| Objective:This study is aimed to explore whether electroacupuncture(EA)could improve the spatial learning and memory function of VCI rats to promote synaptic plasticity of the entorhinal cortex-hippocampal neural circuit by regulating mi R-219 a.Also,this study can enrich experimental data for evidence of the efficacy of EA in improving VCI symptoms.Methods:A total of 70 SPF male Sprabue-Dawley(SD)rats(280g±30g)were included,10 rats were randomly selected as the sham operation group,and the rest rats were used for VCI model operation.The VCI rats(n=50)were randomly divided into 5 groups: model group,EA group,model+mi R-219 a overexpression group,EA+mi R-219 a overexpression group and EA+empty vector group.The rats in EA group,EA+mi R-219 a overexpression group and EA+empty vector group were treated with EA at Baihui and Shenting acupoints and density wave was1/20 Hz.The rats were treated once a day for 30 mins and once a day,5 days a week for 4weeks.The rats in the other groups were captured equally and fixed for 30 min without any treatment.(EA therapy was performed on Baihui and Shenting acupoints of rats with density wave of 1/20 Hz.Based on the control group,the other non-EA treated rats were equally grabbed and fixed for 30 minutes without any treatment).(1)Neuron-specific adeno-associated virus overexpressing mi R-219 a was constructed and r AVV-h Syn-e GFP-2A-CRE-WPRE-PA retrotracer virus was injected into the hippocampal CA1 region,and r VAA-CMV-Dio-mi R-219a-m Cherry-p A,r VAA-CMV-Dio-m Cherry-p A virus as a control.(2)Barnes maze and Morris water maze were used to detect the spatial learning and memory function of rats in each group.(3)Magnetic resonance spectroscopy was used to analyze the changes of excitatory neurotransmitters glutamate(Glu),inhibitory neurotransmitters aminobutyric acid(GABA)and N-acetylaspartate(NAA)in the entorhinal cortex and hippocampal CA1 region of rats in each group.(4)Golgi staining was used to observe the synaptic morphological changes in the entorhinal cortex and hippocampal CA1 area of rats in each group.(5)The in vitro patch clamp electrophysiological technique was used to detect the field potential long-term potentiation(LTP),whole-cell voltage clamp pattern of the endolfactory cortex-hippocampal CA1 neural circuit to detect the changes of spontaneous excitatory postsynaptic current(s EPSC)and N-methyl-D-aspartate receptor(NMDAR)ion channel current in the entorhinal cortex and hippocampus.(6)The laser confocal microscope was used to observe the fluorescence expression of mi R-219 a in the entorhinal cortex-hippocampal CA1 neural circuit.(7)RT-q PCR was used to detect the expression of mi R-219 a in the entorhinal cortex and hippocampus of rats in each group.Results:(1)Modeling tests were carried out using Barnes maze : Compared with the sham operation group,the escape latency of the other five groups was significantly increased(P<0.01),but the contact times at the correct hole and the target quadrant time were significantly decreased(P<0.01),and there was no significant difference in pairwise comparison between the five groups(P>0.05).(2)Electroacupuncture at Baihui and Shenting acupoints affected spatial learning and memory function of VCI model rats by regulating mi R-219 a.In the learning stage,compared with the sham operation group,the escape latency of the model group was longer than that of the sham operation group(P<0.05),the escape latency of the EA group and the EA+empty vector group was shorter than that of the model group,and the escape latency of the model+mi R-219 a overexpression group was significantly longer than that of the model group(P<0.05).Compared with the EA group,the escape latency was significantly increased in the EA+mi R-219 a overexpression group.In the memory test stage,compared with the sham operation group,the number of crossing the platform in the model group was significantly lower than that in the target quadrant(P<0.01),and the proportion of crossing the platform and target quadrant in the EA group was significantly higher than that in the model group(P<0.05).Compared with the electroacupuncture group,the number of times crossing the platform in the EA+mi R-219 a overexpression group decreased(P<0.05).(3)Electroacupuncture at Baihui and Shenting acupoints affected neurosubstance metabolism in the entorhinal cortex and hippocampus CA1 region of VCI model rats by regulating mi R-219 a.As the results of magnetic resonance spectroscopy showed,compared with the sham operation group,the content of NAA in the olfactoric cortex and hippocampus CA1 region decreased(P<0.01),the content of excitatory glutamate neurotransmitter decreased(P<0.01),and the content of GABA increased(P<0.01)in the model group;Compared with model group,the content of NAA increased(P<0.05),excitatory Glu increased(P<0.05),and GABA decreased(P<0.05)in both EA group and EA+empty vector group.Compared with the model group,the NAA content in the model+mi R-219 a overexpression group showed a downward trend(P>0.05),excitatory Glu showed a downward trend(P>0.05),and GABA content showed an upward trend(P>0.05).Compared with the electroacupuncture group,the content of NAA decreased in the EA+mi R-219 a overexpression group(P<0.05),the content of excitant Glu decreased(P>0.05),and the content of GABA increased(P<0.05).(4)Electroacupuncture at Baihui and Shenting acupoints improved synaptic morphology in the endoorhinal cortex and hippocampus CA1 region in VCI model rats by regulating mi R-219 a.In the endoorhinal cortex and hippocampus CA1 region,the dendritic spines in the sham operation group were highly distributed and densely arranged,while the dendritic spines in the other five groups were abscissed and atrophied,the number of dendritic spines in model group was significantly decreased(P<0.01).Compared with the model group,the number of dendritic spines in the model+mi R-219 a overexpression group was further decreased(P<0.05).The number of dendritic spines in EA group and EA+empty vector group was significantly increased(P<0.05).Compared with the EA group,the number of dendritic spines in the EA+mi R-219 a overexpression group decreased(P<0.05).(5)Electroacupuncture at Baihui and Shenting acupoints improved the field potential of the endoorhinal cortical and hippocampal CA1 neural circuit in VCI model rats by regulating mi R-219 a.Compared with the sham operation group,the f EPSP slope of the CA1 neural circuit in the endoorhinal cortex and hippocampus in the model group was significantly decreased(P<0.01),while the f EPSP slope of the CA1 neural circuit in the EA group was increased compared with the model group(P<0.05).The f EPSP slope of the circuit was further decreased in the model+mi R-219 a overexpression group(P<0.05),while the f EPSP slope was decreased in the EA+mi R-219 a overexpression group compared with the EA group(P<0.05).(6)Electroacupuncture at Baihui and Shenting acupoints affected the firing pattern of pyramidal neurons in the entorhinal cortex and hippocampus CA1 region of VCI model rats by regulating mi R-219 a.Compared with the sham operation group,the amplitude and frequency of s EPSC in the endoorhinal endoorhinal cortex and hippocampal CA1 region in the model group were significantly decreased(P<0.01),and the amplitude and frequency of s EPSC in the EA group were increased compared with the model group(P<0.05).Compared with the model group,the amplitude and frequency of s EPSC in the endoorhinal cortical and hippocampal CA1 region were decreased in the model+mi R-219 a overexpression group(P<0.05).Compared with the EA group,the amplitude and frequency of the s EPSC in the EC-hip region were decreased in the EA+mi R-219 a overexpression group(P<0.05).(7)Electroacupuncture at Baihui and Shenting acupoints upregulates the current intensity of NMDAR ion channels in the hippocampus of VCI model rats by regulating mi R-219 a.Compared with sham operation group,the current of NMDAR channel in hippocampus of model group was significantly decreased(P<0.01).Compared with the model group,the current of NMDAR channel in hippocampal area was increased in the EA group(P<0.05).Compared with model group,the NMDAR channel current in the hippocampus of model+mi R-219 a overexpression group was significantly decreased(P<0.05);Compared with the electroacupuncture group,the NMDAR channel current in the hippocampus of the EA+mi R-219 a overexpression group was down-regulated(P<0.05).(8)Effects of electroacupuncture at Baihui and Shenting acupoints on mi R-219 a expression in entorhinal cortex and hippocampus CA1 region.As the RT-q PCR results showed,compared with the sham operation group,the expression levels of mi R-219 a in the entorhinal cortex and hippocampus in the model group were increased(P<0.01).Compared with model group,the expression of mi R-219 a in EA group was significantly decreased(P<0.05).Compared with the model group,the expression of mi R-219 a in the entorhinal cortex and hippocampal CA1 region of rats in the mi R-219 a overexpression group was significantly increased(P<0.05).(9)Entorhinal cortex-hippocampal CA1 neural circuit projection and mi R-219 a localization and expression.Laser confocal microscopy showed that there was direct nerve fiber projection from the entorhinal cortex to hippocampal CA1,and mi R-219 a fluorescent localization expression was seen in the entorhinal cortex and hippocampal CA1 area.Conclusion:Electroacupuncture at Baihui and Shenting acupoints can regulate mi R-219 a to promote synaptic plasticity of entorhinal cortex-hippocampal neural circuit and improve spatial learning and memory function of vascular cognitive impairment model rats.The electrophysiological mechanism is related to enhancement of NMDAR ion channel current,the promotion of neuronal excitatory discharge,and the induction of the formation of LTP in the entorhinal cortical and hippocampal CA1 neural circuit. |
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