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

描述(由申请人提供)：该项目旨在利用先进的显微镜来描述与阿尔茨海默病(AD)相关的钙调节和大脑代谢的变化。AD对全球2000多万人及其家庭造成了毁灭性的影响，发病率预计每20年翻一番。了解阿尔茨海默病相关的钙稳态和线粒体代谢紊乱的机制，对于开发新的诊断标准和有效的预防治疗具有重要意义。光学显微镜可以在人类病理的动物模型中以高空间和时间分辨率无损地测量代谢标记物。双光子/寿命显微镜最近被证明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，它能够以前所未有的分辨率区分参与糖酵解的NADH和参与氧化代谢的NADH。通过使用新型树枝状钯-卟啉荧光粉，还可以使用双光子显微镜以高分辨率测量脑组织和血管中的脑氧分压(PO2)。这个项目的中心焦点是使用双光子/寿命显微镜在不同疾病阶段的AD小鼠模型体内测量大脑NADH、[Ca]和PO2。表征这些代谢标志物之间的关系，随着疾病在体内的进展，将提供详细的洞察复杂的代谢变化参与AD的发病机制。本项目的具体目标是：1.开发和验证用于近同时测量PO2和[Ca]或NADH的2P的方法和仪器。双光子成像系统将进行升级和验证，配备两个激发源、四个检测通道和定制的控制软件，从而能够对体内的大脑代谢指标进行无损测量。2.应用显微镜鉴定大脑中NADH的种类，并验证它们与健康小鼠特定代谢过程的关系。荧光寿命成像可以在特定的酶结合的NADH配方之间进行分辨。将对这些酶结合制剂的具体性质进行全面的评估和表征。3.应用显微镜观察AD发病过程中转基因小鼠暴露皮质中脑代谢的变化利用AIMS 1和AIMS 2所获得的技术和知识，评价和比较APPsWE：PS1dE9转基因小鼠不同发展阶段的脑代谢。该项目将以前所未有的细节深入了解AD的发病机制，并将促进新的治疗技术的开发，这些技术广泛适用于日益增长的高危老龄化人口。

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.