What can we see with diceCT? Comparison of iodine-enhanced brains and histological atlases in a rat model

Major advancement and refinement of neurological imaging modalities have the potential to enable new understanding in nervous system organization. One recent example, diffusible iodine-based contrast-enhanced computed tomography (diceCT), is an iodine staining and X-ray μCT-imaging technique that allows for the differentiation of myelinated from unmyelinated nervous tissues at finer scales of spatial resolution than standard 3D brain-imaging tools allow. DiceCT is versatile, capable of imaging specimens across several orders of magnitude in size and enabling large and complex structures such as human brains to be studied alongside those of much smaller organisms like fish, frogs, and birds. The staining agent can also be removed, allowing the subsequent application of acetate, fluorescent, and immunofluorescent histology following diceCT imaging. However, because of its relative newness, ground-truthing diceCT by determining its reliably for distinguishing precise boundaries between neuroanatomical structures is critical for its potential broad application by anatomists, neuroscientists, and organismal biologists. For example, some tissue-level neuroanatomical features are poorly differentiated due to similar make-up of adjacent structures or obscure boundaries (such as the thalamus, which houses many small white-matter tracts). To address this, we compare 3D orientation-matched image stacks of diceCT juvenile Sprague Dawley rat brains to well-established, standard 2D brain atlases. We score the consistency, repeatability, and error associated with documenting atlas-neuroanatomical features in diceCT images. Visual identification of brain structures was attempted for all brain regions, structure grayscales were documented, and results were exclusively categorized as: (i) visibly distinct and replicable, (ii) visible but with ambiguous boundaries; (iii) visibly distinct and replicable after image enhancement, (iv) visibly distinct but non-replicable (e.g., right/left staining asymmetry), or (v) visibly non-distinct and non-replicable. We used this scheme to evaluate which neuroanatomical structures are distinguishable using diceCT and what proportion of all Paxinos and Watson atlas structures they make up. Preliminary results show that diceCT rat brain sections align well with standard cresyl violet histological sections, and visibility of neuroanatomical features is similar to tissue-based, 3D magnetic resonance imaging atlases of comparable resolution. We examine the potential future for diceCT as a powerful neuroanatomical imaging standard.