卢非酰胺选择性抑制C纤维介导的伤害性初级传入发挥镇痛作用

发布时间：2018-09-01 07:58

【摘要】：疼痛(pain)是由伤害性刺激引起的复杂生理心理活动,在正常情况下,是机体重要的警告信号,当伤害性刺激发生时,机体通过全身皮肤和有关组织中分布的的伤害性感受器将各种形式的刺激转换成神经冲动的电活动信号,沿着传入神经,背根神经节到达脊髓背角或三叉神经脊束核中的相关神经元,再由对侧的腹外侧索传至丘脑、其他脑区以及大脑皮质,产生痛觉,从而对有害刺激做出回避反应。另一方面,过度的疼痛会产生负面影响,长期的剧烈疼痛会对机体产生难以忍受的精神和躯体的折磨,所以减轻异常疼痛,提高人类生活质量始终是医学研究的关注点。介导疼痛的传导通路及分子作用复杂,在疼痛机制的研究长河中各种学说层出不穷。其中,作为疼痛四大学说之一的闸门控制学说相对比较确切的阐述了病理性疼痛的发生机制,认为在整个痛觉的传导过程中,脊髓背角胶状质(Substantia gelatinosa,SG)中的某些神经细胞控制着痛觉信息的传递,作用机理类似闸门的开放,这些神经元本身就受粗、细纤维传入活动和高级中枢下行控制作用的影响,本学说的提出为揭示疼痛的发生机制开辟了新的方向,基于此理论,我们意识到,脊髓背角胶状质作为疼痛传导的中枢起着信息转换的重要作用,必然也是研究开发新型镇痛药物的重要靶点。癫痫是因脑部神经元异常放电引起的一组临床综合征,由已知或未知病因引起,临床特征表现为反复、短暂、刻板的神经系统功能失常。随着人类医学研究的不断进步,新时期提出的整合医学认为将相关各领域最先进的医学发现加以整合可以形成更加全面的医学知识体系。随着人们对癫痫和疼痛机制研究的不断深入,循着整合医学的方向,查阅资料后我们发现癫痫和疼痛的的发病机制部分类似。新型抗癫痫药物卢非酰胺属于三唑类衍生物,目前临床作为辅助治疗药物应用于4岁以上儿童和成人Lennox-Gastant综合征(LGS)相关癫痫发作。研究发现它不仅在癫痫的治疗上作用显著,而且具有潜在的镇痛作用,与此同时,它具有稳定情绪的独特优势。近年来,卢非酰胺已经成功用于癫痫疾病的治疗,但是镇痛机制尚不明确,尤其是对疼痛重要调节位点的脊髓背角胶状质神经元及其突触传递的作用机制缺乏有力的研究报道。本课题主要利用动物行为学测试技术、膜片钳全细胞记录探讨卢非酰胺对脊髓背角SG神经元的兴奋性以及对伤害性刺激突触传递的影响,为阐明卢非酰胺的脊髓背角镇痛机制提供实验依据。实验一:卢非酰胺对腰5脊神经结扎(Spinal nerve ligation,SNL)神经病理性疼痛大鼠模型的镇痛作用目的:观察抗癫痫药物卢非酰胺对腰5脊神经结扎神经病理性疼痛大鼠模型(SNL)的机械、热痛觉超敏的影响。方法:选取180~220g雄性SD大鼠,于SNL模型制作前3d给予连续的相同时间同一环境适应,前1d测定大鼠疼痛基础阈值。选取疼痛阈值在正常范围内的大鼠制备腰5脊神经结扎(SNL)神经病理性疼痛动物模型。术后5d同一时间测试机械性缩足反射阈值、热痛缩足反射潜伏时间,若疼痛阈值未下降或出现后足偏瘫,行为异常视为造模失败,剔除出组。神经病理性疼痛动物模型被随机分为三组:(1)大剂量实验组:将50 mg/kg卢非酰胺溶入1%DMSO中,生理盐水稀释至1ml,单次腹腔注射。(2)小剂量实验组:25 mg/kg卢非酰胺溶入1%DMSO中用生理盐水稀释至1ml单次腹腔注射。(3)对照组:与实验组等体积、等比例的1%DMSO单次腹腔注射。分别于注射后20 min、40 min、60 min、4 h、12 h、24 h进行行为学评估。观察卢非酰胺对大鼠L5神经损伤后形成的神经病理性疼痛的镇痛效果。结果:SNL术前对大鼠基础疼痛阈值进行测定,左、右后足机械性缩足反射阈值(PWMT)分别为(21.87±5.69)g和(18.33±4.18)g,热痛缩足反射潜伏时间(TWL)为(24.43±3.32)s和(22.31±4.28)s,无统计学差异。术后第5 d,形成稳定神经病理性疼痛,与术前相比,左后足的阈值显著降低至(6.00±2.13)g(P0.001,one-way ANOVA,n=22),热痛缩足反射潜伏时间降为(13.45±2.17)s(P0.001,one-way ANOVA,n=22),证实模型建立成功。当腹腔注射不同浓度的卢非酰胺后,测试机械、热痛阈值发现两种浓度的卢非酰胺均可以显著缓解已形成的慢性疼痛,并且镇痛作用具有明显的浓度依赖性,并在给药后1 h作用达到高峰,与溶剂组相比可显著提升大鼠术侧机械性缩足反射阈值至(19.99±7.17)g和(17.00±7.32)g(P0.001,one-way ANOVA,n=8),增加热痛缩足反射潜伏时间至(16.23±3.14)s和(19.90±2.41)s(P0.001,one-way ANOVA,n=8)这一作用可维持至注药后12 h。实验二:卢非酰胺对脊髓背角胶状质(SG)神经元兴奋性的影响目的:应用膜片钳全细胞记录技术,在脊髓矢状位切片上观察卢非酰胺对SG神经元动作电位(Action potential,AP)发放频率的影响。方法:取4~5周龄雄性SD大鼠,制备厚度为400-500μm带后根的脊髓腰骶膨大段矢状位切片。低倍镜下确定脊髓背角浅层,高倍镜下选择状态较好的SG神经元做全细胞记录,钳制电流为0 p A,待细胞状态稳定5 min后。观察卢非酰胺对SG神经元动作电位发放频率的影响。结果:抗癫痫药物卢非酰胺可显著减少脊髓背角SG神经元动作电位的发放频率(P0.01,paired t-test),给予一段时间洗脱,卢非酰胺的抑制作用被成功逆转(P0.01,paired t-test)。实验三:卢非酰胺对伤害性感觉通路突触传递的选择性抑制作用目的:全细胞记录,电压钳模式下给予后根刺激,观察抗癫痫药物卢非酰胺对中等直径Aδ纤维和小直径C纤维介导的脊髓背角SG神经元的兴奋性突触后电流(e EPSCs)及自发性兴奋性突触后电流(s EPSCs)的影响。方法:同实验二,制备SD大鼠脊髓切片,选取SG神经元,钳制电压为-70 m V,给予从小到大的后根恒压电刺激,先记录给药前诱发的兴奋性突触后电流(e EPSCs)15条曲线,平均后作为对照,浴槽内不间断灌流200μM卢非酰胺1 min后相同方法再次记录,根据文献报道不同的纤维传导具有的电生理特异性,对记录的细胞进行分类统计。(1)观察卢非酰胺对Aδ纤维介导的e EPSCs的作用。(2)观察卢非酰胺对C纤维介导的e EPSCs的作用。(3)观察卢非酰胺对脊髓背角SG神经元s EPSCs的影响。结果:(1)单次灌流不同浓度的卢非酰胺对Aδ纤维介导的e EPSCs几乎不产生影响(P0.05,paired t-test)。(2)卢非酰胺可显著抑制C纤维介导的e EPSCs的峰值(P0.01,paired t-test)。(3)卢非酰胺灌流可以明显抑制SG神经元s EPSCs的发放频率(P0.001,paired t-test),但对其幅度没有明显影响(P0.05,paired t-test)。结论:1.单次腹腔注射不同浓度的卢非酰胺均可以有效缓解已形成的慢性疼痛,镇痛作用在给药后1h达到高峰且具有浓度依赖性。2.卢非酰胺可以显著抑制脊髓背角SG神经元动作电位的发放频率,这一抑制作用可以在给药后10 min内被有效洗脱。3.不同浓度的卢非酰胺几乎不影响Aδ纤维支配的单突触SG神经元所产生的e EPSCs峰值,与对照组e EPSCs相比,无统计学意义。卢非酰胺可以显著抑制C纤维介导e EPSCs的峰值,作用时间大于10 min。持续灌流卢非酰胺1 min还可以显著降低SG神经元s EPSCs的频率,但不影响s EPSCs的幅度。
[Abstract]:Pain is a complex physiological and psychological activity caused by noxious stimuli. Normally, it is an important warning signal of the body. When noxious stimuli occur, the body converts various forms of stimuli into electrical signals of nerve impulses through noxious receptors distributed in the skin and related tissues of the body, along the afferent nerves. The dorsal root ganglion (DRG) reaches the neurons in the dorsal horn of the spinal cord or the nucleus of the spinal trigeminal tract, and then passes from the contralateral ventrolateral cord to the thalamus, other brain regions, and the cerebral cortex, producing a sense of pain, thus avoiding harmful stimuli. On the other hand, excessive pain can have a negative impact, and long-term severe pain can be difficult to produce. It is always the focus of medical research to alleviate abnormal pain and improve the quality of human life because of the mental and physical torture endured. The pathways and molecular functions that mediate pain are complex. Various theories emerge in endlessly in the study of pain mechanism. The mechanism of pathological pain is expounded. It is believed that some neurons in the spinal dorsal horn gelatinosa (SG) control the transmission of pain information during the whole process of pain transmission. The mechanism of action is similar to the opening of gates. These neurons themselves are thick, thin fiber afferent activities and advanced central downward control. Based on this theory, we realize that the gelatinous substance of the spinal dorsal horn, as the center of pain transmission, plays an important role in information conversion and is also an important target for the development of new analgesic drugs. Epilepsy is caused by abnormal release of neurons in the brain. A group of clinical syndromes, caused by known or unknown causes, characterized by repeated, transient, and rigid dysfunction of the nervous system. With the continuous progress of human medical research, integrated medicine proposed in the new era considers that the integration of the most advanced medical discoveries in related fields can lead to a more comprehensive medicine. Knowledge system. With the deepening of research on the mechanism of epilepsy and pain, following the direction of integrated medicine, we found that the pathogenesis of epilepsy and pain is similar in part. Lufenamide, a new antiepileptic drug, belongs to triazole derivatives. It is currently used as an adjuvant therapy in children and adults over 4 years old. Epilepsy related to human Lennox-Gastant syndrome (LGS). Studies have found that lufenamide has not only a significant role in the treatment of epilepsy, but also a potential analgesic effect. At the same time, it has a unique advantage in stabilizing mood. In recent years, lufenamide has been successfully used in the treatment of epilepsy, but the mechanism of analgesia is still unclear, especially for pain. In order to elucidate the effect of lufenamide on the excitability of spinal dorsal horn SG neurons and the synaptic transmission of noxious stimuli, we investigated the effects of lufenamide on the excitability of spinal dorsal horn SG neurons and the synaptic transmission of noxious stimuli by using animal behavioral testing techniques and patch clamp whole cell recording. Objective: To observe the analgesic effect of lufenamide on lumbar 5 spinal nerve ligation (SNL) induced neuropathic pain in rats. METHODS: Male SD rats of 180-220 g were selected and adapted to the same environment at the same time for three days before the establishment of the SNL model. The basic pain threshold was measured at the first day. The neuropathic pain animal model of lumbar 5 spinal ligation (SNL) was established in rats with normal pain threshold. Neuropathic pain animal models were randomly divided into three groups: (1) High-dose experimental group: 50 mg/kg lufenamide was dissolved in 1% DMSO, normal saline was diluted to 1 ml, single intraperitoneal injection. (2) Low-dose experimental group: 25 mg/kg of lufenamide dissolved in 1% DMSO and diluted to 1 ml of normal saline for a single intraperitoneal injection. (3) Control group: 1% DMSO was injected into the abdominal cavity in the same volume and proportion as the experimental group. Behavioral evaluation was carried out 20 minutes, 40 minutes, 60 minutes, 4 hours, 12 hours and 24 hours after injection, respectively. Results: The mechanical shrinkage reflex thresholds (PWMT) of left and right hind feet were (21.87 [5.69] g and (18.33 [4.18] g respectively, and the latency time (TWL) of hot pain shrinkage reflex was (24.43 [3.32] s] and (22.31 [4.28] s, respectively. There was no significant difference between them. Compared with preoperative neuropathic pain, the threshold of left hind foot was significantly reduced to (6.00 (+ 2.13) g (P 0.001, one-way ANOVA, n = 22) and the latent time of foot reflex was reduced to (13.45 (+ 2.17) s (P 0.001, one-way ANOVA, n = 22), which confirmed the success of the model. Lufenamide at different concentrations could significantly alleviate chronic pain, and the analgesic effect was obviously concentration-dependent, and reached its peak at 1 hour after administration. Compared with the solvent group, Lufenamide significantly increased the threshold of mechanical foot contraction reflex to (19.99 (7.17) g and (17.00 (7.32) g (P 0.001, one-way ANOVA, n=8), and increased the fever. The effect of lufenamide on the excitability of spinal dorsal horn gelatinous (SG) neurons was observed on sagittal sections of the spinal cord using patch clamp whole cell recording technique. Methods: Sagittal sections of lumbosacral enlarged spinal cord with posterior roots were prepared from 4 to 5 weeks old male SD rats. The superficial layer of spinal dorsal horn was determined at low magnification, and the better SG neurons were selected at high magnification for whole cell recording. The clamping current was 0 P A and the cells were waiting for cells. Results: Antiepileptic drug lufenamide could significantly reduce the action potential release frequency of SG neurons in spinal dorsal horn (P 0.01, paired t-test). After a period of elution, the inhibitory effect of lufenamide was reversed (P 0.01, paired t-test). Experiment 3: Selective inhibitory effect of lufenamide on synaptic transmission in nociceptive pathways Objective: Whole cell recording, posterior root stimulation under voltage clamp mode, and observation of excitatory postsynaptic currents (e EPSCs) and spontaneous activity of antiepileptic drug lufenamide on SG neurons in spinal dorsal horn mediated by medium-diameter A delta fibers and small-diameter C fibers METHODS: In the same experiment 2, the spinal cord sections of SD rats were prepared, and SG neurons were selected. The clamping voltage was - 70 m V, and constant voltage stimulation was given to the posterior roots from small to large. Fifteen curves of excitatory postsynaptic currents (e EPSCs) were recorded before administration, and then, on average, were used as controls. The same method was recorded 1 minute later, and the recorded cells were classified according to the electrophysiological specificity of different fiber transduction reported in the literature. (1) To observe the effect of lufenamide on E EPSCs mediated by A Delta fiber. (2) To observe the effect of lufenamide on E EPSCs mediated by C fiber. (3) To observe the effect of lufenamide on spinal cord dorsal part. Results: (1) Single perfusion with different concentrations of lufenamide had little effect on Adelta fiber-mediated EPSCs (P 0.05, paired T-T e s t). (2) Lufenamide significantly inhibited the peak of C-fiber-mediated EPSCs (P 0.01, paired T-T e s t). (3) Lufenamide perfusion could significantly inhibit the development of SG neurons EPSCs. Radiation frequency (P 0.001, paired t-test), but no significant effect on its amplitude (P 0.05, paired t-test). Conclusion: 1. Single intraperitoneal injection of different concentrations of lufenamide can effectively alleviate chronic pain, the analgesic effect reached a peak 1 hour after administration and has a concentration-dependent. 2. Lufenamide can significantly inhibit SG nerves in spinal dorsal horn. The inhibitory effect was eluted within 10 minutes after administration. 3. Different concentrations of lufenamide had little effect on the peak value of e-EPSCs produced by single synaptic SG neurons innervated by Adelta fibers, which was not statistically significant compared with the control group. Continuous perfusion of lufenamide for 1 minute also significantly decreased the frequency of s EPSCs in SG neurons, but did not affect the amplitude of s EPSCs.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614

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