Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain

ABSTRACTThe formation of new blood vessels and the establishment of vascular networks are crucial during brain development, in the adult healthy brain, as well as in various diseases of the central nervous system (CNS). Here, we describe a method that enables hierarchical imaging and computational analysis of vascular networks in postnatal- and adult mouse brains. Resin-based vascular corrosion casting, scanning electron microscopy, synchrotron radiation and desktop µCT imaging, and computational network analysis are used. Combining these methods enables detailed visualization and quantification of the three-dimensional (3D) brain vasculature. Network features such as vascular volume fraction, branch point density, vessel diameter, - length, -tortuosity, and -directionality as well as extravascular distance can be obtained at any developmental stage from the early postnatal to the adult brain. Our method allows characterizing brain vascular networks separately for capillaries and non-capillaries.The entire protocol, from mouse perfusion to vessel network analysis, takes approximately 10 days.Online summaryThis protocol uses vascular corrosion casting, hierarchical synchrotron radiation µCT imaging, and computational image analysis to assess the three-dimensional vascular network architecture.

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Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain

Nature Protocols ◽

10.1038/s41596-021-00587-1 ◽

2021 ◽

Author(s):

Thomas Wälchli ◽

Jeroen Bisschop ◽

Arttu Miettinen ◽

Alexandra Ulmann-Schuler ◽

Christoph Hintermüller ◽

...

Keyword(s):

Mouse Brain ◽

Network Architecture ◽

Computational Analysis ◽

Adult Mouse ◽

Three Dimensional ◽

Vascular Network ◽

Adult Mouse Brain

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Nogo-A regulates vascular network architecture in the postnatal brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x16675182 ◽

2016 ◽

Vol 37 (2) ◽

pp. 614-631 ◽

Cited By ~ 5

Author(s):

Thomas Wälchli ◽

Alexandra Ulmann-Schuler ◽

Christoph Hintermüller ◽

Eric Meyer ◽

Marco Stampanoni ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Network Architecture ◽

Three Dimensional ◽

Nervous System Development ◽

Vascular Network ◽

Negative Regulator ◽

Corrosion Casting ◽

Genetic Ablation ◽

Vessel Morphology

Recently, we discovered a new role for the well-known axonal growth inhibitory molecule Nogo-A as a negative regulator of angiogenesis in the developing central nervous system. However, how Nogo-A affected the three-dimensional (3D) central nervous system (CNS) vascular network architecture remained unknown. Here, using vascular corrosion casting, hierarchical, synchrotron radiation μCT-based network imaging and computer-aided network analysis, we found that genetic ablation of Nogo-A significantly increased the three-dimensional vascular volume fraction in the postnatal day 10 (P10) mouse brain. More detailed analysis of the cerebral cortex revealed that this effect was mainly due to an increased number of capillaries and capillary branchpoints. Interestingly, other vascular parameters such as vessel diameter, -length, -tortuosity, and -volume were comparable between both genotypes for non-capillary vessels and capillaries. Taken together, our three-dimensional data showing more vessel segments and branchpoints at unchanged vessel morphology suggest that stimulated angiogenesis upon Nogo-A gene deletion results in the insertion of complete capillary micro-networks and not just single vessels into existing vascular networks. These findings significantly enhance our understanding of how angiogenesis, vascular remodeling, and three-dimensional vessel network architecture are regulated during central nervous system development. Nogo-A may therefore be a potential novel target for angiogenesis-dependent central nervous system pathologies such as brain tumors or stroke.

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Comparison of high-resolution synchrotron-radiation-based phase-contrast imaging and absorption-contrast imaging for evaluating microstructure of vascular networks in rat brain: from 2D to 3D views

Journal of Synchrotron Radiation ◽

10.1107/s1600577519011688 ◽

2019 ◽

Vol 26 (6) ◽

pp. 2024-2032 ◽

Cited By ~ 1

Author(s):

Hong-Lei Li ◽

Hui Ding ◽

Xian-Zhen Yin ◽

Zhuo-Hui Chen ◽

Bin Tang ◽

...

Keyword(s):

High Resolution ◽

Synchrotron Radiation ◽

Phase Contrast ◽

Three Dimensional ◽

Phase Contrast Imaging ◽

Vascular Networks ◽

Contrast Imaging ◽

Microvascular Networks ◽

Absorption Phase ◽

Absorption Contrast

Conventional imaging methods such as magnetic resonance imaging, computed tomography and digital subtraction angiography have limited temporospatial resolutions and shortcomings like invasive angiography, potential allergy to contrast agents, and image deformation, that restrict their application in high-resolution visualization of the structure of microvessels. In this study, through comparing synchrotron radiation (SR) absorption-contrast imaging to absorption phase-contrast imaging, it was found that SR-based phase-contrast imaging could provide more detailed ultra-high-pixel images of microvascular networks than absorption phase-contrast imaging. Simultaneously, SR-based phase-contrast imaging was used to perform high-quality, multi-dimensional and multi-scale imaging of rat brain angioarchitecture. With the aid of image post-processing, high-pixel-size two-dimensional virtual slices can be obtained without sectioning. The distribution of blood supply is in accordance with the results of traditional tissue staining. Three-dimensional anatomical maps of cerebral angioarchitecture can also be acquired. Functional partitions of regions of interest are reproduced in the reconstructed rat cerebral vascular networks. Imaging analysis of the same sample can also be displayed simultaneously in two- and three-dimensional views, which provides abundant anatomical information together with parenchyma and vessels. In conclusion, SR-based phase-contrast imaging holds great promise for visualizing microstructure of microvascular networks in two- and three-dimensional perspectives during the development of neurovascular diseases.

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Some quantitative applications of vascular corrosion casting

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124094 ◽

1992 ◽

Vol 50 (1) ◽

pp. 738-739

Author(s):

Fred E. Hossler

Keyword(s):

Blood Vessels ◽

Nuclear Orientation ◽

Three Dimensional ◽

Surrounding Tissue ◽

Confocal Laser ◽

Corrosion Casting ◽

Venous Valve ◽

Casting Technique ◽

Low Viscosity ◽

Quantitative Measurements

Preparation of replicas of the complex arrangement of blood vessels in various organs and tissues has been accomplished by infusing low viscosity resins into the vasculature. Subsequent removal of the surrounding tissue by maceration leaves a model of the intricate three-dimensional anatomy of the blood vessels of the tissue not obtainable by any other procedure. When applied with care, the vascular corrosion casting technique can reveal fine details of the microvasculature including endothelial nuclear orientation and distribution (Fig. 1), locations of arteriolar sphincters (Fig. 2), venous valve anatomy (Fig. 3), and vessel size, density, and branching patterns. Because casts faithfully replicate tissue vasculature, they can be used for quantitative measurements of that vasculature. The purpose of this report is to summarize and highlight some quantitative applications of vascular corrosion casting. In each example, casts were prepared by infusing Mercox, a methyl-methacrylate resin, and macerating the tissue with 20% KOH. Casts were either mounted for conventional scanning electron microscopy, or sliced for viewing with a confocal laser microscope.

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