In Vivo Imaging Alzheimers Disease Pathology with 2-Photon/Lifetime Microscopy

描述（由申请人提供）：本项目旨在使用先进的显微镜来表征与阿尔茨海默病（AD）相关的钙调节和脑代谢的变化。AD对全世界2000多万人及其家庭造成破坏性影响，预计发病率每20年翻一番。了解AD相关的钙稳态和线粒体代谢缺陷的机制对于开发新的诊断标准和有效的预防性治疗是重要的。光学显微镜允许在人类病理学的动物模型中以高空间和时间分辨率非破坏性地测量代谢标志物。双光子/寿命显微镜最近已被证明可用于测量绝对钙浓度（[Ca]）和测量内源性电子载体烟酰胺腺嘌呤二核苷酸（NADH）的还原形式，据报道，允许参与糖酵解的NADH物种与参与氧化代谢的NADH物种的区别，具有前所未有的分辨率。通过使用新型树枝状Pd-卟啉磷光体，还可以使用双光子显微镜以高分辨率测量脑组织和脉管系统中的脑氧分压（pO 2）。该项目的中心焦点是使用双光子/寿命显微镜来测量AD小鼠模型在不同疾病阶段的体内脑NADH、[Ca]和pO 2。表征这些代谢标志物之间的关系，随着疾病的进展，在体内将提供详细的洞察复杂的代谢改变参与AD的发病机制。该项目的具体目标是：1。开发并验证用于pO 2和[Ca]或NADH物质的近同步2 P测量的方法和仪器。将对双光子成像系统进行升级和验证，使其具有两个激发源、四个检测通道和定制编写的控制软件，从而能够对体内脑代谢指标进行非破坏性测量。2.应用显微镜识别大脑中的NADH种类，并验证它们与健康小鼠特定代谢过程的关联荧光寿命成像能够分辨特定酶结合的NADH制剂。将对这些酶结合制剂的特定性质进行全面评价和表征。3.应用显微镜表征模拟AD发病机制的转基因小鼠暴露皮质中的脑代谢将利用从目标1和2获得的技术和知识来评价和比较代表AD进展的不同阶段的APPswe：PS1 dE 9转基因小鼠体内的脑代谢。该项目将以前所未有的细节深入了解AD发病机制，并将促进新治疗技术的开发，广泛适用于不断增长的老年高危人群。

DESCRIPTION (provided by applicant): This project aims to characterize the changes in calcium regulation and cerebral metabolism associated with Alzheimer's Disease (AD) using advanced microscopy. AD has a devastating impact on over 20 million people and their families worldwide, and the incidence rate is expected to double every 20 years. Understanding the mechanisms of AD-related deficits in calcium homeostasis and mitochondrial metabolism is important for developing new diagnostic criterion and effective preventative therapies. Optical microscopy permits non- destructive, measurement of metabolic markers with high spatial and temporal resolution in animal models of human pathologies. Two-photon / lifetime microscopy has recently proven useful for measuring absolute calcium concentration ([Ca]) and measuring the reduced form of the endogenous electron carrier nicotinamide adenine dinucleotide (NADH), reportedly allowing the distinction of NADH species involved in glycolysis from NADH species involved in oxidative metabolism, with unprecedented resolution. Through the use of novel dendritic Pd-porphyrin phosphors, cerebral oxygen partial pressure (pO2) in brain tissue and vasculature can also be measured with high resolution using two-photon microscopy. The central focus of this project is to use two-photon / lifetime microscopy to measure cerebral NADH, [Ca], and pO2 in vivo in an AD mouse model at different disease stages. Characterizing the relationship between these metabolic markers as the disease progresses in vivo will provide detailed insight into the complex metabolic alterations involved in AD pathogenesis. The specific aims of this project are: 1. Develop and validate methods and instrumentation for near-simultaneous 2P measurement of pO2, and either [Ca] or NADH species. A two-photon imaging system will be upgraded and validated to feature two excitation sources, four detection channels, and custom-written control software, enabling non-destructive measurement of cerebral metabolic indicators in vivo. 2. Apply the microscope to identify cerebral NADH species and validate their association with specific metabolic processes in healthy mice Fluorescence lifetime imaging enables resolution between specific enzyme-bound formulations of NADH. The specific nature of these enzyme bound formulations will be comprehensively evaluated and characterized. 3. Apply the microscope to characterize cerebral metabolism in the exposed cortices of transgenic mice modeling AD pathogenesis The technology and knowledge obtained from aims 1 and 2 will be utilized to evaluate and compare cerebral metabolism in vivo in APPswe:PS1dE9 transgenic mice representing distinct stages of AD progression. This project will yield insight into the mechanisms of AD pathogenesis with unprecedented detail, and it will facilitate the development of new therapeutic techniques widely applicable to the growing population of at-risk aging citizens.