To improve our understanding of the brain microstructure, high-resolution 3D imaging is used to complement classical 2D histological assessment techniques. X-ray computed tomography allows high-resolution 3D imaging, but requires methods for enhancing contrast of soft tissues. Applying contrast-enhancing staining agents (CESAs) ameliorates the X-ray attenuating properties of soft tissue constituents and is referred to as contrast-enhanced computed tomography (CECT). Despite the large number of chemical compounds that have successfully been applied as CESAs for imaging brain, they are often toxic for the researcher, destructive for the tissue and without proper characterization of affinity mechanisms. We evaluated two sets of chemically related CESAs (organic, iodinated: Hexabrix and CA4+ and inorganic polyoxometalates: 1:2 hafnium-substituted Wells-Dawson phosphotungstate and Preyssler anion), for CECT imaging of healthy murine hemispheres. We then selected the CESA (Hexabrix) that provided the highest contrast between gray and white matter and applied it to a cuprizone-induced demyelination model. Differences in the penetration rate, effect on tissue integrity and affinity for tissue constituents have been observed for the evaluated CESAs. Cuprizone-induced demyelination could be visualized and quantified after Hexabrix staining. Four new non-toxic and non-destructive CESAs to the field of brain CECT imaging were introduced. The added value of CECT was shown by successfully applying it to a cuprizone-induced demyelination model. This research will prove to be crucial for further development of CESAs for ex vivo brain CECT and 3D histopathology.