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节的能量依赖性细胞死亡形式，是神经发育、稳态和神经变性的重要组成部分。它是胎儿酒精综合征、缺血性中风和帕金森病等不同脑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，但一直没有直接的方法来量化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，这个问题：一个转基因小鼠含有荧光标记的半胱天冬酶-3活性-一个关键的“刽子手”酶的凋亡信号级联-将通过细胞形态学和生物化学测定进行验证。这种凋亡报告小鼠将与各种疾病模型一起繁殖，包括多巴胺转运蛋白敲除，microRNA-9缺陷小鼠和torsin A突变体;后代的大脑将首先接受高分辨率磁共振和扩散 张量成像，以阐明整体结构的细节和纤维束，分别。将组织切成几个1- 2 mm厚的矢状切片并在水溶液中澄清后，将通过双光子共聚焦显微镜以约1微米的分辨率对切片进行完全三维扫描。然后，双光子图像将与磁共振和扩散张量图像配准，提供整个大脑的多模式视图。自动分割和细胞计数算法将用于量化每个脑区中的凋亡细胞数量;分析将涉及将细胞死亡增加或减少的区域与结构形态学和纤维束各向异性的畸变相关联。我们方法的最后阶段将是以研究人员和公众易于访问的格式将许多疾病突变模型的多模态图像上传到服务器或云。我们相信，这种方法将是简单的，足以追求任何研究小组与适当的硬件，这种方法的发展将产生一般的工具，为先进的多模态成像的生物现象。

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.