The Function And Molecular Mechanism Of Different Subtypes Of NMDA Receptors In Neonatal Hypoxic-ischemic

Objectives:Hypoxic-Ischemic Encephalopathy(HIE)not only causes neonatal perinatal death but is also a common cause of post-neonatal disability.With the implementation of our country’s second-child policy and the increasing age of pregnant women,the incidence of HIE is likely to increase.Children with severe HIE can observe obvious neurobehavioral abnormalities(such as cerebral palsy,epilepsy,etc)in the early stages of development which accounts for approximately 45%.Even though children with mild HIE are considered to be cured at an early stage,they often develop neurobehavioral problems during school age and adolescence,such as autism,attention deficit,schizophrenia and so on.The impaired cognitive function and social behavior disorder of the children have brought heavy burden to the family and society.Previous studies have shown that NMDA receptors(N-methyl-D-aspartate Acid Receptor,NMDAR)are involved in excitotoxicity caused by hypoxia-ischemia,suggesting that regulating the function of NMDA receptors may possess protective effects.However,the specific role of different NMDA receptor subtype in HIE is not clear yet.This project hopes to explore the pathological differences between neonatal and adult hypoxic-ischemic injury by studying the mechanism of action of different NMDA receptor subtypes in neonatal hypoxic-ischemic injury and protection,so as to provide a new basic theoretical basis for intervening neonatal hypoxia ischemic.Methods:The modified Vannucci method was used to establish a 9-11 day hypoxic-ischemic(HIE)model of neonatal C57 mice.The mice were randomly divided into normal control group,sham group,HIE group and drug treatment group.The drug treatment group was further divided into three groups,which were given GluN2A-NMDAR specific inhibitor TCN-201 or GluN2B-NMDAR specific inhibitor ifenprodil respectively.Three administration time points were set:before HIE modeling,after modeling,and 24 hours after modeling(n=5 for each treatment).The cortex was collected 72h after modeling,and TTC(2,3,5-triphenyltetrazolium Chloride)staining was used to detect the area of cerebral infarction.Hoechst staining was used to detect the apoptosis of cells after modeling and drug administration.The expression and phosphorylation changes of proteins of apoptosis related signaling pathway-related(GluN2A,GluN2B,Akt,ERK,PI3K,CREB,GSK,caspase3,were measured by Western Blot.Thus,the functions of different subtypes of NMDA receptor in HIE and their associated signaling pathways can be determined.Afterwards,in order to further explore the molecular mechanism of GluN2B-mediated neuron protection,co-immunoprecipitation(CO-IP)was used to detect the interaction between GluN2B and PSD95 after modeling.Then,the interaction between GluN2B and PSD95 was interfered by giving the transmembrane interference polypeptide TAT-GluN2B-9c,and the interference effect was detected by CO-IP.The mice were randomly divided into four groups,which were sham operation group,model group,normal saline group,and interference peptide group(n=4 in each group).The cortical tissue were taken at 24h and 72h after the treatment.The changes in the interaction between GluN2B and PSD95 after administration were detected by immunoprecipitation,and the changes in the expression of proteins(PI3K,p-PI3K,CREB,p-CREB,Akt,p-Akt)in the protective signaling pathway were detected by Western Blot.Finally,tail suspension test(n=4-5 per group)was used to detect the motor function of mice 8 weeks after HIE.Results:①After hypoxic-ischemic modeling in newborn mice,there were obvious white infarcts in the brain,and the apoptosis of nerve cells was significantly increased,suggesting that the modeling was successful.②Western blot results showed that the expression of GluN2A was down-regulated after HIE,but the expression of GluN2B subunit was not significantly changed.Meanwhile,the phosphorylation levels of Akt and ERK were significantly down regulated.③Hoechst experiment showed that after ifenprodil treatment,which could specifically inhibit GluN2B-containing NMDA receptors,the apoptosis of cortical neurons were intensified.Western blot results showed that the phosphorylation level of Akt in the brain of the ifenprodil-treated mice was significantly reduced,while the expression levels of caspase 3 and cleaved caspase 3 significant increased,suggesting that neuronal apoptosis is intensified.There was no obvious change after inhibiting GluN2A-NMDAR.④The phosphorylation levels of PI3K and CREB in mice increased after HIE,which was inhibited by ifenprodil.There was no obvious change after inhibiting GluN2A-NMDAR.⑤ The interaction between GluN2B and PSD95 increased significantly from 24 hours to 72 hours after HIE(P<0.05),and the interference peptide TAT-GluN2B-9c could effectively inhibit the binding of GluN2B and PSD95.⑥After inhibiting the interaction between GluN2B and PSD95,the phosphorylation levels of Akt,PI3K,and CREB in the mice brain were significantly down-regulated compared with HIE model mice.The phosphorylation levels of the three proteins in the saline treatment control group were not significantly different from those in the HIE group.⑦Compared with the control mice,the expression of caspase-3 in the interference peptide treatment group increased significantly after HIE.⑧ The behavioral experimental scores of mice in the HIE model and ifenprodil treatment groups were significantly lower than those of the control group.In addition,compared with the model,the behavioral scores of the mice in the TAT-GluN2B-9c treatment group were higher,suggesting that the peptide could rescue the behavioral impairment of HIE mice.Conclusions:① The brain damage of HIE model mice is obvious,that cell apoptosis is intensified,and the expression of GluN2A of NMDA receptor is significantly down-regulated.In addition,signal pathways related to neuronal apoptosis were activated.② GluN2B-NMDAR couples with synaptic protection signaling pathway in HIE-induced neuronal apoptosis and mediates neuronal survival.③PSD95 mediates the protective effect of GluN2B-NMDAR.Interfering with the binding of PSD95 and GluN2B will aggravate neuronal apoptosis caused by HIE,but can prevent the long-term behavioral impairment.