Date Published: March 2, 2017
Publisher: Public Library of Science
Author(s): Rene Markovič, Julien Peltan, Marko Gosak, Denis Horvat, Borut Žalik, Benjamin Seguy, Remi Chauvel, Gregoire Malandain, Thierry Couffinhal, Cécile Duplàa, Marko Marhl, Etienne Roux, Arie Horowitz.
Quantitative analysis of the vascular network anatomy is critical for the understanding of the vasculature structure and function. In this study, we have combined microcomputed tomography (microCT) and computational analysis to provide quantitative three-dimensional geometrical and topological characterization of the normal kidney vasculature, and to investigate how 2 core genes of the Wnt/planar cell polarity, Frizzled4 and Frizzled6, affect vascular network morphogenesis. Experiments were performed on frizzled4 (Fzd4-/-) and frizzled6 (Fzd6-/-) deleted mice and littermate controls (WT) perfused with a contrast medium after euthanasia and exsanguination. The kidneys were scanned with a high-resolution (16 μm) microCT imaging system, followed by 3D reconstruction of the arterial vasculature. Computational treatment includes decomposition of 3D networks based on Diameter-Defined Strahler Order (DDSO). We have calculated quantitative (i) Global scale parameters, such as the volume of the vasculature and its fractal dimension (ii) Structural parameters depending on the DDSO hierarchical levels such as hierarchical ordering, diameter, length and branching angles of the vessel segments, and (iii) Functional parameters such as estimated resistance to blood flow alongside the vascular tree and average density of terminal arterioles. In normal kidneys, fractal dimension was 2.07±0.11 (n = 7), and was significantly lower in Fzd4-/- (1.71±0.04; n = 4), and Fzd6-/- (1.54±0.09; n = 3) kidneys. The DDSO number was 5 in WT and Fzd4-/-, and only 4 in Fzd6-/-. Scaling characteristics such as diameter and length of vessel segments were altered in mutants, whereas bifurcation angles were not different from WT. Fzd4 and Fzd6 deletion increased vessel resistance, calculated using the Hagen-Poiseuille equation, for each DDSO, and decreased the density and the homogeneity of the distal vessel segments. Our results show that our methodology is suitable for 3D quantitative characterization of vascular networks, and that Fzd4 and Fzd6 genes have a deep patterning effect on arterial vessel morphogenesis that may determine its functional efficiency.
Precise and comprehensive measurements of vascular network anatomy are crucial steps for the analysis of normal and pathologic vascular networks, and is of paramount importance for the understanding of several aspects of the vasculature structure and function . Microcomputed Tomography (microCT) has emerged in recent years as the preferred modality for vascular studies, because it provides high-resolution three-dimentional (3D) volumetric data suitable for visualization and analysis at the level of an organ or set of tissues . We have previously used this microCT technique specifically to study the arterial network in a hind limb, heart and kidney [3–5]. However, in most of the studies about 3D analysis of a vascular network, including our previous ones, quantitative characterization of the vessel network pattern suffered several limitations. Except for some general parameters, such as vascular volume, most morphometric parameters are computed primarily after dimensional reduction of the 3D micro-CT volumes to 2D sections, and quantification lacks topographical analysis of the vascular network.
In this study, we have used microCT scanning of mouse kidneys, followed by computational skeletonization and topographical analysis to characterize the 3D pattern of the normal arterial vasculature. Applying this methodology to genetically modified mice, we have shown that the cellular arrangements responsible for vascular morphogenesis were coordinated, in part, by two PCP core genes, Fzd4 and Fzd6. MicroCT vascular scanning is the best tool to screen transgenic mice for 3D vascular impairment . However, in most cases, the analysis is limited to the visualization of the vasculature with few, if any, quantitative evaluations of the vascular structure. Skeletonization of mCT is a powerful tool to go beyond the mere visual observation of the vasculature and to provide a detailed study of the changes in structure and their potential effects on hemodynamics. The method was found to generate data with sufficient accuracy and reproducibility to reveal new quantitative information on the pathophysiology of vessel morphogenesis. Algorithms for such reconstructions, based on detailed anatomical data, have been applied to the porcine coronary arterial vasculature [38, 41] and to the cerebral circulation [1, 42, 43]. The accuracy of such procedures depends on the correct identification of the continuity of the vascular tree. During the segmentation, this continuity may be artefactually lost because of inhomogeneous contrast agent filling or pixel discretization of the vessels. Our methodology includes the application of a morphological closing operator that minimalizes these artefactual discontinuities and, hence, allows a more accurate skeletonization of the arterial tree.