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

项目摘要 脊髓损伤(SCI)不仅可引起感觉运动障碍，还可导致慢性、重度和多发性脊髓损伤 持续的疼痛(SCI-疼痛)，出现在多达85%的患者中。SCI-Pain具有神经病理性特征和 通常对传统的疼痛疗法有抵抗力。后者可能在一定程度上反映了对 伤害机制。识别损伤后神经病理性疼痛的机制可以提供靶点 进行更有效的治疗干预。我们确定了一个有前景的新治疗靶点trkB.T1，a 脑源性神经营养因子受体-原肌球蛋白相关激酶B(TrkB)的截短亚型。在……里面 包括脊髓损伤在内的神经病理性疼痛的小鼠模型，trkB.T1基因缺失降低了机械性疼痛和 热过敏症。然而，这一发现背后的确切细胞机制并不完全 明白了。本研究的目的是研究trkB.T1是如何通过 并验证星形胶质细胞trkB.T1作为后遗症关键机制的假说。 损伤反应性星形胶质细胞增生症，通过改变转录程序控制细胞运动和 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活体中使用星形细胞trkB.T1敲除(KO)、NOX2 KO和trkB.T1-KFG敲打 以及体外创新技术以确定脊髓损伤后trkB.T1上调的机制。 伤害性过敏症。目标1将确定trkB.T1/[Ca2+]I/NOX2的功能和机制 脊髓损伤后星形胶质细胞的通路。对星形胶质细胞增生症的多项定量评估将与 针对trkB.T1的基因干预以检验脊髓损伤触发星形胶质细胞trkB.T1升高的假设 增加[Ca~(2+)]i和NOX2的活性，导致神经炎症和过敏。Aim 2将澄清 星形胶质细胞NOX2信号在损伤后亢进中的作用。我们将利用基因干预来 删除trkB.T1依赖的星形胶质细胞NOX2上调，并评价其对星形胶质细胞NOX2的影响 脊髓损伤后神经病理性疼痛。目标3将确定trkB.T1上的KFG结构域在调节中的作用 星形胶质细胞功能和脊髓疼痛的关系。将使用免费的细胞、分子和遗传方法 检验trkB.T1的KFG结构域调节trkB.T1功能对BDNF的响应的假设，以及 KFG结构域的突变消除了损伤后的神经病理性疼痛。 我们的研究将首次将星形细胞trkB.T1介导的[Ca~(2+)]i/NOX2信号与病理生理联系起来 关于SCI的。我们的数据应该建立了与trkB.T1上的细胞内KFG结构域结合的第二个信使 一种重要的生理机制，调节trkB.T1介导的疼痛信号。这些观察结果 可能导致在广泛的疾病状态下治疗神经病理性疼痛的新靶点。

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.