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Qian Garrett, Sharon M. Shih, Berry J. Beard, Peter Simmons, Joseph Vehige, Mark D. Willcox; Hyperosmotic Stress-induced Damage Of Human Corneal Epithelial Cells Is Mediated Initially Through The Intrinsic Apoptotic Pathway. Invest. Ophthalmol. Vis. Sci. 2011;52(14):290.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effect of hyperosmotic stress on human corneal limbal epithelial (HCLE) cell survival and activation of apoptosis.
HCLE cells were exposed to hypertonic medium (450 or 600 mOsm) for various time periods. Cell viability was determined by measuring mitochondrial activity and cell number. Cells were stained with Annexin V, Propidium Iodide (PI), or Hoechst to assess early apoptotic, necrotic or total cells, respectively. Apoptosis was also detected by measuring the activity of caspase-8, -9 or -3/7.
The increase in osmotic strength and/or exposure time reduced cell mitochondrial activity and cell numbers, with 600 mOsm demonstrating deletrious effects from 4 h and 450 mOsm from 16 h. Hyperosmolality induced both apoptosis and necrosis. At 450mOsm cells exhibited a greater percentage of early apoptotic cells than necrotic cells (p >0.05). The apoptotic pathway was activated initially via caspase 9 and 3/7 after 8h exposure to hyperosmolar solution, followed by Annexin V (16h) and caspase 8 (24h). The initial effects of hyperosmolality at 450mOsm were through the intrinsic apoptotic pathway (caspase 9 and 3/7) followed by decreases in cell numbers (16 h) and finally changes to the cell mitochondrial activity (after 24 h exposure).
Hyperosmotic damage of HCLE cells, under constant stress conditions, is dependent on the strength of osmotic stress and exposure time. It involves both the intrinsic and extrinsic apoptotic pathways with the initial effects mediated through the intrinsic pathway followed by decreases in cell numbers and finally changes to the cell mitochondrial activity. Also, use of 450mOsm solutions appears to be a more appropriate challenge condition than 600mOsm solutions.
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