Abstract 13256: Efficient and Precise Phenotyping of Murine Fetal Hearts by Means of Synchrotron-Based Phase-Contrast Micro-Computed Tomography

Abstract

Mouse models are routinely utilized to investigate the role of genes in congenital heart disease. Cardiovascular phenotyping is, however, quite challenging because of the small size of fetal mouse hearts. The most used method is histological assessment, which typically provides only two-dimensional information and thus might overlook important structural defects. Three-dimensional (3D) methods with sufficient resolution are either time-consuming, require specific sample preparation and/or selective staining, or cause destruction of the sample. Here, synchrotron radiation-based phase-contrast micro-computed tomography (SRμCT) was evaluated for fast and precise phenotyping of unstained, intact murine fetal hearts in a non-destructive way. Embryonic day (E) 18.5 hearts from control mice and mice that lack the secreted protease ADAMTS6 were formalin-fixed, paraffin-embedded and then scanned with SRμCT at the TOMCAT beamline (Swiss Light Source) with a 1.63 μm effective pixel size. Following SRμCT, the hearts were sectioned and processed for standard hematoxylin and eosin staining (H&E), and for specific immunofluorescence staining. SRμCT enabled evaluation of the hearts with a high level of detail. A full E18.5 mouse heart was scanned in less than 3 minutes, followed by virtual sectioning along arbitrary slicing planes and 3D rendering for morphological assessment. The resolution proved sufficient to trace minute features like the coronary arteries. In the Adamts6 knockouts, malformations such as a ventricular septal defect and double outlet right ventricle could be readily diagnosed. We also explored the potential of merging stained sections with SRμCT. In summary, SRμCT has the potential to become a powerful tool for fast phenotyping of mutant mouse models. The combination of 3D imaging with subsequent sectioning and methods like histology, immunohistochemistry and in situ hybridization will uncover pathophysiological mechanisms in 3D space.