Synaptotagmin ? Is Involved In Electroacupuncture-mediated Alleviation Of Neuropathic Pain

Neuropathic pain,as one of chronic pains,is caused by central or peripheral nerve damage or disease,including some syndromes such as allodynia,hyperalgesia and spontaneous pain.And the health of animals and people are strongly affectd by the long-term pain.At present,drug administration is principally applied to relieve neuropathic pain,but it is not effective and has side effects.Electroacupuncture?EA?analgesia,combined with electric stimulation on the basis of traditional hand-needle stimulation of specific acupoints,has the advantages of less side effects,obvious analgesic effect,simple operation and strong controllability.EA is widely used in veterinary clinic and human medical as a modern therapy.Researches have shown that EA analgesia has a good analgesic effect on neuropathic pain,but the underlying mechanism of EA stimulation in neuropathic pain is still unclear.Researches have indicated that the central sensitization of the dorsal horn of the spinal cord is due to the excitatory neurotransmitters,especially glutamate?Glu?,are increased the release after peripheral nerve injury,which enhances the excitability of postsynaptic neurons through acting on the corresponding receptor,eventually leads to the development of abnormal pain in neuropathic pain.Synaptotagmin??Syt??is a presynaptic vesicle membrane protein in mammal,as one of Ca²⁺receptor proteins,its main function is to promote the release of neurotransmitters and participate in the regulation of synaptic plasticity.Furthermore,studies have demonstrated that Syt?can regulate the release of glutamate.As well as,the differential expression of Syt?gene was observed in the nerve center of goats with EA stimulation by high-throughput sequencing technology in our laboratory.Therefore,we speculate that Syt?may be involved in the formation of neuropathic pain and EA may relieve neuropathic pain by regulating the expression of Syt?.In order to investigate the role of Syt?on neuropathic pain,we established the model of neuropathic pain by a surgery of the spared nerve injury?SNI model?in rats and then detected the changes of the paw withdrawal threshold?PWT?after intrathecal injection of different doses of Syt?antibody.Thirty SD rats were randomly divided into five groups:Sham group?sham surgery group?,SNI group?nerve injury group?,SNI+different doses of anti-Syt?group?1?g,SNI+anti-Syt-1?g;4?g,SNI+anti-Syt-4?g;8?g,SNI+anti-Syt-8?g?.The Sham group kept nerve intact with sham surgery,while other four groups were performed with a surgery of the SNI and intrathecal cannulation.At day 8 after surgery,all rats were given intrathecal injection of different doses of Syt?antibody once every two days and 7 times in total.The PWT was measured by paw tactile apparatus at day 0?before surgery?and at day 7,day 8,day 14 and day 20 after surgery,respectively;and the effects on neuropathic pain rats with different doses of Syt?antibody were simultaneously observed.To investigate the effect of EA on Syt?expression in spinal cord in the SNI rats,another fifity four SD rats were randomly divided into three groups:Sham group,SNI group,and SNI+EA group?nerve injury+EA group?.The SNI group and SNI+EA group were established by a surgery of the SNI,whereas the Sham group was performed with the same procedure but retained the intact sciatic nerve.The PWT of all rats were measured at day 0?before surgery?and at day 7,day 8,day 20 after surgery,respectively.At day 8 after surgery,EA stimulation was performed for 30 min on bilateral“Zusanli”and“Sanyinjiao”acupoints in the SNI+EA group?2 Hz,1³ mA?once every two days and 7 times in total.The spinal L4⁶ segments in rats were immediately collected after measuring the PWT in all groups at day 8,14 and 20 after the surgery,respectively.Meanwhile,the expressions levels of Syt?were detected with the methods of the Real-time quantitative PCR,Western Blot and immunohistochemistry?IHC?in the spinal cord.At the experiment of using intrathecal injection of Syt?antibody,the results displayed that the PWT of the SNI rats continuously reduce from day 7 to day 20,on the other hand,the PWT of the injection of 4?g or 8?g Syt?antibody group was significantly higher than those of the SNI group and SNI+anti-Syt-1?g group?p<0.05?at day 8,14 and 20 after the surgery.Meanwhile,the PWT of the SNI+anti-Syt-8?g group showed the most significant increase in all groups,which was dramatically lower than that in Sham group?p<0.05?.These results indicated that the SNI rat model could induce allodynia,which can be dose-dependently relieved by Syt?antibody.The results of EA stimulation showed that the PWT in SNI+EA group was significantly higher than that in SNI group at day 14 and 20?p<0.05?,whereas there was no difference with the Sham group?p>0.05?,and the strongest analgesic effect was observed at day 20.Those results indicated that the EA stimulation could remarkably reverse the PWT caused by nerve injury in rat,and leading to an analgesic accumulative effect.The results of qPCR showed that the mRNA expression levels of Syt?had no significant difference at day 8,14 and 20?p>0.05?.And the results of Western Blot showed that the protein expression levels of Syt?in the SNI group was significantly higher than that in the Sham group at day 8,14 and 20 after surgery?p<0.05?.However,the expression levels of Syt?protein in spinal cord was significantly lower than that in SNI rats at day 14 and 20?p<0.05?in SNI+EA rats,and there was no difference with the Sham group?p>0.05?,indicating EA stimulation significantly reduce Syt?protein expression levels in spinal cord of SNI rats.At the same time,the results of IHC showed that the Syt?protein had differently expressed levels in the dorsal horn?and?layers of the spinal cord,which was highly consistent with the results of the Western Blot.The SNI rat models were successfully established in the present research.Intrathecal injection of Syt?antibody to investigate the role of Syt?in neuropathic pain of rats,and the effect of EA stimulation on PWT and Syt?expression levels changes in spinal cord of SNI rats after EA stimulation.The results showed that Syt?was involved in the formation of neuropathic pain,and EA stimulation by down-regulating the expression levels of Syt?protein relieved the neuropathic pain.This research can help to elucidate the central mechanism of EA treatment for neuropathic pain in the future.