Volume 137, Issue 5, Pages 1757-1767.e1 (November 2009) Oxazolone-Induced Enterocolitis in Zebrafish Depends on the Composition of the Intestinal Microbiota  Sylvia Brugman, Kit–Yeng Liu, Dicky Lindenbergh–Kortleve, Janneke N. Samsom, Glenn T. Furuta, Stephen A. Renshaw, Rob Willemsen, Edward E.S. Nieuwenhuis  Gastroenterology  Volume 137, Issue 5, Pages 1757-1767.e1 (November 2009) DOI: 10.1053/j.gastro.2009.07.069 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Total enterocolitis score and bacterial environment. (A) Total enterocolitis score of zebrafish in continuous flow tanks (white bars) (n = 4–27/group) and zebrafish in stand-alone (static) tanks (black bars) (n = 6–19/group). *P < .05; ***P < .001. (B) Representative pictures of the maximum score (intestinal bulb) for bowel-wall thickening (top left panel), intestinal-fold architecture disruption (top right panel), goblet cell depletion (bottom left panel), and infiltrating periodic acid Schiff–positive eosinophils (bottom right panel). Gastroenterology 2009 137, 1757-1767.e1DOI: (10.1053/j.gastro.2009.07.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Scores per segment and per criterion of zebrafish transferred to the stand-alone (static) tank. (A) Histological score per segment (white bars: intestinal bulb, gray bars: anterior mid-intestine, black bars: posterior mid-intestine). (B) Total enterocolitis score at indicated time points at baseline (gray bars), after injection with ethanol (50%) (white bars) or oxazolone (0.2% in 50% ethanol) (black bars), (C) bowel-wall thickening, (D) intestinal-fold architecture disruption, (E) goblet cell depletion, (F) infiltrating periodic acid Schiff–positive eosinophils. *P < .05; **P < 0.01; ***P < 0.001. Gastroenterology 2009 137, 1757-1767.e1DOI: (10.1053/j.gastro.2009.07.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Histology and electron microscopy of intestinal infiltrates. Electron microscope images of infiltrating cells in the intestine (intestinal bulb) 5 hours after oxazolone (stand-alone tank). (A) Mixed infiltrate located at the base of the fold (700×), (B) enlargement of (A) (2950×), (C) eosinophilic granulocyte and neutrophil in infiltrate (8900×), (D) higher-power view of cigar-shaped, electron-dense neutrophil cytoplasmic granules (21000×), (E) eosinophilic granulocyte (15500×), and (F) macrophage-like cell (3900×). Gastroenterology 2009 137, 1757-1767.e1DOI: (10.1053/j.gastro.2009.07.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Flow cytometry on intestinal cell suspensions. (A) representative flow cytometry plots of intestinal cell suspensions of zebrafish Tg(mpx::eGFP)i114 under static stand-alone conditions at 5 hours after ethanol (top panels) or oxazolone (bottom panels) injection (SSC, side scatter [granularity]; FSC, forward scatter [size]), (B) percentage of enhanced green fluorescent protein (EGFP)–positive cells in the intestines of untreated (gray bars), ethanol-(white bars), or oxazolone-(black bars) injected zebrafish at 5, 1 days and 7 days after administration (n = 6–13/group). Gastroenterology 2009 137, 1757-1767.e1DOI: (10.1053/j.gastro.2009.07.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Relative expression of cytokines in the intestine. Interleukin (IL)-1β (A), tumor necrosis factor–α (TNF-α) (B), and IL-10 (C) compared to β-actin on whole intestinal tissue RNA of untreated zebrafish (gray bars), ethanol-(white bars), or oxazolone-(black bars) injected zebrafish 5, 24 hours, and 1 week after administration (n = 3–6/group) all under static stand-alone tank conditions. *P < .05. Gastroenterology 2009 137, 1757-1767.e1DOI: (10.1053/j.gastro.2009.07.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Phylum composition and bacterial load of intestinal bacteria. (A) Phyla composition of intestinal bacteria by16S rRNA sequencing of flushed intestinal content (2 pools per group, consisting of 2–3 animals/pool; 45–182 clones analyzed/group). Proteobacteria (white), Firmicutes (black), Fusobacteria (dark gray), and Bacteroides (light gray), (B) colony-forming unit (CFU) counts of flushed intestinal contents on Columbia blood agar, MacConkey agar, and Tryptic soy agar under aerobic and anaerobic conditions (3–5 zebrafish per group, not pooled, 18 hours). Gastroenterology 2009 137, 1757-1767.e1DOI: (10.1053/j.gastro.2009.07.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Effect of antibiotic treatment on enterocolitis score and infiltrate composition. (A) Total colitis score of zebrafish kept in stand-alone tanks treated with antibiotics colistin sulphate or vancomycin with or without oxazolon (n = 7–19/group), (B) bowel-wall thickening, (C) intestinal-fold architecture disruption, (D) goblet cell depletion, (E) infiltrating periodic acid Schiff–positive eosinophils, (F) percentage of enhanced green fluorescent protein (EGFP)–positive cells present in the intestines of zebrafish as determined by flow cytometry (n = 11–20/group). *P < .05; **P < .01; ***P < .001. Gastroenterology 2009 137, 1757-1767.e1DOI: (10.1053/j.gastro.2009.07.069) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Effect of antibiotic treatment on the relative expression of cytokines in the intestine. Relative expression compared to β-actin of interleukin (IL)-1β (A) and IL-10 (B) in the intestines of oxazolone-injected control, colistine sulphate–treated, and vancomycin-treated zebrafish (n = 5–10/group). *P < .05; **P < .01). Gastroenterology 2009 137, 1757-1767.e1DOI: (10.1053/j.gastro.2009.07.069) Copyright © 2009 AGA Institute Terms and Conditions