The Role Of ?7nAChR In Spinal Cord Stimulation Treatment Of Neuropathic Pain In Rat Models

Objective:Neuropathic pain(NP)is a kind of intractable chronic pain.The pathogenesis of NP is extremely complicated.Conventional analgesics are not effective enough and frequently associated with the occurrence of adverse effects,such as opioid dependence and overdose toxicity.At present,the role of neuroinflammation in the pathogenesis of NP has attracted more and more attention.Neuroinflammation refers to the inflammation of the nervous tissue initiated by infection,injury,toxic metabolites,and autoimmune reactions.Immune cells and glial cells are the main cells involved in neuroinflammation.After activation,these cells can release many inflammatory mediators which contribute to peripheral and central sensitization,which are essential for the emergence and maintenance of NP.Spinal cord stimulation(SCS)is currently one of the main treatment options for intractable neuropathic pain.The neuronal mechanism of spinal cord stimulation has been well studied,but its effect on neuroinflammation is still unclear.?7nAChR plays a crucial role in the cholinergic anti-inflammatory pathway.?7nAChR participates in the regulation of systemic inflammatory response through neuro-immune interactions.Studies have shown that in animal models of peripheral nerve injury,the expression of ?7nAChR in the ipsilateral spinal cord was down-regulated.Intrathecal administration of ?7nAChR agonists could relieve inflammatory pain and chronic pain,indicating that ?7nAChR is involved in the regulation of central inflammatory response.Recent studies have confirmed that SCS can protect neurons from damage by activating?7nAChR in the spinal ischemia-reperfusion injury model.In this study,we established a rat neuropathic pain model and a spinal cord stimulation model to explore whether spinal cord stimulation can control the neuroinflammatory response by activating ?7nAChR,thereby alleviating neuropathic pain in rats.Method:Part ?:Changes in the expression of ?7nAChR in the spinal dorsal horn of rats with neuropathic pain and its effect on pain behavior.1.Establish CCI neuropathic pain model with rats,which were randomly allocated to 3 groups,namely(1)Control group,(2)Sham CCI group,(3)CCI group.Paw withdrawal threshold was detected in each group;Double-labeling immunofluorescence was used to observe microglia activation in spinal dorsal horn and p38 phosphorylation;RT-PCR and ELISA were performed to detect inflammatory factors expression in spinal cord;Immunofluorescence staining and Western Blot were applied to detect spinal cord?7nAChR expression.2.Intrathecal injection of ?7nAChR agonist PNU-282987 into CCI rats,which were randomly allocated to 6 groups,namely(1)Control group,(2)CCI+Vehicle group,(3)CCI+PNU 0.1 mg/Kg group,(4)CCI+PNU 0.25 mg/Kg group,(5)CCI+PNU 0.5 mg/Kg group,(6)CCI+PNU 0.5 mg/Kg+?-BGT 0.5 ?g/Kg group;Paw withdrawal threshold was measured before and 1h,3h and 5h after administration in each group.Part ?:Spinal cord stimulation relieves neuropathic pain by inhibiting p38 MAPK phosphorylation in microgliaEstablish spinal cord stimulation model and randomly allocate rats to 3 groups,namely(1)Control group,(2)CCI+Sham SCS group,(3)CCI+SCS group.The mechanical hyperalgesia were detected in each group with von Frey filaments;Immunofluorescence staining was used to observe the microglia activation and p38 phosphorylation in spinal dorsal horn;RT-PCR and ELISA were used to detect the expression of inflammatory factors in the spinal cord;Western Blot was applied to detect the expression of spinal?7nAChR.Part ?:Spinal cord stimulation inhibits microglia-mediated neuroinflammation by promoting the expression of ?7nAChRIntrathecal administration of ?7nAChR selective inhibitor ?-BGT before spinal cord stimulation,the rats were randomly allocated to 4 groups,namely(1)Control group,(2)CCI+Sham SCS group,(3)CCI+SCS+Vehicle group,(4)CCI+SCS+?-BGT group.The changes of paw withdrawal threshold in each group were detected;Immunofluorescence staining was used to observe the microglia activation and p38 phosphorylation in spinal dorsal horn;RT-PCR and ELISA were used to detect the expression of inflammatory factors in the spinal cord;Immunofluorescence staining and Western Blot were used to detect the expression of spinal ?7nAChR.Results:Part ?:A stable neuropathic pain model was established after CCI surgery,and the paw withdrawal threshold of rats in CCI group was significantly reduced(P<0.001);Immunofluorescence results showed increased CD11b and P-p38 fluorescence intensity in the ipsilateral spinal dorsal horn 3 weeks after CCI surgery;RT-PCR and ELISA results showed that compared with the Naive and sham groups,the mRNA and protein expression levels of IL-1? and TNF-? in the spinal cord of the CCI group were significantly increased(P<0.01);Immunofluorescence and WB showed that the expression of ?7nAChR in the ipsilateral spinal cord was reduced 3 weeks after CCI surgery;On the 14th day after CCI,the ?7nAChR agonist PNU-282987 was administered intrathecally,and results showed that the mechanical hyperalgesia was significantly relieved within 3 hours.What's more,the analgesic effect of ?7nAChR agonist PNU-282987 was dose-dependent.Part ?:Explore the effect of spinal cord stimualtion on CCI induced neuropathic pain by measuring the paw withdrawal threshold in each group.The results showed that there was no significant difference in the paw withdrawal threshold among the three groups before CCI surgery.Compared with the control group,the PWT of the CCI+Sham SCS group and the CCI+SCS group was significantly lower before SCS(P<0.05);Compared with the CCI+Sham SCS group,CCI+SCS group exhibited significant increase in PWT for the entire SCS treatment period(P<0.05).What's more,the PWT remained significantly higher for 5 days after the termination of SCS treatment,but it gradually returned to the same degree as that in CCI+Sham SCS rats at day 14;Double-labeling immunofluorescence showed that compared with the control group,the CD11b fluorescence reactivity and the number of P-p38 in the dorsal horn of the spinal cord in the CCI+Sham SCS group were significantly increased after peripheral nerve injury(P<0.001);Compared with CCI+Sham SCS group,the CDllb fluorescence reactivity and the number of P-p38 in the ipsilateral dorsal horn of spinal cord were significantly decreased in CCI+SCS group(P<0.001);RT-PCR and ELISA results showed that compared with the CCI+Sham SCS group,the mRNA and protein expression of inflammatory factors TNF-? and IL-1? in the spinal cord of the CCI+SCS group were significantly attenuated(P<0.05);WB results showed that the protein level of spinal?7nAChR in CCI+Sham SCS group was significantly reduced(P<0.001);However,the protein expression of ?7nAChR in the spinal cord of the CCI+SCS group was significantly higher than that of the CCI+Sham SCS(P<0.01).Part ?:Double-labeling immunofluorescence revealed expression of ?7nAChR in the neurons(NeuN),microglia(CDllb)and astrocytes(GFAP)in the spinal dorsal horn;Compared with CCI+sham SCS rats,the PWT of CCI+SCS+Vehicle group was significantly increased during the entire stimulation period(P<0.001);However,intrathecal injection of ?-BGT significantly reversed the increase of PWT induced by SCS on CCI rats(P<0.01);Double-labeling immunofluorescence showed that compared with the CCI+Sham SCS group,the CD11b fluorescence intensity and the number of P-p38 positive microglia in the spinal dorsal horn were significantly reduced in the CCI+SCS+Vehicle group(P<0.001);Compared with the CCI+SCS+Vehicle group,the CD11b fluorescence intensity and the number of P-p38 positive microglia in the spinal dorsal horn of the CCI+SCS+?-BGT group were increased significantly(P<0.01);The results of RT-PCR and ELISA showed that compared with CCI+Sham SCS group,the mRNA and protein expression levels of pro-inflammatory cytokines IL-1? and TNF-? in the spinal cord of CCI+SCS+Vehicle were significantly deceased(P<0.001).However,in the ?-BGT treatment group,the expression of inflammatory factors was significantly higher than that in the CCI+SCS+Vehicle group(P<0.05).Conclusion:Part ?:CCI induced persistent mechanical hyperalgesia in rats;After sciatic nerve injury,the microglia in the spinal dorsal horn of rats showed significant proliferation and activation.CCI could induce local inflammation in the ipsilateral dorsal horn of spinal cord,which is characterized by the activation of microglia,the increase of p38 phosphorylation,and the increased expression of IL-1? and TNF-?,suggesting that neuroinflammation is involved in the regulation of neuropathic pain;After peripheral nerve injury,the expression of ?7nAChR on the ipsilateral spinal dorsal horn was significantly reduced.Intrathecal administration of ?7nAChR agonist PNU-282987 could relieve CCI-induced mechanical hyperalgesia,suggesting that ?7nAChR can be regarded as a potential target for neuropathic pain.Part ?:SCS ameliorated neuropathic pain in CCI rats and pain inhibition extended beyond the stimulation period;SCS inhibited CCI-induced spinal microglia activation,P-p38 increase and pro-inflammatory cytokines production,suggesting that SCS could regulate neuropathic pain by inhibiting neuroinflammatory response;SCS promoted the expression of a7nAChR in the ipsilateral spinal dorsal horn of CCI rats.Part ?:?7nAChR is the main receptor of the cholinergic anti-inflammatory pathway,which is expressed in neurons,microglia and astrocytes in the spinal dorsal horn of rats;Intrathecal injection of ?7nAChR selective inhibitors could antagonize the inhibitory effects of SCS treatment on microglia activation,p38 phosphorylation and the release of inflammatory factors.The regulatory effect of SCS on neuroinflammation is related to the promotion of spinal ?7nAChR expression.