脊髓背角神经元上调Nav1.8通道参与缺血再灌注损伤后痛觉过敏的机制

Mechanisms of ischemia-reperfusion induced hyperalgesia via up-regulation of neuronal Nav1. 8 channel in spinal dorsal horn

目的观察鞘内注射钠通道抑制剂619C89对脊髓缺血再灌注损伤引起的痛觉过敏大鼠的痛阈、脊髓背角神经元中Nav1.8通道表达的影响。方法 SD大鼠随机分为3组：假手术组（S组）、痛觉过敏组（H组,缺血再灌注前3 d鞘内注射30μL生理盐水）、钠通道抑制剂组（I组,缺血再灌注前3 d鞘内注射619C895μg/30μL）。S组仅暴露主动脉弓而不结扎,其他各组开胸后无创动脉夹夹闭主动脉弓14 min后再开放,建立SCIRI引起的痛觉过敏模型。各组手术前均于L_（5-6）鞘内置管并连续注射3 d。术后1、3、5、7、14 d分别测定各组大鼠的热痛阈及机械性痛阈;取第4~6节腰段脊髓,采用免疫双荧光法观察背角神经元状态及其Nav1.8的表达,Real time-PCR法检测各组大鼠脊髓组织Nav1.8表达。结果与S组相比,术后各观察点（尤以第7天为著）H组大鼠热痛阈和机械性痛阈降低,损伤后7 d脊髓组织中Nav1.8 mRNA的表达增加（P〈0.05）;I组大鼠热痛阈和机械性痛阈值明显提高（P〈0.05）,脊髓组织中Nav1.8 mRNA的表达降低（P〈0.05）。免疫双荧光染色显示,损伤后7 d,H组大鼠脊髓背角Nav1.8的荧光强度明显增加,且主要表达在NeuN表达阳性的神经元的胞浆中;且与S组相比,H组中NeuN/Nav1.8双阳性的细胞数量明显增多,而I组双阳性的细胞数量减少（P〈0.05）。结论脊髓背角神经元通过上调Nav1.8通道参与缺血再灌注损伤后痛觉过敏的形成。

Objective To observe the effects of intrathecal injection （IT） of Navl. 8 channel inhibitor 619C89 on hyperalgesia and spinal cord levels of neuronal Navl. 8 expressions in rat model of spinal cord ischemia-reperfusion injury （SCIRI）. Methods Male Sprague-Dawley rats were randomly divided into three groups：group S, group H （ SCIRI ＋ IT NS） and Navl. 8 channel inhibitor group （ group I,SCIRI ＋ IT 5 μg/30 μL 619C89）. The lumbar intrath- ecal catheters were implanted in L5-6 of rats and SCIRI models were established by aortic arch occlusion for 14 min. The thermal and mechanical nociceptive thresholds were assessed by paw withdrawal latency （PWL） to radiant heat and von Frey filaments. The 619C89 was administered intrathecally for 3 days before surgery. The spinal mRNA expression of Navl. 8 was assessed by Real time-PCR and double immunofluorescence staining was performed for examination of the distribution of neurons and Navl. 8 and also quantification Of NeuN/Navl. 8 positive cells of dorsal horn at 1,3,5, 7 and 14 days after surgery. Results Compared with group S, animals in group H had significantly lower mechanical and thermal pain thresholds ,but higher spinal mRNA expression of Navl. 8 （P 〈 0. 05 ）. Rats in group I had signifi- cantly higher mechanical and thermal pain thresholds and significantly lower mRNA expression of Navl. 8 compared with those in group H （ at any observed time points after IR, but most significantly at 7 days, P 〈 0. 05 ）. Double fluo- rescent staining showed the distribution of increased fluorescence intensity of Navl. 8 was similar to that of fluorescent staining of NeuN （neuronal marker）. The number of NeuN/Navl. 8 positive cells was greatly increased in group H, whereas the number was obviously decreased in group I （ P 〈 0. 05 ）. Conclusion Up-regulation of neuronal Navl. 8 channel in spinal dorsal horn plays a role in IR-induced hyperalgesia.