• Journal of Korean Ophthalmic Optics Society
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• v.4 no.1
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• pp.1-6
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• 1999

The corneal epithelium is constantly shed and apoptosis may play an important role in this turn-over. We sought to define that serum-free medium was able to induce apoptosis of corneal epithelial cells. SV-40 transfected human corneal epithelial(HCE) cells were grown to 70% confluency in culture. Serum-free medium was added to cells and the cells incubated for 1, 2, 3, or 6 days. Apoptosis of cells at different times was assessed by staining cells with Giemsa or Hoechst 33342 and measuring DNA fragmentation using the TUNEL assay. HCE cells exposed to serum-free medium demonstrated a high incidence of apoptosis, which increased over time to $50{\pm}4%$ after 3 days. They also stained positively with TUNEL assay. Serum-free medium caused time dependent apoptosis of HCE cells. Thus, serum-like nutrient might be important in corneal epithelial cell homeostasis.

• Journal of Life Science
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• v.32 no.9
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• pp.712-720
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• 2022

The purpose of this study was to investigate the efficacy of rat corneal-derived epithelial cells as an in vitro model to evaluate the harmfulness of the cornea caused by particulate matter 2.5 (PM_2.5). To establish an experimental model for the effect of PM_2.5 on corneal epithelial cells, it was confirmed that primary cultured cells isolated from rat eyes were corneal epithelial cells through pan-cytokeratin staining. Our results showed that PM_2.5 treatment reduced cell viability of primary rat corneal epithelial (RCE) cells, which was associated with the induction of apoptosis. PM_2.5 treatment also increased the generation of reactive oxygen species due to mitochondrial dysfunction. In addition, the production of nitric oxide and inflammatory cytokines was increased in PM_2.5-treated RCE cells. Furthermore, through heatmap analysis showing various expression profiling between PM_2.5-exposed and unexposed RCE cells, we proposed five genes, including BLNK, IL-1RA, Itga2b, ABCb1a and Ptgs2, as potential targets for clinical treatment of PM-related ocular diseases. These findings indicate that the primary RCE cell line is a useful in vitro model system for the study of PM_2.5-mediated pathological mechanisms and that PM2.5-induced oxidative and inflammatory responses are key factors in PM2.5-induced ocular surface disorders.

• Journal of Korean Ophthalmic Optics Society
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• v.5 no.1
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• pp.83-87
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• 2000

To investigate exhausted-medium-induced apoptosis in human corneal epithelial(HCE) cells, this study was performed DNA gel electrophoresis, M30 CytoDEATH staining and FAS-FAS ligand ELISA. SV-40 transfected cells were grown to confluency in culture for 7days. The supernatant was harvested and filtered with $0.22{\mu}m$ filter paper. Fresh HCE cells were exposed to the filtered exhausted medium for 1~2 days. Apoptotic cells were prepared for DNA extraction and run the agarose gel for DNA ladder pattern. M30 CytoDEATH was used a tool for easy and reliable determination of very early apoptosis in HCE cells. The control and exhausted medium were assayed for soluble FAS/FAS ligand protein by ELISA. HCE cells exposed to exhausted medium showed a typical DNA ladder pattern. Sporadic M30 CytoDEATH positive cells were detected among HCE cells exposed to exhausted medium. Soluble FAS/FAS ligand levels were not elevated in the exhausted medium compared to the fresh medium control. This study suggests that possible mechanism of exhausted medium induced apoptosis does not include the FAS-FAS ligand system.

• Journal of Korean Ophthalmic Optics Society
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• v.12 no.2
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• pp.79-86
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• 2007

The aim of the present study was to determine mechanisms of corneal epithelial cell apoptosis in vitro following exposure to anti-FAS and anti-FAS ligand antibody and during infection with mycoplasma sp.. A cultured human corneal epithelial(HCE) cell line was treated with anti-FAS antibody or anti-FAS ligand antibody for 2 and 4 days. The original cell line was found to be contaminated by mycoplasma removal agent(MRA) was used to eliminate the bacterium from the cell line. MRA($0.5{\mu}{\ell}$ tissue culture medium) was added to the cell line and incubated for 1 week. The cell line underwent multiple passages in media not contaminating MRA and cells were grown to 50-80% confluency on coverslips and stained using the Hoechst stain provided in the kit to ensure mycoplasma removal. Apoptosis experiments were performed before and after mycoplasma removal. The apoptotic index of anti-FAS and anti-FAS ligand antibody on mycoplasma contaminated cell line was studied using Hoechst 33342 staining and Annexin V-FITC and Propidium Iodide Staining. In conclusion, anti-FAS antibody induces apoptosis in HCE cells in a time and concentration-dependent mechanism. Cell lines contaminated with mycoplasma have an incresed susceptibility to FAS induced apoptosis.

• Journal of Korean Ophthalmic Optics Society
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• v.20 no.1
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• pp.51-60
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• 2015

Purpose: To determine the effect of marketed multipurpose contact lens solutions (MPSs) on human corneal epithelial cells (HCEpiCs) toxicity by using image analysis. Methods: HCEpiCs were exposed six MPSs (product A-F) at 0.05~50% for 2h, 12h, 24h, and 48h respectively. HCEpiCs were fixed and stained with Draq5 after exposure with MPSs, and the cell viability and apoptosis were evaluated by using confocal microscope and ImageXpress UltraTM. Results: Viabilities of HCEpiCs exposed to MPS A-F for a 2h were not affected, while reductions (52~75%) in cell viability over a 12h exposure of MPS B, MPS C, MPS D and MPS F, and significant more reductions (29~73%) over a 24h and 48h-exposure. Apoptosis of HCEpiC was not affect over a 12h MPS exposure, however was significantly increased (199~526%) over 24h and 48h MPS exposure. Among the products MPS D, E and F reduced viability of HCEpiCs and apoptosis increased more than MPS A (p<0.05). Conclusions: Lower concentration of MPSs have not an cytotoxic effect on HCEpiCs, however higher concentration of MPSs induce apoptosis and reduce viability of HCEpiCs. Therefore, it need to develop MPS having antimicrobial effectiveness with low cytotoxicity.

• Journal of Veterinary Clinics
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• v.18 no.4
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• pp.418-423
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• 2001

To investigate the modulation of nerve growth factor (NGF) during corneal epithelial wound healing and the effect of topical NGF on corneal epithelial wound healing in dogs. An axial epithelial defect was created in the right eye using 6mm axial corneal mechanical debridement while the left served as an unwounded control. The tears were collected from both eyes during 1 week and the corneal epithelium was processed for the measurement of NGF at day 0 and 7. The NGF content of tears and corneal epithelium was determined by enzyme-linked immunosorbent assay. In another experiment, the animals were divided into 3 groups. The right eyes in each group were treated every six hours with 200 ug/ml of recombinant human (rh) NGF, murine NGF, or 600 ug/ml of anti-NGF blocking antibody. The left eye of each animal was treated with bovine serum albumin (BSA) to serve as controls. Wound healing was analyzed using NIH image software. Tear NGF was markedly increased in the wounded eyes, relative to tears from control eyes during the early healing period. The NGF content of the corneal epithelium was elevated in the wounded eye (p=0.024). Time to wound closure and rate of epithelial migration were not significantly different between the NGF treated or the NGF antibody treated, and the control BSA treated eyes. Corneal epithelial wounding increased NGF content only on the wounded side during the early healing period. Neither topical recombinant human or murine NGF affected corneal epithelial wound healing in the normal dog.

• The Korean Journal of Vision Science
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• v.20 no.4
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• pp.531-541
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• 2018

Purpose : To evaluate the acute cytotoxic effect of polyhexamethylene biguanide (PHMB), epigallocatechin gallate (EGCG) and PHMB/EGCG mixture on the cultured human corneal epithelial cell (HCEpiC). Methods : HCEpiCs were cultured in the media of HCEpiC containing 0.00001~0.005% PHMB, 0.001~5% EGCG and 0.00005% PHMB/0.05% EGCG mixture respectively for 30, 60, 120 and 240 min. Cultured HCEpiCs were fixed and stained with Draq5 and cell viability and apoptosis were evaluated using confocal microscope and ImageXpress $Ultra^{TM}$. Results : Cultured HCEpiC did not show cytotoxic effect at below 0.00005% PHMB and below 0.05% EGCG concentration. In the media containing 0.00005% PHMB/0.05% EGCG, acute cytototoxic effect was not found, whereas damaged HCEpiCS were increased and survival cells were decreased in the media incubated for 240 min. Conclusion : The mixture of 0.00005% PHMB/0.05% EGCG showed non acute cytotoxic effect on the cultured HCEpiCs, however it is needed to investigate its chronic cytotoxic effect.

• Journal of Korean Ophthalmic Optics Society
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• v.10 no.1
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• pp.63-69
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• 2005

The effects of R products of B company and natural disinfectant naringin, well-known chemical disinfectant on Corneal endothelium and epithelium of rabbit were observed by scanning electron microscope. The main component of naringin is extract of grapefruit seed, which is one of the flavonoid widely recognized as natural antioxidants and used as preservative agent of food and cosmetics. The chemical disinfectants cytotoxicity in cell cultures were announced by MIT assay and LDH leakage assay. But there has been no study about that chemical disinfectants and natural disinfectants is inserted drops on rabbit's cornea directly. In this test R products B company and natural disinfectants has been dropped on the cornea of rabbits for one weeks and observed cytotoxiciy by light camera after rose bengal staining. Also, we analysed the damage of corneal epithelium and endothelium morphologically after enucleating the cornea by scanning electron microscope.

• Journal of Korean Ophthalmic Optics Society
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• v.1 no.2
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• pp.19-35
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• 1996

This study was performed on the mouse to estimate the effect of UV radiation on the cornea in UV clean bench by LM & SEM. The results are as follows In the control groups, The cornea tissue have relatively compact and each layer have well identify, and the thick of cornea have constant. In the increasing experimental time, the experimental result have very different. The early experimental groups results have not severely degeneration. But, some substrate layer have a swelling and some epithelial tissue have not normal shape. The middle experimental groups results have very swelling of the stroma, the vacoule of some region the condensation of the epithelium, and the irregular arrangement of the endothelium. The last experimental groups results have shirinking of cornea tissue, the swelling and vacoule of the end endothelium, the partially disruption of epithelium, the irregular thick of the corneal tissue.

• Journal of Veterinary Clinics
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• v.28 no.6
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• pp.562-565
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• 2011

A total of 29 eyes (25 dogs: one eye, 2 dogs: both eyes) with indolent corneal ulcer were treated with grid keratotomy from January 2008 to March 2010. The corneal lesions were reevaluated at 7-14 day intervals. The treatments had been repeated until fluorescein dye was not retained on the cornea and the epithelium did not appear to be loosely attached to the stromal layer. The healing rate of the corneal ulcers was 86.2%. The mean healing time ($mean{\pm}SD$) was $15.92{\pm}9.19$ days, ranged from 7 to 39 days. The lesions of remaining 4 eyes had deteriorated or not improved for more than 6 weeks. In those cases, $3^{rd}$ eyelid flap following grid keratotomy was applied. After 2 weeks, all of the eyes healed by the treatment. The results in this study suggest that grid keratotomy could be an excellent choice as an initial treatment for superficial corneal ulcers in dogs. In the cases of recurrence or to promote healing of the lesions, however, $3^{rd}$ eyelid flap following grid keratotomy is recommended.