Role of Opiates in Alcohol-Induced Neurotoxicity

说明(申请人提供)：怀孕期间饮酒是一个严重的公共卫生问题，可能会给孩子带来一系列不利后果。这些胎儿酒精暴露的儿童中，许多表现出较差的应激耐受性，免疫系统功能低下，内分泌系统功能异常。使用大鼠作为动物模型，我们在前一个资助期的工作表明，早期生命中的酒精暴露会对下丘脑中的β-内啡肽神经元产生神经毒性，并导致该神经元对应力轴和免疫系统功能的永久性缺陷。生命早期的乙醇暴露是如何对β-内啡肽神经元产生神经毒性作用的，目前还不完全清楚。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。为了验证这一假说，我们将确定不同过程在细胞外和细胞内产生的高活性氧物种和活性氮物种是否启动和促进乙醇诱导的新生大鼠下丘脑β-内啡肽神经元的凋亡死亡。此外，我们将评估乙醇是否通过激活小胶质细胞来诱导活性氧的产生和炎症反应。此外，我们将研究已知的调节β-内啡肽功能的营养因子是否抑制炎症反应，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。我们将在体内和体外研究中使用大鼠作为动物模型。我们将使用标准的组织学方法通过细胞凋亡或自噬来确定细胞死亡，并使用生化方法来确定导致氧化应激和神经炎症的酶活性。我们还将使用最先进的技术，如结合激光捕获显微切割和定量实时聚合酶链式反应来确定体内β-内啡肽细胞基因表达的变化。拟议的一系列研究应该会继续产生有价值的数据，从而更好地理解乙醇对发育中的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，这可能是防止β-内啡肽神经元死亡的关键。公共卫生相关性使用大鼠作为动物模型，我们在前一个资助期的工作表明，在生命早期接触酒精会对下丘脑中的β-内啡肽神经元产生神经毒性，并导致该神经元对应激轴和免疫系统功能的永久性缺陷。这项建议中提议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了解，并确定防止β-内啡肽神经元死亡的化合物，以便开发药物治疗，以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。

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.