Vitality of epithelial cells after alcohol exposure during laser-assisted subepithelial keratectomy flap preparation

Purpose: To evaluate the vitality of epithelial cells after various exposure times to 20% ethanol and epithelial flap preparation in laser-assisted subepithelial keratectomy (LASEK) using the trypan blue dye test. Setting: University Eye Clinic Regensburg, Regensburg, Germany, and the Rayne Institute, Department of Ophthalmology, St. Thomas' Hospital, London, United Kingdom. Methods: Five human cadaver eyes were exposed to 20% ethanol for 15, 30, 45, 60, and 120 seconds, respectively. After an epithelial flap (as in LASEK) was prepared, the flap was deliberately cut off. The flaps were soaked in a trypan blue 0.1% solution at 37°C. After 3 washes with phosphate-buffered saline (PBS), the specimens were reincubated for 30 minutes in culture medium containing 10% fetal calf serum at 37°C. After an additional wash with PBS, the cells were observed with a standard inverted light microscope. Results: After 15- and 30-second exposure to 20% ethanol, most epithelial cells were vital. This changed substantially after 45 seconds, when vital and dead cells were approximately equal. Longer exposure times (60 seconds and 120 seconds) showed predominantly dead epithelial cells. Conclusions: Exposure to 20% ethanol should be 20 to 30 seconds as the number of vital epithelial cells rapidly decreased after that. Vitality of the epithelial flap is probably a crucial factor in the dampened wound response in LASEK compared to that in photorefractive keratectomy.