Poster Session II – Poster of Distinction II A320 3D IMAGING UNCOVERS STRUCTURAL ALTERATIONS IN THE MUSCULARIS MACROPHAGES AND ICC NETWORK ASSOCIATED WITH MICROBIOTA-MEDIATED GUT DYSFUNCTION

Abstract Background Clostridioides difficile infection (CDI) is common and often triggered by gut microbiota disruption following antibiotic exposure. Up to 25% of patients develop persistent gut dysfunction after CDI eradication, including constipation, the mechanisms of which remain unclear. Healthy colonic peristalsis results from complex interplay between interstitial cells of Cajal (ICC), muscle and enteric nervous systems, with input from immune cells, including muscularis macrophages (MMØ). We found that microbiota from patients who developed severe constipation following CDI induces slow colonic transit in germ-free mice, with MMØ playing an important role. Here we use confocal imaging to investigate whether microbiota from pCDI patients alter the ICC network and MMØ morphology. Aims To determine whether pCDI microbiota induce structural alterations in the MMØ and ICC network. Methods Germ-free NIH Swiss mice (n = 27) were colonized with fecal microbiota from 3 pCDI patients with severe constipation or 3 healthy controls. Three weeks later, colonic transit was assessed via bead expulsion. Colonic tissues were stained for c-kit+ ICC and CD68+ macrophages and imaged by 3D confocal microscopy (Nikon A1R). Image quantification and morphological analyses were performed using FIJI (ImageJ) and RStudio pipelines, with statistical analyses in GraphPad Prism (v10.4.1). Data are expressed as mean ±SEM and analyzed by Mann-Whitney or Kolmogorov-Smirnov tests. Results Mice with pCDI microbiota exhibited slower colonic transit than those with control microbiota (398 ±51.5 vs. 150 ±30.2 seconds; p