Functional and genetic characterization of human DRG and spinal cord at single cell resolution

单细胞分辨率下人类 DRG 和脊髓的功能和遗传特征

• 批准号: 10707419
• 负责人: Robert W Gereau
• 金额: $ 48.83万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707419
• 关键词: Address; American; Analgesics; Anatomy; Axon; Biological Assay; Catalogs; Cells; Central Nervous System; Data; Data Set; Development; Diagnosis; Electrophysiology (science); Gene Expression Profile; Genetic; Genetic Transcription; Genetic study; Goals; Helping to End Addiction Long-term; Human; Knowledge; Location; Maintenance; Maps; Membrane; Molecular; Morphology; Nervous System; Neurons; Opioid; Pain; Pain management; Patients; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Population; Positioning Attribute; Posterior Horn Cells; Preparation; Property; Protocols documentation; Recording of previous events; Research; Resolution; Resources; Risk; Slice; Spinal; Spinal Cord; Spinal Ganglia; Structure; Synapses; Tissues; United States National Institutes of Health; Vertebral column; Work; addiction; aspirate; cell type; chronic pain; chronic pain patient; chronic painful condition; evidence base; novel; opioid epidemic; opioid misuse; opioid mortality; opioid use; opioid use disorder; patch clamp; patch sequencing; reconstruction; single-cell RNA sequencing; substance misuse; substance use; tool; transcriptome sequencing; transcriptomics; transmission process

Specific Aims: Tens of millions of Americans suffer from chronic pain. Unfortunately for these patients, there is very little we can offer in the way of treatments. Opioids represent the main tool for treating pain, but their use in chronic pain conditions suffers from a poor evidence base and the inherent risk of addiction. The current crisis of opioid-related deaths highlights the risk associated with widespread opioid use. The parallel crises of chronic pain and opioid-related deaths has led the NIH to launch the HEAL Initiative, with one of the major goals being the identification of novel analgesics for the treatment of pain, with a focus on drugs that are non-addicting. The PRECISION Human Pain Network seeks to provide foundational data on the diversity among cell types that comprise the pain neuraxis. Goals for our proposed PRECISION Human Pain Network U19 center include the development of optimized experimental protocols and functional assays utilizing primary human cells and tissue (including dorsal root ganglion neurons and spinal cord slices), and the elucidation of comprehensive molecular/cellular phenotypes that underlie human pain transduction, transmission, and processing under different pain conditions. This project seeks to increase our understanding of the physiological properties of human neurons in the pain neuraxis. The three aims here will begin to catalog the functional profiles and transcriptinoal signatures of DRG and spinal neurons from donors with and without a history of pain or opioid use. We will continue our work to optimize protocols for cutting edge functional and genetic studies in human nervous system tissue. We will work with other centers to optimize protocols to assess physiological, transcriptional, and morphological features of human DRG and spinal cord neurons at cellular resolution, and how these properties are impacted in tissues recovered from patients with a history of pain. Because our donor population also includes patients with substance use and misuse, and in some cases this overlaps with chronic pain conditions, we will also characterize similar properties of DRG and spinal cord neurons in patients with and without a recent history of opioid use. These studies will provide foundational knowledge of human neurons in the pain neuraxis by mapping electrophysiological features of neurons in the human DRG and spinal cord onto genetically-defined subpopulations of human DRG or morphological classes of spinal neurons. The study will also profile tissues derived from donors with and without a history of chronic pain or opioid use/misuse, providing opportunity for new understanding of the impact of pain and substance use on these neurons in the human pain neuraxis. Another major deliverable from the present study will be the development of optimized protocols for key aspects of the proposed analyses, including protocols for RNA sequencing from patch clamped neurons (Patch-seq) and for the preparation, maintenance, and recording form human spinal cord slices.

具体目的：数以千计的美国人遭受慢性疼痛。不幸的是对于这些患者， 我们几乎无法提供治疗方式。阿片类药物代表治疗疼痛的主要工具，但它们使用 在慢性疼痛状态下，证据基础差和成瘾的固有风险。当前的危机 阿片类药物相关的死亡人数突出了与广泛使用阿片类药物相关的风险。慢性的平行危机 疼痛和与阿片类药物相关的死亡导致NIH发起了Heal Initiative，主要目标之一是 新型镇痛药治疗疼痛的鉴定，重点是不依赖的药物。这 精密人类痛苦网络旨在提供有关细胞类型多样性的基本数据 包括疼痛神经。我们提出的精确的人类痛苦网络U19中心的目标包括 使用原代人细胞和组织开发优化的实验方案和功能测定 （包括背根神经神经元和脊髓切片），并阐明综合 人类疼痛转导，传播和加工的分子/细胞表型 不同的疼痛条件。该项目旨在提高我们对 疼痛神经中的人类神经元。这里的三个目标将开始对功能配置文件进行分类和 有或没有疼痛史或阿片类药物病史 使用。我们将继续我们的工作，以优化人类最前沿功能和遗传研究的方案 神经系统组织。我们将与其他中心合作，以优化协议以评估生理， 人DRG和脊髓神经元在细胞分辨率下的转录和形态特征，以及 这些特性如何影响从患有疼痛病史的患者中回收的组织。因为我们的捐助者 人口还包括使用和滥用药物的患者，在某些情况下，这与慢性重叠 疼痛条件，我们还将表征DRG和脊髓神经元在患者和 没有最近使用阿片类药物的历史。这些研究将为人类神经元的基础知识提供 通过绘制人DRG和脊髓中神经元的电生理特征来疼痛神经性 人类DRG或脊柱神经元形态学类别的遗传定义的亚群。研究将 还介绍了有或没有慢性疼痛或阿片类药物使用/滥用病史的供体的组织 新理解疼痛和药物对这些神经元在人类疼痛中的影响的机会 神经。本研究的另一个主要可交付是开发优化协议 提出的分析的关键方面，包括贴片夹神经元的RNA测序方案 （Patch-seq）以及用于制备，维护和记录的人脊髓切片。