Human corneal epithelial cell viability and morphology after dilute alcohol exposure.
OBJECTIVE
To determine the effect of dilute alcohol on human corneal epithelial cellular morphology and viability. Dilute alcohol is used for epithelial removal during photorefractive keratectomy (PRK) and laser subepithelial keratomileusis (LASEK).


METHODS
Corneal epithelial sheets harvested from human eyes after alcohol application during PRK were examined by light and electron microscopy (specimens I-IV). In addition, tissue cultures of human epithelial sheets were monitored for epithelial migration and attachment (specimens V-VII). To determine the effect of dilute alcohol on epithelial cell viability, simian virus (SV)40-immortalized human corneal epithelial cells were exposed to dilute alcohol in distilled water (EtOH-H2O) or to keratinocyte serum-free medium (EtOH-KSFM) for incubation periods of 20 to 45 seconds and concentrations of 10% to 70%. Cell membrane permeability and intracellular esterase activity were analyzed by calcein-acetoxymethyl ester (AM)/ethidium homodimer assay. TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to detect apoptotic cells at 0, 8,12, 24, and 72 hours.


RESULTS
Electron microscopy showed varying degrees of basement membrane alterations after alcohol application, including disruptions, discontinuities, irregularities, and duplication (specimens I-IV). Cellular destruction and vacuolization of basal epithelial cells associated with absent basement membrane were also observed (specimen III). One of three cultured epithelial sheets showed attachment and outgrowth in the tissue culture until day 15 (specimen V). Twenty-second exposure of cultured immortalized human cells to various concentrations of EtOH-H2O showed significant reduction of viable cells when EtOH-H2O concentration exceeded 25% (P = 0.005). Increasing the duration of application of 20% EtOH-H2O beyond 30 seconds resulted in a significant reduction in viable cells (69.69% +/- 16.34% at 30 seconds compared with 2.14% +/- 2.29%, 10.45% +/- 7.11%, and 11.09% +/- 15.73% at 35, 40, and 45 seconds, respectively; P = 0.01). TUNEL assay of cultured human corneal epithelial cells exposed to 20% EtOH-H(2)O for 20 and 40 seconds showed maximal labeling at 24 hours (58.05% +/- 33.10%) and 8 hours (94.12% +/- 1.21%), respectively. Exposure to 20% EtOH-KSFM for 20 and 40 seconds resulted in substantially lower TUNEL positivity (3.51% +/- 0.20% at 24 hours and 7.11% +/- 0.49% at 8 hours).


CONCLUSIONS
The viability and electron microscopic findings in the basement membrane zone showed significant variation after treatment of the epithelium in vivo with dilute alcohol. The application of dilute alcohol on the monolayer of cultured corneal epithelial cells resulted in increasing cell death in a dose- and time-dependent manner.
Chen
Chun Chen
CC
Massachusetts Eye and Ear Infirmary and the Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Chang
Jin-Hong
JH
Lee
Jae Bum
JB
Javier
Joel
J
Azar
Dimitri T
DT
D000199:Actins / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D001485:Basement Membrane / Q000187:drug effects / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D024102:Collagen Type VIII / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D005452:Fluoresceins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D007633:Keratins / Q000378:metabolism; D013539:Simian virus 40 / Q000502:physiology; D013997:Time Factors; D014746:Vimentin / Q000378:metabolism
D000199:Actins; D024102:Collagen Type VIII; D005452:Fluoresceins; D005456:Fluorescent Dyes; D014746:Vimentin; C085925:calcein AM; D000431:Ethanol; D007633:Keratins; D004950:Esterases