μCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry

Abstract

The small size of the adult and developing mouse heart poses a great challenge for imaging in preclinical research. The aim of the study was to establish a phosphotungstic acid (PTA) ex-vivo staining approach that efficiently enhances the x-ray attenuation of soft-tissue to allow high resolution 3D visualization of mouse hearts by synchrotron radiation based μCT (SRμCT) and classical μCT. We demonstrate that SRμCT of PTA stained mouse hearts ex-vivo allows imaging of the cardiac atrium, ventricles, myocardium especially its fibre structure and vessel walls in great detail and furthermore enables the depiction of growth and anatomical changes during distinct developmental stages of hearts in mouse embryos. Our x-ray based virtual histology approach is not limited to SRμCT as it does not require monochromatic and/or coherent x-ray sources and even more importantly can be combined with conventional histological procedures. Furthermore, it permits volumetric measurements as we show for the assessment of the plaque volumes in the aortic valve region of mice from an ApoE-/- mouse model. Subsequent, Masson-Goldner trichrome staining of paraffin sections of PTA stained samples revealed intact collagen and muscle fibres and positive staining of CD31 on endothelial cells by immunohistochemistry illustrates that our approach does not prevent immunochemistry analysis. The feasibility to scan hearts already embedded in paraffin ensured a 100% correlation between virtual cut sections of the CT data sets and histological heart sections of the same sample and may allow in future guiding the cutting process to specific regions of interest. In summary, since our CT based virtual histology approach is a powerful tool for the 3D depiction of morphological alterations in hearts and embryos in high resolution and can be combined with classical histological analysis it may be used in preclinical research to unravel structural alterations of various heart diseases.

Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice

“…PTA staining has been used on both embryonic and adult mice to visualize internal organ systems, including the heart and kidneys . Our protocol builds upon these advances, permitting in situ study of these and other developing embryonic organs.…”

“…Compared with potential deformations in soft tissue morphology resulting from iodine staining, water‐based PTA staining solutions have demonstrated no systematic alteration to osseous and soft tissue structures of middle ear samples, despite the potential for soft tissue shrinkage . Ethanol‐based PTA staining solutions have also reported low rates of shrinkage . Notably, studies of excised murine hearts and kidneys stained with 0.7% PTA in 70% ethanol actually resulted in a slight swelling of the organs, causing a 9% to 12% increase in volume, while subsequent chemical drying and embedding procedures resulted in shrinkage and a 7% to 19% decrease in volume .…”

Enhanced contrast in X‐ray microtomographic images of the membranous labyrinth using different X‐ray sources and scanning modes

“…Sometimes, PCI increases the CNR sufficiently in order to alleviate the need for soft tissue staining (Dullin et al. ). In other cases, however, this proved not to be sufficient and, in these cases, better results were obtained using both PCI and a staining procedure (Dullin et al.…”

“…In other cases, however, this proved not to be sufficient and, in these cases, better results were obtained using both PCI and a staining procedure (Dullin et al. ; Saccomano et al. ).…”

“…) and because it has been shown to yield especially good results using synchrotron‐based X‐ray μCT scanning (Dullin et al. ; Saccomano et al. ).…”