1 In vivo spinal cord monitoring

Real Time Monitoring of Mitochondrial NADH and

Microcirculatory Blood Flow in the Spinal Cord

Maryana Simonovich M.Sc., Efrat Barbiro-Michaely Ph.D.,

Avraham Mayevsky Ph.D.

The Mina & Everard Goodman Faculty of Life Sciences

and the Leslie and Susan Gonda Multidisciplinary Brain Research Center,

Bar-Ilan University, Ramat-Gan 52900, Israel

Running title: In vivo spinal cord monitoring

Corresponding Author:

Prof. Avraham Mayevsky

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University

The Leslie and Susan Gonda Multidisciplinary Brain Research Center

Ramat-Gan 52900, Israel

Tel: 972-3-5318218

Fax: 972-3-6354459

E-mail: mayevsa@mail.biu.ac.il

2 In vivo spinal cord monitoring

Introduction

Postoperative spinal cord injuries following abdominal aortic aneurysm repair and

surgeries under sclerosis of spinal cord blood vessels, are common and usually result

from ischemia, reperfusion injury, and postoperative hemodynamic damage.1;2

The

patients whose postoperative complications include paraplegia or paresis not only suffer

severe physical disability, but their long-term survival is also known to be shorter.2 To

learn more about the pathology of spinal cord injury following abrupt SCBF changes,

several animal models were developed. The most common methods include the occlusion

of the abdominal aorta, first introduced by Niels Stensen in 1667,3 balloon inflation in the

spinal extradural space 4 and photochemical injury of the spinal cord's vascular

endothelium with or without laminectomy.5;6

In the past two decades, spinal cord monitoring during surgery has been a desirable

tool for preventing potential intra-operative neurological injury, leading to a considerable

increase in its use. Traditional intraoperative neurophysiological assessment methods,

such as Direct Motor Pathway Stimulation Technique (DMPST) and Somatosensory

Evoked Potential (SSEP) monitoring, have been used to evaluate proximal nerve activity.

However, it is clear that neurological injury following operation of the spinal cord or

thoracic and abdominal operations is mostly due to intraoperative aortic cross-clamping,

or due to an abrupt (change or decrease) of blood flow in the blood vessels which are

critical to spinal cord function and not a direct impact of the spinal cord 7. Furthermore,

false monitoring outcomes, such as in cases of definite neurologic deficits despite stable

SEP, can occur during surgery Hence, the ability to early detect changes in the

3 In vivo spinal cord monitoring

hemodynamic and metabolic state of the tissue is extremely important and may contribute

to the reliability of monitoring in clinical practice.8

In the present study, we offer a new approach for spinal cord monitoring which

includes the simultaneous monitoring of spinal cord blood flow and mitochondrial

NADH level. NADH, a major component of the respiratory chain, is one of the most

sensitive indicators of oxygen deficiency.9 A decrease in oxygen supply to the spinal cord

tissue is followed by a decrease in ATP levels, a decrease in Na+/K

+ ATPase activity and

an increase in extracellular K+ levels.

10 Only a few studies measured energy metabolism

after spinal cord injury, demonstrating a rapid decrease in high-energy phosphates.1

Moreover, the monitoring of mitochondrial NADH in the spinal cord is rare in animal

experiments and apparently never performed in patients.

In view of these limitations of the available monitoring techniques, our new approach

for the evaluation of the spinal cord tissue integrity, if used clinically, will provide

essential complementary information that may improve the patients’ outcome. Up to date,

monitoring of the spinal cord energy metabolism in patients during surgical procedures is

not available.

The aim of the present study is to evaluate in real-time the spinal cord hemodynamic

and mitochondrial redox state using laser Doppler flowmetry and NADH fluorometry

simultaneously, during transient global ischemia in a rat model and during local ischemia.

4 In vivo spinal cord monitoring

Methods

The Tissue Vitality Monitoring System (TVMS)

In order to achieve real time monitoring of the metabolic and hemodynamic state of

the spinal cord, we used the Tissue Vitality Monitoring System (TVMS), developed in

our laboratory and previously applied in other studies 11

. The TVMS includes optical

fibers for the measurements of spinal cord blood flow (SCBF) and mitochondrial NADH

redox state as demonstrated in Figure 1. The inner diameter of the monitoring probe was

2mm and the depth of monitoring was 0.5-1mm for NADH and 1-1.5mm for SCBF.

Spinal cord blood flow (SCBF)

Spinal cord blood flow (SCBF) was monitored by the laser Doppler flowmetery (LDF)

approach (Perimed Inc.)12;13

applied in the TVMS. The laser Doppler flowmetry is

principally based on the Doppler shift which results from the amount and velocity of red

blood cells movement. The tissue is illuminated by red light at 632.8nm wavelength, to

the depth of 1-1.5mm. This light is shifted by moving red blood cells. The number of

shifted waves is affected by tissue blood volume and the degree of the shift is influenced

by the blood flow velocity. These two parameters (volume and velocity) are used for the

calculation of the relative blood flow level in which 0% is measured at death and the

basal level is presented as 100%. The LDF is significantly correlated to other two

quantitative monitoring approaches: the micro-sphere method and H2 clearance.14-16

NADH Surface Fluorometry

The principle of NADH monitoring from the surface of the spinal cord is that

excitation light (366nm) is passed from the fluorometer to the spinal cord surface (to a

5 In vivo spinal cord monitoring

depth of 0.5-1mm) via a bundle of quartz optical fibers. The emitted light (450 nm),

together with the reflected light at the excitation wavelength, are transferred to the

fluorometer via another bundle of fibers and appropriate filters. The changes in the

reflected light are correlated to changes in tissue blood volume in such a way that an

increase in tissue blood volume decreases the reflected light, due to an increase in tissue

light absorption, and vice versa. Since these changes in the reflectance affects also the

fluorescent light emitted from NADH molecules a correction of the signal is made by

subtracting the reflectance from the fluorescence value at a 1:1 ratio yielding the

corrected fluorescence which express the mitochondrial NADH redox state.17;18

.

Animal Preparation and Protocols

All experiments were carried out according to the NIH guidelines for the care and use

of laboratory animals and approved by the Institutional animal care review board. Adult

male Wistar rats (250-350gr) were anesthetized by intraperitoneal (IP) injection of

Equithesin solution (each ml contains: Chloral Hydrate 42.51mg, Propylene Glycol

44.34%, Pentobarbital 9.72 mg, Magnesium Sulphate 21.25 mg, Alcohol 11.5% water)

0.3 ml/100g body weight. The right femoral artery was cannulated using polyethylene

tubes (PE-50) for arterial blood pressure (MAP) monitoring. The animals' body

temperature was kept at 37±0.5°C during the entire experimental period.

Laminectomy was performed at L3 vertebra, and a 3mm hole was gradually drilled in

the vertebra until the dura was revealed. Than the TVMS was located above the spinal

cord tissue (leaving the dura matter intact) using a micromanipulator, avoiding extra

pressure on the monitored tissue, as was ensured by the initial level of SCBF which

remained stable throughout the entire procedure of placing the TVMS on it. In the

6 In vivo spinal cord monitoring

abdominal aorta occlusion model, the probe was fixated to the monitoring site by acryl

cement, whereas in the compression model no fixation was used. At the end of the

experiments, the rats were sacrificed by pure N2 (100%) inhalation.

Experimental protocols

The following animal groups were used:

A) Control group (n=8): Following laminectomy and probe fixation, the rats were

exposed to pure N2 to induce short (15 sec) anoxia, and then the animals were re-

exposed to room air and continuously monitored for approximately 1.5 hours to

ensure steady monitoring through the entire experimental period.

Short anoxia was also induced in each animal in the study, as this is a routine

procedure used for the assessment of spinal cord tissue viability at the beginning of

each experiment, as well as for the verification of proper probe fixation to the

monitored site.

B) Ischemic group (n=11): Thirty minutes after a short anoxia, animals underwent

transient spinal cord ischemia, which was induced by a 5 min occlusion of the

abdominal aorta, just distal to the left kidney, followed by a reperfusion and a

recovery period of 1.5 hours.

C) Compression model group (n=6): Following laminectomy, the fiber optic probe

was placed on the monitoring site (with no fixation) using a micromanipulator.

SCBF was continuously monitored through this procedure making sure that blood

flow remains intact through the entire procedure of probe placement on the spinal

cord tissue. Compression was induced by lowering the probe on the spinal cord

7 In vivo spinal cord monitoring

tissue using a micromanipulator and compressing the spinal cord tissue until SCBF

was completely abolished (0%). The spinal cord tissue remained compressed for a

period of 5 min, followed by elevation of the probe back to its initial height.

Monitoring continued for another 1.5 hours to evaluate tissue recovery. This

protocol included two sessions of spinal cord compression with an interval of 90

min.

Statistical Analysis:

The Student paired two-tailed t test was used to examine the significance of changes in

the various parameters as related to the initial level. The Student unpaired two-tailed t test

was used to examine the effects of the transient ischemia or compression model on the

physiological parameters measured from the rat’s spinal cord and the systemic blood

pressure in each minute. A value of p<0.05 was considered to be significant. In each

experiment, values were obtained at intervals of 60 sec and mean ± SE values were

calculated for each parameter. Correlation tests were used to determine the relation

between mean SCBF and NADH monitored by the TVMS.

8 In vivo spinal cord monitoring

Results

In order to ensure proper fixation of the TVMS probe on the spinal cord surface, as

well as validate spinal cord tissue integrity, all animals were subjected to short anoxia for

15 seconds while the parameters were recorded. Figure 2 presents the mean ± SE levels

of the various parameters monitored by the TVMS during short anoxia (N2 100%). The

significance of each value was examined as compared to the basal level. At the start of

anoxia, SCBF decreased to 84.4±0.37% and MAP reached the level of 67±0.3 mmHg.

These changes were associated with a significant (p<0.001) increase in the fluorometric

parameters, namely, the Reflectance (5.6±0.2%), Fluorescence (12.5±0.2%) and NADH

(7.1±0.2%). Following exposure to room air, SCBF hyperemia was noted (100-170sec

post N2, p<0.001), which was followed by full recovery. All the recorded metabolic

parameters (SCBF and NADH) returned to the basal levels. However, MAP remained

elevated (p<0.01) for 280 sec.

Eleven rats underwent lumbar spinal cord ischemia, induced by 5 min abdominal

aortic occlusion just distal to the renal arteries, as described in Zivin’s work.19

As

demonstrated in Figure 3, abdominal aortic occlusion resulted in a significant MAP

decline to extremely low levels (10.8±0.5 mmHg, p<0.001), which served as an

indication for the efficacy of the abdominal aorta occlusion (since MAP was monitored in

the Femoral artery). In addition, SCBF levels dropped to 19.9±6.1% and remained low

through the entire ischemia period. These changes were associated with a significant

increase of NADH components to maximum levels, namely: Reflectance (25±9.3%),

Fluorescence (70±17.2%) and NADH (39±11.1%). These changes were significantly

9 In vivo spinal cord monitoring

higher than the basal level, as well as compared to the levels measured at the same time

points in the control group (p<0.01).

With the release of the occlusion (open) MAP was restored (114.8±6.5 mmHg) and

SCBF reached hyperemic levels (p<0.01) in the first 10 min of reperfusion. The

Reflectance, Fluorescence and NADH also gradually returned to their initial levels.

The effects of spinal cord compression are presented in Figure 4. As seen, the

compression of the spine (comp. on) yielded a primary decrease of SCBF to 0%,

followed by a partial recovery, within the compression phases, up to the levels of

24.0±6.4% (1st compression) and 19±3.3% (2

nd compression). In association with the

SCBF changes, there was a significant rise (p<0.001) in the reflectance (196±27.5%),

fluorescence (265.6±43.0%) and NADH (64.4±26.9%) during the first compression

session. Similarly, the second compression caused an increase (p<0.001) of the

parameters to the levels of 115.7±25.1%, 219.4±32.8% and 69.6±18%, respectively.

There were no significant differences between the responses of the parameters during the

two compression periods.

As the TVMS probe was elevated back to the initial height, a short hyperemia of

160.4±16.6% and 199.3±25.9% was observed in the two compression sessions

respectively, after which SCBF returned to the initial levels. Consistently with the SCBF

responses, the reflectance, fluorescence and mitochondrial NADH returned to their basal

levels.

MAP levels monitored in the first two minutes of compression, were significantly

lower

10 In vivo spinal cord monitoring

(59.3±2.5 mmHg and 56.4±3.9 mmHg in the first and second compression, respectively)

than initial MAP levels (77.1±2.4 mmHg and 72.6±4.1 mmHg, respectively). At 7-9 min,

there were observed significant differences (p<0.05) between MAP levels during the two

compression periods. Nevertheless, during the rest of the experiment, MAP recovered

and stabilized at the basal levels. To better understand the relationship between the

various parameters monitored by the TVMS, during treatments where the SCBF

dramatically changed, we performed several correlation tests. The relationship between

SCBF and the mitochondrial redox state (NADH) was described as a polynomic

regression line (p<0.05), shown in Figure 5. Under low SCBF levels, mitochondrial

NADH increased, while under SCBF elevation NADH became oxidized. The correlation

coefficients between NADH and SCBF in all the three experimental groups were

significantly high: R = 0.95 for anoxia, R = 0.94 for ischemia, R = 0.95 for the 1st

compression and R = 0.98 for the 2nd

compression.

Discussion

Although intraoperative monitoring has been increasingly used during the past

decades, there is still a great need for devices that enable not only an early warning for

complications but can also evaluate spinal cord integrity during the procedure in order to

improve the recovery. In the current study, we present a new real-time monitoring

approach for the evaluation of spinal cord vitality. Although the monitoring portion of the

spinal cord by our probe is relatively small (a diameter of 2mm and a depth of

approximately 1mm) we are sure that this area supplies reliable information on the cord

viability. This statement is based on our vast experience in real time monitoring of the

brain showing that even when monitoring takes place simultaneously in different

11 In vivo spinal cord monitoring

locations of the brain under a specific perturbation, using even smaller probes (1mm in

diameter), the responses of all monitored parameters are very similar. Hence the

monitoring of tissue blood flow and mitochondrial NADH level, even in a small area

provides reliable information on the tissue metabolic state 18

.

Spinal cord monitoring technology must include a number of important characteristics:

accuracy, an optimal monitoring site and minimal complication risks. Over the years,

wide experience has been gathered in the intraoperative neurophysiological monitoring of

somatosensory or motor evoked potential, however, there is no consensus among the

physicians as to the reliability and efficiency of these methods during surgeries.14;20-24

During the last years, the new monitoring technique of Near Infrared Spectroscopy

(NIRS) has begun to be implemented. This technique enables a continuous monitoring of

oxy/deoxy hemoglobin levels and cytochrome aa3 in the tissue. When used during spinal

surgeries 25;26

it may allow an early detection of spinal ischemia. However, even though

desirable for its non-invasive features, this technique has not yet been accepted into

routine clinical practice, due to its low spatial resolution,27

namely, the monitored volume

includes a variety of tissue types,28;29

decreasing its accuracy. In addition, the monitoring

is influenced by age, hemoglobin concentration at the measured site, and sensor

location.30

In view of these facts, it is extremely important to suggest an alternative monitoring

approach for the real time assessment of the spine during surgical procedures that may

carry a risk of ischemic injury.

The first step was to develop a monitoring model which would enable the induction of

spinal cord ischemia concomitantly with its monitoring. For this purpose, we chose two

12 In vivo spinal cord monitoring

animal models, inducing a decrease of blood supply: transient abdominal aortic occlusion

and spinal cord compression. In addition, since the decrease in tissue blood supply mainly

involves oxygen deficiency, we also tested the effect of complete oxygen deficit induced

by exposing the animals to pure N2 (anoxia). The anoxia model was selected also in order

to verify the reliability of the monitoring technique, insofar as our vast experience in

brain monitoring using the same techniques has shown its effects on tissue blood flow

and mitochondrial NADH.17;31

As expected, short anoxia caused a rapid oxygen level

drop, triggering wide systemic vasodilation resulting in a blood pressure drop as well as a

decrease in SCBF. This apparently decreased the tissue blood volume, leading to an

elevation in the reflectance level. Moreover, the lack of oxygen led to an interruption of

mitochondrial electron flow and an elevation in NADH levels. When the animals

resumed breathing air, all the parameters returned to their basal levels, indicating that no

irreversible damage to the spinal cord tissue occurred.

The global ischemia model, used in the present study, corresponded to the rat model

reported by Kanellopoulos, Ueno et al, and described by Marsala as “low flow.”32

As

presented in Figure 3, MAP dropped immediately, indicating a complete abdominal

artery occlusion. Additionally, aortic occlusion caused a drastic reduction in SCBF, but

did not completely block it. This was presumably due to additional blood supply sources,

such as posterior spinal arteries and other intercostal branches, namely arteria radicularis

magna (the artery of Adamkiewicz). At the same time, the blood volume in the spinal

cord tissue decreased, changing the absorption characteristics of the tissue and leading to

an increase in the reflectance levels. Simultaneously, the levels of Fluorescence and

NADH rose, indicating tissue metabolic stress and mitochondrial dysfunction due to the

13 In vivo spinal cord monitoring

deficit of oxygen, glucose and other metabolites, shifting the energy equilibrium towards

anaerobic metabolism, as was previously reported.22;33

Once the reperfusion started, all of

the fluorometric parameters fully recovered. The observed hyperemia was probably

produced by auto-regulation mechanisms activated by the sympathetic nervous system, as

was previously reported for ischemia models in pigs and mice.32;34;35

Concerning the focal ischemia model induced by the compression of the spinal tissue,

we had the advantage of directly monitoring the site of primary injury. Indeed, in this

model, SCBF fully decreased to the "no flow" status, which was followed by a partial

recovery towards “low flow” perfusion within the compression phase, and fully

recovered during reperfusion. These changes were inversely correlated to changes in the

mitochondrial NADH levels, indicating that focal ischemia of the spinal cord, during 5

minutes, caused no irreversible damage to the spinal tissue.

The fact that the correlation between the blood supply and the metabolic state of the

spinal cord is not linear can be explained by the action of autoregulatory mechanisms that

are activated when SCBF exceeds its limits of autoregulation. The lower limit of SCBF

seems to be below 30%, yielding an increase of 50% in the mitochondrial NADH level.

The TVMS monitors relative hemodynamic and metabolic changes in a restricted

tissue volume within the posterior area of the spinal cord and is not influenced by the

various layers of the spinal cord. Therefore, the superiority of the present technique lies

in its high spatial and temporal resolution36

as opposed to NIRS which is characterized by

a low spatial resolution.27

Nevertheless, the use of the TVMS in the clinic is suggested

only as a monitoring tool complementary to other neurophysiological monitoring

techniques.

14 In vivo spinal cord monitoring

During the past decade, the connection between the mitochondria and neuronal

survival has become apparent in many pathophysiological processes and clinical

conditions.37

However, in vivo NADH monitoring has not been implicated. It is well

known that mitochondrial NADH is the most stable and representative parameter of

tissue energy metabolism. It is affected by substrate and O2 availability and ATP

turnover, determined by the metabolic activity of the tissue. Nevertheless, very little has

been done to monitor mitochondrial function in vivo in clinical environments. The

monitoring of microcirculatory tissue blood flow (TBF) together with mitochondrial

NADH, reveals that, with the greater number of parameters, a better interpretation can be

given to the very complex pathophysiological situations.38

In conclusion, the use of the TVMS has a great advantage for spinal cord tissue

monitoring during surgical procedures, as it allows an immediate detection of

hemodynamic and metabolic deteriorations of the tissue and, when applied together with

other currently used methods, may help prevent secondary damage to the spinal tissue

and improve the final outcome.

15 In vivo spinal cord monitoring

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20 In vivo spinal cord monitoring

Figure Legends

Figure 1:

Schematic representation of the TVMS placed on the spinal cord surface (A) and a cross

section view of the fiber optic bundle tip (B). Ex - Excitation, Em - Emission optical

fibers for the monitoring of NADH redox sate. LDout/LDin – optical fibers for laser

Doppler blood flow monitoring.

Figure 2:

The effect of a short anoxia (15 sec) on the mean arterial pressure (MAP) and the

recorded metabolic and hemodynamic parameters. Anoxia was induced by N2 (100%)

inhalation. The graph presents average data ± SE (n=29). The arrows mark significant

differences between each value versus the values recorded during the first 30 sec, p<0.05-

(^), p<0.01-(^^), p<0.001(^^^).

Figure 3:

Responses of the various parameters monitored by the TVMS to a 5 minutes abdominal

aortic occlusion. The graph presents mean data ± SE. The arrows mark significant

differences between the values of the ischemic group (n=11) versus the control group

(n=8) for each minute of monitoring, p<0.01-(**), p<0.001(***).

Figure 4:

Responses of the mean arterial pressure, metabolic and hemodynamic parameters, to two

sessions of spinal cord compression (5 min). Calibration was performed 3 minutes before

each compression. The graph presents average data ± SE, n=6. The arrows mark the time

21 In vivo spinal cord monitoring

(each minute) in which the values were significantly different between the two

compression sessions, p<0.05-(*).

Figure 5:

Correlation between SCBF and NADH under short anoxia (N2), ischemia and two

sessions of compression (1st and 2nd).

22 In vivo spinal cord monitoring

3mm

Vertebral body

Spinal cord

Neuroforamen

Nerve root

Laser Doppler Laser Doppler

FlowmeterFlowmeter

Red lightRed light

(632.8nm)(632.8nm)

Spinal Cord Blood FlowSpinal Cord Blood Flow

(Doppler shift)

Back scattered lightBack scattered light

(Blood volume)

Probe

FluorometerFluorometer

UV lightUV light

(366nm)(366nm)

NADH NADH redox redox statestate

(450nm fluorescence)

366nm reflectance366nm reflectance

(Blood volume)

1

A

Figure 1:

23 In vivo spinal cord monitoring

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-10

0

10

20^

-20

-10

0

10

20 ^^^

-20

-10

0

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20

^^^

0

50

100

150^^^ ^

0

50

100

150

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450Time (sec)

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ance

(%

) F

luo

resc

ence

(%

) N

AD

H (

%)

SC

BF

(%

) M

AP

(m

mH

g)

Figure 2:

24 In vivo spinal cord monitoring

Figure 3:

-100

-50

0

50

100ISCHEMIA CONTROL

*

-100

-50

0

50

100 **

-100

-50

0

50

100

**

0

50

100

150

200

***

0

50

100

150

200

0 10 20 30 40 50 60 70 80 90Time (min)

***

MA

P (

mm

Hg

) S

CB

F (

%)

NA

DH

(%

) F

luo

resc

ence

(%

) R

efle

ctan

ce (

%)

Occlusion

Open

25 In vivo spinal cord monitoring

Figure 4:

-100

0

100

200

300

-100

0

100

200

3001st Compression Injury 2nd Compression Injury

-100

0

100

200

300

* *

0

50

100

150

200

0

50

100

150

0 10 20 30 40 50 60 70 80 90Time (min)

MA

P (

mm

Hg

) S

CB

F (

%)

NA

DH

(%

) F

luo

resc

ence

(%

) R

efle

ctan

ce (

%)

Comp.on

Comp. off

*

26 In vivo spinal cord monitoring

Figure 5:

SCBF (%)

NA

DH

(%

)

-10

10

30

50

70

90

0 30 60 90 120 150 180

N2

Ischemia

Compression 1

Compression 2