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

糖尿病和缺血应激期间脑动脉的线粒体结构和功能

基本信息

批准号：
10337298
负责人：
DAVID W BUSIJA
金额：
$ 64.83万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-15 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10337298
关键词：
Affect Aging Agonist Animal Model Architecture Area Arteries Blood - brain barrier anatomy Blood Vessels Brain Brain Injuries Caliber Cell physiology Cerebrovascular Disorders Cerebrovascular system Cerebrum Characteristics Data Dementia Development Diabetes Mellitus Diabetic mouse Disease Distant Effectiveness Endothelial Cells Endothelium Event Female Functional disorder Glycolysis Harvest Health High Fat Diet Impaired cognition Impairment Insulin Resistance Ischemia Label Laboratories Lead Link Location Maintenance Measures Mediating Memory impairment Metabolic Methods Microcirculation Microvascular Dysfunction Middle Cerebral Artery Occlusion Mitochondria Mitochondrial Proteins Modality Modeling Morphology Mus Nervous System Trauma Non-Insulin-Dependent Diabetes Mellitus Normal Cell Pathology Persons Play Prediabetes syndrome Preparation Production Property Protective Agents Proteins Proteomics Publishing Reactive Oxygen Species Recovery Respiration Rhodamine Rodent Model Role Sampling Sex Differences Signal Pathway Signal Transduction Site Stimulation of Cell Proliferation Stress Stroke Structure Testing Therapeutic Therapeutic Agents Time Vascular Endothelium angiogenesis base cell type cerebral artery cerebral microvasculature cerebrovascular density differential expression improved in vivo innovation male meetings metabolic rate mitochondrial K(ATP) channel mouse model multiphoton imaging multiphoton microscopy neurovascular unit novel novel strategies preservation prevent repaired response sex stroke recovery therapy development transcriptome sequencing

项目摘要

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.

2 型糖尿病 (T2D) 引起的小脑血管的不良变化会导致认知障碍，记忆缺陷和痴呆，并加剧脑血管意外引起的脑损伤。机制尚不完全清楚，但内皮线粒体的有害变化似乎起着关键的、起始的作用角色。我们已经生成了试点数据并开发了新模型来研究大脑微循环 T2D 和中风。我们的研究在概念上具有创新性，基于我们实验室的发现：（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 T2D 中微血管与动脉的比较，(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脉管系统，以及（5）远处血管线粒体特征的重大变化来自脑损伤。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，我们开发了一个可以进行基因标记的小鼠模型线粒体仅存在于内皮细胞中，具有 Dendra2 绿色/红色光可切换荧光蛋白。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部位同时测量大脑微循环，持续时间长达每次测定均需麻醉小鼠 12 个月，使用多光子显微镜。其次，我们有开发了一种高通量方法，首次允许测定线粒体从小鼠新鲜收获的脑微血管制剂中的呼吸。我们将延长此通过 OXPHOS 和糖酵解比较同一样品中 ATP 产量的方法或使用替代方法由线粒体提供燃料。第三，我们将使用 RNAseq 和蛋白质组学来阐明潜在的机制在衰老和 T2D 过程中观察到的变化。这些方法提供了有关信号传导的新颖信息途径。我们还将检查线粒体定向疗法在限制损伤方面的有效性和/或改善 T2D 和中风的微循环恢复。我们的总体假设是内皮细胞中的线粒体代表了性别特异性和疾病特异性治疗的新靶点。我们有 2 目标。目标 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 T2D 发展过程中。我们将： a) 确定线粒体和使用低脂肪或高脂肪饮食小鼠体内多光子成像研究血管特征，b) 调查 T2D 进展过程中收获的微血管中线粒体和血管的变化，c) 阐明影响 T2D 期间线粒体和血管动力学的机制，以及 d) 探索治疗方式。目标 2：研究男性和女性糖尿病患者的线粒体动力学和脉管系统结构短暂性脑缺血后的小鼠。我们将： a) 使用体内测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和离体方法应激，b) 阐明线粒体和血管动力学变化的机制，以及 c) 探索改善 T2D 小鼠缺血后线粒体和血管功能的治疗方法。