Molecular Pathways of Pain Generation in Osteoarthritis

描述（由申请人提供）：疼痛是骨关节炎（OA）的主要症状，也是美国老年人群活动能力受损的主要原因之一。我们对慢性疼痛的机制缺乏了解，特别是与OA相关的慢性疼痛，这是目前可用治疗方案普遍无效的原因。缓解严重OA疼痛仍然是一个未满足的医疗需求，也是寻求手术干预的主要原因。该提案的长期目标是确定OA疼痛的起源和机制，从而能够识别新的靶点，并开发OA疼痛的新疗法和生物标志物。使用复杂的慢性进行性小鼠膝关节OA模型，结合最先进的方法和有效的选择性抑制剂，该提案旨在系统地解决中心假设：OA关节中的病理性基质周转慢性激活关节伤害感受器并引起神经系统的可塑性变化，导致疼痛的开始和持续。目的1旨在通过对进行性实验性OA过程中支配DRG（外周神经系统）和背角（中枢神经系统）的变化进行纵向分析，确定趋化因子MCP-1和fractalkine在慢性OA疼痛中的时空作用。为了深入了解DRG（外周神经系统）中的OA疼痛调制和向中枢神经系统的过渡，我们的目标是记录在进行性OA过程中以时空方式激活DRG和DH中细胞亚群的分子标志物。此外，我们将测试干扰MCP-1/CCR 2信号传导对疼痛行为和相关通路的影响。目的2旨在评估模式识别受体（PRR）及其配体（损伤相关分子模式，DAMP）对关节伤害感受器激活和OA相关疼痛的贡献。我们将使用新型Nav1.8-tdTomato报告基因小鼠，观察DMM手术后进行性膝关节OA过程中关节伤害感受器上的TLR 2/4和TLR 4表达。我们将使用细胞内Ca 2+动员和MCP-1释放作为结果标志物，评估选定DAMP激活感觉神经元的能力。为了评估DAMP信号传导对该模型中疼痛的贡献，我们将评估新型条件性敲除小鼠中疼痛相关行为，这些小鼠在疼痛感受神经元中缺乏Myd 88或TLR 4。最后，目标3旨在差异靶向OA关节的病理成分，以评估对疼痛和相关通路的影响。我们的目标是通过在疾病进展期间的不同时间进行药理学调节来区别靶向软骨或软骨下骨，并监测与详细关节组织病理学相关的疼痛/疼痛通路结局标志物。这将阐明关节OA疼痛的来源和调节。这些目标将导致在理解疼痛是如何在OA中产生和维持方面取得相当大的进展，并为更有针对性和更安全的治疗方法铺平道路。

DESCRIPTION (provided by applicant): Pain is the major symptom in osteoarthritis (OA) and one of the leading causes of impaired mobility in the elderly population in the US. Our lack of understanding of the mechanisms underlying chronic pain in general, and chronic pain associated with OA in particular, accounts for the general ineffectiveness of currently available treatment options. Relief from severe OA pain remains an unmet medical need and a major reason for seeking surgical intervention. The long-term goal of this proposal is to define origins and mechanisms of pain in OA, thus enabling identification of new targets, and development of new therapies and biomarkers for OA pain. Using a sophisticated chronic progressive murine model of knee OA, in combination with state-of-the-art approaches and potent, selective inhibitors, this proposal aims to systematically address the central hypothesis: pathological matrix turnover in the OA joint chronically activates joint nociceptors and causes plasticity changes in the nervous system, resulting in the initiation and the persistence of pain. Aim 1 seeks to define a temporo-spatial role for the chemokines, MCP-1 and fractalkine, in chronic OA pain through longitudinal analysis of changes in the innervating DRG (peripheral nervous system) and dorsal horn (central nervous system) during the course of progressive experimental OA. In order to gain insight into OA pain modulation in the DRG (peripheral nervous system) and transition to the central nervous system, we aim to document molecular markers of activation of cellular subsets in DRG and DH in a temporo-spatial manner over the course of progressive OA. Further, we will test the effect of interfering with MCP-1/CCR2 signaling on pain behaviors and associated pathways. Aim 2 seeks to evaluate the contribution of Pattern Recognition Receptors (PRR) and their ligands (Damage Associated Molecular Patterns, DAMPs) to joint nociceptor activation and OA-associated pain. We will visualize TLR2/4 and RAGE expression on joint nociceptors in the course of progressive knee OA after DMM surgery, using novel Nav1.8-tdTomato reporter mice. We will evaluate the ability of selected DAMPs to activate sensory neurons, using intracellular Ca2+ mobilization and MCP-1 release as outcome markers. In order to assess the contribution of DAMP signaling to pain in this model, we will evaluate pain-related behaviors in novel conditional knock-out mice that lack Myd88 or TLR4 in pain-sensing neurons. Finally, Aim 3 seeks to differentially target pathological components of the OA joint in order to assess effects on pain and associated pathways. We aim to differentially target cartilage or subchondral bone through pharmacological modulation at different times during progressive disease, and monitor pain/pain pathway outcome markers in association with detailed joint histopathology. This will clarify sources and regulation of OA pain in the joint. These aims will lead to considerable gains in understanding how pain is in generated and maintained in OA, and pave the way for a more targeted and safer therapeutic approach.