A型肉毒毒素对神经病理性疼痛大鼠钠离子通道Nav1.3和钠电流的影响

目的 探讨A型肉毒毒素（BoNT/A）对神经病理性疼痛大鼠背根神经节（DRG）神经元中钠通道Nav1.3和功能性钠电流的影响。 方法 建立保留性神经损伤（SNI）病理性疼痛模型,将造模成功大鼠根据注射溶液的不同按随机数字表法分为生理盐水注射组（注射生理盐水）和BoNT/A注射组（注射BoNT/A）,每组9只;另取9只大鼠设为假手术组,只分离暴露坐骨神经分支,但不做神经结扎手术。术后第5天在一侧足底皮下注射BoNT/A（7 U/kg和15 U/kg）或等量的生理盐水,检测注射后不同时间点（给药后第3天、第7天和第14天）大鼠机械撤足阈值的变化。采用Western Blot检测观察给药后第7天和第14天BoNT/A对各组大鼠DRG神经元中Nav1.3蛋白表达的影响,用电生理膜片钳检测BoNT/A对各组大鼠功能性河豚毒素敏感型（TTX-S）钠电流的影响。 结果 足底皮下注射BoNT/A可显著升高外周神经损伤后下降的机械触痛阈（P＜0.001）,缓解神经病理性疼痛。SNI术后DRG神经元中Nav1.3蛋白表达明显上调（P＜0.001）,BoNT/A干预后第7天和第14天的Nav1.3蛋白表达显著下调（P＜0.01）;BoNT/A可降低SNI术后增大的TTX-S钠电流（P＜0.05）。 结论 BoNT/A可调控Nav1.3钠通道蛋白表达,影响功能性TTX-S钠电流,发挥改善神经病理性疼痛的作用。     

A型肉毒毒素关节腔注射对佐剂性关节炎疼痛大鼠背根神经节中降钙素基因相关肽表达的影响

目的观察A型肉毒毒素(BoNT-A)关节腔注射对佐剂性关节炎疼痛大鼠镇痛效果与背根神经节(DRG)中降钙素基因相关肽(CGRP)表达的关系,并检测其逆向轴浆运输的情况。 方法SD大鼠90只按随机数字表法分为A、B、C、D和E五组,每组18只大鼠。分组后A组注射50μL生理盐水作为对照组,而B、C、D、E组大鼠左后肢踝关节腔注射50μL完全弗氏佐剂(CFA)建立佐剂性关节炎疼痛模型作为模型组。建模3周关节炎疼痛模型逐渐稳定后,A组和B组大鼠关节腔内注射20μL生理盐水,而C、D和E组大鼠关节腔内注射不同剂量20μL的BoNT-A,即A组为对照组+关节腔内注射20μL生理盐水;B组为模型组+关节腔内注射20μL生理盐水;C组为模型组+关节腔内注射1U的20μL BoNT-A;D组为模型组+关节腔内注射3U的20μL BoNT-A;E组为模型组+关节腔内注射10U的20μL BoNT-A 。分别于造模前、造模后1、5、14和21d和给药后1、3、5和14d,对各组大鼠进行机械痛阈和肌力评分测试;应用免疫印迹和免疫荧光法观察DRG中CGRP蛋白表达水平和阳性细胞数变化,以及观察BoNT-A的水解产物裂解的突出相关蛋白-25kDa(cl-SNAP-25)是否存在于DRG中。 结果与A组相比,B、C、D、E组大鼠的机械痛阈值和肌力评分均显著降低(P<0.001),大鼠DRG中的CGRP蛋白表达水平和阳性细胞数均显著增加(P<0.001);与B组相比,BoNT-A注射5d后D组和E组大鼠的机械痛阈显著提高(P<0.001),而14d后C组的机械痛阈亦显著提高(P<0.01);BoNT-A注射3d后,D组和E组大鼠DRG中的CGRP蛋白表达水平和阳性细胞数显著降低(P<0.01),C组14d后DRG中的CGRP蛋白表达水平和阳性细胞数显著降低(P<0.01);D组与E组相比,机械痛阈、CGRP蛋白表达水平和阳性细胞数组间差异均无统计学意义(P>0.05);与B组相比,C组和D组肌力评分组间差异均无统计学意义(P>0.05);E组BoNT-A注射5d后,肌力评分显著下降(P<0.001);免疫荧光检测到DRG中存在cl-SNAP-25。 结论BoNT-A通过减少DRG中CGRP的表达,减轻关节炎疼痛且存在剂量依赖性;BoNT-A可以通过逆向轴浆运输到达DRG。     

A型肉毒毒素在慢性疼痛中的应用进展

A型肉毒毒素因松弛麻痹肌肉的药理作用,曾广泛应用于神经肌肉过度活跃性疾病。近年来随着人们发现其在外周和中枢神经系统的去敏化作用,其临床应用范畴已由肌痉挛相关性疼痛逐步扩展到慢性偏头痛乃至神经病理性疼痛。但目前A型肉毒毒素镇痛机制尚需进一步完善,临床应用仍处起步阶段,其镇痛适应证、剂量、注射部位、治疗时机和远期疗效等仍需探索。本文就A型肉毒毒素治疗慢性疼痛的镇痛机制和应用进展进行综述。     

肉毒毒素A对神经病理性疼痛小鼠施旺细胞髓鞘形成的影响

目的:观察不同给药途径的肉毒毒素A(Botulinum neurotoxin type A,Bo NT/A)对慢性坐骨神经缩窄性损伤(Chronic constriction injury,CCI)疼痛模型小鼠的镇痛效果;研究Bo NT/A对施旺细胞髓鞘标志蛋白以及调控髓鞘形成的转录因子表达的影响。方法:通过CCI建立神经病理性疼痛模型小鼠,并随机平均分成4组:生理盐水皮下注射(intraplantar injection,i.pl.)组、Bo NT/A皮下注射组、生理盐水神经旁注射(peripheral nerve injection,i.pn.)组、Bo NT/A神经旁注射组,此外设置未造模的空白对照组。在造模后第5天给药,测量不同时间点各组小鼠机械缩足阈值和热缩足潜伏期的变化。在术后14天和28天分别对Bo NT/A皮下注射组、生理盐水皮下注射组及空白对照组小鼠进行坐骨神经采样,应用蛋白印迹检测坐骨神经髓鞘相关蛋白MBP、P0、Krox-20及C-jun表达量的变化,应用免疫荧光技术检测坐骨神经P0的表达情况。结果:皮下注射或神经旁注射Bo NT/A均可增加神经病理性疼痛模型小鼠的机械缩足阈值和热缩足潜伏期(P<0.05)。Bo NT/A皮下注射组坐骨神经MBP、P0、Krox-20及C-jun表达量与生理盐水皮下注射组对比无显著性差异。结论:皮下注射和神经旁注射Bo NT/A均可改善神经病理性疼痛小鼠的机械痛敏和热痛敏,但皮下注射Bo NT/A不能促进神经病理性疼痛小鼠坐骨神经施旺细胞髓鞘的形成,提示Bo NT/A并不是通过促进髓鞘形成这一机制来促进运动功能恢复以及缓解神经病理性疼痛。     

A型肉毒毒素治疗三叉神经痛

目的:观察A型肉毒毒素治疗三叉神经痛(TN)的临床疗效。方法:选取57例TN患者,随机分为A、B 2组。A组28例患者口服卡马西平片治疗;B组29例在疼痛部位及板机点周围皮下注射肉毒素治疗。治疗后1,3及6个月时随访,行简式McGill疼痛问卷表(SF-MPQ)及生活质量评价量表(SF-36)评分,并观察不良反应。结果:治疗中脱失7例,A组3例,B组4例。与治疗前3个月SF-MPQ及SF-36平均分作为基础水平比较,治疗1,3及6个月后2组SF-MPQ评分明显下降,SF-36明显上升(P<0.01),B组表明更明显(P<0.05,P<0.01)。治疗过程中,A组出现不适患者多于B组。结论:A型肉毒毒素疼痛点皮下注射治疗TN发作作用高峰1～3个月,维持时间6个月,且临床疗效显著,不良反应轻微。     

A型肉毒毒素治疗痉挛的研究进展

A型肉毒毒素(Botulinum toxin type A,BoNT-A)出现之前,局部肌张力增高的治疗方法主要包括口服抗痉挛药物以及注射酚类物质。口服抗痉挛药物包括巴氯芬、丹曲林、氯硝安定等,容易出现全身不良反应,且效果较差[1]。酚类物质注射操作困难,可引起感觉异常的并发症,效果持续时间及有效性并不确定[2]。痉挛可以造成患者日常生活各方面的困难,对有残存肌力的患者也可造成主动运动功能的障碍[3]。还可以引起疼痛等不适感,影响患者的形象,造成自卑等心理障碍[4]。BoNT-A的出现彻底改写了局部肌张力增高治疗的历史。     

背根神经节解剖及其参与神经病理性疼痛机制的研究进展

背根神经节是感觉通路的初级神经元,在神经病理性疼痛的发生和维持阶段起着关键作用,背根神经节因而成为疼痛治疗的重要靶点。近年来,对背根神经节解剖细节的掌握及其参与神经病理性疼痛机制的研究成为热点。本文就背根神经节解剖及其参与神经病理性疼痛的主要分子和通路研究进展进行综述。     

脉冲射频对大鼠痛行为及背根神经节电压门控性钠离子通道的影响

目的:探讨脉冲射频(pulsed radiofrequency,PRF)对正常大鼠痛行为的影响以及对背根神经节(dorsal root ganglion,DRG)神经元电压门控性钠离子通道(voltage-gated sodium channels,VGSC)的电流密度和动力学特性的影响。方法:80~100 g雄性SD大鼠63只,随机分为对照组,sham组及PRF组。对照组不做任何处理,将sham组及PRF组大鼠经腹腔麻醉后暴露其右侧坐骨神经,PRF组坐骨神经接受2 Hz,2 min,42℃的脉冲射频处理,而sham组不接受脉冲射频处理。在脉冲射频前2 h,脉冲射频后1 d,3 d,5 d,7 d分别测定各组大鼠的机械刺激缩足反射阈值(paw withdrawal mechanical threshold,PWMT)和热辐射缩足反射潜伏期(paw withdrawal thermal latency,PWTL)。脉冲射频后1 d取L4~5段DRG细胞,应用单细胞膜片钳技术检测大鼠DRG细胞钠电流密度和动力学特性的改变。结果:sham组和PRF组大鼠术后不同时间点的PWMT值和PWTL值与自身手术前的基础阈值相比无差异,各组大鼠之间相同时间点的PWMT值和PWTL值均无统计学差异。PRF组大鼠DRG细胞电压依赖性钠电流密度、半数激活电压(V1/2act)、半数失活电压(V1/2inact)与sham组和对照组相比也均无显著差异。结论:该参数的脉冲射频未影响正常大鼠痛行为。DRG神经元钠电流密度以及通道动力学特性也未受该参数脉冲射频的影响,提示该参数的脉冲射频可能并不影响大鼠生理状态下伤害性感觉传导功能。     

A型肉毒毒素治疗肌痉挛

我科从 1994年 11月以来应用 A型肉毒杆菌毒素治疗肌张力障碍疾患 67例,并已随访 8个月～ 5年,结果报告如下。 1资料与方法 1.1资料 67例中男 31例,女 36例,年龄 13～ 80岁,平均 49.9岁 ;病程 0.5～ 5年。其中完全型 Meige综合征 55例,非完全型 Meige综合征 7例,颈肌痉挛 4例,全身型扭转痉挛 1例。全部病例既往曾分别行针灸、理疗、镇静、心理疗法等治疗, 3例曾行手术治疗均无效。痉挛程度按 Scott痉挛程度分级, 0级：无痉挛 ;Ⅰ级：外部刺激时小幅痉挛 ;Ⅱ级：轻度痉挛,无功能障碍 ;Ⅲ级：明显痉挛,轻度功能障碍 ;Ⅳ级：严重…     

背根神经节钠通道与疼痛

慢性疼痛是周围神经、组织损伤或炎性刺激所诱导的周围神经性病变的一种主要症状。最近一些研究表明背根神经节(dorsalrootganglion,DRG)钠通道表达及位置的改变与某些病理性疼痛有关,钠通道基因表达的可塑性及电生理的改变导致DRG细胞呈高兴奋性、产生自发动作电位及异常高频电活性,DRG钠通道在疼痛的病理生理中起做重要的作用。通过选择性地影响伤害性神经元产生动作电位及阻滞特异性钠通道可缓解神经性及炎性疼痛,有希望成为疼痛治疗的又一新领域。     

Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain

Nociceptive neurons within dorsal root ganglia (DRG) express multiple voltage-gated sodium channels, of which the tetrodotoxin-resistant (TTX-R) channel Na(v)1.8 has been suggested to play a major role in inflammatory pain. Previous work has shown that acute administration of inflammatory mediators, including prostaglandin E2 (PGE2), serotonin, and adenosine, modulates TTX-R current in DRG neurons, producing increased current amplitude and a hyperpolarizing shift of its activation curve. In addition, 4 days following injection of carrageenan into the hind paw, an established model of inflammatory pain, Na(v)1.8 mRNA and slowly-inactivating TTX-R current are increased in DRG neurons projecting to the affected paw. In the present study, the expression of sodium channels Na(v)1.1-Na(v)1.9 in small (< or = 25 micromdiameter) DRG neurons was examined with in situ hybridization, immunocytochemistry, Western blot and whole-cell patch-clamp methods following carrageenan injection into the peripheral projection fields of these cells. The results demonstrate that, following carrageenan injection, there is increased expression of TTX-S channels Na(v)1.3 and Na(v)1.7 and a parallel increase in TTX-S currents. The previously reported upregulation of Na(v)1.8 and slowly-inactivating TTX-R current is not accompanied by upregulation of mRNA or protein for Na(v)1.9, an additional TTX-R channel that is expressed in some DRG neurons. These observations demonstrate that chronic inflammation results in an upregulation in the expression of both TTX-S and TTX-R sodium channels, and suggest that TTX-S sodium channels may also contribute, at least in part, to pain associated with inflammation.     

背根神经节血红素加氧酶1过表达缓解小鼠神经病理性疼痛

目的:探讨背根神经节(dorsal root ganglion,DRG)中血红素加氧酶1(heme oxygenase 1,HO-1)的表达分布及在DRG中过表达HO-1对神经病理性疼痛的作用。方法:构建保留性坐骨神经损伤(spared nerve injury,SNI)诱导的神经病理性疼痛模型;行为学检测机械性触诱发痛;免疫荧光染色检测DRG中HO-1在神经元中表达和分布;免疫印迹检测DRG中HO-1表达。结果:免疫荧光染色显示DRG中HO-1阳性神经元在SNI后数量显著减少;DRG中HO-1阳性神经元主要是IB4阳性的小型神经元(<300μm2);连续5天腹腔注射原卟啉IX氯化钴(cobalt protoporphyrin-IX,CoPP)能增加神经病理性疼痛小鼠的机械缩足阈值;鞘内注射过表达HO-1的腺病毒相关病毒(adenovirus associated virus,AAV)能特异性浸染DRG神经元,并上调HO-1表达;同时能有效提高神经病理性疼痛小鼠的机械缩足阈值,此效应持续21天以上。结论:DRG中HO-1主要表达在IB4阳性的小型神经元;过表达DRG神经元中HO-1能有效缓解SNI诱导的神经病理性疼痛。     

新生大鼠背根神经节机械敏感性离子通道的电生理研究

目的:探讨新生大鼠背根神经节(DRG)的机械敏感性离子通道(MS通道)电生理特性。方法:将新生大鼠DRG细胞培养2—4d后,应用细胞贴附式和内面向外式膜片钳技术记录细胞膜上的MS通道电流,对通道的电生理性质,如压力-电流关系、通道的影响因素和通道在DRG细胞上的分布进行了分析。采用的机械刺激方式为负压抽吸。结果:在培养的大鼠DRG细胞膜上发现一种对机械刺激敏感的电流,开放压力阈值为-12—-15mmHg,P1/2=-37mmHg。压力恒定时,电流恒定;去除压力,电流回到基线水平。在施加压力的30s内,电流无明显衰减趋势。同一电位下,随压力的增大,通道活性也增大,但电流的幅度不变。钳制膜电位为-60mV,在-50mmHg负压下电流的平均幅度为(-3.40±0.13)pA,平均开放概率为0.448±0.125。该通道对机械刺激的反应可被钆和秋水仙素阻断,河豚毒素可阻断大直径神经元上的外向电流,对小直径细胞无效。阿米洛利对该电流无效。该通道主要存在于中、小直径神经元(≤30!m)上。结论:大鼠DRG细胞膜的MS通道参与机械信号,特别是伤害性刺激信号的转导,对其电生理性质的研究为理解机械信号转导的机制提供了理论依据。     

A subpopulation of capsaicin-sensitive porcine dorsal root ganglion neurons is lacking hyperpolarization-activated cyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to stabilizing resting membrane potential, thus controlling neuron excitability. Subclasses of nociceptive neurons differ in their excitability, therefore, these channels could be a distinguishing marker.We investigated isolated dorsal root ganglion neurons from a non-rodent species, the pig, Sus scrofa domesticus. Single labeling revealed capsaicin-induced cobalt-uptake in 54.3% and transient receptor potential V1 (TRPV1) immunoreactivity in 55.1% of all neurons. Ruthenium red and capsazepine suppressed capsaicin-induced cobalt-uptake. HCN-1 and HCN-2 channel isoform immunoreactivity was detected in 82.6% and 88.3%, respectively, and binding of IB4 in 29.4% of all neurons. Double labeling revealed that out of the capsaicin-positive neurons, 42.3% were IB4-positive, 80.0% immunoreactive for the HCN-1, and 77.3% for the HCN-2 channel isoform, respectively. Neurons lacking HCN-1 or HCN-2 channel isoforms were mostly capsaicin-positive and IB4-negative. The soma size of neurons lacking HCN-1 and/or HCN-2 channels was small to medium.Western blot analysis showed protein products of sizes similar to those of HCN-1 and HCN-2 channel isoforms. Functionally, in patch-clamp experiments, some neurons were unresponsive to membrane hyperpolarization, thus, probably lacking HCN channels.In conclusion, in porcine dorsal root ganglion neurons there is a subset of capsaicin-positive, IB4-negative neurons lacking HCN-1 and/or HCN-2 channel isoforms.     

Kv1.1 channels of dorsal root ganglion neurons are inhibited by n-butyl-p-aminobenzoate, a promising anesthetic for the treatment of chronic pain

In this study, we investigated the effects of the local anesthetic n-butyl-p-aminobenzoate (BAB) on the delayed rectifier potassium current of cultured dorsal root ganglion (DRG) neurons using the patch-clamp technique. The majority of the K(+) current of small DRG neurons rapidly activates and slowly inactivates at depolarized voltages. BAB inhibited the whole-cell K(+) current of these neurons with an IC(50) value of 228 microM. Dendrotoxin K (DTX(K)), a specific inhibitor of Kv1.1, reduced the DRG K(+) current at +20 mV by 34%, consistent with an important contribution of channels incorporating the Kv1.1 subunit to the delayed rectifier current. To further investigate the mechanism of BAB inhibition, we examined its effect on Kv1.1 channels heterologously expressed in mammalian tsA201 cells. BAB inhibits the Kv1.1 channels with an IC(50) value of 238 microM, similar to what was observed for the native DRG current. BAB accelerates the opening and closing of Kv1.1, but does not alter the midpoint of steady-state activation. BAB seems to inhibit Kv1.1 by stabilizing closed conformations of the channel. Coexpression with the Kv beta 1 subunit induces rapid inactivation and reduces the BAB sensitivity of Kv1.1. Comparison of the heterologously expressed Kv1.1 and native DRG currents indicates that the Kv beta 1 subunit does not modulate the gating of the DTX(K)-sensitive Kv1.1 channels of DRG neurons. Inhibition of the delayed rectifier current of these neurons may contribute to the long-duration anesthesia attained during the epidural administration of BAB.     

大鼠背根神经节机械敏感性离子通道的生物物理学性质

目的探讨新生大鼠背根神经节(DRG)的机械敏感性离子通道(MS通道)生物物理学性质。方法取出新生大鼠DRG细胞,培养2~4d后,应用细胞贴附式和内面向外式膜片钳技术对细胞膜上的MS通道电流进行记录,对通道生物物理学性质,如压力-电流关系、电位-电流关系、动力学和离子选择性等进行了分析。采用的机械刺激方式为负压抽吸。结果压力可引起大鼠背根神经节上的机械敏感性非选择性阳离子通道开放,压力恒定时,电流恒定;去除压力,电流回到基线水平。在平衡溶液中,通道的电位-电流关系近似为直线。在膜电位为正值时,通道电流表现为外向电流,同时表现出外向整流特性,+40~+60mV电流的弦电导为(96.2±3.6)pS;当膜电位为负值时,通道电流表现为内向电流,-60~0mV斜率电导为(62.5±0.4)pS。通道的平均逆转电位为(-2.3±0.8)mV。通道的动力学分析表明压力可使短开放时间和长开放时间都明显升高,长关闭时间明显减少,而短关闭时间变化不大。结论分析了大鼠DRG神经元细胞膜上的MS通道的生物物理学性质,有助于进一步了解大鼠背根神经节神经元细胞电活动的机制。     

Key role of the dorsal root ganglion in neuropathic tactile hypersensibility.

Cutting spinal nerves just distal to the dorsal root ganglion (DRG) triggers, with rapid onset, massive spontaneous ectopic discharge in axotomized afferent A-neurons, and at the same time induces tactile allodynia in the partially denervated hindlimb. We show that secondary transection of the dorsal root (rhizotomy) of the axotomized DRG, or suppression of the ectopia with topically applied local anesthetics, eliminates or attenuates the allodynia. Dorsal rhizotomy alone does not trigger allodynia. These observations support the hypothesis that ectopic firing in DRG A-neurons induces central sensitization which leads to tactile allodynia. The question of how activity in afferent A-neurons, which are not normally nociceptive, might induce allodynia is discussed in light of the current literature.     

Calcitonin gene-related peptide enhances TTX-resistant sodium currents in cultured dorsal root ganglion neurons from adult rats

The neuropeptide calcitonin gene-related peptide (CGRP) binds to a subpopulation of dorsal root ganglion (DRG) neurons, elevates intracellular calcium, and causes inward currents in about 30% of lumbar DRG neurons. Using whole-cell patch clamp recordings, we found in the present study that application of CGRP to isolated and cultured DRG neurons from the adult rat enhances voltage-gated TTX-resistant (TTX-R) Na(+) inward currents in about 30% of small- to medium-sized DRG neurons. During CGRP, peak densities of Na(+) currents increased significantly. CGRP shifted the membrane conductance of the CGRP-responsive cells towards hyperpolarization without changing the slope of the peak conductance curve. The effect of CGRP was blocked by coadministration of CGRP8-37, an antagonist at the CGRP receptor. The effect of CGRP was also blocked after bath application of PKA14-22, a membrane-permeant blocker of protein kinase A, and PKC19-31, a PKC inhibitor, in the recording pipette. These data show pronounced facilitatory effects of CGRP on TTX-R Na(+) currents in DRG neurons which are mediated through CGRP receptors and intracellular pathways involving protein kinases A and C. Thus, in addition to prostaglandins, CGRP is another mediator that affects TTX-R Na(+) currents which are thought to occur mainly in nociceptive DRG neurons.