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

描述(由申请人提供)：细胞特异性代谢活动的活体成像对于了解广泛的临床情况至关重要。其中包括中风后的血液灌流受损，以及阿尔茨海默氏症和帕金森氏症等神经退行性疾病中出现的单细胞呼吸过程效率下降。然而，目前还没有足够灵敏的方法在体内测量单细胞代谢，以解决与正在进行的神经元电活动和神经元对刺激的反应有关的快速代谢事件。为了迎接这一挑战，我们将采用现有的双光子激光扫描显微镜技术，利用代谢辅因子2-烟酰胺腺嘌呤二核苷酸(NADH)的内在荧光，对单个脑细胞的功能代谢进行高分辨率显微成像。该项目将生物工程工作与生物假说测试结合在一起，并汇集了神经科学、物理学、工程学和计算科学的跨学科专家团队。从工程学的角度，我们建议(1)发展和验证NADH的双光子成像，该成像具有足够的灵敏度来检测单个皮质神经元和星形胶质细胞对感觉刺激的反应的功能变化；(2)优化光学设计和实验方案，以高空间和时间分辨率同时进行体内代谢、神经元和血管活动的双光子成像。利用这些技术发展，我们将探索关于神经-血管-代谢耦合机制的新概念。具体地说，我们将(1)研究氧化磷酸化和糖酵解在神经元和星形胶质细胞瞬时代谢反应中的相对贡献，(2)通过星形细胞钙依赖机制来测试星形细胞代谢反应和血流调节之间的关系，以及(3)建立功能性NADH成像作为缺氧和线粒体功能障碍的生物标志物。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，并通过使用体内药理学来实现。拟议项目的主要成果--代谢、神经元和血管活动的同时双光子成像工具--有可能改变人类大脑疾病的啮齿动物模型的研究，因为它打开了一个前所未有的机会，研究活着的大脑中神经元、胶质细胞和毛细血管之间的稳态和功能相互作用。未来，这种在疾病不同阶段重复使用的三方成像方法将允许建立一组明确的体内成像生物标记物，表征神经-血管-代谢病理的进展，可用于客观筛选潜在的治疗方法。 与公共健康相关：我们将采用现有的双光子激光扫描显微镜技术，利用代谢辅因子2-烟酰胺腺嘌呤二核苷酸(NADH)的内在荧光，可视化活动物单个脑细胞的功能代谢。此外，我们将NADH成像与神经元和血管活动的双光子测量相结合，并将NADH确立为人类疾病小鼠模型中缺氧或线粒体效率下降的显微成像生物标记物。

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.