Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: Role of TLR4 in hypoxic microglia

Background: hypoxia induces microglial activation which causes damage to the developing brain. microglia derived inflammatory mediators may contribute to this process. toll-like receptor 4 (tlr4) has been reported to induce microglial activation and cytokines production in brain injuries; however,its role in hypoxic injury remains uncertain. we investigate here tlr4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury.methods: one day old wistar rats were subjected to hypoxia for 2 h. primary cultured microglia and bv-2 cells were subjected to hypoxia for different durations. tlr4 expression in microglia was determined by rt-pcr,western blot and immunofluorescence staining. small interfering rna (sirna) transfection and antibody neutralization were employed to downregulate tlr4 in bv-2 and primary culture. mrna and protein expression of tumor necrosis factor-alpha (tnf-α),interleukin-1 beta (il-1β) and inducible nitric oxide synthase (inos) was assessed. reactive oxygen species (ros),nitric oxide (no) and nf-κb levels were determined by flow cytometry,colorimetric and elisa assays respectively. hypoxia-inducible factor-1 alpha (hif-1α) mrna and protein expression was quantified and where necessary,the protein expression was depleted by antibody neutralization. in vivo inhibition of tlr4 with cli-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining.results: tlr4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. in vitro,tlr4 protein expression was significantly increased in both primary microglia and bv-2 cells post-hypoxia. tlr4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of tnf-α,il-1β and inos. sirna knockdown of tlr4 reduced hypoxia-induced upregulation of tnf-α,il-1β,inos,ros and no in bv-2 cells. tlr4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and bv-2 cells was accompanied by the suppression of nf-κb activation. furthermore,hif-1α antibody neutralization attenuated the increase of tlr4 expression in hypoxic bv-2 cells. tlr4 inhibition in vivo attenuated the immunoexpression of tnf-α,il-1β and inos on microglia post-hypoxia.conclusion: activated microglia tlr4 expression mediated neuroinflammation via a nf-κb signaling pathway in response to hypoxia. hence,microglia tlr4 presents as a potential therapeutic target for neonatal hypoxia brain injuries. © 2013 yao et al.; licensee biomed central ltd.