| Objective: we observe the effect of electroacupuncture on behavior，brain tissue pathological morphology of hippocampal CA1area,single amineneurotransmitter and the oxygen free radical of VD (Vascular Dementia, VD)model mice after cerebral ischemia reperfusion.And we compare thedifferences between different electroacupuncture intervention times to explorethe effect of electroacupuncture and possible mechanism for the treatment ofVD.Methods:1Model replicationWe divided sixty kunming mice into the first batch and the second batchand each batch had thirty mice. There were shamoperation group, model groupand electroacpuncture group in each batch and each group had ten mice. Withthe reference to the literature, the method of replication ofischemia-reperfusion of bilateral common carotid artery was used to make VDmodel. Intraperitoneal injection of0.35g/kg with10%chloral hydrateanesthesia was used to narcotize the mice.The central incision of the neck wasroutineiy disinfected,and then we separated the bilateral common carotidartery, tighted wire clasp with the silk buckle to block blood flowing within20min, and at the same time1cm from the tail was cutted to put out0.3mlblood with stopping blooding by heat. And next we loosed the screw thread tomake the blood perfusion within10min and then blocked the blood flowingwithin20min, and second observation was30min after reperfusion.In theshamoperation group we only separated the bilateral common carotid arteryand threaded wire,but not tighted the wire and the tail was not bleeded.The observation time of the shamoperation group was the same as the other groups.After the operation every group was injected penicillin daily2000U, andcontinued3d.In the process of the experiment anal temperature wasmaintained at36±0.5℃in orde to prevent the low temperature protection ofischemic brain injury.2TreatmentThe first batch of electroacupuncture group began electroacupunctureintervention on the day after awakening and the second batch beganelectroacupuncture intervention on third day after awakening. Each batch ofelectroacupuncture group selected “dazhui”,“baihui” and “geshu”on bothsides,“zusanli” on both sides with filiform needle of0.35mm x13mmpricked into, chose dilatational wave of2/80hz, which just made the animalbody gently tremble but not struggle bray for degrees.The acupuncturetreatment continued10min per day and continued15d.The shamoperationgroup and model group in the each batch were fixed as the same position for10min.3Detection methodsAfter the treatment both groups chose platform experiment as behavioraltest in order to observe the learning and memory ability of mice in the eachgroup. After the behavioral test,we put all of the mice to death underanesthesia, fast cutting the brain tissue on ice tray, abandoning the cerebellumand olfactory bulb, and fixed the left side of the optic chiasma to the nippletissues into4%paraformaldehyde, conventional HE staining, and thenobserved hippocampal CA1pyramidal cell morphology changes of braintissue under light microscope.and took the right hippocampus of mice intophysiological saline of precooling after weighing, and the tissue was madeinto the hippocampal tissue homogenate of10%, centrifuged for5min at4℃w ith3600r/min,to extract supernatant on standby.Fluorescencespectrophotometry was used to determine the content of single amineneurotransmitter NE, DA and5-HT, SOD activity was evaluated by xanthineoxidase method, while glucosinolates barbituric acid colorimetric analysis neurotransmitter NE, DA and5-HT, method was used to determine thecontent of MDA.Results:1Results of behavioral testsAfter the platform experiments, the reaction time of model group in thetwo batches were both extended, the incubation period were both shorten,andthe error number of the two batch increased, compared with shamoperationgroup in the same batch. And the differences were statistically significant (P<0.01); The reaction time of electroacupuncture groups in the two batches wereboth shortened, the incubation period are both extended,and number of errorswere significantly lower, compared with the same batch of model group, thedifferences had significant difference(P<0.01);And there were also differencebetween electroacupuncture groups in the two batches,which was that thestudy results has no difference and the memory performance of the first batchis better than that of the second batch (P<0.05or P <0.01).2The results on the pathological morphologyGeneral morphology observation under light microscopy showed that thehippocampal CA1area pyramidal cell of shamoperation groups in the twobatches were arranged closely, the neat cells were round,ucleus were largeand round, cell nucleolus was clear and the uniform nuclear was equality.CA1area pyramidal cells of the model group in the two batches arranged disorder,osteoporosis,And pyramidal cell was depigmentation serious, part of thecellular structure was not complete, owe rules, smaller volume pyknotic nuclei,nucleoli were triangular, much Angle, nucleoplasm uneven, and could found alarge number of dead cells in the field of vision.Hippocampal CA1pyramidalcells of the electroacupuncture group of the first batch were only a littledepigmentation, there were a few nuclei pyknosis of triangle or polygon; andthe second batch hippocampal CA1pyramidal cells of the electoacupuncturegroup has only a small amount of depigmentation, cellular structure were clear,cell arrangement were more closely, the nuclei were round and big, nucleoluswere clear and there are only a few nuclei pyknosis of triangle. 3The detection results of hippocampus single amine neurotransmitterThe result showed that the content of hippocampus NE, DA and5-HTof model group mice in the both batches were significantly lower, comparedwith shamoperation group in the same batch, and the difference werestatistically significant (P <0.01).The content of NE, DA and5-HT ofelectroacupuncture group in the two batches were significantly increased,compared with model group in the same batch and the resules had significantdifference (P<0.01); And there were also differences between theelectroacupuncture groups in the two batches and the differencese is thatcontent of DA and5-HT of the second batch was highter than that of the firstbatch electroacupuncture group (P <0.05or P <0.01).4The detection results of hippocampus oxygen free radicalsCompared with shamoperation group, the hippocampal SOD activity ineach model group were significantly decreased, the concent of MDA wassignificantly increased, and the difference were statistically significant(P<0.01).Compared with model group, hippocampal SOD activity of the twoelectroacupuncture groups in each batch were significantly increased, thecontent of MDA were significantly lower and there were significantdifferences(P<0.01).And there were also differences between theelectroacupuncture groups in the two batches. The SOD activity of the secondbatch was higher than electroacupuncture group of the first batch (P <0.05).Conclusion: The experiment demonstrated that electroacupuncture canimprove the cognitive impairment of the VD model mice caused byischemia-reperfusion as we inserts the acupuncture points of "dazhui","baihui" and "geshu","zushanli". Electroacupuncture can improve the NE,DA and5-HT contents in hippocampus of VD model mice and the same timecan enhance SOD activity and reduce the content of MDA.Electroacupuncture can effectively recover metabolic disorders of singleamine neurotransmitter after ischemia injury of the hippocampus and enhancethe ability of scavenging free radicals, which can bring about damage to the nervous system. And the same time we have preliminary results that thereexists different effects between different acupuncture intervention times. |
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