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00003226-200705000-0000200003226_2007_26_390_mazzotta_ultrastructural_4article< 91_0_6_9 >CorneaCopyright © 2007 Wolters Kluwer Health, Inc. All rights reserved.Volume 26(4)May 2007pp 390-397Treatment of Progressive Keratoconus by Riboflavin-UVA-Induced Cross-Linking of Corneal Collagen: Ultrastructural Analysis by Heidelberg Retinal Tomograph II In Vivo Confocal Microscopy in Humans[Clinical Science]Mazzotta, Cosimo PhD; Balestrazzi, Angelo PhD; Traversi, Claudio MD; Baiocchi, Stefano PhD; Caporossi, Tomaso MD; Tommasi, Cristina MD; Caporossi, Aldo MDFrom the *Department of Ophthalmology and Neurosurgery, University of Siena, Siena, Italy; and the †Ophthalmology Institute, Catholic University, Rome, Italy.Received for publication June 5, 2006; revision received November 8, 2006; accepted November 21, 2006.The authors state that they have no proprietary interest in the products named in this article.Reprints: Angelo Balestrazzi, Dipartimento di Scienze Oftalmologiche e Neurochirurgiche, Università di Siena, Viale Bracci 1, 53100 Siena, Italy (e-mail: balestrazzi@unisi.it ).AbstractPurpose: To assess ultrastructural stromal modifications after riboflavin-UVA-induced cross-linking of corneal collagen in patients with progressive keratoconus.Methods: This was a second-phase prospective nonrandomized open study in 10 patients with progressive keratoconus treated by riboflavin-UVA-induced cross-linking of corneal collagen and assessed by means of Heidelberg Retinal Tomograph II Rostock Corneal Module (HRT II-RCM) in vivo confocal microscopy. The eye in the worst clinical condition was treated for each patient. Treatment under topical anesthesia included corneal deepithelization (9-mm diameter) and instillation of 0.1% riboflavin phosphate-20% dextran T 500 solution at 5 minutes before UVA irradiation and every 5 minutes for a total of 30 minutes. UVA irradiation was 7 mm in diameter. Patients were assessed by HRT II-RCM confocal microscopy in vivo at 1, 3, and 6 months after treatment.Results: Rarefaction of keratocytes in the anterior and intermediate stroma, associated with stromal edema, was observed immediately after treatment. The observation at 3 months after the operation detected keratocyte repopulation in the central treated area, whereas the edema had disappeared. Cell density increased progressively over the postoperative period. At ∼6 months, keratocyte repopulation was complete, accompanied by increased density of stromal fibers. No endothelial damage was observed at any time.Conclusions: Reduction in anterior and intermediate stromal keratocytes followed by gradual repopulation has been confirmed directly in vivo in humans by HRT II-RCM confocal microscopy after riboflavin-UVA-induced corneal collagen cross-linking.Keratoconus is a degenerative, noninflammatory disease of the cornea, with onset generally at puberty. It is progressive in 20% of cases and can be treated by lamellar or penetrating keratoplasty. Its incidence in the general population is reported to be ∼1/2000.1 Changes in corneal collagen structure,2,3 organization,4 and intercellular matrix5 and apoptosis6 and necrosis of keratinocytes,7 prevalently or exclusively involving the central anterior stroma and the Bowman layer, are documented in the literature.4-8 These findings are evidence of structurally weakened corneal tissue, typical of keratoconus.The technique of corneal collagen cross-linking consists in the photopolymerization of the stromal fibers by the combined action of a photosensitizing substance (riboflavin or vitamin B2) and UV light from a solid-state UVA source.9 Photopolymerization increases the rigidity of the corneal collagen and its resistance to keratectasia.The first studies in photobiology began in the early 1990s, with attempts to identify biologic glues that could be activated by heat or light to increase the resistance of stromal collagen.10 It was discovered that the gluing effect was mediated by an oxidative mechanism associated with hydroxyl radical release. A similar mechanism of hardening and thickening of collagen fibers has been shown in corneal aging,11 related to active glycosylation of age-dependent collagen molecules.The idea to use this conservative approach to treat keratoconus was conceived in Germany in the 1990s by a research group at Dresden Technical University.12 The aim was to slow or arrest progression to avoid the need for penetrating keratoplasty. The basis for its use finds clinical and scientific support in the fact that young patients with diabetes never have keratoconus; in the few exceptions, it predated the onset of diabetes and did not progress because of the natural cross-linking effect of glucose, which increases corneal resistance in these patients.13-15The biomechanical properties of the cornea depend on the characteristics of the collagen fibers,2,3 interfibril bonds,5 and their spatial-structural disposition.4 The biomechanical resistance of the cornea in patients with keratoconus is one half the normal value. The technique of corneal collagen cross-linking has been used experimentally to block progression of keratoconus in the refractive phase at least temporarily. Collagen turnover is ∼2 to 3 years. Cross-linking “freezes” stromal collagen, increasing the biomechanical stability of the cornea.The use of confocal microscopy has been reported for various corneal diseases.16 This device allows for real-time in vivo qualitative examination of all layers of the cornea and thus visualization of microscopic alterations.In recent years, several studies have investigated the microscopic abnormalities visible by confocal microscopy in eyes affected by keratoconus.17,18In this prospective study, the modifications induced by riboflavin-UVA-induced cross-linking of corneal collagen are investigated by in vivo Heidelberg Retinal Tomograph II Rostock Corneal Module (HRT II-RCM) confocal microscopy.MATERIALS AND METHODSTen eyes of 10 patients with keratoconus of low or moderate degree were included in this prospective nonrandomized study.Subjects younger than 18 and older than 60 years were excluded and patients with corneal thickness <400 μm; a history of herpetic keratitis and corneal scarring; severe eye dryness; and current corneal infections or concomitant autoimmune diseases.The study design was approved by the ethical committee of the University of Siena and the Siena University Medical Hospital and was conducted in accordance with the ethical standards set in the 1964 Declaration of Helsinki, as renewed in 2000. All patients gave their written informed consent before inclusion in the study.The operation was performed on a day-surgery basis under aseptic conditions and topical anesthesia (4% lidocaine). Each operation took, on average, 30 minutes. After applying the blepharostat, a 9-mm-diameter marker was used to remove the corneal epithelium in a central circle with a blunt spatula. Photosensitizing solution (0.1% riboflavin-20% dextran, Sooft, Montegiorgio, Italy) was instilled 5 minutes before beginning UVA irradiation and every 5 minutes thereafter for 30 minutes.The UVA source was a solid-state device (CBM X-linker; CSO, Firenze, Italy), consisting of a 7-mm UVA LED array (370-nm wavelength) with a potentiometric voltage regulator.Irradiated energy was dosed through a UVA power meter (Lasermate Q-Coherent, Auburn, CA). Wavelength was 370 nm, and power was 3 mW/cm2 or 5.4 J/cm2, at 1.5 cm from the cornea. After treatment, patients were medicated 4 times a day with topical antibiotic (Levofloxacin, Oftaquix, Tubilux, Pomezia) and the eye was dressed with a soft therapeutic contact lens for 4 days.Postoperative follow-up was performed at 1, 3, and 6 months and included ultrastructural analysis by HRT II-RCM confocal microscopy (Cornea Module HRT II; Rostock, Heidelberg, Germany), ultrasound pachymetry, biomicroscopy, and photography of the anterior segment (Eye Image System; CSO).RESULTSThe mean preoperative corneal thickness in the observed population was 441 ± 29 (SD) μm.The mean postoperative corneal thickness, 1 month after the cross-linking treatment, was 463 ± 43 μm. Six months after surgery, the mean pachymetry value was 453 ± 39 μm.One month after the cross-linking therapy, the treated stroma was analyzed by in vivo confocal biomicroscopy at a depth of 80 to 90 μm (Fig. 1). 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, we detected the presence of activated keratocytes, indicative of an initial repopulation of the anterior stroma. However, it was not until the sixth month that a dense cell population of activated keratocytes was observed, with regenerated nerve fibers and increased tissue density without edema. Laterally, beyond the 8-mm-diameter irradiated area, keratocytes had a normal appearance, with a minimal spread of edema (Fig. 1).FIGURE 1. Anterior corneal stroma 1, 3, and 6 months after collagen cross-linking by in vivo real-time HRT II confocal microscopy (depth, 80-90 μm). A, Rarefaction of keratocytes with spongy or honeycomb-like edema. B, Initial repopulation with activated keratocytes 3 months after the operation. C, No changes in cell density in the peripheral untreated area. D, Dense population of activated keratocytes, regenerated nerve fibers, and increased tissue density at 6 months.One month after treatment, confocal analysis at a depth of 130 to 150 μm (Fig. 2) showed a rarefaction of keratocytes associated with stromal edema. In the dense trabecular network, some elongated, probably masquerade keratocytes were observed, together with other smaller nuclei. These were perhaps necrotic residues and keratocyte apoptotic bodies in the process of disappearing. After 3 months, the edema began to decrease together with an initial keratocyte 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.FIGURE 2. Corneal stroma after collagen cross-linking by HRT II in vivo real-time confocal microscopy (depth, 130-150 μm). A, Rarefaction of keratocytes and edema after the treatment with trabecular network. Inside the trabecular network, elongated (masked necrotic keratocytes) nuclei and smaller nuclei (keratocyte apoptotic bodies) are detectable. Gradual keratocyte stromal repopulation at this depth after 3 months (B), almost completed at 6 months (C), and with good corneal biomicroscopic clinical appearance (D).At a depth of 170 to 180 μm, the edema was visible at 1 month in the intermediate stroma. It presented ghost nuclei (apoptosis bodies) in the fibrillar network, elongated nuclei, and the absence of keratocytes. Few ghost cells were still visible. 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 (Figs. 3, 4). This increase seemed compatible with a subclinical, microscopically detectable haze that did not seem to impair patient 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.FIGURE 3. Intermediate stroma after combined riboflavin-UVA-induced corneal collagen cross-linking by HRT II in vivo real-time confocal microscopy (depth, 170-180 μm). A, Corneal edema at 1 month in the intermediate stroma with ghost nuclei (apoptosis bodies) in the fibrillar network, elongated nuclei, and absence of keratocytes. B, Initial keratocyte repopulation after 3 months. C, Dense extracellular matrix, cell repopulation with activated keratocytes, and dark microstriae at 6 months.FIGURE 4. A, Subclinical wound healing is detectable by in vivo HRT II confocal microscopy in a case of advanced keratoconus with large dark microstriae and deep stromal Vogt striae. B, Corneal edema at 1 month. C, Initial keratocyte repopulation after 3 months. D, Activated keratocytes nuclei (green arrows) with increased stromal reflectance (red arrows) compatible with subclinical, microscopically detectable haze.At a depth of 270 to 300 μm (Fig. 5), cell necrosis and stromal edema were evident at 1 month, with ghost cells or keratocyte apoptosis bodies in the fibrillar network. Initial signs of cell repopulation were observed at 3 months. Activated oval nuclei and elongated nuclei increased the reflectance of the stroma at 3 and 6 months.FIGURE 5. Intermediate corneal stroma after riboflavin-UVA-induced corneal collagen cross-linking in vivo HRT II confocal microscopy (depth, 270-300 μm). A, Keratocyte disappearance and stromal edema were evident, with small nuclei (keratocyte apoptosis bodies, arrows) in the fibrillar network. B, Initial signs of cell repopulation were observed at 3 months. Keratocyte repopulation was clearly evident and complete at 6 months (C), with good corneal clinical aspect (D).Confocal microscopy allowed for the assessment of treatment penetration. In this case, it revealed in all patients an evident vertical and lateral transition area (VTA and LTA, respectively; Figs. 6, 7), where an edematous zone-poor in cells and with low reflectivity-merges with a deeper zone that presents lighter edema and is regularly populated in keratocytes (Figs. 6, 7). The average treatment depth was ∼320 μm (range, 270-350 μm), assessed by in vivo human confocal evaluation (Fig. 6).FIGURE 6. Evaluation of real treatment depth in humans after corneal collagen cross-linking. The depth of treatment is more frequent at 320 μm (range, 270-350 μm). In the vertical transition area (VTA), the edematous zone-poor in cells and with low reflectivity (A)-merges with the deeper stromal zone, which presents lighter edema and is regularly populated in keratocytes (B-D).FIGURE 7. In vivo HRT II confocal microscopy after corneal collagen cross-linking. Evaluation of lateral transition area (LTA). Laterally, beyond the 7-mm-diameter irradiated area, keratocytes had a normal appearance (A and B, green arrows,) with minimal spread of edema (A and B, yellow arrows). Typical keratoconus dark microstriae were evident in both section (A and B, red arrows). Lateral transition (LTA) consists of a crossing lateral area regularly populated with keratocytes and a slight edema with a central edematous zone poor in cells, with low reflectivity (B, blue arrows).The deep stroma beyond 350 μm was not reached by the cross-linking, except for the limited stromal edema that did not extend beyond 10 to 20 μm from the transition zone (Fig. 8). In our population, the endothelium had regular cell density and morphology during follow-up (Fig. 9).FIGURE 8. In vivo HRT II confocal microscopy after corneal collagen cross-linking. A, Evaluation of the deep stroma. The deep stroma beyond 350 μm was not reached by cross-linking except for a slight spread of stromal edema that did not extend >10-20 μm beyond the vertical transition zone. Activated keratocytes (yellow arrows) are evident with slight edema (red arrows) at 1 (B), 3 (C), and 6 months (D).FIGURE 9. In vivo real-time HRT II confocal microscopy after collagen cross-linking. A, At 1 month, corneal thickness is increased by edema. Endothelium had regular cell density and morphology at 1 (B), 3 (C), and 6 months (D).DISCUSSIONPreclinical studies ex vivo and in vivo in animals19-22 have shown that cross-linking induces an increase in bonding between fibrils having the diameter of collagen fibers in the anterior and intermediate corneal stroma to a depth of ∼300 μm, without any endothelial damage.19 This is caused by the radical formation effect of the photosensitizing agent riboflavin. It also increases the resistance to pepsin breakdown because of the enhancement of the corneal anticollagenase activity. After treatment ex vivo has been described from a histological point of view21: an increase in the diameter of the collagen fibers, greater resistance to enzyme breakdown (anticollagenase effect), apoptosis of keratocytes in the anterior and intermediate stroma, followed by a gradual repopulation by deep keratocytes.Although the epithelial debridement can cause a necrosis of the keratocytes at a depth of 50 to 60 μm,23 the significant cytotoxic effect of cross-linking seems concentrated in the anterior part of the cornea because of the high absorption of UVA by riboflavin. This behavior prevents UVA from reaching deeper levels, thus preserving the endothelium and lens.With the exception of a transient corneal edema and the sensation of a foreign body for 24 to 48 hours, together with burning and lacrimation (as in photorefractive surgery for common sight defects), there were no clinically evident side effects, such as endotheliopathy, persistent epithelial deficit, or cataract or glaucoma in the anterior or posterior segments.To reduce the transitory postoperative pain, some authors have suggested to proceed with the corneal cross-linking without the propedeutic epithelial debridement. However, other published studies9,12 have shown the relevance of epithelial debridement for good penetration of the riboflavin solution. Riboflavin concentration in the corneal layers is important for the high absorption coefficient, and it is necessary not only for cross-linking but also for UVA blocking. The lower the concentration of riboflavin in the cornea, the higher the penetration of UVA, with risks of damage in the lens or the retina.Our results show for the first time in vivo in humans that the cytotoxic effects of corneal collagen cross-linking used to treat keratoconus are significant but concentrated in the first 350 μm of the stroma. Real-time HRT II confocal microscopy in vivo detects an incomplete disappearance of keratocytes in the anterior and intermediate corneal stroma immediately after the operation, associated with high stromal edema with a sponge or honeycomb appearance. The edema was persistent, attenuating over a period of 3 months. We observed a dense fibrillar network with trabecular appearance, harboring ghost cell bodies, presumably apoptotic, masked by the edema, and denser, elongated nuclei, probably of keratocyte origin.This study showed that activated keratocytes repopulate the stroma from the deeper layers, starting at 2 to 3 months after the operation, as witnessed by the observation of activated cell nuclei in all intermediate and anterior stromal layers. Six months after the operation, stromal repopulation was almost complete.In some cases with more advanced keratoconus, we found other relevant aspects, such as an increase in the density of the extracellular stromal matrix starting 3 months after the operation, evident as a microscopically detectable haze, which did not seem to impair vision.HRT II confocal microscopy showed clear vertical and horizontal transition zones with a slight spread of edema and a regular number of cells in the untreated corneal periphery and at depths >345 μm (range, 275-345 μm). The deep corneal stroma beyond 350 μm did not show any changes in endothelial density or morphology.These results indicate that this first confocal microscopy study in humans largely confirms preclinical studies in animal models ex vivo and in vivo.19,21,22 It showed some variability in the penetration of the treatment that does not, however, seem to pose risks for the endothelium. We observed a stromal scar component, especially in patients with more dark microstriae and Vogt striae, namely in cases with more advanced keratoconus. This component is currently being studied in the longer follow-up.The results obtained through confocal microscopy suggest that the cross-linking technique is a safe method for treating progressive keratoconus that cannot be corrected by contact lenses and for avoiding or at least delaying the recourse to corneal transplant.Further clinical and ultrastructural studies and a longer follow-up in a larger patient population are needed. These preliminary results together with clinical and surface topo-aberrometric diagnostics will allow monitoring for any side effects. Although advanced cases of keratoconus were also treated in the pilot study, in our opinion, cross-linking is most indicated in refractive forms at early stages that show signs of progression, that cannot be optically corrected, and that are eligible for lamellar or perforating keratoplasty on clinical and topographic grounds.The method of corneal cross-linking using riboflavin and UVA is technically simple and less invasive than all other therapies proposed for keratoconus. Unlike other mini-invasive methods, such as intrastromal rings (INTACS) and excimer laser surgery, which do not block keratectasia but mainly treat the refractive effects of the disease, this method can treat and prevent some of the underlying pathophysiologic mechanisms.REFERENCES1. Rabinowitz YS. Keratoconus. Surv Ophthalmol. 1998;42:297-319. [CrossRef] [Medline Link] [Context Link]2. Tuori AJ, Virtanen I, Aine E, et al. The immunohistochemical composition of corneal basement membrane in keratoconus. 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