| Acupuncture can alleviate both acute and chronic pain effectively,one acupuncture treatment of acute disease can achieve a good and immediate therapeutic effect.In order to strengthen and obtain a reliable clinical efficacy,repeated treatment is necessary,particularly for chronic conditions.The research on the cumulative effect of repeated acupuncture treatment possesses an important significance.Our research results have revealed that when there is a EA-induced ccumulative analgesic effect,the expression of hypothalamic and hippocampal neuronal vesicles acetyltransferase (VAChT),choline acetyltransferase(ChAT),Protein kinase C(PKC) and Protein kinase A(PKA) increased.The memory impairment caused by ovariectomy(OVX) may weaken the effects of acupuncture.This experiment from the perspective of neural memory,combined with synaptic plasticity,further analyzes the mechanism of cumulative effects of acupuncture analgesia.Objectives:To observe the cumulative analgesic effect of electroacupuncture(EA) and to analyze its relation to the plasticity of hippocampal and hypothalamic neurons in chronic neurophathic pain rats,so as to explore its underlying biological mechanism of acupuncture in alleviating chronic pain.Methods:A total of 98 Wistar rats were randomized into control,CCI,CCI+EA 2 day(D), CCI+EA2 week(W),OVX+CCI,OVX+CCI+EA2D and OVX+CCI+EA2W groups,with 14 cases respectively.For rats of the latter 3 groups,subcutaneous injection of D-galactose was given once daily for 7W.Morris water maze test was conducted to evaluate the rats' memory-learning ability.Chronic pain model was established by ligating the left sciatic nerve with a piece of catgut.EA(2/15Hz,1-2 mA) was applied to bilateral "Yanglingquan(GB 34)"-"Zusanli (ST 36)",for 30minutes,once daily for 2 days and for 2 weeks respectively.Radiant heat irradiation-induced paw withdrawal latency(PWL,pain threshold) was detected and PWL difference values between the two paws were used as the hyperalgesia score(HAS).Eighteen days after CCI,the rats were perfused transcardially with 4%Polyoxymethylene for immunohistochemical experiments,and 2%Polyoxymethylene plus 2%glutaraldehyde for transmission electron microscopic observation.Transmission electron microscopy was used to observe the ultrastructure changes of the synapses of the hypothalamic and hippocampal neurons,including the synaptic cleft width,thickness of the postsynaptic densities(PSD),length of the active zones and curvature of the synaptic interface after EA,Synaptophysin(SYN) and calcium/calmodulin-dependentprotein kinaseⅡ(CaMKⅡ) expression of paraventricular nucleus (PVN) of hypothalamus,and CA1 and CA3 regions of hippocampus were detected by immunohistochemistry.Results:1) Effect of EA on pain threshold In simple CCI rats,compared with CCI control group,18 days after CCI,the absolute values of HAS of CCI+EA2W group reduced significantly(P＜0.05).In comparison with CCI group, the absolute values of HAS in CCI+EA2W group lowered significantly(P＜0.05),suggesting a cumulative analgesic effect after repeated EA for 2 weeks,Two days' EA had no significant changes(P＞0.05).In OVX+CCI compound model rats,the tendency of HAS was similar.In comparison with CCI +EA2W group,HAS of OVX+CCI+EA2W group was significantly higher(P＜0.05),suggesting an attenuation of the analgesic effect after OVX.2) Effect of EA on synaptic structural parameters of neurons in the hippocampus and hypothalamusIn simple CCI rats,compared with nolmal group,18 days after CCI,the thickness of the postsynaptic densities(PSD),length of the active zones and curvature of the synaptic interface in both hippocampus and hypothalamus of CCI control group decreased significantly,and the synaptic cleft width increased significantly in CCI control group and CCI+ EA2D group(P＜0.05),Compared with CCI group,the PSD value,length of the active zones and curvature of the synaptic interface in CCI+EA2D group increased to a certain degree,and the synaptic cleft width decreased slightly.In comparison with CCI group,PSD value,length of the active zones and curvature of the synaptic interface of neuron in both hippocampus and hypothalamus in CCI+ EA2W group increased remarkably(P＜0.05),and its synaptic cleft width decreased markedly(P＜0.05),while no significant changes were found in CCI+EA2D group.It suggests that CCI may weaken the synaptic transmission of information,and EA may adjust the relevant parameters of the synaptic interface and thus to rebuild the synapse,which has a cumulative effect.In OVX+CCI rats,the tendency was similar,Compared with CCI+EA2W,the PSD, length of the active zones and curvature of the synaptic interface of hippocampal and hypothalamic neurons in OVX+CCI+EA2W group decreased considerably(P＜0.05),while the synaptic cleft width increased markedly(P＜0.05).The findings indicate that in the aged rats, the synaptic plasticity of hippocampal and hypothalamic neurons and the cumulative effect of acupuncture analgesia were weakened.3) Effect of EA on the expression of SYN and CaMKⅡin the hippocampus CA1,CA3 and hypothalamic paraventricular nucleus(PVN)SYN and CaMKⅡimmunoreaction positive product integral grey values(IGV) in rat hippocampal CA1,CA3 areas and hypothalamic PVN of CCI+EA2W were significantly lower (upregulation of expression) than those of CCI group(P＜0.05),but CCI+EA 2D had no significant changes.SYN and CaMKⅡof hippocampal CA1 and CA3 areas and hypothalamic PVN in OVX+CCI+EA2W group were significantly lower than those of OVX+CCI group (P＜0.05),but obviously higher than those in CCI+EA2W group(P＜0.05),suggestting that the expression of SYN and CaMKⅡin the hippocampal CA1 and CA3 areas and hypothalamic PVN was closely related to the cumulative effect of acupuncture.In rats with neural memory impairment the cumulative effect of acupuncture analgesia was reduced. Conclusion:1) EA of "Yanglingquan"-"Zusanli" can significantly reduce pain in CCI rots and repeated EA has a cumulative effect and OVX may lower the cumulative effect of EA.2) EA of "Yanglingquan"-"Zusanli"can remodel the synaptic interface changes of hippocampus and hypothalamus neurons in CCI rats by increasing the PSD,length of the active zones and curvature of the synaptic interface,and reducing the synaptic deft width,and has a cumulative effect.OVX plus administration of D-galactose may reduce the synaptic plasticity of hippocampus and hypothalamus neurons and weaken the cumulative effect of EA.3) EA of "Yanglingquan"-"Zusanli" can increase the expression of SYN and CaMKⅡin hippocampus and hypothalamus neurons and repeated EA has a cumulative effect,which may contribute to its effect in improving the synaptic plasticity.Therefore,repeated EA of "Yanglingquan"-"Zusanli" points can relieve chronic pain,and has a cumulative effect which may be closely associated with its efficacy in upregulating the expression of presynatic SYN and intracellular CaMKⅡin hippocampal and hypothalamic neurons;and the neuronal plasticity decline by the memory impairment impacts the cumulative effect of EA.The innovation of this experiment is analysis of electro-acupuncture "Yanglingquan"-"Zusanli" cumulative effect of analgesic mechanism from the perspective of neural memory with synaptic plasticity.
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