Neuropathic Pain Elevates Functional Cav3.2 Channels In Superficial Spinal Dorsal Horn

Objective:Neuropathic pain（NP）is a clinical problem with high incidence and its therapeutic effect is often ineffective,which bring a heavy burden on patients and society.Lamina II（substantia gelatinosa,SG）in the superficial spinal dorsal horn（SDH）is the primary center of pain signal transduction and local processing.Low-voltage activated calcium channels,also named as T-type calcium（Cav3）channels,that modulates the excitability of cells,which is crucial for the pathogenesis of chronic pain and been used as an analgesic target.Although it has been shown that the expression of Cav3 channels in SDH was upregulated after nerve ligation-or paclitaxel-treatment,the functional changes of Cav3 channels under the NP state have not been studied yet.Here,we examined the effect of partial sciatic nerve ligation（PSNL）on either expression or electrophysiological properties of Cav3 channels in superficial SDH,further clarify pain modulation mechanism of Cav3 channels in spinal,which may provide new theoretical basis for the research and development of drugs to treat neuropathic pain.Methods:Sprague-Dawley（SD）rats（6-8 w,150-250 g）,C57/BL6 wild-type（WT）and Cav3.2 knockout（KO）mice（8-12 w,20-30 g）were used to generate PSNL-induced neuropathic pain models in this study.The paw withdrawal threshold（PWT）and paw withdrawal latency（PWL）were recorded before the operation,and at day 1,3,7,10and 14 after the operation.First,PSNL rats received a continuous intrathecal or intermittent intraperitoneal injection of Cav3 channel blockers（NiCl₂,TTA-A2 and ascorbic acid）at the surgery day with PWT and PWL were observed.The equivalent volume of saline was used as the control.Second,the comparison of pain behavior from PSNL models between WT and Cav3.2 KO mice was made.Third,qRT-PCR and western blot were performed to observe the expression of Cav3 in superficial SDH of PSNL rats at 14 day after operation.Finally,transverse slices（400-450μm thickness）of spinal cords from rats or mice models were prepared in vitro,and the electrophysiological properties of Cav3 channels were recorded in superficial SDH of PSNL models by the electrophysiological technique.Results:1.PWT and PWL of PSNL group on 1,3,7,10 and 14 day after operation were significantly lower than that of sham group.The admission of Cav3 channel blockers either by continuous intrathecal or intermittent intraperitoneal injection to PSNL rats elicit a strong and stable analgesic effect until postoperation day 14.2.PSNL increased both mRNA and protein levels of Cav3.2 in rat superficial SDH of rats,but not Cav3.1 or Cav3.3.3.PSNL triggered pain behavior in both WT and Cav3.2 KO mice.PSNL-induced mechanical allodynia,but not thermal hyperalgesia,in Cav3.2 KO-PSNL mice was partly alleviated compared to WT-PSNL mice.4.Both the overall proportion of Cav3 current（I_T）-expressing neurons（sham:59.4%;PSNL:76.7%）,amplitude（sham:-78.8±12.3 pA;PSNL:-124.3±14.9 pA）and the current density of I_T were elevated in SG neurons from PSNL rats;the kinetics of I_T in SG neurons were not significantly altered after PSNL.5.There was no significant difference of proportion of I_T-expressing neurons,amplitude and the current density of I_T between sham and PSNL Cav3.2 KO mice.6.NiCl₂ blocked I_T in SG neurons from sham and PSNL rats with similar potency.Conclusions:In summary,these data for the first time demonstrated that the augment of functional Cav3.2 channels in superficial SDH neurons may underlie the central mechanisms of mechanical allodynia in PSNL-induced neuropathic pain,which might provide new insights into the contribution of Cav3.2 in spinal as an analgesic target in pain.