The Roles Of CXCL13 And CXCR5 In The Anterior Cingulate Cortex In Neuropathic Pain In Mice

Objective To study the expression and cell distribution of chemokine CXCL13 and its receptor CXCR5 in the anterior cingulate cortex(ACC)of mice with neuropathic pain induced by spinal nerve ligation(SNL),and investigate their regulating effects on neuropathic pain.Method 1.The neuropathic pain model was induced by left L5 spinal nerve ligation(SNL).2.The mechanical allodynia and heat hyperalgesia were evaluated by behavioral test..3.The place escape/avoidance paradigm(PEAP)was applied to determine the extent of pain-related aversion.4.The expressions of CXCLl3 and CXCR5 mRNA were detected by the Real-time PCR method.5.The change of CXCL13 protein was detected by ELISA.6.The change of CXCR5 protein was detected by western blot.7.The cell type of CXCLl3 mRNA was detected by hybridization in situ and double-labelling immunofluorescence methods.8.The cellular distribution of CXCR5 was dertermined by the immunofluorescence staining.9.The CXCR5 lentiviral(LV-CXCR5 shRNA)was injected in ACC,so as to investigate the extents of SNL on mechanical hyperalgesia and pain-related aversion.10.The change of sEPSCs in ACC ?/? layer pyramidal neurons was recorded using the whole-cell patch clamp.Result 1.SNL had no effect on the growth,development and motor coordination ability of mice,but could induce the neuropathic pain of ipsilateral hindpaw.Moreover,the mechanical allodynia and thermal hyperalgesia were observed 1 d after SNL and could last for more than 21 d.2.SNL induced upregulation of CXCL13 mRNA and protein in ACC;The expressions of CXCL13 mRNA and protein were increased 1 d later(p<0.01)and was maintained for more than 10 d(p<0.001);The in-situ hybridization result showed that CXCLl3 mRNA was distributed in ACC neurons.3.SNL induced upregulation of CXCR5 mRNA and protein in ACC.Meanwhile,CXCR5 mRNA showed no significant change 1 d later(p>0.05),was significantly increased in 3 d(p<0.001)and was maintained for more than 10 d(p<0.001);The CXCR5 protein showed no significant upregulation in 3 d(p<0.001);The double-labelling immunofluorescence result showed that CXCR5 was expressed in ACC neurons.4.Microinjection of CXCR5 interfering lentivirus(LV-CXCR5 shRNA)in ACC did not alleviate the mechanical hyperalgesia induced by SNL,but alleviated pain related aversion induced by SNL.5.SNL did not affect the discharge frequency of sEPSCs pyramidal neurons on ?/? layer(p>0.05),but the amplitude of sEPSCs was increased significantly(p<0.05);The acute CXCL13 treatment significantly increased the amplitude of s EPSC in neurons(p<0.01),but did not increase the discharge distribution frequency of s EPSC,(p>0.05).Conclusion 1.The neuropathic pain model was induced by left L5 spinal nerve ligation(SNL).SNL induced the neuropathic pain in mice and could be used as a reliable neuropathic pain model.2.SNL induced the increased expressions of CXCLl3 and CXCR5 mRNA and protein in ACC;CXCLl3 and CXCR5 were mainly expressed in ACC neurons.3.The inhibition of CXCR5 in ACC alleviated the pain-related aversion induced by SNL.4.SNL induced pain affect might be related to the AMPA receptor function of pyramidal neurons excitatory synapse postsynaptic afference in ACC ?/? layer;The increase of CXCL13 in ACC might directly induce the enhanced function of excitatory synaptic transmission of pyramidal neurons on ?/? ACC layer after SNL.