Development of barium‐based low viscosity contrast agents for micro CT vascular casting: Application to 3D visualization of the adult mouse cerebrovasculature

Abstract

Recent advances in three-dimensional (3D) fluorescence microscopy offer the ability to image the entire vascular network in entire organs, or even whole animals.However, these imaging modalities rely on either endogenous fluorescent reporters or involved immunohistochemistry protocols, as well as optical clearing of the tissue and refractive index matching. Conversely, X-ray-based 3D imaging modalities, such as micro CT, can image non-transparent samples, at high resolution, without requiring complicated or expensive immunolabeling and clearing protocols, or fluorescent reporters. Here, we compared two "homemade" barium-based contrast agents to the field standard, lead-containing Microfil, for micro-computed tomography (micro CT) imaging of the adult mouse cerebrovasculature. The perfusion pressure required for uniform vessel filling was significantly lower with the barium-based contrast agents compared to the polymer-based Microfil. Accordingly, the barium agents showed no evidence of vascular distension or rupture, common problems associated with Microfil. Compellingly, perfusion of an aqueous BaCl 2 /gelatin mixture yielded equal or superior visualization of the cerebrovasculature by micro CT compared to Microfil.However, phosphate-containing buffers and fixatives were incompatible with BaCl 2 due to the formation of unwanted precipitates. X-ray attenuation of the vessels also decreased overtime, as the BaCl 2 appeared to gradually diffuse into surrounding tissues. A second, unique formulation composed of BaSO 4 microparticles, generated in-house by mixing BaCl 2 and MgSO 4 , suffered none of these drawbacks. These microparticles, however, were unable to pass small diameter capillary vessels, conveniently labeling only the arterial cerebrovasculature. In summary, we present an affordable, robust, low pressure, non-toxic, and straightforward methodology for 3D visualization of the cerebrovasculature.

3D visualization and morphometric analysis of spinal motion segments and vascular networks: A synchrotron radiation‐based micro‐CT study in mice

“…To visualize the intact spinal structures, including the vascular, bone, and cartilage tissues, MICROFIL® contrast agent was used to enhance the attenuation of the contrast of the vessels. The viscosity of the contrast agent results in insufficient vascular perfusion, which is another factor that limits the depiction of the intramedullary microvasculature of the spinal cord within the spine vertebral bony column (Hong et al, 2020). Our study also indicated that proper specimen fixation is important in the visualization of the anatomic features of spinal motion segments through SRμCT, including soft tissue and hard tissue that can be simultaneously visualized (Barbone et al, 2021; Jiang et al, 2021b).…”

Innovative high-resolution microCT imaging of animal brain vasculature

“…Furthermore, it minimizes sample dehydration/ shrinkage and corresponding imaging artifacts during the long-lasting high-resolution microCT scans. On the contrary, outside the brain cage, the brains are very susceptible to dehydration, which results in sample shrinkage and imaging artifacts, which are evident especially in the higher magnifications as a feathering (or shadowing) of the images (Hong et al 2020). To mitigate this problem, the researchers often use wet paper tissue or wrap the samples with cling film.…”

“…To mitigate this problem, the researchers often use wet paper tissue or wrap the samples with cling film. This reduces the dehydration, but wrapping and unwrapping of the exposed brain often damage the surface vessels (Hong et al 2020).…”

“…By employing contrast agents such as barium sulfate suspension in gel or Microfil, it is both possible to overcome the destructiveness of the approach and to enhance the signal-to-noise ratio. Unfortunately, these contrast agents are known for inducing changes in vascular anatomy like vascular distension or ruptures (Microfil) (Hong et al 2020) and other artifacts such as discontinuous/incomplete filling of the capillaries and microvessels (Perrien et al 2016) and inhomogeneous or weak signal, also due to particle aggregations (e.g., in the case with BaSo 4 suspension) (Hlushchuk et al 2018;Krucker et al 2006;Perrien et al 2016).…”

High resolution micro-CT imaging in mice stroke models: from 3D detailed infarct characterization to automatic area segmentation

“…Use of these contrast agents has been applied to the brain either by perfusion or immersion, allowing for cerebrovascular or cellular visualization in micro-CT 30 . The vasculature can be studied using polymerising compounds or agents that penetrate the vessels lumen 31–33 34–36 , meanwhile, neuronal tissue contrast can be achieved through immersion of the whole brain Different contrasts can be achieved because of the different binding affinities of the stains to the different brain cells 23 . The staining of soft tissues with ionic contrast agents has been used for imaging in the micro-CT of brain 37 and whole-body 38 of zebrafish, as well as, rodent embryos, brains and other organs 28, 29, 38 .…”