Simple methodology to visualize whole-brain microvasculature in three dimensions

Abstract

. Significance: To explore brain architecture and pathology, a consistent and reliable methodology to visualize the three-dimensional cerebral microvasculature is beneficial. Perfusion-based vascular labeling is quick and easily deliverable. However, the quality of vascular labeling can vary with perfusion-based labels due to aggregate formation, leakage, rapid photobleaching, and incomplete perfusion. Aim: We describe a simple, two-day protocol combining perfusion-based labeling with a two-day clearing step that facilitates whole-brain, three-dimensional microvascular imaging and characterization. Approach: The combination of retro-orbital injection of Lectin-Dylight-649 to label the vasculature, the clearing process of a modified iDISCO+ protocol, and light-sheet imaging collectively enables a comprehensive view of the cerebrovasculature. Results: We observed increase in contrast-to-background ratio of Lectin-Dylight-649 vascular labeling over endogenous green fluorescent protein fluorescence from a transgenic mouse model. With light-sheet microscopy, we demonstrate sharp visualization of cerebral microvasculature throughout the intact mouse brain. Conclusions: Our tissue preparation protocol requires fairly routine processing steps and is compatible with multiple types of optical microscopy.

High-Resolution Digital Panorama of Multiple Structures in Whole Brain of Alzheimer's Disease Mice

“…Serial two-photon microscopy (Delafontaine-Martel et al, 2018 ) and light sheet microscopy (Di Giovanna et al, 2018 ) combined with intracardiac perfusion of FITC-gelatin perfusate achieved whole-brain vascular imaging at high resolution. Except the FITC-gelatin gel perfusion method, whole-mount immunolabeling (Kirst et al, 2020 ), fluorescent tagged transgenic mice (Jing et al, 2018 ), and conventional vascular-labeling dyes such as fluorescent lectin (Khouri et al, 2021 ) or lipophilic tracers (Konno et al, 2020 ) were also been adopted in light sheet microscopy technique for whole-brain vascular imaging. Interestingly, a novel method named “SeeNet” integrating multiple technologies (vascular casting with intravascular perfusion of fluorescent cross-linker, tissue clearing, and light sheet microscopy) achieved whole-brain visualization of cerebral vasculatures at the single-micro-vessel level (Miyawaki et al, 2020 ).…”

Vascular persistence following precision micropuncture

“…However, we recognize that true 3D microvascular architecture may be more precisely assessed with other established methods, such as scanning electron microscopy corrosion casting or newer emerging approaches. Specifically, we are in the process of developing light sheet microscopic approaches in this context which will build upon recent advances with this technology 66,67 . This will provide our future studies with true volume imaging capabilities and be immensely valuable in further understanding the anatomical aspects of microvascular development and regression following MP.…”

Semi-automated protocol to quantify and characterize fluorescent three-dimensional vascular images

“…We currently use iDISCO (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) as our primary method of tissue clearing (Khouri et al, 2021; Renier et al, 2014). Briefly, iDISCO consists of methanol dehydration, lipid removal with dichloromethane, and refractive index matching with dibenzyl ether.…”

“…It is compatible with many exogenous labels often used for traditional immunohistochemistry. We previously demonstrated the effectiveness of iDISCO in combination with lectin-DyLight-649 for 3D visualization of vasculature in a mouse brain (Khouri et al, 2021). We also have used Prussian blue labelling of hemosiderin (Crouzet et al, 2021; Lo et al, 2016; Sumbria et al, 2018), a by-product of cerebral microhemorrhages, with iDISCO-cleared brains and lectin-DyLight-649 labelling.…”

“…It is compatible with many exogenous labels often used for traditional immunohistochemistry. We previously demonstrated the effectiveness of iDISCO in combination with lectin-DyLight-649 for 3D visualization of vasculature in a mouse brain (10).…”