神经病理性痛大鼠脊髓背角缝隙连接蛋白表达变化及鞘内注射甘珀酸的镇痛作用

Changes of Connexin 43 and 32 expression in the dorsal horn of spinal cord of rat with neuropathic pain and analgesic effect of carbenoxolone by intrathecal injection

目的研究慢性神经病理性痛大鼠脊髓背角内缝隙连接蛋白家族（Cx）中Cx43和Cx32的表达变化,以及鞘内注射缝隙连接阻断剂甘珀酸（CBX）的镇痛作用。方法成年SD大鼠50只,分为正常对照组、假手术组和坐骨神经分支选择性损伤组（SNI）。手术前1d、术后3d、5d、10d2、0d和30d,观察大鼠行为并检测机械刺激缩足反射阈值（PWMT）。15只大鼠于术后10d2、0d、30d取脊髓腰段进行免疫印迹检测,另15只大鼠于术后10d、20d、30d取脊髓腰段进行免疫荧光染色,检测腰段脊髓背角内Cx43和Cx32表达的变化。有10只大鼠先进行鞘内插管,后行SNI手术,术后20d向鞘内注射生理盐水或CBX,观察大鼠PWMT的变化。结果SNI大鼠手术侧PWMT阈值较非手术侧或假手术组明显降低,术后20d达最低值。SNI大鼠手术侧脊髓背角内Cx43、32表达增多,明显高于非手术侧和假手术组背角。鞘内注射CBX 3h后,PWMT平均阈值由（2.5±1.0）g上升到（20.0±3.2）g,有抑制效应,而生理盐水组则无抑制效应。结论脊髓背角内的缝隙连接在因外周神经损伤引起的神经病理性痛中起重要作用。

Objective To study the changes of connexin 32 （Cx32） and Cx43 signals in the dorsal horn of spinal cord of rat with neuropathic pain induced by spared nerve injury （SNI） to sciatic nerve, and the analgeaic effect of 10μl carbenoxolone （CBX, a gap junction blocker, 1mmol/L）on neuropathic pain via intrathecal administration. Methods Adult SD rats were randomly divided into 3 groups, normal group （the animals did not receive any treatment）, sham group （incised the skin and only exposed the sciatic nerve） and the SNI group, exposed three branches of the sciatic nerve, i.e. the tibial nerve, the common peroneal nerve and the sural nerve, and cut the tibial nerve and the common peroneal nerve but remained sural nerve. The paw withdrawal mechanical threshold （PWMT） of normal, sham and SNI groups was measured at lday before SNI and at 3, 5, 10, 20 and 30 days after SNI, then the animals were killed and the spinal cords （IA-S1 segments） were removed and were used for Western blotting and anti-Cx32 and anti-Cx43 immunofluorescent staining. CBX was intrathecally injected at 20 days after SNI, and measured PWMT induced with an innocuous mechanical yon Frey filaments stimulation. Results The PWMT of left behind leg in normal and sham groups did not reduce, while the left behind leg in SNI animals developed a conspicuous hypersensitivity to innocuous mechanical yon Frey filaments stimulation. The PWMT of SNI rat decreased significantly as compared with normal and sham groups, and peaked at 20 days after SNI, it could maintain for as long as the animals were monitored. Anti-Cx32 and anti-Cx43 immunofluoreseent reaction in the dorsal horn of spinal cord of SNI side increased significantly as compared with that in normal and sham group or the non-SNI side. After intratheeal injection of CBX 3hours, the PWMT of SNI side increased from the （2.5 ± 1.0）g to （20.0 ± 3.2）g, CBX inhibited the mechanical allodynia. In NS intrathecal injection group did not affect PWMT on SNI rats. Conclusion The gap junction plays an important role in the pathophysiologie process of chronic neuropathic pain.