RESUMEN
Vascular corrosion casting is a method used to visualize the three dimensional (3D) anatomy and branching pattern of blood vessels. A polymer resin is injected in the vascular system and, after curing, the surrounding tissue is removed. The latter often deforms or even fractures the fragile cast. Here, a method is proposed that does not require corrosion, and is based on in situ micro computed tomography (micro-CT) scans. To overcome the lack of CT contrast between the polymer cast and the animals' surrounding soft tissue, hafnium oxide nanocrystals (HfO2 NCs) are introduced as CT contrast agents into the resin. The NCs dramatically improve the overall CT contrast of the cast and allow for straightforward segmentation in the CT scans. Careful design of the NC surface chemistry ensures the colloidal stability of the NCs in the casting resin. Using only 5 m% of HfO2 NCs, high-quality cardiovascular casts of both zebrafish and mice can be automatically segmented using CT imaging software. This allows to differentiate even µ $\umu$ m-scale details without having to alter the current resin injection methods. This new method of virtual dissection by visualizing casts in situ using contrast-enhanced CT imaging greatly expands the application potential of the technique.
RESUMEN
High-frequency cardiac ultrasound is the only well-established method to characterize in vivo cardiovascular function in adult zebrafish noninvasively. Pulsed-wave Doppler imaging allows measurements of blood flow velocities at well-defined anatomical positions, but the measurements and results obtained using this technique need to be analyzed carefully, taking into account the substantial baseline variability within one recording and the possibility for operator bias. To address these issues and to increase throughput by limiting hands-on analysis time, we have developed a fully automated processing pipeline. This framework enables the fast, unbiased analysis of all cardiac cycles in a zebrafish pulsed-wave Doppler recording of both atrioventricular valve flow as well as aortic valve flow without operator-dependent inputs. Applying this automated pipeline to a large number of recordings from wild-type zebrafish shows a strong agreement between the automated results and manual annotations performed by an experienced operator. The reference data obtained from this analysis showed that the early wave peak during ventricular inflow is lower for female compared with male zebrafish. We also found that the peaks of the ventricular inflow and outflow waves as well as the peaks of the regurgitation waves are all correlated positively with body surface area. In general, the presented reference data, as well as the automated Doppler measurement processing tools developed and validated in this study will facilitate future (high-throughput) cardiovascular phenotyping studies in adult zebrafish ultimately leading to a more comprehensive understanding of human (genetic) cardiovascular diseases.
Asunto(s)
Corazón , Pez Cebra , Animales , Masculino , Adulto , Femenino , Humanos , Pez Cebra/fisiología , Corazón/diagnóstico por imagen , Ecocardiografía/métodos , Ventrículos Cardíacos/diagnóstico por imagen , Ultrasonografía Doppler , Velocidad del Flujo SanguíneoRESUMEN
OBJECTIVE: The zebrafish is increasingly used as a small animal model for cardiovascular disease, including vascular disorders. Nevertheless, a comprehensive biomechanical understanding of the zebrafish cardiovascular circulation is still lacking and possibilities for phenotyping the zebrafish heart and vasculature at adult - no longer optically transparent - stages are limited. To improve these aspects, we developed imaging-based 3D models of the cardiovascular system of wild-type adult zebrafish. METHODS: In vivo high-frequency echocardiography and ex vivo synchrotron X-ray tomography were combined to build fluid-structure interaction finite element models of the fluid dynamics and biomechanics inside the ventral aorta. RESULTS: We successfully generated a reference model of the circulation in adult zebrafish. The dorsal side of the most proximal branching region was found as the location of highest first principal wall stress and was also a location of low wall shear stress. Reynolds number and oscillatory shear were very low compared to mice and humans. SIGNIFICANCE: The presented wild-type results provide a first extensive biomechanical reference for adult zebrafish. This framework can be used for advanced cardiovascular phenotyping of adult genetically engineered zebrafish models of cardiovascular disease, showing disruptions of the normal mechano-biology and homeostasis. By providing reference values for key biomechanical stimuli (including wall shear stress and first principal stress) in wild-type animals, and a pipeline for image-based animal-specific computational biomechanical models, this study contributes to a more comprehensive understanding of the role of altered biomechanics and hemodynamics in heritable cardiovascular pathologies.
Asunto(s)
Enfermedades Cardiovasculares , Pez Cebra , Adulto , Humanos , Animales , Ratones , Proyectos Piloto , Sincrotrones , Aorta/diagnóstico por imagen , Hemodinámica , Tomografía Computarizada por Rayos X , Modelos Cardiovasculares , Estrés MecánicoRESUMEN
Zebrafish have come to the forefront as a flexible, relevant animal model to study human disease, including cardiovascular disorders. Zebrafish are optically transparent during early developmental stages, enabling unparalleled imaging modalities to examine cardiovascular structure and function in vivo and ex vivo. At later stages, however, the options for systematic cardiovascular phenotyping are more limited. To visualise the complete vascular tree of adult zebrafish, we have optimised a vascular corrosion casting method. We present several improvements to the technique leading to increased reproducibility and accuracy. We designed a customised support system and used a combination of the commercially available Mercox II methyl methacrylate with the Batson's catalyst for optimal vascular corrosion casting of zebrafish. We also highlight different imaging approaches, with a focus on scanning electron microscopy (SEM) and X-ray microtomography (micro-CT) to obtain highly detailed, faithful three-dimensional reconstructed images of the zebrafish cardiovascular structure. This procedure can be of great value to a wide range of research lines related to cardiovascular biology in small specimens.