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M.C. Kenney, M. Chwa, S.R. Atilano; Increased Production of Reactive Oxygen Species by Keratoconus Fibroblasts in vitro . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4962.
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Purpose: To determine the reactive oxygen species (ROS) burden in normal and keratoconus (KC) fibroblasts and to evaluate the response to an exogenous hydrogen peroxide (H2O2) challenge. Methods:KC (n=3) and age–matched normal (n=3) corneal fibroblast cultures were incubated 1 hour in Dulbecco’s phosphate–buffered saline (Invitrogen, Carlsbad, CA) or serum free media with H2O2 (50 µM, 100µM, 200µM or 400µM). ROS production was assayed using the fluorescent dye 2’,7’–dichlorodihydrofluorescein diacetate (H2DCFDA reagent; Molecular Probes, Eugene, OR). Catalase activities were measured in triplicate by a fluorescent substrate assay (Amplex Red Catalase kit, Molecular Probes, OR). Cell viability was analyzed by the WST–1 colorimetry assay that measures mitochondrial dehydrogenase activity (Roche Diagnostics, Indianapolis, IN). The linear regressions for normal and KC cultures were calculated by GraphPad Prism Software. Results: The relative catalase activities in the untreated, 200µM H2O2 and 400µM H2O2 were 100%, 120% and 142%, respectively for normal corneal fibroblast cultures and 100%, 256% and 294%, respectively for KC cultures. The slope for catalase activity in the KC cultures increased significantly (p<0.01) while in the normal cultures it did not (p=0.16). Untreated KC fibroblasts had a 4–fold increase in ROS production compared to normal fibroblasts (p<0.02). The relative cell viabilities for untreated, 200µM H2O2 treated and 400µM H2O2 treated cultures were 100%, 118% and 71% respectively in normal and 100%, 101% and 71% in KC cultures. Conclusions: KC fibroblast cultures have an elevated ROS burden at baseline and an increased (hypersensitive) catalase response to exogenous H2O2 compared to normal fibroblast cultures. The higher levels of ROS found in KC cells may play a role in the oxidative damage found in KC corneas.
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