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，与认知和精神运动功能的老化有关。由于酗酒者会经历反复的乙醇戒断（EW），我们建议使用反复的乙醇戒断来研究衰老是否会通过线粒体的氧化损伤加剧乙醇戒断对认知和精神运动功能的影响。我们还建议调查雌激素的减少是否会导致这个问题。这项工作可能对女性酗酒者有特别的意义，她们在老年时同时经历EW和雌激素缺乏。我们的初步研究表明，雌激素对年轻和老年雌性大鼠的EW毒性都有保护作用。在这项研究中，我们将使用三个年龄组的卵巢完好的雌性大鼠：5个月大，12个月大，16个月大，当乙醇饮食开始时。我们建议采用一个饮食周期，即25天的乙醇，然后5天的停药，重复5个周期。这些周期将被定时，使大鼠处于发情期（高雌激素水平），此时停止饮食，EW体征达到峰值。在Aim 1中，我们将确定EW对衰老过程中认知和精神运动功能的影响。在发情期的大鼠将从间歇性乙醇饮食中退出，并使用既定的行为方法测试精神运动功能。在目的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。我们将测量氧化标记（活性氧，脂质过氧化）和随之而来的线粒体功能障碍（线粒体膜电位崩溃和ATP损失）。在Aim 3中，我们将表征氧化修饰的线粒体酶细胞色素c氧化酶（COX），我们假设它介导年龄和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COX和脂质过氧化修饰COX的羰基化。在Aim 4中，我们将确定衰老过程中雌激素缺乏是否会导致ew诱导的线粒体氧化损伤和精神运动缺陷。同样的精神运动和氧化标志物将在卵巢完整和卵巢切除的大鼠中测量，无论是否使用雌激素替代。这些研究可能最终有助于更好地理解女性酗酒者在脑衰老过程中接受EW治疗的策略。

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 Rats at the estrus phase will be withdrawn from an intermittent ethanol diet and tested for psychomotor function using established behavioral methods.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.