淋巴細胞介導(dǎo)的免疫反應(yīng)在神經(jīng)病理性疼痛發(fā)病機制中的作用

1、淋巴細胞介導(dǎo)的免疫反應(yīng)在神經(jīng)病理性疼痛發(fā)病機制中的作用李晶 蘭蘊平 左云霞*四川大學(xué)華西醫(yī)院麻醉科，四川成都 610041*通訊作者：左云霞(1963-)，女，四川省人，博士，教授，博士生導(dǎo)師，主要從事小兒麻醉與神經(jīng)病理性疼痛的基礎(chǔ)研究。通訊地址：四川大學(xué)華西醫(yī)院麻醉科，成都610041。聯(lián)系電話：(028) 85422520。E-mail: zuoyunxiahxa yahoo. com. cn 基金項目：國家自然科學(xué)基金資助項目(30571786)摘 要目的：神經(jīng)性病理性疼痛的發(fā)病機制不清楚導(dǎo)致臨床治療困難。外周和中樞神經(jīng)系統(tǒng)的炎癥和免疫機制在神經(jīng)損傷后的神經(jīng)病理性疼痛的發(fā)生發(fā)展中起了重要

2、作用。本實驗通過比較淋巴細胞缺失的裸鼠和野生型小鼠神經(jīng)慢性神經(jīng)壓迫性損傷（Chronic Constrictive Injury, CCI）后疼痛行為學(xué)改變和神經(jīng)損傷局部炎癥反應(yīng)程度、L4-6脊髓節(jié)段膠質(zhì)細胞的活化程度的區(qū)別；通過提取神經(jīng)病理性疼痛大鼠坐骨神經(jīng)、相應(yīng)后根神經(jīng)節(jié)和脊髓免疫正常大鼠并觀察被免疫動物疼痛行為學(xué)改變。從而探討神經(jīng)損傷后T淋巴細胞介導(dǎo)的特異性免疫反應(yīng)在神經(jīng)病理性疼痛發(fā)病機制中的作用。方法：在第一個研究中，Balb/c-nu/nu雄性裸鼠及其野生型小鼠各20只，右側(cè)坐骨神經(jīng)制作CCI模型。其中裸鼠及其野生型小鼠各5只用于持續(xù)觀察疼痛行為學(xué)包括熱痛耐受時間和機械觸誘發(fā)痛閾值，

3、觀察時間長達損傷后3月。另分別有5只動物在損傷后28、56、84天處死后分別獲取損傷坐骨神經(jīng)和L4-6脊髓節(jié)段，并用免疫組化方法檢測坐骨神經(jīng)周圍巨噬細胞數(shù)量、脊髓小膠質(zhì)細胞、IL17陽性記憶淋巴細胞。在第二個研究中，雄性SD大鼠72只。24只大鼠隨機實施CCI 模型或者假手術(shù)(Sham)，于術(shù)后3周CCI 模型大鼠神經(jīng)病理性疼痛明顯時，取其坐骨神經(jīng)、后根神經(jīng)節(jié)和脊髓，制成50%組織勻漿，加入等體積的完全弗氏佐劑，注射到正常大鼠坐骨神經(jīng)走行區(qū)皮下(6只/組)。于免疫當(dāng)天和免疫后3，5，7，14，21天測量熱耐受時間和機械痛閾。以Sham手術(shù)動物和正常大鼠坐骨神經(jīng)、脊髓、神經(jīng)節(jié)提取物為對照免疫組。

4、結(jié)果：T淋巴細胞缺失的裸鼠神經(jīng)損傷后熱痛過敏程度明顯輕于淋巴細胞正常野生型小鼠，且恢復(fù)更快；同時野生型鼠坐骨神經(jīng)損傷側(cè)L4-6脊髓節(jié)段有大量記憶淋巴細胞，裸鼠也出現(xiàn)但數(shù)目明顯降低(P<0.05)；損傷神經(jīng)局部巨噬細胞的數(shù)量和脊髓活化膠質(zhì)細胞的數(shù)量野生型鼠明顯高于裸鼠(P<0.05)。用神經(jīng)損傷后21天CCI 模型大鼠的受損外周神經(jīng)組織勻漿皮下接種免疫其它正常大鼠觀察到接受免疫的大鼠出現(xiàn)熱痛過敏和觸誘發(fā)痛，L4-6脊髓小膠質(zhì)細胞、IL17陽記憶淋巴細胞數(shù)目升高(P<0.05)，但CCI模型大鼠后根神經(jīng)節(jié)和脊髓提取液免疫正常大鼠后沒有觀察到類似現(xiàn)象。Sham 手術(shù)大鼠和正常大鼠的

5、坐骨神經(jīng)、后根神經(jīng)節(jié)和脊髓提取液免疫動物后也沒有觀察到熱痛過敏和觸誘發(fā)痛現(xiàn)象。結(jié)論：神經(jīng)損傷可能通過誘發(fā)T淋巴細胞介導(dǎo)的特異性免疫反應(yīng)而導(dǎo)致慢性神經(jīng)病理性疼痛的產(chǎn)生。關(guān)鍵詞：神經(jīng)病理性疼痛；T淋巴細胞；免疫機制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.