HIF-1 Regulation of macrophages in Injured Spinal Cord

描述(申请人提供)：脊髓损伤(SCI)是一种毁灭性的创伤性损伤，目前尚无治愈方法。脊髓损伤可引发以活化的中枢神经系统巨噬细胞为主的强烈炎症反应。数据表明，调节脊髓损伤后巨噬细胞的活性可以限制延迟性神经退变，促进功能恢复。这项建议中的研究将检查病变微环境如何驱动巨噬细胞炎症功能。HIF1是一种普遍存在的转录因子，调节细胞对低氧的反应。然而，在巨噬细胞中，它也被证明对静息代谢和炎症功能都是至关重要的。AIM 1的研究将提供有关HIF1a及其下游效应因子在培养的巨噬细胞中对缺氧和炎症刺激的反应的调节，以及这种调节在巨噬细胞介导的神经突起生长和神经毒性中所起的作用的新数据。使用巨噬细胞特异性缺失HIF1a的基因敲除小鼠将使我们能够从其他炎症信号级联中梳理HIF1a依赖的调节。细胞因子和其他炎症介质的产生将通过实时RT-PCR和Western blotting进行检测。巨噬细胞介导的神经再生和神经毒性我们通过已建立的轴突生长和神经元死亡试验在体外进行评估。同时，AIM 2的研究人员将使用临床相关的挫伤小鼠模型来比较表达WT或KO小胶质细胞/巨噬细胞的小鼠之间的损伤进展和功能恢复。HIF1a在巨噬细胞对脊髓损伤反应中的作用将通过损伤体积、髓鞘储备和巨噬细胞聚集的体视学测量来评估。免疫组织化学和激光捕获显微镜将被用来定义和定量HIF1a、其主要下游靶点和致炎细胞因子在损伤和未损伤的脊髓切片中的细胞特异性表达。渗透性研究将用于确定巨噬细胞表达的HIF1a效应下游基因是否会影响血脊髓屏障的完整性。将进行运动行为测试，以检测两组之间功能恢复的差异。了解HIF1a在脊髓损伤特有的微环境中的复杂和多方面的调节，将使研究人员和临床医生深入了解脊髓损伤后炎症的作用。我们相信，旨在减轻或操纵亚急性和慢性疾病过程的治疗，如缺血再灌注损伤和炎症，将改善脊髓损伤患者的功能结局，从而提高他们的生活质量，并减少与这种疾病相关的巨大社会和经济负担。

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) is a devastating traumatic injury for which there currently is no cure. SCI triggers a robust inflammatory response that is dominated by activated CNS macrophages. Data suggests that modulating macrophage activity after SCI can limit delayed neurodegeneration and promote functional recovery. Studies in this proposal will examine how the lesion microenvironment drives macrophage inflammatory function. HIF1 is a ubiquitous transcription factor regulating the cellular response to hypoxia. Within macrophages however, it has also been shown to be crucial for both resting metabolism and inflammatory function. Studies in Aim 1 will provide novel data on the regulation of HIF1a and its downstream effectors in cultured macrophages in response to hypoxic and inflammatory stimuli, and what role this regulation plays in macrophage-mediated neurite outgrowth and neurotoxicity. Use of knockout mice with macrophage-specific deletion of HIF1a will allow us to tease HIF1a-dependent regulation from other inflammatory signaling cascades. Production of cytokines and other inflammatory mediators will be assayed via real-time RT-PCR and Western blotting. Macrophage-mediated neuroregeneration and neurotoxicity we be assessed in vitro using established neurite outgrowth and neuron death assays. In parallel, studiles in Aim 2 will use a clinically relevant mouse model of contusive SCI to compare lesion progression and functional recovery between mice expressing WT or KO microglia/macrophages. HIF1a's role in the macrophage response to SCI will be assessed through stereological measurements of lesion volume, myelin sparing, and macrophage accumulation. Immunohistochemistry and laser capture microscopy will be used to define and quantify cell-specific expression of HIF1a, its major downstream targets, and proinflammatory cytokines in injured and uninjured spinal cord sections. Permeability studies will be used to determine if macrophage-expressed downstream genes of HIF1a effect affect blood-spinal cord barrier integrity. Locomotor behavior tests will be performed to detect differences in functional recovery between the groups. Understanding the complex and multi-faceted regulation of HIF1a in the unique microenvironments present in the SCI lesion will give researchers and clinicians insight into the roles of inflammation post-SCI. We believe that therapies designed to attenuate or manipulate subacute and chronic disease processes such as ischemia-reperfusion injury and inflammation will improve functional outcome in SCI patients, thus improving their quality of life and reducing the immense social and economic burden associated with this disorder.