Mechanisms of Muscle Afferent Sensitization after Ischemia

描述（由申请人提供）：由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/再灌注发生在镰状细胞病等血液疾病和外周血管疾病等心血管疾病中。缺血/再灌注也被认为是复杂局部疼痛综合征的根本原因。虽然人们对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如何导致疼痛了解甚多，但对III和IV组肌肉传入神经的功能特性及其在肌肉疼痛发展中的作用知之甚少。本提案的主要目的是确定肌肉传入敏感化的分子机制，这可能是缺血和组织再灌注后肌肉疼痛的基础。我们假设这些不同的阶段导致了肌肉传入神经中热、机械和化学敏感性的不同变化，这些变化是通过缺血时嘌呤能受体的上调和再灌注后酸感离子通道的上调介导的，从而导致肌肉疼痛。为了增加我们对肌肉传入神经的认识，我们开发了一种新的离体前爪肌肉、正中和尺神经、背根神经节（DRG）、脊髓记录制备，使我们能够在小鼠中全面地表型这些传入神经。我们还能够分析中枢解剖结构，以及这些传入事件的神经化学或分子表型，使用体外记录与免疫细胞化学和单细胞RT-PCR分析相结合。在Specific Aim 1中，我们将通过体外sirna介导的单个外周神经中嘌呤能受体P2Y1和P2X5基因的敲除，结合体外记录制剂，确定在缺血期间嘌呤能受体P2Y1和P2X5的上调是否分别调节肌肉传入神经中观察到的热和化学敏感性的变化。接下来，在Specific Aim 2中，我们将采用与SA1类似的方法，除了我们将确定ASIC1和ASIC3的上调是否分别调节短暂缺血与组织再灌注后肌肉传入神经中机械和代谢物反应的新变化。最后，在Specific Aim 3中，我们将通过分析受体敲低对缺血期肌肉疼痛行为测试的影响，确定P2Y1和P2X5上调是否调节缺血期肌肉疼痛，而ASIC1和ASIC3上调是否调节再灌注期肌肉疼痛。这项研究将使我们能够描述缺血/再灌注后非伤害性和伤害性肌肉传入神经的变化，并确定肌肉传入神经致敏的独特机制，这是肌肉疼痛发展的基础。这可能会导致制定更合适的治疗方法，以适当的疼痛受体或原发性传入亚群为目标，治疗与缺血/再灌注相关的肌肉骨骼疼痛。

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).