The Utility of DiceCT Imaging for High-Throughput Comparative Neuroanatomical Studies

Abstract

Advancements in imaging techniques have drastically improved our ability to visualize, study, and digitally share complex, often minute, anatomical relationships. The recent adoption of soft-tissue X-ray imaging techniques, such as diffusible iodine-based contrast-enhanced computed tomography (diceCT), is beginning to offer previously unattainable insights into the detailed configurations of soft- tissue complexes across Metazoa. As a contrast agent, dissolved iodine diffuses deeply throughout preserved specimens to bind fats and carbohydrates that are natural ly present within metazoan soft tissues, increasing the radiodensities of these tissues in predictable ways. Like the current “gold standard” of magnetic resonance imaging, diceCT does not require physical dissection and can differentiate between the lipid content of myelinated versus nonmyelinated tissues, thereby offering great potential for neuroanatomical studies. Within the brain, for example, diceCT distinguishes myelinated fiber tracts from unmyelinated cortices, nuclei, and ganglia and allows three-dimensional visualization of their anatomical interrelationships at previously unrealized spatial scales. In this study, we illustrate the utility of diceCT for the rapid visualization of both external and internal brain anatomy in vertebrates – alongside the intact bones of the skull and the complete, undisturbed pathways of peripheral nerves, up to and including the target organs that they innervate. We demonstrate the transformative potential of this technique for developing high-resolution neuroanatomical datasets and describe best practices for imaging large numbers of specimens for broad evolutionary studies across vertebrates.

ViceCT and whiceCT for simultaneous high-resolution visualization of craniofacial, brain and ventricular anatomy from micro-computed tomography

“…We envisage a wide array of possible applications where this novel methodology covers an unmet need, such as when planning for targeting a specific brain region with stereotactic injection, which currently needs a brain µMRI co-registered with a skull µCT as a reference 18 , 59 , 60 . Other examples where viceCT and/or whiceCT could be instrumental are phylogenetic and evolutionary studies 27 , as well as studies in neuroscience research that involve precise phenotyping and experimental procedures with (transgenic) animal models of neurodevelopmental disorders with simultaneous affectation of the brain and craniofacial structure.…”

“…Diffusible iodine-based contrast-enhanced computed tomography (diceCT) could provide an alternative to image the brain and the craniofacial complex within a single acquisition. DiceCT is a time and cost efficient ex vivo imaging procedure that uses iodine as contrast agent, thereby allowing the visualization of both skeletal and soft tissues at the high resolution from a µCT scan 22 – 27 . However, diceCT procedures for adult mice only manage to deliver the contrast to the brain at the expense of the integrity of the craniofacial complex.…”

diceCT: A Valuable Technique to Study the Nervous System of Fish

“…Lugol’s iodine appears to be the best performing staining solution for brain tissues of larger, postembryonic specimens (>1–2 cm), as it shows good tissue penetration and differentiation throughout samples (including deeper brain areas), despite the shrinkage observed (although less shrinkage seems to occur in denser brain regions, such as the cerebellum; Buytaert et al, 2014 ). Lugol’s iodine is of specific interest in neurobiological research, as it enables one to differentiate myelinated and unmyelinated neuronal tissue within both the central and peripheral nervous systems in vertebrates ( Efimova et al, 2013 ; Gignac and Kley, 2014 , 2018 ). μCT imaging using Lugol’s solution has been increasingly used in recent years, offering diverse applications across taxa and/or tissues of interest ( Metscher, 2009a ; Descamps et al, 2014 ).…”

“…However, few studies have undertaken a rigorous assessment of stain penetration using Lugol’s iodine. And, only the cephalic region of some species of freshwater fish, reptiles, birds, and mammals have been studied using diceCT ( Holliday et al, 2006 , 2013 ; Gignac and Kley, 2014 , 2018 ; Anderson and Maga, 2015 ; Clement et al, 2015 ; Li et al, 2016 ). Staining procedures (length of time in stain and stain concentration) have been found to vary significantly in different animal groups, with sample size and the mitigation of tissue shrinkage being the major drivers.…”

“…Staining procedures (length of time in stain and stain concentration) have been found to vary significantly in different animal groups, with sample size and the mitigation of tissue shrinkage being the major drivers. To date, no marine and/or cartilaginous fish species have been examined using this method, and there are limited published data available on how diceCT can be used to investigate neuroanatomy in fish ( Gignac et al, 2016 ; Gignac and Kley, 2018 ).…”