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Background: As the innate immune cell of the central nervous system (CNS), microglia are poised to be the first cellular responders to ischemic stroke. Microglia are capable of polarizing from a resting (M0) phenotype to a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype depending on the signals these cells receive from the microenvironment. Typically cell damage through neurological injury results in a pro-inflammatory phenotype. Paradoxically, despite the cell death that occurs following ischemic stroke that presumably polarizes microglia to a pro-inflammatory phenotype, our lab and others have shown that microglia in mice are predominantly anti-inflammatory after ischemic stroke.Hypothesis: To possibly explain this anomaly, we hypothesized that hypoxia itself is a stimulus of anti-inflammatory microglia M2 polarization following ischemic stroke that overrides pro-inflammatory polarizing signals from cellular damage.Methods: We developed a novel method of culturing primary microglia from individual male and female neonatal mice. We then exposed isolated microglia to oxygen glucose deprivation (OGD, 95% nitrogen / 5% carbon dioxide) and examined the expression of M1 and M2 genes by quantitative PCR. Media was collected at 24hr and analyzed by lactate dehydrogenase (LDH) assay and ELISA.Results: We found that microglia are resistant to OGD-induced cytotoxicity, with LDH assay showing only 16% cell death after 8hr of OGD (p<0.05). OGD induced a robust up-regulation of anti-inflammatory M2 genes (Interferon Regulatory Factor 4 (IRF4), 2.08±0.08-fold, p<0.05; Vascular Endothelial Growth Factor (VEGF), 1.76±0.05-fold, p<0.0001) immediately following OGD and also down-regulated expression of pro-inflammatory M1 genes (IRF5, 0.385±0.018-fold, p<0.0001; Interleukin-1β (IL1B), 0.018±0.005-fold, p<0.01; Tumor Necrosis Factor-α (TNFA), 0.345±0.024-fold, p<0.0001) in both male and female microglia (n=7). ELISA of cell culture supernatant following OGD demonstrated that microglia secrete VEGF, suggesting OGD-treated microglia have anti-inflammatory functions.Conclusion: Collectively, our data suggest that hypoxia drives both male and female microglia to polarize to an anti-inflammatory M2 phenotype.