Protocol for Post-Mortem Micro-CT Imaging of Coronary Arteries in Low-Mass Neonatal Puppy Hearts Using Barium-Based...

Aim: We aimed to provide a structured ex vivo protocol for cardiopulmonary micro-CT that combines gelatin-barium sulfate (gelatin-BaSO₄) contrast medium with agar embedding in neonatal canine cardiopulmonary specimens. Materials and Methods: Heart-lung specimens from 23 puppies that died shortly after birth were collected, stored at -20 °C, and then slowly thawed prior to imaging. Before perfusion, body mass and heart-lung complex mass were recorded. Body mass ranged from 140 to 951 g, and heart-lung complex mass ranged from 1.2 to 51.2 g. The cranial and caudal venae cavae, the brachiocephalic trunk, and the left subclavian artery were ligated. A catheter was introduced into the thoracic aorta. Contrast was prepared by dissolving porcine gelatin in hot water and mixing with a commercial BaSO₄ suspension. The mixture was maintained at a warm temperature to remain free-flowing and was delivered at low pressure until uniform opacification of the coronary and pulmonary arteries was observed. After in situ gelation, the organs were embedded in warm agar and sealed to limit motion and dehydration. Scans were performed on a benchtop system (120 kV, ~83 µA, ~1200 projections, ~2 s exposures; voxel ~40 µm). Reconstruction was performed in XMReconstructor, with post-processing in Falcon and RadiAnt. The reconstructed micro-CT datasets were reviewed anatomically by a medical cardiologist and a veterinary cardiologist, whereas vascular filling was evaluated semi-quantitatively by three observers with expertise in veterinary anatomy and cardiology. Results: In all specimens examined, the main coronary artery course was assessable. Conclusions: The gelatin-BaSO₄ contrast medium combined with agar immobilization provides a simple, lead-free, and affordable approach for structured cardiopulmonary micro-CT in very small post-mortem specimens. In the examined specimens, the workflow provided visually consistent low-pressure vascular opacification without gross evidence of vessel rupture or motion-related acquisition failure under the conditions of this study. Practical mitigations included temperature/viscosity control, avoidance of phosphate buffers, container sealing, and minimization of particle aggregation, bubbles, and dehydration. The protocol may complement conventional autopsy in very small post-mortem specimens in similar ex vivo research settings.