Experimental Study On Rat MCAO Model Of Dexmedetomidine On Brain Protection By Inhibiting Microglial Activation

Background:During the perioperative period,the risk of cerebrovascular accidents in patients with cerebrovascular disease is greatly increased,which may cause serious consequences such as disability and death,and threaten life safety.In cerebrovascular diseases,ischemic cerebrovascular disease is the most common.After ischemia and reperfusion,the brain tissue causes a series of pathophysiological changes,which lead to further damage,called cerebral ischemia-reperfusion injury.Proactive and effective measures can alleviate cerebral ischemia-reperfusion injury and prevent further deterioration.The mechanism of cerebral ischemia-reperfusion injury involves multiple factors,mutual promotion,and is very complicated.Inflammatory response is considered to be one of the important causes of ischemia-reperfusion injury,and reducing inflammation is one of the effective strategies for the treatment of ischemia-reperfusion injury.After cerebral ischemia,microglia are rapidly activated,activated microglia produce a large number of pro-inflammatory factors,leading to increased damage,inhibition of microglia activation can reduce ischemia-reperfusion injury.Dexmedetomidine（Dex）,a highly selectiveα₂ receptor agonist,has sedative,analgesic,antisympathetic and excitatory effects and is widely used in clinical practice.A large number of clinical and basic research results show that dexmedetomidine has protective effects on brain,liver,kidney,spinal cord,lung,heart and other vital organs,and may involve multiple mechanisms,which is still unclear.Studies have found that dexmedetomidine can reduce spinal cord injury by inhibiting the release of inflammatory factors by microglia activation.Whether or not dexmedetomidine can affect cerebral ischemia-reperfusion injury through microglia in vivo study has not been reported,and it is worthy of further study.Objective:In this experiment,a model of rat middle cerebral artery occlusion and a mouse microglia BV2 cell oxygen glucose deprivation/reoxygenation model were established.The ischemia-reperfusion injury was simulated at the animal and cell level.From the perspective of microglia activation,the brain protection of dexmedetomidine and its possible mechanism were explored,which provided a theoretical basis for clinical application.Methods:The rat MCAO model was established by the suture method.Sixty male Sprague-Dawley rats were randomly divided into three groups（n=20）:sham operation group（Sham group）,MCAO model group（I/R group）and Dex+I/R group.After 24 hours of reperfusion,each group was simultaneously tested for various indicators.The Longa’s score was used to evaluate the neurological function.HE staining was used to observe the pathological changes of the infarcted cerebral cortex.TUNEL staining was used to detect the apoptosis of cells in the penumbra.Tissue immunofluorescence staining was us ed to observe the activation of microglia in the penumbra.ELISA kit was used to detect the level of TNF-αin the infarctal cortex.An Oxygen glucose deprivation/reoxygenation（OGD/R）model was established.The cultured BV2 microglia were divided into a co ntrol group（Control group）,a model group（OGD/R group）,1μM Dex+OGD/R group,10μM Dex+OGD/R group.After 24 hours of reoxygenation culture,various indexes were detected.The concentration of TNF-αin the supernatant was detected by ELISA kit.The expres sions of CD68,NF-κB p65 and phosphorylated NF-κB p65（p-NF-κB p65）in BV2 cells were detected by Western blot.Results:1.Effect of Dex on neurological function score in MCAO model ratsCompared with the Sham group,the neurological function scores in the I/R group were significantly increased（P<0.01）,and the neurological function scores in the Dex+I/R group were lower than those in the I/R group（P<0.05）.2.Effect of Dex on the microstructure of infarcted cortical brain tissue in MCAO model ratsCompared with the Sham group,the number of cells in the I/R group was reduced,a large number of cells were necrotic,the cells were vacuolated,the interstitial edema was loose,and the nucleus was partially condensed,a small number of cells showed vacuolar degeneration and interstitial edema was alleviated after Dex intervention.3.Effect of Dex on apoptosis of penumbra cells in infarcted cortex of MCAO model ratsCompared with Sham group,the apoptotic rate of penumbra cells in infarcted cortex of rats in I/R group increased significantly（P<0.01）,and the apoptotic rate after Dex intervention was lower than that in I/R group（P<0.05）.4.Effects of Dex on the activation of microglia in the penumbra and the level of TNF-αin the infarcted cortex of MCAO model ratsCompared with the Sham group,the number of activated microglia in the I/R group was significantly increased（P<0.01）,and the concentration of TNF-αin the brain tissue was significantly increased（P<0.01）.Compared with the I/R group,the activation rate of microglia in the Dex+I/R group was decreased（P<0.05）,and the concentration of TNF-αin the brain tissue was decreased（P<0.05）.5.Effect of Dex on activation and secretion of TNF-αby OGD/R BV2 cellsThe expression of CD68 was significantly increased in BV2 cells after OGD/R（P<0.01）.The lower concentration（1μM）Dex had no significant effect on the expression of CD68 in OGD/R BV2 cells,while the expression of CD68 in OGD/R BV2 cells treated with 10μM Dex was decrease（P<0.05）.Compared with the Control group,the concentration of TNF-αin the supernatant of the OGD/R group increased（P<0.01）,compared with the OGD/R group,the concentration of TNF-αin the supernatant of the 1μM Dex+OGD/R group and the 10μM Dex+OGD/R group was decreased（P<0.05）.6.Effect of Dex on the expression and activation of NF-κB in OGD/R BV2 cellsCompared with the Control group,the expression of NF-κB p65 and p-NF-κB p65 in BV2 cells of OGD/R group increased（P<0.01）.Compared with OGD/R group,there was no significant difference in NF-κB p65 expression in 1μM Dex+OGD/R group or 10μM Dex+OGD/R group（P>0.05）,and expression of p-NF-κB p65 in 10μM Dex+OGD/R group was reduced（P<0.05）.Conclusion:Dexmedetomidine protects against cerebral ischemia-reperfusion injury by inhibiting microglial activation,which may be related to the inhibition of NF-κB activation in microglia.