Enteric Glial Networks Visualized using SOX10 Fluorescent Reporter in Optically‐Cleared Full Thickness Intestinal Tissues

Abstract

Enteric glia are important components of the enteric nervous system (ENS) that control critical functions of the alimentary canal including motility, fluid and ion transport, and immune barrier. Anatomical assessments of enteric glia to date have utilized microdissected whole‐mount tissue or thin sections to visualize cells in the optically dense matrix of the bowel. The aim of this study was to assess the morphological features of enteric glia throughout the small and large intestines of the mouse in intact full‐thickness preparations. B6.Cg‐Polr2atm1(CAG‐GCaMP5g,‐tdTomato)Tvrd/J mice were bred to homozygosity with B6.Cg‐Tg(SOX10‐cre/ERT2)Vpa mice which were maintained as hemizygous. Mice received a gavage of tamoxifen (5mg in 0.2ml corn oil) for five days at seven to eight weeks of age to induce tdTomato (tdT) expression in glia. Mice were euthanized by CO2 inhalation at 10–20 weeks of age. Jejunum and mid colon segments were opened on the mesenteric border, fixed in 4% paraformaldehyde, immunostained for anti‐S100β, and cleared with Ce3D solution. Tissues were imaged using confocal microscopy and 3D reconstructions were made using Analyze. SOX10‐tdT cells were found in the bowel throughout all layers. In the muscularis externa of both the jejunum and colon, there were extensive networks within myenteric ganglia, intraganglionic fiber tracts, within the circular muscle layer, along fiber tracts ascending through the circular muscle, and rare cells were observed in the longitudinal muscle layer. In the submucosal layer of both the jejunum and colon, SOX‐tdT cells were in submucosal ganglia, along intraganglionic fibers and formed a regular network of processes that extended within the lamina propria surrounding the base of intestinal crypts. In the mucosa, SOX10‐tdT distribution differed between the jejunum and colon. In the colonic mucosa, there were two distinct bands of SOX10‐tdT networks oriented perpendicular to and surrounding all colonic glands which were approximately 120μm in height. These bands were located approximately 20μm and 60μm from the base of the glands. SOX10‐tdT cells were very rarely observed near the luminal surface of the colonic glands. In the jejunal mucosa, there were extensive SOX10‐tdT networks throughout the lamina propria surrounding crypts and extending to the tips of villi. Overlap of SOX10‐tdT and S100β immunoreactivity was nearly 100% throughout all glial populations with two exceptions, only 10–20% of SOX10‐tdT cells had S100β immunoreactivity in the jejunum mucosa and there were submucosal neurons that expressed S100β that did not contain SOX10‐tdT. Collectively, these data demonstrate significant complexity of the glial networks of the intestines with distinct morphological differences between mucosal glia of the colon and jejunum that may implicate different functions of enteric glia in the small and large intestines.Support or Funding InformationSupported by NIH grants R01DK106011, P30DK084567 and DP2AT010875 and Department of Defense grant W81XWH‐18‐1‐0218.Representative SOX10‐tdT fluorescence in projections of 409 or 139 1.22μm‐thick confocal images through the full thickness of either jejunum (left) or colon (right), respectively.Figure 1