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Sci Rep. 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.

Author information

1
Robarts Research Institute, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
2
Department of Medical Biophysics, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.
3
Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.
4
Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.
5
Departments of Medicine and Biochemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
6
Robarts Research Institute, The University of Western Ontario, London, Ontario, N6A 5B7, Canada. mdrangova@robarts.ca.
7
Department of Medical Biophysics, The University of Western Ontario, London, Ontario, N6A 5C1, Canada. mdrangova@robarts.ca.

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.

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