A型肉毒毒素治疗大鼠三叉神经痛的中枢作用机制研究

发布时间：2018-05-20 10:12

  本文选题：A型肉毒毒素 + 三叉神经痛　； 参考：《郑州大学》2016年博士论文

【摘要】：三叉神经痛是临床上最为痛苦和常见的神经病理性疼痛之一。临床表现为局限于三叉神经分布区域的单侧短暂性电击样或刀割样剧痛。它的首选治疗为口服药物治疗,在口服药物治疗无效的情况下可以考虑手术治疗,然而有不少患者经过多种治疗后效果仍不理想。A型肉毒毒素是革兰氏阳性厌氧芽孢肉毒杆菌释放的一种外毒素,广泛应用于肌张力障碍治疗和美容领域。近年来,它在疼痛治疗方面的研究越来越多,2012年我们改进了A型肉毒毒素治疗三叉神经痛的技术路线,在国际上首次用随机、双盲、安慰剂对照的试验方法证实了A型肉毒毒素能够长期有效的缓解三叉神经痛的疼痛且不良反应轻微。随后的多项临床研究也得出了相似的结论。但是A型肉毒毒素治疗三叉神经痛的机制仍不清楚。为此,我们建立大鼠眶下神经慢性缩窄环术三叉神经痛模型,探讨A型肉毒毒素对三叉神经痛的治疗作用、作用部位和作用机制。为A型肉毒毒素治疗三叉神经痛和其他疼痛性疾病提供基础理论依据。第一部分大鼠眶下神经慢性缩窄环术建立三叉神经痛模型的行为学和组织学研究目的建立大鼠眶下神经慢性缩窄环术三叉神经痛模型,模拟经典三叉神经痛,观察术后大鼠的疼痛行为学和眶下神经组织学改变。方法实验组大鼠经口建立眶下神经慢性缩窄环术三叉神经痛模型,对照组只暴露眶下神经而不结扎。用Von Frey filaments对两组术前和术后每2天大鼠的疼痛学行为进行评价;采用HE染色和MBP免疫组织化学染色对术前和术后14天眶下神经组织学改变进行评价。结果实验组术后第4天出现一明显的不应期,同侧面部疼痛阈值显著上升,第6天疼痛阈值达到高峰,为24.6±8.6g,与术前相比有统计学差异(P0.05)。随后疼痛阈值急剧下降,在术后第14天达到5.5±2.3g(P0.05),这一趋势至少持续至术后34天。对照组术前和术后各时间点疼痛阈值相比无统计学差异(P0.05)。实验组和对照组术前疼痛阈值分别为13.5±3.6g和12.6±5.5g,两者相比无统计学差异(P0.05)。两组间疼痛阈值在术后第10天分别为5.5±3.2g和13.6±4.8g,组间比较有统计学差异(P0.05),这一差异至少持续至术后第34天。HE染色和MBP免疫组织化学染色表明实验组术后第14天眶下神经存在明显的脱髓鞘改变,而对照组与术前相比无显著变化。结论大鼠眶下神经慢性缩窄环术三叉神经痛模型符合经典三叉神经痛的临床特点和组织学改变。该模型简单易行,我们可以利用该模型探讨三叉神经痛的发病机制,研究新的治疗方法。第二部分A型肉毒毒素对大鼠三叉神经痛模型的抗伤害感受作用研究目的用A型肉毒毒素对三叉神经痛模型进行干预,观察大鼠疼痛行为学的改变。方法大鼠眶下神经慢性缩窄环术建立三叉神经痛模型术后第14天,实验组在手术同侧面部须垫部皮下注射A型肉毒毒素(3U/Kg和10U/Kg),对照组在同样部位注射等量的生理盐水。用Von Frey filaments对各组术前和术后不同时间点的疼痛学行为进行评价。结果A型肉毒毒素治疗后第4天(术后第18天),3U组和10U组痛觉阈值均显著升高,与对照组相比有统计学差异(P0.05),这一差异至少持续至治疗后20天(术后34天)。10U组同一时间点疼痛阈值高于3U组,但两组间痛觉阈值对比无统计学差异(P0.05)。结论A型肉毒毒素须垫部皮下注射可以显著升高大鼠三叉神经痛模型的疼痛阈值,提高其抗伤害感受能力。第三部分A型肉毒毒素治疗大鼠三叉神经痛的作用部位研究目的在前两部分研究的基础上,通过对A型肉毒毒素的特异性标记物c SNAP-25的检测,研究A型肉毒毒素治疗三叉神经痛的作用部位,探讨轴突运输在A型肉毒毒素治疗三叉神经痛中的作用。方法大鼠眶下神经慢性缩窄环术后13.5天,实验组在三叉神经节内注射轴突运输抑制剂秋水仙碱,对照组注射等量生理盐水。术后14天在手术同侧面部须垫部皮下注射A型肉毒毒素(10U/Kg)或生理盐水溶液。观察不同时间点各组大鼠对疼痛刺激的变化,探讨轴突运输在A型肉毒毒素治疗三叉神经痛中的作用。用Western blot和免疫荧光对A型肉毒毒素的特异性标志物c SNAP-25进行检测,观察A型肉毒毒素在三叉神经痛模型大鼠的作用部位。结果实验组用秋水仙碱阻断轴突运输降低了A型肉毒毒素的抗伤害感受作用,A型肉毒毒素治疗后第4天实验组疼痛阈值显著低于对照组,差异有统计学意义(P0.05),这一差异持续至实验结束。A型肉毒毒素治疗后7天,通过western-blot和免疫荧光检测到大鼠三叉神经脊束核尾侧亚核c SNAP-25水平明显增高。三叉神经节注射秋水仙碱阻断轴突运输后,可以阻断其增高。Western blot表明三叉神经节注射秋水仙碱阻断轴突运输后三叉神经脊束核尾侧亚核c SNAP-25较对照组显著降低,差异有统计学意义(P0.05)。结论秋水仙碱阻断轴突运输后使A型肉毒毒素的抗伤害感受作用显著减弱。A型肉毒毒素通过轴突运输至三叉神经脊束核尾侧亚核发挥作用。第四部分TRPs在A型肉毒毒素治疗大鼠三叉神经痛中的作用机制研究目的探讨痛觉相关TRPs(TRPA1、TRPV1、TRPV2及TRPM8)在A型肉毒毒素对大鼠三叉神经痛模型发挥抗伤害感受作用的机制。方法TRPA1、TRPV1、TRPV2和TRPM8是TRPs家族中可能与疼痛相关的离子通道,为了进一步明确A型肉毒毒素对三叉神经痛抗伤害感受的机制,采用Western blot对三叉神经痛模型三叉神经脊束核尾侧亚核不同时间点的TRPA1、TRPV1、TRPV2和TRPM8蛋白表达进行检测,观察这些蛋白在三叉神经痛模型中的动态变化。同时用Western blot检测A型肉毒毒素同侧面部须垫部皮下注射(3U/Kg和10U/Kg)对三叉神经痛模型三叉神经脊束核尾侧亚核中TRPA1、TRPV1、TRPV2和TRPM8蛋白表达的影响。结果Western blot表明三叉神经脊束核尾侧亚核中TRPA1和TRPV1的表达水平在眶下神经慢性缩窄环术后第14天明显增高(P0.05),在术后第28天仍显著增高(P0.05)。TRPV2在术后第7天表达明显增高(P0.05),术后第28天仍然持续增高(P0.05)。TRPM8在术后第7天开始增高,在术后第14天达到高峰,术后第28天与对照组相比仍有统计学差异(P0.05)。A型肉毒毒素治疗后第14天,Western blot表明大鼠三叉神经脊束核尾侧亚核中TRPA1和TRPV1的表达较对照组显著降低(P0.05),A型肉毒毒素降低TRPA1和TRPV1的表达有剂量依赖的效果,10U组的TRPA1和TRPV1的表达与3U相比有统计学差异(P0.05)。A型肉毒毒素(10U/Kg)治疗后第14天显著降低了TRPV2的表达(P0.05),3U组TRPV2的表达水平与对照组无统计学差异(P0.05)。与对照组相比,A型肉毒毒素(3U/Kg和10U/Kg)不影响TRPM8的表达(P0.05)。结论大鼠眶下神经慢性缩窄环术后三叉神经脊束核尾侧亚核TRPA1、TRPV1、TRPV2和TRPM8的表达显著升高,且有一动态变化过程。A型肉毒毒素治疗三叉神经痛的机制可能是抑制三叉神经脊束核尾侧亚核中TRPA1、TRPV1和TRPV2的高表达,降低中枢敏化,从而发挥抗伤害感受作用。
[Abstract]:Trigeminal neuralgia is one of the most painful and common neuropathic pain in the clinic. Clinical manifestations are unilateral transient electric shock or knife cut pain limited to the trigeminal distribution area. Its first choice is oral medication, and surgical treatment can be considered in the case of ineffective oral medication. However, there are many patients. After a variety of treatments, the effect of botulinum toxin type.A is a kind of exotoxin released by Gram positive anaerobic bacillus botulinum. It is widely used in the field of dystonia and Cosmetology. In recent years, more and more studies have been made on the treatment of pain. In 2012, we changed the technology of botulinum toxin type A to treat trigeminal neuralgia. Route, for the first time, a randomized, double blind, placebo-controlled trial for the first time confirmed that botulinum A can effectively alleviate the pain and slight adverse reactions of trigeminal neuralgia for a long time. Several subsequent clinical studies have drawn similar conclusions. However, the mechanism of A botulinum toxin for the treatment of trigeminal neuralgia is still unclear. We established a rat model of trigeminal neuralgia with chronic suborbital coarctation of the suborbital nerve to explore the therapeutic effect, location and mechanism of botulinum toxin A on trigeminal neuralgia. It provides the basis for the treatment of trigeminal neuralgia and other painful diseases by botulinum toxin type A in the first part of the rats. The behavior and histology of the trigeminal neuralgia model aim to establish a rat model of trigeminal neuralgia with chronic constrictive ring of the suborbital nerve, to simulate the classical trigeminal neuralgia, to observe the pain behavior and the histological changes of the suborbital nerve in the rats after the operation. Von Frey filaments was used to evaluate the pain behavior of the two groups of rats before and after the operation. HE staining and MBP immunohistochemical staining were used to evaluate the histological changes of the suborbital nerve 14 days before and after the operation. The results showed an obvious refractory period in the experimental group fourth days after operation. The threshold of pain in the same side was increased significantly, the sixth day pain threshold reached a peak of 24.6 + 8.6g, which was significantly different from that before the operation (P0.05). Then the threshold of pain decreased sharply and reached 5.5 + 2.3g (P0.05) fourteenth days after the operation. This trend was at least 34 days after the operation. The pain threshold of the group before and after the operation was not statistically significant. The pain threshold of the experimental group and the control group was 13.5 + 3.6g and 12.6 + 5.5G, respectively. There was no statistical difference between the two groups (P0.05). The pain threshold between the two groups was 5.5 + 3.2g and 13.6 + 4.8g after the operation, and there was a statistically significant difference between the groups (P0.05). The difference lasted to at least thirty-fourth days after the operation,.HE and MBP immunity. Histochemical staining showed that there were obvious demyelination changes in the suborbital nerve in the experimental group fourteenth days after operation, but there was no significant change in the control group. Conclusion the model of trigeminal neuralgia for the chronic suborbital constriction ring operation of the rat's suborbital nerve conforms to the clinical characteristics and histological changes of the classical trigeminal neuralgia. This model studies the pathogenesis of trigeminal neuralgia and studies new treatment methods. Second part of the anti nociceptive effect of botulinum toxin type A on the trigeminal neuralgia model in rats. Aim to intervene the trigeminal neuralgia model with A botulinum toxin and observe the change of pain behavior in rats. Method the chronic constriction ring of the suborbital nerve in rats. Fourteenth days after the operation of the trigeminal neuralgia model, the experimental group was subcutaneously injected with botulinum toxin A (3U/Kg and 10U/Kg) and the control group was injected with equal amount of physiological saline in the same part. Von Frey filaments was used to evaluate the pain and pain behavior of each group before and after the operation. Results the treatment of A botulinum toxin was found. Fourth days after treatment (eighteenth days after operation), the pain threshold of both group 3U and group 10U was significantly higher than that of the control group (P0.05). This difference lasted for at least 20 days after the treatment (34 days after the operation) and the pain threshold of the.10U group was higher than that of the 3U group, but there was no statistical difference between the two groups (P0.05). Conclusion A botulinum toxin must be found. Subcutaneous injection can significantly increase the pain threshold of the rat trigeminal neuralgia model and improve its anti nociceptive ability. Third Research on the role of botulinum toxin type A in the treatment of trigeminal neuralgia in rats, based on the study of the first two parts and the detection of C SNAP-25, a specific marker for botulinum toxin type A, study A The role of botulinum toxin in the treatment of trigeminal neuralgia and the role of axon transport in the treatment of trigeminal neuralgia by botulinum toxin type A. Methods 13.5 days after the operation of the chronic constrictive ring of the suborbital nerve in rats, the experimental group injected the axon transport inhibitor colchicine in the trigeminal ganglion and injected the same amount of normal saline to the group for 14 days after operation. A botulinum toxin (10U/Kg) or saline solution was injected subcutaneously on the same side of the facial facial pad. The changes of pain stimulation in each group of rats at different time points were observed and the role of axon transport in the treatment of trigeminal neuralgia by botulinum toxin type A was investigated. C SNAP-25, a specific marker for botulinum toxin type A, was examined by Western blot and immunofluorescence. Test, observe the role of botulinum toxin type A in the rat model of trigeminal neuralgia. Results the experimental group used colchicine blocking axonal transportation to reduce the anti nociceptive effect of type A botulinum toxin. The pain threshold of the experimental botulinum toxin type A was significantly lower than that of the control group at fourth days after the treatment, and the difference was statistically significant (P0.05). The difference continued to be sustained. 7 days after the treatment of botulinum toxin type.A, the level of C SNAP-25 in the nucleus caudal subnucleus of the trigeminal nucleus of the rat was significantly increased by Western-blot and immunofluorescence. After blocking the axon transport by injection of colchicine, the trigeminal ganglion could block the.Western blot Biao Mingsan ganglion injection of colchicine to block axonal transportation The C SNAP-25 in the posterior trigeminal nucleus caudal subnucleus was significantly lower than that in the control group (P0.05). Conclusion colchicine inhibited the anti nociceptive effect of A type botulinum toxin by colchicine blocking axon transport and the effect of.A type botulinum toxin on the nucleus caudal subnucleus of the trigeminal nucleus through axon. Fourth part TRPs was observed. Study on the mechanism of A botulinum toxin in the treatment of trigeminal neuralgia in rats. Objective to explore the mechanism of pain related TRPs (TRPA1, TRPV1, TRPV2 and TRPM8) in the anti nociceptive effect of botulinum toxin type A on rat trigeminal neuralgia model. Methods TRPA1, TRPV1, TRPV2 and TRPM8 are the ion channels associated with pain in the TRPs family, for the purpose of The mechanism of the anti nociceptive sensitivity of botulinum toxin A to trigeminal neuralgia was further clarified, and the expression of TRPA1, TRPV1, TRPV2 and TRPM8 in the trigeminal nucleus caudal nucleus of trigeminal neuralgia model was detected by Western blot, and the dynamic changes of these proteins in the trigeminal neuralgia model were observed. At the same time, Western blot was used. The effects of subcutaneous injection (3U/Kg and 10U/Kg) on the expression of TRPA1, TRPV1, TRPV2 and TRPM8 in the trigeminal nucleus caudal nucleus of the trigeminal neuralgia model of botulinum toxin type A botulinum toxin (3U/Kg and 10U/Kg). Results Western blot showed that the expression level of TRPA1 and TRPV1 in the caudal nucleus of the trigeminal nucleus of the spinal trigeminal tract was performed by chronic constrictive ring of the orbital nerve After fourteenth days (P0.05), the expression increased significantly (P0.05) (P0.05) at the seventh day after operation (P0.05), and continued to increase on the twenty-eighth day after operation (P0.05).TRPM8 began to increase on the seventh day after the operation, and reached the peak at the fourteenth day after the operation, and the twenty-eighth day after the operation was still statistically different (P0.05).A type botulinum toxin compared to the control group (P0.05). On the fourteenth day after treatment, Western blot showed that the expression of TRPA1 and TRPV1 in the nucleus caudal nucleus of the trigeminal nucleus of the rat was significantly lower than that of the control group (P0.05). The expression of TRPA1 and TRPV1 of the A type botulinum toxin was dose-dependent. The expression of TRPA1 and TRPV1 in the 10U group was statistically different from that of 3U. The expression of TRPV2 was significantly reduced in the last fourteenth days (P0.05), and the expression level of TRPV2 in group 3U was not significantly different from that in the control group (P0.05). Compared with the control group, A type botulinum toxin (3U/Kg and 10U/Kg) did not affect the expression of TRPM8 (P0.05). Conclusion the caudal nucleus of the trigeminal nucleus of the trigeminal nucleus after the operation of the suborbital nerve of the rat is TRPA1, TRPV1, and The mechanism of botulinum toxin type.A in the treatment of trigeminal neuralgia may be to inhibit the high expression of TRPA1, TRPV1 and TRPV2 in the caudal nucleus of the trigeminal nucleus, and reduce the central sensitization, thus exerting anti nociceptive effect.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R745.11
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