Intervertebral Disc Degeneration and Cross-Talk with the Nervous System

椎间盘退变和与神经系统的交互作用

基本信息

批准号：
10454431
负责人：
Lori A. Setton
金额：
$ 63.77万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10454431
关键词：
Action Potentials Acute Affect Afferent Neurons Algorithms Anatomy Animal Model Animals Behavior Behavior monitoring Behavioral Brain imaging Calcium Cells Chronic Confocal Microscopy Dissociation Distant Exposure to Fluorescence Microscopy Generations Genetic Transcription Goals Harvest Image Impairment In Vitro Infiltration Inflammation Mediators Injury Intervention Intervertebral disc structure Ion Channel Knowledge Low Back Pain Measures Methods Modeling Molecular Molecular Target Monitor Morphology Motion Mus Nerve Nerve Fibers Nervous system structure Neuraxis Neurons Operative Surgical Procedures Organ Organ Culture Techniques Pain Pathology Pattern Phenotype Population Pre-Clinical Model Puncture procedure Rattus Role Site Sodium Channel Blockers Spinal Cord Spinal Ganglia TRPV1 gene Testing Tetrodotoxin Time Tissues Transgenes Visualization Work afferent nerve arm cohort contrast enhanced disability discogenic pain electric field fluorophore in vivo in vivo fluorescence injured intervertebral disk degeneration microCT mouse model neurotransmission neurotrophic factor novel novel strategies predictive test promoter protein expression response second harmonic sensory stimulus spinal disk injury two-photon voltage

项目摘要

Intervertebral disc (IVD) degeneration is one of the greatest contributors to low back pain, yet how the IVD can generate pain remains poorly understood. To date, our knowledge of “cross-talk” between degenerating IVD and sensory nerves involved in transmitting pain is limited to findings of altered protein and RNA expression in tissues of the IVD, the spinal cord or dorsal root ganglia (DRG). Recent advances in the imaging of sensory neuron activation via recording of Ca2+ sensitive fluorescent indicators, together with pre-clinical models of IVD degeneration, now enable the study of temporal and spatial changes in neuronal function and their “cross-talk” to changes in the degenerating IVD. We propose to evaluate action potential-driven Ca2+ transients and molecular changes in sensory neurons in a mouse model of injury-induced IVD degeneration. In Specific Aim 1, we will document temporal changes to pain-related behaviors and sensitivity in Thy1-GCaMP6s mice following puncture of a lumbar IVD to induce IVD degeneration. These mice carry a transgene for the calcium-sensitive fluorophore, GCaMP6s, that is expressed in sensory nerves of the DRG. We will also evaluate the presence of neuronal markers and key ion channels in innervating DRGs, and anatomic changes and nerve fiber infiltration in IVDs, to test for changes with IVD degeneration as compared to sham controls. This work will document molecular changes to IVD and DRGs for this model from 6 to 52 weeks of IVD degeneration, and test for relationships between injured IVD and the innervating lumbar DRGs as a first measure of “cross-talk.” In Specific Aim 2, we will evaluate action potential-driven Ca2+ transients in lumbar DRG neurons in the IVD degeneration model of Specific Aim 1. We will record Ca2+ transients in intact DRG of Thy1-GCaMP6s mice in vitro following electric field stimulation, and measure threshold voltage, 50% maximum (IC50), and numbers of responding DRG neurons and their nearest neighbor response. DRGs will also be tested before and after incubation with sodium channel blockers to screen for remodeling of specific ion channel function with periods of IVD degeneration. Our goal is to identify temporal and spatial changes in DRG function and “cross-talk” with changes of IVD degeneration. Finally, in Specific Aim 3, we will evaluate sensory stimuli-induced responses in the DRG of living mice using in vivo fluorescence microscopy. Working with 2-photon confocal microscopy and motion correction algorithms developed for brain imaging, we will identify the threshold response of DRG neurons in Thy1-GCaMP6s mice with and without IVD degeneration, following in vivo stimulation of brush, pinch, heat and cold. Our goal is to test for relationships between in vivo activation of lumbar DRG neurons with behavioral and sensitivity changes following onset of IVD degeneration. Completion of this study would identify functional changes to sensory neurons at sites distant to the degenerated IVD and reveal new information about IVD- nervous system “cross-talk” that may suggest novel interventions for treatment of discogenic pain.

椎间盘 (IVD) 退变是腰痛的最大诱因之一，但 IVD 如何迄今为止，我们对退化 IVD 与疼痛之间的“串扰”的了解仍然知之甚少。参与传递疼痛的感觉神经仅限于组织中蛋白质和 RNA 表达的发现 IVD、脊髓或背根神经节 (DRG) 感觉神经元成像的最新进展。通过记录 Ca2+ 敏感荧光指示剂以及 IVD 临床前模型进行激活退化，现在可以研究神经功能的时间和空间变化及其“串扰” 退化 IVD 的变化。我们建议评估动作电位驱动的 Ca2+ 瞬变和感觉中的分子变化在特定目标 1 中，我们将记录损伤引起的 IVD 变性小鼠模型中的神经元。 Thy1-GCaMP6s 小鼠在穿刺后疼痛相关行为和敏感性的暂时变化腰椎 IVD 诱导 IVD 变性这些小鼠携带钙敏感荧光团的转基因， GCaMP6，在 DRG 的感觉神经中表达，我们还将评估神经元的存在。神经支配 DRG 中的标记物和关键离子通道，以及 IVD 中的解剖变化和神经纤维浸润，测试与假对照相比 IVD 变性的变化。这项工作将记录分子。在 IVD 退化 6 至 52 周期间对该模型的 IVD 和 DRG 进行更改，并测试关系在受伤的 IVD 和支配腰椎 DRG 之间作为“串扰”的第一个衡量标准。将评估 IVD 变性模型中腰部 DRG 神经元中动作电位驱动的 Ca2+ 瞬变具体目标 1. 我们将在电刺激后体外记录 Thy1-GCaMP6s 小鼠完整 DRG 中的 Ca2+ 瞬变场刺激，并测量阈值电压、最大 50% (IC50) 和响应 DRG 的数量神经元及其最近邻 DRG 的反应也将在与钠一起孵育之前和之后进行测试。通道阻断剂可筛选具有 IVD 变性周期的特定离子通道功能的重塑。目标是识别 DRG 功能的时间和空间变化以及与 IVD 变化的“串扰” 最后，在具体目标 3 中，我们将评估 DRG 中的感觉刺激引起的反应。使用活体荧光显微镜观察活体小鼠，使用 2 光子共聚焦显微镜和运动。为大脑成像开发的校正算法，我们将识别 DRG 神经元的阈值响应在体内刷、捏、热和刺激后，有或没有 IVD 变性的 Thy1-GCaMP6s 小鼠我们的目标是测试腰部 DRG 神经元的体内激活与行为和冷之间的关系。 IVD 变性发生后敏感性发生变化，完成本研究将确定功能。远离退化 IVD 部位的感觉神经元发生变化，揭示了有关 IVD 的新信息神经系统“串扰”可能会建议治疗椎间盘源性疼痛的新干预措施。