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/cm(2)) 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 mu m) 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 mu m. Using DAPI staining, an anterior and concentrated displacement of cell nuclei due to collagen compaction was observed after cross-linking (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, Treatment of the cornea with riboflavin and other groups. 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.