Rapid 3D Phenotyping of Cardiovascular Development in Mouse Embryos by Micro-CT With Iodine Staining

Abstract

Background-Microcomputed tomography (micro-CT) has been used extensively in research to generate high-resolution 3D images of calcified tissues in small animals nondestructively. It has been especially useful for the characterization of skeletal mutations but limited in its utility for the analysis of soft tissue such as the cardiovascular system. Visualization of the cardiovascular system has been largely restricted to structures that can be filled with radiopaque intravascular contrast agents in adult animals. Recent ex vivo studies using osmium tetroxide, iodinated contrast agents, inorganic iodine, and phosphotungstic acid have demonstrated the ability to stain soft tissues differentially, allowing for high intertissue contrast in micro-CT images. In the present study, we demonstrate the application of this technology for visualization of cardiovascular structures in developing mouse embryos using Lugol solution (aqueous potassium iodide plus iodine). Methods and Results-We show the optimization of this method to obtain ex vivo micro-CT images of embryonic and neonatal mice with excellent soft-tissue contrast. We demonstrate the utility of this method to visualize key structures during cardiovascular development at various stages of embryogenesis. Our method benefits from the ease of sample preparation, low toxicity, and low cost. Furthermore, we show how multiple cardiac defects can be demonstrated by micro-CT in a single specimen with a known genetic lesion. Indeed, a previously undescribed cardiac venous abnormality is revealed in a PlexinD1 mutant mouse. Conclusions-Micro-CT of iodine-stained tissue is a valuable technique for the characterization of cardiovascular development and defects in mouse models of congenital heart disease. (Circ Cardiovasc Imaging. 2010;3:314-322.)Key Words: micro-CT Ⅲ iodine Ⅲ mouse Ⅲ development Ⅲ PlexinD1 Ⅲ congenital heart disease T he ability to genetically manipulate the mouse has resulted in a powerful model system for the investigation of many disease processes. In particular, genetic studies in the mouse have enhanced our understanding of embryonic development, and by extension, of congenital defects. In humans, cardiac defects are the most common serious anomalies among live births with an estimated frequency of 0.6%. 1 Numerous mouse models of congenital heart disease have been generated and characterized, adding greater insight into the molecular and cellular origins of these defects. 2 In addition, current research in the area of targeted gene deletions holds great promise to further elucidate mechanisms of cardiac development. Editorial see p 228 Clinical Perspective on p 322Although structurally similar to the human, the significantly reduced size of the murine cardiovascular system presents a number of technical challenges when attempting to stage anatomic features such as vascular structures. Identification and characterization of the phenotype of cardiovascular defects in mice traditionally has relied on histological analysis of sectioned specimens. Histology,...

Quantitative Three‐Dimensional Analysis of Embryonic Chick Morphogenesis Via Microcomputed Tomography

“…A persistent challenge, however, was the limited ability to visualize and quantify soft tissues using Micro-CT. We and others have overcome this limitation with the optimization of osmium tetroxide (OT) as a CT dense contrast agent, showing dramatic visualization capability in mouse embryos without altering specimen geometry (Johnson et al, 2006;Metscher, 2009;Degenhardt et al, 2010). This study complements these by quantitatively analyzing embryonic chick morphogenesis.…”

“…More recently, we demonstrated using osmium tetroxide (OT) as a diffusive CT contrast agent that high resolution detailed visualization and segmentation of rodent embryos to newborns was possible with Micro-CT; these previous studies included early stage mouse and rabbit embryos (Johnson et al, 2006;Carney et al, 2007;Martinez et al, 2009). Investigators have since followed with related studies in model organisms to explore the effects of different chemical agents on soft tissue contrast enhancement (Metscher, 2009;Degenhardt et al, 2010).…”

X‐ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation

“…Metscher (2009a) suggested that stains are usually not tissue‐specific and the final quality depended mainly on the fixative. However, comparisons between different stains have shown that indeed some compounds can stain lipidic tissue more intensely (e.g., Lugol's solution) (Degenhardt et al., 2010); thus, recognition of different tissues can be greatly influenced by the chosen stain (Smith et al., 2016). …”

“…PTA failed to stain the complete sample (neither head nor abdomen), and the few stained regions had very strong contrast, precluding adequate tissue recognition (Figure 4a‐d). The unstained parts of the samples exposed to PTA had minor contrast, resembling tissues that had not been exposed to any stain (Degenhardt et al., 2010; Metscher, 2009a), thus indicating that the tissue penetration was insufficient and samples would have to be exposed for more days to attain the desired stain.…”

High-Resolution CT for Small-Animal Imaging Research

“…Alternatively, rapid 3D phenotyping of cardiovascular development in mouse embryos has been achieved by ex vivo micro-CT with iodine staining (Degenhardt et al, 2010). In vivo micro-CT-based cardiac morphological and functional imaging in mice and rats has been reported using both PG and RG approaches (Badea et al, 2005(Badea et al, , 2008cBartling et al, 2007a,b;Detombe et al, 2008;Drangova et al, 2007;Nahrendorf et al, 2007;Song et al, 2007).…”