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.
Highlights
The development of advanced imaging techniques for phenotyping the heart and circulatory system in small animal models has provided novel insights into cardiovascular pathophysiology [1]
In order to assess the feasibility of μCT imaging to visualize hearts ex-vivo in great detail, hearts were excised from healthy female adult mice (Table 1) and stained with phosphotungstic acid (PTA) for 6 days before embedding in agarose
In order to demonstrate that the approach is not limited to synchrotron radiation based μCT (SRμCT), explanted mouse hearts stained in the same way were scanned with a classical benchtop μCT (S1 Fig)
Summary
The development of advanced imaging techniques for phenotyping the heart and circulatory system in small animal models has provided novel insights into cardiovascular pathophysiology [1]. To pursue this research avenue new developments are necessary to combine high resolution organ imaging with the characterization of the analysed tissue. Histological analysis is well established, and allows the depiction and discrimination of various tissue and cell types as well as extracellular matrix at high spatial resolution. Analysing the heart by histology and IHC provides only planar information about the sample and the loss of tissue as well as implementation of artefacts due to the cutting procedure leads to a fragmentary depiction [2]. CT imaging of soft-tissue like the heart muscle or vessels produces poor contrast, but which can be enhanced by applying staining solutions or contrast agents containing heavy ions such as iodine, barium or tungsten [4]. PTA, among other contrast agents such as Lugol, has recently been used for highcontrast imaging of insects and embryonic tissues of mouse and chicken at histological resolutions using commercial μCT systems [7,8,9]
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