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

综述：AD/ADRD模型中皮质和白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 AD/ADRD，其机制还不是很清楚。在AD/ADRD小鼠模型中，减少的几个原因 在微血管网络的不同水平上运行的血流和调节功能障碍 已确认身份。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，神经血管偶联障碍， 周细胞引起的毛细血管管腔变窄，以及白细胞受阻使毛细血管流动受阻。 据报道。然而，仍有许多未知之处，因为目前量化微血管血流的方法 对网络中单个微血管中发生的事件不敏感(即，它们测量 许多血管错过了像毛细血管失速这样的事件)，或者无法评估网络血流和血流灌注的变化 由微血管事件引起(即一次测量的血管太少，无法量化上行和下行 毛细血管失速引起的血流或区域血流灌注改变)。血流和灌注量减少，不均质性 可能在神经退行性疾病的进展中发挥重要作用，如 持续或反复出现网络低灌注期的网络微域--“少血微囊” -可能是脑细胞功能障碍、淀粉样蛋白堆积和微梗塞的热点。流量测量 需要通过连接网络的每个微血管的速度来研究瞬时微血管如何 事件影响网络血流量和组织灌注量。这项提议寻求开发和测试一个范例-- 双光子和三光子(2P和3P)激发荧光成像的移位方法实现速度和深度 同时测量新皮质中~300个微血管或深层~50个微血管中的血流所需的穿透性 小鼠皮质下白质(WM)。一种产生飞秒激光脉冲的自适应激励源 按需扫描与快速3D栅格扫描同步，并被编程为仅在血液 船只停靠。因为可以传输到大脑的最大激光功率是速率限制的，所以AES限制了 2P/3P激励脉冲仅适用于血管，从而能够测量速度、直径和来自 来自皮质中体积为300x300x300微米的所有微血管的附加细胞类型特定的荧光标记 或在200×200×100微米的深度WM(100赫兹体积成像，体素大小为1x1×10微米)中(目标1)。 这一创新的成像能力将用于探索集体影响和选定的 阿尔茨海默病模型小鼠大脑皮质脑血流异常的分子和细胞机制 假设少血微囊是淀粉样蛋白沉积的部位(目标2)。AES成像的能力 探讨网络血流和血流灌注对小鼠深部白质的影响 而心血管危险因素与AD/ADRD相关的WM微血管网络提供了新的研究方向 洞察全球低灌流如何在微观水平上导致白质损伤(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).