cross-linking of scleral collagen in the rabbit using riboflavin and uva

Cross-linking of scleral collagenin the rabbit using riboflavin andUVA

Gregor Wollensak,1 Elena Iomdina,1 Dag-Daniel Dittert,2

Olga Salamatina3 and Gisela Stoltenburg4

1Moscow Helmholtz Research Institute of Eye Diseases, Moscow, Russia2Institute of Pathology, University Clinic Carl Gustav Carus, Dresden, Germany3Semenov Institute for Chemical Physics, Russian Academy of Sciences, Moscow,

Russia4Institute of Neuropathology, Charite Berlin, Berlin, Germany

ABSTRACT.

Purpose: Scleral biomechanical weakness and thinning is known to be one of the

main factors in the pathogenesis of progressive myopia. We tried to strengthen

rabbit sclera by cross-linking scleral collagen using ultraviolet A (UVA) and the

photosensitizer riboflavin.

Methods: Circumscribed 10· 10mm sectors of the posterior- equatorial sclera of

six chinchilla rabbit eyes were treated in vivo using a UVA double diode with

4.2 mW/cm2 UVA at 370nm and applying 0.1% riboflavin-5-phosphate drops as

photosensitizer for 30min. 1 day postoperatively biomechanical stress�strain meas-

urements of three treated scleral strips were performed using a microcomputer-

controlled biomaterial testing device and compared to non-treated contralateral

control sclera. In addition, three treated eyes were examined histologically by light

microscopy, TUNEL staining and electron microscopy to evaluate side-effects.

Results: Following the cross-linking treatment, the ultimate stress was

11.87– 1.8MPa versus 3.63– 0.40 in the controls (increase of 227.9%, p= 0.014),Young’s modulus 27.67– 4.16MPa versus 4.9– 2.15MPa in the controls (increase of

464.7%, p= 0.021) and ultimate strain 92.2– 7.43% versus 165.63– 19.09% in the

controls (decrease of 54.52%, p= 0.012). Histologically, serious side-effects were found

in the entire posterior globe with almost complete loss of the photoreceptors, the outer

nuclear layer and the retinal pigment epithelium (RPE).

Conclusions: Our new method of scleral collagen cross-linking proved very effective

in increasing the scleral mechanical strength; the new treatment may represent an

option for strengthening scleral tissue in progressive myopia. However, serious side-

effects were observed in the outer retina. In future studies these side-effects could be

avoided by reducing the irradiation dose below the cytotoxic level of the retina. Before

its clinical application, the new method should be tested in a myopia animal model.

Key words: progressive myopia – sclera – UVA – riboflavin – collagen cross-linking – Young’s modulus

Acta Ophthalmol. Scand. 2005: 83: 477–482Copyright # Acta Ophthalmol Scand 2005.

doi: 10.1111/j.1600-0420.2005.00447.x

Introduction

Progressive myopia is one of the mostimportant unsolved problems inophthalmology.

Myopia is reported to affect around30% of the general population in theUSA and Europe and up to 60% inAsian countries (Saw et al. 2002;McBrien & Gentle 2003). Myopic pro-gression occurs in up to 50% of myopes,usually at a rate of around�0.5 dioptresover a 2-year interval (Bullimore et al.2002). The pathogenesis of myopia isstill controversial. One of the importantfeatures of severe myopia is a patho-logical change of the sclera with pro-gressive thinning of the sclera, probablydue to a disturbed feedback mechanismof emmetropization after visual depri-vation, such as in cataract or ptosis(Weiss 2003) or due to some metabolicdisorder of the sclera, such as inEhlers–Danlos syndrome (Mechanic1972). Ultimately, both mechanismslead to stretching and thinning of thesclera, retina and choroid due to struc-tural abnormalities of the myopic sclerasuch as a decreased collagen fibre dia-meter (Curtin et al. 1979; Liu et al.1986) and disturbances of the fibrillo-genesis (Funata & Tokoro 1990). Fromanimal experiments with tree shrews(McBrien & Norton 1994) and fromdiseases with cross-linking disorders(Mechanic 1972) we know that

ACTA OPHTHALMOLOGICA SCANDINAVICA 2005

477

impaired collagen cross-linking is animportant factor in the weakening pro-cess of the myopic sclera (Iomdina et al.1993). Several methods of surgicalscleroplasty, such as injections of apolymeric composition forming afoamed gel under Tenon’s capsule andinducing scar tissue or so-called scleralreinforcement operations where donorsclera, fascia lata or synthetic bands areplaced around the back of the globeand sutured onto the sclera to providescleral support and reduce the progres-sion of axial elongation (Avetisov et al.1997), have been applied for the pur-pose of sclera strengthening. However,these methods do not regenerate theinternal structure and cross-linkingproperties of the weakened sclera.

Recently, collagen cross-linkinginduced by the photosensitizer ribofla-vin and ultravioletA (UVA) irradiationat 370 nm has been found to be success-ful in treating keratoconus, where asimilar thinning process and loss of bio-mechanical stability occurs in the cor-nea, as in the myopic sclera (Wollensaket al. 2003). In vitro experiments haveshown that the cross-linking methodalso leads to a significant increase inYoung’s modulus of treated porcineand human sclera by up to 157%(Wollensak & Sporl 2004). Therefore,this newly developed cross-linkingmethod might also be a novel wayof strengthening the sclera, therebypreventing myopic progression. The pre-sent pilot study was designed to test thenew scleral collagen cross-linkingmethod in vivo in rabbits. In particular,we aimed to develop the practical feasibil-ity of cross-linking the posterior sclera, toinvestigate the biomechanical efficiency ofin vivo cross-linking and to investigatepossible side-effects.

Material and Methods

Animals

Six female chinchilla rabbits (3.5 kg)were treated. The cross-linked righteyes were used for biomechanical meas-urements (n¼ 3) and histological exam-ination (n¼ 3). The contralateral left eyesserved as controls.

Treatment procedure

Premedication was performed withsubcutaneous injection of a mixture ofdiazepam and atropine (1mg). For gen-eral anaesthesia 1.5ml ketamine hydro-

chloride 10% (35mg/kg) and 0.5mlxylazine hydrochloride (5mg/kg) wereused. For local anaesthesia procaracaineyedrops were instilled. In each rabbitonly the right eye was treated. A lidspeculum was placed into the fornix.The conjunctiva was incised in theupper anterior quadrant of the righteye using a pair of scissors. The retractorbulbi muscle and the superior obliquemuscle were dissected at their scleralinsertion. Two scleral sutures (Dacron5–0) were placed in the pre-equatorialsclera of the treatment quadrant andused like reins to move and hold theeyeball. Using the scleral sutures, the eye-ball was rotated to expose the posteriorand equatorial sclera of the upper ante-rior quadrant. Photosensitizer solutioncontaining 0.1% riboflavin-5-phosphatewas dropped onto the posterior sclera5min before the irradiation and every5min during the irradiation. Ultravio-letA irradiation (370nm) was appliedusing a double UVA diode (RoithnerLasertechnik, Vienna, Austria) with asurface irradiance of 4.2mW/cm2 for30min at a distance of 1 cm from thesclera (7.6 J/cm2). Three 1.3V accumu-lators were used as power supply. Beforethe treatment, the desired irradiance of4.2mW/cm2 was controlled with a cali-brated UVA meter (Lasermate-Q,Laser 2000, Wessling, Germany) at1-cmdistance and, if necessary, regulatedwith a potentiometer. The treatment areawas 10� 10mm and included the equa-torial and posterior sclera of the upperanterior quadrant. After irradiation, thescleral sutures were removed, chloram-phenicol solution was applied and theconjunctiva closed using a vicryl 7–0thread. The animals were killed 1 daypostoperatively under general anaesthe-sia using an overdose of intravenous

sodium phenobarbital. All animal pro-cedures conformed with the ARVOResolution on the Use of Animals inOphthalmic and Vision Research.

Specimen preparation for the

biomechanical measurements

After making a complete circular inci-sion 2mm behind the limbus, the ante-rior eye segment was removed from theenucleated eyes. The posterior eye cupswere turned around with the help of aforefinger and the retina and choroidremoved. A 4� 10mm scleral stripwas dissected sagitally from the treat-ment area using a scalpel. A controlstrip was taken from the contralateralleft eye at the same position. The scleralthickness of the strips was determinedusing a mechanical micrometer caliper.

Biomechanical measurements

Stress�strain measurements were per-formed for three sclera specimens andtheir contralateral controls. The4� 10mm scleral strips were clampedhorizontally with a distance of 6mmbetween the jaws of a commerciallyavailable microcomputer-controlled bio-material tester (Minimat 2000; Rheo-metric Scientific GmbH, Bensheim,Germany) (Fig. 1). Strain was increasedlinearly at a velocity of 1mm/min andstress was measured up to tissue rupture.The parameters ultimate stress s (MPa)and ultimate strain e (%) of the sampleswere used for analysis. Young’s modulusE (MPa) was determined as the slope ofthe stress�strain graph at 50% strain.

Statistical evaluation

The data for ultimate stress, ultimatestrain and Young’s modulus werestatistically compared between the

Fig.1. Biomechanicalmaterial testerwith rabbit scleral strips between the clamps that are drawn apart.


478

cross-linked treatment group and theuntreated control group, using Student’st-test.

Temperature measurements

To exclude a thermal effect we mea-sured the temperature rise in six post-mortem scleral specimens which wereexposed to UVA light (4mW/cm2)plus riboflavin for 30min. We applieda contact method using a copper-constantan-thermocouple with a probetip diameter of 80 mm (Therm 2280–8;Ahlborn Mess- und Regelungstechnik,Holzkirchen, Germany).

Light and electron microscopy

Three treated and three control rabbiteyes were used for histological sections.The eyes were bisected. One half wasfixed in 4% neutral-buffered formalinfor light microscopy and the other halfwas immersed in 2% glutaraldehydefor transmission electron microscopy(TEM). For light microscopy, 4mmthin paraffin sections were stained withhaematoxylin-eosin and periodic Schiffreagent (PAS). The specimens wereevaluated using a Zeiss light microscope(Axiomat; Zeiss, Oborkochen, Germany)at 40–1000-fold magnification.

For TEM, small, full-thicknesspieces of the treatment area includingretina, choroid and sclera were post-fixed in 4% osmium tetroxide, dehy-drated and embedded in Epon resin.After observation of semithin sectionsstained with toluidine blue, 50–70 nmthick sections of the relevant areaswere prepared, mounted on coppergrids, contrasted with uranyl acetateand lead citrate and assessed using theelectron microscope EM-902 (Zeiss) ata magnification of 1100–34 000.

TUNEL

To detect apoptosis, terminal deoxy-nucleotidyl transferase (TdT) medi-ated biotin�dUTP nick-end labelling(TUNEL) was performed. After quench-ing the endogenous peroxidase, sectionswere incubated with TdT buffer (30mNTRIS, 140mM sodium cacodylate,1mM cobalt chloride) at pH7.2 andincubated with 0.3 eu/ml TdT (Sigma,Munich, Germany) and biotinylated-dUTP (1 : 200; Boehringer, Mannheim,Germany) in TdT buffer for 60min at37 �. Labelled nuclei were detected withVectastain ABC (Vector Laboratories,

Burlingame, California, USA) and per-oxidase activity was visualized by3-amino-9-ethylcarbazole (AEC) toyield a brownish reaction product. Thesections were lightly counterstainedwith haematoxylin. As a positive con-trol, tissue sections of follicular hyper-plasia of the appendix were used andgave the expected positive staining oftingible bodies in the germinal centres.

Results

Scleral thickness

The rabbit scleral thickness was deter-mined to be 0.44� 0.12mm for the trea-ted eyes (n¼ 3) and 0.41� 0.1mm forthe contralateral controls (n¼ 3). Therewas no statistically significant differencebetween the two groups (p¼ 0.2).

Biomechanical measurements

A comparison of the biomechanicalparameters of experimental and controlsamples of the rabbit sclera showed thatsignificant changes occurred in all thecharacteristics tested (Tables 1 and 2).In the cross-linked specimens theultimate stress showed an increase of228% (p¼ 0.01), Young’s modulus by465% (p¼ 0.02), and the ultimate straina decrease of 31.07% (p¼ 0.01).

Temperature measurements

We measured a temperature rise of1.52� 0.21 � after 30min UVA expos-ure (4mW/cm2) plus riboflavin so thata thermal effect was excluded.

Light microscopy

In the cornea there was considerablestromal oedema, loss of keratocytesand corneal epithelium in the cornealhalf of the treated side (Fig. 2) in thefirst two rabbits, where the diode hadnot been kept perpendicularly all thetime. The endothelium, however, wasintact. In addition, there was almost

complete loss of the outer nuclear andplexiform layer and the photoreceptorsegments in the posterior portion of thetreated eyes with a sharp, abrupt tran-sition towards the undamaged, non-irradiated retina (Fig. 3). Remarkably,there were no inflammatory cells pre-sent in the area of the retinal irradiationdamage as is typical in apoptotic celldeath. The retinal pigment epithelium(RPE) cells were massively rarefied,irregularly shaped and plump (Fig. 3).The outer retinal damage was also pre-sent at the opposite side because theUVA light passed through the vitreousbut not in the peripheral anteriorretina. There was some fibrin and amild inflammatory infiltrate at theouter sclera in the treatment area.About 5% of the scleral fibrocytesappeared pyknotic.

TUNEL

TUNEL-positive cells were identified inless than 10% of the cells in the adja-cent inner nuclear layer and less than5% in the sclera.

Electron microscopy

There were only a few remaining RPEcells in the irradiated area. The photo-receptor inner and outer segments andtheir cell bodies in the outer nuclearlayer were almost completely absent,with large vacuoles and some cellulardebris instead. Inflammatory cells werealso absent. A few plump melanin-laden macrophages could be identified.A few scattered cells of the innernuclear layer and of the scleral fibro-cytes showed sign of apoptosis likepyknosis, margination and condensa-tion of chromatin, cytoplasmic shrink-age, nuclear fragmentation andapoptotic bodies. There were no obviouschanges of scleral collagen fibres,especially no signs of a heat effectlike shrinkage, coagulation scars ordistortion.

Table 1. Primary data of the biomechanical parameters.

Animal no. Ultimate stress

(MPa)

Young’s modulus

(MPa)

Ultimate strain

(%)

1 Treated 12 31 90.3

1 Control 3.2 5 162.5

2 Treated 10 29 100.4

2 Control 3.65 2.7 186.1

3 Treated 13.6 23 85.9

3 Control 4 7 148.3


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Discussion

This study has demonstrated animpressive stiffening effect of cross-linking by riboflavin and UVA on invivo rabbit scleras. The ultimate stresswas increased by 228% (p¼ 0.01),Young’s modulus by 465% (p¼ 0.02),and the ultimate strain decreased by31.07% (p¼ 0.01). Thus, the biomech-anical tests showthat theproposedmethodof sclera reinforcement induces a substan-tial improvement of the stress� strainparameters of the sclera. As the treatmentbrings about the formation of a morestable internal structure of the sclera byinducing intra- and interfibrillar collagencross-links (Kato et al. 1994; Wollensak& Sporl 2004), the creation of anentirely new and promising technique ofprevention and treatment of progressivemyopiamay become possible. The ribofla-vin-sensitized photoreaction generates freeradicals and so-called reactive oxygen spe-cies such as singlet oxygen, superoxide or

superoxide anion radicals, which inducecross-linking of collagen with, therefore,increased molecular weight. The aggrega-tion of collagen is at least partially due tothe formation of dityrosine (Kato et al.1994). In addition, a simple temperatureeffect was excluded in our study with ascleral temperature rise of only 1.5 � andabsence of histological signs of tissueshrinkage or coagulation. In comparisonwith our in vitro results of human andporcine sclera, the relative increase inYoung’s modulus by 464.7% in the rabbitis even more than in human (29%) andporcine sclera (157%). This is probablypartially due to differences in the baselinevalues of untreated sclera in the variousspecies, which is about the same for por-cineandrabbit sclerabuthigher inhumansby a factor of 3 (Wollensak& Sporl 2004).

Our experiment has shown that thein vivo application of the new cross-linkingmethod is also technically feasible forthe posterior sclera by using two scleralsutures to rotate and hold the globe

while irradiating the posterior sclera.Infiltration of the sclera with ribofla-vin-5-phosphate (MW 456) is possiblebecause the sclera is permeable to mole-cules as large as 150 000Da, as has beendemonstrated with rabbit sclera(Ambati et al. 2000). In contrast toother cross-linking methods the extentof the treatment area was easily con-trollable with UVA irradiation becauseof the visible UVA light-induced fluor-escence. The target area at the posteriorand equatorial sclera was chosenbecause in myopia these scleral areasare mainly affected. Usually the equa-torial sclera is affected in the beginningand to a lesser degree and the posteriorsclera in the late phase and more dra-matically (Awetissow 1980; McBrien &Gentle 2003).

On the other hand, our study hasshown severe side-effects of the UVAirradiation on the outer layers of theretina and on keratocytes and epithe-lium in the cornea. From other studies,it is known that selective destruction ofthe photoreceptors can be observed at380 nm and combined damage of bothphotoreceptors and RPE with highUVA doses, as was the case in our ser-ies. It is supposed to be due to thephotosensitizing action of the rhodop-sin bleaching product all-trans–retinolat 380 nm (Noell 1980; Zuclich 1989;Gorgels & van Norren 1995). Remark-ably, the opposite side of the retina wasalso affected because the UVA irradia-tion passed the vitreous space andreached the opposite retina as well. Inthe future, we must find the thresholddose for the retinal UVA-induceddamage (‘action spectrum’) so that theUVA dose can be lowered accordingly.The keratocyte damagewas only observedwhen the diode was slightly tilted towardsthe cornea so that the UVA light waspropagated in the cornea as in a fibreoptic. Interestingly, the endotheliumwas not damaged, possibly due to thereflection of the UVA light at Descemet’smembrane. Keratocyte damage is alsoknown from UVA irradiation passingthe cornea from the anterior (Zuclich1989; Wollensak et al. 2004). However,the corneal side-effects can be easilyavoided by holding the diode strictly per-pendicular to the sclera so that no scatter-ing to the anterior eye segment and corneacan occur. In addition, UVA protectioncan be achieved by laying an aluminiumfoil on the cornea to shield it from UVAirradiation. Remarkably, there was only

Table 2. Statistical analysis of the biomechanical parameters.

Ultimate stress

(MPa)

Young’s modulus

(MPa)

Ultimate strain

(%)

Mean (control) 3.62� 0.40 4.9� 2.15 166.63� 19.09

Mean (cross-linked) 11.87� 1.8 27.67� 4.16 92.20� 7.43

Student’s t-test p¼ 0.014 p¼ 0.021 p¼ 0.013

Percentage 227.9%" 464.7%" 31.07%#

Fig. 2. Photomicrograph of the corneal half on the treatment side with mild inflammatory

infiltration, stromal oedema, loss of keratocytes and epithelium. The endothelium was intact,

however. (Haematoxylin-eosin stain; original magnification �400).


480

minor damage to the scleral fibrocytes,demonstrating the low sensitivity of thesecells towards UVA light.

The rationale for the cross-linkingtreatment in myopia is well founded.Previous biomechanical studies haverevealed the fact that the stress�strainparameter values and Young’s modulusof the sclera of myopic human eyes aresignificantly lower than the age norm(Awetissow 1980). The tensile strengthof the scleral tissue is especially low inthe equatorial and the posterior poleareas. Young’s modulus has beenfound to be 1.2–1.3 times lower than incontrols (Iomdina et al. 1993). In parti-cular, the reduction in the number ofcross-links in myopic sclera of about15% in the equatorial area and about12% in the posterior pole area as meas-ured by nuclear magnetic resonance isclear evidence for a substantial decreasein the level of cross-linking stability ofthe respective areas of myopic sclera(Iomdina et al. 1993). Similar resultswere found in myopic tree shrews(McBrien & Norton 1994). The reduc-tion of the stress�strain parameters ofmyopic sclera is related primarily to aninsufficient formation of stabilizingintra- and intermolecular cross-links inthe scleral collagen, or to their destruc-tion (Funata & Tokoro 1990). Interest-ingly, high myopia, blue sclerae andkeratoconus have been described in

Ehlers–Danlos syndrome, where a lackof intermolecular cross-links of collagenis known (Mechanic 1972; Bell 1993).Conversely, in diabetes patients whereglucose induced collagen cross-linkingis known, axial myopia is very rare(Pierro et al. 1999). Collagen cross-link-ing is also increased by ageing(Bailey 1987). This may be the reasonwhy myopic progression and buphthal-mus are not observed in elderly people(Bell 1978). Accordingly, in the treeshrew, a reduction in creep extensibilityhas been found with age (McBrien &Gentle 2003). Moreover, recent animalexperiments in tree shrews have shownan important influence of collagen cross-linking on the development of experi-mental myopia (McBrien & Norton1994). Therefore, the formation ofcross-links in the sclera might be thekey to an efficient treatment of progres-sive myopia (Bell 1978, 1993). The devel-opment of methods for collagen cross-linking of the sclera is an importantpractical task as it can contribute to theprevention and treatment of myopia.

In the future, we want to test the newcross-linking treatment in a suitablemyopia model, such as the tree shrew(McBrien & Norton 1994; McBrienet al. 2003) or monkey (Troilo et al.2000). If successful, the cytotoxic doselevel for the human or, at least, the mon-key retina must be determined and the

dose level chosen accordingly before itsclinical application. It is to he hoped thatit will then be possible to treat patientswith progressive myopia in order to haltmyopia, one of the oldest and greatest, butstill unsolved, problems in ophthalmology.

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Received on June 28th, 2004.

Accepted on January 15th, 2005.

Correspondence:

Gregor Wollensak

Wildentensteig 4

DE-14195 Berlin

Germany

Tel:þ 49 30 826 4499

Fax:þ 49 30 826 4499

Email: [email protected]


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cross-linking of scleral collagen in the rabbit using riboflavin and uva

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