Mapping the joint-nerve interactome of the knee

绘制膝关节的关节神经相互作用组图

• 批准号: 10607479
• 负责人: Martin K Lotz
• 金额: $ 663.31万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-23 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607479
• 关键词: 3-Dimensional; Afferent Neurons; Age; Aging; Analgesics; Anatomic Models; Anatomy; Arthralgia; Atlases; Biology; C Fiber; C57BL/6 Mouse; Cell Communication; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Communication; Communities; Data; Data Set; Databases; Degenerative polyarthritis; Development; Disease; Elements; Environment; Event; Exhibits; Fatty acid glycerol esters; Gene Expression Profile; Genes; Genetic Transcription; Health; Histology; Histopathology; Human; Hyperplasia; Imaging Techniques; Joints; Knee; Knee Osteoarthritis; Knee joint; Label; Laboratories; Lead; Ligands; Maps; Medial; Mediator of activation protein; Meniscus structure of joint; Microscopy; Molecular; Morphology; Mus; Nerve; Neuronal Plasticity; Neurons; Neurosciences; Nociceptors; Operative Surgical Procedures; Pain; Parvalbumins; Pathologic; Pathology; Patients; Pattern; Process; Property; Proprioceptor; Reporter; Reporting; Resolution; Resources; Sampling; Scanning; Sensory; Standardization; Synovial Cell; Synovial Membrane; Synovitis; Testing; Therapeutic; Time; Tissues; base; differential expression; holistic approach; human disease; improved; joint injury; knee pain; knee replacement arthroplasty; molecular phenotype; mouse model; multidisciplinary; nerve supply; new therapeutic target; novel; novel therapeutics; osteoarthritis pain; osteochondral tissue; programs; quadriceps muscle; receptor; sex; single-cell RNA sequencing; three-dimensional modeling; transcriptome sequencing; transcriptomics; translational scientist

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.

项目概要 我们的多学科团队汇集了具有联合生物学专业知识的基础和转化研究人员 神经科学，提出了一种整体方法来绘制小鼠和人类膝盖的感觉神经支配 我们将使用最先进的成像技术，结合转录组学来构建关节的 3D 模型。 膝盖的感觉神经支配，组成一个细胞图谱，其中膝盖传入神经的转录特征为 单细胞分辨率，并在转录水平记录神经关节细胞相互作用组。 目的是精确描述膝关节的感觉神经支配，以及膝关节发生的动态变化 衰老、关节损伤和骨关节炎（OA）这将为联盟提供丰富的解剖学和分子学知识。 研究关节疼痛机制并指导新型镇痛策略开发的资源。 目标 1. 记录健康和患病小鼠膝关节的感觉神经支配：解剖学和分子学 使用荧光报告小鼠来标记伤害感受器、C 纤维亚群和本体感受器，我们将 绘制 (a) 不同年龄的幼鼠；(b) 关节损伤后的小鼠膝关节的解剖神经分布图； 我们将使用带状扫描共焦和具有透明功能的光片显微镜来进行手术诱导的 OA。 构建关节神经支配的高分辨率 3D 解剖模型 我们将反向标记膝盖神经支配传入神经。 并使用空间转录组学来描述其与其他非膝神经支配的分子表型 目标 2：记录健康和患病人类膝盖的感觉神经支配：解剖学 我们将使用来自 (1) 健康膝盖的一组独特的尸检膝盖/DRG 样本， 20-40 岁（n=15/性别）；（2）来自 70 岁以上捐赠者的膝盖（n=15/性别），我们预计其中 80-90% 会表现出 OA 将以标准化方式收集膝关节组织，包括滑膜、骨软骨塞。 （内侧胫骨平台）、半月板、ACL、脂肪垫和股四头肌，我们将执行 (1)。 组织病理学；(2) 感觉神经支配的 IHC；(3) 体积和 scRNAseq；(4) 空间转录组学。 DRG 将用于批量 RNAseq，以识别组之间的差异表达基因 (DEG)，从而提供 目标 3. 识别膝关节滑膜中驱动疾病的介质。 (1)我们将重建滑膜细胞和DRG之间的细胞相互作用组。 使用匹配滑膜和 DRG 的 scRNAseq 在小鼠衰老、关节损伤和 OA 模型中的神经元 (2) 我们将 TKR 时 OA 膝关节疼痛的患者报告与匹配的滑膜组织学进行比较， 包括内膜增生的程度、单细胞转录变化和神经支配总体而言，该项目。 将为社区提供神经关节环境的综合数据库，这些数据库可以 开采的目的是（1）进行抑制病理性神经可塑性的机制研究以及（2）识别和测试新的 该策略将为开发新颖的、有针对性的、非成瘾性的药物靶点铺平道路。 用于治疗关节疼痛的安全镇痛疗法。