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AbstractDO.05.08 Triamcinolone Acetonide Protects RPE Tight Junctions from Oxidative Stress Miura Y., Roider J.
Institut für Ophthalmologie, Universitätsklinikum Schleswig-Holstein, Campus Kiel Objective: Oxidative stress is known to disrupt the integrity of RPE tight junctions. The goal of this study is to evaluate the effect of triamcinolone acetonide (TA) on the junctional integrity of retinal pigment epithelium (RPE) under oxidative stress and to identify the underlying mechanisms.
Methods: Porcine RPE cells were cultured on 6-well membrane inserts until 4 weeks after reaching confluence. Cells were incubated with TA (10-5M) for 30 minutes. FITC-dextran was added to the upper chamber and then cells were challenged with 2mM Hydrogen Peroxide (H2O2). The medium from the lower chamber was collected after 5 hours and the fluorescence intensity was measured using a fluorescence spectrofluorophotometer. The transepithelial flux of FITC-dextran was measured until the 21st day. For immunohistochemistry, RPE cells were cultured on collagen-coated cover glass, and the cells were incubated with H2O2, with or without the 30-minute preincubation of TA (10-5M). The immunolocalization of occludin and actin was examined with fluorescence microscope. Reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio was determined by a colorimetric assay kit.
Results: Oxidative stress by 2mM H2O2 led to a 3 times increase in transepithelial flux of FITC-dextran. TA inhibited this increase significantly and preserved the lower level flux through the whole experimental period. This permeability change by H2O2 was reversible and recovered to normal levels within 3 weeks. The administration of TA also prevented H2O2-induced disruption of occludin junctional assembly. Glutathione assay demonstrated that intracellular GSH/GSSG ratio decreased significantly by H2O2, while TA preserved this ratio by up-regulating GSH synthesis.
Conclusions: TA has a protective effect against oxidative stress-induced disruption of RPE tight junction by preserving cellular redox state.
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