TRAUMATIC PAINFUL NEUROPATHY AND CALCIUM SIGNALING

神经损伤导致手术后的慢性疼痛，如截肢术、开胸术和乳房切除术，以及癌症、退行性疾病和代谢性疾病。 疼痛通常是强烈的，持续的，对目前可用的治疗反应不良。 在理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，但是对细胞膜和离子通道机制的直接检查很少。初级传入神经元的神经元胞体的兴奋性增加是神经损伤后疼痛产生过程的一个组成部分。 细胞内Ca ~（2+）是调节神经活动（包括电发生、突触传递、基因表达、细胞生长和死亡）的主要第二信使，但对神经损伤后细胞膜Ca ~（2+）电流（伊卡）和细胞内Ca ~（2+）水平的研究尚未见报道。 使用来自神经创伤后表现出神经病理性疼痛行为的动物的组织，我们来自全细胞膜片钳实验的新的初步发现揭示了背根神经节（DRG）神经元中具有投射到坐骨神经损伤部位的轴突的膜伊卡减少。 我们还证实，在完整的DRG中，减少伊卡显著提高神经元的兴奋性。该建议的目的是通过确定神经损伤对可能介导痛觉过敏的初级传入神经元中的伊卡和细胞内Ca 2+的影响来检查神经病理性疼痛的细胞机制。 我们将采用神经损伤后疼痛的临床相关模型来表征感觉神经元中改变的钙通道功能，鉴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Ca 2+微荧光法详细检查细胞内Ca 2+的时空调节，并证明了Ca 2+通量降低对解离细胞和完整组织中膜兴奋性的影响。 拟议的研究将测试的假设，在DRG神经元的一个亚组，轴突损伤减少内向钙电流，这反过来又降低细胞内钙浓度直接和通过减少钙诱导的钙释放。细胞内Ca 2+的减少减少了Ca 2+激活的K+电流，从而降低了膜后超极化，并最终产生升高的初级传入兴奋性。以前还没有探讨过作为神经损伤后感觉变化的机制的伊卡减少。 这项转化研究将是有价值的，在确定神经和解剖学上的具体网站的应用程序的代理，以治疗神经性疼痛，同时保留所需的感觉和运动功能。

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.