HIF-1 Regulation of macrophages in Injured Spinal Cord

描述（由申请人提供）：脊髓损伤（SCI）是一种目前无法治愈的毁灭性创伤性损伤。SCI触发由活化的CNS巨噬细胞主导的强烈炎症反应。数据表明，SCI后调节巨噬细胞活性可以限制迟发性神经变性并促进功能恢复。本研究将探讨病变微环境如何驱动巨噬细胞炎症功能。HIF 1是一种普遍存在的调节细胞对缺氧反应的转录因子。然而，在巨噬细胞内，它也被证明对静息代谢和炎症功能至关重要。目标1中的研究将提供有关HIF 1a及其下游效应物在培养的巨噬细胞中响应缺氧和炎症刺激的调节的新数据，以及这种调节在巨噬细胞介导的神经突生长和神经毒性中起什么作用。使用巨噬细胞特异性缺失HIF 1a的敲除小鼠将使我们能够从其他炎症信号级联中梳理HIF 1a依赖性调节。将通过实时RT-PCR和Western印迹法测定细胞因子和其他炎症介质的产生。巨噬细胞介导的神经再生和神经毒性，我们在体外评估使用建立的轴突生长和神经元死亡测定。同时，目标2中的研究将使用临床相关的挫伤性SCI小鼠模型，比较表达WT或KO小胶质细胞/巨噬细胞的小鼠之间的病变进展和功能恢复。HIF 1a在巨噬细胞对SCI反应中的作用将通过对损伤体积、髓鞘保留和巨噬细胞积聚的体视学测量来评估。免疫组织化学和激光捕获显微镜将用于定义和定量HIF 1a的细胞特异性表达，其主要下游靶点，以及损伤和未损伤脊髓切片中的促炎细胞因子。渗透性研究将用于确定巨噬细胞表达的HIF 1a效应下游基因是否影响血脊髓屏障完整性。将进行运动行为测试，以检测组间功能恢复的差异。了解HIF 1a在SCI病变中独特微环境中的复杂和多方面调节将使研究人员和临床医生深入了解SCI后炎症的作用。我们相信，旨在减轻或控制亚急性和慢性疾病过程（如缺血-再灌注损伤和炎症）的治疗将改善SCI患者的功能结局，从而改善他们的生活质量，减轻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.