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John P. Wood, Glyn Chidlow, Teresa Mammone, Robert J. Casson; Chemically-induced Anoxia in Retinal Cell Cultures: Mechanisms of Injury and Neuroprotection. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4445.
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To use the mitochondrial inhibitor, sodium azide (NaN3), which blocks electron transfer between cytochrome C oxidase and oxygen, to induce a reproducible injury to cultured retinal cells by chemically mimicking anoxia. Furthermore, to determine and attempt to prevent the pathological intracellular processes which result from this insult.
Retinal cultures were prepared from 2 day old Sprague-Dawley rat pups via a mechanical and enzymatic digestion procedure. After 7 days, cultures were treated with NaN3 (range of concentrations from 1µM up to 100mM) for 24 hours. Toxicological effects to retinal neurons and glia were delineated by immunocytochemistry, immunoblotting, TUNEL assay and cell viability determinations. Attempts were made to abrogate the toxic effects of NaN3 by using a range of inhibitory agents.
Retinal cells in culture were dose-dependently killed by NaN3, with neurons being much more sensitive to the treatment than glia (Muller cells, astrocytes and microglia), as assessed by GABA- tau-, PGP9.5-, βIII-tubulin-, nestin-, vimentin-, GFAP- and ED-1-immunolabelling and confirmed by immunoblotting and viability assays. After a 24 hour incubation, 50% of neurons were killed by 245µM and 50% of glia by 10mM NaN3. Death of neurons but not glia was via apoptosis as assessed by TUNEL assay. The determined neuronal death could be partially blocked by the antioxidant, trolox (10µM), the NOS inhibitor, L-NAME (10µM), sodium pyruvate (5mM), the ryanodine receptor blocker, dantrolene (10µM) and by the calpain inhibitor, MDL38170 (1µM).
Treatment with NaN3 proved to provide a reproducible means to mimic an anoxic insult to retinal cells, and particularly neurons, in culture. Co-incubation with specific classes of inhibitory compounds delineated that neuron death occurred through an apoptotic process involving free radical production, release of calcium from intracellular stores and calpain activation. These data have implications for the treatment of energetically-compromised retinal neurons.
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