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.

项目摘要 我们的多学科团队汇集了基础和翻译研究人员，他们在联合生物学和 神经科学，提出了一种绘制小鼠和人膝关节感觉神经分布的整体方法 关节。我们将使用最先进的成像技术，结合转录切割技术来构建 膝关节的感觉神经支配组成了一个细胞图谱，在该图谱中，膝关节的传入被转录成轮廓 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。我们最重要的是 目的是准确地描述膝关节的感觉神经支配和发生的动态变化 衰老、关节损伤和骨关节炎(OA)。这将为该财团提供丰富的解剖和分子 资源，以研究潜在的关节疼痛的机制，并指导开发新的止痛策略。 目的1.记录健康和患病小鼠膝关节的感觉神经分布：解剖学和分子生物学 透视。使用荧光报告鼠标记伤害性感受器、C纤维亚群和本体感受器，我们将 绘制(A)不同年龄幼鼠的膝关节解剖神经图；(B)关节损伤后；(C)在 手术诱发的骨性关节炎。我们将使用带状扫描共聚焦和透明光片显微镜来 建立关节神经支配的高分辨率三维解剖模型。我们将对支配膝盖的传入神经进行反向标记 并使用空间转录组学来描述它们与其他非膝部神经支配相比的分子表型 背根神经节神经元。目的2.记录健康人和患病人膝关节的感觉神经支配：解剖学 和分子视角。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)70岁以上捐赠者的膝盖(n=15/性别)，我们预计有80%-90%的人表现为骨性关节炎 病理学。将以标准化的方式收集膝关节组织，包括滑膜、骨软骨塞 (胫骨内侧平台)、半月板、前交叉韧带、脂肪垫和股四头肌。在每个组织中，我们将执行(1) 组织病理学；(2)感觉神经支配的IHC；(3)Bulk和ScRNAseq；(4)空间转录。匹配的 DRGs将用于批量RNAseq，以识别提供的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.确定膝关节滑膜中导致疾病的介质- 相关的神经可塑性。(1)重建滑膜细胞与背根节细胞间的相互作用 应用匹配滑膜和背根节的单链RNA序列研究衰老、关节损伤和骨关节炎小鼠模型中的神经元 样本。(2)我们将TKR时膝关节疼痛的患者报告与匹配的滑膜组织学进行比较。 包括衬里增生程度、单细胞转录改变和神经支配。总体而言，这个项目 将为社区提供全面的神经关节环境数据库，可以 挖掘以(1)进行机制研究以抑制病理性神经可塑性和(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、不会上瘾的和 治疗关节疼痛的安全止痛疗法。