Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jan 24;9(1):698.
doi: 10.1038/s41598-018-36905-z.

3D vessel-wall virtual histology of whole-body perfused mice using a novel heavy element stain

P Joy Dunmore-Buyze  1 , Charmainne Cruje  1   2 , Zengxuan Nong  1 , Jason J Lee  1   2 , John A Kiernan  3 , J Geoffrey Pickering  1   2   4   5 , Maria Drangova  6   7
Affiliations

3D vessel-wall virtual histology of whole-body perfused mice using a novel heavy element stain

P Joy Dunmore-Buyze et al. Sci Rep. .

Abstract

Virtual histology - utilizing high-resolution three-dimensional imaging - is becoming readily available. Micro-computed tomography (micro-CT) is widely available and is often coupled with x-ray attenuating histological stains that mark specific tissue components for 3D virtual histology. In this study we describe a new tri-element x-ray attenuating stain and perfusion protocol that provides micro-CT contrast of the entire vasculature of an intact mouse. The stain - derived from an established histology stain (Verhoeff's) - is modified to enable perfusion through the vasculature; the attenuating elements of the stain are iodine, aluminum, and iron. After a 30-minute perfusion through the vasculature (10-minute flushing with detergent-containing saline followed by 15-minute perfusion with the stain and a final 5-minute saline flush), animals are scanned using micro-CT. We demonstrate that the new staining protocol enables sharp delineation of the vessel walls in three dimensions over the whole body; corresponding histological analysis verified that the CT stain is localized primarily in the endothelial cells and media of large arteries and the endothelium of smaller vessels, such as the coronaries. The rapid perfusion and scanning protocol ensured that all tissues are available for further analysis via higher resolution CT of smaller sections or traditional histological sectioning.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Coronal slices from micro-CT volume images (50 µm isotropic voxels) of whole-body perfused mice. The images illustrate the difference in contrast enhancement based on the stain perfused: (a) saline, (b) PTA, (c) I2KI, (d) AlumHemFeI and (e) AlumHemFeI-T. The image slices shown were selected to demonstrate uptake by the aorta (arrowheads), heart (circle), and the liver (arrow) in the top row, and kidneys (asterisk) in the bottom row, which are labeled in (e) for reference.
Figure 2
Figure 2
Thick maximum intensity projections (1 mm thick) of the thorax and abdomen of mice perfused with the 4 different stains: PTA (a), I2KI (b), AlumHemFeI (c), and AlumHemFeI-T (d). It can be seen that the AlumHemFeI-T stain provides a clear delineation of arterial walls; the arrow points to the aortic arch.
Figure 3
Figure 3
Coronal (a,b) and sagittal (c) slices of an AlumHemFeI-T whole-body perfused mouse. Slices are taken from images acquired with a 5-minute scan protocol (90 kVp; 50 μm voxels). The images are 1-mm maximum intensity projections selected to highlight the coronary vasculature (circle in a), aortic arch (*in a,b), common carotid arteries (arrows in a,b), the internal and external carotid arteries (arrowheads in b), the cerebral vasculature (arrow in c), and the small lumbar vessels (arrowheads in c). All images can bee seen by scrolling through the Supplementary Videos SV1, SV2 and SV3.
Figure 4
Figure 4
Sections of aortas from mice perfused with PTA (a), I2KI (b), AlumHemFeI (c) and AlumHemFeI-T (d). The images in (e) and (f) are magnifications of the regions outlined in c, and d respectively. The purple colour in (a) indicates the presence of PTA within the aortic wall. The iodine within the aortic wall is orange-brown in colour (frozen section). Both AlumHemFeI (c,e) and AlumHemFeI –T (d,f) stain the interlamellar spaces, but AlumHemFeI-T provides more intense staining of the endothelium (arrow) and the endothelial cell nuclei (arrowheads in f). For negative controls see Supplementary Fig. S2.
Figure 5
Figure 5
Sections of myocardium from mice perfused with PTA (a), I2KI (b), AlumHemFeI (c) and AlumHemFeI-T (d). The images in (e) and (f) are magnifications of the regions outlined in c, and d respectively. The purple colour in (a) indicates the presence of PTA within the myocardium and coronary wall. Intense iodine staining of the myocardium is seen in (b) (frozen section). Intense Prussian blue reaction product is seen in the coronary wall (d and f) when AlumHemFeI–T is used. The endothelial and medial layers (arrow in f) and endothelial cell nuclei (arrowheads in f) appear intensely stained. For negative controls see Supplementary Fig. S3.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Elkerton JS, Xu Y, Pickering JG, Ward AD. Differentiation of arterioles from venules in mouse histology images using machine learning. J Med Imaging (Bellingham) 2017;4:021104. doi: 10.1117/1.jmi.4.2.021104. - DOI - PMC - PubMed
    1. Xu Y, Pickering JG, Nong Z, Ward AD. Segmentation of digitized histological sections for quantification of the muscularized vasculature in the mouse hind limb. J Microsc. 2017;266:89–103. doi: 10.1111/jmi.12522. - DOI - PubMed
    1. Albers, J., Markus, M. A., Alves, F. & Dullin, C. X-ray based virtual histology allows guided sectioning of heavy ion stained murine lungs for histological analysis. Sci Rep8, 10.1038/s41598-018-26086-0 (2018). - PMC - PubMed
    1. Arpino JM, et al. Four-dimensional microvascular analysis reveals that regenerative angiogenesis in ischemic muscle produces a flawed microcirculation. Circ Res. 2017;120:1453–1465. doi: 10.1161/circresaha.116.310535. - DOI - PubMed
    1. Sakaue T, et al. Perivascular adipose tissue angiotensin II type 1 receptor promotes vascular inflammation and aneurysm formation. Hypertension. 2017;70:780–789. doi: 10.1161/hypertensionaha.117.09512. - DOI - PubMed

Publication types

Substances

Grants and funding