淋巴细胞介导的免疫反应在神经病理性疼痛发病机制中的作用

1、淋巴细胞介导的免疫反应在神经病理性疼痛发病机制中的作用李晶 兰蕴平 左云霞*四川大学华西医院麻醉科，四川成都 610041*通讯作者：左云霞(1963-)，女，四川省人，博士，教授，博士生导师，主要从事小儿麻醉与神经病理性疼痛的基础研究。通讯地址：四川大学华西医院麻醉科，成都610041。联系电话：(028) 85422520。E-mail: zuoyunxiahxa yahoo. com. cn 基金项目：国家自然科学基金资助项目(30571786)摘 要目的：神经性病理性疼痛的发病机制不清楚导致临床治疗困难。外周和中枢神经系统的炎症和免疫机制在神经损伤后的神经病理性疼痛的发生发展中起了重要

2、作用。本实验通过比较淋巴细胞缺失的裸鼠和野生型小鼠神经慢性神经压迫性损伤（Chronic Constrictive Injury, CCI）后疼痛行为学改变和神经损伤局部炎症反应程度、L4-6脊髓节段胶质细胞的活化程度的区别；通过提取神经病理性疼痛大鼠坐骨神经、相应后根神经节和脊髓免疫正常大鼠并观察被免疫动物疼痛行为学改变。从而探讨神经损伤后T淋巴细胞介导的特异性免疫反应在神经病理性疼痛发病机制中的作用。方法：在第一个研究中，Balb/c-nu/nu雄性裸鼠及其野生型小鼠各20只，右侧坐骨神经制作CCI模型。其中裸鼠及其野生型小鼠各5只用于持续观察疼痛行为学包括热痛耐受时间和机械触诱发痛阈值，

3、观察时间长达损伤后3月。另分别有5只动物在损伤后28、56、84天处死后分别获取损伤坐骨神经和L4-6脊髓节段，并用免疫组化方法检测坐骨神经周围巨噬细胞数量、脊髓小胶质细胞、IL17阳性记忆淋巴细胞。在第二个研究中，雄性SD大鼠72只。24只大鼠随机实施CCI 模型或者假手术(Sham)，于术后3周CCI 模型大鼠神经病理性疼痛明显时，取其坐骨神经、后根神经节和脊髓，制成50%组织匀浆，加入等体积的完全弗氏佐剂，注射到正常大鼠坐骨神经走行区皮下(6只/组)。于免疫当天和免疫后3，5，7，14，21天测量热耐受时间和机械痛阈。以Sham手术动物和正常大鼠坐骨神经、脊髓、神经节提取物为对照免疫组。

4、结果：T淋巴细胞缺失的裸鼠神经损伤后热痛过敏程度明显轻于淋巴细胞正常野生型小鼠，且恢复更快；同时野生型鼠坐骨神经损伤侧L4-6脊髓节段有大量记忆淋巴细胞，裸鼠也出现但数目明显降低(P<0.05)；损伤神经局部巨噬细胞的数量和脊髓活化胶质细胞的数量野生型鼠明显高于裸鼠(P<0.05)。用神经损伤后21天CCI 模型大鼠的受损外周神经组织匀浆皮下接种免疫其它正常大鼠观察到接受免疫的大鼠出现热痛过敏和触诱发痛，L4-6脊髓小胶质细胞、IL17阳记忆淋巴细胞数目升高(P<0.05)，但CCI模型大鼠后根神经节和脊髓提取液免疫正常大鼠后没有观察到类似现象。Sham 手术大鼠和正常大鼠的

5、坐骨神经、后根神经节和脊髓提取液免疫动物后也没有观察到热痛过敏和触诱发痛现象。结论：神经损伤可能通过诱发T淋巴细胞介导的特异性免疫反应而导致慢性神经病理性疼痛的产生。关键词：神经病理性疼痛；T淋巴细胞；免疫机制The contribution of lymphocyte-mediated immune response to the mechanism of chronic neuropathic pain followed by nerve injuryJing Li, Yun-Ping Lan, Yun-Xia ZuoDepartment of Anesthesiology, West-C

6、hina Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. ChinaCorrespondences to:Yun-Xia Zuo，MD&PhD, Department of Anesthesiology, West China Hospital, Sichuan University. Chengdu, Sichuan 610041, P. R. ChinaTel: +86-28-85423591; Fax: +86-28-85423591. Email: zuoyunxiahxa . cn This study

7、 is supported by a grant from the National Natural Science Foundation of China by Project No. 30571786AbstractObjective: Neuropathic pain is difficult to treat because the underlying mechanism is unclear. Inflammatory and immune mechanisms both in the periphery and the central nervous system play an

8、 important role in neuropathic pain following nerve injury. To elucidate the contribution of T lymphocyte-mediated specific immune response to the mechanism of chronic neuropathic pain，the pain behaviors measured by thermal latencies and mechanical thresholds were observed for as long as three month

9、s in Balb/c-nu/nu nude mice and their heterozygous littermates followed by nerve injury (CCI model). The extent of inflammatory reaction at the site of injured sciatic nerve and memory lymphocytes infiltration as well as the activation of microglia in lumbar spinal cord were also evaluated. Pain beh

10、aviors of SD rats immunized with tissue homogenate of sciatic nerve, L4-6 dorsal root ganglion and L4-6 spinal cord obtained from CCI rats were assessed in another study.Methods: In the first study, the chronic constriction injury (CCI) of the right sciatic nerve was carried out in Balb/c-nu/nu nude

11、 mice (n=20) and their heterozygous littermates (n=20). Five pairs were used for the observation of thermal latencies and mechanical thresholds up to 84 days post-operation. Five pairs at each timepionts (28, 56, and 84 days post-injury) were sacrificed and their sciatic nerve and L4-6 spinal cord w

12、ere harvested for immunohistochemical detecting macrophages or interleukin-17(IL-17) positive memory T cells and microglia. In the second study, 24 rats were randomly divided into 2 groups either accepted CCI or Sham operation respectively. Animals were sacrificed at 21 days after surgery. Sciatic n

13、erve, L4-6 dorsal root ganglion and L4-6 spinal cord were harvested and homogenized. Six rats in each group were immunized subcutaneously with a mixture of 50% homogenizing tissues liquid emulsified in an equal volume of complete Freunds adjuvant (CFA). Thermal latencies and mechanical thresholds of

14、 each rat were measured on the immunization day and on post-immunization day 3, 5, 7, 14 and 21. Rats immunized with homogenizing tissues obtained from Sham group and normal rats were used as the control groups.Results: Balb/c-nu/nu nude mice with T cell deficiency had less hyperalgesia and faster r

15、ecovery following nerve injury than that of heterozygous littermates (P<0.05). A great number of memory T cells have been found in L4-6 sections of spinal cord in heterozygous littermates and far fewer of them to be found in nude mice(P<0.05). Moreover, the numbers of macrophages at the nerve

16、injury site and microglia in spinal cord L4-6 were much fewer in nude mice than those in the heterozygous littermates(P<0.05). Normal rats developed hyperalgesia and showed a great number of memory T cells and microglia in spinal cord L4-6 when homogenized with injured nervous tissues from CCI ra

17、ts at 21 days post-injury were used to immunize them. However, rats immunized with homogenizing tissues of CCI dorsal root ganglia and CCI spinal cords did not show this phenomenon. Animals immunized with homogenizing tissues obtained from Sham group and normal rats did not display thermal hyperalgesia and mechanical hyperalgesia.