Molecular probing for the mechanism of neuropathic pain

描述（由申请人提供）：拟议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，初级感觉神经元的构形变化导致抗河豚毒素钠通道Nav1.8的外周异常积聚，从而诱发异常性疼痛、痛觉过敏和感觉不良等神经病变症状。然而，令人困惑的是，在这些神经元的体细胞中，NaV1.8 mRNA水平被报道为降低、不变或略有增加。Pi’s组最近观察到，大鼠坐骨神经压迫（SNE）损伤后，NaV1.8 mRNA显著升高，而脊髓神经结扎（SNL）损伤后，NaV1.8 mRNA无显著升高。这表明选择性操纵感觉神经元基因表达可能为神经病发病机制的鉴别研究提供了一个独特的机会。本应用程序提出开发一种新的非侵入性基因转移到感觉神经元使用生物相容性阳离子明胶（CG）载体复配质粒DMA。皮下注射CG/DNA复合物到无毛的后爪会导致外周摄取并通过坐骨神经逆行运输到背根神经节（DRG）。初步研究在L4/L5 DRG中实现了报告基因的表达。目标1是一个技术开发项目，将建立CG/DNA神经基因转移的时空动力学。小干扰RNA （siRNA）的质粒表达也将在该模型中建立。目的2将探讨坐骨周围神经中NaV1.8 mRNA的净增加在一定程度上参与神经性疼痛发病机制的机制假设。在大鼠SNE模型中检测NaV1.8 mRNA在DRG和坐骨神经中的空间分布。NaV1.8靶向siRNA将用于研究NaV1.8抑制对神经性疼痛行为的影响。该项目的结果将为未来的研究提供一个新的切入点，并为神经性疼痛提供一个假定的治疗方式。

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.