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


METHODS
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 degrees 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 degrees 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.
Gabler
Bernhard
B
University Eye Clinics Regensburg, Regensburg, Germany. gabler@eye-regensburg.de
Winkler von Mohrenfels
Christoph
C
Dreiss
Alexandra K
AK
Marshall
John
J
Lohmann
Chris P
CP
D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects*; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D020731:Keratomileusis, Laser In Situ; D013524:Surgical Flaps; D013997:Time Factors; D014343:Trypan Blue
D004396:Coloring Agents; D000431:Ethanol; D014343:Trypan Blue