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

cornea.39,40 The lens material (siloxy-fluoro- methacrylate Dk 100, Boston XO, hexafocon-A) has shown to induce minimal edematous swelling of the stroma. Moreover, the morning lens removal allows for a rapid recovery,41 as well as proper cleansing and elimination of debris and waste products. The fitting procedure requires a perfect centration that is critical for the efficacy of the mold but not easy to obtain. The movement observed after each blink must be higher than 0.2 mm and lower than 1.0 mm, always inferior to a conventional RGP-CL fitting. The ideal fluorescein pattern shows an image with concentric rings: the dark center (minimal apical clearance, some times minimal touches) corresponds to the bulging conicoid area to be molded; the surrounding green area, a variable thickness tear reservoir (depending on the intended corrective effect, between 30 and 80 microns). A midperipheral dark ring (the alignment zone) with a minimal clearance, sometimes a slight touch and a thin green ring (the edge lift, 80 to 100 micron) follow (Figures 17.7A to C). The transition between the different zones should be blended and smooth. After the first adaptation, there should be no air bubbles, indicating an excessive lift in the tear reservoir zone.

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Dealing with safety of corneal molding, we observed some cases of grade 1 fluorescein staining of the epithelium, which disappeared in the evening, but no significant corneal infiltrates or ulcers or other adverse 104 events. Minimal or no changes were observed in the

central thickness of the cornea, as measured with an ultrasound pachymeter (Allergan Humphrey 850, Carl Zeiss Meditec). Confocal microscopy (Confoscan 3000, NIDEK, Japan) showed no measurable changes in the endothelium, sub-basal nerve plexus or in the anterior, intermediate and deep stroma (overall density and activation of keratocytes were not modified) while the basal layer of the epithelium showed larger and less regular cells after the molding (Figure 17.8). A slight increase in reflectivity of the matrix was observed and can be explained by a mild increase of corneal glycosaminoglycans production, that is a reversible phenomenon probably due to an aspecific reaction of an already altered corneal parenchyma (mild cellular edema due to hypoxia and/or mechanical effects).45-55

To try to explain the biomechanical working mechanism (the negligible flattening observed in the central cornea was counterintuitive), we hypothesize the role of mid-peripheral forces induced by the displacement of the epithelium that results from a proper compression by the CL alignment zone. This hypothesis certainly deserves further confirmation to accurately explain the achieved regularization of both corneal surfaces.

Phase 3 (for Group B only): Corneal Collagen Cross-linking with Riboflavin (C3-R) was performed after 3 months of CL corneal molding. Immediately after CL removal, 30 minutes of Ultraviolet A exposure (5.4 J/cm2 at 370 nm) with the VEGA CBM X-LinkerTM (CSO, Firenze, Italy)63 were applied to the central cornea (after a minimum diameter of 7 mm of epithelial

debridement). 0.1% riboflavin-5-phosphate and dextran solution (RicrolinTM, SOOFT, Montegiorgio, AP, Italy) was applied every 3 minutes. A 65% hydration, 8.7-mm base curve soft bandage contact lens was applied. Then a regimen of antibiotic-steroid combination plus an aggressive use of lubricant drops was recommended for a month. Overnight CL wear was stopped for 1 month, and then reinitiated.

Outcome measurement data (uncorrected and spectacle best corrected visual acuities, refraction, videokeratographic average simulated keratometry and indices of corneal regularity, including coma aberration (CSO topographer, Firenze, Italy), corneal epithelial thickness as measured with VHF echography (Artemis 2, Ultralink, St. Petersburg, FLA, USA) were collected at baseline and at the overall 6-month gate.

RESULTS

Since there is a potential for further change overtime, as it has been showed to occur after C3-R alone, the reported results must be considered preliminary.

Both groups showed statistically significant improvement of all parameters. The mean outcome of group B (intrastromal corneal ring segments + CL molding with C3-R) had a significantly greater reduction in cylinder and sim-K and improvement of the corneal surface regularity indices than group A

(intrastromal corneal ring segments + CL molding without C3-R) (Table 17.1). UCVA and BSCVA significantly improved in both groups. In Group A, all eyes showed reversibility to the pre-molding conditions (both functional and morphological) after CL wear suspension; in Group B, however, 9 eyes (60%) did not return to visual acuity baseline levels, although similar topographic regression was observed. In this phase of knowledge, this phenomenon is unexplainable. A possible factor might be due to the remodelling of the epithelium that thins over the top of the regions corresponding to the segments and thickens in the flattest zones (Figures 17.9A to D).67

Two anecdotal examples of outcome after the triple procedure (group B) are showed in Figures 17.11 and 17.12.

Table 17.1: Summary of postoperative change (at 6 month gate)

DISCUSSION AND CONCLUSION

Keratoconus is the most frequent reason for corneal transplantation surgery in the western world. It usually affects young patients, generally leading to significant visual handicap. According to the literature, its incidence is continuously growing, up to 1 out of a thousand, thanks to the recent diagnostic advancements (corneal topography in particular). Despite the longstanding interest of the ophthalmic community and the involvement of international organizations in education and clinical research, the exact etiopathology of keratoconus remains obscure,60,61 as well as its prognosis and cure. Examples of inheritance coupled with a higher incidence in closed ethnic communities provide circumstantial evidence that both genetic and environmental factors may be involved. However, neither inheritance mechanisms nor transmission patterns have been elucidated and keratoconus appearance is sporadic in most situations, leaving no pharmacologic or genetic way to prevent its evolution to advanced stages. Evolution is unpredictable. Some patients experience a slow progression for years; others record a rapid escalation in 6-12 months, followed by a process of stabilization with very small changes for the rest of their life. Some are diagnosed only late in life, i.e. after the age of 40, when the condition seldom worsens. About 20% of patients are destined to surgery, mostly penetrating keratoplasty (PKP)62, although the cornea does not show significant opacities (apical scarring) in the vast majority of cases. In our opinion, the decision to entirely replace a transparent cornea in a young patient, as is the case for the average keratoconus patient, must be made carefully. Among the decisive factors, the degree of achievable visual performance and the patient’s life-style play a central role. For some people, a BCVA of 20/30 may be insufficient, though it is judged satisfactory by most patients. The chance of being successful with PKP is high (grafts achieve good results in about 95% of cases, the percentage ranging from 85 to 99% in literature). However, the risk of sight-threatening postoperative complications (from 1 to 10 cases out of 100) must be considered. Functional recovery following corneal transplantation is usually long, often lasting more than one year. The sutures are generally removed at 18 months and the patient might be compelled to use steroids for months. During

this period there is a risk of microbial keratitis and traumatic wound dehiscence. Initially the donor cornea is swollen and often, during the healing process, it remains thicker than the remaining host tissue. Most of patients are between 20 and 35 years of age with a long life expectancy. Considering that Optisolpreserved and cultured donor corneas lose on average 50% of endothelial cells in the first year and graft cell loss continues overtime, a 20-year-old patient will face a limited duration of the graft vitality. The peripheral ring of the recipient cornea remains at all times a potential source of recurrence, as statistical data show a general trend towards an increase in astigmatism overtime.

Immunologically-mediated graft rejection, endothelial failure and high post-PKP ammetropia are not uncommon. Even in uneventful surgeries, with timely suture removal or adjustment, it is common for the edge of the graft to be a little raised or tilted in comparison with the surrounding tissue. For this reason, the graft is usually steeper than the host cornea, inducing various degrees of myopia and regular or irregular astigmatism. Only about 40% of eyes have less than three diopters of astigmatism. At least 25% of cases show more than 5.00 diopters of astigmatism and around 30% have cylinder between 3.00 and 5.00 D. This percentage increases over time - at ten years of follow-up, more than 30% of patients show more than 5 diopters, more than 40% have between 3.00 and 5.00 D. In fact, about 60% of transplant recipients require RGP contact lens wear or additional surgeries like LASIK, PRK-PTK, relaxing or wedge incisions to correct post-PKP refractive error. If a contact lens is needed, it may take up to one year to fit the lens, considering that it is better to wait until the sutures are removed. Also, the prescription may vary for several months after surgery.

It is our feeling that a well informed patient is generally reluctant to undergo PKP, while surgeons should consider it as the last resort. Both should be very interested in more conservative alternatives, capable of delaying the need for a graft. Among the different options available, the combination of intrastromal corneal ring segment implantation plus overnight contact lens corneal moulding has demonstrated excellent safety and efficacy rates. Corneal collagen cross-linking with riboflavin may be associated (the “triple procedure”) to further enhance 107

and stabilize the postoperative improvements of visual performances.

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INTACS EFFECT ON KERATOCONIC EYES

Keratoconus is a corneal ectatic disease that is characterized by non-inflammatory progressive thinning of the paracentral and/or inferior corneal stroma. It is with progressive deformation of the cornea, in the form of irregular astigmatism, which may lead to a significant decrease in visual acuity. Although keratoconus is almost always bilateral, asymmetry between eyes is frequently observed.1,2

Intracorneal ring segments (ICRS), which was first proposed by Fleming and Reynolds3 for the correction of low degrees of myopia, have been recently investigated to correct ectatic corneal diseases. The effect of intracorneal ring segments on the soft corneal keratoconic tissue is much greater than that on normal corneas in case of myopia. The aim from implanting ICRS is not to treat or eliminate the existing disease or should not be considered as a traditional refractive surgical procedure. However, ICRS is a surgical alternative aiming to decrease the astigmatism and corneal abnormality, and thus to increase the visual acuity to acceptable limits as a way to at least delay the need of corneal grafting.4,5

According to the postulates of Barraquer and Blavatskaya, intracorneal ring acts as tissue addition leading to a flattening in the cornea periphery. The diameter of the ring is proportionally inverse to the flattening intensity thus, the smaller the diameter, the more tissue added (ring thickness) with the higher myopic correction.6

In 1987, intrastromal rings introduced as synthetic intracorneal implants for the correction of various degrees of myopia.

An elastic modulus, or modulus of elasticity, is the mathematical description of an object or substance’s tendency to be deformed elastically (i.e. nonpermanently) when a force is applied to it. In keratoconus, the corneal elastic modulus is reduced due to pathology in the corneal stroma.7,8 From a biomechanical perspective, the resistance to deformation is reduced in relation with the reduction of the elastic modulus that leads to increased strain and protrusion in the cornea. The consequence is increased curvature and corneal thinning, the hallmarks of keratoconus. Since stress is defined as applied force divided by cross-sectional area, stress focally increases in the zone of corneal thinning

Figure 18.1: A uniform corneal thickness (TOP) produces a uniform stress distribution. A nonuniform corneal thickness (BOTTOM) produces a stress concentration in the thinnest region

(Figure 18.1).9 The placement of intracorneal ring segments generates both an immediate response that interrupts the biomechanical disease progression in keratoconus, and a time-dependent biomechanical response that allows subsequent improvement of vision over 6 months.10 The immediate response governed by the elastic properties and the long-term response is by viscoelastic properties.9 Intracorneal ring placement results in a reduction of astigmatism and improved visual acuity.5,10 This is accomplished by shortening the path length of the portion of the collagen lamellae which are central to the segments. Redistribution of corneal curvature leads to a redistribution of corneal stress, interrupting the biomechanical cycle of keratoconus disease progression and in some cases (Figure 18.2).9

CROSS-LINKING EFFECT IN EYES WITH INTACS

The structural properties of collagen framework in the corneal stroma determine the biomechanical and optical properties of tissue.

Optimal corneal optics requires a smooth, regular surface with a healthy tear film and epithelium. The regular arrangement of stromal cells and macromolecules is necessary for a clear vision. The lattice arrangement of collagen fibrils embedded in the 111

Figure 18.2: The red biomechanical cycle reflects disease progression in keratoconus. The blue biomechanical cycle reflects the impact of INTACS placement. Once the segments are inserted, the curvature is decreased centrally, including the region of the cone. As curvature is decreased in this region, the stress is redistributed, and the decompensatory biomechanical cycle of keratoconus is broken

extracellular matrix acts as a diffraction grating to reduce light scattering by means of destructive interference. Scattering is greater anteriorly, resulting in a higher refractive index that decreases from 1.401 at the epithelium to 1.380 in the stroma and 1.373 posteriorly. In normal collagen regulation we can see clear, because size of lattice elements is smaller than the wavelength of the visible light.11 In keratoconic corneas include loss of arangement of fibrils in the anterior stroma, decrease in the number of collagen lamellae, separation of collagen bundles.12,13 For the first time, a new treatment based on collagen crosslinking (CXL) has been introduced by Wollensak. 14,15 This new treatment is aimed at the pathogenic cause of keratoconus and changes intrinsic biomechanical properties of corneal collagen.

This treatment creates additional chemical bonds inside the corneal stroma by means of a photopolymerization in the anterior stroma while minimizing exposure to the surrounding structures of the eye.16 CXL is a widespread method in the polymer industry to harden materials and also in bioengineering to stabilize tissue. For example, chemical CXL with glutaraldehyde is used in the preparation of prosthetic heart valves and physical CXL by UVA is often used in

112 dentistry to harden filling materials.15,17 Tissue

specimens are preserved and hardened by glutaraldehyde or formaldehyde in pathology using same method.

The photosensitizer is excited into its triplet state generating so-called reactive oxygen species (ROS) being mainly singlet oxygen and to a much lesser degree superoxide anion radicals using UVA at 370 nm and the photosensitizer riboflavin. The reactive oxygen species can react further with various molecules inducing chemical covalent bonds bridging amino groups of collagen fibrils (type II photochemical reaction). The wavelength of 370 nm was chosen because of an absorption peak of riboflavin at this wavelength.18

A significant challenge in drug delivery is the local administration of drugs to the eye.19,20 To be effective, most drugs must penetrate across the eye’s tissue barriers (e.g. cornea, sclera and conjunctiva) to reach therapeutic targets within the globe. Often, these tissues present the rate limiting step to effective delivery. Thus, the ability to predict rates of drug transport across ocular tissues would be a powerful tool in the development of new drugs and drug delivery strategies.20

Epithelium and Cornea Permeability

A number of centers around the world are now performing CXL treatment with removal of epithelium as first described by the authors.

The cornea contains three primary layers, which are stacked sequentially from the outer to inner surface: epithelium, stroma, and endothelium. In the human eye, the epithelium contains 5-7 layers of cells each connected by tight junctions, which is expected to provide a large barrier to anything but small lipophilic compounds. In normal eyes, the stroma is a thick fibrous, largely acellular tissue composed mostly of water, which should not provide a lipophilic barrier. Finally, the endothelium is a monolayer of cells with large intercellular junctions, which should present a leaky lipophilic barrier. The resistance to transport across the whole cornea can be thought of as a sum of resistances to transport across each of the individual corneal layers, where the resistance to transport (R) is the inverse of permeability (P):

R cornea: R epithelium + R stroma + R endothelium Using this “sum of resistances” approaches allows us to determine which layers of the cornea provide

rate-limiting barriers by comparing the permeability of full cornea to the permeability of cornea with one or more of its layers removed. For example, if the permeability of full cornea was found to be smaller than that of de-epithelialized cornea, it would suggest that the epithelium presents a significant barrier to transport. In contrast, if the permeability of full cornea was found to be equal to that the epithelium does not present a significant barrier to transport. When the stromal layer of cornea isolated, its permeability shows no apparent dependence on molecular radius as expected for its anatomical structure. Because whole cornea and corneal stroma have such different permeability properties, it at first appears that the stroma is not a rate-limiting barrier within the cornea. Permeability of just the endothelial layer of cornea displays a strong dependence on both distribution coefficient and molecular size. This indicates that both the lipophilic pathway across cells and the hydrophilic pathway between cells are important. To determine if endothelium is a rate-limiting step for transport across the full cornea, the permeability of endothelium can be compared to that of the cornea. For molecules with same distribution coefficient, endothelial permeability is generally larger than that of cornea, which indicates that the endothelium is more permeable and, thus, not a rate-limiting barrier. Neither stroma nor endothelium is uniquely rate-limiting but each can play a role in limiting transport of small, lipophilic compounds. By process of elimination, this leaves the epithelium as the dominant barrier in the cornea. Almost no permeability data exist in the literature for corneal epithelium alone. If we accept that epithelium dominates cornea’s barrier properties, it still remains unclear which of the other layers (stroma, endothelium) is the second most important barrier.20

Non-removal of epithelium has considerable benefits in terms of postoperative pain and more rapid healing. Some complications have been reported in the literature after CXL treatment with removal epithelium such as herpetic keratitis with iritis.21 Chemically epithelial distriubtion can be created instead of removal of epithelium. Figures 18.3 and 18.4 show confocal biomicroscopic views of normal epithelium and change after 20% alcohol application. In Figure 18.4, there is no intact epithelial membrane and we are expecting no tight junctions. The intact

Figure 18.3: Confocal microscopic view of normal epithelium (Courtesy of Kaufman H)

Figure 18.4: Corneal epithelium after 20% alcohol application for 25 sec (Courtesy of Kaufman H)

epithelium is a barrier that slows the absorption of riboflavin (molecular weight 376,37 g/mol) into the cornea so it penetrates slowly and incompletely. For that reason chemically disturbed epithelium (20% alcohol) or debrided epithelium removes diffusion barrier for riboflavin molecule and speeds saturation of the corneal stromal tissue.20,22

An important point to remember is that while riboflavin reduces UV penetration by absorbing it, the absorption then results in the very reaction which causes cytotoxic reaction. Infact the presence of riboflavin makes the cornea 10 times more UV sensitive. It would be ideal if the riboflavin penetration could be limited to the first 300 micron of the cornea as this would limit the photochemical reaction to this

level and thus ensure protection of the endothelium.23 113