Effect Of Electroacupuncture Combined With Rehabilitation Training On Angiogenesis Related Factors VEGF/VEGFR₂/bFGF/CD34⁺ In Acute MCAO Rats

ObjectiveTo explore the mechanism of angiogenesis in rats with acute cerebral artery occlusion?MCAO?by Electroacupuncture combined with enrichment Rehabilitation Training/Therapy method?ER?and try to explained the possible mechanism of angiogenesis after ischemic stroke by ER In order to provide basic experimental basis for enriching clinical interventions.Methods1.165 male SD?Sprague Dawley?rats?clean Grade?were randomly divided into sham operation group?n=27?and model group?n=148?.The model module was copied to the right MCAO by Zea Longa et al.R model.According to the neurological deficit score?NDS?,30 failed and dead rats were excluded.The rats after successful modeling were randomly divided into model group,Electroacupuncture combined with Rehabilitation?ER?group,Electroacupuncture?EA?group and Rehabilitation Therapy?RT?group.2.The model group and the sham operation group were not treated;the ER group and the EA group were given electroacupuncture treatment 4 hours after the operation,and the acupoints were selected by Baihui?DU20?,Shuigou?DU26?,Neiguan?PC6?,TheER group,RT group performed a rich rehabilitation environment?sound,light,color,etc.?stimulation and rich Rehabilitation training?running platform,roller,balance beam,screen?4 hours after surgery,once a day,2 days off for 7 days of treatment.3.The five groups of animals were treated with modified neurological severity scores?mNSS?for 1,3,7 and 14 days to observe the degree of neurological deficit in each group.Laser Doppler flowmetry was used.Laser-Doppler flowmetry?LDF?was used to detect regional cerebral blood flow?rCBF?at 3,7 and 14 days after insertion of the animals in each group.TTC was taken at interventions 3,7,and 14d.The cerebral infarction volume of MCAO rats in different groups was measured by staining.The expression of CD34⁺was detected by immunohistochemistry?IHC?.Microvessel Density?MVD?was measured by Weidner method.The expression of protein and gene in hippocampus of cerebral infarction group was detected by Western blot and RT-qPCR.Results1.mNSSThe mNSS of the rats in the sham operation group at1,3,7,14d were 0 score.Compared with the sham operation group,the MCAO model group was treated at four time points.The mNSS of the rats was decreased?P<0.01?,indicating that the model was successful.Compared with the MCAO model group,the ER group were divided into four groups at 1,3,7,and 14 days?P<0.05 or P<0.01?;There was no significant difference between the EA group and the RT group and the model group at 1 and 3 days?P>0.05?.The difference was statistically significant at the 7th and 14th day?P<0.05 or P<0.01?.There was no significant difference in mNSS between the ER group,EA group and the RT group at the 1st intervention period?P>0.05?.The difference was statistically significant at 3,7 and 14 days?P<0.05 or P<0.01?.2.Regional cerebral blood flow?rCBF?The results showed that compared with the sham operation group,the rCBF of the model group decreased at four time points?5 min,3,7,14 d after insertion??P<0.01?;compared with the model group,the intervention of the ER At 3,7,and 14d,the recovery was significant?P<0.01?.There was no significant difference between the EA group and the RT group and the model group.There was no significant difference between the 5 min after insertion and 3days interventions?P>0.05?.Statistical significance ?P<0.01?;there was no significant difference in rCBF between the ER group and the EA group and the RT group?P>0.05?,and the difference was statistically significant at 7and 14 days after intervention.P<0.05 or P<0.01);there was no significant difference in rCBF between the EA group and the RT group at four time points?P>0.05?.3.Measurement of cerebral infarction volume?TTC staining method?After TTC staining,the boundary between the ischemic area and the normal tissue is clearly pale,and infarction can occur in the frontal,apical,temporal,and basal ganglia.The cerebral infarction volume of the sham operation group was 0 points at three time points?3,7,14d?,and the cerebral infarction volume of the model group at three time points was higher than that of the sham operation group.The model was also successful;compared with the model group,the infarct volume of the ischemic side was significantly decreased at 3,7 and 14 days after intervention?P<0.01?;the EA group and the RT group were compared with the model group:intervention 3,7,14d The infarct volume was decreased in the EA group,the RT group and the model group?P<0.01?.There was a statistically significant difference in the volume of cerebral infarction between the ER group and the EA group and the RT group at 3,7 and 14 days?P<0.01?,there was no significant difference in cerebral infarction volume between the EA group and the RT group at 3,7 and 14 days?P>0.05?.4.CD34⁺count microvessel density?MVD?The results showed that only a small amount of brown-yellow-stained CD34⁺expression was observed under the light microscope in the sham operation group.A certain number of CD34⁺positive expressions were observed around the cerebral ischemic area in the model group,but the number was small.In the treatment group,the positive expression of CD34⁺was clearly observed under light microscope.The number of CD34⁺was gradually increased with the prolongation of treatment time.Among them,the trend of the needle group was more obvious than that of the electroacupuncture group and the rehabilitation group.At 3,7 and 14 days,the model group was compared with the sham operation group,the ER group and the model group,the EA group,the RT group and the model group,the ER group and the EA group,and the RT group.The number of CD34⁺positive cells increased around the ischemic penumbra?P<0.05 or P<0.01?.There was no significant difference in the number of CD34⁺positive cells between the EA group and the RT group at three time points?P>0.05?.5.Western blot detection of VEGF/VEGFR₂/bFGF/CD34⁺proteinCompared with the sham operation group,the model group had vascular endothelial growth factor?VEGF?/vascular endothelial growth factor receptor 2?VEGFR₂?/basic fibroblast growth factor?bFGF?on the ischemic side at 3,7 and 14 days after operation.The relative expression of CD34⁺was significantly increased?P<0.01 or P<0.05?.Compared with the model group,the relative expression of VEGF/VEGFR₂/bFGF/CD34⁺in the ischemic side of rats in the ER group was significantly increased at 3,7 and 14 days.The increase was?P<0.01?.The expression of VEGF/VEGFR₂/bFGF/CD34⁺in the model group and the EA group and the RT group was significantly different at 3,7 and 14 days?P<0.01 or P<0.05?.The expression of VEGF/VEGFR₂/bFGF/CD34⁺was significantly increased in the EA group and the RT group at 7 and 14 days?P<0.01 or P<0.05?.6.RT-qPCR detection of VEGF/VEGFR₂/bFGF/CD34⁺mRNACompared with the sham operation group,the relative expression of VEGF/VEGFR₂/bFGF/CD34⁺mRNA in the ischemic side of the model group was significantly increased at 3,7 and 14 days after operation?P<0.01 or P<0.05?;Compared with the model group,the ER group relative expression of VEGF/VEGFR₂/bFGF/CD34⁺mRNA in the ischemic side of the rats was significantly increased at 3,7 and 14 days?P<0.01?.There were significant differences in the expression of VEGF/VEGFR₂/bFGF/CD34⁺mRNA between the model group and the EA group and the RT group at 3,7 and 14 days.?P<0.01 or P<0.05?.Compared with the EA group and the RT group at 3,7 and 14 days after intervention.The expression level of The VEGF/VEGFR₂/bFGF/CD34⁺mRNA was significantly increased?P<0.01?.Conclusions1.ER can promote neurological function reconstruction and improve regional cerebral blood flow in rats with acute focal cerebral ischemia?MCAO?,and combined therapy is superior to electroacupuncture and rehabilitation therapy.2.ER can reduce the volume of infarct in acute MCAO rats,and combined therapy is superior to simple electroacupuncture and rich rehabilitation therapy.3.ER can promote the expression of VEGF,VEGFR₂,bFGF and CD34⁺in the ischemic penumbra of acute MCAO rats,thereby increasing the microvessel density,possibly promoting the homing of endothelial progenitor cells by accelerating microangiogenesis,thereby promoting microvessels.Freshmen,this may be one of the related mechanisms of electroacupuncture combined with rich rehabilitation training to promote neurological remodeling after cerebral ischemia.