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

The transmission spectrum of corneas following application of 20% Alcohol solution applied for 40 seconds and treated with riboflavin and UV-A (Figure 7.5, red diamonds) were similar to controls (Figure 7.5, yellow diamonds), with no significant differences between the groups for wavelengths between 400 nm and 510nm, corresponding to one of the absorption peaks of riboflavin (Figure 7.4). The transmission spectrum of corneas with grid pattern epithelial trauma treated with riboflavin and UV-A (Figure 7.5, green diamonds) showed a dip in transmission between 400490 nm (p<0.03) compared to controls, which may be attributed to the presence of riboflavin in the stroma. This dip in transmission was less than that seen in the complete epithelial debridement group (Figure 7.5, blue diamonds), with significant differences between these two groups between 400nm and 490nm (p<0.001). This is very likely to be attributable to increased stromal uptake of riboflavin in the presence of complete epithelial debridement. Figure 7.2 shows a cornea with grid pattern epithelial trauma following UV-A/riboflavin treatment, although areas of yellow discoloration in a grid pattern can be seen beneath the areas of full-thickness epithelial trauma, there is lack of homogeneous absorption compared to full epithelial debridement as seen in Figure 7.1.

DISCUSSION

Riboflavin/UV-A corneal collagen crosslinkage is the 3 4 first therapeutic modality that may halt the progression

Figure 7.4: Light transmission spectrum of riboflavin solution

of the ectatic process in keratoconus and postkeratorefractive surgery ectasia.6 Riboflavin is a key component of the photochemical cross-linking treatment as it increases corneal absorption of UV-A to approximately 95%8 and thereby protects the deeper ocular structures especially the endothelium from UV- A damage.13-16

Published clinical and laboratory studies of corneal collagen crosslinkage therapy have generally advocated the complete removal of the epithelium to allow adequate penetration of the riboflavin into the corneal stroma.6-16,21,22 However, in an attempt to reduce the early postoperative discomfort experienced by the patient (caused as a result of epithelial removal), some clinicians have elected to perform the procedure with the epithelium intact.17 They postulate that topical anesthetic drops can loosen epithelial tight junctions allowing penetration of riboflavin into the corneal stroma.

In our studies we used spectrophotometry to investigate the importance of complete epithelial debridement by assessing the ability of riboflavin to penetrate the stroma of corneas treated with topical anesthetic eyedrops (tetracaine 1%), after superficial epithelial trauma (in which the basal epithelium remained intact), following a 20% Alcohol solution applied for 40 seconds, after a grid pattern fullthickness epithelial debridement and compared them to corneas in which the central 10 mm of epithelium had been completely debrided.

Our results have shown that in the immediate postoperative period, the light transmission spectrum of fully de-epithelialized porcine corneas treated with riboflavin eyedrops is altered by the presence of riboflavin within the stroma (Figure 7.1) and the subsequent exposure of the cornea to UV-A light does not produce any further changes to the transmission spectrum. It is of note that riboflavin is an decomposes in the presence of light at wavelengths below 500 nm, so that any acute changes in light transmission due to riboflavin absorption will be short-lived and in the clinical setting the yellow discoloration of the cornea due to riboflavin, which is clearly visible following the treatment has cleared 24 hours later.14

The normality compared to controls of the transmission spectra of corneas which underwent superficial epithelial trauma but had an intact basal epithelium, clearly suggests the need to remove all epithelial cell layers prior to treatment to permit stromal penetration of riboflavin (Figure 7.3A). Similarly, the concomitant use of repeated tetracaine 1% eyedrops over the 35-minute treatment period did not appear in our study to allow stromal penetration of riboflavin through an intact corneal epithelium even18 (Figure 7.3B). It appears that the presence of an intact basal

epithelial layer acts as an effective barrier to riboflavin absorption by the corneal stroma and that this barrier is not sufficiently altered either by superficial epithelial trauma or repeated tetracaine eyedrop administration.

Similarly, with 20% Alcohol solution applied for 40 seconds we found no differences in transmission spectra between 400 nm and 510 nm compared to non-treated control corneas (Figure 7.5). Once again, this suggests that the usage of an application of 20% alcohol in the presence of an intact epithelium is not sufficient to allow adequate riboflavin penetration into the corneal stromal and correspondingly alter its normal light transmission spectrum.

A grid pattern of full thickness epithelial debridement did appear to allow some riboflavin stromal penetration, indicated by a significant dip in the transmission spectrum between 400 nm and 490 nm compared to controls (Figure 7.5). This dip in transmission, however, was significantly less compared to that seen after complete central epithelial removal (Figure 7.5). This suggests that whilst partial fullthickness epithelial debridement does allow riboflavin stromal absorption it is less than with complete removal. Indeed examination of the corneas

immediately following treatment revealed that whilst 3 5

to Chan et al.15 they reported fluorescence in the anterior chamber which they attributed to riboflavin penetration although direct measurements were not made. Whilst such findings are of interest we do not believe they give direct evidence that riboflavin can be significantly absorbed into the corneal stroma without removal of all layers of the corneal epithelium prior to topical administration and they are not supported by the spectrophotometric measurements in this study and the recent findings of others.23

Such considerations are important as failure to achieve adequate stromal absorption of riboflavin is likely to limit the crosslinkage process.12,16

It is important to note, however, that spectrophotometic analysis does not provide direct assessment of stromal riboflavin stromal uptake and whilst the absence of any significant alteration of light transmission spectra suggests limited riboflavin absorption it does not preclude small amounts of riboflavin uptake. Further studies directly measuring stromal riboflavin levels are indicated. It may also be of interest to investigate the UV range from 350 to 380 nm where the treatment wavelength is situated, although our analysis of the visible spectrum does include one of the peak absorption spectra of Riboflavin at 400 to 490 nm. It is also important to remember that in eyes with advanced keratoconus the basal epithelial layer is often broken and may behave differently in terms of its barrier functions in comparison to a normal healthy cornea,24 although this procedure is typically only performed on eyes with mild to moderate keratoconus with central pachymetric thicknesses of 400μm or greater in which the basal layer is usually intact.

CONCLUSION

Complete removal of the corneal epithelium is an essential component of riboflavin/UV-A crosslinkage therapy as superficial epithelial trauma, tetracaine administration and 20% alcohol application for 40 seconds alone is not sufficient to permit the penetration of riboflavin into the corneal stroma. Whilst partial grid-pattern epithelial removal does allow some riboflavin penetration, uptake is limited and nonhomogeneous unlike full-thickness complete central epithelial debridement. Such considerations are important as failure to achieve adequate stromal absorption of riboflavin may impair the efficacy of the cross-linkage process.

REFERENCES

1.Krachmer JH, Feder RS, Belin MW. Keratoconus and related non-inflammatory corneal thinning disorders. Surv Ophthalmol 1984;28:293-322.

2.Javadi MA, Motlagh BF, Jafarinasab MR, Rabbanikhah Z, Anissian A, Souri H, Yazdani S. Outcomes of penetrating keratoplasty in keratoconus. Cornea 2005;24(8):941-46.

3.Reeves SW, Stinnett S, Adelman RA, Afshari NA. Risk factors for progression to penetrating keratoplasty in patients with keratoconus. Am J Ophthalmol 2005;140(4):607-11.

4.Mamalis N, Anderson CW, Kreisler KR, Lundergan MK, Olson RJ. Changing trends in the indications for penetrating keratoplasty. Arch Ophthalmol 1992;110(10):1409-11.

5.Al-Yousef N, Marvrikakis I, Mavrikakis E, Daya SM. Penetrating keratoplasty: indications over a 10-year period.

Br J Ophthalmol 2004;88(8):998-1001.

6Wollensak G. Cross-linking treatment of progressive keratoconus: new hope. Curr Opin Ophthalmol

2006;17(4):356-60.

7.Wollensak G, Spoerl E, Seiler T. Stress-strain measurements of human and porcine cornea after riboflavin/ultraviolet-A-induced cross-linking. J Cataract Refract Surg. 2003;29:1780-85.

8.Spoerl E, Schreiber J, Hellmund K, Seiler T, Knuschke P.. Untersuchungen zur Verfestigung der Hornhaut am Kaninchen. Ophthalmologe 2000;97:203-06.

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

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

11.Wollensak G, Aurich H, Pham DT, Wirbelauer C. Hydration behavior of porcine cornea crosslinked with riboflavin and ultraviolet A. J Cataract Refract Surg 2007;33(3):516-21.

12.Andley U. Photo-oxidative stress. In: Albert DM, Jakobiec FA, eds. Principles and Practice of Ophthalmology, vol. 1, Philadelphia: WB Saunders 1992;575-90.

13.Wollensak G, Spoerl E, Wilsch M, Seiler T. Endothelial cell damage after riboflavin-ultraviolet-A-treatment in the rabbit. J Cataract Refract Surg 2003;29:1786-90.

14.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.

15.Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. Safety of UVA-riboflavin cross-linking of the cornea. Cornea 2007;26:385-89.

16.Spoerl E, Huhle M, Seiler T. Induction of crosslinks in corneal tissue. Exp Eye Res 1998;66:97-103.

17.Chan CK, Sharma M, Boxer-Walcher BS. Effect of inferiorsegment intacs with and without C3-R on keratoconus. J Cataract Refract Surg 2007;33:75-80.

18.Hayes S, O°Brart DP, Lamdin LS, Doutch J, Samaras K, Meek KM, Marshall J. An investigation into the importance of complete epithelial debridement prior to Riboflavin/ Ultraviolet A (UVA) corneal collagen cross-linkage therapy. J Cat Ref Surg 2008;34:557-61.

19.Samaras K, O°Brart DPS, Doutch J, Hayes S, Marshall J, Meek K. Effect of Epithelial Retention and Removal on Riboflavin Absorption in Porcine Corneas. J Ref Surg 2008 (in press).

20.Kostyuk O, Navolina O, Mubard TM, et al. Transparency of the bovine corneal stroma at physiological hydration and its dependence on concentration of ambient ion. Journal of Physiology 2002;543:633-42.

21.Wollensak G, Wilsch M, Spoerl E, Seiler T. Collagen fiber diameter in the rabbit cornea after collagen-cross-linking. Cornea 2004;23:503-7.

22.Kohlhaas M, Spoerl E, Schilde T. Biomechanical evidence of the distribution of cross-links in corneas treated with riboflavin and ultraviolet A light. J Cataract Refract Surg 2006;32(2):279-83.

23.Bottos KM, Dreyfuss JL, Regatieri, Lima-Filho AA, Schor P, Nader HB, Chamon W. Immunofluorescence confocal microscopy of porcine corneas following collagen crosslinking treatment with riboflavin and ultraviolet A. J Refract Surg 2008;24:S715-9.

24.Hollingsworth JG, Bonshek RE, Efron N. Correlation of the appearance of keratoconic cornea in vivo by confocal microscopy and in vivo by light microscopy. Cornea 2005;24:397-405.

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.