Cytokine and MAP Kinase Involvment in Neuropathic Pain

描述(申请人提供)：神经损伤后，在背根节(DRG)和脊髓内，促炎细胞因子增加，在那里它们激活丝裂原激活蛋白激酶(MAPK)，包括p38激酶(P38)。P38的激活改变了初级传入纤维和背角细胞中的多种蛋白质，其抑制可以减少神经损伤后的疼痛行为。这项应用建议解剖p38激活的上游和下游的分子通路，以响应脊髓神经结扎，这是一种神经病理性疼痛的模型。目标1将集中在神经损伤引起的p38及其亚型的变化。目标2将着眼于上游监管机构，目标3将着眼于潜在的下游影响因素。我们将系统地研究疼痛行为(机械和热敏性)与细胞因子(TNF和IL-1)、MKK3、MKK6、p38亚型和p38底物激酶MAPKAP-2激活和/或表达的变化。蛋白表达的变化(蛋白印迹)之后，在某些情况下，将通过激酶分析来验证活性，并通过免疫组织化学来确定细胞定位。将分别检查脊髓和背根节，以确定这两个位置之间的级联是否不同。对级联中个别元素的抑制或损失将使我们能够确定封锁“下游”的哪些元素减少了。利用对单个p38亚型的反义、敲除小鼠和受体特异性药理拮抗剂，将发生对单个元件的阻断。检测疼痛行为的平行实验将使我们的发现扩大到过去的协方差，并确定在DRG或脊髓内阻断任何物质是否必要或足够减少疼痛行为。这些模式和机制因果关系的描述将通过为我们提供更多选择性靶点，提高我们从方法上开发合理治疗神经病理性疼痛的能力。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.