Immune Regualtion of Neuronal Injury and Repair

描述(由申请人提供)：这是一个基于我们发现外周免疫系统在小鼠面部运动神经元(FMN)修复过程中的有益作用的项目的修订竞争续签申请。在最初的资助阶段，确定了所涉及的免疫细胞和分子成分：面神经损伤后，通过需要外周激活和中枢重新激活的抗原依赖过程，CD4效应器Th2细胞是FMN存活所必需的。脑源性神经营养因子(BDNF)是一种必不可少的分子。重要的是，对切断轴突的小鼠的淋巴结细胞进行的FACS分析表明，周围神经损伤时产生了THI/Th2细胞因子分泌细胞。在免疫缺陷小鼠中，挤压伤引起的面瘫的功能恢复受到损害，但通过免疫重建可以恢复到野生型(WT)。推测CD4效应T细胞在运动神经元修复过程中发挥着不同的作用，其中Tha细胞通过中枢BDNF依赖的过程介导FMN的存活，Thi细胞通过参与病变部位的促炎反应来介导功能恢复。4 AIMS将检验这一假设。目的1：探讨面神经损伤后CD4效应器T细胞定位于面神经运动核的机制。实验将利用表达GFP的CD4T细胞重组的免疫缺陷小鼠，THI/Th2细胞因子核探针的原位杂交组织化学，趋化因子RT-PCR，以及趋化因子中和抗体/基因敲除小鼠。目的2探讨面神经损伤后CD4T细胞介导FMN存活的机制，并利用WT和嵌合BDNF阴性小鼠检测CD4T细胞来源的BDNF在FMN修复中的作用。目的#3确定免疫细胞介导的脑运动神经元(FMN)从轴突切断诱导的细胞死亡中的拯救是否可以推广到脊髓MN。WT、免疫缺陷和重组免疫缺陷小鼠将被用来确定免疫系统对坐骨神经损伤后坐骨神经MN活性的影响。目的#4是确定THI效应细胞是否介导周围神经损伤所致瘫痪的功能恢复。STAT4、T-bet和STAT6缺陷小鼠将被用于选择性重建实验，以确定Thi/Th.2效应细胞在面部或坐骨神经挤压损伤后通过行为测试评估的运动功能恢复中的作用。免疫系统在神经系统疾病(如肌萎缩侧索硬化症，一种致命的MN疾病)和/或创伤(如脊髓损伤)中既有保护作用，也有破坏性作用，但这种相互矛盾的作用的调节性质尚未确定。了解免疫系统如何使受损的神经系统受益，对于制定有效的治疗策略来抵消疾病或损伤的进展，减少残疾具有很大的希望。

DESCRIPTION (provided by applicant): This is a revised competitive renewal application of a project based on our discovery of a beneficial role for the peripheral immune system in mouse facial motoneuron (FMN) reparative processes. In the initial funding period, the immune cellular and molecular components involved were identified: the CD4+ effector Th2 cell is necessary for FMN survival after facial nerve injury, via an antigen-dependent process requiring peripheral activation and central re-activation. & brain-derived neurotrophic factor (BDNF) is an essential molecule. Importantly, FACS analysis of lymph node cells taken from axotomized mice indicates that Thi/Th2 cytokine-secreting cells are generated in response to peripheral nerve injury. Functional recovery from facial paralysis induced by crush injury is impaired in immunodeficient mice, but can be restored to wildtype (WT) with immune reconstitution. It is hypothesized that CD4+ effector T cells play distinct roles in motoneuron reparative processes, with the Tha cell mediating FMN survival through a central BDNF-dependent process and the Thi cell mediating functional recovery by participation in the lesion site pro-inflammatory response. 4 aims will test this hypothesis. Aim #1 is to determine the mechanism underlying CD4+ effector T cell localization within the facial motor nucleus after facial nerve injury. Experiments will utilize immunodeficient mice reconstituted with GFP-expressing CD4+T cells, in situ hybridization histochemistry with Thi/Th2 cytokine riboprobes, chemokine RT- PCR, and chemokine neutralization antibodies/knockout mice. Aim 2 is to determine the mechanism underlying CD4+ T cell-mediated FMN survival after facial nerve injury, and will use WT and chimeric BDNF-negative mice to test the role of CD4+T cell-derived BDNF in FMN rescue. Aim #3 is to determine if immune cell-mediated rescue of cranial motoneurons (FMN) from axotomy-induced cell death can be generalized to a spinal MN. WT, immunodeficient, and reconstituted immunodeficient mice will be used to determine the impact of the immune system on sciatic MN viability after sciatic nerve injury. Aim #4 is to determine if the Thi effector cell mediates functional recovery from peripheral nerve injury-induced paralysis. STAT4, T-bet, and STAT6 deficient mice will be used with selective reconstitution experiments to determine the role of Thi/Th.2 effector cells in recovery of motor function, assessed with behavioral tests, after a facial or sciatic nerve crush injury. The immune system can have both protective and destructive effects in neural disease (such as amyotrophic lateral sclerosis, a fatal MN disease) and/or trauma (such as spinal cord injury), but the regulatory nature of such contradictory actions has yet to be determined. Understanding how the immune system benefits the injured nervous system holds great promise in the development of effective treatment strategies to offset disease or injury progression, and reduce disability.