脊神经损伤后钠电流的变化及其离子通道机制

目的：检测脊神经切断大鼠背根节（DRG）神经元重复放电能力和钠电流的变化,并研究介导其电流变化的钠通道亚型的表达情况。方法：脊神经切断术后2～8d慢性痛大鼠模型背根节急性分离,对中等直径DRG神经元运用全细胞膜片钳技术记录神经元放电和钠电流的变化。对背根节神经元进行RT-PCR检测,分析其钠通道亚型的表达情况。结果：电流钳下,实验组DRG神经元在电流刺激下产生重复放电,而对照组神经元多诱发单个动作电位,电压钳记录发现实验组背根节神经元快钠电流和持续性钠电流幅值均明显大于对照组,PCR结果显示,Nav1.3、Nav1.7和Nav1.8通道亚型mRNA表达显著增高。结论：钠通道介导了脊神经受损模型的DRG神经元兴奋性增高,持续性钠电流可能通过调节阈下膜电位振荡的产生调节神经元兴奋性。     

缓激肽对背根节神经元钠通道电流的作用

目的：观察缓激肽(bradykinin,BK)对大鼠背根节神经元电压依赖性钠通道电流的作用。方法：采用全细胞膜片钳技术,记录钠通道电流。结果：缓激肽剂量依赖性(0．01～10μmol／L)增高小细胞背根节神经元诱发放电频率;缓激肽剂量依赖性(O．01～10μmol／L)增加小细胞背根节神经元的河豚毒素不敏感(TTX—resistant,TTX—R)钠电流,对TTX敏感(TTX—sensitive,TTX-S)钠电流无明显影响。结论：缓激肽引起炎性痛的机制可能与TTX-R钠通道电流有关。     

人参皂甙Rd通过调节SNI大鼠背根神经节钠、钾电流抑制痛敏

目的:观察人参皂甙Rd(ginsenoside Rd)对大鼠坐骨神经分支选择性损伤(spared sciatic nerve injury,SNI)引起的痛敏的影响及其作用机制。方法:坐骨神经分支选择性损伤术后7天,观察腹腔注射不同浓度人参皂甙Rd后大鼠后足的机械性缩足反应阈值(paw withdrawl mechanical threshold,PWMT)的变化;在术后7天,急性分离并取出大鼠腰4和腰5段背根节,对整节DRG上的中小型神经元运用全细胞膜片钳技术进行记录。结果:坐骨神经分支选择性损伤术后7天,大鼠出现明显的机械性痛敏,腹腔注射5 mg/ml和10 mg/ml的人参皂甙Rd能剂量依赖性的翻转大鼠机械性痛敏;坐骨神经分支选择性损伤能明显地增大SNI大鼠DRG中小型神经元上的钠电流以及减小电压依赖性钾电流,而100μM人参皂甙Rd能有效翻转该钠、钾电流的变化。结论:人参皂甙Rd能有效地改善坐骨神经分支选择性损伤引起的机械性痛敏,其机制可能与人参皂甙Rd明显地调节SNI大鼠DRG中小型神经元上的电压依赖性钠、钾电流有关。     

人参皂甙Rd通过调节SNI大鼠背根神经节钠、钾电流抑制痛敏

目的：观察人参皂甙Rd（ginsenoside Rd）对大鼠坐骨神经分支选择性损伤（spared sciatic nerve injury,SNI）引起的痛敏的影响及其作用机制。方法：坐骨神经分支选择性损伤术后7天,观察腹腔注射不同浓度人参皂甙Rd后大鼠后足的机械性缩足反应阈值（paw withdrawl mechanical threshold,PWMT）的变化;在术后7天,急性分离并取出大鼠腰4和腰5段背根节,对整节DRG上的中小型神经元运用全细胞膜片钳技术进行记录。结果：坐骨神经分支选择性损伤术后7天,大鼠出现明显的机械性痛敏,腹腔注射5 mg/ml和10 mg/ml的人参皂甙Rd能剂量依赖性的翻转大鼠机械性痛敏;坐骨神经分支选择性损伤能明显地增大SNI大鼠DRG中小型神经元上的钠电流以及减小电压依赖性钾电流,而100μM人参皂甙Rd能有效翻转该钠、钾电流的变化。结论：人参皂甙Rd能有效地改善坐骨神经分支选择性损伤引起的机械性痛敏,其机制可能与人参皂甙Rd明显地调节SNI大鼠DRG中小型神经元上的电压依赖性钠、钾电流有关。     

瞬时外向钾电流的降低介导了慢性压迫背根神经节细胞兴奋性的升高

慢性压迫大鼠背根神经节（chronic compression of the dorsal root,ganglion,CCD）后,背根神经节细胞兴奋性升高,但引起神经元兴奋性改变的离子通道机制还需进一步探索。本实验采用胞内记录以及全细胞膜片钳记录方法,研究急性分离的大鼠背根神经节细胞兴奋性改变与瞬时外向钾电流（A-type potassium current,ⅠA）的关系。结果表明,CCD术后背根神经节细胞兴奋性升高,在急性分离的体外细胞中仍继续存在,表现为对辣椒素敏感的背根神经节细胞产生动作电位的最小电流刺激强度,即阈电流（current threshold）及阈电位（voltage threshold）降低;给予正常对照组神经元（未压迫损伤）瞬时外向钾通道阻断剂4-氨基吡啶,出现了类似CCD术后兴奋性升高的改变。进一步用两步电压钳方法分离ⅠA,研究CCD术后神经元ⅠA的变化,结果表明,CCD组神经元的ⅠA比对照组神经元ⅠA降低,并且与其阈电位的改变一致。以上结果提示,背根神经节压迫受损后,神经节细胞ⅠA降低可能参与介导了神经节细胞兴奋性的升高。     

藜芦碱和乌头碱在受损背根节神经元诱发不同的放电模式

为了研究钠通道失活门阻断后受损背根节神经元放电模式的变化特征,在大鼠背根节慢性压迫模型上采用单纤维技术记录A类神经元的自发放电。藜芦碱和乌头碱是钠通道失活门的抑制剂,但二者作用于不同的位点,前者结合于D2-S6,后者结合于D3-S6。我们比较了这两种试剂引发的放电模式。结果发现,在同一神经元,藜芦碱(1．5～5．0μmol／L)可以引起放电峰峰间期的慢波振荡,即峰峰间期由大逐渐减小,然后又逐渐增大,形成重复的振荡波形,每个振荡持续约数十秒至数分钟：而乌头碱(10～200μmol／L)则引起强直性放电,即峰峰间期逐渐减小,然后维持在一个稳定的水平。这两种不同的放电模式不因背景放电或试剂浓度的不同而发生明显的改变。实验结果表明,藜芦碱和乌头碱在受损的背根节神经元可以引发不同的放电模式,这可能与它们结合于钠通道上不同位点的抑制作用有关。     

神经病理痛大鼠DRG神经元GABAA受体激活电流的变化

目的：观察坐骨神经慢性压榨损伤（CCI）致神经病理痛后,大鼠背根节神经元GABAA受体（γ-氨基丁酸A受体）激活电流的变化。方法：运用全细胞膜片钳技术记录CCI模型手术侧、手术对侧及假手术组大鼠背根神经节细胞GABAx受体激活电流,比较坐骨神经慢性压榨损伤后GABAA受体激活电流的变化。结果：①CCI模型组大鼠手术侧DRG神经元在不同浓度（0．1-1000μmol／L）GABAA受体激活电流幅值均显著小于假手术组。②CCI模型组大鼠手术对侧DRG神经元在不同浓度（0．01-1000μmol／L）GABAA受体激活电流幅值均显著大于手术同侧及假手术组。结论：在坐骨神经慢性压榨损伤的过程中,不仅损伤侧的DRG神经元GABAA受体激活电流显著减小,这种损伤同时还引起了手术对侧的DRG神经元GABA激活电流代偿性的增强,GABAA受体功能的改变导致的突触前抑制作用的减弱可能是神经病理痛产生的根本原因之一。     

右美托咪啶通过抑制超极化激活内向电流减轻机械性触诱发痛

本文旨在研究右美托咪啶(dexmedetomidine,DEX)对慢性压迫背根神经节(chronic compression of dorsal root ganglion,CCD)引起的机械性触诱发痛的作用及机制。制备Sprague Dawley(SD)大鼠CCD模型,用痛行为学方法检测机械性触诱发痛,用全细胞膜片钳技术检测背根神经节(dorsal root ganglion,DRG)C类和A_δ类神经元兴奋性和超极化激活内向电流(hyperpolarization-activated inward currents,I_h)的变化。结果显示:鞘内注射DEX显著减轻CCD大鼠机械性触诱发痛(P0.05);在CCD大鼠DRG C类和A_δ类神经元上,DEX显著提高细胞基强度和后超极化电位,降低I_h电流密度。上述结果提示,DEX可能通过抑制C类和A_δ类DRG神经元I_h电流,降低神经元兴奋性,从而减轻神经病理性疼痛。     

敬钊毒素-Ⅲ对Nav1.8通道的抑制作用

敬钊毒素-Ⅲ(JingzhaotoxinⅢ,JZTX-Ⅲ)是从敬钊缨毛蛛毒液中分离到的一种门控调节型毒素,能选择性抑制钠通道亚型Nav1.5激活,但对其他6种钠通道亚型(Nav1.1 Nav1.4 Nav1.6和Nav1.7)无抑制作用。为了更好地研究钠通道结构与功能之间的关系,采用全细胞膜片钳技术检测了JZTX-Ⅲ对表达在ND7123细胞上的Nav1.8画道的影响。结果显示,JZTX-Ⅲ抑制Nav1.8电流,并且这种抑制作用具有时间和浓度依赖性,抑制时间常数和IC_(50)值分别为41.15±0.6 s和1.4±0.23μmol/L;1μmol/JZTX-Ⅲ使Nav1.8画道的电流-电压关系曲线和激活曲线分别向去极化方向漂移10 mV和9mV,使Nav.1.8通道的稳态失活曲线向超极化方向漂移16 mV,明显改变Nav1.8通道的激活和稳态失活动力学。此外,钠通道序列比对结果提示JZTX-Ⅲ可能通过结合Nav1.8通道DIIS3~S4连接环上的Lys(K)残基抑制Nav1.8通道。以上研究结果为进一步探索钠通道结构与功能之间的关系奠定了基础。     

藜芦碱引起神经元放电峰峰间期慢波振荡

为了研究Na通道失活门与受损背根节神经元放电型式的关系,在大鼠背根节慢性压迫模型上记录单纤维自发放电,观察与分析了Na通道失活门抑制剂藜芦碱（veratridine）引起峰峰间期慢波振荡的型式的特征。结果表明：在阻断Na通道失活门之后,受损背根节神经元产生的慢波振荡具有变化幅度大和振荡时程长的特征,可分成Ⅴ,倒П,整数倍,弥散和复合等5种基本形式。     

Up-regulation of Cavβ3 subunit in primary sensory neurons increases voltage-activated Ca2+ channel activity and nociceptive input in neuropathic pain

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.     

肝细胞生长因子在正常大鼠腰段脊髓和背根神经节的表达

目的:观察肝细胞生长因子(hepatocyte growth factor,HGF)在大鼠脊髓和背根神经节(dorsal root ganglion,DRG)的表达。方法:取健康成年6只SD大鼠运用免疫组织化学染色技术检测HGF在腰段脊髓、背根神经节内的表达和分布。结果:在L4-6段脊髓,HGF免疫阳性产物可见于各板层神经元,尤以脊髓前角运动神经元明显;在DRG中,HGF免疫阳性物质可见于以大、中型为主的神经元的胞浆及突起中。结论:脊髓和背根神经节内的HGF通过与受体c-Met结合可能在神经再生及突触可塑性方面起一定作用。     

The tetrodotoxin-insensitive sodium current in rat dorsal root ganglia is unlikely to involve the expression of the tetrodotoxin-resistant sodium channel,SkM2

Tetrodotoxin-insensitive (TTX-I) sodium currents have been recorded from newborn and adult rat sensory neurons, but the sodium channel gene(s) responsible for the TTX-I current are unknown. Because SkM2, one of six voltage-sensitive sodium channel genes cloned from rat, encodes the only cloned channel that is relatively resistant to tetrodotoxin, we sought to test whether the TTX-I current in rat sensory neurons is due to the SkM2 channel. We hypothesized that the TTX-I current might be generated from (1) an RNA splicing variant of SkM2, (2) post-translational modification of the SkM2 protein, or (3) interaction with altenate additional channel subunits. SkM2 mRNA expression was examined in newborn rat dorsal root ganglia (DRG) by RNase arotection assay. No SkM2 expression was detected. Therefore, we conclude that the TTX-I sodium current in DRG is unlikely to result from the expression of the SkM2 gene.     

Plasticity and emerging role of BKCa channels in nociceptive control in neuropathic pain

Large‐conductance Ca²⁺‐activated K⁺ (BK_Ca, MaxiK) channels are important for the regulation of neuronal excitability. Peripheral nerve injury causes plasticity of primary afferent neurons and spinal dorsal horn neurons, leading to central sensitization and neuropathic pain. However, little is known about changes in the BK_Ca channels in the dorsal root ganglion (DRG) and spinal dorsal horn and their role in the control of nociception in neuropathic pain. Here we show that L5 and L6 spinal nerve ligation in rats resulted in a substantial reduction in both the mRNA and protein levels of BK_Ca channels in the DRG but not in the spinal cord. Nerve injury primarily reduced the BK_Ca channel immunoreactivity in small‐ and medium‐sized DRG neurons. Furthermore, although the BK_Ca channel immunoreactivity was decreased in the lateral dorsal horn, there was an increase in the BK_Ca channel immunoreactivity present on dorsal horn neurons near the dorsal root entry zone. Blocking the BK_Ca channel with iberiotoxin at the spinal level significantly reduced the mechanical nociceptive withdrawal threshold in control and nerve‐injured rats. Intrathecal injection of the BK_Ca channel opener [1,3‐dihydro‐1‐[2‐hydroxy‐5‐(trifluoromethyl)phenyl]‐5‐(trifluoromethyl)‐2H‐benzimidazol‐2‐one] dose dependently reversed allodynia and hyperalgesia in nerve‐ligated rats but it had no significant effect on nociception in control rats. Our study provides novel information that nerve injury suppresses BK_Ca channel expression in the DRG and induces a redistribution of BK_Ca channels in the spinal dorsal horn. BK_Ca channels are increasingly involved in the control of sensory input in neuropathic pain and may represent a new target for neuropathic pain treatment.     

Involvement of hyperpolarization-activated, cyclic nucleotide-gated cation channels in dorsal root ganglion in neuropathic pain

Dorsal root ganglion(DRG)neurons have peripheral terminals in skin,muscle,and other peripheral tissues,andcentral terminals     

Experimental colitis modulates the functional properties of NMDA receptors in dorsal root ganglia neurons

N-methyl-D-aspartate (NMDA) receptors (NMDARs) on spinal afferent neurons regulate the peripheral and central release of neuropeptides involved in the development of hyperalgesia. We examined the effect of experimental colitis on the molecular and functional properties of NMDARs on these neurons. Lumbosacral dorsal root ganglia (DRG) were collected from adult rats 5 days after the induction of colitis for whole cell patch-clamp recording, Western blot analysis, and quantitative RT-PCR. Compared with neurons from control rats, those taken from animals with colitis had a threefold higher density of NMDA currents in both retrograde-labeled, colon-specific, and unlabeled DRG neurons. Increased current densities were not observed in DRG neurons taken from thoracic spinal levels. There was no significant change in NMDA or glycine affinity or in voltage-dependent Mg2+ inhibition; however, there was a 10-fold decrease in sensitivity to the NR2B subunit-selective antagonist ifenprodil. Quantitative RT-PCR and Western blot analysis indicated a 28% increase in the expression of NR2B with little or no change in the other three NR2 subunits. The addition of the Src family tyrosine kinase inhibitor PP2 (10 microM) decreased NMDAR currents in neurons from colitis but not control rats. Conversely, pretreatment of DRG neurons from control animals with 100 microM sodium orthovanadate increased NMDAR currents and decreased ifenprodil sensitivity to levels similar to those observed in neurons from animals with colitis. In conclusion, colonic inflammation upregulates the activity of NMDARs in all DRG neurons within ganglia innervating this tissue through mechanisms involving increased expression and persistent tyrosine phosphorylation.     

Pannexin-1 Up-regulation in the Dorsal Root Ganglion Contributes to Neuropathic Pain Development

Pannexin-1 (Panx1) is a large-pore membrane channel involved in the release of ATP and other signaling mediators. Little is known about the expression and functional role of Panx1 in the dorsal root ganglion (DRG) in the development of chronic neuropathic pain. In this study, we determined the epigenetic mechanism involved in increased Panx1 expression in the DRG after nerve injury. Spinal nerve ligation in rats significantly increased the mRNA and protein levels of Panx1 in the DRG but not in the spinal cord. Immunocytochemical labeling showed that Panx1 was primarily expressed in a subset of medium and large DRG neurons in control rats and that nerve injury markedly increased the number of Panx1-immunoreactive DRG neurons. Nerve injury significantly increased the enrichment of two activating histone marks (H3K4me2 and H3K9ac) and decreased the occupancy of two repressive histone marks (H3K9me2 and H3K27me3) around the promoter region of Panx1 in the DRG. However, nerve injury had no effect on the DNA methylation level around the Panx1 promoter in the DRG. Furthermore, intrathecal injection of the Panx1 blockers or Panx1-specific siRNA significantly reduced pain hypersensitivity induced by nerve injury. In addition, siRNA knockdown of Panx1 expression in a DRG cell line significantly reduced caspase-1 release induced by neuronal depolarization. Our findings suggest that nerve injury increases Panx1 expression levels in the DRG through altered histone modifications. Panx1 up-regulation contributes to the development of neuropathic pain and stimulation of inflammasome signaling.