Corneal Epithelial Cell Survival and Electron Microscopic Changes after Ethanol Treatment

ABSTRACT

PURPOSE: In LASEK models, the viability of corneal epithelial cells after ethanol treatment was investigated in addition to morphological analysis. METHODS: Cell viability was assayed using a confocal laser scanning microscopy(CLSM) and MTT assay for in vivo and cultured rabbit corneal epithelial cells after treatment with various concentrations of ethanol. Ethanol treated human corneas were compared with control in terms of structural changes. RESULTS: CLSM assay showed that cells were viable at the ratio of 78.5, 75.0, 57.3, 43.3, 23.9, 4.3% for 0, 10, 20, 30 40, 60 % ethanol groups, respectively. The relative survival rates in comparison to 0% ethanol-treated (control) corneas were 95.5(10%), 72.9(20%), 55.2(30%), 30.4(40%), 5.5(60%). On MTT assay after 10, 20, 25, 30, 40, 60% ethanol treatment, cultured epithelial cells were still alive at the percentage of 92.7, 92.2, 36.6, 30.7, 5.4, 5.1% (20 sec), 90.8, 57.1, 29.0. 28.6, 5.0, 4.7% (40 sec) at 0 hr with decreasing cell survival over time in 20% ethanol group. TEM showed multiple vacuole-like loosenings along the intercellular junction of superficial squamous and wing cells. The loss and break-up of hemidesmosome and basement membrane were also demonstrated in conjunction with the loosening of sub-basal interface. CONCLUSIONS: After 20% ethanol exposure for 40 seconds, 72.9% and 57.1% of in vivo and cultured rabbit corneal epithelial cells were in viable state with decreasing cell survival over increasing ethanol concentration and time. The loss and break-up of hemidesmosome and basement membrane, resulting in loosening of sub-basal interface, might be the mechanism for the detachment of LASEK flap en bloc.