A Practical Guide to the Automated Analysis of Vascular Growth, Maturation and Injury in the Brain

Abstract

The distinct organization of the brain's vasculature ensures the adequate delivery of oxygen and nutrients during development and adulthood. Acute and chronic pathological changes of the vascular system have been implicated in many neurological disorders including stroke and dementia. Here, we describe a fast, automated method that allows the highly reproducible, quantitative assessment of distinct vascular parameters and their changes based on the open source software Fiji (ImageJ). In particular, we developed a practical guide to reliably measure aspects of growth, repair and maturation of the brain's vasculature during development and neurovascular disease in mice and humans. The script can be used to assess the effects of different external factors including pharmacological treatments or disease states. Moreover, the procedure is expandable to blood vessels of other organs and vascular in vitro models.

Keywords: angiogenesis; blood vessels; central nervous system (CNS); development; image processing; quantification; stroke.

FIGURE 1

Visualization and quantification of cortical vasculature in the developing mouse brain. (A) Experimental design. (B) Visualization of the vasculature with immune- or lectin-histofluorescence, transcardial perfusion with Lectin-Dylight594 or the use of Cldn5-GFP reporter mice at the age of p10 (development) and 3 months (adult). Scale bar 50 μm. (C) Step-by-step analysis pipeline for vascular quantification. (D) Heatmap and quantification of vascular area fraction, length, branching and nearest distance between blood vessels in cortical brain sections. Scale bar: 2 mm. Data are represented as means ± SD. Each dot represents one animal.

FIGURE 2

Vascular changes in the peri-infarct ischemic border zone around the stroke core in the mouse cortex. (A) Experimental design. (B) Representative images of intact cortex vasculature (left panels) and endothelial network (CD31+; red) and pericytes (CD13+; blue) in the ischemic border zone. Scale bar: 100 μm (overview), 20 μm (close-up). (C) Quantitative assessment of vascular parameters (area fraction, length, branching, distance) and pericyte coverage of the vasculature. Data are represented as boxplots (median, two hinges and two whiskers); the upper and lower hinges correspond to the first and third quantiles. The whiskers extend until 1.5 × IQR from the hinges; data beyond the end of the whiskers are defined as outlying points. Each dot represents one animal, and significance of mean differences between the groups was assessed using two-tailed unpaired one-sample t-test. Asterisks indicate significance: ^∗P < 0.05, ^∗∗∗P < 0.001. IQR, interquartile range.

FIGURE 3

Vascular changes in human Alzheimer’s disease brains. (A) Experimental design. (B) Representative images of Alzheimer’s brain sections. Scale bar 100 μm (overview), 20 μm (close-up). (C) Quantitative assessment of brain vasculature and pericyte coverage in cortical gray and white matter sections of AD patients and healthy controls. Data are represented as boxplots (median, two hinges and two whiskers); the upper and lower hinges correspond to the first and third quantiles. The whiskers extend until 1.5 × IQR from the hinges; data beyond the end of the whiskers are defined as outlying points. Each dot represents one subject and significance of mean differences between the groups was assessed using a two two-tailed unpaired one-sample t-test. Asterisks indicate significance: ^∗∗P < 0.01. GM, gray matter; WM, white matter; AD, Alzheimer’s disease; Ctrl, control; n.s., not significant; IQR: interquartile range.