The Mechanism Of Microglia Contribute To Quinolinic Acid-induced Excitotoxicity

Objective: To elucidate the potential intra-cellular mechanism of neural injure in neural degeneration disease, we analyzed the role of microglia played in quinolinic acid-induced excitotoxicity model, and investigated the possible mechanism by which microglia contribute to neuronal excitotocity.Method: BV-2 microglia were cultured in DMEM complete medium with 1 mM quinolinic acid for 12 h. Supernatant was collected as BCMq (BV-2 microglia conditioned medium with quinolinic acid); then challenged cultured primary cortical neurons with quinolinic acid or BCMq, to establish the excitotoxic model.Neuronal nuclear and neuronal cytoskeletal integrity were determined by Horchest orβ-tubulin staining; NF-κB activation and subsequent nuclear translocation in microglia was determined by immunofluorescence; the synthesis and secretion changes of cytokines induced by NF-κB nuclear translocation were determined by sqRT-PCR assay and ELISA assay.Result: In this excitotoxic model, 1 mM quinolinic acid added to cultured primary cortical neurons did not elicit apparent neuronal damage. However, addition of BCMq to cultured cortical neurons caused intense toxicity.According to the Western blot results, 1 mM quinolinic acid induced IκB-αphosphorylation and subsequent degradation, NF-κB nuclear translocation. Immunofluorescence assays also show a highly expression of NF-κB in quinolinic acid treated microglial nuclei, and this effect may be blocked by inhibitors of microglial activation. The changes in mRNA levels of pro-inflammation factors in quinolinic acid-treated microglia with SqRT-PCR, we found that three pro-inflammation factors mRNAs significantly changed in animal excitotoxic model: iNOS mRNA showed a transient decrease after quinolinic acid treatment, then increased to the basal levels gradually; IL-1βmRNA showed a transient increase after quinolinic acid treatment, then decreases to basal levels gradually; TNF-αmRNA showed a persistent increase after quinolinic acid treatment.In addition, the inhibitors of microglia and TNF-αantagonist attenuated neural injury confirmed that TNF-αis the main cytokine in mediating microglial contribution to quinolinic acid-induced excitotoxicity. Moreover, supplementing extrinsic TNF-αto culture medium showed an intense neural injury resembling BCMq-induced neural injury.Conclusion: Our previous research had proved quinolinic acid intra-striatal injection led to increases in TNF-αand Fas L, and this effect may be blocked by blocking NF-κB activation. Here we provided evidence elucidating a mechanism by which microglia contributed to neural injury in quinolinic acid-induced excitotoxicity. These suggest that inhibition of microglia activation or reducing pro-inflammation cytokines releasing, such as TNF-α, may promote neurons survival, thereby suggest new strategy for treatment of certain neurodegenerative diseases where excitotoxicity has been implicated in pathogenesis.