A Revised View on Growth and Remodeling in the Retinal Vasculature - PubMed (original) (raw)

A Revised View on Growth and Remodeling in the Retinal Vasculature

Ruslan Rust et al. Sci Rep. 2019.

Abstract

The mouse retina provides an excellent model for studying angiogenesis. Recent advancements in high-throughput microscopy and image analysis provide great tools to visualize and describe the complexity of the retinal vascular architecture in a detailed and comprehensive way. Most developmental studies have focused on only a few parameters mostly in the inner-most layers that do not describe the entirety of the three-dimensional vascular network. Here, we analyzed the entire three-dimensional retinal vascular architecture and its growth and remodeling starting from the age of postnatal day 3 to 4 months in mice. We show plexus specific characteristics of the vasculature in terms of vascular tissue fraction, branching and length of the blood vessels, and distance and distribution between single capillaries. Such detailed knowledge is of particular interest, as it has become apparent that disease-specific mechanisms and treatments affect the retinal vasculature often in a plexus specific way.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Figure 1

Visualization and image-processing of retinal vasculature. (A) Representative images of retinal flatmounts of Cldn5-eGFP reporter mice stained for CD31 and IB4. Scale bar 200 µm. (B) Representative images of Cldn5-eGFP+ retinal flatmounts after transcardial perfusion with tomato Lectin. Scale bar 100 µm. (C) Illustration of the image processing procedure in ImageJ (FIJI). (D) Schematic representation of vascular parameters that are assessed in a retinal flatmount. Vascular area fraction: Ratio of area covered by vasculature to total area in the retina.

Figure 2

Figure 2

Development of the mouse retinal vasculature from postnatal day p3 to p120. (A) Retinal flat mounts of p3, p7, p10, p30, p60 and p120 old mice with close up. Scale bar overview: 1 mm; close up: 100 µm. (B) Binarized images of Cldn5-eGFP+ retinal flatmounts from p3 to adulthood (p120) (left sides) and corresponding heatmaps of the vascular area fraction (right sides). Scale bar 1 mm. (C) Histograms of retinal heatmaps. (D) Quantitative evaluation of vascular outgrowth, area fraction, segment length, number of branches, nearest neighbor distance (NND) and variability of NND from p3 to adulthood. The animal number is n = 4 (p3, p7, p10) and n = 3 (p30, p60, p120).

Figure 3

Figure 3

Development of the primary, intermediate and deep vascular plexus. (A) Schematic drawings and binarized microscopic images (orthogonal view of retinal wholemount 60x image stack) illustrate the sequential development of the three-layered vascular network in the mouse retina. (B) Representative images of the three retinal plexuses at different developmental and adult stages. Scale bar 100 µm. (C) Quantitative assessment of vascular parameters reflecting growth and remodeling processes in all three vascular layers. The animal number is n = 4 (p3, p7, p10) and n = 3 (p30, p60, p120).

Figure 4

Figure 4

Single plexus analysis reveals more accurate measure of vasculature than whole retina analysis. (A) Schematic illustration of whole retina and single plexus analysis. (B) Quantification and comparison of vascular outgrowth, area fraction, length and branching between single plexus and whole retina. (C) Tabular overview of vascular parameters with deviations in %. The animal number is n = 4 (p3, p7, p10) and n = 3 (p30, p60, p120).

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