| Objective: Vascular Dementia (VD hereinafter) is a kind of acquired intelligence damage syndrome caused by brain dysfunction resulted from various kind of Vascular diseases. It subordinates to the disease scope of dementia of old age in the light of TCM (tradditional Chinese medincine). In the recent years, the prevalence of vascular dementia is on the rise and affects seriously the people's working ability as well as living quality. We made deep research on the etiology and pathogenesis of VD according to the traditional knowledge of Chinese medicine and modern research achievement and concluded that the fundamental pathogenesis of VD lies in Qi deficiency, blood stasis and collateral-obstruction of the brain. Therefore, we made the treatment principle of"strengthening qi, promoting blood flow", clinical treatment with electroacupuncture, dazhui,baihui,geshu and zusanli are selected as the main points, we got highly effect in treating VD. In order to make probe into its treatment action mechanism, the experiment observed its effect on praxiology, pathomorphology, oxygen free radicals, cell apoptosis of hippocampus. Methods:Part One The effect of electroacupuncture on praxiology and cerebral pathomorphology of VD miceWe duplicated the model of VD mice with the cerebral ischemic-reperfusion operation with the ligation both side carotid artery. 80 Kunming mice were divided into four groups at random, which were sham-operation group, model group, electroacupuncture group and Nimodipine group. After anesthesia by abdominal injection(350mg/kg)with chloral hydrate, the mouse was fixed on the operation platform in supine, after routine sterilization, the incision was done at the center of neck and bilateral common carotid arteries were separated. And the blood flow was occluded with No.4 silk suture for 20min, meanwhile, 1cm length of tail was clipped off to bleed about 0.3mL and the bleeding was stopped with heat cure. Then released for 10min and occluded again for another 20min, after the second reperfusion, the mouse was observed for 30min and the incision was sutured. In sham-operated group, bilateral common carotid arteries were only separated but not occluded and the tail was not clipped off to bleed. The observing time was same to the other groups.The treatment started after the mouse analepsia that day, dazhui,baihui,geshu and zusanli are selected as the main points in the electroacupuncture group, with No.30 acupuncture pin of 0.5cun pricked into, use electric acupuncture apparatus, adopt thin dense wave with the frequency of 2~80Hz, at the strength of the mouse limbs trill lightly but not struggle and vocalization, 10min every time; The Nimodipine group were given Nimodipine(30mg/kg); Except the electroacupuncture group, the other groups were fixed with same position 10min; Except the Nimodipine group, the other groups were given physiological saline(10ml/kg). The treatment run by same person, one time per day, lasted 15 days. To observe the learning and memory ability of the mouse, the praxiology experiment (jumping stand)was performed on the end of the treatment. After that, the mice were beheaded and the brains were taken out on the ice plate, the olfactory bulb and cerebellum were thrown away. The left part of which was fixed by 10% formaldehyde solution. The tissue was stained by HE(Hematoxylin)and Nissl. We observed constitutional morphologic changes of cerebral tissue under light microscope.Part Two The effect of electroacupuncture on oxygen free radicals in brain of VD miceThe way to make model and the treatment were same to part one. The mice were beheaded, the right brain was make into 10% homogenate. The homogenate was centrifugated for 5min at 4℃, 3600r/min, the supernatant was extracted. The experiment used biuret method to determin protein level, and thio-barbituric acid compared color analytical method to detect the MDA content, xanthine oxidase method to detect the activity changes of SOD in cerebral tissue of Kunming mice.Part Three The effect of electroacupuncture on Apoptosis of hippocampus cell in brain of VD micewe used the method of Terminal Deoxynucleotidyl Transferase-mediated dUTP-Nick End-labeling (TUNEL)to label the apoptosis cells. The way to make model and the treatment were same to part one. The mice were beheaded, the left part of which was fixed by 10% formaldehyde solution. Embed with paraffin,slice,take off paraffin at rule, digest Proteinase K; Application of sample,reaction,coloration were conducted strictly according to the directions in its kit; Mounting,test under microscope and image analysis at rule after reaction, observe the number of apoptosis cell in each group.Results:Part One The effect of electroacupuncture on praxiology and cerebral pathomorphology of VD mice1 Results of praxiology experimentModel group had longer reaction time(the time of jumping up the stand)(51.87±7.79), shorter latency time(the time of jumping down the stand)(72.99±10.53)and more wrong times(8.00±1.96,8.19±2.21)than sham-operation group (16.72±3.26,262.10±35.29,1.52±0.23,1.38±0.20)(P<0.01). At the same time, electroacupuncture group(43.10±4.38,167.71±23.36,4.54±0.54,4.08±0.52)and Nimodipine group(47.31±7.01,130.17±27.79,5.14±0.78,5.11±0.74)had shorter reaction time, longer latency time and less wrong times than model group(P<0.05 or P<0.01). Compared with Nimodipine group, the electroacupuncture group had better memory(P<0.05 or P<0.01), but had no difference in learning(P>0.05).2 The effect of electroacupuncture on Hippocampus pathomorphology in brain of VD miceGeneral morphology of brain tissues was shown under light microscope: In the sham-operated group, the hippocampal pyramid cells in CA1 area were closely arranged by HE staining, the karyons were round and big and the nucleoli were clear; Rich Nissl bodies were seen by Nissl staining and dense, well-arranged nerve fibers were observed as well. In the model group, the layers of hippocampal pyramid cells were not obvious by HE staining, the cells were evidently lost and the cell line became indistinct, the structure of many cells turned incomplete and the gap emerged among confused, sparsely arranged cells; nucleus volume shrank,dark staining, unclear structure, karyopyknosis appeared; Nissl bodies inside cytoplasm disappeared and nerve fibers were in disorder by Nissl staining. In all treatment groups, the cell lines in CA1 area of hippocampus were relatively clear by HE staining, the arrangement of cells was relatively compact and the amount of cells increased obviously; Nissl bodies inside cytoplasm were relatively abundant seen by Nissl staining and only a few cells were found degenerative and necrotic. The electroacupuncture group was similar and slightly better than that in the Nimodipine group in both HE and Nissl staining.Part Two The effect of electroacupuncture on oxygen free radicals in brain of VD miceThe result manifested that the content of MDA in cerebral tissue of model group(10.37±2.19)was raised significantly than that in sham-operation group(3.22±0.39)(P<0.01); At the same tine, the MDA content of electroacupuncture group(3.87±0.57)and Nimodipine group(5.03±0.75)were lower than that of model group(P<0.01); The MDA content in cerebral tissue of electroacupuncture group was lower than that Nimodipine group(P<0.05).The result manifested that the activity of SOD in cerebral tissue of model group(13.43±2.44)was reduced significantly than that in sham-operation group(35.15±4.11)(P<0.01); The SOD activity of electroacupuncture group(33.35±4.47)and Nimodipine group(30.14±3.06)were higher than that of model group(P<0.01); The SOD activity of cerebral tissue of electroacupuncture group was higher than that of Nimodipine group(P<0.05).Part Three The effect of electroacupuncture on Apoptosis of hippocampus cell in brain of VD miceThe sham-operation group(8.74±1.58)had a small quantity of apoptosis cell in disorder, the model group(32.77±6.58)had a great quantity of apoptosis cell, it was higher significantly than that in sham-operation group(P<0.01); The electroacupuncture group(25.79±5.57)and Nimodipine group(28.75±5.70)also had a small quantity of apoptosis cell, they were lower than that of model group(P<0.05 or P<0.01), but had no difference between themselves(P>0.05).Conclusion : The experiment demonstrated that electroacupuncture had therapeutical effect on VD mice and relieved pathological lesion of cerebral ischemia-reperfusion, and it could improve learning and memory ability of VD mice significantly; Electroacupuncture could reduce MDA content and increase the activity of SOD in cerebral tissue of VD mice significantly, it could resist the oxidative injury to alleviate the physical injury from lipid peroxidation and free radical; Electroacupuncture could restrain excessive apoptosis of hippocampus cell in brain of VD mice, At the same time it promoted nerve cell.
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