The vasa vasorum (VV) have gathered considerable interest over the last decade due to its role in vascular wall biology and pathology
however, while the coronary VV are relatively well studied, the anatomy of peripheral VV, such as those of the aorta, remains poorly described, hampering knowledge of their role in peripheral vascular diseases. Through careful retrieval of the porcine thoracic aorta and successful microthrombi removal, the thoracic aorta was perfused with BriteVu™ followed by micro-computed tomography (micro-CT) scanning to image the VV and obtain a 3D reconstruction. We used commercially available software and its thresholding algorithms. The arterial segments were further dehydrated and stained in an iodine solution to delineate the tunica adventitia and tunica media on imaging. A meticulous technique of harvest, clearing and perfusion of the porcine aorta was developed. BriteVu™ perfusion enabled clear visualization of the VV structure, branching pattern and distribution with a voxel size of 9 μm. Additional iodine staining differentiated VV residing in the tunica media versus the tunica adventitia of the vessel. The technique we have developed allows detailed knowledge of peripheral VV anatomy and distribution to be obtained where previous information was scarce. Detailed anatomical study of the VV and its relation to branches will further our understanding of its relationship to vascular disease processes.