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(Investigative Ophthalmology and Visual Science. 2002;43:2593-2602.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Human Corneal Epithelial Cell Viability and Morphology after Dilute Alcohol Exposure

Chun Chen Chen, Jin-Hong Chang, Jae Bum Lee, Joel Javier and Dimitri T. Azar

From the Massachusetts Eye and Ear Infirmary and the Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. 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-H2O 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.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Excimer laser technology provides the capacity for tissue ablation with a high degree of precision and minimal damage to adjacent structures.1 Applied before laser application to reshape the cornea, dilute alcohol is one of the most widely used methods to remove the epithelium during the procedures of photorefractive keratectomy (PRK) and laser subepithelial keratomileusis (LASEK).2 3 4 5 6 Other methods of corneal epithelial removal include mechanical debridement,7 laser transepithelial ablation,8 9 and a rotating brush.10

The corneal epithelium is a nonkeratinized, mucosal epithelial multilayer that covers the front surface of the cornea11 and provides an initial barrier to tears and the intraocular environment. Corneal epithelial integrity is essential to maintaining balanced epithelial–mesenchymal interactions, which play an active role in the chemokinetics of corneal wound healing,12 13 keratocyte apoptosis,14 15 myofibroblast transformation,16 and corneal neovascularization.17

For centuries, alcohols have been used for their antimicrobial properties. Ethanol induces apoptosis in a variety of tissues, including liver,18 buccal mucosa,19 salivary gland,20 gastric mucosa,21 brain,22 thymus,23 and spleen,24 but only limited information is available about the effect of alcohol on the corneal epithelium.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Keratinocyte serum-free medium (KSFM), Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12, L-glutamine, penicillin-streptomycin, and trypsin-EDTA solution were obtained from BioWhittaker (Walkersville, MD) and fetal bovine serum (FBS) from HyClone Laboratories (Logan, UT). Falcon tissue culture flasks, tissue culture plates, pipettes, and other routine plastics were obtained from Becton Dickinson Labware (Franklin Lakes, NJ); glass coverslips from VWR Scientific (San Francisco, CA); and neutral protease (Dispase II) from Roche Molecular Biochemicals (Indianapolis, IN). Monoclonal antibody AE1/AE3, AE5 were from ICN (San Francisco, CA) and monoclonal vimentin antibody from Sigma Chemical Co. (St. Louis, MO). Collagen VIII antibody was the gift of E. Helene Sage (University of Washington School of Medicine, Seattle, WA). Fluorescein isothiocyanate (FITC)-labeled donkey anti-mouse IgG antibody and mouse IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and absolute alcohol (EtOH) from Sigma-Aldrich. A live–dead viability kit was purchased from Molecular Probes (Eugene, OR) and an in situ cell death detection kit for TdT-mediated dUTP nick-end labeling (TUNEL) assay from Roche Diagnostics Corp. (Indianapolis, IN). All other chemicals, unless otherwise specified, were obtained from Sigma.

Tissue Preparation for Electron Microscopy and Cultures
The study was conducted in compliance with the provisions of the Declaration of Helsinki. Informed consent was obtained from all subjects undergoing PRK at the Massachusetts Eye and Ear Infirmary. The Institutional Review Board approved the study protocol. The method of creating an epithelial flap by dilute alcohol before refractive surgery has been described.2 In brief, the cornea was marked with overlapping 3-mm circles around the corneal periphery, simulating a floral pattern. A 7-mm optical zone marker was used to delineate the area centered on the pupil. Gentle pressure was applied on the cornea while the barrel of the optical zone marker was filled with two drops of 20% ethanol (dehydrated alcohol, 1-mL ampules; American Reagent Laboratories, Shirley, NY). After 20 seconds, the ethanol was absorbed with a dry sponge (Weckel or Merocel; Xomed, Jacksonville, FL) to prevent alcohol spillage onto the epithelium outside the marker barrel. One arm of the scissor was then inserted under the epithelium and traced around the delineated margin of the epithelium, leaving 2 to 3 clock hours of intact margin. The loosened epithelium was peeled as a single sheet using a sponge (Merocel; Xomed). Preoperative slit lamp examination of all corneas showed no evidence of corneal abnormalities.

Electron Microscopy
The epithelial sheets (specimens I–IV) were fixed in half-strength Karnofsky fixative (2% paraformaldehyde and 2.5% glutaraldehyde) in 0.2 M sodium cacodylate buffer (pH 7.4) overnight and postfixed in 1% osmium tetroxide in 0.2 M sodium cacodylate for 1.5 hours. After dehydration in graded alcohol, the eyes were embedded in epoxy resin (Epon-Araldite). Thick sections (1 µm) were stained with toluidine blue, and a suitable area containing basal layers was chosen. The blocks were trimmed accordingly, thin sectioned (80–90 Å), stained with 2% uranyl acetate-Reynold lead nitrate, and examined with a transmission electron microscope (model 410; Philips, Eindhoven, The Netherlands).

Tissue Culture of Harvested Human Corneal Epithelial Sheets
The epithelial sheets (specimens V–VII) were placed directly, epithelium side up in a tissue culture well. The culture well contained just enough medium to cover the bottom of the well, so that the tissue would receive nutrients through surface tension. The medium consisted of KSFM supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin, 5 ng/mL epidermal growth factor (EGF), 2.5 mg/mL bovine pituitary extract, and 0.03 mM calcium chloride. The epithelial sheet in culture medium was incubated at 37°C in a 5% CO2, 95% air incubator for up to 28 days, with the medium changed every 2 days. Migration and attachment were assessed every day by phase-contrast microscopy.

Human Corneal Epithelial Cell Culture
Primary human corneal epithelial cells were obtained from corneoscleral trims after trephination of corneal grafts from the New England Eye Bank and cultured as previously described.25 Briefly, after incubation at 37°C for 2 hours with 1.2 IU/mL neutral protease (Dispase II; Roche Molecular Biochemicals), the epithelium was stripped off with gentle scraping from peripheral areas (1–2 mm from the limbus) to the center into phosphate-buffered saline (PBS; 145 mM NaCl, 7.3 mM NaH2PO4, 2.7 mM NaH2PO4 [pH 7.2]; BioWhittaker). This preparation was centrifuged at 100g for 5 minutes, and then the cells were suspended in supplemented hormone epithelial medium (SHEM) that consisted of a 1:1 mixture of DMEM and Ham’s F12 with 10% FBS, 5 µg/mL insulin, 0.5 mg/mL cholera toxin, 10 ng/mL human recombinant epidermal growth factor, 0.5% dimethyl sulfoxide, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin.26 The cells were cultured at 37°C with 5% CO2 in 95% humidified air until 80% confluent (Fig. 2D) . The medium was changed every 2 days. The cells were routinely passaged by trypsinization of confluent, adherent cells with 0.05% trypsin-0.53 mM EDTA. In addition, the SV40-immortalized human corneal epithelial cell lines27 (the gift of Hiroshi Handa, Tokyo Institute of Technology, Tokyo, Japan) were cultured.



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  		Figure 2. Inverted phase-contrast micrographs of the tissue culture from one of the three freed epithelial sheets generated after 20% ethanol treatment for 25 seconds (A–C, specimen V) and of primary corneal epithelial cell cultures (D). Arrowheads: original sheet border; arrows: outer border of culture. (A) Epithelial outgrowth was observed at day 1, extending from the original sheet border to the outer border. (B) Cell attachment and epithelial outgrowth were persistent until day 15. (C) Higher magnification showing intact cellular contours and attachment of the cells to the culture plate at day 15. (D) More elongated cells observed in primary cultures. The epithelial cells became confluent at 1 month. Bar, 50 µm.

 
Immunocytochemistry
Cultured epithelial cells examined for immunocytochemistry, live–dead viability (calcein-acetoxymethyl ester [AM])/ethidium homodimer) assay, and TUNEL assay were grown on glass coverslips, rinsed in PBS, and fixed in 100% methanol for 15 minutes at room temperature before they were returned to fresh PBS. Fixed cells were incubated in blocking buffer of 1% BSA and 0.2% Triton X-100 in PBS for 30 minutes at room temperature. Cells were then incubated with the following dilutions of primary antibodies for 1 hour at room temperature: (1) AE1/AE3, which recognizes most acidic (type I) keratins and known basic (type II) keratins, was diluted 1:100 in 1% BSA-PBS; (2) AE5, specific for the basic 64-kDa (number 3) keratin, was diluted 1:50 in 1% BSA-PBS; (3) antibody to human vimentin was diluted 1:200 in 1% BSA-PBS; (4) collagen VIII antibody was diluted 1:100 in 1% BSA-PBS; and (5) {alpha}-smooth muscle actin ({alpha}-SMA) antibody was diluted 1:200 in 1% BSA-PBS. The secondary antibodies, conjugated to FITC, were diluted 1:200 in 1% BSA-PBS and incubated for 1 hour at room temperature. Coverslips were washed three times in PBS, mounted (Vectashield; Vector Laboratories, Burlingame, CA), and viewed with a fluorescence microscope (Eclipse E 800; Nikon, Tokyo, Japan). DNA-intercalating dye 4'-6-diamidino-2-phenylindole (DAPI) was used for nuclear labeling. For the negative control, preimmune mouse serum was substituted for the primary antibody.

Preparation of Test Drug
EtOH was diluted in distilled H2O or defined KSFM to yield EtOH-H2O, or EtOH-KSFM. 10%, 20%, 22%, 24%, 25%, 26%, 28%, 30%, 40%, 50%, 60%, and 70% EtOH-H2O and 20% EtOH-KSFM were used. Cells in the control group were treated with KSFM only.

Effect of Alcohol on Cell Survival
The cells were plated for morphologic evaluation on six-well plastic tissue culture wells. Cells were plated at a density of 8 x 104 cells/well. The cells were incubated for at least 24 hours with KSFM supplemented by bovine pituitary extract. When approximately 60% confluence was reached, the medium was withdrawn and the wells were washed twice with PBS. The cells were then incubated for a further 18 hours with defined KSFM supplemented only with penicillin-streptomycin. The wells were washed twice with 200 µL PBS and incubated with 200 µL of the test drug. The following exposure periods were used with each concentration of the test drug: 20, 25, 30, 35, 40, and 45 seconds.

Live–Dead Viability Assays
The live–dead assay permitted the simultaneous determination of viable and nonviable cells. Calcein-AM indicates intracellular esterase activity, and the ethidium homodimer indicates membrane integrity.28 After the defined exposure period, the test drug was removed and the wells were washed twice with PBS. For live–dead viability staining, each coverslip was incubated with 200 µL of 4 µM ethidium homodimer-0.5 µM calcein-AM for 30 minutes in a moist chamber at room temperature. Then the cells were immediately viewed with a fluorescence microscope at 485-nm excitation and 515-nm emission wavelength. The nonfluorescent calcein-AM is converted into green fluorescent polyanionic calcein by intracellular esterase, indicating active cell metabolism. Ethidium homodimer is excluded by viable cells but permeates damaged cell membranes, binds to nucleic acids, and results in red fluorescence.25 The number of green, red, and bicolored cells was counted per 10 fields at 400-fold magnification. The percentage of cells with exclusive green fluorescence (interpreted as viable cells) was calculated.

Detection of Apoptosis
To determine whether a high percentage of the surviving epithelial cells after alcohol exposure would undergo subsequent DNA breaks in situ we performed the TUNEL method,29 30 an approach based on specific binding of terminal deoxynucleotidyl transferase (TdT) to 3'-OH ends of DNA, thus ensuring synthesis of a polydeoxynucleotide polymer. Cultured human corneal epithelial cells were grown on the coverslip at a density of 8 x 104 cells/well. When approximately 60% confluence was reached, the cells were washed with PBS, as described earlier. They were then incubated for a further 18 hours with defined KSFM supplemented with penicillin-streptomycin and exposed to the test drug for 20- to 40-second intervals. Cells were washed twice with PBS and then incubated for 0, 8, 12, 24, or 72 hours in KSFM. They were fixed with a freshly prepared paraformaldehyde solution (4% in PBS, pH 7.4) for 1 hour at room temperature. After the cells were rinsed with PBS, blocking of endogenous peroxidase by 3% H2O2 in methanol was performed for 10 minutes at room temperature. The slides were rinsed again with PBS and incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 minutes on ice (4°C). The cells were rinsed twice with PBS, and 50 µL TUNEL reaction mixture was added to each coverslip, followed by incubation in a humidified chamber for 1 hour at 37°C. The cells were then rinsed three times with PBS and examined immediately under a fluorescence microscope at an excitation wavelength of 450 to 500 nm. In the positive control experiments, collective cells were incubated with DNase I (Roche Molecular Biochemicals) for 15 minutes at room temperature; in the negative control experiments, cells were incubated with fluorescein-tagged dNTP without TdT enzyme. The total cell number and the positively stained cell number were counted per 10 fields at 400-fold magnification.

Statistical Analysis
Data are presented as the mean ± SD. Data were entered into a computer spreadsheet (Excel 7.0, Microsoft, Seattle, WA) and imported into statistical software (Statview [1992–1998], SAS Institute, Cary, NC). The Friedman test and the Wilcoxon signed rank test for nonparametric data were used to determine the significance of difference between various exposure periods and concentrations of dilute alcohol. Comparisons of epithelial survival between different solutions of dilute alcohol were performed by the Kruskal-Wallis and Mann-Whitney tests. Probabilities were considered significant if P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Electron Microscopic Analysis of Epithelial Sheets Removed with 20% Alcohol
Normal corneal epithelium is nonkeratinizing, stratified, squamous epithelium, five to seven layers thick. Desmosomes are present along all cell membranes abutting other cell membranes. The cells of the basal layer are columnar, and hemidesmosomes are present along their basal plasma membrane adjacent to the basement membrane. Beneath the epithelium is an unilamellar basement membrane that overlies a thick collagen stroma through which anchoring fibers extend from the lamina densa.31 32 Figure 1 shows the effect of 20% alcohol (EtOH-H2O) treatment on the human corneal epithelium. All four freed epithelial sheets displayed normal stratification. The basal epithelial surface of isolated epithelial sheets showed blebbing of the basal cell membrane and autophagic vacuoles within the cytoplasm of the epithelial basal cells of the freed sheet in specimens II and III. Various basement membrane complex configurations were observed beneath the epithelial basal cells: unilamellar basement membrane with focal disruptions (specimen I), irregular and discontinuous basement membrane with intact hemidesmosome (specimen II), disruptions of basal cell membranes with absent basement membrane (specimen III), and duplicated basement membrane containing dense bundles of anchoring fibrils (specimen IV).



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  		Figure 1. Transmission electron micrographs of freed epithelial sheets after 20% alcohol application for 25 seconds (specimen I: A, B; II: C, D; III: E; and IV: F). Varied separation of the basement membrane zone was seen. (A) Specimen I showing basal epithelial cells with intact basement membrane complex (arrows) with intact electron-dense hemidesmosome attachment (arrowheads). (B) Higher magnification exhibited a localized area of irregular basement membrane zone (arrow) and basal cell membrane disruption (arrowheads). (C) The basal epithelial layer in specimen II showed autophagic vacuoles (arrows). (D) Discontinuous basement membrane zone beneath the basal epithelial cells (arrows), evident at higher magnification, was associated with decreased number of electron-dense hemidesmosomes (arrowheads). (E) The basal cell membranes and the basement membrane (arrows) were disrupted in specimen III. Formation of autophagic vacuoles (arrowheads) was extensive in the cytoplasm. (F) Specimen IV: the freed epithelial sheet retained a duplicated basement membrane zone. Pockets of cross-banded anchoring fibrils were arranged in a network between the layers of basal lamina (arrows). Electron-dense hemidesmosomes (arrowheads) were present along the basal cell membrane. Magnifications: (A) x2,500; (B, F) x17,750; (C) x6,000; (D) x30,000; (E) x1,650. Bar, 1 µm.

 
Tissue Cultures of Epithelial Sheets
We obtained three human epithelial sheets after 20% alcohol application for approximately 25 seconds (specimens V–VII). One specimen showed outgrowth of epithelial cells from the epithelial sheet from day 1 and areas of cellular attachment and outgrowth at days 1 to 15 (specimen V; Fig. 2 ). The other two specimens (VI and VII) detached from the tissue culture well at days 7 and 12, respectively. There was no evidence of epithelial outgrowth from these epithelial sheets.

Effect of Dilute Alcohol on Immortalized Human Corneal Epithelial Cells
To confirm the absence of other cell types in the immortalized corneal epithelial cells used in our experiments, we performed immunocytochemical localization of the following markers: AE1/AE3, AE5, vimentin, collagen VIII, and {alpha}-SMA. The cytoplasm of human corneal epithelial cells stained positively for AE1/AE3 and AE5, but not for vimentin, collagen VIII, or {alpha}- SMA, confirming the epithelial characteristics of the cells (Figs. 3A 3B 3C 3D 3E) . Live–dead viability assays showed that all cells were calcein-AM positive and ethidium homodimer negative (Fig. 3G) . As a control, we performed similar studies on the fifth passage of primary cultured corneal epithelial cells and found all cells to be calcein-AM negative (Fig. 3H) and ethidium homodimer positive (Fig. 3I) .



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  		Figure 3. Immunohistochemicalcharacterization (A–F) and live–dead viability assays (G–I) in corneal epithelial cell cultures. After primary antibody incubation (A–F): FITC-conjugated secondary antibodies were used and counterstained with DAPI. The corneal epithelial cells in the culture expressed (A) AE1/AE3 and (B) AE5 antibodies, but not (C) vimentin, (D) collagen VIII, and (E) {alpha}-SMA antibodies. There was no staining in the control group, in which preimmune mouse serum was substituted for primary antibody (F). Cellular survival of immortalized (G) and primary human corneal epithelial cell cultures (H) at passage 5 were demonstrated with a live–dead cytotoxicity assay. The immortalized cells showed homogeneous viability (G, calcein-AM positive-green fluorescence and ethidium homodimer negative), whereas the primary cultured cells were nonviable (H, calcein-AM negative; I, ethidium homodimer–binding red fluorescence). Bar, 50 µm.

 
Effect of Various Concentrations of EtOH-H2O on Immortalized Human Corneal Epithelial Cell Survival.
Cellular viability was defined as the maintenance of intracellular esterase activity and cell membrane permeability. Figure 4 shows corneal epithelial cell survival after 20 seconds of incubation with different concentrations of EtOH-H2O (10%, 20%, 22%, 24%, 25%, 26%, 28%, 30%, 40%, 50%, 60%, and 70%). Alteration of cell membrane permeability was synchronized with loss of esterase activity. The percentage of cells with esterase activity (cellular survival) at the several concentrations of EtOH-H2O were as follows: 10%, 96.12% ± 3.86%; 20%, 94.47% ± 1.68%; 22%, 96.66% ± 3.50%; 24%, 92.60% ± 3.56%; 25%, 33.86% ± 19.15%; 26%, 1.87% ± 1.60%; and at the concentrations from 28% to 70%, 0% showed esterase activity. Cellular survival rate was significantly different between 24% and 25% (P = 0.005) and between 25% and 26% (P = 0.005) concentrations of EtOH-H2O. The control group (KSFM) showed 100% survival of epithelia. There was no significant difference in cellular survival between concentrations of 10%, 20%, 22%, and 24% EtOH-H2O. There were no viable cells when EtOH-H2O concentrations exceeded 28%. Epithelial survival data formed an S curve, and 25% was the inflection point of the curve.



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  		Figure 4. Fluorescein viability stain with calcein AM/ethidium homodimer of the cells after (A) 10%, (B) 20%, (C) 24%, (D) 25%, (E) 26%, and (F) 40% EtOH-H2O treatment for 20 seconds. Metabolically active cells converted nonfluorescent calcein-AM into green fluorescent polyanionic calcein and excluded ethidium homodimer (A). Damaged cell membranes allowed permeation of ethidium homodimer and its binding to nucleic acids, resulting in red fluorescence (F). Bar, 50 µm. (G) Cellular survival after different concentrations of alcohol treatment for 20 seconds. The percentage of viable cells (with exclusively green fluorescence) was calculated by counting cells per 10 fields at x400 magnification.

 
Effect of Various Periods of Exposure to EtOH-H2O on Survival of Immortalized Human Corneal Epithelial Cells.
In the control group (100% KSFM), there was no change of cellular survival (100%) in the different exposure periods. However, a significant difference was observed between all exposure periods and the control in epithelial cells treated with 20% EtOH-H2O (P < 0.05). The percentage of cellular survival at exposure periods of 30 seconds or less were as follows: 20 second, 59.10% ± 11.40%; 25 seconds, 53.96% ± 22.96%; and 30 seconds, 69.69% ± 16.34%. This was significantly higher than the percentage of cellular survival at 35 (2.14% ± 2.29%), 40 (10.45% ± 7.11%), and 45 (11.10% ± 15.73%) seconds in EtOH-H2O (P = 0.01; Fig. 5 ).



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  		Figure 5. Fluorescein viability stain with calcein-AM/ethidium homodimer of cells exposed to 20% EtOH-H2O for (A) 20, (B) 25, (C) 30, (D) 35, (E) 40, or (F) 45 seconds. Calcein-positive green fluorescence indicated metabolically active cells, and ethidium homodimer–positive red fluorescence indicated damage to the cell membranes and binding to nucleic acids. Bar, 50 µm. (G) Cellular survival in different exposure periods. The percentage of viable cells was calculated from the number of green, red, and bicolored cells counted per 10 fields at x400 magnification. Control group was treated with 100% KSFM (0% ethanol). Bar, 50 µm.

 
Effect of Various Periods of Exposure to EtOH-KSFM on Survival of Immortalized Human Corneal Epithelial Cells.
In cells treated with 20% EtOH-KSFM, there was no significant difference in any defined exposure periods (Fig. 6) . The percentage of cellular survival was as follows: 20 seconds, 96.27% ± 2.71%; 25 seconds, 92.35% ± 12.36%; 30 seconds, 95.35% ± 4.95%; 35 seconds, 98.21% ± 1.77%; 40 seconds, 82.00% ± 21.46%; and 45 seconds, 85.35% ± 8.72%. There were no statistically significant differences between cellular survival at any of these exposure periods and those of the control group (100% KSFM; 0% EtOH). Significant differences in cellular survival were observed between 20% EtOH-KSFM and 20% EtOH-H2O (P = 0.0002) for all exposure periods.



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  		Figure 6. Fluorescein viability stain with calcein AM/ethidium homodimer of cells exposed to 20% EtOH-KSFM for (A) 20, (B) 25, (C) 30, (D) 35, (E) 40, or (F) 45 seconds. Bar, 50 µm.

 
TUNEL Staining
To determine the subsequent programmed cellular death after exposure of 20% EtOH-H2O or EtOH-KSFM for 20 and 40 seconds, we performed a TUNEL assay immediately or after 8, 12, 24, or 72 hours of incubation (Fig. 7) . No nuclear labeling was evident after 72 hours of incubation and in the control at 0, 8, 12, 24, and 72 hours. In immortalized human corneal epithelial cells treated with 20% EtOH-H2O for 20 (Figs. 7A 7B 7C) and 40 (Figs. 7D 7E 7F) seconds, a peak of apoptosis occurred after 24 (58.05% ± 33.10%) and 8 (94.12% ± 1.21%) hours of incubation, respectively (Fig. 7J) . No significant increase was detected in cells treated with 20% or 40% EtOH-KSFM after 8, 12, 24, 72 hours of incubation in varying exposure periods (Fig. 7J) . In the negative control, no TUNEL-positive nuclei could be detected, whereas in the positive control (DNase I–treated) all nuclei were TUNEL positive.



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  		Figure 7. TUNEL labeling of cultured corneal epithelial cells exposed to 20% EtOH-H2O for 20 seconds (A–C) and 40 seconds (D–F) and to EtOH-KSFM for 40 seconds (G–I). TUNEL positivity was evaluated after 8 (A, D, G), 12 (B, E, H), and 24 (C, F, I) hours of incubation. Maximum TUNEL positivity after 20 seconds of EtOH-H2O exposure was detected at 24 hours of incubation (C; 58.05% ± 33.10%) and after 40 seconds of EtOH-H2O exposure at 8 hours of incubation (D; 94.12% ± 1.21%). Substantially lower TUNEL positivity was seen after 8, 12, and 24 hours of incubation with EtOH-KSFM for 40 seconds (G, 0.65% ± 0.02%; H, 7.11% ± 1.49%; I, 4.52% ± 1.05%). Bar, 50 µm. (J) TUNEL positivity after 8, 12, and 24 hours of incubation of 20% EtOH-H2O for 20 and 40 seconds and 20% EtOH-KSFM for 20 and 40 seconds compared with controls. Control groups were treated with 100% KSFM for 20 seconds. Bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Dilute alcohol is frequently used for removal of the epithelium during PRK and LASEK.2 3 4 5 6 Our data show that the conventional concentrations and duration of alcohol treatment (20%, 25 seconds) result in varying morphologic changes in the basement membrane zone by electron microscopy and varying viability in standard tissue culture conditions. Our electron microscopic findings show morphologic differences in the plane of cleavage among several patients, in whom the same technique was used for creating the epithelial flap. This may be due to variability between individuals in relation to the adhesion of the epithelium to the basal membrane or to the variability of the effect of alcohol on adhesion of epithelial cells.

Our in vitro studies suggest a dose- and time-dependent effect of alcohol on epithelial cells. The 25% concentration of EtOH-H2O was the inflection point of epithelial survival. Significant increase in cellular death occurred after 35 seconds of EtOH-H2O exposure. Forty seconds of exposure further increased apoptosis after 8 hours of incubation. These findings are consistent with the clinical observations of varied epithelial attachment to the stromal bed after LASEK surgery.

The first stage of wound healing of the cornea after PRK and LASEK is epithelial migration, followed by epithelial hyperplasia and subsequent stromal regeneration. By cytokine induction, the epithelium can activate the process of keratocyte apoptosis and myofibroblast transformation. Therefore, it is important to preserve epithelial viability and integrity during the refractive surgery to achieve uneventful wound healing and optimal visual recovery. If the in vitro monolayered results apply to in vivo multilayered epithelium, the critical alcohol concentration and duration of exposure may frequently be exceeded during surgery. This is not always unintentional. Increased duration of alcohol application can be used intentionally to weaken the epithelial adhesion, which contributes to the variation in alcohol-induced toxicity that is observed in vivo.

The viability of the epithelial cells in vivo after application of dilute alcohol is ambiguous. The assessment of cell viability cannot be achieved merely by morphology. Bias of interpretation can occur because of variations among individual patients and surgical techniques. In the present study, we attempted to understand the effect of dilute alcohol on corneal epithelial cells by means of an in vitro model. Cultured, homogeneous epithelial cells were used in qualitative and quantitative assays of live, dead, and apoptotic cells.

The results of our study emphasize the importance of combining short-term toxicity studies with long-term apoptosis studies. For instance, our results with a 20% EtOH-H2O solution for 40 seconds show that 10% of the epithelial cells were viable (representing a 90% decrease in cell survival). Furthermore, epithelial cells treated in the same manner and examined 8 hours later showed a 94% apoptotic cell rate, indicating that the final survival was less than 1%.

Cultured epithelial cells of animal33 34 and human35 corneal and conjunctival36 origins have been used to reduce the number of animals used in the preclinical evaluation of ocular toxicity of various substrates. Monolayered cultures of corneal epithelial cells have been shown to be equally sensitive as three-dimensional corneal constructs for evaluation of acute toxicity.25 37 These tests should be performed under standardized conditions, and undefined supplements in the culture medium should be avoided. Limited human corneal donor material for experimental purposes and human corneal epithelial cells tend to become senescent after passage 5, which can significantly increase variability and introduce bias.35

Fluorescent viability staining provides information beyond morphology, because it visualizes functional alterations in cell membrane integrity and cell metabolism. It has previously been used to evaluate the viability of corneal donor tissue,38 the in situ organization of keratocytes,39 apoptosis in normal surface corneal epithelial cells,28 Fas /Fas ligand–stimulated keratocytes,14 toxicity of natural tear substitutes,25 and surfactant-induced cell death.40 In this study, cell survival was reduced when dilute alcohol concentration exceeded 25% or exposure period exceeded 35 seconds. Accompanied by characteristic ultrastructural change, apoptosis is a controlled form of cell death that occurs during tissue development, homeostasis, response to infection, and wound healing.41 Apoptosis in the corneal epithelium has been detected after shedding,28 42 mechanical injuries,15 infection, UV exposure, and steroid treatment.26 It has also been proposed as an initiating factor in wound healing.14 Apoptosis can be identified morphologically by the presence of apoptotic bodies (blebs) in the cell membrane or biochemically by the characteristic manner in which the nucleus breaks into nucleosomes.29 43 44

In the present study, we detected TUNEL-positive cells after 8 and 24 hours of incubation after 40 and 20 seconds’ exposure to 20% alcohol. Most of the nuclei in TUNEL-positive cells were round without cellular condensation. The TUNEL-positive cells with condensed nuclei showed the characteristic morphology of apoptosis. The condensed nuclei were shown to be apoptotic bodies by light or electron microscopy. In the liver, TUNEL-positive nuclei without condensation are considered to be the early stage of apoptosis in cells.45 They have also been reported in tissues with a high turnover rate, such as epithelium of the small intestine29 and the Henle layer of hair follicles,46 and in shed corneal epithelial cells.42

The relevance of this in vitro study may be challenged because culture conditions cannot substitute for the complex physical and molecular interactions of tear film and ocular surface in vivo.25 Furthermore, this approach cannot tackle the important question of endothelial toxicity, especially the facts that the epithelium is capable of regeneration, but the endothelium is not. Although cell culture models cannot provide neural pathways that are important in epithelial integrity in the long term, the model used in the current study may allow extrapolation to the acute toxicity of alcohol treatment substitutes on human corneal epithelium in vivo. Alcohol diluted in KSFM had no effect on cellular survival and apoptosis. At this time, it is not clear whether modification of the preparation of dilute alcohol, used during LASEK and PRK, would allow for better cell survival and adhesion in vivo.


   Acknowledgements
 
The authors thank Pat Pearson and Marie A. Shatos for assistance with electron microscopy and cell cultures.


   Footnotes
 
Supported by National Eye Institute Grant EY10101, the New England Corneal Transplant Research Fund, the Massachusetts Lions Eye Research Fund, a Research to Prevent Blindness Lew R. Wasserman Merit Award (DTA), and the Municipal Jen-Ai Hospital, National Yang-Ming University, Taipei, Taiwan (CCC).

Submitted for publication February 1, 2002; revised March 8, 2002; accepted March 22, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Dimitri T. Azar, Director, Corneal, External Disease, and Refractive Surgery Services, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114; dazar{at}meei.harvard.edu.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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