Investigation Of The Therapeutic Effect Of Acupuncture On The Behavior And Neural Angiogenesis In Rats With Cerebral Ischemia Reperfusion Injury

ObjectiveAfter cerebral ischemia and reperfusion, ischemic half dark zone is formed, and reperfusion injury impairs the brain tissue. Hypoxia and inflammation are particularly prominent. Patients clinically display severe partial body sensory, motor, and emotional disorders. This study is to investigate the therapeutic effects of acupuncture at Baihui and Zusanli sites on the behavior, nerve inflammation, hypoxia response as well as nerve vascular regeneration in the rat model of middle cerebral artery occlusion (MCAO) monitored by Doppler Blood flowmetry. We also applied phenotypic characterization and molecular biological methods to explore the mechanisms underlying acupunctural neural protective function against ischemic cerebral vascular diseases.MethodsAdult Sprague-Dawley rats were randomly divided into the following groups:the sham (S) group, the middle cerebral artery occlusion (MCAO) model groups of 2h MCAO followed by 1,3,5,7 d of reperfusion, the acupuncture (A) groups of 2h MCAO followed by 1,3,5,7 d of reperfusion, the electro-acupuncture (EA) groups of 2h MCAO followed by 1,3,5,7 d of reperfusion and the Drug groups of 2h MCAO followed by 1,3,5,7 d of reperfusion.Preparation of cerebral ischemia reperfusion injury model by using reforming longa method to occlusion the right middle cerebral artery of rats. When dropped about 70%, Blood flow in brain was measured by laser Doppler flowmetry.After the model is successfully buillt, The acupuncture group was given acupuncture Baihui and Zusanli on left after injection in 24h after reperfusion Groups ED were based on groups EA. Intraperitoneal injection of Edavarone everyday among the Drug group after the model is successful.Each group according to reperfusion time to score Bederson and mNSS evaluation nerve function to validate the model in rats. After 72h reperfusion, all brains of the rats were taken after anesthesia.After TTC staining, calculation the cerebral infarction volume with ImageJ. Application of immuno-histochemical staining and Western blot method to detect the expression of VEGF, Hif-la, Thorase, GFAP, TSPO expression in the cerebral ischemia rats. Detection of the expression of VEGF, Hif-la, Thorase and GFAP mRNAs in cerebral ischemia rats by rt-pcr method.Results1. Bederson Score shows that the score of the rats in the S group is near to O.No neurological deficit was founded while other groups exist different degree of neurological deficit after reperfusion 24h later. But as the treatment goes, the score of neurological deficit is lower, the apoptosis percentage in the MCAO groups significantly increased than those in the A group, the EA group and the Drug group.; no significant differences in apoptosis percentage were detected between A group, the EA group and the Drug group.2. TTC staining showed that the area of infarction core was in white color compared with the common red brain tissue. Among the five slices of brain, the slice around optic chiasma area had the most stable infarction region. EA and edaravone both significantly decreased the volume of the MCAO-evoked infarct region (compare groups EA and Edaravone group with the MCAO groups); no significant differences in infarct volume were detected between the EA groups and the Drug groups.3. Results related to Behavioral Function:mNSS Score shows:the scores in the sham group were 0; No neurological deficit and ischemia was founded while other groups exist different degree of neurological deficit after reperfusion 24h later. But as the treatment goes, the degree of neurological deficit is lower. In all 1,3,5,7 d four time points,the score of group EA and Drug group is lower than the model group; no significant differences in apoptosis percentage were detected between the A group,the EA group and the Drug group. Grip strength test showed, there is no muscle force damage in the sham group while there are different degrees of muscle force damage in the rest four groups. In the day 3、5、7,the muscle force of groups A, groups EA and the Drug groups were significantly higher than the MCAO groups; no significant differences between groups A groups EA and the Drug groups were detected.Cylinder test showed that there was no asymmetry on limb use score in sham group. The asymmetric index of using limb in EA group and drug group at each time point were lower than the model group, the difference statistically was significant. Compared to the model group, acupuncture group was significantly lower at 3d and 5d, the difference was statistically significant; Corner test showed that there was no bias in the use of limbs in sham group, its lateral index tends to 0. Forelimb lateral index in the acupuncture group and acupuncture group were lower than model group at each time point, the difference was statistically significant. Forelimb lateral index in drug group was higher than the model group at 3,5,7d, the difference was statistically significant Sucrose preference test showed sham-operated rats tend index were higher at each time point sugar; 5d and 7d on the two points in time, sugar and drug group EA group preference index were higher than the model group, the difference was statistically significance, the acupuncture group and model group no significant difference. There was no significant difference in each of the treatment groups. Open-field test showed that locomotor activity observed in open field did not differ between group son the total distance and mean velocity among all the groups. The cumulative duration and frequency of each group both in the center and in the border of the field at different time points was even not significantly different.4.Indicators on neurogenesis and angiogenesis:Immuno-histochemistry showed: VEGF, Hif-la, GFAP and Thorase protein in model group overall showed an increasing trend. Compared with the model group, EA group at 5d and 7d, the difference was statistically significant; Acupuncture group was significantly higher on VEGF protein than model group only at 7d time point, the difference statistically significant. Hif-la protein peak appeared at 1d, it was lower than acupuncture group, EA group, medication group at 3d and 7d, the difference was statistically significant. Compared with model group, acupuncture group, EA group, Drug group were higher on thorase protein at 1d and 7d, the difference was statistically significant. Compared to the model group, EA group was significantly higher than the model group on GFAP protein at 1d and 7d:c the difference was statistically significant, the drug group was significantly higher than the model group at 3d and 7d. the difference was statistically significant. WB results showed that acupuncture group was higher than the model group on VEGF protein at 1,3,7d, the difference was statistically significant in the EA group was significantly higher than when the 7d model group, the difference was statistically significance. The expression in the drug group 1,3,7d three time points higher than the model group, the difference was not statistically significant. Acupuncture group, EA group were significantly lower than model group on Hif-1a protein at 3d, the difference was statistically significant. Compared with model group, acupuncture group, EA group, drug group were higher than the model group on the thorase protein at 1d and 7d,the difference was statistically significant. Compared with model group, acupuncture group and EA group were significantly higher on GFAP protein at 1,3,5,7d, the difference was statistically significant, significantly higher in the drug group 1,5,7d, the difference was statistically significant. RT-PCR results showed that EA group was significantly higher than the model group on VEGF mRNA, the difference was statistically significant when compared with model group. Acupuncture group, EA group, drug group were reduced and then increased after the first rising trend for the peak of expression, but compared with the model group on Hif-la mRNA at 3d time, At the 3d, the expression of each treatment group and the untreated group no significant difference, drug group at 7d only significantly higher than the model group, the difference was statistically significant. Compared with model group, acupuncture group showed lower level at 1,3,5, on Thorase mRNA, the difference was statistically significant. EA group and drug group were significantly lower than the model only at 7d,the difference was statistically significant. Compared with model group, acupuncture group showed low GFAP mRNA expression at 1,3,5,7d, the difference was statistically significant. EA group and drug group were significantly lower than model group, only at 7d, the difference was statistically significant.Conclusion:1. Infarction area of ischemia area at 72h rats was stable after cerebral ischemia and reperfusion, acupuncture, electroacupuncture and drug therapy can save the ischemic lesion portion penumbra region.2. After cerebral ischemia and reperfusion, all rats showed significant barriers tend partial body and emotional disorders, acupuncture, electroacupuncture and drug therapy can effectively improve the neurological function, improve muscle strength and limb rats usage bias, ameliorate rats’ depression.3. All these factors about neurogenesis and angiogenesis were all up-regulated after cerebral injury. Acupuncture and electro-acupuncture intervention at rats Baihui and Zusanli can increase VEGF and GFAP, while reduce Hif-la, Thorase protein.InnovationThe copy of the MCAO model is based on the classic improved sature occlude method with intuitive verification under the test of CBF. After the models were to successfully established, we stratified five groups randomization according to different time points of neural function and behavioral function of rats to verify the effectiveness of acupuncture apparently. Finally, from the level of molecular biology to validation the mechanism of acupuncture of Baihui and Zusanli in the rat with cerebral ischemia reperfusion injury through the detection of VEGF, Hif-la, Thorase and GFAP on nerogenesis and angiogenesis related pathways. Research on the intervention effect of behavioral and neural protective function in the rat with cerebral ischemia reperfusion injury providing more thinking for clinical. There have not been found any researches on intervention mechanism of thorase in brain tissue of rat with cerebral ischemia reperfusion injury when acupuncture at Baihui and Zusanli at the same time through the literature retrieval.