Mechanisms of Muscle Afferent Sensitization after Ischemia

描述（由申请人提供）：组织缺血再灌注引起的肌肉骨骼疼痛是影响美国数百万人的主要健康问题。外周缺血/再灌注发生在血液疾病如镰状细胞病和心血管疾病如外周血管疾病中。缺血/再灌注也被认为是复杂区域疼痛综合征的根本原因。虽然很多人都知道皮肤伤害感受器的功能特性和可塑性外周损伤后，这些纤维如何有助于疼痛，相对较少的是知道的功能特性的第III组和第IV组肌肉传入和它们在肌肉疼痛的发展中的作用。这项建议的主要目标是确定肌肉传入敏化的分子机制，可能是缺血期间和组织再灌注后肌肉疼痛的基础。我们推测，这些不同的阶段导致热，机械和化学敏感性的肌肉传入，这是由上调嘌呤受体在缺血和酸敏感离子通道再灌注后导致肌肉疼痛介导的差异变化。为了增加我们对肌肉传入的认识，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）、脊髓记录制备物，使我们能够在小鼠中全面地表型这些传入。我们还能够分析中央解剖，和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分析的组合。在具体目标1中，我们将确定是否上调嘌呤受体，P2 Y1和P2 X5，调节观察到的变化，在肌肉传入的热和化学敏感性，分别在缺血期间使用在体内siRNA介导的敲低这些基因在单个外周神经与离体记录准备。接下来，在具体目标2中，我们将利用与SA 1类似的方法，除了我们将确定在短暂缺血和组织再灌注后，ASIC 1和ASIC 3的上调是否分别调节肌肉传入中机械和代谢物反应的新变化。最后，在具体目标3中，我们将通过分析受体敲低对这两个阶段之间公认的肌肉疼痛行为测试的影响，确定P2 Y1和P2 X5的上调是否调节缺血期间的肌肉疼痛，而ASIC 1和ASIC 3的上调是否调节再灌注后的肌肉疼痛。这项研究将使我们能够表征缺血/再灌注后非伤害性和伤害性肌肉传入神经的变化，并确定与肌肉疼痛发展相关的肌肉传入敏感化的独特机制。这可能会导致制定更合适的治疗与缺血/再灌注相关的肌肉骨骼疼痛，目标是适当的疼痛受体或初级传入亚群。

DESCRIPTION (provided by applicant): 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 are 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).