Effects of Age on Ethanol Withdrawal Toxicity: Mechanisms and Therapy

描述（由申请人提供）：酒精中毒是长期酒精滥用的结果，并与认知和精神功能的老化有关。由于酗酒者经历了反复的乙醇戒断（EW），我们建议使用重复的EW来研究衰老是否会通过对线粒体的氧化损伤加剧EW对认知和精神功能的影响。我们还建议调查是否雌激素的损失有助于这个问题。这项工作可能有特殊意义的女性酗酒者，谁同时经历EW和雌激素缺乏症在他们的高龄。我们的初步研究表明，雌激素保护免受EW毒性在年轻和年老的雌性大鼠。在拟议的研究中，我们将使用三个年龄组的卵巢完整的雌性大鼠：5个月大，12个月大，16个月大，当乙醇饮食开始。我们建议使用25天的乙醇饮食周期，然后5天的停药，重复5个周期。这些周期将被定时，使得大鼠将处于发情期（高雌激素水平），此时饮食被移除并且EW体征达到峰值。在目标1中，我们将确定EW对衰老过程中认知和心理功能的影响。将处于发情期的大鼠从间歇性乙醇饮食中撤出，并使用已建立的行为方法测试精神功能。在目标2中，我们将描述年龄和EW之间有害相互作用的线粒体氧化机制。我们将测量氧化标志物（活性氧、脂质过氧化）和随之发生的线粒体功能障碍（线粒体膜电位崩溃和ATP损失）。在目标3中，我们将表征氧化修饰的线粒体酶细胞色素c氧化酶（考克斯），我们假设介导年龄和EW之间的有害相互作用。我们将使用液相色谱-质谱法和串联质谱法定量考克斯和脂质过氧化物修饰的考克斯的羰基化。在目标4中，我们将确定在衰老过程中雌激素缺乏是否有助于EW诱导的线粒体氧化损伤和精神缺陷。将在有或没有雌激素替代的卵巢完整和卵巢切除大鼠中测量相同的精神和氧化标志物。这些研究可能最终有助于更好地理解和策略，女性酗酒者在大脑老化过程中进行EW。

DESCRIPTION (provided by applicant): Alcoholism is a consequence of prolonged ethanol abuse and has been associated with the aging of cognition and psychomotor function. Because alcoholics go through repeated rounds of ethanol withdrawal (EW), we propose using repeated EW to investigate whether aging with exacerbates the effects of EW on cognition and psychomotor function through oxidative insults to mitochondria. We also propose to investigate whether a loss of estrogen contributes to this problem. This work may have particular significance for female alcoholics, who simultaneously undergo EW and estrogen deficiency in their advanced age. Our pilot study has demonstrated that estrogen protects against EW toxicity in both young and old female rats. In the proposed study, we will use three age groups of ovary-intact female rats: 5 months old, 12 months old, and 16 months old, when the ethanol diet begins. We propose to use a diet cycle of 25 days of ethanol followed by 5 days of withdrawal repeated for 5 cycles. These cycles will be timed such that rats will be at the estrus phase (high estrogen levels) when diet is removed and EW signs peak. In Aim 1, we will determine the effects of EW on cognition and psychomotor functions during aging. Rats at the estrus phase will be withdrawn from an intermittent ethanol diet and tested for psychomotor function using established behavioral methods. In Aim 2, we will characterize the mitochondrial oxidative mechanisms underlying the deleterious interaction between age and EW. We will measure oxidative markers (reactive oxygen species, lipid peroxidation) and consequent mitochondrial dysfunction (collapse of mitochondrial membrane potential and ATP loss). In Aim 3, we will characterize oxidatively modified mitochondrial enzyme cytochrome c oxidase (COX) which we hypothesize mediates the deleterious interaction between age and EW. We will quantify carbonylation of COX and lipid-peroxide-modified COX using liquid chromatography-mass spectrometry and tandem mass spectrometry. In Aim 4, we will determine whether estrogen deficiency during aging contributes to EW-induced oxidative damage to mitochondria and psychomotor deficit. The same psychomotor and oxidative markers will be measured in ovary-intact and ovariectomized rats with or without estrogen replacement. These studies may ultimately contribute to a better understanding of and strategy for female alcoholics undergoing EW during brain aging.