang J Clement D and Taunton J (2000) The efficacy of Farabloc an electromagnetic shield in attenuating delayed-onset muscle soreness Clin J Sport Med 10(1)15-21
22 Fitzgerald GK Rothstein JM Mayhew TP and Lamb RL (1991) Exercise-Induced Muscle Soreness After Concentric and Eccentric Isokinetic Contractions Phys Ther 71(7)505-513
Delayed-onset muscle soreness
109
23 Nosaka K and Clarkson PM (1997) Influence of Previous Concentric Exercise on Eccentric Exercise-Induced Muscle Damage J Sports Sci 15(5)477-483
24 Jensen MP Karoly P and Braver S (1986) The Measurement of Clinical Pain Intensity - a Comparison of 6 Methods Pain 27(1)117-126
25 Price DD Mcgrath PA Rafii A and Buckingham B (1983) The Validation of Visual Analog Scales as Ratio Scale Measures for Chronic and Experimental Pain Pain 17(1)45-56
26 Revill SI Robinson JO Rosen M and Hogg MIJ (1976) Reliability of a Linear Analog for Evaluating Pain Anaesthesia 31(9)1191-1198
27 Nussbaum EL and Downes L (1998) Reliability of Clinical Pressure-Pain Algometric Measurements Obtained on Consecutive Days Phys Ther 78(2)160-169
28 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46
29 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295
30 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118
31 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in biomedical optics and imaging 3(28 Proceedings of SPIE 4903)156-165
32 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18(2)95-99
33 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2005) Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level J Photomed and Laser Surg 23(2)167-171
34 Pollo A Amanzio M Arslanian A Casadio C Maggi G and Benedetti F (2001) Response Expectancies in Placebo Analgesia and Their Clinical Relevance Pain 93(1)77-84
35 Shellock FG and Prentice WE (1985) Warming-up and Stretching for Improved Physical Performance and Prevention of Sports-Related Injuries Sports Med 2(4)267-278
36 Safran MR Seaber AV and Garrett WE (1989) Warm-up and Muscular Injury Prevention an Update Sports Med 8(4)239-249
37 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
38 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
GENERAL DISCUSSION
General discussion
113
SUMMARY
As outlined in the general introduction the overall objective of this doctoral thesis is to
develop the current knowledge about the mechanisms of LED action in view of the
eventual provision of evidence-based support for the clinical use of LED as a
biostimulatory and analgesic treatment modality especially in the field of
physiotherapy
Part I Wound healing
The investigations described in chapter 1 and 2 were conducted to gain insight into the
potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As
fibroblasts are principal cells for biostimulation (in view of growing and dividing in
healing wounds) the influence of LED irradiation on fibroblast proliferation was
assessed1
The first investigation consisted of a pilot study performed in order to evaluate the
appropriateness of the cell isolation technique cell culture protocol and proliferation
analysis as well as to appraise the feasibility of the light source properties and
illumination procedure
Data analysis revealed no statistically significant differences between the infrared LED
irradiated and control petri dishes for the used parameters (table 1) Considering this
outcome other experimental findings disclose that the absence of stimulatory effects of
LED irradiation on fibroblast proliferation can partly be attributed to the use of
inappropriate light source properties However the applied external dosimetric
parameters are well within the recommended spectrum described by previous studies
investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be
excluded that changes in the illumination procedure (such as the use of lower power
shorter exposure times wavelengths with finer coverage of the absorption spectrum of
the irradiated cells and a longer incubation period between the last irradiation and cell
counting) could still result in an increased fibroblast proliferation467 Of equal
importance in interpreting the lack of distinctive results are the imperfections of the
applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails
114
considerable intervention from the investigator compromising the reliability of the
method It is also a time-consuming technique with an insufficient sensitivity for some
purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11
To avoid contamination of the results by these modifiable remarks a similar
experiment (chapter 2) was performed in which wavelength power and output mode of
the infrared LED source were not modified (table 1) only the exposure time was
reduced resulting in a lower radiant exposure In addition the effect of two other
emission spectra was evaluated These probes emitting red and green light had a
shorter wavelength than the infrared LED source and the power was half or a
sixteenth of the power from the infrared probe Consequently the red LED irradiation
occurred with a different exposure time than the infrared one in order to attain the
same radiant exposure (053 Jcm2) With respect to the green LED it was not
endeavoured to achieve the same radiant exposure as 16 min of irradiation is not
feasible for in vitro or clinical application
Finally also an LLL light source was integrated Although it was not attempted to
analyse the effectiveness of LED in comparison to LLL enclosure of this modality was
interesting in order to join in with the available literature covering mostly LLL studies
To bypass the described problems regarding analysis of fibroblast proliferation
counting of the cells was carried out this time by means of a colorimetric MTT assay
This method provides more accurate cell counts in a short period of time and therefore
can be considered as a more reliable alternative to Buumlrker hemocytometer11
MTT assay 24 h after the last irradiation revealed a significantly increased number of
cells in the irradiated wells in comparison to their (respective sham-irradiated) controls
Although the study supplied experimental support for a significantly increased cell
proliferation by all external dosimetric properties based on the results of the
comparative trial with an incubation period of 24 hours irradiation with the green
LED source yielded the highest number of fibroblasts Thus it can be concluded that
the wavelength of the green LED is probably within the bandwidth of the absorption
spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that
General discussion
115
the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast
cultures in vitro612
Table 1 External dosimetric properties summarized for each chapter
Wavelength Power Exposure
time Output mode
Radiant exposure
PART I Chapter 1
In vitro part
LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2
LED-infrared 950 nm 160 mW 1 min continuous 053 Jcm2
LED-red 660 nm 80 mW 2 min continuous 053 Jcm2
LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2
Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2
PART II Chapter 4
LED-infrared 950 nm 160 mW 2 min continuous 107 Jcm2
Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2
The next aim of the first part of this doctoral thesis was to explore whether LED
treatment could ameliorate in vitro cell proliferation under conditions of impaired
healing In the pursuit of this aim fibroblasts were cultured in medium supplemented
with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a
pilot study the amount of glucose necessary to inhibit normal growth was determined
In order to attain an important reduction of cell viability and decreased proliferation
rate a relatively high concentration of glucose (1667 mM) was necessary in
comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia
(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity
between both circumstances in vitro limited to 72 h whereas the human tissue of a
diabetic patient in vivo is chronically exposed to glucose
Treatment of the fibroblasts occurred in respect of the previously described results
with the same irradiation parameters and illumination procedure (chapter 2)
Accordingly green LED irradiation labelled as the most appropriate treatment for
116
irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in
the previous study (table 1)
Analysis of the cell proliferation by means of MTT measurements yielded a
significantly higher rate of proliferation in hyperglycaemic circumstances after
irradiation than in the control conditions (ie hyperglycaemic circumstances without
irradiation) Thus this outcome supported the stimulatory potential of green LED
irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a
considerable destructive hyperglycaemic medium
Finally although the results of the in vivo part of chapter 1 were persuasive and
encouraging they will not be further discussed in this summary of part I as it was not
aimed in this doctoral thesis to investigate the wound healing process in vivo However
the results of this case study can be a valuable hold for future in vivo research
The possible clinical implications of these results and future research directions in the
scope of wound healing will be discussed below
Part II Analgesia
In the second part two studies investigated the effects of LED irradiation as a
potential intervention mode in one of the most important fields in physiotherapy
practice analgesia Chapter 4 describes the influence of LED treatment on changing
sensory nerve conduction characteristics of a human superficial peripheral nerve
Altering nerve conduction characteristics may not be the sole beneficial purpose to
attain with LED irradiation in view of analgesia but the advantage of nerve conduction
characteristics is that they are objective measurable physical variables and changes in
these characteristics provide a potential explanatory mechanism of pain inhibition by
LED treatment17
The results showed that percutaneous LED irradiation at feasible clinical parameters
can generate a significant decrease in NCV and increase in NPL for all recordings post-
treatment in comparison to the baseline measurement The data in the placebo group
did not reveal any significant difference in the same course of time Statistical analysis
General discussion
117
revealed significant differences between the experimental and the placebo group for
NCV as well as for NPL at all time-points of observation with exception of the NPL
recording immediately after finishing irradiation
It was also observed that the noted effects did not weaken as time progressed It can
be concluded that post-treatment conduction measurements should be extended in
time which is in accordance with the findings of some previous studies18-21 Clarifying
the point of time at which the effect extinguishes is necessary and clinically relevant
when treating pain by means of LED irradiation Besides obtaining the desired
neurophysiological effects ideally the optimal irradiation parameters should be
applied The most favourable dosimetric properties are not yet determined but based
on this study and previously described assays it can be speculated that the dosimetric
window is quite large
Regardless of these clinically important remarks the present findings allow to draw the
following conclusion LED irradiation at clinically applied densities can generate an
immediate and localized effect upon conduction characteristics in underlying nerves as
LED treatment results in lowering the NCV and augmenting the NPL Therefore the
outcome of this in vivo experiment assumes a potential pain relief by means of LED
treatment and justifies further research regarding its clinical effectiveness in laboratory
settings and at a clinical level
The fourth and final aim was to determine the efficacy of LED irradiation as an
analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5
illustrates a clinical study observing the effect of LED treatment on a model
comprising experimentally induced DOMS in a healthy population The progress of
pain perception and peak torque was evaluated during 4 consecutive days commencing
on the day of DOMS induction The effect of infrared LED treatment at the light
parameters described (table 1) was assessed with regard to three different factors time
(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group
(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures
(VAS MPT and IPT) revealed no significant interactive effects of the main interaction
118
(timegrouppre-post) For the remaining interactions and for the main effects only a
few significant findings were relevant in view of the postulated hypothesis
Notwithstanding the absence of an apparent and overall statistically significant finding
the present results indicate favourable trends of LED treatment on pain as the means
of all VAS and MPT variables show a statistically nonsignificant general analgesic
effect of infrared LED irradiation expressed by lower subjective pain rates and higher
MPT values in the irradiated group In addition to the analgesic influence of LED an
augmented restoration of muscle strength was noted The lack of solid statistically
significant evidence for these beneficial effects of LED treatment upon DOMS-
associated pain can possibly be attributed to the small sample size in this study or even
to the size of the treatment effect in relation to the severity of the induced DOMS as
induction of severe DOMS can mask relatively small but apparent treatment
effects2223 A final possibility is that the results only become significantly different after
a prolonged treatment and follow up period as previous research demonstrated that
recuperation subsequent to DOMS induction can last up to 10 days24
It should also be noted that the described general analgesic effect of LED irradiation
was identical for the treated as well as for the control arm in the irradiated group
proposing that infrared LED might induce systemic effects 2526 However it needs to
be stressed that these results were not statistically significant
Regardless of the absence of statistically significant findings the mean values suggest a
potential role for infrared LED irradiation in the management of pain and functional
impairment associated with DOMS Notwithstanding this postulation future research
is absolutely required to establish the effectiveness of LED treatment to reduce pain as
well at the applied densities as for other dosimetric parameters
CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS
In the course of the past years during the process of the genesis of this thesis
therapeutic physical agents in general and phototherapeutic modalities in particular
became less important as physiotherapeutic modes of treatment than during the
preceding two decades The diminished use of these treatment modalities in the
General discussion
119
physiotherapy practice is to a certain degree a consequence of the controversial
research findings regarding the use of these physical agents This issue of controversy
led to less support for the use of these treatment modalities and a growing scepticism
regarding the effectiveness of these physical agents within the scope of the growing
climate of evidence-based practice A second responsible protagonist for the loss of
popularity of physical agents is linked with the current tendency within physiotherapy
emphasising active remedial therapy The establishment of this development was based
on various experiments mainly performed during the last decade demonstrating that
active treatment modalities are for numerous impairments and disabilities preferable to
more passive forms of therapy In Belgium the prevailing nomenclature which came
into use on 1 May 2002 went along with this tendency In the appendix to the Royal
decree of 14 September 1984 towards settlement of the nomenclature of medicinal
treatments concerning compulsory insurance for medical care and allowances the
personal involvement of the physical therapist during the physiotherapeutic session
was emphasized and it was even defined that massage physical techniques within the
framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal
application can only be remunerated when they are applied supplementarily and not as a sole therapy
This implies that passive treatment modalities should not be used as sole method of
treatment and should always be considered as an adjunct to an active treatment
program This development needs to be applauded in many cases such as various
painful musculoskeletal problems functional instability rehabilitation of neurological
patients re-activation of the elderly population psychomotor rehabilitation
cardiovascular and respiratory convalescence Nevertheless it would be erroneous to
entirely reject physical agents including LED treatment Based on the findings of the
above described experiments it needs to be stressed that for some purposes especially
within the scope of impaired wound healing LED irradiation could be a suitable
therapeutic measure This statement is founded on the results of part I of the present
thesis they provided satisfactory fundamental evidence for the advantageous effects of
LED treatment on a crucial exponent of the wound healing process namely fibroblast
proliferation The beneficial findings are the result of basic in vitro research As it is
120
inaccurate to simply extrapolate these results to the clinical practice the clinical use of
LED irradiation for wound healing needs to be preceded by purposive and specific in
vivo investigations to substantiate these basic research findings27
The case study described in chapter 1 indicates a foundation for further in vivo research
Visual appraisal of the surgical incision revealed (from the 65th day in the course of the
reparative process onwards) that the irradiated area -which initially showed inferior
epithelialization and wound contraction- showed a more appropriate contracture than
the control area characterized by less discoloration at scar level and a less hypertrophic
scar These clear beneficial effects of LED treatment on a human cutaneous wound
can serve as preliminary impetus for further research into the clinical applicability of
LED therapy although this case study is insufficient in order to guarantee a safe
correct and effective use of LED as a therapeutic modality
Despite these remarks it tentatively can be concluded that based on a detailed analysis
of the available data of the present in vitro studies and the given case report in
combination with the small number of previously published human studies the
beneficial effects of LED irradiation at the cellular level are obvious and therefore a
potentially favourable outcome can be assumed in clinical practice28-30 LED-
modulated stimulation of wound healing can be gradually and vigilantly implemented
clinically Nevertheless the real benefits of LED irradiation within the scope of wound
healing can only be established by additional clinical trials as thus far clinical
application and stipulation of dosimetry still occurs on a trial-and-error basis which is
not conducive to a generally accepted clinical use of LED To lend more credibility to
the treatment of wounds by means of LED irradiation and to expel the existing
controversy and scepticism surrounding this topic in vivo investigations on wound
healing using a number of different animal models and adequately controlled human
studies are necessary In addition these studies should be performed preferably on a
population suffering from impaired healing as a consequence of diabetes mellitus or as
a result of any other debilitating reason because as posed by Reddy et al3132 and as
mentioned above light has possible optimal clinical effects in the treatment of healing-
resistant wounds
General discussion
121
Drawing general conclusions and formulating clinical implications for analgesia is
obviously less manifest first of all because only a limited number of possible
mechanisms of action in order to obtain analgesia were highlighted and secondly
because both studies did not come to a joint or complementary conclusion The
outcome of the first study revealed that LED treatment lowers the NCV and augments
the NPL resulting in a slower stimulus conduction and consequently a reduced number
of sensory pulses per unit of time Thus it could be assumed that LED induces pain
relief but the results of the study describing the effect of LED treatment on
experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy
In addition it needs to be emphasised that the first study (chapter 4) measured the effect
of LED irradiation on the large myelinated Aβ afferents A noteworthy question and
meanwhile a stimulus for future investigation is whether the measured effects can be
extrapolated from these sensory nerve fibres to the actual nociceptive afferents
notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of
these nociceptive pathways relies on laser-evoked potentials which selectively activate
Aδ-fibres and C-fibres3334 This technique was presently not available therefore a
standard sensory nerve conduction study was performed
Whereas stimulation of wound healing by means of LED irradiation can be cautiously
implemented in the clinical practice at this stage it is too early to promote LED
irradiation as a treatment modality for pain To make this possible it is essential to
conduct numerous studies with regards to the use of LED in the field of analgesia
Future research should focus on fundamental investigations in order to discover the
underlying mechanisms and physiological basis of pain modulation utilizing LED
treatment Furthermore the evaluation of the appropriateness of DOMS as an
experimental model of pain is an important prospect to consider as validation of this
model would enhance the ability to study various modalities for their potential effects
on pain Irrespective of the difficulties regarding standardisation of the research
population and evaluation of soreness inextricably linked with clinical pain studies the
122
ultimate objective of future research should be the establishment of the effectiveness
of LED irradiation to reduce pain of miscellaneous origin in a clinical setting
Regardless of the encouraging results of the described studies and besides the earlier
proposed specific directions for future research (directed towards wound healing or
pain relief) it is necessary in the interest of the patientrsquos well being and to the
advantage of the prospective clinical use of LED to highlight a few more issues for
future research Therefore one has to deal with some limitations of the performed
investigations A first limitation concerns the fact that only two mechanisms of LED
action were investigated (notably changed fibroblast proliferation and alteration of the
nerve conduction characteristics) So one can conclude that for further and better
understanding of the mechanisms of action it is necessary to perform more basic
research Answering the questions regarding the functioning of LED irradiation will
simplify the evaluation and reinforce the interpretation of the obtained results and
ultimately contribute to a more widespread and well definded acceptance of the use of
LED in clinical settings
A second general limitation of this doctoral thesis is the substantial difference in the
used external dosimetric parameters between the different chapters and even within
one and the same study (illustrated in table 1) this complicates the comparison
between the different trials In each trial the dosimetry was individually ascertained
based on previous studies within the given field As not for every application the same
dosimetry is suggested in literature a range of dosages were used Another important
factor in deciding on the dosimetry was the clinical applicability of the dosage as it is
useless to investigate the appropriateness of a treatment modality at a clinically
unrealistic dose As a result of this limitation the current findings do not fully
contribute to the explanation regarding the ideal parameters one should use although
this was not set as a principal purpose Based on this thesis and previously described
assays it can be speculated that the possible window for these parameters is quite large
the ideal irradiation parameters and proper timing or sequencing of LED irradiation
General discussion
123
for example to the various phases of wound healing and to different painful conditions
are therefore possibly unattainable
The establishment of an appropriate dosimetry should also consist of investigating the
absolute and relative penetration depth of LED irradiation into human tissue This is
less crucial within the scope of wound healing but it is of key importance while
treating deep-seated tissue (eg nerve fibres muscles circulatory components et
cetera)
Finally this thesis only investigated the efficiency of LED in a very limited number of
conditions notebly wound healing and pain Within the scope of physiotherapy and
medicine in general there are numerous other purposes for which LED irradiation is
promoted such as oedema arthritis miscellaneous orthodontic applications seasonal
affective disorder neonatal jaundice photodynamical therapy et cetera2835-41
In summary additional work on establishing proper dosimetry and identifying the
biochemical or photobiologic phenomena that are responsible for improving wound
healing and reducing pain or even other effects within a broader spectrum of
conditions remains to be done in order to answer unreciprocated questions Until that
time the potential clinical usefulness and actual value of LED irradiation for wound
healing and even to a larger extent for analgesia should always be approached with
appropriate professionalism and even caution
FINAL CONCLUSION
LED devices are promoted for clinical use but the currently available scientific
documentation regarding effectiveness of this physical agent is rather scarce Through
providing scientific support for the biostimulatory and analgesic effectiveness of LED
irradiation this doctoral thesis attempted to bridge in some degree this gap
The conducted studies revealed that LED irradiation undeniably has potential
beneficial effects on wound healing and to a lesser degree within the scope of
analgesia However based on the present results it can be corroborated that light
124
therapy in the guise of LED irradiation is not magic but these results can raise some
corrective doubts in fundamental disbelievers and antagonists
Nevertheless we have to join the queue of scientists who have found beneficial results
but cannot elucidate with certainty how this outcome was established Thus although
the present results are encouraging a continuing development and integration of new
knowledge based on further research is necessary in various domains of intervention
Therefore several directions for future investigations were proposed in order to cover
as many existing gaps and to answer the utmost number of remaining questions as
possible Still one ought to be aware not to carry future fundamental research at a
disproportional level and the inevitable quest for mechanisms of LED action should
not hypothecate the potential clinical value implying that at a certain point it should be
appropriate to make the transfer from science to the application of the available
knowledge in clinical practice
The described findings regarding LED irradiation are comparable to the results of
previously published studies performed with other light sources Consequently as
postulated by some LED providers it can be speculated that the biological response of
tissue to light irradiation can probably not be equated merely to a light source but
rather to a broad photo-energy window
General discussion
125
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20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200
126
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22 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347
23 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39
24 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230
25 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246
26 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees DG (1989) Systemic effects of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182
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G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43
29 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum
30 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74
31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255
32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287
33 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-28
34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856
35 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428
36 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557
37 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809
38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263
39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64
40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74
General discussion
127
41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press
NEDERLANDSTALIGE SAMENVATTING
Nederlandstalige samenvatting
131
NEDERLANDSTALIGE SAMENVATTING
Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de
tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te
vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie
van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen
Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest
enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is
ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als
op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang
van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve
interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken
Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van
ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen
moeilijk te verantwoorden
Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin
de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere
overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit
methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan
consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in
de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen
laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in
de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag
aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies
inclusief het gebruik van licht
Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat
de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde
doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en
dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht
komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt
geloosd
132
De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo
lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en
gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te
anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te
voorkomen lijkt een gerichte en rationele a priori aanpak conform de
wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen
Te meer daar grondige literatuurstudie leert dat men ten behoeve van de
werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie
zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser
De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de
extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is
geboden De introductie van alternatieve lichtbronnen in de huidige
kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk
weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus
eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood
naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based
gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder
binnen de domeinen van haar potentieel beloftevolle klinische toepassingen
wondheling en analgesie
Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de
wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op
prominente protagonisten van de wondheling de fibroblasten werd getracht het
fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten
zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de
proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd
respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand
waarbij de normale celgroei werd verstoord
In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het
effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische
Nederlandstalige samenvatting
133
data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden
verklaard door het gebruik van een inadequate LED dosering en anderzijds een
methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie
vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de
precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en
inter-tester variabiliteit tot gevolg
In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde
onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de
bestralingsparameters (dosering) De effecten van de drie verschillende LED
golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten
werden hierbij geanalyseerd door middel van een meer betrouwbare en minder
subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-
yl)-25-diphenyl tetrazolium bromide (MTT)
De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir
effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden
tevens een basis voor meer coherente en relevante inzichten aangaande de globale
bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)
Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het
wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend
karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp
rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde
vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De
fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met
extreem hoge concentraties glucose Deze modificatie van het medium staat model
voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische
praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan
vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de
gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en
proliferatie
134
Het tweede deel van dit proefschrift exploreert het domein van het potentieel
analgetisch effect van LED binnen de kinesitherapie aan de hand van twee
fundamentele onderzoeken
In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere
sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele
hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en
een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale
verklaring van een analgetisch effect van het medium zou kunnen belichamen
Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)
meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf
verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen
aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke
significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de
negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele
hypothese
Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend
fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale
pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp
van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische
krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED
behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier
keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED
op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd
(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde
isokinetische krachtmeting en een registratie van de waargenomen spierpijn De
spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met
behulp van een kwantitatieve hand-hold algometer
Analyse van de bekomen data bracht geen significante verschillen tussen de
controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden
Nederlandstalige samenvatting
135
kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED
behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van
de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de
proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene
afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine
proefgroep die werd onderzocht enof door de grootte van het behandeleffect in
verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers
een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up
kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel
absolute omzichtigheid geboden en moet deze visie louter als speculatief worden
beschouwd
Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de
kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de
laatste jaren aanzienlijk afgenomen
De positieve resultaten van de verschillende in vitro studies in het kader van wondheling
vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing
vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel
gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een
belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek
Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie
Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme
onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch
complementair resultaat Verder onderzoek ter exploratie van de mogelijke
onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom
onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op
termijn wetenschappelijk te rechtvaardigen
136
LED tovenarij trend of therapie
LED mag geen magische krachten worden toegemeten maar verdient het lot van een
kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een
wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen
zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke
indicaties
Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds
the darkness has always got there first and is waiting for it
(Terry Pratchett Reaper Man 1991)