| Objective:To observe the effect of electroacupuncture combined with two different acupoint combinations on the expression of vascular endothelial growth factor(VEGF)and brain-derived neurotrophic factor(BDNF)in ischemic stroke rats.By regulating angiogenesis to promote the functional recovery of ischemic brain injury,the efficacy and possible mechanism of the aforementioned therapy on stroke are discussed.Comparing the differences in the therapeutic effect of different acupoint combinations on post-stroke angiogenesis provides an experimental basis and theoretical reference for the treatment of this disease by electroacupuncture with different acupoint combinations.Methods:Referring to Zea Longa,Belayev and other right external carotid artery,a total of72 healthy male clean Sprague-Dawley(SD)rats were selected to establish focal middle cerebral artery ischemia model.After 4h,the rats were selected and divided into four groups randomly,which were sham operation group,model group,electroacupuncture group and electroacupuncture group(two groups).A group of electroacupuncture acupoints "Shenmen" Neiguan "Xinshu" and "Baihui",two groups of electroacupuncture acupoints "Quchi" "Hegu","Zusanli" ditch "".18 rats in each group were divided into three subgroups of 3D,7d and 14 d at different time points after the treatment.Each group had 6 rats each.At the first postoperative day,a 0.20×13mm needle was used to puncture the needle.The parameters of the EA were: density wave,5to 100 times/s.Time 20 min,once a day,the strength of rats can be tolerated as degree(usually 3 ~ 5V),each group were treated for 3d,7d,14 d.According to the different symptoms of nerve function defect score of rats in each group to observe the motor function and the recovery of nerve injury,and the application of hematoxylin and eosin staining(HE staining)to observe pathological changes in hippocampus of rats with ischemia,immunohistochemical method and Weidner method to the ischemic area(Microvessel Density,Microvessel density MVD)were measured to observe the ischemic area around angiogenesis,immunohistochemistry and chromogenic Western blotting(Western Blot)detection and analysis of expression of VEGF and BDNF related factors in cerebral infarction group ischemia rats died in hippocampus area.The effect of Electroacupuncture on VEGF mRNA and BDNF mRNA in the hippocampus of acute ischemic rats was observed by real-time fluorescence quantitative PCR(RT-qPCR).Results:1.The neural function defect scoreSham operation group at different time after operation(4h,3d,7d,14d)neurological deficit score was 0;model group at different time(4h,3d,7d,14d)of the rat neurological severity scores were significantly higher than those in sham operation group,statistically significant differences(P<0.01);and 3D after 4h,EA group and the score defect degree of EA group two nerve function were lower than the model group,but the difference was not statistically significant(P>0.05);postoperative 7d score,EA group and EA group two nerve function defect degree were lower than the model group,with statistical significance the difference(P<0.05);postoperative 14 d,electroacupuncture group neurological deficit score was lower than that of model group,the difference was statistically significant(P<0.01),EA group two neurological impairment score lower than the model group,with statistical difference Learning significance(P<0.05),compared with the two treatment groups,the score of nerve function defect in electroacupuncture group was lower than that in electroacupuncture group two,and the difference was statistically significant(P<0.05).2.Histopathological observationThe sham operation group,brain tissue morphology and structure were complete,the number of nerve cells,nucleus center clear,oval or circular;the rest of the group at3 d after operation,there are different degrees of brain edema,loose structure,the number of nerve cells significantly reduced the infarct area,arrangement disorder,nuclear pyknosis degeneration and other pathological changes.After 7d,compared with the model group,the pathological changes of the ischemic hippocampal area in the electroacupuncture group were relieved in varying degrees.At 14 d after operation,the pathological changes of hippocampus in the ischemic side of rats decreased,and the degree of disorder of cell arrangement and degeneration of nuclei was also significantly reduced in the two groups.3.Expression of VEGF protein and BDNF protein(1)VEGF: data analysis showed that there was a small number of VEGF positive cells in the hippocampus in the sham operation group after cerebral ischemia,while the number of VEGF positive cells in the model group was higher than that in the sham operation group,and the difference was statistically significant(P<0.05).The number of VEGF positive cells in two treatment groups was significantly expressed at 3d,7d and14 d after operation,and reached the peak at 7d.The number of positive cells in treatment group at each time point was higher than that in sham operation group,the difference was statistically significant(P<0.05).In the Western blot analysis further and real-time fluorescence quantitative PCR method,we found that the sham operation group after cerebral ischemia in ischemic hippocampus VEGF protein and VEGF mRNA cells,and the model group the expression of VEGF protein and VEGF mRNA expression were significantly higher than the sham group,the difference was statistically significant(P<0.05).The expression of VEGF protein and the expression level of VEGF mRNA in the two treatment groups were higher than that in the model group,and the difference was statistically significant(P<0.05).(2)BDNF: data analysis showed that there was a small number of BDNF positive cells in the hippocampus in the sham operation group after cerebral ischemia,while the number of BDNF positive cells in the model group was higher than that in the sham operation group,and the difference was statistically significant(P<0.05).The number of BDNF positive cells in the two treatment groups showed significant expression at 3d,7d and 14 d after operation,and reached the peak at 7d.In the Western blot analysisfurther and real-time fluorescence quantitative PCR method,we found that the sham operation group the expression of BDNF protein after cerebral ischemia in ischemic hippocampus and BDNF mRNA cells,and the model group the expression of BDNF protein and BDNF expression of mRNA was higher than that of sham operation group,and the difference was statistically significant(P<0.05).The expression of BDNF protein and the expression level of BDNF mRNA in the two treatment groups were higher than that in the model group,and the difference was statistically significant(P<0.05).4.Observation of angiogenesis in ischemic areaThe sham operation group did not find the number of vessels or only a very small amount of staining under microscope,staining around a certain number of blood vessels of rats in model group were observed under microscope in cerebral ischemic area,but the number is less.The treatment group could see clearly new blood vessels in the microscope,and the number of vessels increased with the prolongation of treatment time.Among them,the electroacupuncture group was more obvious than the electroacupuncture two group.Conclusion:1.Electroacupuncture treatment can improve the neurological deficits after focal cerebral ischemia in rats,and the therapeutic effect of Naoxintongzhi group is better than the traditional acupoint combination.2.Electroacupuncture can improve the pathological changes of ischemic tissue in rats,and the therapeutic effect of Naoxintongzhi group is better than the traditional acupoint combination.3.Electroacupuncture can increase the protein expression and mRNA expression of VEGF and BDNF in the ischemic hippocampus of rats.The interaction between nerve and blood vessels can promote the angiogenesis after cerebral ischemia,so as to achieve the protective effect on brain injury.4.Different acupoint combination therapy may have different treatment effect oncerebral ischemia rats.Through the relationship between heart and brain to guide the distribution of points,the effect may be better than the traditional acupoint combination. |
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