| Objective:To explore the regulatory effect of electroacupuncture combined with rich rehabilitation training on angiogenesis at different time points,this experiment takes neovascularization as the breakthrough point and chooses AKT/eNOS signal pathway as the intervention target,which can help us enrich the experimental basis and theoretical basis for clinical treatment of ischemic stroke and enrich clinical treatment.Methods:1.A total of 160 healthy male SD(Sprague Dawley)rats(SPF grade)weighing280g-320 g were randomly selected as sham operation group(Sham group),and the other rats were used to establish the model of middle cerebral artery occlusion(middle cerebral artery occlusion,MCAO).According to the complete random principle,they were divided into model(Model)group,electroacupuncture(EA)group,rehabilitation(RT)group and acupuncture combined with rich rehabilitation training(EA+RT)group.Each group was divided into three subgroups according to the three time points of 3d,7d and 14 d,with 9 rats in each subgroup and 27 rats in each group.2.The rats in the Sham group and the Model group were routinely fed in the animal room without other treatment.The rats in the EA group were treated with electroacupuncture at four acupoints "Baihui","Fengfu","Xinshu" and "Neiguan"(selected points on the hemiplegic side)once a day,20 minutes per time and rich rehabilitation training(including sound,light,touch and other sensory stimulation and rehabilitation training)once a day for 14 days.The EA+RT group was intervened by electroacupuncture and rich rehabilitation training.3.According to the longa5 score system,the neurological deficit score was recorded at 4 hours after modeling and 3rd day,7th day and 14 th day after treatment;the cerebral infarction volume was measured by TTC staining,the pathological changes of brain tissue were observed by hematoxylin-eosin(HE)staining;the expression of AKT/p-AKT/eNOS/p-eNOS protein in ischemic cortex was detected by Western blot;the expression of AKT/eNOSmRNA was detected by RT-q PCR;the microvessel density(Microvessel Density,MVD)of ischemic penumbra was counted by Weidner method.Results:1.There were no signs of neurological deficit at all time points in the Sham group and theNDS was 0.Compared with the Sham group,theNDS of the Model group increased significantly at 4h,3th day and 7th day after operation(P<0.01),but there was no significant difference between the Model group and the EA group at 4h and 3rd day after operation(P>0.05).Compared with Model group,there was no significant difference in EA,RT and RA+RT groups at 4h after operation and 3rd day after intervention,but NDS decreased significantly at 7th day and 14 th day after intervention(P<0.01 or P<0.05).Compared with EA or RT groups,there was no significant difference in NDS at 4h after operation,3rd day,7rd day after operation in EA+RT group,but NDS decreased significantly at 14 th day after operation(P<0.01),suggesting that the therapeutic effect gradually accumulated with time went by after operation.Comparison of the different time points in the same group: in the EA group,compared to 3th day,theNDS was significantly decreased(P<0.01 or P<0.05)and the signs of neurological deficit were improved on the 7th day in the EA group;there was no significant difference between the 14 th day and the 7th day in the RT group.In the EA+RT group,compared with the 3rd day,theNDS decreased significantly on the 7th day(P<0.01 or P<0.05),and there was no significant difference between the 14 th day and the 7th day.2.Brain histopathological observation(HE staining)showed that in the Sham group,the structure of the cells in the ischemic cortex was intact,the cytoplasm was evenly distributed,the nucleus was purple and regular,and the cells showed normal morphology,and there was no significant change between 3rd day,7th day and 14 th day after operation.In Model group,a large number of shrinking cells were observed in the ischemic cortex,the arrangement was disordered,the nucleus deformed,dissolved or even disappeared,the cell structure was disordered,and the cytoplasm was blue-purpleand uneven,and the cumulative pathological changes weakened gradually with time.Compared with the Model group,the ischemic cortex of EA group,RT group and EA+RT group had less pathological changes,more complete cell structure,more uniform staining and more orderly arrangement of cells at three time points after operation,and the degree of improvement in EA+RT group was more obvious than that in EA group and RT group.3.The volume of cerebral infarction(TTC staining)was stained by TTC,the ischemic area of brain tissue was pale and the normal tissue was red,and the boundary between them was obvious.The results showed that the infarct volume of Sham group was 0 at 3rd day,7th day and 14 th day after operation.Compared with Sham group,the infarct volumes of 3rd day,7th day,14 th day in Model group,EA group,RT group and EA+RT group increased significantly after operation(P<0.01),which indicated that the model was replicated successfully.The infarct volume in EA group was significantly smaller than that in Model group at 3rd,7th and 14 th day after intervention(P<0.01).The infarct volume in RT group was significantly smaller than that in Model group at3 rd,7th and 14 th day after intervention(P<0.01);the infarct volume in EA+RT group was significantly smaller than that in EA group at 3rd,7th and 14 th day after treatment(P<0.01 or P<0.05);the infarct volume in EA+RT group was significantly smaller than that in RT group at 3rd,7th and 14 th day after intervention(P<0.01).There was no significant difference between EA group and RT group at each time point(P>0.05).4.The relative expression of AKT/p-AKT/eNOS/p-eNOS protein was detected by Western.The expression of AKT/p-AKT/eNOS/p-eNOS protein in the ischemic cortex of Sham group rats is normal.Compared with the Sham group,The AKT/p-AKT/eNOS/p-eNOS protein in the ischemic side of the treatment group was significantly increased on the 3rd,7th and 14 th days(P<0.01)in Model group;compared with the Model group,the expression of AKT/p-AKT/eNOS/p-eNOS protein increased significantly on the 3rd,7th and 14 th day after treatment in EA group,RTgroup,EA + RT group(P<0.01);compared with the EA group,the expression of AKT/p-AKT/eNOS/p-eNOS protein on the 3rd,7th and 14 th day were significantly increased in EA + RT group(P<0.01 or P<0.05);compared with RT group,the expression of AKT/p-AKT/eNOS/p-eNOS protein on the 3rd,7th and 14 th day were significantly increased in EA + RT group(P<0.01 or P<0.05);there was no significant difference between the EA group and the RT group at each time point.5.The expression of AKT/eNOS mRNA detected by RT-q PCR.Compared with the Sham group,the expression of AKT/eNOS mRNA in the ischemic cortex of the Model group rats increased significantly at three time points after surgery(P<0.01);compared with the Model group,the expression of AKT /eNOSmRNA in the ischemic cortical area of ??the EA,RT,EA + RT group increased significantly at three time points after intervention(P<0.01 or P<0.05);compared with the EA group,the expression of AKT/eNOSmRNA in RT group has no statistically significant difference while the expression of AKT / eNOSmRNA in the EA + RT group increased significantly(P<0.01);compared with the RT group,the expression of AKT/eNOSmRNA in the EA + RT group also increased significantly(P<0.01 or P<0.05).6.Observation of angiogenesis in the ischemic area.Compared with the Sham group,the microvessel density(MVD)of the Model group increased slightly at 3rd,7th,and 14 th day after surgery(P<0.05 or P<0.01);compared with the Model group,EA,RT,EA + RT group MVD increased significantly at three time points after surgery(P<0.01).Compared with EA group,the MVD in EA +RT group increased significantly at each time point after surgery(P<0.01).Compared with the RT group,the number of MVD in the EA + RT group increased significantly at each time point after surgery(P<0.01).There was no significant difference between the EA group and the RT group at each time point.Comparison of different time points in the same group: the MVD of the EA group was significantly increased from 3rd day to7 th day(P<0.01),and the MVD was also significantly increased from 7th day to 14thday(P<0.05).The MVD of the RT group was significantly increased from 3rd day to7 th day(P<0.01),the MVD also increased significantly from 7th day to 14 th day(P<0.05).The MVD increased significantly from 3rd day to 7th day in the EA + RT group(P<0.01),and the MVD also increased significantly from 7th day to 14 th day(P<0.05).Conclusion1.Electroacupuncture combined with rich rehabilitation training can effectively improve the pathological changes of MCAO rat brain tissue and improve cell morphology.2.Electroacupuncture combined with rich rehabilitation training can significantly improve the signs of neurological deficits and reduce the volume of cerebral infarction in MCAO rats at different time points after operation,and combined therapy is superior to monotherapy.3.Electroacupuncture combined with rich rehabilitation training can effectively regulate the expression of angiogenesis-related factors AKT/p-AKT/eNOS /p-eNOS protein and AKT/eNOSmRNA in the ischemic cortex of MCAO rats and increase microvessels density,promotes angiogenesis at different time points after operation,and the effect is better than monotherapy.It is speculated that the peak curative effect may be between 7d and 14 d after surgery. |
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