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/再灌流发生在血液紊乱中，如 镰状细胞病，以及心血管疾病，如外周血管疾病。缺血/再灌流 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 周围损伤后皮肤伤害性感受器的功能特性和可塑性以及这些纤维是如何 导致疼痛，对III和IV组肌肉传入的功能特性知之甚少 以及它们在肌肉疼痛发展中的作用。这项提议的主要目标是确定分子 肌肉传入敏化可能是缺血期间和组织后肌肉疼痛的基础机制 再灌流。我们假设，这些不同的阶段会导致热、机械和热的不同变化 肌肉传入的化疗敏感性，这是由嘌呤能受体上调所介导的。 再灌流后的缺血和酸敏离子通道导致肌肉疼痛。为了增加我们的 在肌肉传入的知识，我们开发了一种新的体外前爪肌肉，正中神经和尺神经，背部 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。我们还能够分析中枢解剖结构，以及神经化学或分子 免疫细胞化学体外记录与单细胞记录相结合研究这些传入细胞的表型 细胞RT-PCR分析。在特定的目标1中，我们将确定嘌呤能受体、P2Y1和 P2X5，分别调节观察到的肌肉传入的热和化学敏感性的变化 利用体内siRNA介导的联合敲除单个周围神经中这些基因的方法进行缺血 用体外记录准备。接下来，在具体目标2中，我们将使用与SA1类似的方法，但 我们将确定ASIC1和ASIC3的上调是否调节机械和 组织短暂性缺血再灌流后肌肉传入的代谢物反应。最后， 在特定的目标3中，我们将确定P2Y1和P2X5的上调是否调节缺血时的肌肉疼痛 而上调ASIC1和ASIC3则通过分析ASIC1和ASIC3对再灌流后肌肉疼痛的影响 这两个阶段之间公认的肌肉疼痛行为测试中的受体敲除。这项研究将 使我们能够描述缺血后非伤害性和伤害性肌肉传入的变化。 再灌流并确定与肌肉传入敏化相关的独特机制 疼痛的发展。这可能会导致制定出更适合肌肉骨骼疼痛的治疗方法。 与以适当痛觉感受器(S)或初级传入为靶点的缺血/再灌注有关 亚群(S)。

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).