TRAUMATIC PAINFUL NEUROPATHY AND CALCIUM SIGNALING

神经损伤会导致手术后的慢性疼痛，如截肢、开胸和乳房切除术，以及癌症、退行性疾病和代谢性疾病。疼痛通常是剧烈的，持续的，对目前可用的治疗反应不佳。在了解神经病理性疼痛的病理生理学方面已经取得了重大进展，但对细胞膜和离子通道机制的直接检查还很少。初级传入神经元胞体兴奋性的增加是神经损伤后疼痛产生过程的一个组成部分。细胞内钙离子是调节神经活动的主要第二信使，包括电发生、突触传递、基因表达以及细胞的生长和死亡，但有关神经损伤后膜钙电流(ICA)和细胞内钙离子水平的研究尚未见报道。使用神经创伤后表现出神经病理性疼痛行为的动物组织，我们新的全细胞膜片钳实验的初步发现显示，背根神经节(DRG)神经元的膜ICA减少，轴突投射到坐骨神经损伤部位。我们还证实，在完整的背根节中，ICA的减少大大提高了神经元的兴奋性。本研究的目的是通过确定神经损伤对初级传入神经元ICA和细胞内钙离子的影响来研究神经病理性疼痛的细胞机制，这些神经元可能介导痛觉过敏。我们将使用一个临床相关的神经损伤后疼痛模型来表征感觉神经元钙通道功能的改变，确定受损伤影响的通道亚型，用免疫细胞化学描述钙通道表达的变化，用钙离子显微荧光法检测细胞内钙离子调节的空间和时间细节，并展示钙离子通量降低对游离细胞和完整组织膜兴奋性的影响。这些研究将检验这样一个假设，即在DRG神经元的一个亚群中，轴突损伤减少了内向钙电流，进而直接或通过减少钙离子诱导的钙释放来降低细胞内钙离子浓度。细胞内钙离子的减少减少了钙激活的K+电流，从而减少了膜后超极化，最终导致初级传入兴奋性增加。ICA降低作为神经损伤后感觉改变的机制以前还没有被探索过。这项转译研究将有助于确定药理学和解剖学上的特定部位，以便在保留所需感觉和运动功能的同时，应用药物治疗神经病理性疼痛。

Nerve injury results in chronic pain following surgery, such as amputation, thoracotomy and mastectomy, as well as in cancer, degenerative conditions and metabolic diseases. The pain is typically intense, persistent and poorly responsive to currently available therapies. Significant progress has been made in understanding the pathophysiology of neuropathic pain, but there has been minimal direct examination of cell membrane and ion channel mechanisms. Increased excitability of neuronal somata of primary afferent neurons is a component of the pain generating process following nerve injury. Intracellular Ca2+ is the dominant second messenger regulating neural activity including electrogenesis, synaptic transmission, gene expression, and cell growth and death, yet no studies of membrane Ca2+ current (ICa) and intracellular Ca2+ levels following nerve injury have been reported. Using tissue from animals showing neuropathic pain behavior following nerve trauma, our novel preliminary findings from whole-cell patch clamp experiments reveal decreased membrane ICa in dorsal root ganglion (DRG) neurons with axons projecting to a sciatic nerve injury site. We have also confirmed in intact DRGs that decreased ICa substantially elevates neuronal excitability. The aim of this proposal is to examine cellular mechanisms of neuropathic pain by determining the effects of nerve injury on ICa and intracellular Ca2+ in primary afferent neurons that may mediate hyperalgesia. We will employ a clinically relevant model of pain following nerve injury to characterize altered calcium channel function in sensory neurons, identify the channel subtype affected by injury, describe the changes in calcium channel expression with immunocytochemistry, examine intracellular Ca2+ regulation in spatial and temporal detail using Ca2+ microfluorimetry, and demonstrate the effect of decreased Ca2+ flux on membrane excitability in dissociated cells and intact tissue. The proposed studies will test the hypothesis that, in a subgroup of DRG neurons, axonal injury decreases inward Ca2+ current, which in turn decreases intracellular Ca2+ concentration both directly and through diminished Ca2+-induced Ca2+ release. The decrease in intracellular Ca2+ diminishes the Ca 2+-activated K+ current, thereby decreasing membrane afterhyperpolarization and ultimately producing elevated primary afferent excitability. Decreased ICa has not previously been explored as a mechanism of sensory change following nerve injury. This translational research will be valuable in identifying pharmacologically and anatomically specific sites for application of agents to treat neuropathic pain while preserving desired sensory and motor function.