High-speed imaging of cortical and white matter microvascular flow in AD/ADRD models

总结：AD/ADRD模型中皮质和白色物质微血管血流的高速成像 尽管已知血管危险因素和脑血管功能障碍与发病有关， AD/ADRD，其机制尚不清楚。在AD/ADRD小鼠模型中， 在微血管网络的不同水平上运作的血流和调节功能障碍已经被 鉴定在过度表达导致AD的突变基因的小鼠以及具有遗传和心血管风险的小鼠中 神经变性的因素，脑血流量减少，神经血管耦合受损， 周细胞导致毛细血管管腔变窄，白色血细胞停滞导致毛细血管流动停滞， 报道然而，由于目前用于量化微血管流量的方法是 对网络中的单个微血管中发生的事件不敏感（即，它们测量跨微血管的平均流量）。 许多血管因此错过了像毛细血管失速这样的事件）或不能评估网络流动和灌注变化 由微血管事件引起（即，它们一次测量的血管太少，无法量化上游和下游 由于毛细血管停滞导致的流量或局部灌注变化）。血流和灌注减少， 由这些事件引起的炎症可能在神经退行性疾病的进展中起重要作用， 具有持续或重复的网络灌注不足时期的网络微域-“少血微囊” - 可能是脑细胞功能障碍、淀粉样蛋白积聚和微梗塞的热点。测量沥青的流动 需要在连接的网络中的每个微血管中的速度来研究瞬时微血管 事件影响网络血流和组织灌注。这项建议旨在发展和测试一种模式- 2-和3-光子（2 P和3 P）激发的荧光成像的移位方法，以实现速度和深度 同时测量新皮层中约300条微血管或深部约50条微血管的血流所需的穿透力 小鼠皮质下白色物质（WM）。产生飞秒激光脉冲的自适应激励源 “按需”与快速3D光栅扫描同步，并被编程为仅在血液 船只停留。因为可以输送到大脑的最大激光功率是速率限制的，所以AES限制了 2 P/3 P激励脉冲仅用于血管，能够测量速度、直径和来自血管的信号。 来自皮质中300 × 300 × 300 µm3体积中所有微血管的额外细胞类型特异性荧光标记 或在深部WM中的200 x 200 x 100 µm3体积中（100 Hz体积成像，体素大小为1 x 1 x 10 µm3）（目标1）。 这种创新的成像能力将用于探索选定的 AD小鼠模型大脑皮层CBF异常的分子和细胞机制，以及检测 假设少血微囊是淀粉样蛋白沉积的位点（目的2）。AES成像的能力 探讨网络血流灌注对小鼠脑深部白质使能检查遗传的影响 以及与AD/ADRD相关的心血管危险因素， 深入了解全局灌注不足如何在微观水平上诱导WM损伤（目标3）。

Summary: High-speed imaging of cortical and white matter microvascular flow in AD/ADRD models Although vascular risk factors and cerebrovascular dysfunction are known to be pathogenically linked to AD/ADRD, the mechanisms are not well understood. In AD/ADRD mouse models, several causes of decreased blood flow and regulatory dysfunction that operate at different levels of the microvascular network have been identified. In mice overexpressing mutant genes that cause AD and in mice with genetic and cardiovascular risk factors for neurodegeneration, decreases in cerebral blood flow, impairment of neurovascular coupling, narrowing of capillary lumens by pericytes, and stalling of capillary flow by arrested white blood cells have been reported. However, much remains unknown because current approaches for quantifying microvascular flow are insensitive to events occurring in individual microvessels in a network (i.e. they measure averaged flow across many vessels so miss an event like a capillary stall) or are unable to evaluate network flow and perfusion changes caused by microvascular events (i.e. they measure too few vessels at a time to quantify up- and down-stream flow or regional perfusion changes due to a capillary stall). Flow and perfusion decreases and heterogeneity arising from such events could play an important role in the progression of neurodegenerative disease, as network microdomains with persistent or repeated epochs of network hypoperfusion – “oligemic micropockets” – may be hotspots for brain cell dysfunction, amyloid accumulation, and microinfarcts. Measurement of flow speed in every microvessel across a connected network is needed to investigate how transient microvascular events impact network blood flow and tissue perfusion. This proposal seeks to develop and test a paradigm- shifting approach to 2- and 3-photon (2P and 3P) excited fluorescence imaging to achieve the speed and depth penetration necessary to simultaneously measure flow in ~300 microvessels in the neocortex or ~50 in the deep subcortical white matter (WM) of mice. An adaptive excitation source (AES) generating femtosecond laser pulses “on demand” is synchronized with fast 3D raster scanning and is programmed to fire pulses only where blood vessels reside. Because the maximum laser power that can be delivered to the brain is rate limiting, AES restricts 2P/3P excitation pulses only to blood vessels enabling measurement of the speed, diameter, and signals from additional cell type-specific fluorescent labels from all microvessels in a 300x300x300 µm3 volume in the cortex or in a 200x200x100 µm3 volume in the deep WM (100 Hz volume imaging with 1x1x10 µm3 voxel size) (Aim 1). This innovative imaging capability will be used to explore the collective impact and causal links between selected molecular and cellular mechanisms of CBF abnormality in the cortex of AD mouse models, as well as to test the hypothesis that oligemic micropockets are sites of amyloid deposition (Aim 2). The ability of AES imaging to explore network flow and perfusion in the deep WM of mice enables the examination of the impact of genetic and cardiovascular risk factors associated with AD/ADRD on the WM microvascular network to provide novel insights into how global hypoperfusion induces WM damage at the microscale level (Aim 3).