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

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 stainsthat mark specific tissue components for 3D virtual histology. In this study we describe a new tri-element x-ray attenuating stainand 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 stainare 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 stainand a final 5-minute saline flush), animals are scanned using micro-CT. We demonstrate that the new stainingprotocol enables sharp delineation of the vessel walls in three dimensions over the whole body; corresponding histologicalanalysis verified that the CT stainis 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 histologicalsectioning.