Cerebral Microvascular Bioenergetics and Neurovascular Coupling

摘要 脑微血管在神经血管偶联(NVC)中起重要作用。线粒体是能量感受器 细胞的数量。我的工作首次证明了线粒体去极化和激活 一氧化氮合酶(NOS)。最近，我们新发现了神经元型一氧化氮合酶(NNOS)亚型。 唯一能产生活性氧簇(ROS)的内皮细胞。NNOS与NO-1共表达 内皮细胞产生eNOS及其两种异构体参与血管内皮细胞内皮细胞的双向调节 线粒体。糖尿病(DM)会增加脑血管功能障碍和痴呆症的风险。 重要的是，低血糖是糖尿病治疗，特别是胰岛素治疗的危险副作用。患有疾病的患者 糖尿病通常会出现轻微的低血糖，但在确定 心血管发病率和死亡率。我们利用SeaHorse XFe24实现了技术突破 并测定脑微血管线粒体呼吸和细胞生物能量学。我们做了 五次复发性低血糖损害微血管线粒体的新观察 功能。值得注意的是，单发急性轻度或严重低血糖以及一氧化氮合酶活性受损 被发现介导了RH诱导的细胞生物能量学的改变。因此，我们假设温和的RH 通过促进微血管线粒体功能障碍而破坏NVC，导致认知功能受损。我们 进一步假设nNOS诱导的氧化应激增加与eNOS衍生的NO减少相结合 导致RH后线粒体功能障碍。我们建议用链脲佐菌素治疗C57BL/6小鼠 瘦素受体突变的db/db小鼠作为糖尿病模型，未治疗的小鼠作为对照。此外, 我们将使用eNOS基因敲除和可诱导内皮细胞特异性nNOS基因敲除小鼠来研究 一氧化氮合酶亚型在RH诱导的微血管功能障碍中的作用每只动物都要经历五集 (每天一次)轻度(血糖70-80 mg/dl)或重度(血糖40-54 mg/dl)胰岛素诱导 低血糖或生理盐水对照组。目的1是为了证明轻度和重度RH(体内)可以增加 脑内一氧化氮合酶源性ROS的产生和RH诱导的功能性线粒体呼吸障碍 微血管(体外)。目的2在体内建立RH对NVC的影响。我们将确定RH诱导的 通过测量神经元激活后微动脉和毛细血管直径的变化来研究NVC的缺陷 (胡须刺激)在清醒的小鼠中使用双光子激光扫描显微镜。目标3是确定 RH对认知功能影响的新纹理辨别任务和改进的Y迷宫测试。结果是 这一建议将确定轻度RH和脑微血管线粒体之间的机制联系 并挑战现有教条，证明轻度RH与重度RH同样有害 在糖尿病引起的脑血管功能障碍、NV受损和认知功能障碍中起重要作用。

Summary Brain microvessels play an important role in the neurovascular coupling (NVC). Mitochondria are energy sensors of cells. My work for the first time demonstrated the link between mitochondrial depolarization and activation of nitric oxide synthases (NOS). Recently, we have made a novel discovery of a neuronal NOS (nNOS) isoform in endothelial cells that uniquely produce reactive oxygen species (ROS). The nNOS is co-expressed with NO- producing eNOS in endothelial cells and both isoforms are involved in the bidirectional regulation of mitochondria. Diabetes mellitus (DM) increases the risk of cerebrovascular dysfunction and dementia. Importantly, hypoglycemia is a dangerous side effect of DM treatments, particularly insulin-therapy. Patients with DM often experience mild hypoglycemia, but these episodes are unaccounted for in determining the cardiovascular morbidity and mortality. We achieved a technological breakthrough utilizing Seahorse XFe24 analyzer and determined the mitochondrial respiration and cellular bioenergetics of brain microvessels. We made a novel observation that five episodes of recurrent hypoglycemia (RH) impaired the microvascular mitochondrial function. Notably, single episode of acute mild or severe hypoglycemia as well as Impairments of NOS activity was found to mediate RH-induced alterations of cellular bioenergetics. Thus, we hypothesize that mild RH disrupts NVC by promoting microvascular mitochondrial dysfunction leading to impaired cognitive function. We further hypothesize that increased nNOS-induced oxidative stress coupled with reduced eNOS-derived NO contribute to the mitochondrial dysfunction following RH. We propose to use streptozotocin treated C57Bl/6 mice and db/db mice with leptin receptor mutation as models of diabetes with untreated mice as controls. In addition, we will employ eNOS knockout and inducible endothelial cell specific nNOS knockout mice to investigate the role of NOS isoforms in RH-induced microvascular dysfunction. Each animal will be subjected to five episodes (one per day) of mild (blood glucose 70-80 mg/dl) or severe (blood glucose 40-54 mg/dl) insulin-induced hypoglycemia or saline control. Aim 1 is to demonstrate that mild and severe RH (in vivo) can increase the production of NOS-derived ROS and display RH-induced functional mitochondrial respiration deficits in cerebral microvessels (ex vivo). Aim 2 is to establish the impact of RH on NVC in vivo. We will determine the RH-induced deficits in NVC by measuring the changes in arteriolar and capillary diameter in response to neuronal activation (whisker stimulation) in awake mice using two-photon laser scanning microscopy. Aim 3 is to determine the impact of RH on cognitive function using novel texture discrimination task and modified Y-maze test. The results of this proposal would identify the mechanistic link between mild RH and the cerebral microvascular mitochondria dysfunction and challenge the existing dogma to demonstrate that mild RH is equally detrimental as severe RH in contributing to the DM-induced cerebrovascular dysfunction, impaired NV, and cognitive dysfunction.