Whole brain multimodal microscopy of an apoptosis reporter mouse

描述（由申请人提供）：细胞凋亡是一种精确调控和能量依赖的细胞死亡形式，是神经发育、体内平衡和神经退行性变的重要组成部分。它是胎儿酒精综合征、缺血性中风和帕金森病等多种脑部疾病的关键组成部分；它可能在精神分裂症、重度抑郁症和创伤后应激障碍等行为障碍中发挥作用。虽然有许多神经和精神疾病的小鼠模型，但还没有直接的方法来定量和定位整个小鼠大脑中的所有凋亡细胞。我们建议开发和优化以下技术解决方案：一个含有caspase-3活性荧光标记的转基因小鼠，caspase-3是凋亡信号级联中关键的“刽子手”酶，将通过细胞形态学和生化分析进行验证。这种凋亡报告小鼠将与多种疾病模型杂交，包括多巴胺转运蛋白敲除、microrna -9缺陷小鼠和torsin A突变体；后代的大脑将首先进行高分辨率磁共振和扩散

DESCRIPTION (provided by applicant): Apoptosis, a precisely regulated and energy-dependent form of cell death, is an important part of neurodevelopment, homeostasis, and neurodegeneration. It is a key component of disparate brain disorders including fetal alcohol syndrome, ischemic stroke, and Parkinson's disease; and it may play a role in behavioral disorders such as schizophrenia, major depression, and posttraumatic stress disorder. Although there are numerous mouse models of neurological and psychiatric disease, there has been no straightforward method for quantifying and locating all of the apoptotic cells in whole mouse brains. We propose to develop and optimize the following technical solution to this problem: A transgenic mouse containing a fluorescent marker of caspase-3 activity - a pivotal "executioner" enzyme in the apoptosis signaling cascade - will be validated by cell morphological and biochemical assays. This apoptosis reporter mouse will be bred with various disease models, including dopamine transporter knockouts, microRNA-9-deficient mice, and torsin A mutants; the brains of offspring will first be submitted to high-resolution magnetic resonance and diffusion tensor imaging, to elucidate overall structural details and fiber tracts, respectively. After the tissue has been sliced into several 1- to 2-mm-thick sagittal sections and clarified in an aqueous solution, the sections will be scanned completely in three dimensions by two-photon confocal microscopy at a resolution of approximately 1 micron. The two- photon images will then be registered to the magnetic resonance and diffusion tensor images, providing a multimodal view of the entire brain. Automatic segmentation and cell counting algorithms will be used to quantify the number of apoptotic cells in each brain region; and analysis will involve correlating areas of increased or decreased cell death with aberrations in structural morphology and fiber tract anisotropy. The final stage of our method will be to upload the multimodal images of numerous mutant models of disease to a server or cloud in a format that is easily accessible to researchers and the public. We believe that this methodology will be straightforward enough to be pursued by any research group with the appropriate hardware; and that the development of this method will give rise to general tools for the advancement of multimodal imaging of biological phenomena. PUBLIC HEALTH RELEVANCE: A method will be developed to locate every dying cell in a mouse brain. This will help us understand how diseases like Alzheimer's, stroke, and autism arise.