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Purpose: There has been a recent explosion in the variety of techniques used to accomplish corneal cross-linking (CXL) for the treatment of ectatic corneal diseases. To understand the success or failure of various techniques, we review the physicochemical basis of corneal CXL and re-evaluate the current principles and long-standing conventional wisdom in the light of recent, compelling, and sometimes contradictory research., Methods: Two clinicians and a medicinal chemist developed a list of current key topics, controversies, and questions in the field of corneal CXL based on information from current literature, medical conferences, and discussions with international practitioners of CXL., Results: Standard corneal CXL with removal of the corneal epithelium is a safe and efficacious procedure for the treatment of corneal ectasias. However, the necessity of epithelium removal is painful for patients, involves risk and requires significant recovery time. Attempts to move to transepithelial corneal CXL have been hindered by the lack of a coherent understanding of the physicochemistry of corneal CXL. Misconceptions about the applicability of the Bunsen-Roscoe law of reciprocity and the Lambert-Beer law in CXL hamper the ability to predict the effect of ultraviolet A energy during CXL. Improved understanding of CXL may also expand the treatment group for corneal ectasia to those with thinner corneas. Finally, it is essential to understand the role of oxygen in successful CXL., Conclusions: Improved understanding of the complex interactions of riboflavin, ultraviolet A energy and oxygen in corneal CXL may provide a successful route to transepithelial corneal CXL.

Purpose: To improve the safety, reproducibility, and depth of effect of corneal cross-linking with the ultraviolet A (UV-A) exposure time and fluence customized according to the corneal thickness., Methods: Twelve human corneas were used for the experimental protocol. They were soaked using a transepithelial (EPI-ON) technique using riboflavin with the permeation enhancer vitamin E-tocopheryl polyethylene glycol succinate. The corneas were then placed on microscope slides and irradiated at 3 mW/cm for 30 minutes. The UV-A output parameters were measured to build a new equation describing the time-dependent loss of endothelial protection induced by riboflavin during cross-linking, as well as a pachymetry-dependent and exposure time-dependent prescription for input UV-A fluence. The proposed equation was used to establish graphs prescribing the maximum UV-A fluence input versus exposure time that always maintains corneal endothelium exposure below toxicity limits., Results: Analysis modifying the Lambert-Beer law for riboflavin oxidation leads to graphs of the maximum safe level of UV-A radiation fluence versus the time applied and thickness of the treated cornea. These graphs prescribe UV-A fluence levels below 1.8 mW/cm for corneas of thickness 540 μm down to 1.2 mW/cm for corneas of thickness 350 μm. Irradiation times are typically below 15 minutes., Conclusions: The experimental and mathematical analyses establish the basis for graphs that prescribe maximum safe fluence and UV-A exposure time for corneas of different thicknesses. Because this clinically tested protocol specifies a corneal surface clear of shielding riboflavin on the corneal surface during UV-A irradiation, it allows for shorter UV-A irradiation time and lower fluence than in the Dresden protocol.

Purpose: To evaluate the stromal interface quality after femtosecond laser full lamellar cuts in gamma-irradiated corneas (VisionGraft sterile cornea) and to determine the limits of the cut depth using the VisionGraft as donor corneas for laser-assisted lamellar anterior keratoplasty., Methods: Fourteen VisionGraft corneas underwent full lamellar cuts using the femtosecond laser. The percent cut depth was 17% to 21% (100 μm, n = 2), 31% to 35% (n = 3), 38% to 40% (n = 3), 45% to 48% (n = 3), and 50% (n = 3) of the total stromal thickness (not including the epithelium). The cap and stromal bed surfaces were imaged with a scanning electron microscope. The quality of cut surfaces was graded by 2 masked observers based on two indices: ridge and roughness. Ridge grading indicated macroscopic irregularity. Roughness grading indicated microscopic irregularity. The grading was done on a subjective integer scale of 1 to 5 (1 = best and 5 = worst), which was used in a previous study of cut quality in fresh corneas., Results: The ridge grading ranged from 1.5 for the shallowest cut to 2.2 for the deepest cut and weakly (r = 0.279) but significantly (P = 0.037) correlated with the percent cut depth. The roughness grading ranged from 2.63 to 2.56 and showed no trend with the percent cut depth (r = 0.006, P = 0.968)., Conclusions: Compared with previously published results of fresh corneas, in which ridge grading exceeded 3 for cuts deeper than 31%, cut quality was better for the VisionGraft. Even at depths up to 48% of the total stromal thickness, ridge grading was not worse than shallow cuts. Thus, gamma-irradiated corneas could provide a smoother interface than do fresh eye bank corneas for laser-assisted lamellar anterior keratoplasty.

Purpose: To compare the physical and biological characteristics of commercial gamma-irradiated corneas with those of fresh human corneas and to determine suitability for transplantation., Methods: The physical properties of gamma-irradiated and fresh corneas were evaluated with respect to light transmittance, hydration (swelling ratio), elastic modulus (compressive modulus by the indentation method), matrix organization (differential scanning calorimetry), and morphology (light and transmission electron microscopy). The biological properties of the gamma-irradiated cornea, including residual cell content and cellular biocompatibility, were evaluated by quantifying DNA content and measuring the proliferation rate of human corneal epithelial cells, respectively., Results: The hydration, light transmittance, elastic modulus, and proliferation rate of human corneal epithelial cells were not significantly different between fresh and gamma-irradiated corneas. However, differences were observed in tissue morphology, DNA content, and thermal properties. The density of collagen fibrils of the gamma-irradiated corneal sample (160.6 ± 33.2 fibrils/μm) was significantly lower than that of the fresh corneal sample (310.0 ± 44.7 fibrils/μm). Additionally, in the gamma-irradiated corneas, cell fragments-but not viable cells-were observed, supported by lower DNA content of the gamma-irradiated cornea (1.0 ± 0.1 μg/mg) than in fresh corneas (1.9 μg/mg). Moreover, the denaturation temperature of gamma-irradiated corneas (61.8 ± 1.1 °C) was significantly lower than that of fresh corneas (66.1 ± 1.9 °C)., Conclusions: Despite structural changes due to irradiation, the physical and biological properties of the gamma-irradiated cornea remain similar to the fresh cornea. These factors, combined with a decreased risk of rejection and longer shelf life, make the gamma-irradiated tissue a viable and clinically desired option in various ophthalmic procedures.

Purpose: Peak toxicity for in vivo ultraviolet radiation (UVR) exposure to the lens is in the 300-nm wavelength region. However, little is known about corneal cell damage at 300 nm. The purpose of the study was to determine the time evolution of apoptosis in the cornea after in vivo exposure to 300-nm UVR., Methods: Altogether, 16 Sprague Dawley rats were divided into 4 groups and unilaterally exposed to 5 kJ/m UVR (λmax: 300 nm; λ0.5: 10 nm) for 15 minutes. After a predetermined latency period of 1, 5, 24, and 120 hours, depending on the group, the animals were killed and eyes were enucleated. Eye globes were further cryosectioned in 10-μm thick midsagittal sections. For the detection of apoptosis, the TUNEL method was applied., Results: TUNEL-positive signals were observed in the superficial epithelial cells in the exposed and control eyes at all latency periods. At 5 hours, TUNEL staining was detected in the exposed corneas in epithelial cells, keratocytes, and endothelial cells with a maximum signal at 24 hours. At 120 hours, no TUNEL staining was found in endothelial cells and only occasionally in keratocytes in exposed corneas. Signs of ulceration and stromal thinning were observed at 120 hours., Conclusions: UVR in the 300-nm wavelength region induces TUNEL staining in all 3 corneal layers. TUNEL staining of all 3 corneal layers is an early postexposure event observed after a 5-hour latency period. Corneal sterile keratolysis occurs in the time window of 24 to 120 hours probably induced by neutrophils.

Purpose: To assess corneal thermal profile during combined riboflavin and accelerated UV corneal collagen cross-linking (A-CXL) using in vivo surface thermographic analysis., Methods: In this open-label, nonrandomized, prospective pilot study, 28 eyes of 28 patients were included. The study was conduced at the Department of Surgery and Translational Medicine, University of Florence, Italy, in collaboration with the Ophthalmic Operative Unit of Siena University, Italy. All patients underwent high-fluence A-CXL in pulsed light and continuous light UV-A exposure at 30 and 18 mW/cm. Patients were divided into 4 groups: 7 for continuous light A-CXL, 7 for pulsed light A-CXL at 30 mW/cm with 7.2 J energy, 7 for continuous light A-CXL, and 7 for pulsed light A-CXL at 18 mW/cm with 5.4 J energy. Corneal surface temperature measurements were recorded using an infrared FLIR thermocamera (FLIR 320A; FLIR Systems). Corneal temperature values were detected in the surface area exposed to UV-A light irradiation, selecting it in the acquired thermographic image. The maximum temperature value detected in the area studied was recorded and considered for comparative analysis., Results: Infrared thermocamera measurements of the corneal surface during A-CXL treatments showed an average temperature of 31.5°C during the entire procedure in all groups and UV-A powers (30 mW/cm at 7.2 J/cm and 18 mW/cm at 5.4 J/cm) and light exposure modality remained under the threshold of collagen thermal injury., Conclusions: Accelerated corneal collagen cross-linking did not cause thermal rise over the threshold of thermal injury to the corneal surface, demonstrating a safe thermal profile both at 30 mW/cm with 7.2 J and 18 mW/cm with 5.4 J energy dose.

Purpose: The aim of this study was to evaluate the clarity of gamma-irradiated sterile corneal donor lenticules., Methods: Broadband UV, visible, and near-infrared (200-850 nm) light transmission was measured through gamma-irradiated, sterile partial-thickness, and full-thickness donor lenticules and fresh corneal tissues and compared with standard acrylic intraocular lens (IOL) implants using a conventional spectrophotometer technique., Results: All tissues had high light transmission (≥ 90%) in the visible and near-infrared regions and very low (<2%) transmission below 290 nm. Differences in light transmission between irradiated and fresh cornea types were observed between 300 and 450 nm, which mirrored differences in light transmission through their respective storage solutions. Light transmission through partial-thickness irradiated donor lenticules was greatest across all wavelengths. All corneal tissues exhibited higher transmission than acrylic IOL implant across all wavelengths., Conclusions: Gamma-irradiated donor lenticules are comparable with fresh corneas regarding light transmission, with both partial-thickness and full-thickness lenticules having greater transmission than standard IOL. We would expect the optical performance of gamma-irradiated donor lenticules to be comparable to fresh cornea if used for lamellar corneal procedures that do not require a viable endothelium.

Purpose: The Boston keratoprosthesis (KPro) is the most commonly used KPro worldwide. There are limited data on the outcomes when irradiated corneas are used as KPro carriers. We report a retrospective analysis of corneal transplantations performed in a regular surgical mission setting in Beirut, Lebanon, using the Boston KPro type 1 and gamma-irradiated carrier corneas, and we describe visual outcomes, complications, and retention percentage., Methods: We conducted a retrospective analysis of 17 consecutive eyes from 16 patients who underwent Boston KPro type 1 implantation at the Beirut Eye Specialist Hospital between December 2010 and July 2012. Patient medical records were reviewed for preoperative, intraoperative, and postoperative details., Results: Postoperatively, 9 (52.9%), 5 (29.4%), and 2 (11.7%) eyes had a corrected visual acuity of 20/400 or better, 20/100 or better, and 20/40 or better, respectively, at the most recent follow-up visit. A total of 16 eyes (94.1%) improved in corrected visual acuity over the course of follow-up. Overall, 13 eyes (76.4%) developed at least 1 complication after surgery. Retroprosthetic membrane formation was the most common complication, occurring in 10 eyes (58.8%). Neither infectious keratitis nor corneal stromal necrosis was noted during the follow-up period. The retention percentage was 94.1%., Conclusions: The visual acuity outcomes, incidence of complications, and retention percentage of the KPro using gamma-irradiated carrier corneas are comparable with the outcomes of KPro implantation reported in the literature using fresh grafts as carriers. KPro with irradiated corneal carrier grafts seems to be an effective option to increase the supply of transplantation suitable corneas in remote areas, where fresh corneal grafts may be scarce.

Purpose: The depth of corneal crosslinking (CXL) does not seem homogeneous within the treatment area but shows a reduction toward the periphery of the cornea. This study was undertaken to investigate this reduction effect and to look for possible solutions., Methods: Ten corneas were investigated by means of an optical coherence tomography system (SS-100; Tomey, Nagoya, Japan) 1 month after standard CXL (epithelium off, 0.1% riboflavin for 30 minutes, ultraviolet A radiation 365 nm, 3 mW/cm). The depth of the demarcation line was measured as a function of the radial distance from the apex. These curves were compared with a theoretical curve derived from a standard model of photopolymerization., Results: The CXL depth 3 mm away from the center decreases on average to 65% of the central depth (range: 52%-78%). Polymerization theory predicts this decay, however, underestimates the effect., Conclusions: The intended depth of CXL using current light sources is achieved only within the central area of the cornea. To provide CXL to the peripheral cornea, the ultraviolet beam either should have an improved intensity profile or may have to be decentered.

Purpose: Evaluation of stability and functional response after riboflavin-UVA–induced cross-linking in a population of patients younger than 18 years with progressive keratoconus after 36 months of follow-up., Methods: Prospective nonrandomized phase II open trial conducted at the Department of Ophthalmology, Siena University, Italy. The "Siena CXL Pediatrics" trial involved 152 patients aged 18 years or younger (10–18 years) with clinical and instrumental evidence of keratoconus progression. The population was divided into 2 groups according to corneal thickness (>450 and <450 μm) at the time of enrollment. The riboflavin-UVA–induced corneal cross-linking was performed in all patients according to the standard epi-off protocol. Parameters recorded preoperatively and postoperatively were as follows: uncorrected visual acuity, best spectacle–corrected visual acuity, corneal topography and surface aberrometry (CSO Eye Top topographer; Florence, Italy), optical pachometry (Visante OCT; Zeiss Meditec, Jena, Germany), and HRT II confocal microscopy (Rostock Cornea Module, Heidelberg, Germany)., Results: Functional data at 36 months showed an increase of +0.18 and +0.16 Snellen lines for uncorrected visual acuity and best spectacle-corrected visual acuity, respectively, in the thicker group (corneal thickness >450 μm) and +0.14 and +0.15 Snellen lines, respectively, in the thinner group (corneal thickness <450 μm). Patients in the latter group already showed a better and faster functional recovery than the thicker group at 3-month follow-up. Topographic results showed statistically significant improvement in K readings and asymmetry index values. Coma reduction was also statistically significant., Conclusions: The study demonstrated significant and rapid functional improvement in pediatric patients younger than 18 years with progressive keratoconus, undergoing riboflavin-UVA–induced cross-linking. In pediatric age, a good functional response and keratoconus stability was obtained after corneal cross-linking in a 36-month follow-up.

Purpose: To record visual and refractive data in eyes with previous radial keratotomy (RK) and corneal instability undergoing corneal cross-linking (CXL)., Methods: Case series of eyes with RK and refractive instability undergoing CXL with riboflavin and UV-A radiation. The following variables were recorded before 3 and 6 months after CXL: sphere, spherical equivalent (SE), uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), topographic astigmatism, mean topographic K (Km), pachymetry, and Q values., Results: Seven eyes of 4 patients with pre-CXL variables included mean sphere [-0.7 ± 3.2 diopters (D)], astigmatism (2.5 ± 1.17 D), SE (-2.1 ± 3.1 D), UCVA (0.3 ± 2.0), BSCVA (0.63 ± 0.36), Km (38.75 ± 5.74 D), pachymetry (522 ± 36.8 μm), and Q (1.73 ± 1.34). Three months after CXL, mean sphere was +1.0 ± 3.0 D (P = 0.00); astigmatism, 2.5 ± 1.2 D (P = 0.95); SE, 0.3 ± 2.2 D (P = 0.008); UCVA, 0.41 ± 2.7 (P = 0.22); BSCVA, 0.80 ± 0.27 (P = 0.14); Km, 37.5 ± 5.7 D (P = 0.3); pachymetry, 491 ± 33.74 μm (P = 0.00); and Q, 2.27 ± 1.8 (P = 0.22). Six months after CXL, mean sphere was 0.53 ± 3.5 D (P = 0.00); astigmatism, 1.62 ± 1.76 D (P = 0.13); SE, 0.52 ± 2.9 D (P = 0.023); UCVA, 0.60 ± 2.0 (P = 0.018); BSCVA, 0.9 ± 0.15 (P = 0.03); Km, 38.3 ± 4.10 D (P = 0.64); and Q, 1.7 ± 0.78 (P = 0.80)., Conclusions: Short-term follow-up of RK patients treated with CXL showed clinical and statistically significant improvement.

Purpose: To analyze the influence of corneal cross-linking (CXL) using ultraviolet-A and riboflavin on corneal drug penetration of topically applied drugs., Methods: In an ex vivo porcine eye model, eyes were randomly assigned to CXL or control treatment. Central corneal thickness and anterior chamber depth were measured with a Pentacam device. In the CXL group, eyes were treated with CXL using ultraviolet-A (370 nm) and riboflavin, whereas in the control group only riboflavin was applied without irradiation. Subsequently, 0.3% ofloxacin (n = 40 eyes) or 1% voriconazole (n = 40 eyes) eye drops were applied to the cornea every 5 minutes for 30 minutes. Aqueous humour samples were obtained performing an anterior chamber tap. The concentrations of ofloxacin and voriconazole were determined with high-pressure liquid chromatography. Groups were compared performing a Mann-Whitney test., Results: In the CXL group, the mean concentration of ofloxacin (13.33 ± 4.67 μg/mL) and voriconazole (52.70 ± 8.76 μg/mL) was significantly lower than in the untreated control group (ofloxacin: 18.51 ± 6.08 μg/mL, P = 0.005; voriconazole: 62.43 ± 13.5 μg/mL, P = 0.01). This corresponds to a reduction in permeability of 27.98% for ofloxacin and 15.59% for voriconazole. Central corneal thickness and anterior chamber depth were comparable in the CXL and control groups (P > 0.05, each)., Conclusions: CXL reduces the corneal permeability of ofloxacin and voriconazole. This may be of clinical significance, for example, in keratitis treatment.

Purpose: To assess possible cellular damage in the corneal and conjunctival epithelium after corneal cross-linking treatment., Methods: Riboflavin-dextran solution was applied every 5 minutes to the right eyes of 3 rabbits 10 minutes before and 30 minutes during the irradiation with UVA light of 370 nm and an irradiance of 3 mW/cm at the 8- to 10-o'clock position, including conjunctival, limbal, and central corneal epithelium. In addition, 3 rabbits were treated with UVA light only. The rabbits were killed 24 hours later. The treated eyes were examined histologically using hematoxylin-eosin and periodic acid-Schiff staining, immunohistochemistry, and a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) apoptosis assay and compared with the left fellow eyes, which served as controls., Results: There was no epithelial defect on fluorescein staining. No apoptosis was found in the corneal limbal epithelium, keratocytes, or endothelial cells in the irradiated area. In the adjacent conjunctival epithelium, rare superficial conjunctival epithelial cells were positive in TUNEL staining in all animals. Anti-multicytokeratin-positive staining was demonstrated in both the limbal corneal and the conjunctival epithelium. The proliferation marker Ki-67 was identified in the basal cell layer of the limbal epithelium. The frequency and distribution pattern of goblet cells, multicytokeratin, and the proliferation marker Ki-67 were the same in all eyes compared with the untreated fellow control eyes., Conclusions: Standard corneal cross-linking does not induce significant cellular epithelial damage as assessed by histological methods. Further studies on possible genotoxic and long-term changes would be helpful to complete the risk assessment.

Purpose: To report the short-term outcome of gamma-irradiated sterile cornea in corneal and glaucoma patch graft surgeries when viable endothelium is not necessary., Methods: Surgeons who have used the irradiated corneas were asked to rate their impression from 1 (fair) to 4 (very good). Specific intraoperative questions included availability, ease of use, clarity, thickness, tensile strength, and suturing. Postoperative outcomes included epithelialization, biological incorporation, and clarity of tissue, as well as any adverse outcomes., Results: Irradiated sterile cornea has been used in more than 150 surgeries so far. The major uses for irradiated cornea were as glaucoma patch graft (65.3%), as corneal patch graft (14%), in anterior lamellar keratoplasty (10.7%), and in keratoprosthesis (10%). The mean score for packaging of tissue was 3.75 (SD, 0.45), ease of use 3.83 (SD, 0.39), clarity 3.62 (SD, 0.51), thickness 3.67 (SD, 0.49), tensile strength 3.64 (SD, 0.50), and suturing 3.62 (SD, 0.65). Postoperative outcomes were also favorable, including epithelialization (mean ± SD, 3.46 ± 0.69), biological incorporation (mean ± SD, 3.46 ± 0.69), and clarity (mean ± SD, 3.43 ± 0.85). There were 4 episodes of corneal melt (all occurred in patients with previous corneal melt). There were no reported cases of tissue rejection or infection., Conclusions: Gamma-irradiated sterile cornea is a promising new development that may help increase the supply of donor cornea tissue. It virtually eliminates the risk of infection while providing a long shelf life. It is particularly helpful in emergency situations or in remote areas. However, longer follow-up and a prospective study comparing the outcomes of the irradiated corneas with those of the optisol-stored corneas are needed.

Purpose: To report a case of severe corneal endothelial damage after collagen cross-linking treatment in a 37-year-old man with progressive keratoconus with a corneal thickness of more than 400 μm., Methods: After central epithelial debridement, the left cornea was cross-linked for 25 minutes using dextran-riboflavin solution and UV-A light of 370 nm with an irradiance of 3.0 mW/cm2. One month after cross-linking treatment, the patient presented with massive corneal edema. He was treated with 1% prednisolone and carboxymethylcellulose 1% eyedrops 4 times a day for 3 months. Specular microscopy with endothelial cell counting was performed after resolution of the corneal edema 6 months after cross-linking., Results: Despite intense treatment, a ring-shaped corneal scar remained, and uncorrected visual acuity was finger counting. Cell density after resolution was 1776 cells per square millimeter in the affected eye compared with 2978 cells per square millimeter in the untreated fellow eye., Conclusions: Corneal thickness is not the only factor for corneal endothelial damage after cross-linking procedure.

Purpose: To report a patient with a tumor-like corneal keloid., Methods: Retrospective review of clinical features and histopathologic findings., Results: A 68-year-old woman with a remote history of an orbital tumor treated with radiation developed a corneal tumor. A biopsy of the tumor showed fibrocellular tissue, and her blind painful eye was enucleated. Pathologic findings in the enucleated eye showed that the corneal mass was consistent with a large keloid., Conclusions: A corneal keloid may clinically appear as a large corneal tumor.

Purpose: To determine the protective effect of ascorbic acid (ASA) against corneal damage by ultraviolet B (UVB) irradiation., Methods: Eight rabbits weighing 2.1-2.5 kg were randomly divided into 3 groups: a UVB group, an ASA group, and a control group. The UVB group was made up of 2 rabbits that were exposed to 6.84 J/cm of UVB irradiation at a peak wavelength of 306 nm for 17 min/d for 7 days. The ASA group was made up of 4 rabbits that were pretreated intravenously with ASA before being subjected to UVB irradiation. The control group was made up of 2 rabbits that were not subjected to ASA treatment or UVB exposure. After 7 days, the corneas were clinically evaluated for signs of epithelial damage, corneal haze, and neovascularization. After evaluation, the enucleated eyes were analyzed pathologically for signs of proliferation, nuclear change, inflammation, edema, and neovascularization., Results: More damage was observed in the irradiation groups (UVB and ASA groups) than in the control group for all clinical and pathologic parameters (P < 0.0001). Also, when each of the parameters was considered, more severe changes were observed in the UVB group than the ASA group (P < 0.0001), with the exception of nuclear changes, for which the comparison was not applicable., Conclusions: ASA protected the cornea against UVB irradiation in this pilot study; however, further evaluation using many animals is needed to establish its efficacy.

Purpose: Photodynamic collagen cross-linking by using ultraviolet A (UVA) irradiation and the photosensitizer riboflavin has been recently introduced as a new possible treatment of progressive keratoconus. This is the first study, to our knowledge, investigating biochemical aspects of the new procedure. Its aim was to analyze the possible changes in the electrophoretic pattern of corneal collagen type I after collagen cross-linking treatment., Methods: Twenty fresh postmortem porcine corneas were cross-linked; another 20 porcine corneas treated with physiologic saline were used as controls. After removal of the central 10 mm of the epithelium, the corneas were treated with the photosensitizer riboflavin and UVA irradiation for 30 minutes by using a double UVA diode (370 nm, 3 mW/cm). For biochemical analysis, the central 10-mm corneal buttons were trephined, tissue was homogenized, and collagen type I was extracted. Subsequently, the collagen extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis., Results: In the controls, the typical collagen pattern of normal cornea was found with 1 gamma trimer band, 2 beta dimer bands, and 2 alpha monomer bands. In the cross-linked samples, there was an additional intense polymer band in the stacking gel that was resistant to mercaptoethanol, heat, and pepsin treatment. Its molecular size was estimated to be at least 1000 kDa., Conclusions: In the cross-linked corneas, a strong band of high-molecular-weight collagen polymers was shown as the biochemical correlate of the cross-linking effect, showing the efficiency of the new cross-linking procedure. This polymer band complies well with the morphologic correlate of an increased fiber diameter after cross-linking treatment. Its chemical stability supports hopes of a long-term effect of the new treatment.

Purpose: To study potential damage to ocular tissue during corneal collagen cross-linking (X-linking) by means of the riboflavin/UVA (370 nm) approach., Methods: Comparison of the currently used technique with officially accepted guidelines regarding direct UV damage and the damage created by the induced free radicals (photochemical damage)., Results: The currently used UVA radiant exposure of 5.4 mJ/cm and the corresponding irradiance of 3 mW/cm2 is below the known damage thresholds of UVA for the corneal endothelium, lens, and retina. Regarding the photochemical damage caused by the free radicals, the damage thresholds for keratocytes and endothelial cells are 0.45 and 0.35 mW/cm, respectively. In a 400-microm-thick cornea saturated with riboflavin, the irradiance at the endothelial level was 0.18 mW/cm, which is a factor of 2 smaller than the damage threshold., Conclusions: After corneal X-linking, the stroma is depopulated of keratocytes approximately 300 microm deep. Repopulation of this area takes up to 6 months. As long as the cornea treated has a minimum thickness of 400 microm (as recommended), the corneal endothelium will not experience damage, nor will deeper structures such as lens and retina. The light source should provide a homogenous irradiance, avoiding hot spots.

Purpose: To report the case of a patient with keratoconus who developed a corneal perforation secondary to UV radiation from a tanning lamp. We believe this to be the first case of a corneal perforation secondary to UV radiation., Methods: The presentation and management of the patient and the pathophysiology of UV keratitis are discussed., Results: Our patient developed a full-thickness corneal perforation after 30 minutes of tanning lamp exposure without eye protection. The cornea was temporized with cyanoacrylate tissue adhesive until penetrating keratoplasty could be performed., Conclusion: With an increased understanding of the pathophysiology of UV damage, treatment should be aimed at modulating the disease to reduce the likelihood of a poor outcome.

Purpose: To investigate the effects of mitomycin C (MMC), ceramide, and 5-fluororacil (5-FU) on haze after photorefractive keratectomy (PRK) and exposure to ultraviolet B (UVB) radiation., Methods: The right eyes of 42 New Zealand white rabbits were treated with PRK to correct -10 diopter with a 5-mm optical zone. Sponges soaked in 0.02% MMC, 10 or 40 micromol/L ceramide, or 0.5% 5-FU were applied to the right eyes of 6 rabbits each, and a tarsorrhaphy was performed. Eight weeks after complete healing, topical 0.02% MMC or 0.5% 5-FU was applied twice daily to the right eyes of 6 rabbits that had previously received PRK but no topical medication. The control group of 6 rabbits was treated only with PRK. Three weeks after PRK, all the laser-treated eyes were exposed to 100 mJ/cm UVB radiation. Corneal haze was assessed biomicroscopically every 2 weeks using the Fantes scale. Eyes were enucleated 2, 7, and 13 weeks after PRK, and tissue specimens were stained with hematoxylin and eosin and with Apostain., Results: Corneal haze was observed in all rabbits after PRK and was aggravated by UVB irradiation. When applied immediately after PRK, MMC induced corneal opacity and apoptosis of keratocytes, but, at later times, this reagent significantly suppressed opacity, Apostain-positive keratocytes and reactivation of keratocytes, even after UVB irradiation. In contrast, ceramide and 5-FU suppressed corneal opacity after PRK, but this effect was not sustained after UVB irradiation., Conclusions: MMC is a potent inhibitor of haze induced by PRK and UVB irradiation. Throughout the process of corneal wound healing, the severity of apoptosis and reactivation of keratocytes was closely correlated with haze formation.

Purpose: To investigate long-term changes in the cornea after ionizing irradiation in rabbits., Methods: Mature albino rabbits (n = 4) were unilaterally irradiated with 20 Gy of x-rays. The contralateral eye served as a control. The rabbits were examined with slit-lamp biomicroscopy for 5 years. The eyes were then enucleated for histopathologic examinations with light microscopy and transmission electron microscopy., Results: On biomicroscopy, there was no corneal abnormality during the 5-year follow-up period. On histopathology, no abnormality was observed in the corneal epithelium. In the endothelium, a vacuole-like structure was recognized by light microscopy, and transmission electron microscopy revealed marked enlargement of intercellular space and anamorphosis of the nuclei., Conclusion: Moderate x-ray irradiation to rabbit eye did not induce any long-term damage to the corneal epithelium, but the endothelium demonstrated persistent and irreversible damage, which was observed even 5 years after irradiation.

Purpose: Corneas have the high activity of the pentose phosphate pathway. To clarify the relevance of the pentose phosphate pathway to their antioxidant defense, we examined the activity of glucose-6-phosphate dehydrogenase (G6PDH), the rate-determining enzyme of the pathway, in corneas exposed to ultraviolet light (UV)., Methods: Fresh porcine eye globes were exposed to ultraviolet light A (UVA) or ultraviolet light C (UVC), and the tear film-removed eye globes to UVA. After UV exposure, the corneas were dissected from the eye globes and extracted with a saline solution, and the G6PDH activity in the extract was assayed. The G6PDH activity of unilateral opaque corneas, their paired, transparent corneas, and normal corneas, all of which were obtained from fresh, UV-unexposed eye globes, and the L-lactate dehydrogenase (LDH) activity of these opaque and normal corneas also were assayed., Results: The G6PDH activity of corneas increased with UVA exposure, and decreased with long-term UVC exposure, although it increased with short-term UVC exposure. A UV-blocking contact lens screened corneas from the UVA-induced increase. Removal of the tear film enhanced the UVC-induced decrease. The G6PDH activity of unilateral opaque corneas was lower than that of paired, transparent corneas or normal corneas, all of which were obtained from fresh, UV-unexposed eye globes. The LDH activity of the opaque corneas was much higher than that of the normal corneas., Conclusion: Exposure of corneas to UVA or a small dose of UVC enhances the G6PDH activity, i.e., the pentose phosphate pathway. This activity enhancement may play an important role in corneal antioxidant defense against UV-induced oxidative stress. However, exposure of corneas to large doses of UVC appears to damage the pathway.

Purpose: Keratocytes undergo apoptosis during various pathologic conditions and after exposure to ultraviolet radiation (UVR). It was reported that the Fas/Fas ligand system is involved in modulating keratocyte apoptosis. The expression of Fas ligand (FasL) protein was studied in rabbit corneas after photokeratitis induced by different UV wavelengths., Methods: Six New Zealand albino rabbit corneas were exposed to 280- and 310-nm UVR in 10-nm full wavebands at doses producing biomicroscopically significant keratitis (0.12 J/cm2 for 280 nm and 0.47 J/cm2 for 310 nm). Animals were killed 24 hours after exposure. Immunohistochemistry was performed to localize FasL protein in paraffin sections of rabbit corneas. Primary antibody was polyclonal goat anti-FasL IgG., Results: FasL protein was uniformly detected in epithelial and endothelial layers of all UVR-exposed and control, nonexposed corneas. The positive staining of keratocytes was confined to the anterior stroma of corneas that were exposed to 280-nm UVR. Corneas exposed to 310-nm UVR showed positively stained keratocytes throughout the entire thickness of the stroma., Conclusions: These data strongly suggest that the Fas/FasL system may play an important role in apoptosis of corneal cells after UVR. The FasL expression in the corneal stroma was more extensive after exposures at 310-nm UVR than at 280-nm UVR.

Purpose: Apoptosis was studied in rabbit corneas as a possible mechanism of cell death after photokeratitis induced by different UV wavelengths., Method: Fourteen albino rabbit corneas were exposed to 280- and 310-nm UV radiation (UVR) in 10-nm full wavebands at doses that cause biomicroscopically significant keratitis (0.12 J/cm2 for 280 nm and 0.47 J/cm2 for 310 nm). Animals were killed 24 and 76 h after exposure. Corneas were processed for light and transmission electron microscopy and in situ end labeling of fragmented DNA by using a modification of the TUNEL technique., Results: Corneas exposed to 280-nm UVR showed TUNEL-positive staining only in epithelial cells and superficial keratocytes at 24 and 76 h after irradiation. Twenty-four hours after 310-nm UVR exposure, TUNEL-positive staining was present in the epithelial cells, keratocytes throughout the entire thickness of the central stroma, and in endothelial cells. Seventy-six hours after exposure to 310-nm UVR, keratocytes disappeared throughout the whole thickness of the damaged stroma. Only a few epithelial cells were TUNEL positive at that time. Transmission electron microscopy (TEM) verified the occurrence of apoptotic nuclei and cells., Conclusion: Apoptosis appears to be a mechanism of corneal cell death after UVR. The 310-nm UVR caused more extensive damage to the corneal stroma and endothelium than did the 280-nm UVR.

Purpose: CTLA4, a high-affinity ligand of B7, can, in soluble form, prevent antigen-driven T-cell activation by blocking CD28-B7 interaction and can thereby prevent immune graft rejection. In this study, we tested the capacity of soluble CTLA4-Ig alone or in combination with UV-B irradiation to suppress corneal allograft rejection in rabbits., Methods: Corneas from Dutch belted rabbits were incubated in corneal storage medium containing 0, 1, 10, 25, or 250 microg/ml of CTLA4-Ig for 18 h and were then transplanted into the vascularized or nonvascularized corneas of New Zealand White rabbit recipients. A series of donor corneas were exposed to UV-B irradiation alone or a combination of irradiation and CTLA4-Ig to determine if these two treatments would have an additive effect in prolonging graft survival. The fate and clinical condition of the allografts were evaluated by slit-lamp photomicroscopic observation and corneal-thickness measurements. Grafts that were rejected were processed for histopathologic and immunohistochemical analysis to determine the characteristics of cells infiltrating the grafts., Results: Grafts placed in nonvascularized corneas showed no differences in survival times, regardless of treatment. Among the grafts placed in vascularized corneas, those incubated with CTLA4-Ig at a concentration of 250 microg/ml failed within 7-14 days. Histopathologic and immunocytochemical examination revealed a dense accumulation of immune inflammatory cells, especially class II major histocompatibility complex (MHC)-expressing, antigen-presenting cells, in the failed grafts. Grafts incubated with CTLA4-Ig at concentrations of 1 and 10 microg/ml had mean survival times greater than the control, untreated corneal allografts. Some of the grafts in these two treatment groups survived for the 100-day observation period, whereas none of the grafts in the other treatment groups survived to this end point. UV-B irradiated grafts incubated with CTLA4-Ig at a concentration of 1 microg/ml appeared to have longer survival times and fewer rejections compared with control, untreated grafts and grafts treated with UV-B or CTLA4-Ig alone., Conclusion: The results show that the CTLA4-Ig coreceptor blocking agent can prolong corneal allograft survival in vascularized graft sites and that UV-B irradiation followed by incubation in CTLA4-Ig may prolong graft survival better than either treatment alone. These results suggest that agents that prevent second-signal interaction between antigen-presenting cells and T lymphocytes may be useful for inhibiting corneal allograft rejection.

Purpose: The distribution of hyaluronan (HA) and the cellular response after photokeratitis induced by different ultraviolet (UV) wavelengths in the rabbit cornea was examined to help understand the mechanism of corneal injury and repair after UV damage. HA is a high molecular weight disaccharide polymer capable of binding considerable amounts of water. It is not normally found in the rabbit corneal stroma. The production of HA represents a generalized corneal response to injury., Methods: Twenty-four albino rabbit corneas were exposed to 270, 290, and 310 nm of UV radiant energy in 8-nm full wavebands in doses producing biomicroscopically significant keratitis (three corneal thresholds for keratitis (Hc): 0.016 J/cm2 for 270 nm, 0.04 J/cm2 for 290 nm, and 0.14 J/cm2 for 310 nm) and in subkeratitis doses (0.7 Hc: 0.004 J/cm2 for 270 nm, 0.008 J/cm2 for 290 nm, and 0.03 J/cm2 for 310 nm). The rabbits exposed to 270 and 290 nm of UV radiation were sacrificed 3 days after exposure. The rabbits exposed to 310 nm of UV radiation were sacrificed 3, 7, and 14 days after exposure, respectively. The corneal tissue specimens were double stained and examined morphologically and histochemically for HA by light microscopy., Results: Evaluation of corneas exposed to 270 and 290 nm of UV radiant energy in both subkeratitis and keratitis doses and those corneas exposed to 310 nm of radiant energy in subkeratitis dose showed neither stromal changes nor production of HA by corneal cells. Corneas exposed to 310 nm of UV radiant energy in keratitis dose at 3 days after exposure showed disappearance of keratocytes in entire thickness of central cornea. Cells bordering this damaged area were staining for HA. By 7 days after exposure almost the whole damaged area, except one fourth of anterior stroma, was repopulated by new keratocytes staining positive for HA. The corneal structures became normal and HA almost completely disappeared 14 days after exposure., Conclusions: A keratitis dose of 310 nm of UV light irradiation is needed to cause keratocyte damage. A keratitis dose of the shorter wavelengths does not cause keratocyte cell damage at the light microscopic level. The keratocyte production of HA appears to be a sign of cell readiness to repopulate the damaged stroma devoid of keratocytes.

A 22-year-old female college student from the southwestern United States was first seen with progressive fluffy white growths over both corneas nasally and a history of chronic allergies and extensive sun, wind, and chlorine exposure as a competitive swimmer. The lesions were superficial, elevated, crescent-shaped, vascularized, and extended from the limbal conjunctiva onto the peripheral cornea nasally. The lesions were composed of an unencapsulated thick fibrovascular pannus with numerous fibrocytes, chronic inflammatory cells and mast cells, and abundant linear and wavy elastinophilic profiles, but no adipose tissue. Ultrastructurally, the elastinophilic structures were immature elastic fibers arranged in globules and bundles within a thick collagenous matrix. No mature elastic fibers were found. A few areas of elastotic degeneration were also found. This lesion resembled elastofibromas that have been reported more commonly in the subcapsular region and only once in the ocular region. Previous reports debated whether the elastic material in elastofibromas is derived from excessive production of elastic fibers by activated fibrocytes or from elastotic degeneration of collagen. In our case, both processes occur and are presumed to result from excessive ultraviolet radiation, wind and chlorine exposure, and perhaps, chronic inflammation; features that have been ascribed to the pathogenesis of pingueculae and pterygia.

This study attempts to measure the quantitative contribution of major chemical fractions of the whole bovine cornea to ultraviolet (UV) absorption between 240 and 300 nm, with special attention to the biologically significant range of 290-300 nm. The cornea was divided into water-insoluble, nonprotein small water-soluble, water-soluble protein, and lipid-soluble fractions. The insoluble fraction (largely collagen) was solubilized by enzymatic digestion. Solutions of individual fractions equivalent to a constant mass of fresh cornea were scanned for absorption from 240 to 300 nm. The sum of the absorbances of the individual fractions closely approximated the absorbance of whole corneas at all wavelengths examined. The extinction coefficients of the lipid and water-soluble fractions were several times greater than that of the insoluble fraction throughout the studied spectrum. Yet, because of its large mass (75% of cornea dry weight), the insoluble fraction accounted for 40-50% of UV absorbance between 240 and 280 nm. However, in the range of 290-300 nm, the water-soluble plus lipid-soluble fractions accounted for 60-65% of the total absorption, with the water-soluble proteins alone accounting for 40-45% of the total. The soluble proteins comprised only approximately 17% of the cornea's dry weight. The special contribution of the water-soluble proteins to absorption was attributed to their relatively high tryptophan content (approximately 1.6% by weight). A 54-kDa protein, identified by others as tryptophan-rich, tumor-associated aldehyde dehydrogenase, accounted for approximately 30% of the total soluble protein mass.(ABSTRACT TRUNCATED AT 250 WORDS)

The formation of a pterygium is modelled using a population balance model of corneal and limbal epithelial production. Scattered light incident at the temporal limbus is focused at the nasal limbus with a peak intensity > or = 20 times. We hypothesize that this causes a focal alteration of corneal epithelial stem cells (which play a role in maintaining a barrier between corneal and conjunctival epithelia). Normative rabbit corneal data were used. Light-induced conjunctival epithelial proliferation was implemented by amplifying normal epithelial cell production along a nasal sector of the limbal circumference. A ten-fold normal peak in stimulation was assumed at the nasal zero azimuth (3 o'clock position in the right eye) with a quadratic attenuation to zero stimulation (normal) at the circumferential limits. Adjacent points on the outer limbus were assumed to be independent generators of epithelial cells that migrated in centripetal streams into the cornea. Normalization of the attrition rate for a net surface accumulation of the proliferating cells was embedded into the computational algorithm. The localized conjunctival mass was allowed to travel the predicted distance along the surface before the final shape was computed. The result shows a wing-shaped mass with a curved leading edge (corneal side). It is proposed that the initial biologic event in pterygium pathogenesis is an alteration of limbal stem cells due to chronic ultraviolet light exposure. The concomitant breakdown of the limbal barrier and subsequent conjunctivalization of the cornea explain the shape and formation of a primary pterygium.

Adult male mice from four inbred albino strains (SJL/J, NZW/BL, BALB/c HeA, and SWR/J) were subjected to ultraviolet radiation (UVR) exposure (302 nm peak wavelength, intensity 398 microW/cm2) for 3.25 min and photographed 4 days postexposure to assess corneal clouding. Corneal extracts from control (unexposed) mice from each strain, were also monitored for aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activity and soluble protein content. The SWR/J strain exhibited more extensive corneal clouding after UV exposure than did the other strains, and control SWR/J mice exhibited a low activity variant phenotype for the major ocular ALDH AHD-4, and decreased levels of soluble protein in corneal extracts. These data support earlier proposals for a major role for ALDH in assisting the cornea in protecting the eye against UVR-induced tissue damage.

Adult male C57BL/6J inbred mice were subjected to ultraviolet radiation (UVR) exposure (302-nm peak wavelength; average intensity 282 microW/cm2) for 1 h and monitored for ocular aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activity changes over a period of 25 days. Dramatic reductions in activities were observed by 4-6 days postexposure, resulting in enzyme levels of 15-16% of control animals. Major decreases in corneal enzyme levels were predominantly responsible for these changes. Ocular morphology was observed throughout using a photoslit-lamp biomicroscope, with maximum corneal clouding occurring at days 4-6. These data support earlier proposals for major roles for these corneal enzymes in assisting the cornea in protecting the eye against UVR-induced tissue damage.

To determine whether exposure to the excimer laser beam results in the oncogenic transformation of cornea cells, tissue-cultured corneal keratocytes and intact corneas from an inbred strain of rat were exposed to ablative and subablative energies of the excimer laser beam. No evidence for cellular transformation was found. Neither the corneas nor the stromal keratocytes exposed to the laser beam were transformed into cells with unregulated growth potential. Both treated keratocytes and corneas were implanted subcutaneously in the same strain of rats; neither developed into tumors. In tissue culture, cells from cultures exposed to the excimer laser beam exhibited normal growth patterns, growing at the same rate as control, unexposed cells and exhibiting the same capacity to respond to the contact inhibition of growth as the control cells. These results suggest that the 193-nm excimer laser beam does not transform corneal keratocytes and that the energies emitted by this beam will not cause cell transformation when the excimer laser is used as a surgical tool in human eyes.