Molecular Pathways of Pain Generation in Osteoarthritis

项目摘要 骨关节炎（OA）是关节炎最常见的形式，也是慢性疼痛的主要来源。不是疾病- 患者可获得改良OA药物，并且目前的镇痛方法不能满足患者的需要。 神经生长因子（NGF）已成为OA疼痛的一个有前途的目标。中和抗体可以防止 NGF与其受体原肌球蛋白受体激酶A（TrkA）结合后，在中枢神经系统中显示出强烈的镇痛作用。 OA疼痛的临床试验然而，高达10%的抗神经生长因子治疗的患者迅速进展， OA，需要进行关节置换。神经生长因子阻断对关节的这种有害作用的机制 完整性是未知的，暴露了我们对联合损伤和 OA疼痛在之前的资助周期中，我们开发了检测方法，报告小鼠， 用于监测体内神经元活动以评估疼痛行为的神经解剖学和生物物理学技术 以及OA小鼠模型的潜在神经生物学机制。我们发现了关节损伤， OA伴随着广泛的解剖学和功能性神经元可塑性的伤害性神经支配， 在膝我们将在这一发现的基础上检验中心假设，即NGF-TrkA轴是至关重要的， 通过神经元和非神经元的作用， 机制等我们将（目的1）确定关节中NGF-TrkA“相互作用组”的空间和时间。使用 报告小鼠，背根神经节（DRG）的单细胞RNAseq，RNAscope和免疫组织化学，我们 将详细说明在进行性实验性OA过程中，膝关节和DRG中哪些细胞表达Ngf和Ntrk 1， 神经可塑性和关节病理的时空关系。我们将验证人类膝关节的发现 （目的2）确定NGF-TrkA轴在促进OA关节中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。我们将通过将其注射到 NaV1.8报告小鼠的膝盖，并评估疼痛行为、关节神经支配和关节完整性（组织学和病理学）。 microCT），以及对感觉神经元的功能影响。Advillin-creERT 2小鼠将用于删除Nrtrk 1 来自成年小鼠接受DMM或PMX手术的感觉神经元;疼痛、神经可塑性和关节损伤 （目的3）探索NGF-TrkA轴在OA中通过非神经元途径的作用， 机制等我们将使用Ngf-loxp和Ntrk 1-loxp小鼠从选择的非- 神经元细胞（骨髓细胞、成骨细胞或软骨细胞）。将对小鼠进行手术，并且OA （目的4）评估抗NGF Ab对OA关节的作用。NGF-TrkA的作用 将通过使用中和抗NGF抗体来确定作为器官的整个关节中的轴， 手术后的活动性（0-8周和0-16周）或治疗性（8-16周）。这种方法将澄清 NGF-TrkA轴如何在OA疼痛和关节损伤之间的界面起作用。我们的实验将 我也开始讨论这个轴的疼痛产生和稳态作用是否以及如何被分离， 以确定OA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.