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. 2021 Apr 20;17(4):e1008930.
doi: 10.1371/journal.pcbi.1008930. eCollection 2021 Apr.

Vessel network extraction and analysis of mouse pulmonary vasculature via X-ray micro-computed tomographic imaging

Eric A Chadwick  1 , Takaya Suzuki  2 , Michael G George  1 , David A Romero  3 , Cristina Amon  3 , Thomas K Waddell  2 , Golnaz Karoubi  2 , Aimy Bazylak  1
Affiliations

Vessel network extraction and analysis of mouse pulmonary vasculature via X-ray micro-computed tomographic imaging

Eric A Chadwick et al. PLoS Comput Biol. .

Abstract

In this work, non-invasive high-spatial resolution three-dimensional (3D) X-ray micro-computed tomography (μCT) of healthy mouse lung vasculature is performed. Methodologies are presented for filtering, segmenting, and skeletonizing the collected 3D images. Novel methods for the removal of spurious branch artefacts from the skeletonized 3D image are introduced, and these novel methods involve a combination of distance transform gradients, diameter-length ratios, and the fast marching method (FMM). These new techniques of spurious branch removal result in the consistent removal of spurious branches without compromising the connectivity of the pulmonary circuit. Analysis of the filtered, skeletonized, and segmented 3D images is performed using a newly developed Vessel Network Extraction algorithm to fully characterize the morphology of the mouse pulmonary circuit. The removal of spurious branches from the skeletonized image results in an accurate representation of the pulmonary circuit with significantly less variability in vessel diameter and vessel length in each generation. The branching morphology of a full pulmonary circuit is characterized by the mean diameter per generation and number of vessels per generation. The methods presented in this paper lead to a significant improvement in the characterization of 3D vasculature imaging, allow for automatic separation of arteries and veins, and for the characterization of generations containing capillaries and intrapulmonary arteriovenous anastomoses (IPAVA).

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. A set of mouse lungs filled with liquid contrast agent.
Contrast agent (orange) was injected through the pulmonary artery (black arrow). The second cannulation pictured (*) was for the trachea and was not used in this study. Scale bar represents 10mm.
Fig 2
Fig 2. Radiography images of a whole mouse lung.
a) Sample radiograph from μCT scan demonstrating successful opacification of the pulmonary circuit. b) A cross-sectional reconstructed image where darker regions correspond to higher relative local densities of contrast agent. Reconstructed slices were acquired using NRecon (Bruker Corporation). Scale bar represents 2 mm.
Fig 3
Fig 3. Tomographic slices of mouse lung vasculature, with cropped region shown for illustrative purposes.
a) Reconstructed image slice. b) Binary image resulting from a global auto threshold using Otsu’s method on a). c) Grayscale image after applying the vesselness filter to a) with a sigma range of 1:10. d) Binary image resulting from a global auto threshold using Otsu’s method on c), after the vesselness filter was applied. Scale bar represents 150 μm and applies to all four images.
Fig 4
Fig 4. 3D reconstruction of mouse lung vasculature after filtering, global auto-thresholding, and isolation of the largest voxel cluster.
The branching of the pulmonary circuit is visualized. Large pulmonary arteries and veins are observed as well as micro-vasculature that make up the capillary bed. Scale bar represents 1 mm.
Fig 5
Fig 5. Schematic representation to illustrate defined terms used in branching centreline and junction identification.
a) Definitions of centrelines, centreline points, junction points, and terminal points. Labels also show how vessels can be defined as a set of centreline points connecting a terminal point and a junction point or a set of centreline points connecting two junction points. b) Example of an L-shaped vessel where two pixels could be erroneously labelled as junction points.
Fig 6
Fig 6
Centrelines of branching vessels highlighting removed spurious branches (red) and the final improved centreline (green).
Fig 7
Fig 7. 3D reconstruction of branching vessels where each voxel is coloured to represent vessel diameter.
Larger vessels are darker shades of red and smaller vessels are darker shades of blue. Voxels were assigned to each vessel based on proximity to its centrelines using the fast marching method.
Fig 8
Fig 8. Schematic illustrating the classification of generations.
Each time the vasculature reaches a junction point (solid black dots), a new generation is created including all vessels that sprout from the junction points reached by the previous generation.
Fig 9
Fig 9. Pulmonary circuit morphology of the mouse lungs before the spurious filtering was characterized.
a) average vessel length as a function of generation and b) average vessel diameter as a function of generation. Pulmonary circuit morphology after the spurious filtering was characterized: c) vessel length as a function of generation and d) vessel diameter per generation. Generations start with the left and right pulmonary arteries and main pulmonary vein. Shaded area represents one standard deviation.
Fig 10
Fig 10. All vessels in the full lung classified as arterial, venous, or connecting vessels (including shunts).
a) Average diameter of each generation in the pulmonary circuit of the mouse lungs, where the first generation includes the left and right pulmonary arteries and the main pulmonary vein. The stacked bar graph shows the total number of vessels per generation, differentiating between arterial, venous, and connecting vessels. The shaded region shown is one standard deviation of the vessel diameters. b) Example of an intrapulmonary arteriovenous anastomoses (IPAVA) (green) in the pulmonary circuit represented in a 3D model. The IPAVA connects two larger vessels belonging to the arterial and venous sides of the pulmonary circuit.
Fig 11
Fig 11. 3D representation of the pulmonary circuit of the mouse lungs.
Arterial (blue) and venous (red) sides of the circuit are highlighted. Scale bar represents 1 mm. Capillaries and other parts of the microvasculature were manually removed from Fig 11 in some regions so that they did not obscure the arterial vessels that appear behind the venous vessels.
See this image and copyright information in PMC

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Grants and funding

The authors would like to gratefully acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (AB), the NSERC Canada Research Chairs Program (AB), and the Canada First Research Excellence Fund (CFREF) Medicine by Design Grant (AB, CA, TW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.