Spinal Mechanisms Underlying SCI-Induced Pain: Implications for Targeted Therapy

项目摘要 脊髓损伤（SCI）不仅导致感觉运动障碍，而且还导致慢性、严重和经常性的脊髓损伤。 持续性疼痛（SCI疼痛）发生在多达85%的患者中。SCI疼痛具有神经病理学特征， 通常对传统的疼痛治疗有抵抗力。后者可能部分反映了对 伤害机制。确定损伤后神经性疼痛的机制可以提供靶点 更有效的治疗干预。我们发现了一个有前途的新的治疗靶点trkB.T1， 脑源性神经营养因子受体-原肌球蛋白相关激酶B（trk B）的截短亚型。在 在神经性疼痛包括SCI的小鼠模型中，trkB.T1的基因缺失降低了机械性疼痛和 热过敏然而，这一发现背后的确切细胞机制并不完全 明白本研究的目的是研究trkB.T1如何通过以下途径驱动损伤后神经病理性疼痛： 星形胶质细胞功能障碍和测试的假设，星形胶质细胞trkB.T1功能作为一个关键机制后， 损伤反应性星形胶质细胞增生，通过改变控制细胞运动的转录编程， 免疫功能，从而影响脊髓损伤后的慢性疼痛。 我们将在转基因小鼠和体内使用星形胶质细胞trkB. T1敲除（KO）、Nox 2 KO和trkB. T1-KFG敲除 和体外创新技术，以确定SCI触发的trkB. T1升高的机制， 损伤加重。目的1探讨trkB.T1/[Ca ~（2+）]i/Nox ~（2+）的功能和作用机制 脊髓损伤后星形胶质细胞中的信号通路。星形胶质细胞增生的多种定量评估将与 以trkB.T1为靶点的遗传干预，以检验SCI触发星形胶质细胞trkB.T1升高的假设 增加[Ca 2 +]i和Nox 2活性，导致神经炎症和痛觉亢进。目标2将阐明 星形胶质细胞Nox 2信号在损伤后痛觉过敏中的作用。我们将利用基因干预， 在星形胶质细胞中删除trkB.T1依赖性的Nox 2上调，并评估星形胶质细胞Nox 2对 脊髓损伤后神经痛目的3将确定trkB.T1上的KFG结构域在调控中的作用。 星形胶质细胞功能和SCI-疼痛。将使用补充的细胞、分子和遗传方法 检验trkB.T1的KFG结构域响应BDNF调节trkB.T1功能的假设，和 KFG结构域的突变消除了损伤后神经性疼痛。 我们的研究将是第一个涉及星形胶质细胞trkB.T1介导的[Ca 2 +]i/Nox 2信号在病理生理学中的作用。 SCI的。我们的数据应该建立第二信使与trkB.T1上的细胞内KFG结构域的结合， 一种调节trkB.T1介导的疼痛信号传导的生理学重要机制。这些观察结果 可能导致在广泛的疾病状态下神经性疼痛的新的治疗靶点。

Project Summary Spinal cord injury (SCI) causes not only in sensorimotor deficits, but also in a chronic, severe and often unrelenting pain (SCI-pain) that occurs in as many as 85% of patients. SCI-pain has neuropathic features and is often resistant to conventional pain therapy. The latter may reflect, in part, an incomplete understanding of injury mechanisms. Identifying mechanisms responsible for post-injury neuropathic pain could provide targets for more effective therapeutic interventions. We identified a promising new therapeutic target trkB.T1, a truncated isoform of the brain-derived neurotrophic factor receptor—tropomyosin related kinase B (trkB). In mouse models of neuropathic pain including SCI, genetic deletion of trkB.T1 reduces both mechanical and thermal hypersensitivity. However, the precise cellular mechanisms underlying this finding are not fully understood. The purpose of this study is to investigate how trkB.T1 drives post-injury neuropathic pain via astrocyte dysfunction and test the hypothesis that astrocytic trkB.T1 functions as a key mechanism in post- injury reactive astrogliosis, through altered transcriptional programming that controls cellular movement and immune function, thus affecting chronic pain after spinal cord injury. We will use astrocytic trkB.T1 knock out (KO), Nox2 KO, and trkB.T1-KFG knock in transgenic mice and in vivo and in vitro innovatively technologies to determine the mechanisms of SCI-triggered trkB.T1 elevation on post- injury hyperpathia. Aim 1 will determine the function and mechanisms of the trkB.T1/[Ca2+]i/Nox2 pathway in astrocytes after SCI. Multiple quantitative assessments of astrogliosis will be combined with a genetic intervention targeting trkB.T1 to test the hypothesis that SCI-triggered trkB.T1 elevation in astrocytes increases [Ca2+]i and Nox2 activity, contributing to neuroinflammation and hyperpathia. Aim 2 will elucidate the role of astrocytic Nox2 signaling in post-injury hyperpathia. We will utilize genetic intervention to delete trkB.T1-dependent up-regulation of Nox2 in astrocytes, and evaluate the effects on astrocytic Nox2 on neuropathic pain after SCI. Aim 3 will determine the role for the KFG domain on trkB.T1 in the regulation of astrocyte function and SCI-Pain. Complimentary cellular, molecular, and genetic approaches will be used to test the hypothesis that KFG domain of trkB.T1 regulates trkB.T1 function in response to BDNF, and mutation of KFG domain abolishes post-injury neuropathic pain. Our study will be the first to implicate astrocytic trkB.T1-mediated [Ca2+]i/Nox2 signaling in the pathophysiology of SCI. Our data should establish that second messenger binding to the intracellular KFG domain on trkB.T1 is a physiologically important mechanism that regulates trkB.T1-mediated pain signaling. These observations may lead to novel therapeutic targets for neuropathic pain in a wide range of disease states.