Mapping the joint-nerve interactome of the knee

项目摘要 我们的多学科团队汇集了具有关节生物学专业知识的基础和翻译研究人员， 神经科学，提出了一个整体的方法来映射小鼠和人类膝盖的感觉神经支配 接头.我们将使用最先进的成像技术，结合转录组学来构建3D模型， 膝关节的感觉神经支配，组成了一个细胞图谱，其中膝关节传入神经在转录水平上进行了分析。 单细胞分辨率，并在转录水平上记录神经-关节细胞相互作用组。我们的总体 目的是精确描述膝关节的感觉神经支配，以及随着膝关节的运动而发生的动态变化。 老化、关节损伤和骨关节炎（OA）。这将为财团提供丰富的解剖学和分子生物学证据， 研究关节疼痛的潜在机制，并指导开发新的镇痛策略。 目标1。记录健康和患病小鼠膝关节的感觉神经支配：解剖学和分子生物学 视角使用荧光报告小鼠标记伤害感受器，C-纤维亚群和本体感受器，我们将 绘制（a）不同年龄的未处理小鼠;（B）关节损伤后;（c） 手术诱发的OA我们将使用带扫描共聚焦和清除启用光片显微镜， 构建关节神经支配的高分辨率三维解剖模型。我们将标记膝盖神经传入 并使用空间转录组学来描述他们的分子表型相比，其他非膝神经支配 DRG神经元。目标二。记录健康和患病人类膝关节的感觉神经支配：解剖学 和分子的观点。我们将使用一组独特的尸检膝关节/DRG样本，来自（1）健康膝关节， 年龄20-40岁（n=15/性别）;（2）来自70岁以上供体的膝关节（n=15/性别），我们预计其中80-90%表现为OA 病理将以标准化方式收集膝关节组织，包括滑膜、骨软骨栓 （内侧胫骨平台）、半月板、ACL、脂肪垫和四头肌。在每个组织中，我们将执行（1） 组织病理学;（2）用于感觉神经支配的IHC;（3）体积和scRNAseq;（4）空间转录组学。匹配 DRG将用于批量RNAseq，以鉴定各组之间的差异表达基因（DEG）， 用于配体-受体分析的信息。目标3。确定膝关节滑膜中驱动疾病的介质- 相关的神经可塑性(1)我们将重建滑膜细胞和DRG之间的细胞相互作用组 使用匹配的滑膜和DRG的scRNAseq的小鼠模型中的衰老、关节损伤和OA神经元 样品(2)我们将比较TKR时OA膝关节疼痛的患者报告与匹配的滑膜组织学， 包括衬里增生、单细胞转录变化和神经支配的程度。总的来说，这个项目 将为社区提供神经关节环境的综合数据库， （1）进行机制研究以抑制病理性神经可塑性，（2）识别和测试新的 可下药的目标这一战略将为开发新颖的、有针对性的、不上瘾的、 用于治疗关节疼痛的安全的镇痛治疗剂。

Project Summary Our multidisciplinary team assembles basic and translational researchers with expertise in joint biology and neuroscience, proposing a holistic approach to mapping the sensory innervation of murine and human knee joints. We will use state-of-the-art imaging techniques, combined with transcriptomics to construct 3D models of the sensory innervation of the knee, compose a cell atlas in which knee afferents are transcriptionally profiled at a single cell resolution, and document the nerve-joint cell interactome at the transcriptional level. Our overarching objective is to precisely describe the sensory innervation of the knee, and the dynamic changes occurring with aging, joint injury, and osteoarthritis (OA). This will provide the Consortium with a rich anatomical and molecular resource to study mechanisms underlying joint pain and guide the development of novel analgesic strategies. Aim 1. Documenting the sensory innervation of the healthy and diseased mouse knee: Anatomical and molecular perspectives. Using fluorescent reporter mice to label nociceptors, C-fiber subsets, and proprioceptors, we will map the anatomical innervation of the mouse knee in (a) naïve mice of different ages; (b) after joint injury; (c) in surgically induced OA. We will use ribbon scanning confocal and clearing-enabled lightsheet microscopy to construct high-resolution 3-D anatomical models of joint innervation. We will backlabel knee-innervating afferents and use spatial transcriptomics to describe their molecular phenotypes compared to other non-knee innervating DRG neurons. Aim 2. Documenting the sensory innervation of the healthy and diseased human knee: Anatomical and molecular perspectives. We will use a unique set of post mortem knee/DRG samples from (1) healthy knees, age 20-40 (n=15/sex); (2) knees from donors over 70 (n=15/sex), in which we anticipate 80-90% to exhibit OA pathology. Knee tissues will be collected in a standardized fashion, including synovium, osteochondral plugs (medial tibial plateau), meniscus, ACL, fat pad, and quadriceps muscle. In each tissue, we will perform (1) histopathology; (2) IHC for sensory innervation; (3) bulk and scRNAseq; (4) spatial transcriptomics. Matched DRGs will be used for bulk RNAseq to identify differentially expressed genes (DEG) between the groups provide information for ligand-receptor analysis. Aim 3. Identifying mediators in the knee synovium that drive disease- associated neuroplasticity. (1) We will reconstruct the cellular interactome between synovial cells and DRG neurons in mouse models of aging, joint injury, and OA using scRNAseq of matched synovium and DRG samples. (2) We will compare patient reports of OA knee pain at the time of TKR to matched synovial histology, including extent of lining hyperplasia, single-cell transcriptional changes, and innervation. Overall, this project will provide the community with comprehensive databases of the neuro-articular environment, which can be mined to (1) undertake mechanistic studies to inhibit pathological neuroplasticity and (2) identify and test new druggable targets. This strategy will pave the way for the development of novel, targeted, non-addictive, and safe analgesic therapeutics for the treatment of joint pain.