Molecular probing for the mechanism of neuropathic pain

描述（由申请人提供）：拟议的R21项目的长期目标是阐明周围神经性疼痛的病理机制。据推测，初级感觉神经元发生组成性变化，导致河豚毒素抗性钠通道Nav1.8的异常外周蓄积，从而诱导神经病症状，如异常性疼痛、痛觉过敏和感觉迟钝。然而，令人困惑的是，据报道，在这些神经元的胞体中，NaV1.8 mRNA水平交替地降低、不变或略有增加。Pi's研究小组最近观察到，在坐骨神经卡压（SNE）损伤后，大鼠坐骨神经中NaV1.8 mRNA显著增加，但在脊神经结扎（SNL）损伤后则没有。这表明，感觉神经元基因表达的选择性操纵可能提供了一个独特的机会，神经病理性发病机制的差异调查。本申请提出使用与质粒DMA复合的生物相容性阳离子化明胶（CG）载体开发新的非侵入性基因转移至感觉神经元。将CG/DNA复合物皮下注射到无毛的后爪应导致外周摄取并通过坐骨神经逆行转运到背根神经节（DRG）。初步研究在L4/L5 DRG中实现了报告基因表达。目的1是建立CG/DNA神经基因转移的时空动力学的技术开发项目。还将在该模型中建立小干扰RNA（siRNA）的质粒表达。目的2探讨外周坐骨神经中NaV1.8 mRNA的净增加在神经病理性疼痛发病机制中的作用。将检查大鼠SNE模型中DRG和坐骨神经中NaV1.8 mRNA的空间分布。NaV1.8靶向siRNA将用于研究NaV1.8抑制对神经病理性疼痛行为的影响。该项目的结果将为未来的研究提供一个新的切入点，并为神经病理性疼痛提供一种公认的治疗方式。

DESCRIPTION (provided by applicant): The long-term goal of the proposed R21 project is to elucidate a pathological mechanism underlying peripheral neuropathic pain. It has been postulated that constitutive changes occur in primary sensory neurons leading to abnormal peripheral accumulation of the tetrodotoxin-resistant sodium channel Nav1.8, with a resulting induction of neuropathy symptoms such as allodynia, hyperalgesia, and dysesthesia. It is puzzling, however, that the NaV1.8 mRNA level has alternately been reported to be reduced, unchanged, or slightly increased in the somata of these neurons. The Pi's group recently observed that NaV1.8 mRNA is significantly increased in the rat sciatic nerve after sciatic nerve entrapment (SNE) injury but not spinal nerve ligation (SNL) injury. This suggests that selective manipulation of sensory neuron gene expression may offer a unique opportunity for differential investigations of neuropathic pathogenesis. This application proposes to develop a novel non-invasive gene transfer to sensory neurons using a biocompatible cationized gelatin (CG) vector complexed with plasmid DMA. Subcutaneous CG/DNA complex injection to the glabrous hindpaw should result in peripheral uptake and retrograde transport to the dorsal root ganglion (DRG) via the sciatic nerve. Preliminary studies achieved reporter gene expression in the L4/L5 DRG. Aim 1 is a technology development project which will establish the spatiotemporal kinetics of CG/DNA neural gene transfer. Plasmid expression of small interfering RNA (siRNA) will also be established in this model. Aim 2 will explore the mechanistic hypothesis that the net increase of NaV1.8 mRNA in the peripheral sciatic nerve contributes, in part, to the pathogenesis of neuropathic pain. The rat SNE model will be examined for the spatial distribution of NaV1.8 mRNA in the DRG and sciatic nerve. NaV1.8-targeted siRNA will be applied to study effects of NaV1.8 suppression on neuropathic pain behavior. The outcome of this project will provide a novel entry point for future investigations of, and a putative therapeutic modality for neuropathic pain.