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Anne Katrine Toft-Kehler, Dorte Marie Skytt, Miriam Kolko; Oxidative Stress in Retinal Müller Cells contributes to Dysfunction of Retinal Glutamate Uptake and Altered Protein Expression. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3672.
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© ARVO (1962-2015); The Authors (2016-present)
The viability of retinal ganglion cells (RGC) is essential to maintain the neuronal function of the retina. Müller cells (MC) are assumed to be vital in neuroprotection of the RGC. In this study, we evaluate the ability of oxidative stressed and energy restricted MC to remove glutamate from the extracellular space and evaluate related changes in gene and protein expressions.
The human Müller glial cell line, MIO-M1, kindly provided by Astrid Limb, was used in all experiments. Changes in glutamate uptake were evaluated by kinetic uptake studies using 3H-L-glutamate in oxidative stressed MC. The cell viability and mitochondrial function were evaluated by LDH and MTT assays, respectively. The expression of glutamate receptors as well as apoptotic and oxidative stress genes were evaluated by qPCR. By means of Western blot analysis the gene regulations were confirmed at the protein level. Finally, the ATP production was measured by ATP assay kit.
Generally, glutamate uptake was significantly decreased in cells exposed to oxidative stress. Energy-restricted cells had a significant decrease of ATP production after 1 and 24 hours of exposure to oxidative stress. A minor, though significant, reduction of cell viability was seen after 1 and 24 hours of exposure to oxidative stress. The glutamate transporter, EAAT1, was significantly up-regulated at RNA-level after exposure to oxidative stress, whereas the alterations of superoxide dismutase 2 (SOD2) was time-dependant. Hence, SOD2 was significantly down-regulated after 1 hour and up-regulated after 24 hours of exposure to oxidative stress. Caspase-3 was found to be significantly down-regulated 1 hour after exposure to oxidative stress. A significant up-regulation was found after 24 hours of exposure to oxidative stress in energy restricted cells.
Over all, the present study revealed changes in MC homeostasis during exposure to oxidative stress and/or energy restriction in a time dependent matter. Hence, decreased ability to remove glutamate from the extracellular space was observed in response to oxidative stress. Moreover, stress-induced changes in the regulation of glutamate transporters, oxidative stress genes as well as pro-apoptotic genes indicated that restricted energy supply and oxidative stress may affect Müller cell homeostasis in such ways that their ability to protect RGC may suffer.
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