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

2型糖尿病（T2 D）引起的小脑血管不良变化会导致认知障碍， 记忆缺陷和痴呆，并增强脑血管意外引起的脑损伤。的机制 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， 作用我们已经生成了试点数据，并开发了新的模型来研究脑微循环， T2 D和中风。我们的研究是基于我们实验室的发现而在概念上创新的：（1）主要 正常条件下线粒体丰度的性别差异，（2）对线粒体的优先影响， 与动脉相比，T2 D中的微血管，（3）线粒体破坏性和线粒体保护性的差异表达 男性和女性血管中的蛋白质，（4）大脑中线粒体的性别依赖性反应 中风后的血管，和（5）在血管线粒体特征的主要变化，在网站远离 脑损伤我们的研究在技术上是创新的，基于研究大脑的新方法。 小鼠的微循环。首先，我们开发了一种小鼠模型， Dendra 2绿色/红色可光转换荧光蛋白仅在内皮中存在线粒体。 线粒体密度、内皮细胞位置、血管直径和数量（罗丹明红） 脑微循环可以在多个脑区的相同部位同时测量， 在每次测定时麻醉的小鼠中使用多光子显微镜观察12个月。二是 开发了一种高通量的方法，这使得第一次确定线粒体 呼吸在新鲜收获的脑微血管制剂从小鼠。我们会延长这个 通过OXPHOS和糖酵解或使用替代方法比较相同样品中ATP产生的方法 由线粒体提供能量第三，我们将使用RNAseq和蛋白质组学来阐明潜在的机制， 老化和T2 D期间观察到的变化。这些方法提供了新的信息， 途径。我们还将研究在限制损伤方面， 和/或改善T2 D和中风中微循环的恢复。我们的总体假设是 内皮中的线粒体代表了性别特异性和疾病特异性治疗的新靶点。We have 2 目标。目的1：表征雄性和雌性小鼠的线粒体动力学和血管结构 在基线条件下和T2 D发展期间。我们将：a）确定线粒体和 在低或高脂肪饮食的小鼠中使用体内多光子成像的血管特征，B）研究 在T2 D进展过程中收获的微血管中的线粒体和血管变化，c）阐明 在T2 D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）使用体内方法确定线粒体和血管的变化 短暂性大脑中动脉闭塞（tMCAO）缺血后糖尿病小鼠的体外方法 应激，B）阐明涉及线粒体和血管动力学变化机制，和c）探索 在T2 D小鼠中改善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.