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Review
. 2021 Mar 23;22(6):3263.
doi: 10.3390/ijms22063263.

A Review of Ex Vivo X-ray Microfocus Computed Tomography-Based Characterization of the Cardiovascular System

Lisa Leyssens  1   2 , Camille Pestiaux  1   2 , Greet Kerckhofs  1   2   3   4
Affiliations
Review

A Review of Ex Vivo X-ray Microfocus Computed Tomography-Based Characterization of the Cardiovascular System

Lisa Leyssens et al. Int J Mol Sci. .

Abstract

Cardiovascular malformations and diseases are common but complex and often not yet fully understood. To better understand the effects of structural and microstructural changes of the heart and the vasculature on their proper functioning, a detailed characterization of the microstructure is crucial. In vivo imaging approaches are noninvasive and allow visualizing the heart and the vasculature in 3D. However, their spatial image resolution is often too limited for microstructural analyses, and hence, ex vivo imaging is preferred for this purpose. Ex vivo X-ray microfocus computed tomography (microCT) is a rapidly emerging high-resolution 3D structural imaging technique often used for the assessment of calcified tissues. Contrast-enhanced microCT (CE-CT) or phase-contrast microCT (PC-CT) improve this technique by additionally allowing the distinction of different low X-ray-absorbing soft tissues. In this review, we present the strengths of ex vivo microCT, CE-CT and PC-CT for quantitative 3D imaging of the structure and/or microstructure of the heart, the vasculature and their substructures in healthy and diseased state. We also discuss their current limitations, mainly with regard to the contrasting methods and the tissue preparation.

Keywords: ex vivo; heart; microCT; morphometrics; structural characterization; vasculature.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of the data; in the writing of the manuscript or in the decision to publish the results.

Figures

Figure 1
Figure 1
Schematic representation of the structure of this review. In Section 2, we provide a general description of X-ray microfocus computed tomography (microCT), contrast-enhanced microCT (CE-CT) and phase-contrast microCT (PC-CT). The third section describes the use of microCT for imaging the heart with three different parts: (A) the whole heart, (B) the morphometrical assessment of the myocardium (adapted from Reference [31]) and (C) the heart valves. Section 4 focuses on (D) the spatial distribution and morphometrics of the vasculature (adapted from Reference [5]) and (E) the vessel wall microstructure. We conclude with the current limitations and future perspectives.
Figure 2
Figure 2
Whole-heart CE-CT imaging (Appendix A). Mouse heart stained with 3.5% (wt/v) Hafnium-substituted Wells-Dawson polyoxometalate (Hf-WD POM) (A–D,K), rinsed in phosphate-buffered saline (PBS) and then stained with 3.5% isotonic Lugol’s iodine (E–H,J). (A,F) 3D renderings with the heart valves in orange and (B–E,G–J) orthogonal slices. (K,L) A zoom-in on the tricuspid valve after Hf-WD POM and isotonic Lugol’s iodine staining, respectively. Arrows indicate blood vessels, and asterisks indicate the tricuspid valve. Scale bars are 1 mm (A,B,D–G,I,J), 0.3 mm (C,H,K,L) and 0.2 mm (M,N).
Figure 3
Figure 3
Extraction of the fiber orientation in rat hearts based on PC-CT. First column: quantification of the orientation of myocyte aggregates in (A) WKY and (B) LAD hearts: ventricular helical angle maps in four chambers view. White dashed lines were used for additional illustrations. Last three columns: collagen segmentation in high-resolution images. (C–G) Representative PC-CT image slices from subvolumes in the left ventricular septum of the WKY, SHR and ISO hearts, respectively. (D–H) 3D rendering of collagen segmentation in the same subvolumes, visually showing the increase in density and change in shape and distribution around the tissue. Scar tissues can be seen in the ISO specimen. ISO: isoproterenol-treated rats, LAD: left anterior descending artery ligation model, SHR: spontaneously hypertensive rat and WKY: Wistar Kyoto rat model (adapted from and with kind permission from Reference [31], licensed under the Creative Commons Attribution 4.0 International License [95]).
Figure 4
Figure 4
MicroCT imaging of fresh calcified aortic valves (n = 3) from human patients that were explanted during aortic valve replacement (Appendix A). (A,C) Cross-sectional 2D microCT image (no contrast enhancement) of samples 1.2 and 2.3, respectively, after the region of interest (ROI) selection. (B,D) 3D rendering of samples 1.2 and 2.3, respectively; calcifications are white and soft tissue red. (E) Volume fraction of the calcifications in the entire valve. Semiautomatic segmentation of the soft tissue and the different densities of calcification were done based on greyscale differences. Scale bars represent 1 mm.
Figure 5
Figure 5
Microfil-perfused rat kidney vasculature in progressive ischemia/reperfusion (I/R)-induced renal injury. (A) Representative CE-CT renderings of the sham control and I/R days 14, 21 and 56 (2D cross-sectional images in the transversal (I), coronal (II) and sagittal (III) planes, as well as 3D volume renderings). CE-CT-based quantification of (B) the vascular branching points, (C) mean vessel tortuosity and (D) mean vessel diameter in the sham control and I/R days 14, 21 and 56 for the 4th- (Aa. interlobulares) and 5th (afferent arterioles)-order branching points. Progressive rarefaction of the functional vessels and continuous shrinkage of the fibrotic kidneys, as well as an increased vessel tortuosity over time, can be seen. Scale bars are 200 µm. ** p < 0.01; *** p < 0.001 (adapted from and with kind permission from Reference [5]).
Figure 6
Figure 6
Bone marrow vasculature of the tibial metaphysis stained with Hf-WD POM. (A) Young (YNG), (B) old and (C) high-fat diet mice (HFD). Scale bars represent 250 µm. Quantification of the (D) volume fraction of the blood vessels in the medular open volume, (E) average blood vessel thickness, (F) blood vessel density, (G) total number of branches and (H) average branch length. * p < 0.05; ** p < 0.01 (adapted from and with kind permission from Reference [78] with permission from Elsevier).
Figure 7
Figure 7
Abdominal rat aorta stained with Hf-WD POM (Appendix A). (A) Cross-sectional 2D CE-CT slice, (B) ROI selection and segmentation of aortic wall from background with an indication of the substructures, (C) segmentation of the media and adventitia and elastic fibers on a 2D ROI slice and (D) a 3D view of the segmentation on the ROI of the aortic wall. Scale bars are 100 µm (A,D) and 50 µm (B,C).
Figure 8
Figure 8
Paraffin-embedded rat common carotid artery without CESA. Major arterial substructures are readily identifiable in the vessel wall. (A) Virtual slice extracted from an X-ray tomogram of an intact rat common carotid artery (yellow box indicates the magnified region in panel (B). (B) Major arterial substructures. (C) Rendering showing the output of the segmentation process that enables the medial and adventitial layers to be virtually dissected (adapted from and with kind permission from Reference [113]—licensed under the Creative Commons Attribution 4.0 International License [95]).
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