Wound healing in the rabbit cornea after corneal collagen cross-linking with riboflavin and UVA

PURPOSE: This study was undertaken to investigate the wound healing process of the first 6 weeks after photodynamic cross-linking treatment in the rabbit cornea, using the photosensitizer riboflavin and UVA. METHODS: After removal of the central epithelium, the right corneas of 8 Chinchilla rabbits were cross-linked with a photosensitizing 0.1% riboflavin solution and UVA light (370 nm; irradiance, 3 mW/cm(2); dose, 5.4 J/cm(2)) for 30 minutes. Two animals were euthanized 3 days, 7 days, 4 weeks, and 6 weeks postoperatively. The corneas of the enucleated eyes were evaluated using 4-microm light microscopic sections with routine stains and avidin-biotin complex immunostaining with anti-alpha-smooth muscle actin. RESULTS: By day 3 after treatment, complete apoptotic damage and loss of the endothelial cells and the stromal keratocytes were found in the irradiated area through the entire thickness of the stroma. There was marked stromal edema (850 +/- 66 vs. 332 +/- 43 microm in the untreated controls; P < 0.01). The epithelium was already closed again. At the margins of the lesion, there was a mild inflammatory reaction with scattered macrophages, lymphocytes, and neutrophils. By day 7, the endothelium was already intact again, and keratocyte repopulation of the posterior stroma was noted. By week 4, the keratocyte repopulation of the anterior stroma was observed with some acellular areas between. By week 6, the cytoarchitecture of the cornea seemed normal again. By weeks 4 and 6, alpha-actin-positive keratocytes were identified, especially in the periphery of the irradiated area. CONCLUSIONS: After riboflavin/UVA cross-linking of rabbit cornea, a complete cell loss occurs in the irradiation area with an irradiance of 3 mW/cm(2). The cytotoxic damage is repaired by repopulation after approximately 4-6 weeks. A combination of cross-linking with other procedures such as the implantation of intracorneal rings should be performed only after a sufficient time interval of approximately 2 months, allowing cellular regeneration.     

Endothelial cell damage after riboflavin-ultraviolet-A treatment in the rabbit

PURPOSE: To evaluate the possible cytotoxic effect of combined riboflavin-ultraviolet-A (UVA) treatment on the corneal endothelium. SETTING: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. METHODS: The right eyes of 34 New Zealand White rabbits were treated with riboflavin and various endothelial UVA doses ranging from 0.16 to 0.9 J/cm2 (0.09 to 0.5 mW/cm2, 370 nm) and postoperative enucleation times of 4 hours and 24 hours. The endothelial cells were evaluated in histological sections. The terminal deoxynulceotidyl transferase deoxy-UTP-nick-end labeling (TUNEL) technique and transmission electron microscopy were used to detect apoptosis. RESULTS: There was no endothelial damage in the 6 rabbit eyes enucleated at 4 hours. In those enucleated at 24 hours, there was significant necrosis and apoptosis of endothelial cells in the corneas treated with an endothelial dose of > or =0.65 J/cm2 (0.36 mW/cm2), which is about twice the endothelial UVA dose used in the treatment of keratoconus patients. CONCLUSIONS: In rabbit corneas with a corneal thickness less than 400 microm, the endothelial UVA dose reached a cytotoxic level of > or =0.65 J/cm2 (0.36 mW/cm2) using the standard surface UVA dose of 5.4 J/cm2 (3 mW/cm2). Pachymetry should be routinely performed before riboflavin-UVA treatment; in thinner corneas, irradiation should not be done because of the cytotoxic risk to the endothelium.     

Keratocyte cytotoxicity of riboflavin/UVA-treatment in vitro

PURPOSE: Collagen crosslinking using ultraviolet- A (UVA) -irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. It has been shown to increase effectively the biomechanical strength of the cornea and to stop or even reverse the progression of keratoconus. As part of a safety evaluation, the present study was undertaken to investigate in vitro the possible cytotoxic effect of combined riboflavin/UVA-treatment on corneal keratocytes and to compare it to UVA-irradiation alone. METHODS: Cell cultures established from porcine keratocytes were treated with 0.025% riboflavin solution and various UVA (370 nm)-irradiances ranging from 0.4 to 1.0 mW/cm2 and with UVA alone between 2 and 9 mW/cm2 for 30 min. The cell cultures were evaluated for cell death 24 h after irradiation using trypan-blue and Yopro-fluorescence staining. RESULTS: An abrupt cytotoxic irradiance level was found at 0.5 mW/cm2 for keratocytes after UVA-irradiation combined with the photosensitizer riboflavin, which is 10-fold lower than the cytotoxic irradiance of 5 mW/cm2 after UVA-irradiation alone. CONCLUSIONS: A cytotoxic effect of combined riboflavin/UVA-treatment on keratocytes is to be expected at 0.5 mW/cm2, which is reached in the clinical setting in human corneas down to a depth of 300 microm using the standard surface UVA-irradiance of 3 mW/cm2.     

Increased resistance of crosslinked cornea against enzymatic digestion

PURPOSE: Collagen-crosslinking using combined riboflavin/ UVA treatment has been developed by us as a new treatment for keratoconus by stiffening the collagenous matrix. Recently, we have started to use the same method for the treatment of corneal ulcers. The aim of the present study was to evaluate the influence of the crosslinking treatment on the resistance of the cornea against enzymatic degradation. METHODS: 60 enucleated porcine eyes were treated with the photosensitizer riboflavin and UVA-irradiation (370 nm; irradiance of 1, 2 or 3 mW/cm2) for 30 minutes and compared with 20 untreated control eyes. After crosslinking treatment, the corneal buttons were trephined and exposed to pepsin, trypsin and collagenase solutions. The extent of the corneal digestion was monitored daily. Selected cases were examined by light microscopy. RESULTS: The corneal buttons crosslinked with riboflavin/ UVA at 3 mW/cm2 were dissolved only by day 13 following pepsin digestion and by day 14 following collagenase treatment versus 6 days in the untreated control corneas. Digestion by trypsin was observed on day 5 in buttons crosslinked at 3 mW/cm2 compared to day 2 in the control corneas. Microscopically, a prolonged preservation especially of the anterior portion of the crosslinked corneas could be demonstrated. CONCLUSIONS: Photochemical crosslinking of the cornea using riboflavin and UVA results in a markedly increased resistance versus collagen digesting enzymes. The findings support the use of the new method in the treatment of corneal ulcers.     

Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro

Recently, we have developed collagen crosslinking induced by combined riboflavin/UVA treatment, thus increasing the biomechanical rigidity of the cornea to treat progressive keratoconus. The present safety study was performed to evaluate possible cytotoxic effects of combined riboflavin/UVA treatment on the corneal endothelium in vitro. Endothelial cell cultures from porcine corneas were treated with 500 microM riboflavin solution, exposed to various endothelial UVA irradiances (370 nm) ranging from 0.1 to 1.6 mW/cm2 for 30 min and evaluated 24 h later using trypan blue staining and Yopro fluorescence staining. The effect of either treatment alone (UVA irradiation ranging from 0.2 to 6 mW/cm2) was also tested. An abrupt cytotoxic threshold irradiance level was found at 0.35 mW/cm2 after combined treatment with riboflavin plus UVA irradiation and at 4 mW/cm2 with UVA irradiation alone. Riboflavin alone was not toxic. A cytotoxic effect of the combined riboflavin/UVA treatment on corneal endothelial cells is to be expected with a corneal thickness of less than 400 microm. Therefore, pachymetry should be routinely performed before riboflavin/UVA treatment to exclude patients at risk.     

Safety of UVA-riboflavin cross-linking of the cornea

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.     

Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking

PURPOSE: To evaluate the biomechanical effect of combined riboflavin-ultraviolet A (UVA) treatment on porcine and human corneas. SETTING: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. METHODS: Corneal strips from 5 human enucleated eyes and 20 porcine cadaver corneas were treated with the photosensitizer riboflavin and irradiated with 2 double UVA diodes (370 nm, irradiance = 3 mW/cm2) for 30 minutes. After cross-linking, static stress-strain measurements of the treated and untreated corneas were performed using a microcomputer-controlled biomaterial tester with a prestress of 5 x 10(3) Pa. RESULTS: There was a significant increase in corneal rigidity after cross-linking, indicated by a rise in stress in treated porcine corneas (by 71.9%) and human corneas (by 328.9%) and in Young's modulus by the factor 1.8 in porcine corneas and 4.5 in human corneas. The mean central corneal thickness was 850 microm +/- 70 (SD) in porcine corneas and 550 +/- 40 microm in human corneas. CONCLUSIONS: Riboflavin-UVA-induced collagen cross-linking led to an increase in mechanical rigidity in porcine corneas and an even greater increase in human corneas. As collagen cross-linking is maximal in the anterior 300 microm of the cornea, the greater stiffening effect in human corneas can be explained by the relatively larger portion of the cornea being cross-linked in the overall thinner human cornea.     

Immunofluorescence confocal microscopy of porcine corneas following collagen cross-linking treatment with riboflavin and ultraviolet A

PURPOSE: To assess ultrastructural stromal modifications in porcine corneas after riboflavin and ultraviolet A (UVA) exposure using immunofluorescence confocal imaging. METHODS: Twenty-five freshly enucleated porcine eyes were enrolled in the study. Five eyes served as control (group I). Twenty eyes had their epithelium removed (groups I, II, IV, and V) and five eyes had their epithelium intact (group III). Groups II and III were cross-linked with riboflavin 0.1% solution (10 mg riboflavin-5-phosphate in 10 mL 20% dextran-T-500) and exposed to UVA (365 nm, 3 mW/cm2) for 30 minutes. Group IV included five eyes soaked with riboflavin without posterior irradiation, and group V included five eyes irradiated, without previous exposure to riboflavin. Ultra-thin sections (8 microm) of the corneas were stained with anti-collagen I and DAPI and their fluorescence was revealed under confocal microscopy. RESULTS: Only the cross-linked corneas (group II) showed a pronounced, highly organized anterior fluorescence zone of 182.5 +/- 22.5 microm. Using DAPI staining, an anterior and concentrated displacement of cell nuclei due to collagen compaction was observed after crosslinking (group II). No structural changes were observed in all other groups. CONCLUSIONS: The cross-linking treatment effect can be directly visualized using confocal fluorescence imaging, allowing for a quantitative analysis. Cross-linked corneas showed a pronounced and limited anterior zone of organized collagen fibers, which was not observed in the other groups. Treatment of the cornea with riboflavin and UVA without previous deepithelialization did not induce any cross-linking effect. Consequently, to facilitate diffusion of riboflavin throughout the corneal stroma, the epithelium should be removed as an important initial step in the treatment.     

Thermomechanical behavior of collagen-cross-linked porcine cornea

PURPOSE: Collagen cross-linking using combined riboflavin/UVA treatment has been shown to increase the biomechanical rigidity of the cornea and has been used successfully for the treatment of progressive keratoconus. From morphological and biochemical investigations, a different degree of cross-linking for the anterior and posterior stroma by the treatment is suggested. The present study was undertaken to better evaluate this effect by testing the thermomechanical behavior. METHODS: Ten 10 x 5 mm corneal strips from porcine cadaver eyes enucleated within 5 h post mortem were cross-linked using the photosensitizer riboflavin and UVA irradiation (370 nm, irradiance = 3 mW/cm(2)) for 30 min and compared to ten untreated corneal strips and ten corneal strips cross-linked with 0.1% glutaraldehyde. The temperature in a water bath was raised from 60 to 95 degrees C with temperature increments of 1 degrees C per minute. The hydrothermal shrinkage of the corneal strips was measured in 2.5 degrees C steps using a micrometer. In addition, six 10-mm whole corneal buttons were cross-linked with riboflavin/UVA and immersed into water at 70 or 75 degrees C. RESULTS: The maximal hydrothermal shrinkage for the untreated control specimens and the posterior portion of the riboflavin/UVA-treated corneas was at 70 degrees C, for the anterior portion of the cornea cross-linked by riboflavin/UVA at 75 degrees C and for glutaraldehyde-cross-linked cornea at 90 degrees C. In the cross-linked corneal buttons, a typical mushroom-like shape was observed at 70 degrees C and a cylinder shape at 75 degrees C. CONCLUSIONS: The different degree of collagen cross-linking in the corneal stroma after riboflavin/UVA treatment is reflected by the differences in the maximal shrinkage temperature of the anterior and posterior portion. Therefore, in the corneas cross-linked with riboflavin/UVA a higher shrinkage temperature was observed for the anterior portion of the cornea (75 degrees C) compared to the posterior stroma (70 degrees C) due to the higher degree of cross-linking of the anterior stroma. The anterior localization of the cross-linking effect is advantageous for the endothelium and for the preservation of the anterior corneal curvature.     

Collagen fiber diameter in the rabbit cornea after collagen crosslinking by riboflavin/UVA

OBJECTIVE: Collagen crosslinking of the cornea has been developed recently as a quasiconservative treatment of keratoconus. Biomechanical in vitro measurements have demonstrated a significant increase in biomechanical stiffness of the crosslinked cornea. The aim of the present study was to evaluate the effect of this new procedure on the collagen fiber diameter of the rabbit cornea. METHODS: The corneas of the right eyes of 10 New Zealand White albino rabbits were crosslinked by application of the photosensitizer riboflavin and exposure to UVA light (370 nm, 3 mW/cm2) for 30 minutes. The left fellow control eyes were either left untreated (rabbits 1-4), deepithelialized (rabbits 5-7), or deepithelialized and treated with riboflavin/dextran solution (rabbits 8-10) to exclude an influence of epithelial debridement or hydration changes on the fiber diameter. On ultrathin sections of samples from the anterior and posterior cornea, the collagen fiber diameter was measured semiautomatically with the help of morphometric computer software. RESULTS: In the anterior stroma, the collagen fiber diameter in the treated corneas was significantly increased by 12.2% (3.96 nm), and in the posterior stroma by 4.6% (1.63 nm), compared with the control fellow eyes. In the crosslinked eyes, the collagen fiber diameter was also significantly increased by, on average, 9.3% (3.1 nm) in the anterior compared with the posterior stroma within the same eye. CONCLUSIONS: Collagen crosslinking using riboflavin and UVA leads to a significant increase in corneal collagen diameter. This alteration is the morphologic correlate of the crosslinking process leading to an increase in biomechanical stability. The crosslinking effect is strongest in the anterior half of the stroma because of the rapid decrease in UVA irradiance across the corneal stroma as a result of riboflavin-enhanced UVA absorption.     

The effects of epithelial viability on stromal keratocyte apoptosis in porcine corneas stored in Optisol-GS

PURPOSE: To investigate the effect of corneal epithelium on the viability of corneal stromal keratocytes in Optisol-GS. METHODS: After sterilization, corneoscleral buttons were excised and stored in Optisol-GS for various time periods. Group 1 corneas (n = 40) underwent mechanical corneal epithelial debridement before storage while group 2 corneas (n = 40) were stored with intact epithelium. Changes in corneal thickness, keratocyte density, and keratocyte apoptosis were investigated immediately, at 4 hours, and on days 1, 2, 3, 5, 7, and 14 in the preservation medium. The differences between group 1 and 2 corneas were analyzed. RESULTS: Corneal thickness increased significantly in the second week of preservation in both groups, though more substantially in group 1. Significant corneal epithelial apoptosis was noticed in the first week in group 2 corneas. Corneal stromal keratocyte density decreased with prolonged preservation time. DNA laddering was detected by ligation-mediated polymerase chain reaction throughout the experiment periods in both groups, but the increase of keratocyte apoptosis was more significant after 5 days of preservation, especially in group 1. CONCLUSIONS: Stromal keratocytes underwent apoptosis in Optisol-GS. The absence of corneal epithelium during preservation further increased the stromal keratocyte apoptosis.     

Corneal cross-linking-induced stromal demarcation line

PURPOSE: Corneal collagen cross-linking by UVA/riboflavin (X-linking) represents a new method for the treatment of progressive keratoconus and currently is under clinical study. To avoid UVA irradiation damage to the corneal endothelium, the parameters for X-linking are set in a way that effective treatment occurs only in the first 300 microm of the corneal stroma. Here, X-linking not only strengthens the biomechanical properties of the cornea but also induces keratocyte apoptosis. To date, the effectiveness of treatment could be monitored only indirectly by postoperative follow-up corneal topographies or using corneal confocal microscopy. Here we describe a corneal stromal demarcation line indicating the transition zone between cross-linked anterior corneal stroma and untreated posterior corneal stroma. The demarcation line is biomicroscopically detectable in slit-lamp examination as early as 2 weeks after treatment. METHODS: X-linking was performed in 16 cases of progressive keratoconus, and corneas were examined biomicroscopically and by means of corneal topography and pachymetry before and after treatment. RESULTS: In 14 of 16 cases, a thin stromal demarcation line was visible at a depth of approximately 300 microm over the whole cornea after X-linking treatment. CONCLUSION: This newly observed demarcation line may result from differences in the refractive index and/or reflection properties of untreated versus X-linked corneal stroma and represents an effective tool to biomicroscopically easily monitor the depth of effective X-linking treatment in keratoconus.     

Keratocyte apoptosis associated with keratoconus.

Keratoconus is an ectatic corneal dystrophy associated with stromal thinning and disruption of Bowman's layer. The purpose of this study was to explore a possible association between keratocyte apoptosis and keratoconus. Keratocyte apoptosis was evaluated in corneas of patients with keratoconus, corneas of patients with stromal dystrophies, and normal donor corneas using the transferase-mediated dUTP-digoxigenin nick and labeling (TUNEL) assay. Keratocyte apoptosis was also studied in keratoconus and normal corneas using transmission electron microscopy. TUNEL-stained keratocytes were detected in 60% of corneas with keratoconus, but only 35% of corneas with stromal dystrophies (P =0.03). The number of TUNEL-positive keratocytes detected in the keratoconus, stromal dystrophy, and normal corneas was 7+/-1 (mean+/-standard error, range 0-20), 2+/-0. 8 (range 0-9), and 0+/-0 (range 0-0) TUNEL-positive cells per section, respectively. The differences between the keratoconus and the stromal dystrophy (P =0.0097) or the normal cornea (P =0.01) groups were statistically significant. The difference between the stromal dystrophy and normal cornea groups was not statistically significant (P =0.45). The stromal dystrophy group was included to account for surgery-associated keratocyte apoptosis. No TUNEL-stained keratocytes were detected in normal corneas. Cell morphologic changes consistent with apoptosis were detected by transmission electron microscopy (TEM) in keratocytes of keratoconus corneas, but not in keratocytes in normal corneas. Chronic keratocyte apoptosis associated with ongoing epithelial injury may link risk factors associated with keratoconus such as chronic eye rubbing, contact lens wear, or atopic eye disease. Similarly, increases that have been detected in several different degradative enzymes in keratoconus corneas could be associated with chronic keratocyte apoptosis and less than perfect control of release of intracellular contents.     

Biomechanical evidence of the distribution of cross-links in corneas treated with riboflavin and ultraviolet A light

PURPOSE: To examine to which depth of the cornea the stiffening effect is biomechanically detectable. SETTING: Department of Ophthalmology, University of Dresden, Dresden, Germany. METHODS: Of 40 enucleated porcine eyes, 20 eyes were treated with the photosensitizer riboflavin (0.1%) and ultraviolet A (UVA) light (370 nm, 3 mW/cm2, 30 minutes); the other 20 eyes served as control. From each eye, 2 flaps of 200 microm thickness were cut with a microkeratome, and strips of 5 mm width and 7 mm length were prepared. Stress-strain behavior was measured with a material tester to characterize the stiffening effect. Five pairs of human donor eyes were tested in the same way. RESULTS: In porcine corneas, the stiffening effect was stronger in the anterior-treated flaps than in the posterior-treated flaps and the control flaps (P = .001). A 5% strain was achieved at a stress of 261.7 +/- 133.2 x 10(3) N/m2 in the anterior-treated flaps and 104.1 +/- 52.7 x 10(3) N/m2 in the anterior control flaps. The posterior-treated flaps (105.0 +/- 55.8 x 10(3) N/m2) and the posterior control flaps (103.7 +/- 61.8 x 10(3) N/m2) showed no difference (P = .95). A similar stiffening effect was observed in human eyes, but contrary to findings in porcine corneas, in human corneas the anterior control flaps were stiffer than the posterior control flaps (P = .027). CONCLUSIONS: Treatment of the cornea with riboflavin and UVA significantly stiffened the cornea only in the anterior 200 microm. This depth-dependent stiffening effect may be explained by the absorption behavior for UVA in the riboflavin-treated cornea. Sixty-five percent to 70% of UVA irradiation was absorbed within the anterior 200 microm and only 20% in the next 200 microm. Therefore, deeper structures and even the endothelium are not affected.     

Early keratocyte apoptosis after epithelial scrape injury in the human cornea

Animal studies in mice, rats, rabbits, pigs and hens demonstrated that anterior keratocytes undergo programmed cell death or apoptosis after corneal epithelial injury. Many other wound healing changes subsequently follow the keratocyte apoptosis response. This study evaluated early keratocyte apoptosis after corneal epithelial scrape injury in human eyes scheduled for enucleation for malignancy. Two eyes had corneal epithelial scrape 1 h prior to the enucleation and another eye served as a control and had no corneal scrape prior to enucleation. One additional eye was enucleated, washed with balanced salt solution, and then had the corneal epithelium scraped 1 h prior to processing for analysis. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and confirmed by transmission electron microscopy (TEM). Anterior keratocyte apoptosis was detected in the three corneas that had epithelial scrape injury, but not in the control unwounded cornea. This study confirmed that keratocyte apoptosis is also an early response to corneal epithelial injury in humans and showed that tears are not essential for keratocyte apoptosis to occur in response to epithelial injury.     

Hydration behavior of porcine cornea crosslinked with riboflavin and ultraviolet A

PURPOSE: To examine the influence of a new crosslinking treatment on corneal swelling properties that correlate with the degree of crosslinking. SETTING: Department of Ophthalmology, Vivantes-Klinikum Neuk?lln, Berlin, Germany. METHODS: Twenty freshly enucleated porcine eyes were crosslinked by applying the photosensitizer riboflavin and ultraviolet-A (UVA) light (370 nm, 3 mW/cm2) for 30 minutes. After the eyes were treated and incubated for 24 hours in a moist chamber, 15 eyes were examined by biomicroscopy and optical coherence tomography (OCT); 5 eyes were examined by light microscopy. Five control eyes were included. RESULTS: Using light microscopy, a characteristic swelling pattern with 3 zones was identified in the crosslinked porcine cornea: an anterior intensely crosslinked zone of 242 microm, an intermediate partially crosslinked zone of 238 microm (hydration factor 2.2), and a noncrosslinked posterior zone of 1355 microm (hydration factor 2.7). A condensed OCT signal was demonstrated in the treated portion of the anterior stroma to a depth of 520 microm with a pronounced line at 540 microm, correlating with the combined anterior and intermediate layers after hydration in the histological analysis. In the nonhydrated state of the crosslinked cornea, the anterior zone was deduced to be 242 microm; the intermediate zone, 109 microm; and the posterior zone, 501 microm. Therefore, the maximum depth of the crosslinking effect was 351 microm. CONCLUSIONS: Collagen crosslinking using riboflavin and UVA led to a significant change in the swelling behavior of the anterior stroma, confirming prior findings that the crosslinking effect is strongest in the anterior half of the stroma. Crosslinked cornea did not induce a specific signal on OCT, and OCT is therefore not suited for clinical controls of the crosslinking effect.     

Staged intrastromal delivery of riboflavin with UVA cross-linking in advanced bullous keratopathy: laboratory investigation and first clinical case

PURPOSE: To evaluate the safety and efficacy of staged ultraviolet A (UVA) cross-linking following intrastromal 0.1% riboflavin administration in eyes with advanced corneal edema. METHODS: Ten eye bank corneas divided in two groups (n = 5) were placed on a pressurized artificial anterior chamber following Descemet's membrane stripping. Two consecutive corneal pockets (350- and 150-microm depth) were sequentially created using a femtosecond laser. Sequential intrastromal injections of 0.1% riboflavin (0.2 mL) followed by either UVA irradiation (15 mW/cm2) for 7 minutes or exposure to air were performed for each pocket. Corneal clarity and central thickness were measured before and after the two UVA cross-linking steps. The same steps were clinically applied in an 84-year-old woman with bullous keratopathy prior to corneal transplantation and followed for 6 months. RESULTS: The corneal clarity improved in the treated but not the control eyes. The mean central corneal thickness was significantly reduced by 256 microm (ultrasound, P = .0002) and 273 miccrom (Scheimpflug, P = .0004) in treated eyes, but only 100 microm (ultrasound, P = .048) and 107 microm (Scheimpflug, P = .075) in the control eyes. The clinical treatment of corneal edema showed improved clarity and reduced central corneal thickness from 675 to 550 microm (ultrasound) and 696 to 571 microm (Scheimpflug) at 1 month. Best spectacle-corrected visual acuity improved from finger counting to 20/80 at 1 week and beyond, postponing corneal transplantation for > 6 months. CONCLUSIONS: Staged UVA cross-linking (15 mW/cm2) with femtosecond laser facilitated intrastromal 0.1% riboflavin administration may be a safe (no corneal scarring) and effective (marked reduction of edema) temporizing alternative method for managing bullous keratopathy.     

Enzymatischer Nachweis der Tiefenabhängigkeit der Vernetzungswirkung von Riboflavin/UVA an der Hornhaut

PURPOSE: It has been shown that the treatment of keratoconus with riboflavin/ultraviolet A (UVA) causes significant stiffening of the cornea due to cross-linking. The aim of this study was to evaluate how deep the mechanical stabilization after collagen cross-linking could be shown biochemically. METHOD: Ten out of 20 enucleated porcine eyes were treated with riboflavin as a photosensitizer and UVA (370 nm, 3 mW/cm2, 30 min). The other 10 eyes served as controls. With a Microkeratom device, two flaps with a thickness of 200 microm and a diameter of 8 mm were cut off from each eye and put in a collagenase solution (NaCl plus collagenase A, 1:1). The surfaces of the flaps were measured digitally every day to characterize the dissolving behavior. RESULTS: The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher (p=0.001) compared to those that were cut secondarily and to the control flaps. But even the flaps from deeper layers showed a significant increase in resistance (p=0.02) compared with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220 h; of those cut secondarily, it was 80 h. Both untreated flaps had a half-life of 50 h. CONCLUSIONS: The biochemical study showed that the treatment of the cornea with riboflavin/UVA leads to significant collagen cross-linking not only in the anterior slice of 200 microm but also in the following 200 microm. This locally limited cross-linking effect may be explained by the absorption behavior for UVA of the riboflavin-treated cornea; 65% of UVA irradiation is absorbed in the first 200 microm and only 25-30% in the next 200 microm. Therefore, deeper-lying structures and especially the endothelium are not affected.     

Aquaporin-1-facilitated keratocyte migration in cell culture and in vivo corneal wound healing models

Aquaporin-1 (AQP1) water channels are expressed in corneal keratocytes, which become activated and migrate following corneal wounding. The purpose of this study was to investigate the role of AQP1 in keratocyte migration. Keratocyte primary cell cultures from wildtype and AQP1-null mice were compared, as well as keratocyte cultures from pig cornea in which AQP1 expression was modulated by RNAi knockdown and adenovirus-mediated overexpression. AQP1 expression was found in a plasma membrane pattern in corneal stromal and cultured keratocytes. Osmotic water permeability, as measured by calcein fluorescence quenching, was AQP1-dependent in cultured keratocytes, as was keratocyte migration following a scratch wound. Keratocyte migration in vivo was compared in wildtype and AQP1 knockout mice by histology and immunofluorescence of corneal sections at different times after partial-thickness corneal stromal debridement. AQP1 expression in keratocytes was increased by 24 h after corneal debridement. Wound healing and keratocyte appearance near the wound margin were significantly reduced in AQP1 knockout mice, and the number of neutrophils was increased. These results implicate AQP1 water permeability as a new determinant of keratocyte migration in cornea.     

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.