In vivo 2-photon imaging of NADH in health and disease

健康和疾病状态下 NADH 的体内 2 光子成像

• 批准号: 8028442
• 负责人: Anna Devor
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-15 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8028442
• 关键词: Address; Alzheimer' s Disease; Animals; Astrocytes; Biological Markers; Biological Testing; Biomedical Engineering; Blood Vessels; Blood capillaries; Blood flow; Brain; Brain Diseases; Calcium; Categories; Cells; Characteristics; Citric Acid Cycle; Clinical; Communication; Computational Science; Coupling; Detection; Development; Disease; Dyes; Electrons; Energy Metabolism; Engineering; Event; Experimental Designs; Fluorescence; Functional Imaging; Future; Glycolysis; Goals; Health; Homeostasis; Human; Hypoxia; Image; Intervention; Investigation; Laser Scanning Microscopy; Life; Measurement; Metabolic; Metabolism; Methods; Microscopic; Mitochondria; Monitor; Mus; NADH; Nervous System Trauma; Neurodegenerative Disorders; Neuroglia; Neurons; Neurosciences; Neurosciences Research; Nicotinamide adenine dinucleotide; Optics; Oxidative Phosphorylation; Oxygen; Parkinson Disease; Pathology; Penetration; Pharmacology; Photons; Physics; Physiological; Property; Protocols documentation; Regulation; Relative (related person); Resolution; Respiratory Chain; Respiratory Process; Rodent Model; Screening procedure; Sensory; Signal Transduction; Source; Specificity; Staging; Stimulus; Stroke; Surface; Techniques; Technology; Testing; Time; Tissues; Vasodilation; blood perfusion; brain cell; brain tissue; calcium indicator; capillary; cell type; cellular imaging; cofactor; fluorophore; hemodynamics; human disease; in vivo; inhibitor/antagonist; meetings; metabolic abnormality assessment; mitochondrial dysfunction; mouse model; neurovascular unit; oxidation; research study; response; tool

DESCRIPTION (provided by applicant): Intravital imaging of cell-specific metabolic activity is of key importance for understanding of a wide range of clinical conditions. Among them is compromised blood perfusion following a stroke and a decrease in efficiency of single-cell respiratory processes that occurs in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. However, no methods are available today for in vivo measurement of single cell metabolism with sufficient sensitivity to resolve fast metabolic events related to ongoing neuronal electrical activity and neuronal responses to stimulation. To meet this challenge, we will adapt an existing technology, 2- photon laser scanning microscopy, for high-resolution microscopic imaging of functional metabolism of single brain cells, taking advantage of intrinsic fluorescence of metabolic cofactor 2-nicotinamide adenine dinucleotide (NADH). This project combines a bioengineering effort with testing of biological hypotheses and brings together an interdisciplinary team of experts in neuroscience, physics, engineering and computational science. From the engineering perspective, we propose to (1) develop and validate 2-photon imaging of NADH with sufficient sensitivity to detect functional changes from single cortical neurons and astrocytes in response to sensory stimulation in living animals, and (2) optimize the optical design and experimental protocol for simultaneous in vivo 2-photon imaging of metabolic, neuronal and vascular activity with high spatial and temporal resolution. Using these technological developments, we will explore new concepts concerning the mechanisms of neuro-vascular-metabolic coupling. Specifically, we will (1) address the relative contribution of oxidative phosphorylation and glycolysis to the transient metabolic response in neurons and astrocytes, (2) test the relationship between astrocytic metabolic response and regulation of blood flow through astrocytic calcium-dependent mechanisms, and (3) establish functional NADH imaging as a biomarker for hypoxia and mitochondrial dysfunction. These goals will be achieved by focusing on temporal signal characteristics, by investigation of simultaneously acquired signals, and by using in vivo pharmacology. The main deliverable of the proposed project - a tool for simultaneous 2-photon imaging of metabolic, neuronal and vascular activity - has a potential to transform the investigation of rodent models of human brain disease by opening an unprecedented opportunity to study the homeostasis and functional interactions among neurons, glia, and capillaries of the living brain. In future, this tripartite imaging approach repeated at different stages of disease would allow establishing a defined set of in vivo imaging biomarkers characterizing the progression of neuro-vascular-metabolic pathology that could be used for objective screening of potential therapies. PUBLIC HEALTH RELEVANCE: We will adapt an existing technology, 2-photon laser scanning microscopy, to visualize functional metabolism of single brain cells in living animals taking advantage of the intrinsic fluorescence of metabolic cofactor 2- nicotinamide adenine dinucleotide (NADH). Furthermore, we will combine NADH imaging with 2-photon measurements of neuronal and vascular activity and will establish NADH as a microscopic imaging biomarker for hypoxia or a decrease in mitochondrial efficiency in a mouse model of human disease.

描述（由申请人提供）：细胞特异性代谢活性的插入式成像对于理解广泛的临床状况至关重要。其中的是中风后血液灌注损害，以及在阿尔茨海默氏病和帕金森氏病等神经退行性疾病中发生的单细胞呼吸过程效率的降低。但是，今天没有任何方法可用于在体内测量单细胞代谢具有足够敏感性以解决与持续的神经元电活动相关的快速代谢事件以及对刺激的神经元反应。为了应对这一挑战，我们将适应现有技术，即2光子激光扫描显微镜，以利用代谢辅助剂2-尼古丁氨基酰胺二氨基二核苷酸（NADH）的固有荧光来利用单个脑细胞功能代谢的高分辨率显微成像。 该项目将生物工程的工作与测试生物学假设结合在一起，并汇集了神经科学，物理，工程和计算科学专家跨学科的团队。从工程的角度来看，我们提议（1）具有足够敏感性的NADH的2光子成像，以检测单个皮质神经元和星形胶质细胞的功能变化，以响应活着的动物的感觉刺激，以及（2）优化同步和vast的光学设计和实验方案，并优化了Meturon和Vaston Imalon的高度及速度和vastoin nituron and nituron and abotorial nituron，时间分辨率。使用这些技术发展，我们将探讨有关神经血管代谢耦合机制的新概念。 Specifically, we will (1) address the relative contribution of oxidative phosphorylation and glycolysis to the transient metabolic response in neurons and astrocytes, (2) test the relationship between astrocytic metabolic response and regulation of blood flow through astrocytic calcium-dependent mechanisms, and (3) establish functional NADH imaging as a biomarker for hypoxia and mitochondrial dysfunction.这些目标将通过关注时间信号特征，通过研究同时获得的信号以及使用体内药理学来实现这些目标。拟议项目的主要交付 - 一种同时进行代谢，神经元和血管活性的2光子成像的工具 - 通过为研究人类脑疾病的啮齿动物模型的研究而开放前所未有的机会来研究啮齿动物模型的研究，以研究神经元，神经元，墨西哥膜和毛细血管的稳态和功能相互作用。将来，这种三方成像方法在疾病的不同阶段重复，将允许建立一组定义的体内成像生物标志物，这些生物标志物表征了神经血管替代病理学的进展，可用于对潜在疗法的客观筛查。 公共卫生相关性：我们将适应现有技术，即2光子激光扫描显微镜，以可视化活体动物中单个脑细胞的功能代谢，利用新陈代谢辅助剂2-尼希代谢性腺苷丁胺Dinucleotide（NADH）的固有荧光。此外，我们将将NADH成像与神经元和血管活性的2光子测量结合在一起，并将NADH作为缺氧的显微镜成像生物标志物或人类疾病小鼠模型中线粒体效率的降低。