A Novel Technique for Visualizing and Analyzing the Cerebral Vasculature in Rodents

Abstract

We introduce a novel protocol to stain, visualize, and analyze blood vessels from the rat and mouse cerebrum. This technique utilizes the fluorescent dye, DiI, to label the lumen of the vasculature followed by perfusion fixation. Following brain extraction, the labeled vasculature is then imaged using wide-field fluorescence microscopy for axial and coronal images and can be followed by regional confocal microscopy. Axial and coronal images can be analyzed using classical angiographic methods for vessel density, length, and other features. We also have developed a novel fractal analysis to assess vascular complexity. Our protocol has been optimized for adult rat, adult mouse, and neonatal mouse studies. The protocol is efficient, can be rapidly completed, stains cerebral vessels with a bright fluorescence, and provides valuable quantitative data. This method has a broad range of applications, and we demonstrate its use to study the vasculature in assorted models of acquired brain injury.

Vasculature-staining with lipophilic dyes in tissue-cleared brains assessed by deep learning

“…Lipophilic fluorescent dyes, which are also aldehyde-fixable, are likely to provide a better fluorescent signal since these dyes are better retained in the tissue by fixing to the adjacent lipoproteins (Jensen and Berg, 2016). Here, we tested the CLARITY-compatibility of nine dyes, where some are also fixable, including DiIC variants, and found high-quality vasculaturestaining (Konno et al, 2017;Salehi et al, 2018;Ravnic et al, 2005) with most of the dyes. The dyes were added in the perfusate to stain the endothelium of vessels.…”

“…Previous reports reports show vessel-staining is not only affect by dye aggregration, but also by dye dispersion, which is inhibited by vascular occlusion and constriction during perfusion (Ravnic et al, 2005;Weyers et al, 2012;Ghanavati et al, 2014;Salehi et al, 2018). Vasoconstriction was initially preempted by keeping perfusion fluids at 37 o C. Since there was heterogeneous staining (figure 2 we added an injection of a vasodilator (sodium nitroprusside, SNP) and anticoagulant (heparin) two minutes before euthanasia.)…”

“…The lipophilic dyes are highly hydrophobic and aggregate in the physiological ionic conditions. To reduce dye aggregation in the perfusate, we incorporated them into liposomes, which form small micelles around the dyes and these get integrated into the cellular membranes (MacDonald, 1990;Ertürk et al, 2011) during perfusion (Salehi et al, 2018;. We employed neutral (NL) and anionic liposomes (AL) while omitting cationic liposomes as they increase aggregation for DiIC18 (Konno et al, 2017).…”

“…With exogenous fluorescent molecules, it is possible to stain specific targets, such as tracking the location of electrodes or axonal tracing (Jensen and Berg, 2016). The vasculature can be stained using similar dyes by perfusion, and the tissue can then be cleared using CLARITY, leaving the vasculature visible in 3D Konno et al, 2017;Salehi et al, 2018).…”

“…Lipophilic dyes are prone to aggregation in physiological solutions, which leads to heterogeneous staining and self-quenching (Zimcik et al, 2007;Konno et al, 2017). Incorporating the dyes into the bilayer of liposomes can prevent aggregation and allow the dyes to more easily integrate into the cellular membranes (Blumenthal et al, 2002;Ravnic et al, 2005;Salehi et al, 2018). Especially neutral liposomes (NL) and anionic liposomes (AL) have been found to decrease DiIC18 aggregation and enhance distribution in capillaries (Konno et al, 2017).…”