Sodium Channels in Spinal Cord Injury, Nerve Injury and Neuropathic Pain

描述(由申请人提供)： 这项研究提案包括四个相互关联的项目，力求做到以下几点。具体目标I和II：描述创伤性神经损伤和截肢引起的慢性疼痛的机制，并确定有效的治疗方法。由创伤性神经损伤和创伤性肢体截肢引起的神经瘤可作为异常异位冲动产生的部位，这与神经病理性疼痛有关。在许多情况下，神经瘤引起的疼痛对现有的药物没有反应。我们已经证明，钠通道Nav1.3在疼痛的人类神经瘤和实验性神经瘤中都有积累。Nav1.3的生物物理特性支持其在神经瘤异位冲动产生中的作用。在翻译目标I中，我们将建立在我们的病毒介导的shRNA(AAV-shRNA)击倒大鼠DRG中的Nav1.3的基础上，并测试AAV-shRNA-Nav1.3减轻神经瘤诱导的疼痛的假设。多项研究表明，通道异构体Nav1.7与神经病理性疼痛有关。Nav1.7积聚在人类神经瘤和实验性神经瘤中受损的轴突尖端，并与人类疼痛有遗传联系。作为靶向Nav1.3的替代方案，AIM II将测试AAV-shRNA介导的Nav1.7在减轻神经瘤诱导的疼痛方面的作用。具体目标III：破译负责伤害性神经纤维动作电位传导的特定钠通道亚型。在这一机制目标中，我们将评估Nav1.7、Nav1.8和Nav1.3在DRG神经元小径轴突电发生中的贡献。我们最近发展了在体外膜片钳小直径(1 Mm)背根神经节突起的能力，并证明了TTX-S和TTX-R钠电导在动作电位过程中依次激活。为了促进我们对轴突电发生的理解，合理的下一步是确定潜在的通道亚型，并评估它们对伤害性轴突兴奋性的贡献，正如我们在这一特定目标中所建议的那样。 此外，我们将利用我们的DRG培养/膜片钳系统，评估已知与人类慢性疼痛综合征相关的Nav1.7和Nav1.8功能获得变体对轴突室内电生成的功能影响。特定目的IV：确定钠通道异常表达在脊髓损伤所致痉挛中的作用。脊髓损伤后的痉挛被经典地认为是脊髓反射通路上突触传递增强以及抑制丧失的结果。然而，电路水平的研究和计算机模拟研究表明，运动神经元内在兴奋性的增加和运动神经元内钠通道表达的改变对脊髓损伤后痉挛的发生有贡献。我们已经证实了脊髓损伤后背角神经元中Nav1.3的表达上调，几位研究人员报道了外周神经切断后脊髓和面运动神经元中钠通道的上调表达。此外，我们还发现，在多发性硬化症患者和EAE小鼠中，Nav1.8在人中枢神经系统神经元、小脑浦肯野神经元中错误表达。在这一机制/翻译目标中，我们将使用分子、膜片钳和基因敲除方法来验证运动神经元钠通道表达失调导致脊髓损伤后痉挛的假说，并将确定敲除这种调控失调的通道是否可以改善痉挛。

DESCRIPTION (provided by applicant): This research proposal encompasses four inter-related projects that seek to do the following. Specific Aims I and II: Delineate mechanisms of, and identify effective therapies for, chronic pain resulting from traumatic nerve injury and limb amputation. Neuromas resulting from traumatic nerve injury and traumatic limb amputation can act as sites of abnormal ectopic impulse generation, which are linked to neuropathic pain. In many cases, neuroma-induced pain is not responsive to existing medications. We have shown that sodium channel Nav1.3 accumulates in both painful human neuromas and in experimental neuromas. The biophysical characteristics of Nav1.3 support its contribution to ectopic impulse generation in neuromas. In translational Aim I, we will build upon our viral- mediated shRNA (AAV-shRNA) knockdown of Nav1.3 in rat DRG in vivo and test the hypothesis that AAV- shRNA-Nav1.3 attenuates neuroma-induced pain. Multiple studies have indicated a contribution of channel isoform Nav1.7 to neuropathic pain. Nav1.7 accumulates in injured axon tips within human neuromas and in experimental neuromas, and has been genetically linked to human pain. As an alternative to targeting Nav1.3, Aim II will test AAV-shRNA mediated knockdown of Nav1.7 toward attenuation of neuroma-induced pain. Specific Aim III: Decipher specific sodium channel isoforms responsible for action potential conduction along nociceptive nerve fibers. In this mechanistic aim, we will assess the contribution of Nav1.7, Nav1.8 and Nav1.3 to electrogenesis in small-diameter axons of DRG neurons. We have recently developed the capability to patch-clamp small diameter (<1mm) DRG neurites in vitro and demonstrated that TTX-S and TTX-R sodium conductances are sequentially activated during action potentials. To advance our understanding of axonal electrogenesis, a logical next step is to identify the underlying channel subtypes and assess their contribution to excitability in nociceptive axons, as we propose in this specific aim. In addition, we will assess the functional effect of gain- of-function variants of Nav1.7 and Nav1.8 that are known to be associated with chronic pain syndromes in humans, on electrogenesis within the axonal compartment, utilizing our DRG culture/patch-clamp system. Specific Aim IV: Determine the contribution of aberrant sodium channel expression in SCI-induced spasticity. Spasticity after SCI has been classically thought to be a result of enhanced synaptic transmission along the spinal reflex pathways as well as loss of inhibition. However, studies at the circuit level, and computer simulation studies, suggest a contribution of increased intrinsic excitability of motor neurons and altered expression of sodium channels within motor neurons to spasticity after SCI. We have demonstrated upregulated expression of Nav1.3 within dorsal horn neurons after SCI and several investigators have reported upregulated expression of sodium channels in spinal and facial motor neurons following peripheral axotomy. Additionally, we have shown that Nav1.8 is mis-expressed in human CNS neurons, cerebellar Purkinje neurons) in patients with multiple sclerosis and in mice with EAE. In this mechanistic/translational aim, we will use molecular, patch-clamp, and knockdown methods to test the hypothesis that dysregulated sodium channel expression in motor neurons contributes to spasticity following SCI, and will determine whether knockdown of such dysregulated channels ameliorates spasticity.