Inhibition Of ASIC1a Can Reduce Cerebral Ischemia Reperfusion Injury Caused By NMDA Receptor Excitability

Background:Cerebral ischemia reperfusion injury(CIRI)refers to a more severe process of tissue injury and neurological dysfunction after cerebral ischemia reperfusion.As a secondary injury,inflammatory cells and apoptosis pathways are overactivated,released lots of inflammatory mediators.However,there are many different opinions on the causes of cerebral ischemia reperfusion injury,in which the toxic damage of excitatory amino acids occupies an important place.NMDA receptor,which is high permeability to calcium ions,plays an important role in the toxicity of excitatory amino acids.It regulates intracellular calcium concentration as the second messenger,affecting the activation of multiple signaling pathways,such as NMDA receptor-CaMK II pathway,NMDA receptor-DAPK1 pathway,NMDA receptor-PSD95-nNOS pathway,mediating cell death.The activation of NMDA receptor also aggravates the inflammatory response after reperfusion.These pathways ultimately affect and determine the death or survival of neurons.Therefore,some scholars believe that inhibiting the activation of NMDA receptors is of great significance for the prognosis of cerebral ischemia reperfusion.Objective:1.To demonstrate that NMDA receptor activation aggravates brain injury and inflammatory response after cerebral ischemia and reperfusion.2.To explore the activation of ASIC la mediated NMDA receptor and play an important role in brain injury during cerebral ischemia-reperfusion.3.To compare the mental side effects of ASIC la inhibitors and NMDA receptor inhibitors.Methods:1.Establish transient middle cerebral artery occlusion(tMCAO)model.Assess the degree of brain damage by TTC staining and neurological deficit scores;Detect the expression of inflammatory factor IL-1 beta and TNF-alpha by ELISA method,use Western blotting(WB)to detect the phosphorylation and expression of p-NF-κB/NF-κB,subunits of NMDA receptor(NR2A and NR2B).2.tMCAO group treated with ketamine(ketamine 100mg/kg),a NMDA receptor inhibitor,evaluate the index in the same way.3.Evaluate the expression of ASIC la in the tMCAO model.tMCAO group treated with ASICla inhibitor 10mg/kg amiloride,15mg/kg flurbiprofen,evaluate the index in the same way.4.Use patch clamp method to record excitatory postsynaptic potential(EPSCs)of Pyramidal cells in the hippocampal CA1 region,and the change after apply NMDA receptor inhibitor APV,ASIC la inhibitor PcTX1 and flurbiprofen.5.Use open field test,elevated cross maze and forced swimming test to observe the change on mice’s mental and behavioral after weekly injection of ASIC la inhibitor flurbiprofen and NMDA receptor inhibitor ketamine.Results:1.compared to sham group,tMCAO can definitely increase brain injury area and neurologic deficit score,activate inflammatory pathway by increase p-NF-kB/NF-κB expression,inflammatory factor TNF-alpha\IL-1 beta,increase phosphorylation and expression of NMDA receptor(NR2A\NR2B subunit).NMDA receptor inhibitor,30min advancing intraperitoneal injection of ketamine can inhibit activation of p-NF-κB/NF-κB,expression of TNF-alpha and IL-1 beta,finally alleviate brain injury area and neurological deficit score.2.compared to the sham operation group,tMCAO increased the expression of ASIC la.While 30min advancing intraperitoneal or 120min postpone injection of the ASIC la inhibitor can inhibit the damage area,neurological deficit score,reduce expression of inflammatory factor.The expression of NMDA receptor NR2B subunit also decreased after the use of ASIC la inhibitor,while the expression of NR2A subunit and phosphorylation of NR2A and NR2B had no obvious change or even increased.3.we observed the NMDA receptor mediated EPSCs in pyramidal cells of CA1 area by using voltage clamp technique.APV blocked EPSCs completely,while PcTX1 significantly reduced EPSCs,which was recovered after washing.Similarly,amiloride and flurbiprofen had the same effect.4.NMDA receptor inhibitor ketamine can cause anxiety and depressive behavior in mice,while flurbiprofen has no significant effect.Conclusion:NMDA receptors are involved in brain injury and inflammation after cerebral ischemia reperfusion;at the same time,ASIC la participate in cerebral ischemia reperfusion injury through increase NMDA receptor activation and expression;ASIC la inhibitors have lighter psychiatric side effects than NMDA receptor inhibitor.