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，但对其对小胶质细胞（MG）的影响却知之甚少。在这里，我们将利用体外和体内小鼠脑组织制剂来研究乙醇对MG的激活、运动性、向损伤神经元的迁移以及死亡细胞吞噬清除的影响。我们期望酒精诱导的神经元损伤和凋亡细胞死亡可以在新生小鼠的器官型脑切片培养中重现，从而建立一个实验可处理的体外组织模型，用于研究酒精诱导的神经退行性变的胶质细胞反应。我们假设，在神经元细胞死亡之前，酒精诱导脑内MG快速激活，并且MG通过fractalkine信号激活并招募到受损神经元，促进神经元存活并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，在小鼠海马或皮层切片培养中，通过评估酒精诱导的神经元凋亡起始（cleaved caspase-3标记）和随后的细胞死亡（Sytox/丙啶碘标记），MG激活的时间（MG形态和基因表达的变化）。为了确定MG激活是先于、跟随还是确实需要凋亡神经元死亡，我们将在缺乏BAX（一种凋亡调节蛋白）的小鼠切片中检测MG激活。接下来，我们将在fractalkine受体缺失小鼠（CX3CR1GFP/GFP）的切片中测试fractalkine信号是否调节MG的激活和酒精损伤神经元的募集。最后，我们将使用经颅多光子成像技术在活体酒精处理的GFP报告小鼠（CX3CR1GFP/+）中检测酒精是否会影响MG的基础运动或对发育中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活的、完整的脑组织中神经胶质细胞在高血液酒精和戒断期间和之后的行为。这些研究的结果将为未来旨在阐明MG激活对神经保护或神经毒性的影响的研究奠定基础。这一信息将有助于对酒精对正常脑功能的影响，以及对中枢神经系统损伤的免疫监视和反应受损的影响产生新的想法。

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.