PURPOSE: To determine if the corneal epithelium prevents the collagen cross-linking effect. Using immunofluorescence microscopy after CXL, we indirectly analyzed the role of the epithelium as ultraviolet-A (UVA) shield as well as a barrier to riboflavin penetration. METHODS: Fifteen freshly enucleated porcine eyes were divided into 3 groups. The corneal epithelium was kept intact in all groups. Five eyes served as control (Group 1). On group 2, eyes received tetracaine anesthetic drops and topical 0.1% riboflavin solution (10 mg riboflavin-5-phosphate in 10 mL 20% dextran-T-500). On Group 3, riboflavin was injected into the anterior chamber to allow penetration of the drug through the endothelium. Groups 2 and 3 were exposed to UVA (365 nm, 3 mW/cm²) for 30 minutes. Ultra-thin sections (8 µm) of the corneas were stained with anti-collagen type I and DAPI (4,6-diamidino-2-fenilindole dihydrocloride) and analyzed with fluorescence microscopy. RESULTS: Corneas treated with UVA irradiation and intracameral injection of riboflavin (Group 3) showed greater pattern of collagen organization compared to groups 1 (Control) and 2 (riboflavin and tetracaine eye drops). A yellow stromal staining, which represents the riboflavin diffusion into the stroma, was only observed in eyes injected with riboflavin into the anterior chamber. CONCLUSION: Using immunofluorescence microscopy in porcine corneas, we demonstrated that the corneal epithelium reduces the effectiveness of CXL by preventing the penetration of the drug and not by limiting the UVA transmittance. An inadequate intrastromal concentration of riboflavin may impair CXL effect.
Cross-linking reagents; Riboflavin; Epithelium, corneal; Microscopy, immunofluorescence; Photosensitizing agents; Ultraviolet rays; Swine
