Coronary artery wall imaging in mice using osmium tetroxide and micro‐computed tomography (micro‐CT)

The high spatial resolution of micro-computed tomography (micro-CT) is ideal for 3D imaging of coronary arteries in intact mouse heart specimens. Previously, micro-CT of mouse heart specimens utilized intravascular contrast agents that hardened within the vessel lumen and allowed a vascular cast to be made. However, for mouse coronary artery disease models, it is highly desirable to image coronary artery walls and highlight plaques. For this purpose, we describe an ex vivo contrast-enhanced micro-CT imaging technique based on tissue staining with osmium tetroxide (OsO(4) ) solution. As a tissue-staining contrast agent, OsO(4) is retained in the vessel wall and surrounding tissue during the fixation process and cleared from the vessel lumens. Its high X-ray attenuation makes the artery wall visible in CT. Additionally, since OsO(4) preferentially binds to lipids, it highlights lipid deposition in the artery wall. We performed micro-CT of heart specimens of 5- to 25-week-old C57BL/6 wild-type mice and 5- to 13-week-old apolipoprotein E knockout (apoE(-/-) ) mice at 10 μm resolution. The results show that walls of coronary arteries as small as 45 μm in diameter are visible using a table-top micro-CT scanner. Similar image clarity was achieved with 1/2000th the scan time using a synchrotron CT scanner. In 13-week-old apoE mice, lipid-rich plaques are visible in the aorta. Our study shows that the combination of OsO(4) and micro-CT permits the visualization of the coronary artery wall in intact mouse hearts.