| Objective:To compare the effects of electroacupuncture at different time points on the expression of angiogenesis related factors endostatin(ES)and thrombospondin-1(TSP-1)in rats with focal cerebral ischemia.Taking angiogenesis as the starting point,the therapeutic effect and possible mechanism of electroacupuncture at different time points on ischemic stroke were explored,which to provide new scientific experimental basis for clinical treatment of ischemic stroke.Methods:Twenty-seven rats were randomly selected from 100 male SD rats as the sham operation group,and the remaining 73 rats were established the model of focal middle cerebral artery occlusion(MCAO)by Zea Longa modified right external carotid artery thread embolization.Finally,54 successful rats were randomly divided into model group(27 rats)and electroacupuncture group(27 rats).According to different treatment time,each group was divided into three subgroups,named 3d,7d and 14 d group,with 9 rats in each subgroup.Rats in sham-operated group and model group were not given any treatment.The electroacupuncture group started on the 1st day after operation.Baihui and Shuigou points were selected for continuous treatment according to different time points.The rats were sacrificed to gain materials after 3d,7d and 14 d,respectively.Neurological impairment and recovery of rats in each group were observed at 4h,3d,7d and 14 d after operation with neurological function deficit sign score.Histopathological changes of hippocampus in ischemic area were observed by HE staining.Cerebral infarction volume was measured by TTC staining.The expression of ES and TSP-1 protein in hippocampus of ischemia region of rats in different groups at different time points were detected by immunohistochemistry and Western Blotting.Immunohistochemical method and Weidner method were used to measure microvessel density and observe angiogenesis in cerebral infarction area.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the relative expression of ES and TSP-1 in hippocampus of ischemia region of rats.Results:1.Neurological function deficit scoreAt 4h,7d and 14 d after operation,the scores of neurological deficits in the model group were higher than those in the sham operation group(P<0.01).There was no significant difference in neurological deficits between the electroacupuncture group and the model group(P>0.05);7d,14 d after operation,the degree of neurological deficit in the electroacupuncture group was lower than that in the model group(P<0.01).Compared with the 4h group,the scores of the 3d group decreased(P>0.05);the scores of the 7d group and the 14 d group decreased significantly(P<0.01);compared with the 3d group,the scores of the 7d group and the 14 d group decreased significantly(P<0.01);compared with the 7d group,the scores of the 14 d group decreased significantly(P<0.05).2.Histopathological observationIn the sham operation group,the brain tissue structure was complete,the layers were clear,the neurons were normal,arranged in order,and the clear and intact nuclei were visible;On the 3rd day after operation,the ischemic side of model group and electroacupuncture group showed different degrees of brain interstitial edema.Pathological changes such as disordered cell arrangement,loose structure,pyknosis of some nuclei,were all appeared.After 7d of treatment,the degree of ischemic brain tissue damage in the electroacupuncture group was slightly reduced compared with the model group;after 14 d of treatment,in the electroacupuncture group,the interstitial edema of the ischemic brain tissue was significantly reduced,the necrotic neurons were reduced,the nuclei were clear,and the degree of brain tissue damage was significantly reduced compared with the model group.3.Detection of cerebral infarction volumeThere was no infarction area in the sham operation group,and the infarction area appeared in the model group and the electroacupuncture group in different degrees.After 3d of treatment,compared with model group,the volume of cerebral infarction in electroacupuncture group have no significant difference(P>0.05);after 7d and 14 d of treatment,the volume of cerebral infarction in electroacupuncture group was smaller than that in model group,with significant difference(P<0.01).Compared with the 3d group,the cerebral infarction volume of the electroacupuncture group was smaller than that of the 7d group and 14 d group(P<0.01).Compared with the 7d group,the cerebral infarction volume was reduced in the 14 d group(P<0.01).4.Western Blot results showed that ES and TSP-1 protein were expressed in a small amount in sham operation group.There were significant differences in ES and TSP-1 protein between model group and sham operation group at different time points(P<0.01).After 3d of treatment,there was no significant difference in the expression of ES and TSP-1 between electroacupuncture group and model group(P>0.05).After 7d and 14 d of treatment,the expression of ES and TSP-1 protein in electroacupuncture group was significantly lower than that in model group(P<0.01).Compared with the 3d group,the expression of ES and TSP-1 protein in the 7d and 14 d groups were significantly lower(P<0.01).Compared with the 7d group,there was no significant difference in the expression of ES protein in the 14 d group(P>0.05),the expression of TSP-1 protein in 14 d group was decreased(P<0.05).5.The results of RT-qPCR showed that ES mRNA and TSP-1 mRNA were expressed in a small amount in the sham operation group.The relative expressions of ES mRNA and TSP-1 mRNA in the model group were significantly higher than those in the sham operation group at 3d,7d and 14d(P<0.01).After 3d of treatment,there was no significant difference in the relative expression of ES mRNA between electroacupuncture group and model group(P>0.05),while the relative expression of TSP-1 mRNA in the electroacupuncture group was lower than that in the model group(P<0.01).After 7d of treatment,the relative expression of ES mRNA and TSP-1 mRNA in the electroacupuncture group was significantly lower than that in the model group(P<0.01).Compared with the 3d group,the relative expression of ES mRNA in the 7d group was significantly decreased(P<0.01),and the relative expression of TSP-1 mRNA was not significantly different(P>0.05).The relative expression of ES mRNA and TSP-1 mRNA in the 14 d group was significantly decreased(P<0.05 or P<0.01);compared with the 7d group,the relative expression of ES mRNA decreased in the 14 d group(P<0.05),and the relative expression of TSP-1 mRNA was not significantly different(P>0.05).6.The results of immunohistochemistry showed that ES and TSP-1 positive cells were slightly expressed in the sham operation group.The positive expressions of ES and TSP-1 in the model group were significantly increased at 3d,7d and 14 d compared with the sham operation group(P<0.01).After 3d of treatment,the positive expression of ES in the electroacupuncture group was lower than that in the model group(P<0.05),while there was no significant difference in the positive expression of TSP-1 in electroacupuncture group(P>0.05).After 7d,14 d,the positive expression of ES and TSP-1 in the electroacupuncture group was significantly lower than that in the model group(P<0.01).Compared with the 3d group,the positive expression of ES and TSP-1 was increased in the 7d group(P<0.05 or P<0.01),and the ES positive expression had no significant difference in the 14 d group(P>0.05),the positive expression of TSP-1 increased(P<0.05).Compared with the 7d group,there was no significant difference in the positive expression of ES and TSP-1 in the 14 d group(P>0.05).7.Observation of angiogenesis in ischemic areaThe microvessel density(MVD)of the sham operation group was slightly expressed.The MVD of the model group was higher than that of the sham operation group at 3d,7d and 14d(P<0.01).At each time point after treatment,the MVD of the electroacupuncture group was significantly increased compared with the model group(P<0.01).Compared with the 3d group,the MVD of the 7d and 14 d groups increased significantly(P<0.01).Compared with the 7d group,the MVD of the 14 d group increased significantly(P<0.01).Conclusions:1.Electroacupuncture can improve the signs of neurological deficit and pathological changes of brain tissue in MCAO rats,and reduce the volume of cerebral infarction.2.Electroacupuncture at different time points can down-regulate the expression of angiogenesis inhibitor ES and TSP-1 protein and mRNA in hippocampus of ischemic side of rats,which may be one of the mechanisms of electroacupuncture therapy promoting angiogenesis,thus restoring nerve function after cerebral ischemia and exerting brain protection. |
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