| Objectives : Established a classic autism spectrum disorder(ASD)animal model of Wistar rats with valproic acid(VPA).Electroacupuncture was used to stimulate Baihui and Dazhui points,then,recorded the changes of behavior of autism spectrum disorder rats and analyze the results after the treatment.Moreover,we were studied the mechanism by analysis of pathology changes.We were suggested providing a scientific experimental basis for the clinical treatment of autism spectrum disorders.Methods: The ASD rat model was established according to the method of Schneider’s.The fertilized female rats were divided into three groups that the VPA group,the blank group,and the control group.On the 12.5 day after conception,the VPA group received an injection of VPA by intraperitoneal(600 mg/kg,the VPA was dissolved in saline at a concentration of 250 mg/ml),the control females were injected with normal saline at the same time,and the blank without any dispose of.Moreover,both the weight and eyes opening were recorded,the negative geotaxis and swimming performance of the behavior also were recorded.The eighteen male offspring rats from the VPA group were randomly selected into the model group,the acupoint group(stimulated Baihui point and Dazhui point by electroacupuncture)and the non-acupoint group(stimulated non-acupoint by electroacupuncture),and each group with six offspring rats.Another twelve male offspring were randomly selected from the control group and the blank group into the new control group and blank group by six in each group.Then,compared the five groups with the open field,three-chamber,and Y-maze test after the courses of electroacupuncture intervention.Hematoxylin-eosin staining(HE)was used to detect inflammatory injury.Nissl staining(Nissl)was used to detect the morphology and number of neurons of the hippocampus.Terminal deoxynucleotidyl transferase-mediated d UTP Nick End Labeling(TUNEL)was used to detect the apoptosis of hippocampal.SPSS 24.0 software was used for the analysis of data statistical,Image pro plus 6.0 software was used to measure the apoptosis index of hippocampal cells,and Origin Pro 8.0 was used to draw the map of experimental data.Results:1 Growth and maturation development1.1 weight gain: On PND14 and PND 35,the offspring rats of the VPA group had lower weight gain than the blank group(P<0.01).They also had lower weight gain than the control group(P<0.01).1.2 Eyes opening: The offspring rats of the VPA group were delay in eyes opening than the blank group on PND13(P<0.01).They also were delay in eyes opening than the control group(P<0.01).2 the behavior2.1 Negative geotaxis: The offspring rats of the VPA group were taken more time to turn round 180°than the blank group on PND7,PND8,PND9,and PND10(P<0.01).Similarly,they were taken more time than the control group(P<0.01).2.2 Swimming performance: The offspring rats of the VPA group were delay in swimming function than the blank group and the control group on PND10(P<0.01).2.3 Open field: The offspring rats of the model group had a longer distance compared to the blank group and the control group after electroacupuncture intervention,as the non-acupoint group(P<0.01).Interestingly,they were longer than the acupoint group which was treated by stimulating Baihui and Dazhui with pointselectroacupuncture(P<0.01).The offspring rats of the model group were taken longer time compared to the blank group,the control group and the acupoint group,and the non-acupoint group,too.(P<0.01).The results of the number of times of grooming,running,and standing showed that the offspring rats of the model group and the non-acupoint group were more anxiously than the acupoint group(P<0.01).2.4 Three-chamber:Social ability: For the offspring rats of the model group and the non-acupoint group,they were taken the significantly shorter time of exploring the strange rat number one than that in the blank group,the control group,and the acupoint group(P<0.01).Social preference: For the offspring rats of the model group and the non-acupoint group,they didn’t like to explore the strange rat number one nor the strange rat number two that compared to in the blank group,the control group,and the acupoint group(P<0.01).However,the acupoint group showed the same behavior with the blank group and the control group in social ability,but the function of social preference was weaker than the blank group and the control group(P<0.01).2.5 Y-maze: Compared with the blank group,the control group,and the acupoint group,the number of the offspring rats of the model group and the non-acupoint group entered the three arms increased significantly which compared with the blank group,the control group,and the acupoint group(P<0.01).Similarly,the distance of entering the three arms also increased significantly(P<0.01).3 Hematoxylin-eosin staining:In the field view of the hippocampal tissue,the offspring rats in the acupoint group,we found that the pyknosis of a few pyramidal cells,they with deep staining,small volume and long strip shape and unclear nuclear structure.In the non-acupoint group,more pyramidal cell pyknosis was observed,with deeper staining,smaller volume,and long strip shape and with unclear nuclear structure.In the model group,there were a large number of pyramidal cells with pyknosis,deep staining,small volume,and long strip shape,and unclear nuclear structure.All of them,there was no obvious infiltration of inflammatory cells and glial cells.4 Nissl staining:In the acupoint group,the neurons were arranged neatly,most of the cells were clear in outline,and the nucleoli of the nuclei were blue.A few cells were atrophied with deepened color and irregular changes.In the non-acupoint group and the model group,the distribution of neurons was sparse,the cell contour was fuzzy,the boundary was not clear,and a large number of cells were atrophy in the field of vision,the color was dark blue,the nucleus was irregular or ruptured,and the Nissl corpuscle was reduced.5 TUNEL:More TUNEL positive cells were observed in the non-acupoint group and the model group,while the difference was significant compared with the blank group,the control group,and the acupoint group(P<0.01).However,There were a few positive cells in the acupoint group and the apoptotic index was different from that of the blank group and the control group(P<0.01),even though,the acupoint group was better than the model group.More apoptosis was also observed in the dentate gyrus.Conclusion:1 We found that stimulating Baihui and Dazhui points with electroacupuncture can relieve the emotional anxiety and tension of the ASD rats model,which is helpful for them to adapt to the environment.Moreover,that also can improve their social ability and social preference,and improve the short-term learning and memory ability,which may have important reference significance for clinical treatment.2 The rat model of ASD induced by VPA may have pathological damage of the hippocampus,decreased survival number of neurons,and increased apoptosis.3 Differently,stimulating Baihui and Dazhui points with electroacupuncture may alleviate the pathological damage of hippocampal tissue,improve the survival number of neurons and reduce the apoptosis of the ASD rat model.In general,it is very significant to intensive study. |
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