Mice Experimental Study On Electroacupuncture To Alleviate Cerebral Ischemia-Reperfusion Injury By Regulating TLR4 Signaling Pathway

Objective:By observing the changes of MRI signal combined with microscopic anatomical structure,TLR4 m RNA content and serum inflammatory factor release of electroacupuncture model mice with cerebral ischemia-reperfusion injury,USPIO enhanced MRI imaging technology was used to monitor the changes of inflammatory injury in vivo,and combined with immunohistochemistry,Real time PCR and other means to study the subacute mechanism of electroacupuncture in alleviating inflammatory response of cerebral ischemia-reperfusion in mice.Method:Thirty healthy adult wild-type C57BL/6n mice(20-25g)were randomly divided into three groups: sham operation group,middle cerebral artery occlusion(MCAO)ischemia-reperfusion groupelectroacupuncture,(Electroacupuncture),EA)group;10 healthy adult Toll-like receptor 4 knock out(TLR4 KO)mice were selected.All mice were anesthetized by intraperitoneal injection,and Longa-style thread embolism method was replicated and modified on this basis to establish the right middle cerebral artery occlusion ischemia-reperfusion model of mice.MRI scan was performed on the successfully embolized mouse model,and blood was taken from the brain after completion.The sham-operated mice only separated the common carotid artery,the external carotid artery and the internal carotid artery,and the embolic thread was not inserted into the blood vessel.In MCAO ischemia-reperfusion group mice,the thread plug was inserted into the internal carotid artery about 10-12 mm.After the blood flow stopped for 30 min,the thread plug was withdrawn to the distal end about 10 mm from the bifurcation of internal and external carotid artery to restore the blood flow of the right middle cerebral artery.No electroacupuncture treatment was given to the above two groups,and binding stimulation was fixed with EA group at the same time every day to eliminate the result error caused by stress reaction in mice.The modeling methods of mice in EA group and TLR4 KO group are the same as those in MCAO ischemia reperfusion group.Longa5 score was used to evaluate the neurological function of mice after complete recovery(2 hours)and 48 hours reperfusion.After the successful modeling,all mice were injected with USPIO in the tail vein.Mice in EA group and TLR4 KO group were punctured with "Zusanli" point and "Baihui" point on the right side through "temple" point for 30 minutes/day,once a day.MRI was performed for 48 hours in mice with successful model building.Blood and brain were taken after scanning.The image is uploaded to the post-processing workstation to measure the ratio of the signal value of the coronal infarction side to the normal side of the T2 weighted imaging(T2WI)and the susceptibility weighted imaging(SWI).At room temperature,orbital blood was taken first,whole blood was allowed to stand for 30 min,then centrifuged,upper serum was taken,and relevant inflammatory signal indicators(IL-1?,TNF-?)were detected by ELISA.The brain of mice was taken and immersed in 4% paraformaldehyde solution,and then the brain was dehydrated in graded series of alcohols and embedded in paraffin.Paraffin sections and structural damage of brain neurons were observed by pathological HE staining.Immunohistochemical staining was used to observe the phagocytosis of iron particles in infarct area of brain tissue.Real-time pcr was used to measure the m RNA expression of TLR4 in infarct area of brain tissue.SPSS 21.0 software was used for statistics,and a=0.05 was set as the confidence level.Result:(1)The ischemia-reperfusion model of the right middle cerebral artery in mice was successfully replicated by modified Longa suture method with stable infarct size.The results of neurological function after successful modeling showed that there were significant differences between the sham operation group and the MCAO ischemia-reperfusion group,EA group and TLR4 KO group(p<0.05);There was no significant difference between the model groups(p >0.05).The neurological function score after 48 hours of reperfusion showed that compared with MCAO ischemia reperfusion group,EA group had significant difference in neurological function score.Compared with EA group,the neurological function score of TLR4 KO group also changed significantly.(2)Inflammatory factors(IL-1?,TNF-?)in mice in sham-operation group were different from MCAO ischemia-reperfusion group,EA group and TLR4 KO group(P < 0.05).(3)The ratio of the lowest signal on the right infarction side to the left side of the T2 WI in the EA group was significantly different from that in the MCAO ischemia-reperfusion group(p<0.05).(4)Immunohistochemical staining showed a small number of microglia and no staining of iron particles in the sham-operated mice.In MCAO ischemia-reperfusion group and EA group,there were obvious increase of microglial cells,iron particles scattered in intercellular space and a small amount of iron particles engulfed by microglial cells.(5)the expression of TLR4 m RNA in the passage of mice in sham operation group was different from MCAO ischemia reperfusion group and EA group(p < 0.05).(6)No obvious pathological changes were found in HE staining of mice in sham operation group.MCAO ischemia-reperfusion group and EA group showed structural damage of nerve cells in ischemic brain tissue region of mice.Conclusion: With the USPIO enhanced magnetic resonance monitoring and pathological histology,we found that acupuncture can improve the symptoms of neurological deficits in mice,reduce the number of activated microglia,increase the T2 WI signal value in the coronal position,and down-regulate the TLR4 m RNA in the pathway and signal-related downstream proinflammatory factors IL-1?,TNF-? expression.Therefore,electroacupuncture plays a positive role in the improvement of cerebral ischemic inflammatory damage through the TLR4 signaling pathway.