Molecular Pathways of Pain Generation in Osteoarthritis

项目摘要 骨关节炎是最常见的关节炎形式，也是慢性疼痛的主要来源。没有疾病- 患者可以使用改良的骨性关节炎药物，而目前的止痛方法不能满足患者的需求。 神经生长因子(NGF)已成为治疗骨性关节炎疼痛的有效靶点。中和抗体可以预防 神经生长因子(NGF)与其受体原肌球蛋白受体激酶A(TrkA)结合后显示出较强的镇痛作用。 骨性关节炎疼痛的临床试验。然而，高达10%的接受抗NGF治疗的患者进展迅速。 骨性关节炎，需要关节置换术。神经生长因子阻断对关节有害作用的机制 诚信是未知的，暴露了我们一直缺乏对关节损害和 骨性关节炎的疼痛。在之前的资金周期中，我们开发了分析、报告小鼠和 神经解剖学和生物物理学技术用于监测活体神经元活动以评估疼痛行为 以及在小鼠OA模型中潜在的神经生物学机制。我们已经发现了关节的损坏 骨性关节炎伴随着伤害性神经支配的广泛解剖和功能神经元可塑性 膝盖。我们将在这一发现的基础上检验NGF-TrkA轴对于 神经性和非神经性关节损伤对关节内环境的保护作用 机械装置。我们将(目标1)确定关节中的空间和时间NGF-TrkA“相互作用组”。vbl.使用 报告小鼠，背根神经节单细胞RNAseq，RNAScope，和免疫组织化学，我们 将详细说明在进行性实验性骨性关节炎中，膝关节和背根节中哪些细胞表达NGF和NTRK1 神经可塑性和关节病理学的时空关系。我们将在人类膝盖上验证这些发现 组织；(目的2)确定NGF-TrkA轴在促进骨关节炎关节神经元生长中的作用，以及 这是如何导致疼痛和关节完整性的。我们将评估NGF是否会导致神经可塑性，方法是将其注射到 NAV1.8报告小鼠的膝关节，并评估疼痛行为、关节神经和关节完整性(组织学和 MicroCT)，以及感觉神经元的功能效应。Advilin-creerT2小鼠将被用来删除Nrtrk1 接受DMM或PMX手术的成年小鼠的感觉神经元；疼痛、神经可塑性和关节损伤 将进行长达16周的评估；(目标3)探索NGF-TrkA轴通过非神经元在骨性关节炎中的作用 机械装置。我们将使用NGF-loxP和NTRK1-loxP小鼠有条件地从选择的非 神经细胞(髓系细胞、成骨细胞或软骨细胞)。小鼠将接受手术，而OA 监测至第8周；(目的4)评估抗NGF单抗对OA关节的影响。NGF-TrkA的作用 作为一个器官的整个关节的轴将通过使用中和抗NGF抗体来确定，或者 术后预防性(0-8周和0-16周)或治疗性(8-16周)。这一方法将澄清 NGF-TrkA轴如何在骨性关节炎疼痛和关节损伤之间的交界处工作。我们的实验将 也开始讨论是否以及如何将这一轴的产生疼痛和内环境平衡的行为分离，在 以确定治疗骨性关节炎疼痛的新靶点。

Project Summary Osteoarthritis (OA) is the most common form of arthritis, and a major source of chronic pain. No disease- modifying OA drugs are available to patients, and current analgesic approaches fall short of patients’ needs. Nerve growth factor (NGF) has emerged as a promising target for OA pain. Neutralizing antibodies that prevent NGF from binding to its receptor, tropomyosin receptor kinase A (TrkA), have shown strong analgesic effects in clinical trials for OA pain. However, up to 10% of patients treated with anti-NGF developed rapidly progressive OA, necessitating joint replacement. The mechanism of this deleterious effect of NGF blockade on joint integrity is unknown, exposing our ongoing lack of understanding the relationship between joint damage and pain in OA. During the previous funding cycles, we have developed assays, reporter mice, and neuroanatomical and biophysical techniques for monitoring neuronal activity in vivo to assess pain behaviors and underlying neurobiological mechanisms in mouse models of OA. We have uncovered that joint damage in OA is accompanied by extensive anatomical and functional neuronal plasticity of the nociceptive innervation of the knee. We will build on this discovery to test the central hypothesis that the NGF-TrkA axis is essential for the preservation of joint homeostasis in response to joint injury, through neuronal and non-neuronal mechanisms. We will (Aim 1) Determine the spatial and temporal NGF-TrkA “interactome” in the joint. Using reporter mice, single cell RNAseq of the dorsal root ganglia (DRG), RNAscope, and immunohistochemistry, we will detail which cells in the knee and DRG express Ngf and Ntrk1 during progressive experimental OA, in spatiotemporal relationship to neuronal plasticity and joint pathology. We will validate findings in human knee tissues; (Aim 2) Determine the role of the NGF-TrkA axis in promoting neuronal growth in the OA joint, and how this contributes to pain and joint integrity. We will assess if NGF causes neuroplasticity, by injecting it into knees of NaV1.8 reporter mice and assess pain behaviors, joint innervation, and joint integrity (histology and microCT), as well as functional effects on sensory neurons. Advillin-creERT2 mice will be used to delete Nrtrk1 from sensory neurons in adult mice subjected to DMM or PMX surgery; pain, neuroplasticity, and joint damage will be assessed up to 16 weeks; (Aim 3) Explore the role of the NGF-TrkA axis in OA through non-neuronal mechanisms. We will use Ngf-loxp and Ntrk1-loxp mice to conditionally delete Ngf or Ntrka from select non- neuronal cells (myeloid cells, osteoblasts, or chondrocytes). Mice will be subjected to surgery, and OA monitored up to week 8; (Aim 4) Assess the effect of anti-NGF Abs on the OA joint. The role of the NGF-TrkA axis in the whole joint as an organ will be determined by using neutralizing anti-NGF antibodies, either prophylactically (0-8 and 0-16 weeks) or therapeutically (8-16 weeks) after surgery. This approach will clarify how the NGF-TrkA axis operates at the interface between pain and joint damage in OA. Our experiments will also start to address if and how the pain-producing and homeostatic actions of this axis can be dissociated, in order to identify new targets for OA pain.