| Objective:To study different intervention methods(electroacupuncture,enriched rehabilitation,electroacupuncture combined with enriched rehabilitation)and different period of treatment(3 d,7 d,14 d)in rats with middle cerebral artery occlusion(MCAO),influencing the expression of hypoxia-inducible factor-1α(HIF-1α)and its downstream factor vascular endothelial growth factor(VEGF)and erythropoietin(EPO),to explore the relationship between the ischemic side nerve structure and function recovery with blood vessels,exploring the possible mechanism of its brain protection,providing a theoretical basis and reference significance for the clinical treatment of ischemic stroke.Methods:1.Purchase 200 SPF male SD rats,number them according to the random number table method,randomly select 36 as the sham operation group,and prepare the MCAO model for the rest of the rats according to the modified suture method such as Longa.144successfully modeled rats were kept,and were randomly divided into model group,electroacupuncture group,enrichment rehabilitation group,and electroacupuncture combined enrichment rehabilitation group according to the random number table,with36 rats in each group.According to the duration of treatment,the above five groups of rats were subdivided into three subgroups of 3 d,7 d,and 14 d,with 12 rats in each subgroup.2.The rats in the treatment group(electroacupuncture group,enrichment rehabilitation group,electroacupuncture combined enrichment rehabilitation group)were treated with interventional therapy after modeling anesthesia and awake(approximately 4 hours postoperatively),once a day,and were treated continuously for 3 d,7 d,14 d.The rats in the electro-acupuncture group were stimulated by electro-acupuncture at the"Baihui"and"Shuigou"points and the"Housanli"and"Quchi"points on the left.The rats in the enriched rehabilitation group were placed in a rich rehabilitation environment and supplemented with rehabilitation training.The rats in the electroacupuncture combined enrichment rehabilitation group received rich rehabilitation training first,and rested for10 minutes after completion,and received electroacupuncture at the points"Baihui","Shuigou"and"Housanli"and"Quchi"on the left.Rats in the sham operation group and the model group were raised routinely,and only grasped and fixed under the same conditions without any intervention treatment.3.The modified neurological severity scores(m NSS)was used to determine the signs of neurological deficits in each subgroup at 4 h,3 d,7 d,and 14 d.Laser doppler flowmeter was used to measure the changes of regional cerebral blood flow(r CBF)of rats in each subgroup.Hematoxylin-eosin staining(HE)was used to observe the cerebral cortex of rats on the ischemic side of each group.Immunohistochemical staining(IHC)was used to determine the number of CD34~+positive cells in the cerebral cortex of the ischemic side of each subgroup,and the microvessel density(MVD)of the cerebral cortex of the ischemic side was calculated by Weidner method.Western Blot,RT-q PCR detection of HIF-1α,VEGF,EPO protein and gene expression in the cerebral cortex of the ischemic side of the rats in each subgroup.Results:1.Modified neurological severity scoresIn the four different time courses(4 h,3 d,7 d,14 d)after the operation,the scores of the rats in the sham operation group were all zero.The rats in the model group scores significantly higher than the sham operation group in the four time courses(P<0.01),and with the extension of the treatment time,the score gradually showed a downward trend.Compared with the model group,the difference between the electroacupuncture group and the enrichment rehabilitation group at 4 h and 3 d was not statistically significant(P>0.05),the scores were significantly reduced after 7 days and 14 days of treatment(P<0.01).The scores of the acupuncture group were extremely significant lower than the model group after 3 d,7 d and 14 d(P<0.01).Compared with the electro-acupuncture group and the enrichment rehabilitation group,the acupuncture group had no significant difference at 4 h and 3 d after the operation(P>0.05).After 7 d and 14 d of treatment,the score of the acupuncture group was significantly lower than that of the electro-acupuncture group.There was a significant difference between the acupuncture group and the enrichment rehabilitation group(P<0.05),the electroacupuncture group and the enrichment rehabilitation group had no statistical significance in the above four time courses(P>0.05).2.Regional cerebral blood flow on the ischemic sideThe r CBF of the rats in the sham operation group was normal and stable for the four time courses(5 min,3 d,7 d,14 d)after the operation.Compared with the sham operation group,the r CBF of the rats in the model group was significantly reduced in the four time courses after the operation(P<0.01),and with the extension of the treatment time,r CBF gradually showed an upward trend.Compared with the model group,there was no significant difference in r CBF between the electroacupuncture group and the enrichment rehabilitation group at 5 min and 3 d(P>0.05),r CBF was extremely significant higher than the model group after 7 d and 14 d(P<0.01).The r CBF of the acupuncture group was significantly higher than the model group at the three time courses of 3 d,7 d and 14 d(P<0.01).Compared with the electroacupuncture group and the enrichment rehabilitation group,there was no significant difference in r CBF in the acupuncture group at 5 min after the operation(P>0.05).When the time course was 3 d,7 d,and 14 d,the r CBF of the electroacupuncture group was significantly higher than that of the electroacupuncture group and the enrichment rehabilitation group(P<0.01).Compared with the enrichment rehabilitation group,there was no statistically significant difference in r CBF in each group(P>0.05).3.Histopathological changes in the ischemic side of the brainIn the sham operation group,the neurocytes in the cortex on the ischemic side were normal at 3 d,7 d,and 14 d after the operation,with complete structures,neatly arranged and tightly arranged,with no edema around the cells,and no inflammatory cell infiltration.The four groups(model group,electro-acupuncture group,enriched rehabilitation group,electroacupuncture combined enrichment rehabilitation group)showed loose cell structure and network-like structure,disordered and sparse arrangement,a large number of nerve cells pyknosis,degeneration and necrosis,and obvious interstitial edema 3 days after operation.Capillary swelling and deformation were obvious,glial cell proliferation,nucleolus hyperchromatism,etc,and there was no significant difference in brain pathology between the four groups under light microscope;compared with 3 days after operation,the four groups(model group,electroacupuncture group,Enrichment rehabilitation group,electroacupuncture combined enrichment rehabilitation group)all improved 7 days after operation.Compared with the model group,the brain tissue interstitial edema,cell structure,and arrangement of the three treatment groups(electroacupuncture group,enrichment rehabilitation group,electroacupuncture combined enrichment rehabilitation group)were significantly reduced,the repair was better,and the acupuncture group improved the most.The brain tissue damage of the four groups(model group,electro-acupuncture group,rich rehabilitation group,electroacupuncture combined enrichment rehabilitation group)was significantly reduced on the 14th day after the operation.Compared with the model group,the cell structure in the ischemic cortex of the treatment group was clearer,the shape was more regular,the arrangement was more orderly,the interstitial edema was significantly reduced,and the improvement in the electroacupuncture combined enrichment rehabilitation groupwas the most obvious.4.Microvessel density in ischemic cortexThe rats in the sham operation group showed no significant changes in MVD in the cortical area of the ischemic side at three different treatment time courses(3 d,7 d,14 d)after the operation,and only a small amount of expression was observed in the rats in the model group.The MVD of the cerebral cortex in the ischemic side of the rats in the sham operation group was significantly higher than that of the simultaneous course(P<0.01).Compared with the model group,the electroacupuncture group,the enriched rehabilitation group,and the electroacupuncture combined enriched rehabilitation group were treated for 3 d,7 d and 14 d the MVD of the cortex on the posterior ischemic side increased,and the difference was significant(P<0.05 or P<0.01).Compared with the electroacupuncture group and the enrichment rehabilitation group,the MVD of the electroacupuncture combined enriched rehabilitation group increased after 3 d,7 d,and14 d after treatment(P<0.05).Compared with the electroacupuncture group and the enriched rehabilitation group,there was no significant difference above three time courses(P>0.05).5.HIF-1α,VEGF,EPO protein expression in ischemic cortexIn the sham operation group,there was no significant difference in the expression of HIF-1α,VEGF,and EPO protein in the cerebral ischemic cortex area of rats in the three time courses(3 d,7 d,14 d).Compared with the sham operation group,the expression of HIF-1α,VEGF,and EPO protein in the model group rats extremely significant increased(P<0.01).Compared with the model group,the expression of HIF-1α,VEGF,and EPO protein in the electroacupuncture group,the enriched rehabilitation group,and the electroacupuncture combined enriched rehabilitation group in the same time course of rats were significantly increased(P<0.01).Compared with the electroacupuncture group and the enriched rehabilitation group,the electroacupuncture combined enriched rehabilitation group was treated for 3 d,7 d,the expression of HIF-1α,VEGF,and EPO increased significantly after 14 days(P<0.01).There was no significant difference in the electroacupuncture group and the enriched rehabilitation group in the expression of HIF-1αand VEGF at 3 d,7 d and 14 d.There was no significant difference in the expression of EPO at 3 d and 14 d between the electroacupuncture group and the enriched rehabilitation group,but the treatment time course was 7 d,the expression of EPO protein in the enriched rehabilitation training group was significantly higher than that in the electroacupuncture group(P<0.01).6.Expression of HIF-1αmRNA,VEGF mRNA and EPO mRNA in ischemic cortexThe expressions of HIF-1αmRNA,VEGF mRNA,and EPO mRNA in the cerebral ischemic cortex of rats in the sham operation group were not significantly changed during the three time courses(3 d,7 d,14 d).The expressions of HIF-1αmRNA,VEGF mRNA and EPO mRNA in the three ischemic cortical areas of the same time course increased after operation,and the difference was extremely significant(P<0.01).Compared with the model group,the electroacupuncture group,the enriched rehabilitation group,and the electroacupuncture combined enriched rehabilitation group the expressions of HIF-1αmRNA,VEGF mRNA and EPO mRNA in the three time-course ischemic cortical areas of rats were significantly increased(P<0.01).Compared with the electroacupuncture group and the enriched rehabilitation group,the electroacupuncture combined enriched rehabilitation group was treated for 3 d,7 d,14 d,the expression levels of HIF-1αmRNA,VEGF mRNA,and EPO mRNA all increased,and the difference was extremely significant(P<0.01).Compared with the enriched rehabilitation group,the electroacupuncture group and the enriched rehabilitation group showed that there was no significant difference in the expression of HIF-1αmRNA,VEGF mRNA,and EPO mRNA(P>0.05).Conclusions:1.Electroacupuncture,enriched rehabilitation training,and electroacupuncture combined with enriched rehabilitation training are all effective treatments for ischemic stroke,which can effectively improve the signs of neurological deficits in MCAO rats,increase local cerebral blood flow,and promote the recovery of nerve cell structure,To increase the number of new capillaries.Compared with electroacupuncture and enriched rehabilitation training monotherapy,the effect of electroacupuncture combined with enriched rehabilitation training therapy is more significant,and within fourteen days,the treatment duration is positively correlated with the treatment effect.2.The possible mechanism of the therapeutic effect of electroacupuncture combined with enriched rehabilitation training on MCAO rats is that early intervention further activated the expression of HIF-1αprotein and gene,and then activated the expression of HIF-1αdownstream factors VEGF and EPO,mediating endothelial angiogenesis and playing a brain protective role. |
PDF Full Text Request