Mechanisms of Muscle Afferent Sensitization after Ischemia

描述(申请人提供)：由组织缺血和再灌流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/再灌注可发生在镰状细胞病等血液疾病中，也可发生在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区域疼痛综合征的潜在原因。虽然人们对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如何导致疼痛知之甚少，但对III和IV类肌肉传入的功能特性及其在肌肉疼痛发生中的作用知之甚少。这项建议的主要目的是确定肌肉传入敏化的分子机制，这可能是缺血和组织再灌注后肌肉疼痛的基础。我们假设，这些不同的时相导致肌肉传入的热、机械和化学敏感性的不同变化，这种变化是由缺血期间嘌呤能受体的上调和再灌注后的酸感离子通道导致的，从而导致肌肉疼痛。为了增加我们对肌肉传入的认识，我们开发了一种新型的体外前爪肌、正中神经和尺神经、背根神经节(DRG)和脊髓记录准备，使我们能够在小鼠身上对这些传入进行全面的表型分析。我们还能够使用体外记录结合免疫细胞化学和单细胞RT-PCR分析来分析这些传入细胞的中枢解剖结构和神经化学或分子表型。在特定的目标1中，我们将利用体内siRNA介导的在单个周围神经中下调这些基因的方法，结合体外记录准备，确定嘌呤能受体P2Y1和P2X5的上调是否分别调节了在缺血期间观察到的肌肉传入热和化学敏感性的变化。接下来，在特定的目标2中，我们将使用类似于SA1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和组织再灌注后肌肉传入的机械和代谢物反应的新变化。最后，在特定的目标3中，我们将通过分析受体下调对这两个时相的可识别肌肉疼痛行为测试的影响来确定是否上调P2Y1和P2X5调节缺血时的肌肉疼痛，而上调ASIC1和ASIC3调节再灌注后的肌肉疼痛。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的变化，并确定与肌肉传入敏化有关的独特机制，这些机制是肌肉疼痛发生的基础。这可能导致制定针对适当疼痛感受器(S)或初级传入亚群(S)的与缺血/再灌注相关的肌肉骨骼疼痛的更合适的治疗方案。

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).