Confocal and electron microscopic studies of laser subepithelial keratomileusis (LASEK) in the white leghorn chick eye

Objectives: To evaluate the effect of 20% alcohol on the white leghorn chick cornea and to determine the confocal and electron microscopic findings of laser subepithelial keratomileusis surgery in the white leghorn chick corneal model. Method: Laser subepithelial keratomileusis surgery was performed on chick corneas and the morphologic changes were examined by transmission electron microscopy. Chick corneas were exposed to 20% alcohol for 30 and 45 seconds or 1 and 2 minutes (5 chicks per group) to evaluate the effect on the corneal epithelium. Photorefractive keratectomy using either mechanical or 20% alcohol-assisted debridement (5 chicks per group) was also performed. Keratocyte and epithelial cell deaths were analyzed 4 hours after surgery using terminal deoxynucleotidyl transfer-mediated biotin-dexoyuridine 5-triphosphate nick-end labeling (TUNEL) staining and transmission electron microscopy. Results: Exposure of the corneal epithelium to 20% alcohol for 30 seconds or longer allowed reproducible separation of epithelial flaps in white leghorn chick eyes. Transmission electron microscopy immediately after alcohol treatment showed that exposure to 20% alcohol for 30 seconds or less had minimal adverse effects on the corneal epithelium. The TUNEL staining of corneas obtained 4 hours after surgery revealed TUNEL-positive cells in the central superficial stroma and more abundantly in the peripheral superficial stroma around the epithelial flap margin and in the epithelial flap itself, particularly in the basal epithelial layer. Transmission electron microscopy showed similar evidence of apoptosis in the epithelium and anterior stroma. Conclusions: The white leghorn chick eye seems to be a reasonable model for laser subepithelial keratomileusis surgery. Treatment with 20% alcohol for 30 seconds results in reproducible epithelial flap creation in the chick cornea and in relatively low levels of stromal and epithelial cell death after surgery.