Mechanisms of Muscle Afferent Sensitization after Ischemia

摘要：组织缺血再灌注引起的肌肉骨骼疼痛是一个主要的健康问题， 影响着美国数百万人外周缺血/再灌注发生在血液疾病中， 镰状细胞病和心血管疾病如外周血管疾病。缺血/再灌注 也被认为是复杂区域疼痛综合征的根本原因。虽然我们知道很多关于 外周损伤后皮肤伤害感受器的功能特性和可塑性，以及这些纤维 虽然第三组和第四组肌肉传入纤维的功能特性与疼痛有关，但对它们的功能特性知之甚少。 以及它们在肌肉疼痛发展中的作用。这项提案的主要目标是确定分子 肌肉传入敏化机制可能是缺血期间和组织损伤后肌肉疼痛的基础 再灌注我们假设，这些不同的阶段导致不同的变化，热，机械和 肌肉传入的化学敏感性，这是由嘌呤能受体在 再灌注后的缺血和酸敏感离子通道导致肌肉疼痛。以提高我们 肌肉传入的知识，我们开发了一种新的离体前爪肌肉，正中神经和尺神经，背 根神经节（DRG），脊髓记录准备，使我们能够全面表型这些 小鼠的传入神经我们还能够分析中央解剖结构，神经化学或分子 表型的这些传入使用离体记录与免疫细胞化学和单 细胞RT-PCR分析。在具体目标1中，我们将确定是否上调嘌呤能受体P2 Y1和 P2 X5分别调节肌肉传入中观察到的热和化学敏感性的变化， 使用siRNA介导的这些基因在单个外周神经中的体内敲低联合进行缺血 离体记录制剂。接下来，在具体目标2中，我们将使用与SA 1类似的方法，除了 我们将确定ASIC 1和ASIC 3的上调是否调节了机械和神经系统的新变化， 代谢物的反应，分别在短暂缺血与组织再灌注后肌肉传入。最后， 在具体目标3中，我们将确定在局部缺血期间P2 Y1和P2 X5上调是否调节肌肉疼痛 而ASIC 1和ASIC 3的上调通过分析 在这两个阶段之间的公认的肌肉疼痛行为测试上的受体敲低。本研究将 使我们能够表征缺血后非伤害性和伤害性肌肉传入的变化， 再灌注，并确定与肌肉传入敏化相关的独特机制， 疼痛发展。这可能会导致制定更合适的治疗肌肉骨骼疼痛 与靶向适当疼痛受体或初级传入神经的缺血/再灌注相关 亚群。

ABSTRACT: Musculoskeletal pain resulting from tissue ischemia with reperfusion is a major health issue that affects millions of people in the United States. Peripheral ischemia/ reperfusion occurs in blood disorders like sickle cell disease, and in cardiovascular disorders such as peripheral vascular disease. Ischemia/ reperfusion is also thought to be the underlying cause of complex regional pain syndrome. While much is known about the functional properties and plasticity of cutaneous nociceptors following peripheral injuries and how these fibers contribute to pain, relatively little is known about the functional properties of group III and IV muscle afferents and their role in muscle pain development. The major goal of this proposal is to determine the molecular mechanisms of muscle afferent sensitization that may underlie muscle pain during ischemia and after tissue reperfusion. We hypothesize that these distinct phases cause differential changes in heat, mechanical and chemo-sensitivity in muscle afferents, which are mediated by upregulation of purinergic receptors during ischemia and acid sensing ion channels after reperfusion leading to muscle pain. In order to increase our knowledge of muscle afferents, we developed a novel ex vivo forepaw muscle, median & ulnar nerves, dorsal root ganglion (DRG), spinal cord recording preparation that enables us to comprehensively phenotype these afferents in mouse. We are also able to analyze the central anatomy, and the neurochemical or molecular phenotypes of these afferents using combinations of ex vivo recording with immunocytochemical and single cell RT-PCR analyses. In Specific Aim 1, we will determine if upregulation of purinergic receptors, P2Y1 and P2X5, regulate the observed changes in heat and chemosensitivity in muscle afferents, respectively during ischemia using in vivo siRNA-mediated knockdown of these genes in single peripheral nerves in conjunction with ex vivo recording preparations. Next, in Specific Aim 2, we will utilize a similar approach to SA1 except we will determine if upregulation of ASIC1 and ASIC3 regulate the novel changes in mechanical and metabolite responses in muscle afferents, respectively after transient ischemia with tissue reperfusion. Finally, in Specific Aim 3, we will determine if upregulation of P2Y1 and P2X5 regulate muscle pain during ischemia while upregulation of ASIC1 and ASIC3 regulate muscle pain after reperfusion by analyzing the effects of receptor knockdown on recognized muscle pain behavior tests between these two phases. This study will enable us to characterize the changes in both non-nociceptive and nociceptive muscle afferents after ischemia/ reperfusion and identify unique mechanisms associated with muscle afferent sensitization that underlie muscle pain development. This may lead to the formulation of more appropriate treatments for musculoskeletal pain associated with ischemia/ reperfusion that target the proper pain receptor(s) or primary afferent subpopulation(s).