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

• 批准号: 10534181
• 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
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-15 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10534181
• 关键词: Affect; Aging; Agonist; Anesthesia procedures; Animal Model; Architecture; Area; Arteries; Blood - brain barrier anatomy; Blood Vessels; Brain; Brain Injuries; Cell physiology; Cerebrovascular Disorders; Cerebrovascular system; Cerebrum; Characteristics; Data; Dementia; Development; Diabetes Mellitus; Diabetic mouse; Diameter; Disease; Distant; 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; Platelet aggregation; 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; cell type; cerebral artery; cerebral microvasculature; cerebrovascular; density; differential expression; effectiveness evaluation; 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

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.

由于2型糖尿病（T2D）引起的小脑血管的不利变化导致认知障碍， 记忆缺陷和痴呆症，以及由于脑血管事故而增加的脑损伤。机制 尚不清楚，但内皮中线粒体的有害变化似乎发挥了关键作用 角色。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 T2D和笔触。我们的研究在概念上是基于我们实验室发现的创新性：（1）主要 线粒体丰度在正常条件下的性别差异，（2）对线粒体的优先影响 与T2D中的动脉相比，（3）有线造影和线性保护的差异表达 雄性和雌性血管中的蛋白质，（4）脑中线粒体的性别依赖性反应 中风后的脉管系统，以及（5）远处的位点血管线粒体特性的重大变化 来自脑损伤。我们的研究在技术上是基于研究大脑的新方法的创新性 小鼠的微循环。首先，我们开发了一种小鼠模型，该模型在遗传上标记 线粒体仅在dendra2绿色/红色照片开关荧光蛋白的内皮中。 线粒体密度，内皮中的位置，血管直径和数量（若丹明红） 可以在多个大脑区域的相同部位同时测量脑微循环 在每种测定中麻醉的小鼠中使用多光子显微镜的12个月。第二，我们有 开发了一种高通量方法，该方法首次允许线粒体的测定 小鼠刚收获的脑微血管制剂中的呼吸。我们将扩展此 通过Oxphos和糖酵解在同一样品中比较ATP产生的方法或使用替代方法 线粒体燃料。第三，我们将使用RNASEQ和蛋白质组学来阐明基础机制 衰老和T2D期间观察到的变化。这些方法正在提供有关信号的新信息 途径。我们还将检查线粒体导向疗法对限制损害的有效性 和/或改善T2D和笔触中微循环的恢复。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疗法的新靶标。我们有2个 目标。目标1：表征男性和雌性小鼠的线粒体动力学和血管结构 在基线条件下以及在T2D的发展过程中。我们将：a）确定线粒体和 在低脂肪饮食或高脂肪饮食中使用体内多光子成像的血管特征，b）研究 T2D进展过程中收获微血管的线粒体和血管变化，c）阐明 影响T2D期间线粒体和血管动力学的机制，d）探索治疗方式。 目标2：研究男性和女性糖尿病的线粒体动力学和脉管体结构 瞬时缺血之后的小鼠。我们将：a）使用体内确定线粒体和血管变化 在短暂的脑部动脉闭塞（TMCAO）缺血后，糖尿病小鼠的离体方法和离体方法 压力，b）阐明与线粒体和血管动力学变化有关的机制，以及c）探索 T2D小鼠缺血后，治疗方法改善线粒体和血管功能。

项目成果

Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels.

• DOI: 10.1177/0271678x21999553
• 发表时间: 2021-09
• 期刊: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism
• 作者: Chandra PK;Cikic S;Baddoo MC;Rutkai I;Guidry JJ;Flemington EK;Katakam PV;Busija DW
• 通讯作者: Busija DW

Effects of aging on protein expression in mice brain microvessels: ROS scavengers, mRNA/protein stability, glycolytic enzymes, mitochondrial complexes, and basement membrane components.

• DOI: 10.1007/s11357-021-00468-1
• 发表时间: 2022-03
• 期刊: GeroScience
• 影响因子: 5.6
• 作者: Chandra PK;Cikic S;Rutkai I;Guidry JJ;Katakam PVG;Mostany R;Busija DW
• 通讯作者: Busija DW

Chronic imaging of mitochondria in the murine cerebral vasculature using in vivo two-photon microscopy.

使用体内双光子显微镜对小鼠脑血管系统中的线粒体进行慢性成像。

• DOI: 10.1152/ajpheart.00751.2019
• 发表时间: 2020
• 期刊: American journal of physiology. Heart and circulatory physiology
• 作者: Rutkai,Ibolya;Evans,WesleyR;Bess,Nikita;Salter-Cid,Tomas;Čikić,Siniša;Chandra,ParthaK;Katakam,PrasadVG;Mostany,Ricardo;Busija,DavidW
• 通讯作者: Busija,DavidW

Detrimental effects of transient cerebral ischemia on middle cerebral artery mitochondria in female rats.

短暂性脑缺血对雌性大鼠大脑中动脉线粒体的有害影响。

• DOI: 10.1152/ajpheart.00346.2022
• 发表时间: 2022
• 期刊: American journal of physiology. Heart and circulatory physiology
• 作者: Rutkai,Ibolya;Merdzo,Ivan;Wunnava,Sanjay;McNulty,Catherine;Chandra,ParthaK;Katakam,PrasadV;Busija,DavidW
• 通讯作者: Busija,DavidW