Preoptic/Hypothalamic Mechanisms of Sleep-Wake Regulation

 描述（由申请人提供）： 估计有3960万人，包括850万美国退伍军人（43%），是老年人，有患慢性睡眠障碍的风险。慢性睡眠障碍与许多负面健康结果相关，包括认知能力下降，跌倒风险增加，焦虑和抑郁症，导致生活质量低下。该研究计划将采用多学科的方法来研究：a）视前下丘脑（POAH）睡眠调节系统的生理衰退或功能障碍是否有助于老年人的慢性睡眠障碍; B）慢性POAH炎症和亚硝化应激是否有助于老年人的睡眠障碍，通过对睡眠调节系统产生不利影响;和c）是否可以通过抗炎措施减轻衰老中的睡眠障碍。具体目标1：将确定是否睡眠调节正中视前核（MnPN）和腹外侧视前区（VLPO）神经元的功能活动随着年龄的增长而下降。我们将在年轻大鼠与老年大鼠中比较：a）睡眠活性MnPN和VLPO神经元对自发和稳态睡眠线索的放电活动的变化（实验1）;和B）在不同睡眠压力和睡眠量后VLPO和MnPN GABA能神经元中的c-Fos表达（实验2）。具体目标2：将确定MnPN和VLPO睡眠调节系统是否表现出炎症增加，亚硝化应激和神经元损伤的证据。我们将在年轻和老年大鼠中进行比较：a）小胶质细胞活化，以及TNF-α，iNOS，硝基酪氨酸（亚硝化应激的标志物），以及MnPN和VLPO GABA能神经元中的衰老、凋亡和神经元损伤的标志物，通过免疫组织化学（实验-3）;和B）TNF-α、IL-6、iNOS和硝基酪氨酸通过定量RT-PCR和Western印迹以及NO代谢产物通过ELISA和荧光测定法测定MnPN和VLPO中的硝酸盐和亚硝酸盐（硝酸盐和亚硝酸盐）和细胞因子释放（实验-4）。具体目标3：将确定慢性MnPN和VLPO炎症是否通过对睡眠调节神经系统的不利影响而导致睡眠障碍。我们将量化：a）如果在年轻大鼠中由局部LPS输注诱导的慢性MnPN和VLPO炎症产生睡眠障碍和GABA能神经元的免疫组织化学变化，这是老年的典型特征（实验-5）;和B）如果通过局部输注米诺环素（一种小胶质细胞活性抑制剂和过氧亚硝酸根的选择性清除剂）慢性抑制MnPN和VLPO内的炎症，减少GABA能神经元中的亚硝化应激并改善老年大鼠的睡眠-觉醒组织和睡眠连续性（实验-6）。 拟议的研究计划在概念上是创新的，并将产生新的和基本的数据POAH睡眠调节系统在老龄化的功能状态。更好地了解CNS衰老如何影响睡眠调节系统以及炎症病理性影响睡眠-觉醒功能的机制可能有助于开发新的预防和治疗方案，以优化老年人（包括退伍军人）的睡眠健康。

 DESCRIPTION (provided by applicant): An estimated 39.6 million people, including 8.5 million US Veterans (43%), are elderly and at risk for developing chronic sleep disturbance. Chronic sleep disturbance is associated with a number of negative health outcomes including cognitive decline, increased risk of fall, anxiety, and depressive disorders, contributing to the poor quality of life. The proposed research program will use a multi-disciplinary approach to examine: a) if a physiological decline or dysfunction of the preoptic hypothalamic (POAH) sleep- regulatory systems contribute to chronic sleep disturbance in aging; b) if chronic POAH inflammation and nitrosative stress in aging contribute to sleep-disturbance, by adversely affecting sleep-regulatory systems; and c) if sleep disturbance in aging can be mitigated by anti-inflammatory measures. Specific aim-1: will determine if the functional activity of sleep-regulatory median preoptic nucleus (MnPN) and ventrolateral preoptic area (VLPO) neurons decline with aging. We will compare in young vs. old rats: a) changes in discharge activity of sleep-active MnPN and VLPO neurons to spontaneous and homeostatic sleep cues (experiment-1); and b) c-Fos expression in VLPO and MnPN GABAergic neurons after different sleep pressures and sleep amounts (experiment-2). Specific aim-2: will determine if the MnPN and VLPO sleep-regulatory systems exhibit evidence of increased inflammation, nitrosative stress, and neuronal damage with aging. We will compare in young and old rats: a) microglia activation, and expression of TNF-alpha, iNOS, nitrotyrosine (markers of nitrosative stress), and markers of senescence, apoptosis and neuronal damage in the MnPN and VLPO GABAergic neurons, by immunohistochemistry (experiment-3); and b) levels of TNF-alpha, IL-6, iNOS, and nitrotyrosine by quantitative RT-PCR and Western blots as well as NO metabolites (nitrates and nitrites) and cytokine release in the MnPN and VLPO by ELISA and flourometry (experiment-4). Specific aim-3: will determine if chronic MnPN and VLPO inflammation contribute to sleep disturbance, by adversely affecting their sleep-regulatory neuronal systems. We will quantify: a) if chronic MnPN and VLPO inflammation, induced by local LPS infusion, in young rats produces sleep disturbance and immuno- histochemical changes in GABAergic neurons, that are typical of old age (experiment-5); and b) if chronic suppression of inflammation within the MnPN and VLPO, by focal infusion of minocycline, an inhibitor of microglia activity and a selective scavenger of peroxynitrite, reduces nitrosative stress in GABAergic neurons and improves sleep-wake organization and sleep continuity in aged rats (experiment-6). The proposed research program is conceptually innovative and will generate novel and fundamental data about the functional status of the POAH sleep-regulatory systems in aging. A better understanding of how CNS aging affects sleep-regulatory systems and the mechanism by which inflammation pathologically affects sleep-wake function may contribute to the development of novel preventative and therapeutic options for optimizing sleep health in the elderly including Veterans.