【作者】 孙涛；

【导师】 姚尚龙； 宋文阁；

【作者基本信息】 华中科技大学 ， 麻醉学， 2007， 博士

【摘要】 第一部分脊髓星形胶质细胞和促炎性细胞因子在神经病理性疼痛中的作用机制研究实验一神经病理性疼痛大鼠脊髓星形胶质细胞增殖活化的动态变化目的观察大鼠坐骨神经慢性压迫性损伤(CCI)模型中脊髓星形胶质细胞形态和功能特征的时间变化规律。方法雄性SD大鼠48只,随机分为2组:手术(CCI手术)组和假手术组。于手术前1d、手术后1d、4d、7d、14d和28d动态观察其机械性痛敏和热痛敏的变化。并于相应时段将大鼠处死,免疫组化方法观察脊髓星形胶质细胞在疼痛刺激下形态的转化。结果手术组机械性痛阈在术后第1d开始出现下降(P<0.05),术后7d痛阈下降达到最低(P<0.01),以后逐渐恢复,但至28d仍处于较低水平(P<0.05)。热痛阈在手术组从术后第1d开始出现热痛阈下降(P<0.05),术后4d痛阈下降达到最低(P<0.01),以后逐渐恢复,但至28d仍处于较低水平(P<0.05)。免疫组化结果表明假手术组和手术组大鼠健侧腰脊髓后角没有观察到明显的星形胶质细胞增殖活化现象。手术组术侧脊髓后角星形胶质细胞免疫阳性细胞在术后1d没有明显的变化,术后4d开始增加,术后7d达到高峰,至术后28d虽有下降,但始终维持于高水平(P<0.05)。结论大鼠坐骨神经慢性压迫性损伤可以导致神经病理性疼痛的发生,并且诱发损伤侧脊髓背角星形胶质细胞增殖活化。星形胶质细胞的增殖活化可能是神经病理性疼痛的机制之一。实验二鞘内注射氟代柠檬酸对CCI大鼠的镇痛作用以及脊髓促炎性细胞因子的影响目的观察鞘内注射星形胶质细胞特异性抑制剂氟代柠檬酸(FC)对神经病理性疼痛大鼠的镇痛作用以及脊髓促炎性细胞因子肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的变化。方法雄性SD大鼠32只,随机分为4组:A组(CCI手术,鞘内注射不含FC的配制溶液);B组(CCI手术,鞘内注射FC);C组(假手术,鞘内注射不含FC的配制溶液);D组(假手术,鞘内注射FC)。采用大鼠坐骨神经慢性压迫性损伤(CCI)模型,鞘内注射剂量为1nmol/1μl,1次/d,连续6d。分别于手术前1d,手术后1d,3d,5d,7d观察大鼠机械痛阈和热痛阈的变化,并于手术后7d处死动物,应用RT-PCR和免疫组化技术从基因和蛋白水平观察脊髓TNF-α和IL-6的改变。结果手术前1d各组大鼠机械痛阈和热痛阈无显著性差异(P>0.05)。与C组相比,A组大鼠CCI手术后大鼠机械痛阈和热痛阈明显降低(P<0.05),TNF-α和IL-6mRNA (P<0.01)和免疫阳性细胞平均光密度值(P<0.05)明显增加。B组大鼠鞘内注射FC后与A组相比, CCI大鼠的痛敏状态显著减轻(P<0.05) ,并且TNF-α和IL-6mRNA和蛋白的表达明显抑制(P<0.05)。而D组大鼠鞘内注射FC后对大鼠的痛敏状态和TNF-α和IL-6mRNA和蛋白的表达没有明显的抑制作用。结论鞘内注射FC对大鼠神经病理性疼痛有镇痛作用,其机制可能是与抑制脊髓星星胶质细胞的活性和下调促炎性细胞因子TNF-α和IL-6的表达有关。第二部分脊髓星形胶质细胞NF-κB信号转导通路在神经病理性疼痛中的作用机制研究实验一大鼠神经病理性疼痛模型中脊髓背角GFAP和NF-κBp65的共同表达目的观察大鼠坐骨神经慢性压迫性损伤(CCI)模型脊髓背角GFAP和NF-κB表达间的关系,以研究星形胶质细胞内可能的信号转导通路机制。方法雄性SD大鼠16只,随机分为2组:手术(CCI手术)组和假手术组。于手术前1d和手术后7d观察其机械性痛敏和热痛敏的变化。并应用免疫细胞化学技术,观察大鼠脊髓腰段背角GFAP和NF-κB p65的表达及二者的共存情况。结果与假手术组相比,手术组大鼠手术后7d机械性痛阈和热痛阈均明显降低(P<0.05)。假手术组手术侧背角表层中GFAP和NF-κBp65免疫阳性细胞以及双标细胞表达较少。而手术组手术侧背角表层中GFAP(P<0.05)和NF-κBp65(P<0.01)表达均明显增加。并且NF-κBp65与GFAP均阳性表达的双标细胞高达27.47±4.18,与假手术组(8.39±2.06)相比有显著性差异(P<0.01)。结论脊髓背角星形胶质细胞内NF-κB信号转导通路可能与神经病理性疼痛的调制有关。实验二鞘内注射NF-κBp65反义寡核苷酸治疗CCI大鼠神经病理性疼痛的实验研究目的观察鞘内注射NF-κBp65反义寡核苷酸对CCI大鼠痛敏状态以及促炎性细胞因子TNF-α和IL-6mRNA的影响。方法大鼠随机分为4组(n=8):假手术组,行假手术,鞘内注射生理盐水10μl;手术组,行CCI手术,鞘内注射生理盐水10μl;错义寡核苷酸组,CCI手术,鞘内注射错义寡核苷酸20μg/10μl;反义寡核苷酸组,CCI组,鞘内注射反义寡核苷酸20μg/10μl。鞘内注射从CCI手术后1天开始,每日给药1次,连续5天。于CCI手术前1d,手术后1d、3d、5d、7d、14d测定机械痛阈和热痛阈,连续观察鞘内注射寡核苷酸对大鼠热痛敏和机械性痛敏状态的影响。在手术后14d处死动物,应用RT-PCR、Western blot技术对大鼠脊髓TNF-αmRNA、IL-6 mRNA和NF-κBp65蛋白表达的变化进行检测。结果术前各组机械痛阈和热痛阈无显著性差异。假手术组整个观察期间手术侧机械痛阈和热痛阈无明显改变。手术组从CCI术后第1d开始出现机械痛阈和热痛阈下降,并且持续到14天。CCI手术后鞘内注射反义寡核苷酸后大鼠机械痛阈和热痛阈有明显增加,但是CCI手术后鞘内注射错义寡核苷酸机械痛阈和热痛阈增加不明显。Western blot结果显示CCI手术后NF-κB p65表达量较假手术组有显著升高(p<0.01)。与鞘内注射错义寡核苷酸和生理盐水相比,CCI手术后鞘内注射反义寡核苷酸后NF-κB p65表达量显著降低(P<0.05)。RT-PCR结果显示CCI术后14d TNF-α和IL-6 mRNA与假手术组相比有显著增加(P<0.01)。而CCI手术后鞘内注射反义寡核苷酸后TNF-α和IL-6 mRNA表达量则显著降低(P<0.05)。结论鞘内NF-κB p65反义寡核苷酸后可显著抑制神经病理性疼痛,降低脊髓促炎性细胞因子TNF-α和IL-6 mRNA的表达,提示NF-κB信号通路及其下游促炎性细胞因子可能参与神经病理性疼痛的调节。更多还原

【Abstract】 Part 1 The altervation of spinal astrocyte and pro-inflammatory cytokine in chronic constriction injury model of ratsStudy 1 Dynamic altervation of spinal astrocyte reaction in chronic constriction injury model of ratsObjective To observe the response of astrocytes in lumber spinal cord in chronic constriction injury (CCI) model of rats.Methods Sprague -Dawley rats were randomly divided into 2 groups, which received right sciatic nerve ligation (chronic constriction injury, CCI group) and sham-operation (Sham group) respectively. The mechanical and thermal pain threshold were measured at 1d before the operation and at 1d, 4d, 7d, 14d and 28d after the operation. The expression of GFAP was also assessed by immunohistochemical analysis at the same time point.Results CCI, and not sham surgery, produced significant mechanical allodynia and thermal hyperalgesia. The mechanical threshold began to decrease at 1d after the operation, peaked at 7d and lasted throughout the experiment. The thermal threshold began to decrease at 1d after the operation, peaked at 4d and lasted throughout the experiment. Immunoreactive(IR)-like GFAP protein in the ipsilateral spinal dorsal horn to the injury did not significantly increase until postoperative day 4, reached a peak level on postoperative day 7 and lasted over the duration of the study. Few positive astrocytes were distributed in the contralateral spinal cord or spinal cord of sham group.Conclusion The activation of lumber spinal astrocyte may be involved in the modulation of neuropathic pain arised from chronic constriction injury to the sciatic nerve.Study 2 Downregulation of TNF-alpha, IL-6 mRNA and protein expression in CCI model of rats after intrathecal administration of fluorocitrateObjective To investigate the effect of intrathecal administration of fluorocitrate (FC), an selective astrocyte metabolic inhibitor, on tumor necrosis factorα(TNF-α) and interleukin-6(IL-6) mRNA and protein expression in neuropathic pain model of rats.Methods The CCI model was established and FC was administrated intrathecally (1nmol/1μl) once daily for 6 consecutive days. Negative control for CCI was sham operation and that for FC was the vehicle. The mechanical and thermal pain threshold were measured at 1d before operation and at 1d, 3d, 5d, 7d after the operation. The expression of TNF-αand IL-6 mRNA was measured by reverse transcription polymerse chain reaction (RT-PCR) and the expression of TNF-αand IL-6 protein was measured by immunohistochemistry.Results CCI, not the sham operation, significantly induced the mechanical allodynia and thermal hyperalgesia, and markedly increased the expressions of TNF-αand IL-6 mRNA and immunoreactive(IR)-like TNF-α/IL-6 protein in the ipsilateral spinal dorsal horn to the injury. Intrathecal injection of FC suppressed the increased expressions of TNF-αand IL-6 induced by CCI in the spinal cord, and significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. There were no effects on the mechanical allodynia and thermal hyperalgesia and the expressions of TNF-αand IL-6 by intrathecal FC on the sham operation group.Conclusion Fluorocitrate has the effect of analgesia on neuropathic pain. The activation of astrocyte and upregulation of the expression of TNF-αand IL-6 in the spinal cord may be involved in the modulation of neuropathic pain.Part 2 Study of NF-κB pathway in spinal astrocyte in neuropathic painSyudy 1 Co-expressions of GFAP and NF-κB in spinal cord of CCI model of ratsObjective To determine the possible intracellular signal transduction pathway associated with astrocyte, which have been activated by CCI and clarify the relationship of expression or activation of GFAP and NF-κB in the lumber spinal cord.Methods Sixteen male Sprague-Dawley rats were randomly divided into two groups: the CCI group which received the chronic constriction injury and the sham group which received the sham operation as control. The mechanical and thermal nociceptive thresholds were assessed with paw withdrawal latency (PWL) to von Frey filaments and radiant heat. The expressions of GFAP, NF-κBp65 and co-expressions of GFAP and NF-κB in spinal cord were examined 7d after CCI procedure by using immunocytochemical staining technique.Results At 7d post-lesion, CCI induced mechanical allodynia and thermal hyperalgesia and increased the numbers of GFAP and NF-κBp65 immunoreactive(IR) positive cells in the ipsilateral lumber spinal dorsal horn to the injury. Double immunocytochemical staining also showed the increase of GFAP/NF-κBp65-IR positive cells in the spinal dorsal horn in CCI group, while few positive cells were distributed in the sham group.Conclusion CCI activates NF-κBp65 in astrocytes in the dorsal horn and NF-κB pathways in astrocytes may be involved in the pathogenesis of neuropathic pain.Syudy 2 Alleviation of neuropathic pain by intrathecal injection of antisense oligonucleotides to p65 subunit of NF-κBObjective To investigate the effects of intrathecal administration of NF-κB p65 antisense oligodeoxynucleotides (ODN) on mechanical allodynia and thermal hyperalgesia and the expression of TNF-αand IL-6 mRNA in chronic constriction injury (CCI) model of rats.Methods Lumbar intrathecal catheters were implanted in male Sprague - Dawley rats. The chronic constriction injury (CCI) model was established and thermal and mechanical nociceptive thresholds were assessed with paw withdrawal latency (PWL) to radiant heat and von Frey filaments. The phosphorothioate -modified antisense oligodeoxynucleotides (ODNs) to p65 subunit of NF-κB were administered intrathecally on each of 5 successive days post-CCI. Nuclear NF-κB p65 expression was determined by Western blot. TNF-αand IL-6 mRNA were determined by RT-PCR. Results CCI induced mechanical allodynia and thermal hyperalgesia, and significantly increased the expression of NF-κB p65 protein, TNF-αand IL-6 mRNA. Intrathecal injection of antisense ODNs, but not missense ODNs, markedly suppressed the expression of NF-κB p65 protein, TNF-αand IL-6 mRNA, and significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia.Conclusion The activation of NF-κB pathway and the release of TNF-αand IL-6 may contribute to neuropathic pain in CCI rats. NF-κBp65 is likely to be an effective target for the treatment of neuropathic pain.更多还原