The Impact of Timing of Microglial Activation by Inflammation and Hypoxia Regarding Additive Neurotoxic Effects

Abstract

Introduction: Perinatal cerebral hypoxia and endotoxins are main risk factors for acquired brain injury and neurological disabilities. Here, we investigated the impact of lipopolysaccharide stimulated microglia on inflammation and cytotoxicity in vitro in the context of hypoxia. Methods: BV2 cells were exposed to LPS and hypoxia (1% O2). Expression of the transcription factor hypoxia-inducible factor (HIF)-1α as well as of iNOS, IL-1b, IL-6, and tumor necrosis factor (TNF)-α were analyzed by real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. NO production was quantified by the Griess assay. Results: LPS exposition prior hypoxia increased mRNA and protein expression of HIF-1α, iNOS, and inflammatory cytokines suggesting synergistic regulation of hypoxia and inflammatory signaling pathways in microglial cells. Whereas HIF-1α accumulation was transient depending on the presence of LPS, ongoing cytokine and NO secretion was observed for at least 3 days after LPS removal. Notably, hypoxia potentiated the LPS-induced secretion of IL-1b (22-fold), IL-6 (threefold), TNF-α (threefold), and increased NO production by approximately 60%. Cell-free supernatants derived from LPS exposed BV2 cells were highly cytotoxic. Again, hypoxia enhanced the cytotoxic effect significantly. In contrast, stimulation of BV2 cells by hypoxia prior LPS abolished additive inflammatory neurotoxic effects compared with controls (p < 0.01). Conclusion: Present in vitro data indicate that LPS induces a dose-dependent and long-lasting sensitization of BV2 cells to hypoxia. Enhanced cytokine and NO secretion may contribute to delayed glial damage following cerebral hypoxic insults during the perinatal period. As hypoxia induced accumulation of the neurotrophic transcription factor HIF-1α abolishes cytotoxic inflammatory effects, present observations may have implications for neuroprotective pharmacological strategies.