Ординатура / Офтальмология / Английские материалы / Mastering Corneal Collagen Cross Linking Techniques (C3-R, CCL, CxL)_Garg_2009

4.Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a- induced collagen cross-linking for the treatment of keratoconus. Am J Ophthalmol 2003;135(5):620-27.

5.Wollensak G, Iomdina E. Long-term biomechanical properties of rabbit cornea after photodynamic collagen crosslinking. Acta Ophthalmol 2008;11. [Epub ahead of print]

6.Hafezi F, Kanellopoulos J, Wiltfang R, Seiler T. Corneal collagen cross-linking with riboflavin and ultraviolet A to treat induced keratectasia after laser in situ keratomileusis. J Cataract Refract Surg 2007;33(12):2035-40.

7.Wollensak G. Cross-linking treatment of progressive keratoconus: New hope. Curr Opin Ophthalmol 2006;17(4):356-60.

8.Chan CC, Sharma M, Wachler BS. Effect of inferiorsegment Intacs with and without C3-R on keratoconus. J Cataract Refract Surg 2007;33(1):75-80.

9.Mazzotta C, Balestrazzi A, Baiocchi S, Traversi C, Caporossi A. Stromal haze after combined riboflavin-UVA corneal collagen cross-linking in keratoconus: In vivo confocal microscopic evaluation. Clin Experiment Ophthalmol 2007;35(6):580-82.

10.Wollensak G, Spoerl E, Wilsch M, Seiler T. Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment. Cornea 2004;23(1):43-49.

RELEVANT ANATOMY

The physical strength, constancy of shape and transparency are based on the anatomic and biochemical characteristics of the corneal stroma. Both the epithelium and the endothelium function to maintain corneal transparency.1 The corneal stroma consists of extracellular matrices, keratocytes (corneal fibroblasts) and nerve fibers. Cellular components occupy only 2 to 3% of the total volume of the stroma.2 The rest is occupied by various extracellular matrices mainly collagen and glycosaminoglycans.The collagens in the corneal stroma are primarily collagen type I with lesser amounts of the collagen type III, V and VI. Collagen are stiff fibrous molecules and one of the more abundant proteins throughout the body.

Keratocytes synthesize a pro alpha chain of collagen Three molecules of pro alpha chain are hydroxylated, glycosylated and finally assembled to a procollagen triple helix structure. The characteristic feature of the collagen fibers in the corneal stroma is that they are extremely uniform and constant. This regular arrangement of collagen fibers in the stroma contributes to corneal transparency. The structural properties of the collagen frame work in the corneal stroma determine the biomechanical and optical properties of the tissue.3

BACKGROUND

Keratoconus is a relatively frequent disease often affecting the young. The biomechanical resistance of the cornea in keratoconus patients is half the normal value. Treatment based on collagen cross-linking with the help of ultraviolet (UV) and the photosensitizer riboflavin has been introduced by Wolloensak.4 This treatment is aimed at the pathogenic cause of keratoconus and changes the intrinsic biomechanical properties of corneal collagen. The method of corneal cross-linking using riboflavin and UV-A is technically simple and less invasive than all other therapies proposed for Keratoconus, and unlike other miniinvasive methods, such as intrastromal rings (INTACS) and excimer laser surgery, which do not block keratectasia but merely treat the refractive effects of the diseases, it prevents and treats the underlying pathophysiological mechanism.

2 6 Cross-linking is a common method in the polymer industry to harden materials and in bioengineering to

stabilize tissue. For example, chemical cross-linking with glutaraldehyde is used in the preparation of prosthetic heart valves and physical cross-linking by UV-A is often used in dentistry to harden filling materials. Similarly tissue specimens are preserved and hardened by glutaraldehyde or formaldehyde in pathology. Using UV-A at 370 nm and the photosensitizer riboflavin, the photosensitizer is excited into its triplet state generating so-called reactive oxygen(ROS) and to a much lesser degree superoxide anion radicals. The ROS can react further with various molecules including chemical covalent bonds bridging amino groups of collagen fibrils(type II photochemical reaction).5 The wavelength of 370 nm has been chosen because of an absorption peak of riboflavin at this wavelength.

SURGICAL TECHNIQUE

The treatment is conducted under sterile conditions in the operating room. The patient’s eye is anesthetized with topical anesthetic drops. The central 7 mm of the corneal epithelium are removed to allow better diffusion of riboflavin into the stroma. A 0.1% riboflavin solution (10 mg riboflavin-5-phosphate in 10 ml dextran 20% solution) is applied every 5 min starting 5 min before the irradiation. The irradiation is performed from a 1 cm distance for 30 min using a UVA double diode at 370 nm and an irradiance of 3 mW/cm2 (equal to a dose of 5.4J/cm2).The required irradiance is controlled in each patient directly before the treatment to avoid a potentially dangerous UVA overdose.

STUDIES

The first clinical study on the cross-linking treatment of keratoconus was performed by Wollensak.4 In this 3 year study, 22 patients with progressive keratoconus were treated with riboflavin and UVA. In all the treated eyes, the progression of keratoconus was stopped. In 16, there was a reversal and flattening of keratoconus by two diopters. In the follow-up 5 year study, 60 eyes could be included in the study. No patient had progression of keratoconus. Similar studies have shown stabilization of keratoconus.

Caporossi et al showed a mean K reduction of 2.1+/ - 0.13 D in central 3.0 mm.6 WittigSilva C et al in a series of 66 eyes showed a progressive flattening of

steepest simulated keratometer value over 12 months.7 Kanellopoulos AJ reported significant clinical improvement and apparent stability of more than one year following collagen cross-linking with sequential topography gradual PRK.8 Chan CC reported.

Intacs with collagen cross-linking had a significantly greater reduction in cylinder than the Intacs only group.9

Studies have been conducted to see the biochemical effects, thermomechanical effects and confocal microscopy features. Using a microcomputer– controlled biomaterial testing machine, biomechanical stress-strain measurements showed an increase in corneal rigidity of 71.9% in porcine and 328.9% in human corneas and Young modulus by the factor 1.8 in porcine and 4.5 in human corneas. The cross-linking was maximal only in the anterior 300 microns. The greater biomechanical effect in human corneas is explained by the relatively larger portion of crosslinked stroma because of the lower corneal thickness of 550 microns in human corneas compared with 850 microns in porcine corneas.10

In thermomechanical experiments with porcine corneas, the maximal hydrothermal shrinkage temperature was found to be 70 degree C for the untreated controls , 75 degree C for the cornea cross linked with riboflavin and UV-A and 90 degree centigrade for cross linked with glutaraldehyde, demonstrating the dependence of the shrinkage temperature on the degree of cross-linking . The heat— dependent denaturation of non cross linked collagen could be demonstrated by the loss of birefringence in histological sections.11

Mazzota et al reported the ultrastructural analysis by Heidelberg retinal tomography II and in vivo confocal microscopy in humans.12 One month after the cross-linking therapy, the treated stroma was analyzed by in vivo confocal biomicroscopy at a depth of 80 to 90 microns. A reduction in the keratocyte number associated with a stromal edema (spongy or honeycomb like) was found. Subepithelial and anterior stromal nerve fibers were not found at this depth range. At 3 months, the presence of activated keratocytes, indicative of an initial repopulation of the anterior stroma was seen. However, it was not until the sixthmonth, that a dense cell population of activated keratocytes was observed, with regenerated nerve fibers and increased tissue density without edema.

One month after treatment, confocal analysis at a depth of 130 to 150 microns showed a refraction of keratocytes associated with stromal edema. After 3 months, the edema began to decrease together with an initial ketocyte repopulation and an increase in extracellular fibrillar matrix density. These findings were more accentuated at 6 months, when more activated nuclei and increased stromal density were observed. At this time, the edema had almost disappeared.

At a depth of 170 to 180 microns, the edema was visible at 1 month in the intermediate stroma. It presented ghost nuclei in the fibrillar network, elongated nuclei and the absence of keratocytes. After 2 to 3 months, initial repopulation and reduced edema were evident, aided by the disappearance of the many hyperreflecting oval and elongated nuclei of keratocytic origin. The extracellular matrix had grown denser as the cell population had increased. This increase seemed compatible with a subclinical, microscopically detectable haze that did not seem to impair vision. The haze was greater in patients with more advanced keratoconus, and there were several dark Vogt microstriae. It was not detectable in patients with early-stage disease.

At a depth of 270 to 300 microns, cell necrosis and stromal edema were evident at 1 month, with ghost cells or keratocyte apoptosis bodies in the fibrillar network. Intial signs of cell repopulation were observed at 3 months. Activated oval nuclei and elongated nuclei increased the reflectance of stroma at 3 and 6 months.

The stromal depth of effective cross-linking depends on the concentration of riboflavin solution and the intensity of UV-A light.13 The cross-linking effect seems to localize anterior and collagen fiber diameter is significantly increased only in the anterior half of the stroma, because of the rapid decrease in UV-A irradiation across the corneal stroma as a result of riboflavin enhanced UV-A absorption.14

Kohlhass M et al showed significant stiffing of cornea only in the anterior 200 microns.15

Seiler T et al reported a demarcation line by slit lamp.13 This line was seen in 14 of 16 patients at approximately 60% corneal depth. The line was identified by a thin slit and high illumination levels using a slit lamp that provide high levels of white light.

In the corneal periphery, the line gradually adopts into 2 7

a conical shape because of the increasing total corneal thickness.

The anterior localization of cross-linking treatment is a great advantage because in this way cytotoxic damage of the endothelium is avoided.

Spoerl E reported increased resistence of cross linked cornea against enzymatic digestion supporting this new method in the treatment of corneal ulcers.16 In addition to keratoconus, the other group of patients in whom this treatment seems to work is corneal ectasia following LASIK.17,18 Anecdotal reports of UV cross-linking in the management of corneal edema and its use in the management of corneal ulcers have been presented, but these indications need

careful evaluation.

Risks and Side Effects

UV light in general represents a potential danger to the human eye. UV-induced photochemical damage like sunburn or photokeratitis, both of which are caused, however, by UV-B light. In the cornea UV-B light (290-320nm) is mainly absorbed by the corneal epithelium.

UV-B is also known to mutagenic causing for example, skin cancer. To avoid danger for the endothelium, lens or retina it is mandatory in each patient to perform preoperative pachymetry to exclude extended areas with less than 400 microns stromal thickness, and to check the UV-A irradiance exactly using a UV-A–meter. Stromal haze has been noted and it seems to correlate with the severity of keratoconus.19

Cross-linking treatment for keratoconus is a promising new method of treatment. The treatment is being offered to patients with documented progression of keratoconus. With more experience, prophylactic treatment may be possible at early stage. Additional refractive corrections may be considered if necessary.

In long run, if keratoconus progression is found, a second cross-linking procedure may be the choice.

REFERENCES

4.Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet A-induced collagen cross-linking for the treatment of keratoconus. Am J Ophthalmol 2003;135:620-27.

5.Wollensak G. Cross-linking treatment of progressive keratoconus: new hope. Current Opinion In Ophthalmology 2006;17:356-60.

6.Caporossi A, Baiocchi S, Mazzotta C, Traversi C, Caporossi

T.Parasurgical therapy for keratoconus by riboflavinultraviolet type A rays induced cross-linking of corneal collagen: preliminary refractive results in an Italian study.

JCataract Refract Surg 2007;33:1143-44.

7.Wittig–Silva C, Whiting M, Lamoureux E, Lindsay RG, Sullivan LJ, Lamoureux E, Lindsay RG, Sullivaran LJ, Snibson GR. A randomized controlled trial of corneal collage cross-linking in progressive keratoconus preliminary results. J Refract Surg, 2008;24:S720-25.

8.Kanellopoulos AJ,Binder PS.Collagen cross-linking (CCL) with sequential topography guided PRK a temporizing alternative for keratoconus to penetrating keratoplasty. Cornea 2007;26:891-95.

9.Chan CC, Sharma M, Wachler BS. Effects of inferior segment Intacs with and without C3R on keratoconus. J Cataract Refract Surg 2007;33:75-80.

10.Wollensak G, Spoerl E, Seiler T. Stress strain measurement of human and porcine corneas after riboflavin-ultraviolet

Ainduced cross-linking J Cataract Refract Surg 2003;29:1780-85.

11.Spoerl E, Wollensak G, Dittert DD, Seiler T. Thermomechanical behaviour of collagen-cross-linked porcine cornea. Ophthalmologica 2004;218:136-40.

12.Mazzotta C, Balestrazzi A, Traversi C, Baiocchi S, Caporossi T, Tommasi C, Caporossi A. Treatment of progressive keratoconus by Riboflavin –UVA-induced cross-linking of corneal collagen. Ultra structural analysis by Heidelberg Retinal Tomograph II in vivo confocal microscopy in humans Cornea 2007;26;390-97.

13.Seiler T, Hafezi F. Corneal cross-linking induced stromal demarcation line. Cornea 2006;25:1057-59.

14.Wollensak G, Wilsh M, Spoerl E, Seiler T.Collagen fibre diameter in the rabbit cornea after collagen cross-linking by riboflavin UVA. Cornea 2004;23;503-7.

15.Kohlhass M, Spoerl E, Schilde T, Unger G , Wittig C, Pullinat LE. Biochemical evidence of the distribution of cross-linking in corneas treatment with riboflavin and ultraviolet A light. J Cataract Refract Surgery 2006;32:27983.

16.Spoerl E, Wollensak G, Seiler T. Increased resistance of cross linked cornea against enzymatic digestion. Curr Eye Res 2004;29:35-40.

17.Randleman JB. Post-laser-in-situ keratomileusis ectasia. Curr Opin Ophthalmol 2006;17:406-12.

18.Rabinowitz YS. Ectasia after laser in situ keratomileusis. Curr Opin Ophthalmol 2006;17:421-26.

19.Mazzota C, Blaestrazzi A, Baiochi S, Traversi C, Caporossi

A.Stromal haze after combined riboflavin –UVA cornea collagen cross-linking in keratoconus: in vivo con focal microscopic evaluation: Clin Experiment Ophthalmol 2007;35:580-82.

INTRODUCTION

Keratoconus is the commonest corneal dystrophy affecting 1 in 2000 individuals. It is a degenerative, non-inflammatory disorder of the cornea, characterized by stromal thinning and resultant conical ectasia with associated irregular astigmatism and visual loss.1 It pathophysiology is poorly understood. It is thought to include biochemical, physical and genetic factors. However, no single proposed theory explains the various clinical features and it is likely that the development of keratoconus is the final common pathway for several different disorders. Whilst mild and sub-clinical cases may be corrected with spectacles and soft toric contact lenses, rigid contact lenses provide visual rehabilitation in the majority of cases. However, progressive disease often results in advanced ectasia with associated contact lens intolerance and corneal scarring, which in 10-25% of eyes necessitates surgical intervention usually in the form of corneal transplantation.2-5

In recent years a new therapeutic modality Riboflavin (vitamin B2)/ultraviolet A (UV-A) (370 nm) corneal collagen cross-linkage has been developed, which might be the first treatment available to stabilize the keratoconic process.6 In laboratory studies, it has been shown to increase the stress-strain measurements of corneal stromal tissue, increase its resistance to enzymatic digestion and thermal damage and reduce its hydration rate.7-11 It is thought to induce physical cross-linking of collagen via the lysyl oxidase pathway.12 Riboflavin is essential to this process and has the dual function of acting as a photosensitizer for the production of oxygen free radicals which induce the actual physical cross-linking of collagen12 as well as concentrating and absorbing the UV-A irradiation and preventing damage to deeper ocular structures such as the corneal endothelium, the lens and the retina.13-16 The technique has been shown to be safe with no loss of corneal transparency and no endothelial cell damage, provided the cornea is thicker than 400 μm, and no damage to deeper ocular structures.13-

16

In an initial pre-clinical study, Spoerl et al demonstrated the need for complete central epithelial debridement by a lack of alteration in biomechanical properties of corneal tissue where the technique had 3 0 been performed with the epithelium intact.16 On this

basis the epithelium was removed in the first published clinical studies prior to treatment.14 Despite this recommendation a number of clinicians have elected to perform the technique with the epithelium intact in order to reduce the postoperative discomfort experienced by their patients.17 Some have advocated the use of multiple applications of the topical anesthetic tetracaine 1% in an attempt to loosen the epithelial tight junctions.17 Others have advocated limited full-thickness epithelial debridement in a gridlike pattern, with islands of intact epithelium to facilitate more rapid postoperative epithelial healing (Professor D. Reinstein, personal communication).

In order to investigate the importance of epithelial removal for facilitating the entry of riboflavin into the corneal stroma, we have measured the light transmission spectra of porcine corneas after riboflavin eyedrop administration using spectrophotometry following either complete epithelial debridement, superficial epithelial trauma or no epithelial trauma with the preand perioperative administration of topical tetracaine 1%.18 We have also investigated techniques to assist the entry of riboflavin into the stroma by either loosening the epithelial tight junctions with application of Alcohol solution 20% for 40 seconds or by removing the epithelium in a grid pattern rather than complete debridement.19 These measurements have been compared with untreated control corneas and the absorption spectrum of the riboflavin solution itself. We have also examined the effects of the cross-linkage treatment on light transmission by exposing a number of corneas to UVA irradiation in combination with riboflavin eyedrops.

METHODOLOGY OF OUR STUDIES18,19

One hundred and twenty-five porcine eyes were transported on ice from a local abattoir within 24 hours of death. A visual examination of each specimen for the presence of corneal scarring or opacity resulted in 13 eyes being excluded from the study. The remaining 112 eyes were stored overnight in a sealed bag at 4°C. From these 45 eyes were selected at random for inclusion in the study and divided into the following treatment groups:

1.Controls: The central epithelium (10.00 mm in diameter) was completely removed from 5 corneas using a scalpel blade; the epithelium was left intact on a further 4 corneas.

at a distance of 1cm from the anterior surface of the cornea. The cross-linking treatment lasted 30minutes during which time further riboflavin 0.1% eye drops were applied at 5-minute intervals.

4.Tetracaine plus Riboflavin (no epithelial trauma): Topical tetracaine 1% and riboflavin 0.1% eye drops (10 mg riboflavin-5-phosphate in a 10 ml dextran T-500 20%) were administered to the intact, non-traumatized anterior corneal surface of 6 corneas at 5-minute intervals over a 35minute period, in order to simulate 5 minute preoperative and 30-minute operative time periods.

5.Tetracaine and Riboflavin eyedrops plus UV-A (no epithelial trauma): Tetracaine 1% and riboflavin 0.1% eyedrops were applied to 6 corneas with an intact non-traumatized epithelium. After waiting for 5 minutes the corneas were exposed to a 3 mW/cm2 dose of UV-A (370 nm) at a distance of 1cm from the anterior surface of the cornea. The cross-linking treatment lasted 30 minutes during which time further riboflavin and tetracaine drops were applied at 5-minute intervals.

6.Riboflavin only (epithelium completely removed): Following complete de-bridement of a central 10 mm area of corneal epithelium with a scalpel blade, riboflavin 0.1% drops were applied at 5-minute intervals for 30-minutes in 6 eyes.

7.Riboflavin plus UV-A (epithelium completely removed) (Figure 7.1): Following complete removal of the central epithelium, riboflavin 0.1% drops were applied to the exposed stromal

homogeneous area of yellow discoloration can be seen beneath the area of full-thickness epithelial trauma (black arrow). At the corneal periphery under areas of intact epithelium there is no yellow discolouration (white arrow)

surface. After waiting for 5-minutes the corneas were exposed to a 3mW/cm2 dose of UV-A (370 nm) at a distance of 1cm from the anterior surface of the cornea. The cross-linking treatment lasted 30-minutes during which time riboflavin drops were applied at 5-minute intervals.

8.Alcohol group: In 6 eyes, 20% alcohol solution was applied to the central corneal epithelium with a 9.00 mm laser epithelial keratomileusis (LASEK) well for 40 seconds. Following alcohol administration no attempt was made to remove the epithelium. Riboflavin eyedrops were then administered to the anterior corneal surface. After waiting for 5-minutes the corneas were exposed to a 3mW/cm2 dose of UV-A (370 nm) at a distance of 1cm from the anterior surface of the cornea. The exposure time was 30-minutes during which time further riboflavin drops were applied at 5-minute intervals.

9.Grid pattern epithelial debridement (Figure 7.2): Following a grid pattern full thickness epithelial trauma of the central cornea, at least 7 ° 7 mm in size and with 30 to 40 separate abrasions placed within this area, riboflavin drops were applied to 6 eyes. After waiting for 5-minutes the corneas were exposed to a 3 mW/cm2 dose of UV-A (370 nm) at a distance of 1cm from the anterior surface of the cornea. The cross-linking treatment lasted 30-minutes during which time

riboflavin drops were applied at 5-minute 3 1 intervals.

Figure 7.2: A cornea with grid pattern epithelial trauma following UV-A/riboflavin treatment with application of Riboflavin 5 minutes pre-treatment and every 5 minutes during 30 minutes of ultraviolet light exposure. Although areas of yellow discolouration (black arrow) in a grid pattern can be seen beneath the areas of full-thickness epithelial trauma, there is lack of homogeneous absorption

Immediately following treatment each cornea with a 3 mm scleral rim was dissected from the globe and placed into a specially designed sample holder. The natural curvature of the cornea was maintained by clamping the scleral rim within the sample holder and injecting silicon oil (Dow Corning 200/5cS, BDH Laboratory Supplies, Poole, UK) into the chamber behind it. Silicon oil was also injected into the front chamber of the holder so as to maintain a uniform refractive index and reduce light scatter.20 The sample holder was then positioned into the spectrophotometer (PYE Unicam, SP8-100 UV/VIS) in such a way that light passed through the center of the cornea in the anterior-posterior direction. The optics of the unit and the aperture were set to give a slit of (no larger than) 1 ° 1mm on the surface of the cornea, i.e. at the point where the center of the cell lies, and it was ensured that the cell lay such in the path length that the beam was always incident on the center. A transmission spectrum was measured for each cornea at 10 nm intervals within the range of 400 to 700 nm. Although this spectrum is within the visible spectrum, it is outside the treatment wavelength of 350 to 380 nm. However, it does include one of the peak absorption spectra of Riboflavin at 400 to 490 nm and is therefore relevant to detect changes in light transmission due to stromal absorption of Riboflavin. Using the method detailed

3 2 by Kostyuk and colleagues,20 the transmission

spectrum for each sample was normalized against a baseline transmission spectrum of the chamber filled with silicon oil. A further transmission spectrum over the same wavelength range (400-700 nm) was obtained for riboflavin 0.1% solution (10 mg riboflavin-5- phosphate in a 10 ml dextran T-500 20%) alone.

Student t-tests were used to compare transmission values. Results with p<0.05 were considered statistically significant.

RESULTS FROM OUR STUDIES18-19

Removal of the epithelium had no significant affect on the transmission spectra of control corneas. In each case, a gradual increase in light transmission occurred between 400 and 700 nm. Based on this finding, the spectra of all control corneas (with or without epithelium) were averaged for comparison with the various study groups. Figures 7.3A to C show the average transmission spectra of control corneas, corneas treated with riboflavin only and corneas treated with riboflavin plus UV-A. The standard error bars associated with each spectrum are the result of variations in the hydration of corneas within treatment groups.

The transmission spectrum of corneas with superficial epithelial trauma (but with basal epithelium intact) treated with either riboflavin alone or with riboflavin and UV-A, did not differ from each other or from that of the control corneas (Figure 7.3A). Similarly, the transmission spectrum of corneas with no epithelial trauma treated with either riboflavin and tetracaine alone or with riboflavin, tetracaine and UV- A, did not differ from each other or from that of the control corneas (Figure 7.3B). Complete removal of the epithelium prior to either riboflavin-only or riboflavin plus UV-A treatment did however result in a dramatic reduction in light transmission between 400 and 510 nm (P<0.01) (Figure 7.3C). At 450 nm light transmission was on average 32% lower in riboflavintreated corneas that had experienced complete epithelial removal compared to the untreated control corneas. This dip in light transmission may be attributed to the presence of riboflavin within the tissue which absorbs light between 400-510 nm (Figure 7.4). The light transmission spectrum of corneas with a fully removed epithelium treated with riboflavin plus UV- A did not differ from those treated with riboflavin alone (Figure 7.3C).