The Role Of Pannexin-1channels In Early Brain Injury After Experimental Subarachnoid Hemorrhage

Background:Subarachnoid hemorrhage(SAH)is a catastrophic cerebrovascular disease with high morbidity and mortality worldwide.A range of pathological response that occurs in early brain injury(EBI)period can lead to the blood brain barrier(BBB)dysfunction,inflammatory injury,apoptosis,necrosis and neurological dysfunction following SAH.Accumulating evidence in SAH research suggests that neuroinflammatory injury plays a critical role in EBI.Pannexin-1 channels,as a member of gap junction proteins located on the plasma membrane,releases ATP,second messengers,and neurotransmitters into the extracellular space,when activated.Previous studies identified that the opening of Pannexin-1 channels is essential for cellular energy metabolism,apoptosis,necrosis,differentiation,intercellular signaling,and especially inflammation.Recent research shows that Pannexin-1-mediated inflammatory response participates in the pathomechanism of cardiovascular disease and cerebral ischemia,but its effects on inflammatory response in SAH model have not been explored yet.Given the above,we raised a hypothesis that activation of Pannexin-1 channels could initiate the inflammatory injury and initiates the neuronal apoptosis and necrosis in EBI after SAH.Therefore,the aim of this study was to determine the potential mechanism of Pannexin-1 channels in EBI following experimental SAH.Methods:Expression and cellular localization of Pannexin-1 channels were assessed by western blot and double immunofluorescence staining in vivo SAH model at sequential time points.The expression of ATP ligand protein(P2X7)was evaluated by western blot to reflect the release volumn changes of ATP.Pannexin-1 channels gene knockdown and overexpression were achieved by lentivirus(LV)administration.In addition,western blot,enzyme linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(PCR)and immunofluorescence staining were performed to explore the potential interactive mechanism between Pannexin-1 channels mediated inflammatory response and neuronal apoptosis/necrosis in EBI period after SAH in vivo study.Short term neurological dysfunction was evaluated with modified Garcia score system,and the long term neurocognitive functional change was investigated by the Morris water maze test.Results:According to our result,the expression of Pannexin-1 showed no significant different at sequential time points after SAH;however,the expression of P2X7 was elevated on 24 hours post-SAH.In addition,administration with LV-ShRNA-Panx1 abolished the upregulation of P2X7 after SAH.Meanwhile,administration with LV-ShRNA-Panx1 markedly decreased the expression levels of TLRs/NF-?B pathway-related agents and downstream inflammatory cytokines in the brain cortex and significantly ameliorated short-term neurological dysfuction and long-term neurological cognitive deficits in this SAH model.On the contrary,administration of LV-Panx1-EGFP elevated the levels of TLRs/NF-?B pathway-related agents and inflammatory cytokines,which correlated with augmented neuronal apoptosis and necrosis.Conclusion:Pannexin-1 channels may contribute to inflammatory response and neurobehavioral dysfunction through the TLRs/NF-?B mediated signaling pathway in EBI after SAH,suggesting a potential role of Pannexin-1 channels could be a potential therapeutic target for the treatment of SAH.