Preoptic/Hypothalamic Mechanisms of Sleep-Wake Regulation

 描述(由申请人提供)： 据估计，有3960万人是老年人，其中包括850万美国退伍军人(43%)，他们有患上慢性睡眠障碍的风险。慢性睡眠障碍与许多负面的健康后果有关，包括认知能力下降、摔倒风险增加、焦虑和抑郁障碍，导致生活质量下降。拟议的研究计划将使用多学科方法来检查：a)视前下丘脑(POAH)睡眠调节系统的生理性下降或功能障碍是否导致衰老中的慢性睡眠障碍；b)衰老过程中的慢性POAH炎症和亚硝化应激是否通过对睡眠调节系统的不利影响而导致睡眠障碍；以及c)是否可以通过抗炎措施缓解衰老中的睡眠障碍。具体目标-1：将确定睡眠调节正中视前核(MnPN)和腹外侧视前区(VLPO)神经元的功能活性是否随着年龄的增长而下降。我们将比较年轻大鼠和老年大鼠：a)睡眠活跃的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)；以及b)不同睡眠压力和睡眠时间后VLPO和MnPN GABA能神经元c-Fos的表达(实验2)。具体目标-2：将确定MnPN和VLPO睡眠调节系统是否显示出随着年龄的增长而增加的炎症、亚硝化应激和神经元损伤的证据。我们将对年轻和老年大鼠进行比较：a)用免疫组织化学方法检测MnPN和VLPO GABA能神经元中小胶质细胞的激活和肿瘤坏死因子-α、诱导型一氧化氮合酶、硝基酪氨酸(亚硝化应激的标志)以及衰老、凋亡和神经元损伤的标志物(实验3)；以及b)用定量RT-PCR和Western blot法检测肿瘤坏死因子-α、白介素6、诱导型一氧化氮合酶和硝基酪氨酸的水平，以及用ELISA法和荧光法检测MnPN和VLPO的NO代谢产物(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炎症是否通过不利影响睡眠调节神经系统而导致睡眠障碍。我们将量化：a)局部注射脂多糖诱导的年轻大鼠慢性MnPN和VLPO炎症是否会导致睡眠障碍和GABA能神经元的免疫组织化学变化，这是老年的典型现象(实验5)；以及b)如果通过局部注射米诺环素来抑制MnPN和VLPO内的炎症，是否可以减少GABA能神经元的亚硝化应激，改善老年大鼠的睡眠唤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，将产生关于POAH睡眠调节系统在衰老过程中的功能状态的新颖和基础数据。更好地了解中枢神经系统衰老如何影响睡眠调节系统，以及炎症通过病理方式影响睡眠唤醒功能的机制，可能有助于开发新的预防和治疗方案，以优化包括退伍军人在内的老年人的睡眠健康。

 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.