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

描述（由申请方提供）：细胞特异性代谢活性的活体成像对于了解广泛的临床状况至关重要。其中包括中风后的血液灌注受损，以及阿尔茨海默病和帕金森病等神经退行性疾病中发生的单细胞呼吸过程效率降低。然而，目前还没有方法可用于体内测量单细胞代谢，其具有足够的灵敏度来解决与正在进行的神经元电活动和神经元对刺激的反应相关的快速代谢事件。为了应对这一挑战，我们将采用现有的技术，双光子激光扫描显微镜，利用代谢辅因子2-烟酰胺腺嘌呤二核苷酸（NADH）的固有荧光，对单个脑细胞的功能代谢进行高分辨率显微成像。 该项目将生物工程与生物学假设的测试相结合，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。从工程的角度来看，我们建议（1）开发和验证具有足够灵敏度的NADH的双光子成像，以检测活体动物中单个皮层神经元和星形胶质细胞响应于感觉刺激的功能变化，以及（2）优化光学设计和实验方案，以同时进行代谢，神经元和血管活动的体内双光子成像，具有高的空间和时间分辨率。利用这些技术的发展，我们将探索有关神经血管代谢耦合机制的新概念。具体而言，我们将（1）解决氧化磷酸化和糖酵解对神经元和星形胶质细胞瞬时代谢反应的相对贡献，（2）通过星形胶质细胞钙依赖机制测试星形胶质细胞代谢反应和血流调节之间的关系，（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，通过调查同时获得的信号，并通过使用体内药理学来实现。拟议项目的主要交付成果-代谢，神经元和血管活动的同时双光子成像工具-有可能通过打开一个前所未有的机会来研究神经元，神经胶质细胞和毛细血管之间的动态平衡和功能相互作用来改变人类大脑疾病的啮齿动物模型的研究。将来，在疾病的不同阶段重复的这种三方成像方法将允许建立一组定义的体内成像生物标志物，其表征神经血管代谢病理学的进展，可用于客观筛选潜在的治疗方法。 公共卫生相关性：我们将采用现有的技术，双光子激光扫描显微镜，可视化功能代谢的单个脑细胞在活体动物利用代谢辅因子2-烟酰胺腺嘌呤二核苷酸（NADH）的内在荧光。此外，我们将联合收割机NADH成像与神经元和血管活性的双光子测量相结合，并将建立NADH作为人类疾病小鼠模型中缺氧或线粒体效率降低的显微镜成像生物标志物。

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.