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Darren W. Chan, Jeremy M. Sivak, John G. Flanagan; Time Course of Hypoxia Response in Primary Human Optic Nerve Head Astrocytes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3862.
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To establish an in-vitro model of hypoxia on cultured primary astrocytes of the human optic nerve head (ONH). Both hypoxic responses and astrocyte activation in the ONH are implicated in the pathogenesis of glaucoma. These experiments were designed to determine the time course and optimal conditions to assess hypoxic effects.
Explants prepared from isolated ONHs dissected from healthy human donor eyes were cultured to develop into primary cell lines. Together with these primary cells, SVGp12 cells, a human fetal brain glial cell line, were also used for some preliminary experiments. Hypoxia experiments were run in parallel for 5 time points over 24 hours (2, 5, 8, 18, 24 ). Both the primary astrocytes and SVGp12 cells, were serum deprived for 24 hours upon confluence. They were placed into a hypoxic chamber that was preset for 1% oxygen and 5% carbon dioxide. Control cells were run separately, in parallel, in normoxic conditions. At the end of each time point, samples of conditioned media and cells were harvested in an ice bath, immediately processed for nuclear extracts, and stored at -80°C for future analysis. Some cells were grown on coverslips through hypoxic treatments, and fixed for immunofluorescence analysis.
Nuclear extracts from the experimental samples were used for Western blotting and were analyzed by densitometry. The hypoxia inducible factor HIF-1alpha was found to be stabilized in the nucleus following hypoxia, with maximum levels between 5 and 8 hrs, as compared to normoxic controls. The hypoxia and metabolic response factor, PGC-1alpha, was maximally upregulated around 8 hrs. Increases in the biomarker GFAP were detected from 2 hrs, reflecting astrocyte activation. Immunofluorescent studies showed active translocation of HIF-1alpha from cytoplasm to nucleus.
Primary human ONH astrocytes were activated during hypoxic insult, with typical hypoxic responses peaking between 5 and 8 hours. We propose a model of hypoxia that has the potential of being combined with previous models of biomechanical insult.
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