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U. V. Jurkunas, T. Funaki, M. Dastjerdi, R. Dana; Impact of Oxidative Stress on Corneal Endothelial Cell Pleomorpism, Polymegethism, and Survival. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3891.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelium (CE) is a monolayer of cells that exhibits characteristic age and stress-related changes, denoted as pleomorphism, a variation in cell shape, and polymegethism, a variation in cell size. CE is especially prone to oxidative damage due to life-long exposure to light, high oxygen demand, and non-proliferative nature of the cells. The purpose of this study is to investigate the impact of oxidative stress on endothelial cell viability, monolayer integrity, and apoptosis.
Corneal buttons from BALB/c and C57BL/6 mice were stored in Optisol-GS for 1 hour and then subjected to various concentrations of H2O2 (0-100 µM) for variable time periods (30 minutes to 12 hours) at 37oC. Structural integrity of CE cells was assessed by staining with ZO-1 and analyzing CE cell density, polymegethism and pleomorphism by using Confoscan4 software (NIDEK CO., LTD, U.S.A). The effects of H2O2 on CE cell apoptosis were detected by using propidium iodide (PI), Annexin-V, or TUNEL assays. Corneal buttons were pretreated with ascorbic acid (100ug/ml) prior to H2O2 administration, and cell viability and integrity was assessed with PI and ZO-1 staining.
The number of PI-positive cells increased with increasing H2O2 concentration. Cell density, as assessed by ZO-1 staining, did not differ between 0 and 100 µM treatments for 30 minutes. However, the level of CE cell polymegethism, as measured by coefficient of variation, had a significant increase after exposure to 50 µM H2O2 (p=0.0001) and 100 µM H2O2 (p= 1.4E-06); and CE cell pleomorphism, as measured by percent of hexagonal cells, was significantly decreased after 100 µM of H2O2 exposure (p=0.006). Low-dose (1 µM) H2O2 induced Annexin-V positivity after 2 hours of exposure and TUNEL staining after 6 hours of exposure. CE cell staining with ZO-1 became attenuated after exposure to low-dose H2O2 for 4 hours and completely absent after 6-hour exposure, while the pre-treatment with ascorbic acid at 500ug/ml rescued the cell border integrity.
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