Role of Opiates in Alcohol-Induced Neurotoxicity

描述（由申请人提供）：怀孕期间饮酒是一个重大的公共卫生问题，可能会导致广泛的不良后果的儿童。许多这些胎儿酒精暴露的儿童表现出不良的压力耐受性，免疫系统功能不全，内分泌系统功能异常。使用大鼠作为动物模型，我们在上一个资助期间的工作表明，在生命早期暴露于酒精会对下丘脑中的β-内啡肽神经元产生神经毒性，并导致这种神经元控制应激轴和免疫系统功能的永久性缺陷。在生命早期，乙醇暴露如何对β-内啡肽神经元产生神经毒性作用还不完全清楚。我们假设乙醇对β-内啡肽神经元的神经毒性作用是由氧化应激导致小胶质细胞介导的炎症引起的。为了验证这一假设，我们将确定是否高活性氧和活性氮类产生的细胞外和细胞内的各种过程启动和促进乙醇诱导的β-内啡肽神经元的新生大鼠下丘脑的凋亡。此外，我们将评估乙醇是否通过激活小胶质细胞诱导活性氧的产生和炎症反应。此外，我们将研究已知调节β-内啡肽功能的营养因子是否抑制炎症反应，并纠正基本的氧化剂/抗氧化剂失衡，以防止β-内啡肽神经元死亡。我们将使用大鼠作为体内和体外研究的动物模型。我们将采用标准的组织学方法来确定细胞凋亡或自噬导致的细胞死亡，并使用生物化学方法来确定引起氧化应激和神经炎症的酶活性。我们还将采用最先进的技术，如结合激光捕获显微切割和定量实时聚合酶链反应，以确定体内β-内啡肽细胞中基因表达的变化。拟议的一系列研究将继续产生有价值的数据，从而更好地了解乙醇对发育中的β-内啡肽神经元的神经毒性作用。此外，拟议的研究应该确定作用于新靶点的化合物，以抑制过度激活的小胶质细胞释放广泛的促炎因子，这可能对预防β-内啡肽神经元死亡至关重要。 公共卫生相关性使用大鼠作为动物模型，我们在上一个资助期间的工作表明，在生命早期暴露于酒精会对下丘脑中的β-内啡肽神经元产生神经毒性，并导致这种神经元对应激轴和免疫系统功能的控制出现永久性缺陷。本提案中提出的一系列研究旨在提高我们对乙醇对β-内啡肽神经元的神经毒性作用的理解，并确定用于预防β-内啡肽神经元死亡的化合物，以开发用于控制未来胎儿酒精暴露患者的应激耐受性差和免疫系统功能不全的药物治疗。

DESCRIPTION (provided by applicant): Alcohol consumption during pregnancy is a significant public health problem and may result in a wide range of adverse outcomes for the child. Many of these fetal alcohol exposed children show poor stress tolerance, immune system incompetence, and abnormalities in endocrine system functions. Using the rat as an animal model, our work during the previous funding period demonstrated that alcohol exposure during early life produces neurotoxicity of beta-endorphin neurons in the hypothalamus and causes a permanent defect in this neuronal control of stress axis and immune system functions. How ethanol exposure during early life produces a neurotoxic action on beta-endorphin neurons is not completely well understood. We hypothesize that ethanol's neurotoxic action on beta-endorphin neurons is caused by oxidative stress leading to microglia-mediated inflammation. To test this hypothesis we will determine whether highly reactive oxygen species and reactive nitrogen species generated extracellularly and intracellularly by various processes initiate and promote ethanol-induced apoptotic death of beta-endorphin neurons in the hypothalamus of newborn rats. Furthermore, we will evaluate whether ethanol induces reactive oxygen species generation and inflammatory response through activation of microglia. Additionally, we will study whether trophic factors that are known to regulate beta-endorphin functions inhibit the inflammatory response and correct the fundamental oxidant/antioxidant imbalance to prevent beta-endorphin neuronal death. We will use rats as an animal model in both in vivo and in vitro studies. We will employ standard histological methods to determine cell death by apoptosis or autophagy and use biochemical methods to determine the enzyme activities that cause oxidative stress and neuroinflammation. We will also employ state-of-the-art techniques like combined laser capture microdissection and quantitative real-time polymerase chain reaction to determine changes in gene expression in beta-endorphin cells in vivo. The proposed series of studies should continue to generate valuable data leading to better understanding of ethanol's neurotoxic action on developing beta-endorphin neurons. Additionally, the proposed research should identify compounds acting on novel targets to inhibit the release of a wide range of proinflammatory factors from overactivated microglia that might be critical for preventing of beta-endorphin neuronal death. PUBLIC HEALTH RELEVANCE Using the rat as an animal model, our work during the previous funding period demonstrated that alcohol exposure during early life produces neurotoxicity to beta-endorphin neurons in the hypothalamus and causes a permanent defect in this neuronal control of stress axis and immune system functions. The proposed series of studies in this proposal is aimed at improving our understanding of ethanol's neurotoxic action on beta-endorphin neurons as well as identifying compounds for preventing beta-endorphin neuronal death in order to develop pharmacotherapy for controlling poor stress tolerance and immune system incompetence in fetal alcohol exposed patients in the future.