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Collection: CLARITY and three-dimensional (3D) imaging of the mouse and porcine colonic innervation

Little information is available about 3D anatomical and phenotypical maps of the colonic enteric nerve system (ENS) that are indispensable for understanding the structure-function relationships. CLARITY is a newly developed clearing technique removing lipids while keeping proteins in the tissue. However, its application has been largely restricted to the mouse brain. We aim to establish CLARITY protocols applicable for clearing the mouse and porcine colon and demonstrate 3D imaging and phenotyping of colonic innervation, especially in the porcine whose colonic ENS resembles the human unlike rodents. 4 male adult C57BL/6J mice and 2 male adult Yucatan minipigs were used for CLARITY. The mouse proximal and distal colon (pC and dC), porcine pC, transverse (tC) and dC were dissected. After CLARITY procedures, IHC and multiple labeling of PGP9.5, Hu C/D, neurofilament heavy chain, S100?, peripheral choline acetyltransferase and neuronal nitric oxide synthase were performed. Images acquired with ZEISS LSM710 confocal and SP8 DIVE multi photon microscope were reconstructed into 3D images and videos using Imaris 9.1 for neuroscientists. Colonic samples of both mice and pigs were successfully cleared which enable immunohistochemistry (IHC) and maintenance of fluorophores during imaging. 3D analysis revealed in mice: (i) Segmental differences in innervation (pC>dC); (ii) Connectivity between submucosal and myenteric plexus (SP and MP); (iii) Proximity of enteric neurons and enteric glial cells in SP and MP. In porcines: (i) Connectivity between inner SP (ISP) and outer SP (OSP) or OSP and MP; (ii) Rich innervation of the mucosa primarily from ISP; (iii) Distinct patterns of intrinsic cholinergic innervation with ISP>OSP. We have successfully developed CLARITY/IHC protocols applicable for 3D imaging and phenotyping of colonic innervation in the mouse and porcine colon. The information from 3D images and videos, not readily revealed in 2D, provides foundermental information to distinguish the different classes of enteric neurons, visualize their projections to different layers and compare their differences among the segments.
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