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

corneal collagen.36 Wollensak et al reported an increase of 328.9% in corneal rigidity in human corneas after cross-linking. The stiffening of the corneal collagen flattens the corneal apex which produces a total eye dioptric power reduction. This phenomenon can partially explain the improvement in patients’ uncorrected visual acuity. Changes in the best spectacle-corrected visual acuity are mainly related to the improvement in corneal symmetry demonstrated overall by the early coma reduction in the anterior corneal surface.

The main complication after corneal cross-linking is endothelial cell damage.37 Additionally there have been published cases of DLK38 in post CCL patient with ketatectasia after Lasik and herpetic keratitis with

Given the fact that collagen turnover is 2 to 3 years, more long term studies are essential to determine whether corneal cross-linking with riboflavin and UV- A have a long standing or a transient clinical effect.

INTACS COMBINED WITH CORNEAL COLLAGEN CROSS-LINKING AND IRREGULAR ASTIGMATISM

Intracorneal Ring Segments’ implantation and Corneal Collagen Cross-linking with Riboflavin and Ultraviolet- A as we described are minimally invasive techniques for the treatment of irregular astigmatism. Since ICRS insertion re-shapes the cornea and CCL inhibits or slows the progression of irregular astigmatism, a logical solution would be to combine the two treatment methods in order to synergize their effect (Fig. 14.1).

Chan et al29 studied the combination of Intacs with corneal collagen cross-linking with Riboflavin (C3-R) in 2007, in a retrospective, nonrandomized, comparative cases series comprising of 12 eyes of nine patients who had inferior segment Intacs placement without C3-R (Intacs only group) and 13 eyes of 12 patients who had inferior segment Intacs implantation and afterwards C3-R (Intacs with C3-R group). Intacs with C3-R had a significantly greater reduction in cylinder than the Intacs-only group (p < 0.05). Steep

Figure 14.1: Kerrarings implantation followed by UV-X in a keratoconic eye

and average keratometry were reduced significantly more in the Intacs with C3-R group (p < 0.05).

But which is the right treatment sequence? A pretreatment with ICRS would significantly re-shape the cornea by flattening and regularizing it, which would be followed by CCL in order stabilize the cornea in this newly achieved state. Alternatively, the CCL procedure could be done first, followed by a reshaping procedure.

Segments’ implantation in a “soft” cornea is expected to have a greater flattening effect, than ICRS insertion in a stiff cornea. Therefore, it looks logical to perform first the ICRS placement and afterwards to “freeze” corneal stroma with CCL. Preliminary, unpublished data of a prospective study [keratoconic eyes that underwent both treatments (ICRS and CCL) with different sequence] seems to support this theoretical approach.

CONCLUSION

Intracorneal ring segments and corneal collagen crosslinking with Riboflavin and Ultraviolet-A seems to have a synergic effect as treatments for irregular astigmatism. Implantation of ICRS followed by corneal collagen cross-linking with Riboflavin and Ultraviolet- A possibly have greater keratoconus improvements than\CCL procedure followed by ICRS placement. More, large, comparative studies are needed in order to verify or reject these preliminary results.

REFERENCES

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3.Siganos CS, Kymionis GD, Kartakis N, et al. Management of keratoconus with Intacs. Am J Ophthalmol 2003;135:64-70.

4.Schanzlin DJ, Asbell PA, Burris TE, et al. The intrastromal corneal ring segments’. Phase II results for correction of myopia. Ophthalmology 1997;104:1067-78.

5.Fleming JF, Lovisolo CF. Intrastromal corneal ring segments in a patient with previous laser in situ keratomileusis. J Refract Surg 2000;16:365-7.

6.Lovisolo CF, Fleming JF, Pesando PM. Intrastromal corneal ring segments. Fabiano Edotore, Canelli, Italy, 2002.

7.Colin J, Cochener B, Savary G, et al. Correcting keratoconus with intracorneal rings. J Cataract Refract Surg 2000;26:1117-22.

8.Colin J, Malet FJ. Intacs for the correction of keratoconus: Two-year follow-up. J Cataract Refract Surg 2007;33:6974.

9.Kymionis GD, Siganos CS, Tsiklis NS, et al. Long-term follow-up of Intacs in keratoconus. Am J Ophthalmol 2007;143:236-44.

10.Kanellopoulos AJ, Pe LH, Perry HD, Donnenfeld ED. Modified intracorneal ring segment implantations (INTACS) for the management of moderate to advanced keratoconus: efficacy and complications. Cornea 2006;25:29-33.

11.Colin J. European clinical evaluation: use of Intacs for the treatment of keratoconus. J Cataract Refract Surg 2006;32:747-55.

12.Rabinowitz YS, Li X, Ignacio TS, et al. INTACS inserts using the femtosecond laser compared to the mechanical spreader in the treatment of keratoconus. J Refract Surg 2006;22:764-71.

13.Sharma M, Boxer Wachler BS. Comparison of singlesegment and double-segment Intacs for keratoconus and post-LASIK ectasia. Am J Ophthalmol 2006;141:891-5.

14.Kymionis GD, Siganos CS, Kounis G, et al. Management of post-LASIK corneal ectasia with Intacs inserts: one-year results. Arch Ophthalmol 2003;121:322-26.

15.Kymionis GD, Tsiklis NS, Pallikaris AI, et al. Long-term follow-up of Intacs for post-LASIK corneal ectasia. Ophthalmology 2006;113:1909-17.

16.Boxer Wachler BS, Christie JP, Chandra NS, et al. Intacs for keratoconus. Ophthalmology 2003;110:1031-40.

17.Ruckhofer J, Stoiber J, Alzner E, Grabner G. One year results of European multicenter study of intrastromal corneal ring segments. Part 2: complications, visual symptoms, and patient satisfaction; the Multicenter European Corneal Correction Assessment Study Group. J Cataract Refract Surg 2001;27:287-96.

18.Siganos D, Ferrara P, Chatzinikolas K, et al. Ferrara intrastromal corneal rings for the correction of keratoconus. J Cataract Refract Surg 2002;28:1947-51.

19.Kwitko S, Severo NS. Ferrara intracorneal ring segments for keratoconus. J Cataract Refract Surg 2004;30:812-20.

20.Rabinowitz YS, Li X, Ignacio TS, Maguen E. INTACS inserts using the femtosecond laser compared to the mechanical spreader in the treatment of keratoconus. J Refract Surg 2006;22:764-71.

21.Sugar A. Ultrafast (femtosecond) laser refractive surgery. Curr Opin Ophthalmol 2002;13:246-49.

22.Ertan A, Kamburoglu G, Bahadir M. Intacs insertion with the femtosecond laser for the management of keratoconus: one-year results. J Cataract Refract Surg 2006;32:2039-42.

23.Ertan A, Bahadir M. Topography-guided vertical implantation of Intacs using a femtosecond laser for the treatment of keratoconus. J Cataract Refract Surg 2007;33:148-51.

24.Colin J, Cochener B, Savary G, et al. Intacs inserts for treating keratoconus. One-year results. Ophthalmology 2001;108:1409-14.

25.Hellstedt T, Makela J, Uusitalo R, et al. Treating keratoconus with INTACS corneal ring segments. J Refract Surg 2005;21:236-46.

26.Alio JL, Artola A, Hassanein A, et al. One or 2 Intacs segments for the correction of keratoconus. J Cataract Refract Surg 2005;31:943-53.

27.Alio J, Salem T, Artola A, Osman A. Intracorneal rings to correct corneal ectasia after laser in situ keratomileusis. J Cataract Refract Surg 2002;28:1568-74.

28.Mularoni A, Torreggiani A, Di Biase A, et al. Conservative treatment of early and moderate pellucid marginal degeneration: a new refractive approach with intracorneal rings. Ophthalmology 2005;112:660-66.

29.Chan CC, Sharma M, Wachler BS. Effect of inferior-segment Intacs with and without C3-R on keratoconus. J Cataract Refract Surg 2007;33:75-80.

30.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.

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amino groups in the glycation-mediated cross-linking of γB-cristallin. J Biol Chem 1997;272:14465-69.

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39.Kymionis DG, Portaliou DM, Bouzoukis DI, et al. Herpetic keratitis with iritis after corneal cross-linking with riboflavin and ultraviolet A for keratoconus J Cataract Refract Surg 2007;33:1982-84.

40.Kohlhaas M, Spoerl E, Speck A, et al. Eine neue behandlung der keratektasie nach LASIK durch kollagenvernetzung mit riboflavin/UVAlicht. Klin Monatsbl Augenheilkd. 2005;222:430-36.

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INTRODUCTION

Keratoconus is a non-infiammatory cone-like ectasia of the cornea, which is usually bilateral and progress over time, with consequent central or paracentral thinning of the stroma and irregular astigmatism (Fig. 15.1).

The relevance of keratoconus in the general population seems to be relatively high, with approximately 1 in 20001, even if the diffusion of new diagnostic means will permit to find prevalence rates certainly greater. In nearly all cases both eyes are affected, at least from a topographic point of view.

The cause of keratoconus is unknown, but it seems that enzymatic changes in corneal epithelium, such as decrease of the levels of the inhibitors of proteolytic ezymes and an increase of the lysosomal enzymes can be involved in the cornea degradation.

At the beginning, glasses are sufficient to correct myopia and astigmatism still regular or slightly irregular; successively, in cases of high astigmatism, it becomes necessary to apply hard contact lenses.

Epikeratoplasty is efficacious in patients which do not endure contact lenses and which do not show a significant central corneal opacity, but, due to its visual outcomes not perfect, it was dropped.

Intracorneal rings also can be an option2, but all these described techniques unfortunately only correct

refractive errors and do not treat the cause underlying the corneal ectasia and, therefore, they do not permit to stop the progression of keratoconus.

In 19963, some theoretical studies started investigating more deeply the underlying causes of keratoconus and the possible parasurgical techniques to stop its progression. In all patients affected by keratoconus a reduced degree of cross-links in the corneal collagen fibers has been observed; that is, the aim of those studies was firstly to determine how to increase those cross-links to obtain an improved mechanical stability of the cornea and increase the resistance against enzymatic degradation.

CORNEAL COLLAGEN NETWORKS

Collagen is a structural protein organized in fibers. Those fibers are responsible of limiting the tissue deformation and preventing mechanical brakes. The collagen fibers are chemically stable and have high mechanical properties. Inside the connective tissue, fibroblasts synthetize tropocollagen molecules, the base blocks of collagen fibers. Those molecules have a typical weight of 300 kDa, a length of 280 nm with an average diameter of 1.5 nm. The molecule is composed by 3 helicoidal chains (alpha-chains) interlaced each other like a rope (Figure 15.2).

The factors of stabilization of those collagen molecules are related to the interactions between the 3 helics and are due to Hydrogen links, Ionic links and intra-chain reticulations (cross-links).

Tre stroma, composed mainly by collagen lamellae, gives to cornea 90% of its thickness. Between the lamellae keratocites can proliferate, migrate and turn into their activate state. Integrity of corneal epithelium for the switch of keratocites (resting cells) in fibroblasts (active cells) is very important.

Cheratansulphate type I is the most important mucopolysaccaride present in corneal stroma: it plays an important tole for the orientation of collagen mashes and lamellae (corneal clarity, tensile strength) and for corneal hydration (corneal edema).

PHOTOCHEMICAL CROSS-LINKING

There are many different possibilities of cross-linking:4

•Lysyl oxidase (LOX) cross-links collagen enzymatically

•Transglutaminase (12h, pH=3)

•Sugar aldehydes (diabetes – Advanced Glycation Endproducts AGEs)

•Chemical cross-linking (glutaraldehyde, formaldehyde, DPPA)

•Photochemical cross-linking (UV, ionizating

radiation)

The interaction between organic tissues and radiation depends on the type of radiation used. The ionizing radiation has enough energy to turn out electrons from the atoms of the tissues. Other types of radiation, i.e. UV radiation, have not enough energy to turn out electrons but to make them jump to higher energy levels (exciting radiation).

In the human biologic tissues, water molecule is present at a rate of 70 to 90% so it is clearly the main target of radiation. During the water radiolysis process, the energy applied to water molecules ionizes them and generate free radicals molecules. Free radicals are continuously produced in tissues and quickly inactivated by chemical or enzymatic transformation.

In the eye, ascorbic acid absorbs UV radiation (at cornea, lens and vitreous body districts); it is a cofactor of several enzymes, the best known of which are prlyne hydroxylase, enymes involved in byosinthesis of collagen. In vitreous body, after cataract surgery (absence of glutathione), ascorbic acid (in ascorbate

form) absorbs UV not stopped by lens, resulting in the formation of free radicals, disaggregation of hyaluronic acid and increase an cross-linking of collagen fiber networks.

RIBOFLAVIN-UV-A TREATMENT

A photo sensitizer is a substance which is activated by the absorpion of light at a given wavelight and which can induce free radical reactions in its activate form. This substance can amplify light radiation effect on biologic tissues.

The basic mechanism of the photochemical treatment of keratoconus is to use Riboflavin as a photo sensitizer and apply on it UV irradiation at a determined wavelength to induce free radicals reactions and increase this way the cross-links in the collagen fibers. Riboflavin has a high UV absorption between 360 and 450 nm; due its additional shielding all structures behind the corneal stroma, including corneal endothelium, anterior chamber, iris, lens and retina, are exposed to a residual UV radiant exposure less Than 1J/cm2 (in accordance with safety guidelines). The UV source is typically a group of 3 to 5 Light Emitting Diodes producing a radiation of 370 nm wavelength and 3 mW/cm2 intensity (Figure 15.3).

The cross-linking effect is obtained in three steps (Figure 15.4).

CORNEAL EPITHELIUM

Remove or not remove the epithelium is a matter of a therapeutic range of the X-linking with riboflavin technique. The goal of the treatment is to obtain

radiating energy emitted for the treatment, is blocked before entering the superficial layers of the corneal stroma. Moreover, a part of the energy reaches the superficial corneal stroma, where is located the riboflavin, even if in a small quantity, and then to produce free radicals and cross-links between the collagen fibers.

The news in this treatment is represented by the possibility of realizing cross-linking keeping the epithelium unaltered. This natural barrier protect the cornea but it is not an impermeable stratus: it is an osmotic membrane through which the riboflavin can penetrate to the cornea. Of course, the riboflavin itself cannot penetrate easily so the question is, at this stage, about the real effectiveness of the treatment, compared with the traditional one. If we combine the riboflavin drops with a tense-active substance, we can have a more efficient penetration to the cornea. This substance acts as a vector for riboflavin, with a double effect: reaching the cornea and filling the epithelium, contributing so far to its strengthening (Figure 15.5).

The advantages of this particular technique is that all the macroscopic side effects related to the epithelium-removal technique are not present: no pain, no stromal edema (due to the abrasion) and, more important, the possibility to treat both eyes in the same session (85% of patients has bilateral keratoconus, so the treatment is in most cases necessary in both eyes).

Even if we assume that the riboflavin cannot penetrate efficiently the epithelium, we think that as the photo sensitizer is distributed homogeneously on

Figure 15.5: Patient eye under C3-R treatment

the treated eye, we can at least obtain an increased rigidity of the corneal epithelium, thus a decreased instability in visual acuity of the patient.

Remove or not remove the epithelium is a matter of a therapeutic range of the X-Linking with riboflavin technique. The goal of the treatment is to obtain mechanical X-Linking of collagen fiber networks of the corneal stroma without side effects (edema, demarcation lines in the anterior stroma of the cornea, phlogosis, etc)

The real question is about the effectiveness of the treatment, as the safety issues are not a worry of this technique: keeping the epithelium unalterated mean reducing most of the side effects of the treatment (included the death rate of keratocites and the number of endothelial cells). We continue our studies in this way because we believe that the epithelium removal

is something that could be avoided in the treatment and transepithelial technique will become the standard in Cross-linking treatments.

REFERENCES

1.Rabinowitz YS. Keratoconus – Surv Ophthalmol 1998.

2.Colin J, et al. Correcting keratoconus with intracorneal rings, JCRS 2000.

3.Seiler T, Spoerl E, et al. Conservative therapy of keratoconus by enhancement of collagen cross-links, 1996.

4.Spierl E. Physical background of the riboflavin/UV crosslinking of the cornea. World Vision Surgery Symposium 2007.

5.Wollensak, Spoerl, et al. Keratocyte apoptosis after collagen cross-linking using riboflavin/UVA treatment 2004.

6.Spoerl E. Seiler T, et al. Safety of UVA-Riboflavin Crosslinking of the Cornea. Cornea 2007.

INTRODUCTION

Keratoconus is an ectatic, progressive, noninflammatory disease of the cornea. Reported estimates of the prevalence of keratoconus vary between 50 and 230 per 100 000. In keratoconus, there are normalsized collagen fibers; however, the number of collagen lamellae are abnormally low. The collagen lamellae are released from their interlamellar attachments or from their attachment to Bowman’s layer and become free to slide. The treatment options for keratoconus include glasses, contact lenses, collagen cross-linking, intrastromal ring segments and corneal transplantation.1 Corneal collagen cross-linking is performed by using UV-A at 370 nm and the photosensitizer riboflavin, stiffening the collagen matrix of the cornea.2 Cross-linking treatment is the first and only therapeutic option that has changed the natural course of keratoconus by stopping progression.3

EFFECTS OF CROSS-LINKING ON CORNEAL STROMA

Biomechanical Effects

Tensile strength of cornea is decreased in keratoconus. Biomechanical stress-strain measurements in human corneas showed an increase in corneal rigidity of 328.9% and an increase in Young’s modulus by the factor of 4.5 after cross-linking. The cross-linking effect is maximal in the anterior 300 μm of the cornea.4

Thermomechanical Effects

The maximal hydrothermal shrinkage temperature was found to be 75’C for cross-linked porcine corneas and 70’C for untreated controls. This effect is more pronounced on the anterior stroma of the cornea.5

Morphological Effects

Collagen fiber diameter was increased by 12.2% in the anterior stroma and by 4.6% in the posterior stroma in rabbit eyes. This is because of the induced crosslinks, pushing the collagen polypeptide chains apart, resulting in increased intermolecular spacing.

Increase in collagen fiber diameter and corneal rigidity due to collagen cross-linking is also observed in diabetes mellitus and aging.6