Ординатура / Офтальмология / Английские материалы / Mastering Corneal Collagen Cross-linking Techniques (C3-R CCL CxL)_Garg, Kanellopoulos, O'Brart, Lovisolo, Pinelli_2008

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

The term cross-linking indicates a medical intervention; it was originally used in specialties such as dentistry and orthopedics. Theo Seiler, MD, PhD, of Switzerland, was the first to suggest applying this principle to ophthalmology, more specifically cross-linking corneal collagen fibers.

After researching this idea, Professor Seiler and his colleagues studied the use of riboflavin (vitamin B2) and UVA irradiation, noting that the combination induced a strengthening of the corneal stroma. This effect was obtained by creating new bonds between the collagen fibers—where unstable riboflavin molecules produced these bonds after irradiation with UVA. This early research proved an effective treatment for keratoconus; however, one problem was standardizing the main parameters of the treatment, including riboflavin concentration and penetration, UV fluence, and time of exposure. This standardization was necessary to render the treatment safe and effective.

PRESENT

The corneal collagen cross-linking, or C3-R, treatment initially required epithelial debridement to improve riboflavin penetration in the stroma; however, now the treatment may be performed with or without deepithelialization. There are different opinions regarding epithelial debridement, but we must remember that most complications of the procedure (infections, slow healing, subepithelial haze) arise from de-epithelializa- tion. Epithelial healing in keratoconic corneas is indeed much slower than in healthy corneas, and may take several weeks after C3-R in some eyes (personal observation). Some surgeons argue that leaving the epithelium on the stroma is less efficacious because it slows the penetration of the riboflavin on the stroma; however, our experiences demonstrate the opposite.

Recently, Pinelli et al used fluoroscopy to observe the absorption of riboflavin in the absence of epithelial debridement (Figure 8.1). Riboflavin 0.1% was applied to the cornea via a saturated Merocel sponge and left on the eye for 5 minutes before the start of UVA light administration. We repeated riboflavin applications every 3 minutes. After 6 minutes, the riboflavin penetrated under the epithelium; after 14 minutes, it penetrated the middle of the stroma; and after 30 minutes, we observes its full diffusion. Our research

demonstrated that during C3-R treatments, leaving the epithelium in tact does not significantly limit the penetration of the riboflavin.

PERSONAL EXPERIENCE

Observing via fluoroscopy the riboflavin absorption without epithelial removal, we noticed that the epithelium does not restrict significantly the riboflavin penetration.

Riboflavin 0.1% (PriaLight®, PriaVision, Menlo Park, CA, USA) was applied on the cornea via a saturated Merocel sponge for 5 minutes before the start of UVA light administration. The riboflavin is then applied every 3 minutes during the whole procedure.

After 6 minutes the riboflavin penetrates under the epithelium; after 14 minutes it penetrates in the middle stroma and after 30 minutes we can observe its full diffusion (Fig. 8.1).

On this basis, we conducted a comparative study to evaluate the difference between C3-R with and without disepithelialization on patients affected by keratoconus.

Each group (group A and group B) was composed of five patients each.

The A group was disepithelialization treated monocularly with C3-R without disepithelization; the B group was treated monocularly with C3-R with disepithelization.

Before the treatment, all patients had an assessment of uncorrected visual acuity (UCVA), best spectaclecorrected visual acuity (BSCVA), manifest refraction spherical equivalent (MRSE), bio-microscope evaluation (corneal and lens transparency), intraocular pressure (IOP), corneal computerized topographic examination (Eyesys), linear scan optical tomography (Orbscan II), endothelial cell count and ultrasound pachimetry, and a satisfaction questionnaire was also administrated in order to monitor the level of satisfaction reached by the two different groups. All examinations were repeated at six and nine months after C3-R treatment. Exclusion criteria included pachimetry thinner than 400 µm and aphakic eye.

Each eye was treated with proparacaine 0.5% for < 30 minutes before exposure (i.e. approximately two drops every 5 minutes). Riboflavin was then applied on the cornea for < 25 minutes before irradiation and

was then activated by a 30-minute exposure to the 3 9

UV-A light (i.e. 370 nm fluence at 3 mW/cm2). Riboflavin solution was reapplied on the cornea every 3 minutes during the UV-A irradiation.

Figure 8.1: The penetration of riboflavin under the epithelium

Figure 8.2: Comparative postoperative results

or electronic microscopy; indirect through the study of the molecular properties of collagen). The non-de- epithelized group showed transparent cornea without any stromal abnormality (Figures 8.3 and 8.4).

The postop. therapy for the first group needed topical steroids for two weeks, while the second group of patients only needed artificial tears for one week.

At the Second International Corneal Cross-linking Congress 2006, in Zurich, Switzerland, Pinelli et al reported results and characteristics of our C3-R treatment protocol2:

•No epithelial debridement;

•Two drops of proparacaine 0.5% every 5 minutes for 15 minutes;

•A 5-minute presoak with riboflavin solution (0.1% riboflavin-5-phosphate and dextran);

•Up to 30 minutes of exposure to UV-A light (370 nm fluence at 3mW/cm2) to the central 7 mm of the cornea (with the speculum in place);

•UV-A light combined with reapplication of riboflavin solution every 3 minutes.

The penetration of riboflavin through intact

epithelium can be enhanced by substances increasing its permeability, such as ethylenediaminetetraacetic acid (EDTA)3 and topical gentamicin. Dr Leccisotti is currently pre-treating for 3 hours with these 3 4 2 components, all included in a standard topical

gentamicin industrial preparation (Ribomicin eyedrops, Farmigea, Italy), then by topical anesthetic (oxybuprocaine) for 30 minutes, before instilling riboflavin and irradiating with UV-A. His results at 6 months are encouraging, with BCVA unchanged in 21 eyes, improved in 11 eyes, worsened in 1 eye by 1 Snellen line. Mean BCVA improvement, in decimals, is 0.15. Mean curvature improvement is 1.3 diopters (Figure 8.5). Endothelial safety was tested by specular microscopy, and cell density was unchanged at 1 month and 6 months. This is reassuring, and shows that UV-A penetration is (as expected) under the threshold of endothelial damage.

Pinelli et al have a patented a riboflavin formula (0,1% plus tensioactive) which is at the present time under investigation on rabbits eyes.

Dr Leccisotti and Dr Pinelli truly believe that in the near future the transepithelial procedure will be a new frontier of the treatment for keratoconus; the methods, epi-off and epi-on, can consist of different options for different cases. In the history of refractive surgery similar phenomenon are now routine for everybody (first step with PRK and then LASIK) and

there is the firm belief that these two approaches to the cure of the keratoconus disease can cohabit in the near future.

FUTURE

Although ophthalmologists are still debating whether to remove or keep the epithelium in tact before C3-R treatment, we prefer to avoid de-epithelialization and its associated discomfort, especially until a scientific method or new technology in vivo will demonstrate the opposite.

In our opinion, the C3-R treatment of the future will be a less invasive, painless technique that does not require de-epithelialization. A bilateral option may also be psychologically easier and more accepted by

our patients. Thus far, C3-R treatments are effective, and results and follow-up are very encouraging. The numerous studies on C3-R and its impending CE mark demonstrate its safety.

REFERENCES

1.Pinelli R, “Corneal collagen cross-linking with riboflavin (C3-R) treatment opens new frontiers for keratoconus and corneal ectasia”, Eyeworld, May 2007;34-40.

2.Pinelli R. “The Italian Refractive Surgery Society (SICR) results using C3-R”, paper presented at the Second International Congress of Corneal Cross-linking (CCL), Zurich, 2006.

3.Nakamura T, et al. Electrophysiological characterization of tight junctional pathway of rabbit cornea treated with ophthalmic ingredients. Biol Pharm Bull 2007;30:236064.