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.
Highlights
Advances in vascular research rely heavily on detailed characterization of rodent models of disease
A strategy of using histological stains that mark specific tissue components combined with micro-CT has been applied in “virtual histology” – a technique that can be used to study the microstructure of tissues without disturbing the morphology of an organ or tissue of interest
Several groups have used high resolution micro-CT for virtual histology to visualize soaked excised organs and tissues in iodine-potassium iodide (I2KI)[14,15,16,17,18,19,20], phosphotungstic acid (PTA)[8,9,21], and osmium tetroxide (OsO4)[22] for periods of time ranging from hours to weeks
Summary
Advances in vascular research rely heavily on detailed characterization of rodent models of disease. Several groups have used high resolution micro-CT for virtual histology to visualize soaked excised organs and tissues in iodine-potassium iodide (I2KI)[14,15,16,17,18,19,20], phosphotungstic acid (PTA)[8,9,21], and osmium tetroxide (OsO4)[22] for periods of time ranging from hours to weeks. We describe a novel, tri-element x-ray attenuating stain and perfusion protocol derived from Verhoeff ’s stain that provides micro-CT contrast of the entire vasculature of an intact mouse. We describe our novel stain and demonstrate its ability to provide 3D images of vessel walls throughout the entire mouse vasculature, and its utility in virtual histology; to our knowledge, no other CT staining technique localizes contrast within the structural components of the vessel wall, thereby producing whole-mouse vasculature-specific images using 3D micro-CT, following a perfusion procedure under 30 minutes
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