Intervertebral Disc Degeneration and Cross-Talk with the Nervous System

椎间盘（IVD）退变是下背痛的最大贡献者之一，但IVD如何能够 对疼痛的产生仍然知之甚少。到目前为止，我们对退化的IVD和 参与传递疼痛的感觉神经仅限于组织中蛋白质和RNA表达改变的发现 脊髓或背根神经节（DRG）。感觉神经元影像学研究进展 通过记录Ca 2+敏感荧光指示剂激活，以及IVD临床前模型 退化，现在可以研究神经元功能的时间和空间变化及其“串扰” 退化的IVD的变化。 我们建议评估动作电位驱动的Ca 2+瞬变和感觉神经元的分子变化。 在损伤诱导的IVD变性的小鼠模型中，在具体目标1中，我们将记录 Thy 1-GCaMP 6s小鼠针刺后疼痛相关行为和敏感性的时间变化 腰椎IVD以诱导IVD变性。这些小鼠携带钙敏感荧光团的转基因， GCaMP 6s，其在DRG的感觉神经中表达。我们还将评估是否存在神经元 神经支配DRG的标志物和关键离子通道，以及IVD中的解剖学变化和神经纤维浸润， 检测与假手术对照相比IVD变性的变化。这项工作将记录分子 IVD变性6 - 52周期间该模型的IVD和DRG变化，以及相关性检验 作为“串扰”的第一个测量值。在具体目标2中， 将评价IVD变性模型中腰DRG神经元中动作电位驱动的Ca 2+瞬变， 具体目标1。我们将在体外记录Thy 1-GCaMP 6s小鼠完整DRG中的Ca 2+瞬变， 场刺激，并测量阈值电压，50%最大值（IC 50）和响应DRG的数量 神经元及其最近邻反应。还将在与钠孵育前后检测DRG 通道阻滞剂，以筛选特定离子通道功能随IVD变性时期的重塑。我们 目的是确定DRG功能的时间和空间变化以及与IVD变化的“串扰” 退化最后，在具体目标3中，我们将评估DRG中感觉刺激诱导的反应， 活小鼠使用体内荧光显微镜。使用双光子共聚焦显微镜和运动 校正算法开发的脑成像，我们将确定DRG神经元的阈值反应， Thy 1-GCaMP 6s小鼠伴和不伴IVD变性，在体内刺激刷、捏、热和 冷.我们的目标是测试腰椎背根神经节神经元的体内激活与行为和 IVD变性发作后的敏感性变化。完成这项研究将确定功能 在远离退化IVD的部位感觉神经元的变化，并揭示了关于IVD的新信息- 神经系统的“串扰”，这可能表明新的干预治疗椎间盘源性疼痛。

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.