μCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry.
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.
Institute for Diagnostic and Interventional Radiology, University Medical Center, Goettingen, Germany.|Synchrotron Light Source 'Elettra,' Trieste, Italy.
Christian
http://orcid.org/0000-0003-4297-8077
C
Dullin
Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine, Goettingen, Germany.
Roser
R
Ufartes
Synchrotron Light Source 'Elettra,' Trieste, Italy.
Emanuel
E
Larsson
Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine, Goettingen, Germany.|Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Goettingen, Germany.
Sabine
S
Martin
Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine, Goettingen, Germany.
Marcio
M
Lazzarini
Synchrotron Light Source 'Elettra,' Trieste, Italy.
Giuliana
G
Tromba
Institute for Diagnostic and Interventional Radiology, University Medical Center, Goettingen, Germany.|Clinic for Haematology and Medical Oncology, University Medical Center, Goettingen, Germany.
Jeannine
J
Missbach-Guentner
Clinic for Haematology and Medical Oncology, University Medical Center, Goettingen, Germany.
Diana
D
Pinkert-Leetsch
Institute of Cardiovascular Physiology, University Medical Center, Goettingen, Germany.
Dörthe M
DM
Katschinski
Institute for Diagnostic and Interventional Radiology, University Medical Center, Goettingen, Germany.|Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine, Goettingen, Germany.|Clinic for Haematology and Medical Oncology, University Medical Center, Goettingen, Germany.
Frauke
F
Alves
D000818:Animals; D004622:Embryo, Mammalian / Q000166:cytology* / Q000000981:diagnostic imaging*; D005260:Female; D006321:Heart / Q000000981:diagnostic imaging*; D021621:Imaging, Three-Dimensional; D007120:Immunochemistry; D051379:Mice; D018345:Mice, Knockout; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D058226:Plaque, Atherosclerotic / Q000000981:diagnostic imaging / Q000473:pathology; D055114:X-Ray Microtomography / Q000379:methods