Cytokine and MAP Kinase Involvement in Neuropathic Pain

描述（由申请人提供）：神经损伤后，促炎细胞因子在背根神经节（DRG）和脊髓内增加，在那里它们激活丝裂原活化蛋白激酶（MAPK），包括p38激酶（p38）。p38的激活改变了原代传入纤维和背角细胞中的多种蛋白质，其抑制可减少神经损伤后的疼痛行为。该应用程序旨在剖析p38在脊髓神经结扎反应中的上游和下游分子通路，这是神经性疼痛的一种模型。目的1将集中于神经损伤引起的p38及其亚型的变化。目标2将着眼于上游调控，目标3将着眼于潜在的下游效应。我们将系统地检查疼痛行为（机械和热敏性）与细胞因子（TNF和IL- 1）、MKK3、MKK6、p38亚型和MAPKAP-2 （p38底物激酶）的激活和/或表达变化的协方差。蛋白表达变化（Western blots）之后，在某些情况下，通过激酶测定来验证活性，免疫组织化学来确定细胞定位。将分别检查脊髓和DRG，以确定两个位置之间的级联是否不同。级联中单个元素的抑制或丧失将使我们能够确定阻断的“下游”哪些元素减少。单个元件的阻断将通过对单个p38异构体的反义、敲除小鼠和受体特异性药理拮抗剂来实现。检查疼痛行为的平行实验将允许我们扩展我们的研究结果，超越协方差，并确定在DRG或脊髓内的任何物质的阻断是否必要或充分地减少疼痛行为。通过为我们提供更多选择性靶点，这些模式和机制因果关系的描述将增加我们在方法学上开发合理治疗神经性疼痛的能力。这种方法有可能避免完全抑制p38功能时出现的一些问题。

DESCRIPTION (provided by applicant): Following nerve injury, pro-inflammatory cytokines increase within dorsal root ganglia (DRG) and spinal cord where they activate mitogen-activated protein kinases (MAPK), including p38 kinase (p38). Activation of p38 alters multiple proteins in primary afferent fibers and dorsal horn cells, and its inhibition reduces pain behavior following nerve injury. This application proposes to dissect the molecular pathway upstream and downstream of p38 activation in response to spinal nerve ligation, a model of neuropathic pain. Aim 1 will concentrate on nerve injury-induced changes in p38 and its isoforms. Aim 2 will look at upstream regulators and aim 3 at potential downstream effectors. We will systematically examine co-variance of pain behavior (mechanical and thermal sensitivity) with changes in activation and/or expression of cytokines (TNF and IL- 1), MKK3, MKK6, p38 isoforms and MAPKAP-2, a p38 substrate kinase. Changes in protein expression (Western blots) will be followed, in some instances, by a kinase assays to verify activity, and immunohistochemistry to ascertain cellular localization. Spinal cord and the DRG will be examined separately to determine if the cascade differs between the two locations. Inhibition or loss of individual elements in the cascade will allow us to determine what elements "downstream" of the blockade are reduced. Blockade of individual elements will occur using antisense for individual p38 isoforms, knock out mice and receptor specific pharmacological antagonists. Parallel experiments examining pain behavior will allow us to extend our findings past co-variance and to determine if blockade of any substance, within the DRG or spinal cord, is either necessary or sufficient to reduce pain behavior. Delineation of these patterns and mechanistic cause/effect relationships will increase our ability to methodologically develop rational treatments for neuropathic pain by providing us with more selective targets. This approach has the potential for avoiding some of the problems that occur with complete inhibition of p38 function.