Mitochondrial structure and function in cerebral arteries during diabetes and ischemic stress

2型糖尿病(T2D)引起的小脑血管不良改变导致认知障碍， 记忆障碍和痴呆，并加重脑血管意外造成的脑损伤。其作用机制 目前还不完全清楚，但内皮细胞线粒体的有害变化似乎起着关键作用，启动 角色。我们已经产生了试点数据并开发了新的模型来研究大脑微循环 T2D和笔划。根据我们实验室的发现，我们的研究在概念上是创新的：(1)主要 正常情况下线粒体丰度的性别差异，(2)对线粒体的优先效应 T2D中微血管与动脉的比较，(3)有丝分裂破坏性和保护性的差异表达 男性和女性血管中的蛋白质，(4)脑中线粒体的性别依赖性反应 中风后的血管系统，以及(5)远端血管线粒体特征的主要变化 死于脑损伤。我们的研究是基于研究大脑的新方法而进行的技术创新 小鼠的微循环。首先，我们开发了一种用基因标记的小鼠模型 线粒体仅存在于内皮细胞中，带有Dendra2绿/红色光开关荧光蛋白。 线粒体密度、内皮细胞位置、血管直径和数量(罗丹明红) 可以在多个大脑区域的相同位置同时测量大脑微循环，最多 12个月后在多光子显微镜下麻醉小鼠进行每次测定。第二，我们有 开发了一种高通量方法，首次允许测定线粒体 新鲜采集的小鼠脑微血管制剂的呼吸作用。我们将延长这一期限 方法比较OXPHOS和糖酵解或使用替代物在同一样本中产生ATP的情况 由线粒体提供燃料。第三，我们将使用RNAseq和蛋白质组学来阐明潜在的机制 在老化和T2D期间观察到的变化。这些方法提供了有关信令的新信息 小路。我们还将检验线粒体导向疗法在限制损伤方面的有效性。 和/或改善T2D和卒中患者的微循环恢复。我们的总体假设是 内皮细胞中的线粒体代表了性别和疾病特异性治疗的新靶点。我们有两个 目标。目的1：研究雄性和雌性小鼠线粒体动力学和血管构筑 在基线条件下和在T2D的开发期间。我们将：a)确定线粒体和 使用体内多光子成像技术研究低脂或高脂饮食小鼠的血管特征，b)调查 T2D进展过程中微血管线粒体和血管的变化，c)阐明 T2D期间影响线粒体和血管动力学的机制，以及d)探索治疗方式。 目的2：研究男性和女性糖尿病患者线粒体动力学和血管构筑 小鼠短暂性脑缺血。我们将：a)在体内确定线粒体和血管的变化 糖尿病小鼠短暂性大脑中动脉闭塞(TMCAO)缺血后的体外实验研究 应激，b)阐明线粒体和血管动力学变化的机制，c)探索 改善T2D小鼠缺血后线粒体和血管功能的治疗方法。

Adverse changes in small cerebral blood vessels due to type 2 diabetes (T2D) lead to cognitive impairment, memory deficits and dementias, and potentiate brain injury due to cerebrovascular accidents. The mechanisms are not fully known but detrimental changes in mitochondrial in endothelium appear to play a pivotal, initiating role. We have generated pilot data and developed new models to study the cerebral microcirculation during T2D and strokes. Our studies are conceptually innovative based on discoveries by our laboratory: (1) major sex-differences in mitochondrial abundance under normal conditions, (2) preferential effects on mitochondria in microvessels compared with arteries in T2D, (3) differential expression of mitodestructive and mitoprotective proteins in male and female blood vessels, (4) sex-dependent responses of mitochondria in the cerebral vasculature following strokes, and (5) major changes in vascular mitochondrial characteristics at sites distant from brain injury. Our studies are technically innovative based on new approaches to study the cerebral microcirculation of the mouse. First, we have developed a mouse model that genetically labels mitochondria only in endothelium with Dendra2 green/red photoswitchable fluorescent protein. Mitochondrial density, locations in endothelium, vascular diameters, and numbers (Rhodamine red) in the cerebral microcirculation can be simultaneously measured, at the same sites in multiple brain areas, for up to 12 months with multiphoton microscopy in mice anesthetized for each determination. Second, we have developed a high throughput method, which allows for the first time the determination of mitochondrial respiration in freshly harvested brain microvessel preparations from the mouse. We will extend this method to compare ATP production in the same sample by OXPHOS and glycolysis or the use of alternative fuels by mitochondria. Third, we will use RNAseq and Proteomics to elucidate mechanisms underlying changes observed during aging and T2D. These approaches are providing novel information on signaling pathways. We also will examine effectiveness of mitochondria-directed therapies in limiting damage and/or improving recovery to the microcirculation in T2D and strokes. Our overall hypothesis is that mitochondria in endothelium represent novel targets for sex-specific and disease-specific therapies. We have 2 aims. Aim 1: Characterize mitochondrial dynamics and vascular architecture of male and female mice under baseline conditions and during the development of T2D. We will: a) determine mitochondrial and vascular characteristics using in vivo multiphoton imaging in mice on a low or high fat diet, b) investigate mitochondrial and vascular changes in harvested microvessels during progression of T2D, c) elucidate mechanisms affecting mitochondrial and vascular dynamics during T2D, and d) explore treatment modalities. Aim 2: Investigate mitochondrial dynamics and vasculature architecture of male and female diabetic mice following transient ischemia. We will: a) determine mitochondrial and vascular changes using in vivo and ex vivo approaches in diabetic mice following transient middle cerebral artery occlusion (tMCAO)ischemic stress, b) elucidate mechanisms involved in changes in mitochondrial and vascular dynamics, and c) explore therapeutic approaches to improve mitochondrial and vascular function after ischemia in T2D mice.