Molecular Mechanisms of Pain in Chronic Pancreatitis

描述(申请人提供)：疼痛，慢性胰腺炎的主要特征，是一个困难的，通常是棘手的临床问题，发病机制尚不确定。在人类慢性胰腺炎的躯体疼痛模型和描述性研究中的实验数据表明，神经生长因子在这种情况下的疼痛发病机制中发挥了作用。我们已经建立了一种新的经过验证的慢性胰腺炎大鼠模型，无论是在病理上还是在胰腺中NGF的表达上都与人类相似。它与伤害性敏感化有关，如体内对胰腺刺激的过敏性痛觉行为反应以及牵涉性(躯体)痛觉异常所证明的。它还伴随着胰腺特异性伤害性神经元兴奋性的显著变化，以及由它们表达和释放的神经肽递质的增加。因此，我们的模型非常适合用机械学的方法来理解慢性胰腺炎疼痛的发病机制。我们假设这涉及电压门控钠、钾通道和TRPV1受体的改变，以及神经肽表达/释放的增加，这种变化是由慢性炎症胰腺中过度和异位神经生长因子的表达所介导的。在这方面，我们提出了以下具体目标：(1)确定慢性胰腺炎胰腺特异性初级伤害性神经元兴奋性增加的离子和分子基础；(2)确定慢性胰腺炎对肽类神经递质表达和释放的影响及其在维持伤害性敏感化中的作用；(3)确定NGF在慢性胰腺炎疼痛行为和胰腺特异性感觉神经元反应中的作用。我们将使用各种行为、电生理、细胞和分子技术来实现这些目标。胰腺特异性伤害性感受器将通过逆行标记来识别，NAV、Kv和TRPV1通道电流的变化将通过膜片钳来检测。激光捕获显微切割将收集这些神经元，分析与观察到的电流变化相对应的特定离子通道基因的mRNA表达，蛋白质表达将通过Western blotting和免疫染色得到确认。类似地，将使用蛋白质和RNA分析来检测神经生长因子依赖神经肽SP/NKA、CGRP和BDNF的表达。体外技术将被用来研究背根节制剂中刺激诱发的神经递质释放。这些多肽对疼痛行为的贡献将通过鞘内长期给予拮抗剂来检验，包括药理学和分子(反义)方法。最后，使用中和抗体，我们将检查NGF在调节整体疼痛行为中的作用，以及前两个目标中确定的特定元素。这项研究将为慢性胰腺炎的神经生物学提供重要信息，并确定潜在的新治疗靶点。

DESCRIPTION (provided by applicant): Pain, the cardinal feature of chronic pancreatitis, is a difficult, often intractable clinical problem with uncertain pathogenesis. Experimental data in somatic pain models and descriptive studies in human chronic pancreatitis implicate a role for nerve growth factor in the pathogenesis of pain in this condition. We have developed a novel validated rat model of chronic pancreatitis similar to human condition both pathologically and in the expression of NGF in the pancreas. It is associated with nociceptive sensitization, as demonstrated in vivo by hyperalgesic behavioral responses to pancreatic stimulation as well as referred (somatic) allodynia. It is also accompanied by significant changes in the excitability of pancreas-specific nociceptive neurons, as well as by increases in the expression and release of neuropeptide transmitters by them. Our model therefore is eminently suitable for a mechanistic approach to understanding the pathogenesis of pain in chronic pancreatitis. We hypothesize that this involves changes in voltage-gated sodium and potassium channels as well as TRPV1 receptors, along with increased neuropeptide expression/release and that such changes are mediated by excessive and ectopic nerve growth factor expression in the chronically inflamed pancreas. In this regard, we propose the following specific aims for this proposal: (1) to determine the ionic and molecular basis for increased excitability of pancreas-specific primary nociceptive neurons in chronic pancreatitis, (2) to determine the effects of chronic pancreatitis on expression and release of peptide neurotransmitters and their role in maintaining nociceptive sensitization, and (3) to determine the role of NGF in the pathogenesis of pain behavior and pancreas-specific sensory neuronal responses in chronic pancreatitis. We will accomplish these aims using a variety of behavioral, electrophysiological, cellular and molecular techniques. Pancreas-specific nociceptors will be identified by retrograde labeling and changes in Nav, Kv and TRPV1 channel currents will be examined by patch-clamping. Laser capture microdissection will be used to collect these neurons for analysis of mRNA expression of specific ion channel genes corresponding to observed changes in currents and protein expression will be confirmed by Western blotting and immunostaining. Similarly, the expression of NGF-dependent neuropeptides SP/NKA, CGRP and BDNF will be examined using protein and RNA analysis. Ex vivo techniques will be used to study stimulus evoked neurotransmitter release from dorsal root ganglia preparations. The contribution of these peptides to pain behavior will be examined by chronic intrathecal administration of antagonists including pharmacological and molecular (antisense) approaches. Finally, using a neutralizing antibody, we will examine the role of NGF in mediating overall pain behavior as well as specific elements identified in the first two aims. This study will provide important information on the neurobiology of chronic pancreatitis and identify potentially novel therapeutic targets.