新斯的明对神经病理性痛大鼠脊髓小胶质细胞的影响

Effects of neostigmine on activation of spinal cord microglia in a rat model of neuropathic pain

目的评价新斯的明鞘内给药对神经病理性痛大鼠脊髓小胶质细胞的影响。方法健康雄性SD大鼠，月龄1月，体重200～250g，建立坐骨神经分支选择性损伤（SNI）致神经病理性痛模型。术后5d行鞘内置管，取鞘内置管成功的SNI大鼠40只，恢复2d后行鞘内给药。随机分为4组（n=10）：SNI组、新斯的明组（N组）、阿托品＋新斯的明组（A＋N组）和美加明＋新斯的明组（M＋N组）。SNI组鞘内注射生理盐水20ul，N组鞘内注射新斯的明10ug，A＋N组和M＋N组分别于鞘内注射阿托品10ug或美加明10ug后5min鞘内注射新斯的明10／Lg。分别于造模前2d（基础状态）、造模后即刻（T1）、1d（T1）、3d（T3）、5d（T4）、新斯的明给药前即刻（L）、给药后15min（L）、30min（T7）、45min（B）、60min（Tq）时各组随机取5只大鼠测定机械痛闽，并确定给药后机械痛阈最高点，在机械痛阈最高点时另取5只大鼠麻醉后取脊髓，镜下计数阳性脊髓小胶质细胞。结果与SNI组比较，N组给药后各时点机械痛阈升高，T6时达峰值，A＋N组和M＋N组给药后各时点机械痛阈升高，N组和A＋N组术侧阳性脊髓小胶质细胞数量减少（P〈0．05），M＋N组术侧阳性脊髓小胶质细胞数量差异无统计学意义（P〉0．05）。与N组比较，A＋N组和M＋N组给药后各时点机械痛阈降低（P〈0．05）。结论新斯的明鞘内给药可抑制神经病理性痛大鼠脊髓小胶质细胞的激活，从而发挥镇痛作用，其机制与激活N胆碱能受体有关。

Objective To investigate the effects of intrathecal neostigmine on the activation of spinal cord microglia in a rat model of neuropathic pain. Methods Male SD rats weighing 200-250 g were used in this study. APE 10 catheter was inserted at L4-5 interspace into the epidural space and advanced for 3 cm cephalad. Spinal nerve injury （SNI） was induced by exposure of sciatic nerve and its three branches and ligation and transaction of tibial and common fibular nerves. Forty SD rats in which IT catheter was successfully placed without complication were randomly divided into 4 groups （ n = 10 each） ： group Ⅰ SNI control （SNI） ; group Ⅱ SNI ＋ neostigmine （N）; group Ⅲ SNI ＋ atropine ＋ neostigmine （A＋ N）and group Ⅳ SNI ＋ mecamylamine ＋ neostigmine （M ＋ N）. In group SNI normal saline 20 ul was given IT; in group N neostigmine 10 ug （10 ul） was given IT, while in group A ＋ N （ Ⅲ ） and group M ＋ N （ Ⅳ ） atropine 10 ug and mecamylamine 10 ug were given IT at 5 min before neostigmine 10 ug IT respectively. 50% paw withdrawal tbreshold to yon Frey filament stimulation （50% PWT） was measured at 2 d before SNI, at 0, 1, 3 and 5 d after SNI, before and at 15, 30, 45, 60 rain after IT drug administration. The animals were anesthetized and killed at the time when analgesia induced by IT drug administration was most satisfactory. The spinal cord was removed. The number of positive microglia cells in the spinal cord was determined using immuno-histochemistry. The cell morphology was also examined. Results In group N （group Ⅱ ） 50% PWT on the operated side was significantly increased after IT neostigmine administration as compared with the baseline value before IT drug administration. The number of positive microglia cells on the operated side was significantly larger than that on tire contralateral side in control group, and was significantly decreased by IT neostigmine in group N and by IT atropine ＋ neostigmine in group A ＋ N. There was no significant difference in tire number of positive microglia ceils on the operated side between group C and group M ＋ N.Conclusion N-chotinergic receptor is invbolved in the IT neostigmine-induced inhibition of the activation of spinal cord microglia in a rat model of neuropathic pain.