Abstract
Advancements in tissue visualization techniques have spurred significant gains in the biomedical sciences by enabling researchers to integrate their datasets across anatomical scales. Of particular import are techniques that enable the interpolation of multiple hierarchical scales in samples taken from the same individuals. In this study, we demonstrate that two-dimensional histology techniques can be employed on neural tissues following three-dimensional diffusible iodine-based contrast-enhanced computed tomography (diceCT) without causing tissue degradation. This represents the first step toward a multiscale pipeline for brain visualization. We studied brains from adolescent male Sprague–Dawley rats, comparing experimental (diceCT-stained then de-stained) to control (without diceCT) brains to examine neural tissues for immunolabeling integrity, compare somata sizes, and distinguish neurons from glial cells within the telencephalon and diencephalon. We hypothesized that if experimental and control samples do not differ significantly in morphological cell analysis, then brain tissues are robust to the chemical, temperature, and radiation environments required for these multiple, successive imaging protocols. Visualizations for experimental brains were first captured via micro-computed tomography scanning of isolated, iodine-infused specimens. Samples were then cleared of iodine, serially sectioned, and prepared again using immunofluorescent, fluorescent, and cresyl violet labeling, followed by imaging with confocal and light microscopy, respectively. Our results show that many neural targets are resilient to diceCT imaging and compatible with downstream histological staining as part of a low-cost, multiscale brain imaging pipeline.
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Acknowledgements
We owe special thanks to all UTEP Systems Neuroscience Laboratory members, especially Daniel F. Hughes and Arshad M. Khan, for their valuable comments and feedback. Also, we thank Dr. Daniela Carulli from Neuroscience Otto Lenghi center in University of Turin for the valuable insight in image analysis. We thank Morgan H. Chase and the AMNH Microscopy and Imaging Facility for assistance.
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PMG was supported by NSF (1450850, 1457180, and 1754659) and the American Association for Anatomy. DVS, HDO, and PMG were supported by the OSU-CHS department of Anatomy and Cell Biology. DVS and HDO were also supported by the OSU-CHS Office of the Vice President for Research. JS was supported by pre-doctoral fellowship from the National Mexican Council of Education (CONACyT).
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Gignac, P.M., O’Brien, H.D., Sanchez, J. et al. Multiscale imaging of the rat brain using an integrated diceCT and histology workflow. Brain Struct Funct 226, 2153–2168 (2021). https://doi.org/10.1007/s00429-021-02316-6
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DOI: https://doi.org/10.1007/s00429-021-02316-6