Research Of Mechanism Of NMDAR1on The Traumatic Brain Dema And Its Correlation With AQP-4

Objective:To study the correlation between The N-methyl-D-aspartate receptor(NMDAR) and Aquaporin-4（AQP-4）in the process of the occurrence and developmentof traumatic brain edema,and observe the influence of NMDAR activated or not onAQP-4and effect of AQP-4on NMDAR1.Methods:The model of focal and moderate traumatic brain injury was set up byself-modified Feeney’s weight-dropping brain injury equipment (impact of600g×cm).The experiment includes wild-type AQP4groups and)AQP-4knockoutgroups.1.wild-type AQP4groups (n=60),60male Spraque-Dawley rats were dividedrandomly into five groups as follows:sham-operated groups (n=12),injury groups(n=12),normal saline groups(injury+normal saline n=12),MK-801groups (injury+MK-801n=12),NMDA groups (injury+NMDA n=12). In sham-operated groups,their scalp werecut and skull were opened,they didn’t result in injury;injury groups were not intervented;in normal saline groups,they were received an intraperitoneal injection of0.9%NS(1ml/Kg)60minutes post injury;In MK-801groups, they were received anintraperitoneal injection of NMDA receptor antagonists MK-801(1ml/Kg) at60minutespost injury;In NMDA groups, they were received an intraperitoneal injection of NMDAreceptor agonist NMDA (20mg/Kg) at60minutes post injury,then drew materials at24h post injury. Expression changes of AQP-4protein in the traumatic brain edemawere tested by western blot; Expression changes of AQP-4in the traumatic brainedema range were observed by confocal laser scanning microscopy afterimmunoflurescence labeling.2.AQP-4knockout groups (n=72),72female Spraque-Dawley rats through RNAinterference technique were divided randomly into six sub-groups: normal saline control groups,GFP control groups,LV1control groups,LV2groups,LV3groups,LV4groups,each group had12rats.In normal saline control groups,they werereceived an injection of30μl0.9%NS in local brain cortex throughmicroinjector.Equivalent dose of GFP,LV1,LV2,LV3,LV4interference agents wereinjected to other each group respectively.Brain injury model was set up at48h postusing RNA interference agent,and injury brain tissues were taken out at24h postinjury.The effect of AQP-4knockout and expression changes of NMDAR1mRNAtranscription level were detected by Resl-time quantification PCR and Western bolt.Results:1.After using NMDA receptor antagonists MK-801,the total expression changes ofAQP-4in injury range:expression level of AQP-4decreased obviously(P＜0.05）；2. After using NMDA receptor antagonists MK-801and NMDA receptor agonistNMDA,the expression changes of AQP-4in injury range:（1） Positive signal area percentage of AQP-4expression withimmunohistochemical method in hippocampus CA1,CA2,CA3region:after cerebraltrauma,compare immunopositive signals of AQP-4in CA1,CA2,CA3region of injurygroups with those in sham-operated groups, compare immunopositive signals of AQP-4in CA1,CA2,CA3region of MK-801groups with those in injury groups andsham-operated groups, compare immunopositive signals of AQP-4in CA1,CA2,CA3region of NMDA groups with those in injury groups and sham-operated groups, they allhave significances in statistics(P＜0.05).（2） Positive signal area percentage of AQP-4expression withimmunohistochemical method in injuried ipsilateral cortex:after cerebral trauma,injurygroups and sham-operated groups: after cerebral trauma,compare immunopositivesignals of AQP-4in injuried ipsilateral cortex of injury groups with those insham-operated groups,there were not significances in statistics(P＞0.05).Compareimmunopositive signals of AQP-4in injuried ipsilateral cortex of MK-801with those insham-operated groups,there were not significances in statistics(P＞0.05),but compareimmunopositive signals of AQP-4in injuried ipsilateral cortex of MK-801groups withinjury groups there were significances in statistics(P＜0.05).NMDAgroups,sham-operated groups, there were not significances in statistics(P＞0.05).（3） Positive signal area percentage of AQP-4expression withimmunohistochemical method in dentate gyrus region:there were not there were notsignificances in statistics in each group 3.The changed results of NMDAR1gene expression in vivo experimentalstudy after AQP-4gene interfered by RNA:（1） By Real-time PCR testing AQP-4gene transcriptional levels after RNAinterference,the results showed that viruses with four different sequences had knockouteffect on AQP-4gene, there were significances in statistics(P＜0.05).Viral vector LV1made AQP-4gene down57.9%and viral vector LV3made AQP-4gene down53.3%,there were obvious significances in statistics(P＜0.01).The results tested by WesternBlot showed that AQP-4transcriptional levels were down30.7-44.9%,there wereobvious significances in statistics(P＜0.01).（2） The results tested by Real-time PCR showed that NMDAR1expressionlevels were down19.1-34.2%after RNA interference; The NMDAR1expression levelsof Viral vector LV1and viral vector LV3were down34.2%and30.7%, there wereobvious significances in statistics(P＜0.01).Conclusions:1.Activation and antagonism of NMDA receptor regulate AQP-4definitely,andmore obviously among hippocampus’ each region(CA1,CA2,CA3).2.After AQP-4expression was interfered,NMDAR1expression levels were down,AQP-4and NMDAR also decreased.