The Role Of Cav3.2 In Astrocytes Of Spinal Dorsal Horn In Neuropathic Pain

Objective:Neuropathic pain(NP)is common in clinic and its therapeutic effect is often ineffective,which leads to the necessity to further explore its mechanism to reveal new analgesic targets.Spinal dorsal horn(SDH)is the primary center of pain signal transduction and local processing.T-type calcium channels(Cav3)are the low-voltage activated calcium channels that modulates the excitability of various cells.In NP animal models,Cav3 channels are aberrantly activated in SDH,as increasing the expression of Cav3 and T-type calcium current.Meanwhile,some studies found that the influx of calcium ions in astrocytes subsequently activates the release of intracellular cytokine,which further activates the neurons around and therefore participate in regulating the development of NP.However,it is still largely unknown whether the cell-type specific expression of Cav3 is associated with the generation and maintenance of NP.Therefore,we studied the changes of Cav3 expression in pain by establishing animal pain models.Next,we investigated the modulation of up-regulated Cav3.2 on astrocyte activation.These data may provide new theoretical evidence for the study of NP mechanism and the development of novel analgesic drug.Methods:Male adult SD rats(150-200 g)were used to generate formalin-induced acute inflammatory pain and partial sciatic nerve ligation(PSNL)-induced chronic NP models.Parts of the PSNL-rats received an intrathecal tube implantation at the surgery day.Immunohistochemistry,western blotting,qPCR were used to observe the expression of Cav3 in SDH.The co-staining of Cav3.2 with GFAP,OX42 and NeuN in SDH was observed by double immunohistochemistry in PSNL-rats.Then,Cav3 channel blocker mibefradil and astrocyte inhibitor fluorocitrate were continuous intrathecal injected in PSNL-rats for 14 days,then we used immunohistochemistry to detect the effect of drugs on the expression of Cav3.2 and GFAP in SDH.Results:1.Subcutaneous injection of formalin induced a typical biphasic nociceptive response in rats and the expression of c-Fos was increased significantly in SDH,but no significant changes of Cav3 subtypes staining intensity were observed;2.PSNL induced mechanical allodynia in ipsilateral hind paw.The expression of Cav3.2 protein and mRNA in SDH was increased at day 14 post-surgery,while the staining intensity of Cav3.1 or Cav3.3 showed no significant changes;3.The increased Cav3.2 staining in PSNL-rats was co-localized with astrocyte marker GFAP,but not microglia marker OX42 or neuronal marker NeuN;4.The up-regulation of Cav3.2 is prior to the increase of GFAP staining.Cav3.2 staining began to increase at day 1 post-surgery,while GFAP staining was significantly increased from day 7,and all of these changes continued to day 14,but no notable changes of NeuN staining were observed;5.Chronic intrathecal administration of mibefradil attenuated PSNL-induced allodynia and concomitantly down-regulated the expression of Cav3.2 as well as GFAP.By contrast,although fluorocitrate reduced NP behavior and the expression of GFAP,it had no obvious effect on the expression of Cav3.2 channel.Conclusions:Acute inflammatory pain does not affect the expression of Cav3;the expression of Cav3.2 is increased in SDH in NP,while the expression of Cav3.1 or Cav3.3 is not affected.And the increased expression of Cav3.2 is mainly located in astrocytes,also it is prior to astrocyte activation.Cav3 channel blocker can produce analgesic effect and inhibit the up-regulation of Cav3.2 and GFAP,suggesting that Cav3.2 channel may be a novel regulator of promoting spinal astrocyte activation in the onset and maintenance of NP.