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

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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).