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A. Jayaprakash Patil, A. Sharma, S. Mansoor, M. F. Estrago-Franco, V. Raymond, M. C. Kenney, B. D. Kuppermann; Effects of Dexamethasone on Human Trabecular Meshwork Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4071.
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© ARVO (1962-2015); The Authors (2016-present)
To study the effects of dexamethasone sodium phosphate on human trabecular meshwork (HTM) cells in vitro.
HTM cells were treated with dexamethasone 2 mg/ml (20X clinical dose), 1 mg/ml (10X), 0.5 mg/ml (5X), 0.25 mg/ml (2.5X), 0.1 mg/ml (1X), or 0.05 mg/ml (0.5X) for 24 hours. Cell viability was measured by a trypan blue exclusion test. Caspase-3/7, -8, -9 and -12 activities were measured by Fluorochrome assays to assess apoptosis. Mitochondrial dehydrogenase activity was determined by WST assay to quantify mitochondrial damage.
Mean cell viability of HTM cells exposed to dexamethasone at higher doses 2, 1, 0.5 mg/ml was reduced: 11.9%±3.5 (P<0.001), 31.2%±3.2 (P0.05) and 93.8%±2.3 (P>0.05) compared to untreated HTM cells 97.0%±1.9. Caspase-3/7 activity of HTM cells exposed to dexamethasone 2, 1 or 0.5 mg/ml was 21068±2498 (P<0.001), 26994±3104 (P<0.001) and 20416±1150 (P<0.001) compared to untreated HTM cells 1148±803. Caspase-9 activity of HTM cells after exposure to dexamethasone 2, 1 or 0.5 mg/ml was 14188±1203 (P<0.001), 13256±1564 (P<0.001) and 15041±1584 (P<0.001) compared to untreated HTM cells 1748±524. Lower doses did not significantly increase caspase-3/7 or -9 activities. There was no caspase-8 or -12 activities at any dose. WST assay showed mitochondrial dehydrogenase activity of 14.3±0.7 (P<0.001), 9.6±0.3 (P<0.001) and 56.0±7.6 (P<0.001) at OD 450 nm at 2, 1 and 0.5 mg/ml dexamethasone compared to untreated HTM cells 186.1±15.0. Lower doses did not significantly reduce mitochondrial function. HTM cells that were pre-treated with 4 hours exposure to 3 µg/ml of 0.15% brimonidine as a neuroprotectant prior to dexamethasone exposure at 2, 1 or 0.5 mg/ml showed no significant upregulation in caspase-3/7 or -9, or mitochondrial dehydrogenase impairment, suggestive of a protective effect from brimonidine. However pre-treatment with brimonidine did not prevent decrease in cell viability at any doses tested.
Dexamethasone at 2.5X, 1X and 0.5X clinical dose did not cause significant reduction in cell viability, apoptotic upregulation, or mitochondrial dysfunction of HTM cells in vitro. At high doses (20X, 10X, 5X clinical dose) dexamethasone causes apoptosis via mitochondrial pathways which could be atleast partially prevented by brimonidine pre-treatment.
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