α5亚基γ-氨基丁酸A受体在坐骨神经损伤大鼠认知功能中的作用

Effect ofα5-GABA_(A)Rs in cognitive impairment caused by sciatic nerve injury in rats

目的探讨α5亚基γ-氨基丁酸A受体(α5-GABA_(A)Rs)在坐骨神经损伤(SNI)导致的慢性神经病理性痛大鼠认知功能中的作用。方法选择成年雄性SD大鼠32只,8~10周龄,体重200~230 g。采用随机数字表法将大鼠分为四组:假手术组(N组)、SNI组(S组)、SNI+二甲基亚砜(DMSO)组(P组)、SNI+α5-GABA_(A)Rs反向激动剂组(L组),每组8只。N组仅暴露右侧坐骨神经及其三个终末支,不结扎。S组结扎右侧胫神经和腓总神经并剪断。P组和L组结扎右侧胫神经和腓总神经并剪断,于术后第22天新物体识别实验和术后第23天Y迷宫实验前30 min分别腹腔注射10%DMSO 1 ml和L6557081 mg/kg。于术前1 d和术后第3、7、14、21、22和23天测定四组机械缩足阈值(MWT),术后第22天行新物体识别实验,记录大鼠对新物体和旧物体的探索时间并计算辨别指数,第23天行Y迷宫实验计算自发交替百分比。采用Western blot法检测海马组织α5-GABA_(A)Rs和谷氨酸脱羧酶-67(GAD67)蛋白含量,采用免疫荧光法检测海马组织α5-GABA_(A)Rs与小清蛋白(PV)双阳性细胞数,α5-GABA_(A)Rs与生长抑素(SST)双阳性细胞数。结果与N组比较,S组、P组和L组术后第3、7、14、21、22和23天MWT明显降低(P<0.05),海马组织α5-GABA_(A)Rs蛋白含量明显升高(P<0.05),S组海马组织α5-GABA_(A)Rs与PV双阳性细胞数明显增多(P<0.05),α5-GABA_(A)Rs与SST双阳性细胞数明显增多(P<0.05)。与S组比较,L组对新物体的探索时间明显延长(P<0.05),辩别指数、自发交替百分比和海马组织GAD67蛋白含量明显升高(P<0.05)。结论α5-GABA_(A)Rs反向激动剂L655708可以增加坐骨神经损伤大鼠海马组织GAD67蛋白含量,逆转GABA能系统功能破坏,改善认知功能障碍,进一步显示α5-GABA_(A)Rs参与了慢性神经病理性痛导致的认知功能障碍。

Objective To investigate the role ofα5 containing-GABAAreceptors(α5-GABA_(A)Rs)in cognitive impairment of chronic neuropathic pain caused by sciatic nerve injury(SNI)in rats.MethodsThirty-two male adult SD rats of 8-10 weeks,weighing 200-230 g,were randomly divided into sham group(group N),SNI group(group S),SNI+DMSO group(group P),SNI+α5-GABA_(A)Rs reverse agonist group(group L),8 rats in each group.In group N,the right sciatic nerve and its three terminal branches were exposed without ligation.In group S,right tibial nerve and common peroneal nerve were ligated and clipped.In groups P and L,SNI rats were intraperitoneally injected with 10%DMSO 1 ml orα5-GABA_(A)Rs reverse agonist L6557081 mg/kg,respectively 30 minutes before novel object recognition test on day 22 and the Y maze test on day 23 after surgery.The mechanical withdrawal threshold(MWT)of rats were measured before and on day 3,7,14,21,22,and 23 after surgery,new object recognition test on day 22 after surgery was performed to record exploration time of different objects and calculate recognition index,and Y maze test on day 23 after surgery was performed to calculate percentage of spontaneous alternation.Western blot was used to detect the protein content ofα5-GABA_(A)Rs and GAD67,and immunofluorescence method was used to detect the co-expression number ofα5-GABA_(A)Rs and parvalbumin(PV),the co-expression number ofα5-GABA_(A)Rs and somatostatin(SST)in hippocampus.Results Compared with group N,the MWT was significantly decreased on day 3,7,14,21,22,and 23 after surgery and the protein content ofα5-GABA_(A)Rs were significantly increased in groups S,P and L(P<0.05),the co-expression of hippocampalα5-GABA_(A)Rs and PV,α5-GABA_(A)Rs and SST were significantly increased in group S(P<0.05).Compared with group S,the exploration time of new object was significantly extended(P<0.05),and recognition index,percentage of spontaneous alternation,the protein content of GAD67 were significantly increased in group L(P<0.05).ConclusionIn rats with sciatic nerve injury,theα5-GABA_(A)Rs reverse agonist L655708 upregulates hippocampal protein expression of GAD67,rescues disruption of GABAergic systerm,and improves cognitive impairment,suggesting thatα5-GABA_(A)Rs is involved in the chronic neuropathic pain-related cognitive impairment.