Regulation of Microglial Responses to Alcohol and Alcohol-Induced Neurodegenerati

描述（由申请人提供）：众所周知，酒精对大脑发育和功能有影响，但其对小胶质细胞（MG）的影响还不太清楚。在这里，我们将利用在体外和体内小鼠脑组织制备MG激活，运动，迁移到受伤的神经元，和死亡细胞的吞噬清除研究乙醇的影响。我们期望酒精诱导的神经元损伤和凋亡性细胞死亡可以在来自新生小鼠的器官型脑切片培养物中重现，从而建立实验上易于处理的体外组织模型用于研究胶质细胞对酒精诱导的神经变性的反应。我们假设，酒精诱导大脑居民MG激活迅速，神经元细胞死亡之前，和MG激活和招聘受伤的神经元通过fractalkine信号促进神经元的存活和限制继发性神经元损伤。我们将测试这一假设的各个方面，首先，通过评估MG激活的时间（MG形态和基因表达的变化）相对于酒精诱导的神经元凋亡（切割的半胱天冬酶-3标记）和随后的细胞死亡（Sytox/碘化丙啶标记）在小鼠海马或皮质切片培养。为了确定MG激活是否先于、跟随或确实需要凋亡性神经元死亡，我们将在缺乏BAX（一种凋亡调节蛋白）的小鼠切片中检查MG激活。接下来，我们将测试fractalkine信号传导是否调节MG激活和招募到来自fractalkine受体缺失小鼠（CX 3CR 1GFP/GFP）的切片中的酒精损伤的神经元。最后，我们将在活的、酒精处理的GFP报告小鼠（CX 3CR 1GFP/+）中使用经颅多光子成像来检查酒精是否影响MG基础运动性或体内发育或成年皮质脑组织中受损神经元的动员。这些探索性研究将首次直接观察和分析高血酒精和戒断条件下活的完整脑组织中的胶质细胞行为。这些研究的结果将为未来的研究奠定基础，旨在阐明MG激活的神经保护或神经毒性的后果。这些信息将有助于产生新的想法，酒精对正常大脑功能的影响，以及受损的免疫监视和对中枢神经系统损伤的反应。 公共卫生相关性：人类酒精相关的神经元损伤是一个日益严重的健康问题，也是我们社会经济的一个日益沉重的负担。在神经元发育的关键时期，酒精暴露会诱导神经元的损伤或死亡，并且附近的神经胶质细胞（包括小胶质细胞）的反应可能促进受损神经元的存活或加重损伤。这里产生的数据将有助于确定小胶质细胞对酒精和酒精诱导的神经元损伤的反应，从而有助于指导发展中和成年人酒精诱导的脑损伤后的治疗策略。

DESCRIPTION (provided by applicant): Alcohol has well known effects on brain development and function, but its effects specifically on microglial cells (MG) are much less well understood. Here we will utilize in vitro and in vivo mouse brain tissue preparations to study the effects of ethanol on MG activation, motility, migration to injured neurons, and phagocytic clearance of dead cells. We expect that alcohol- induced neuronal injury and apoptotic cell death can be recapitulated in organotypic brain slice cultures derived from neonatal mice, thereby establishing an experimentally tractable in vitro tissue model for studies of glial responses to alcohol-induced neurodegeneration. We hypothesize that alcohol induces brain resident MG to activate rapidly, prior to neuronal cell death, and that MG activation and recruitment to injured neurons via fractalkine signaling promotes neuronal survival and limits secondary neuronal injury. We will test aspects of this hypothesis, first, by assessing the timing of MG activation (changes in MG morphology and gene expression) relative to alcohol-induced initiation of neuronal apoptosis (cleaved caspase-3 labeling) and subsequent cell death (Sytox/Propidium iodide labeling) in mouse hippocampal or cortical slice cultures. To determine whether MG activation precedes, follows, or indeed requires apoptotic neuronal death, we will examine MG activation in slices from mice lacking BAX, an apoptosis regulator protein. Next, we will test whether fractalkine signaling regulates MG activation and recruitment to alcohol-injured neurons in slices from fractalkine receptor null mice (CX3CR1GFP/GFP). Finally, we will use transcranial multiphoton imaging in live, alcohol- treated GFP reporter mice (CX3CR1GFP/+) to examine whether alcohol affects MG basal motility or mobilization to injured neurons in developing or adult cortical brain tissues in vivo. These exploratory studies will yield the first direct observations and analysis of glial cell behaviors in live, intact brain tissues during and following conditions of high blood alcohol and withdrawal. Results from these studies will lay the groundwork for future studies aimed at elucidating the consequences of MG activation for neuroprotection or neurotoxicity. This information will help generate new ideas on the consequences of alcohol for normal brain function as well as for impaired immune surveillance and response to injury in the CNS. PUBLIC HEALTH RELEVANCE: Alcohol-related neuronal injury in humans is an increasing health concern and a growing burden on the economy of our society. Alcohol exposure during critical periods of neuronal development induces damage to or death of neurons, and the response of nearby glial cells, including microglial cells, may promote the survival of injured neurons or exacerbate the injury. The data generated here will help identify responses of microglia to alcohol and alcohol-induced neuronal injury, and thus help guide therapeutic strategies after alcohol-induced brain injury in developing and adult humans.