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

描述（由申请人提供）： 该研究计划包括四个相互关联的项目，旨在实现以下目标。具体目标一和二：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截肢引起的神经瘤可以作为异常异位冲动产生的部位，其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药物没有反应。我们已经表明，钠通道Nav1.3积累在疼痛的人类神经瘤和实验性神经瘤。Nav1.3的生物物理特性支持其对神经瘤中异位冲动产生的贡献。在翻译目的I中，我们将基于我们的病毒介导的shRNA（AAV-shRNA）在大鼠DRG中的体内敲低，并测试AAV-shRNA-Navl.3减弱神经瘤诱导的疼痛的假设。多项研究表明通道亚型Nav1.7对神经性疼痛的贡献。Nav1.7在人类神经瘤和实验性神经瘤中的受损轴突尖端积累，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敲低对神经瘤诱导疼痛的减弱作用。特定目标III：破译负责沿着伤害性神经纤维传导动作电位的特异性钠通道亚型。在这个机制的目的，我们将评估Nav1.7，Nav1.8和Nav1.3的小直径轴突的DRG神经元的电发生的贡献。我们最近开发了体外膜片钳小直径（<1 mm）DRG神经突的能力，并证明TTX-S和TTX-R钠电导在动作电位期间顺序激活。为了加深我们对轴突电发生的理解，合乎逻辑的下一步是识别潜在的通道亚型并评估它们对伤害性轴突兴奋性的贡献，正如我们在这个特定目标中提出的那样。 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的功能获得变体对轴突隔室内的电发生的功能效应。特定目的IV：确定钠通道表达异常在SCI诱导的痉挛中的作用。SCI后的痉挛通常被认为是脊髓反射通路上沿着突触传递增强以及抑制丧失的结果。然而，在电路水平的研究，和计算机模拟研究，表明增加运动神经元的内在兴奋性和运动神经元内的钠通道的表达改变脊髓损伤后痉挛的贡献。我们已经证明了脊髓损伤后背角神经元内Nav1.3的表达上调，并且一些研究者已经报道了外周轴突切断后脊髓和面部运动神经元中钠通道的表达上调。此外，我们已经表明，Nav1.8在患有多发性硬化症的患者和患有EAE的小鼠中的人CNS神经元、小脑浦肯野神经元中错误表达。在这一机制/翻译的目标，我们将使用分子，膜片钳，敲除方法来测试的假设，运动神经元中的钠通道表达失调，导致痉挛SCI后，并将确定是否敲除这种失调的通道改善痉挛。

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.