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S. Esquenazi, J. He, H. E. P. Bazan; Immunofluoresence of Rabbit Corneas Following Collagen Cross-linking Treatment with Riboflavin and Ultraviolet A. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5497.
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To assess ultrastructural modifications in keratocytes, collagen composition and inflammatory cell response in rabbit corneas after riboflavin and ultraviolet A (UVA) exposure using immunoflurescence microscopy.
Fifteen adult New Zealand albino rabbits weighing 2.0 to 3.0 kg were used in this study. Two rabbits served as controls. The animals had their epithelia removed and were cross-linked with riboflavin 0.1% solution (10mgs riboflavin-5-phosphate in 10ml 20% dextran-T-500) applied every 3 minutes for 30 minutes, and exposed to UVA (360 nm, 3 mW/cm²) for 30 minutes. Three rabbits were humanely euthanized at 1, 3 and 11 days and at 3 and 5 weeks after the procedure. Immunohistochemestry studies of thin sections of each cornea were performed using TUNEL staining, anti-collagen I, III and V, Alpha-smooth muscle actin (-SMA), myeloperoxidase (MPO) antibodies and DAPI counterstained.
Cross-linked corneas showed early stromal edema. By 5 weeks, complete resolution of the edema and a pronounced highly organized anterior 200 µm fluorescent zone was observed. TUNEL and DAPI staining showed keratocyte death by both, necrosis and apoptosis between day 1 and 3 after cross-linking. At day 1 the limbal area close to the cross linking showed some inflammatory cells as well as -SMA positive cells, indicative of the presence of myofibroblasts. By day 3 some myofibroblasts had been migrated to the area beneath the cross linking area, and positive staining for collagen III and V was also detected. Between 3 days and 5 weeks there was an increase in -SMA staining in the area surrounding the cross linking. The area of cross linking remainsed acellular up to 5 weeks.
Collagen cross-linking results in early edema, keratocyte apoptosis and necrosis, appearance of inflammatory cells in the limbal area, and transformation of surrounding keratocytes in myofibroblasts. Compaction of anterior stroma fibers and displacement of cell nuclei, including inflammatory cells, may have implications in the cross-linked corneal immune response.
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