Volume 119, Issue 3, Pages 699-705 (September 2000) Mechanisms of NSAID-induced gastrointestinal injury defined using mutant mice  Paul L. Beck, Ramnik Xavier, Naifang Lu, Nanthakumar N. Nanda, Mary Dinauer, Daniel K. Podolsky, Brian Seed  Gastroenterology  Volume 119, Issue 3, Pages 699-705 (September 2000) DOI: 10.1053/gast.2000.16497 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Inset, Southern blot analysis of wild-type (129/B6)(+/+), FTVII homozygote−/−, and FTVII heterozygote+/− mice. The 8.4-kilobase signal is a result of the insertion of the neomycin cassette into the FTVII gene. Bar graph shows expression of (α1-2/α1-3) FT activity along the length of the mouse gastrointestinal tract. The entire gastrointestinal tract was removed, washed with ice-cold saline, and divided into stomach, duodenum, jejunum, ileum, and colon before membrane fractions were prepared and assayed. The enzyme activity is expressed as total (α1-2/α1-3) FT activity (nmol−1 · h−1 · mg protein−1). Results are given as mean ± SEM. FTVII−/− mice had significantly (P < 0.001) lower total (α1-2/α1-3) FT activity levels in all tissue examined. Analysis used paired 2-tailed t test; tissues were pooled from 3 animals for each tissue and repeated 4 times. Gastroenterology 2000 119, 699-705DOI: (10.1053/gast.2000.16497) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Indomethacin-induced changes in mucosal integrity. Mucosal integrity was assessed by urinary 51Cr-EDTA excretion in control (129/B6) and FTVII−/− mice over a 20-hour period following indomethacin, 2.5–10 mg/kg intraperitoneally (the group that received 0.0 received vehicle alone). Urinary excretion of 51Cr-EDTA was calculated as percent of the oral dose given. Analysis used paired 2-tailed t test (n ≥ 4/group), expressed as mean ± SEM. *P < 0.05, **P <0.01. Gastroenterology 2000 119, 699-705DOI: (10.1053/gast.2000.16497) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Indomethacin-induced changes in mucosal integrity. Mucosal integrity was assessed by urinary 51Cr-EDTA excretion over 20 hours following 5 mg/kg indomethacin intraperitoneally. All mice were of 129/B6 background except for the gp91phox−/−, CGD mice, which were C57/B6. Paired data, gray bars; 129/B6 mice treated with control antibody and antibiotics (129/B6+Abx) vs. 129/B6 mice depleted of neutrophils with Ly6G antibody and antibiotics (anti-Ly6G), and open bars; C57/B6 mice treated with antibiotics (C57/B6+Abx) vs. gp91phox −/−, CGD mice treated with antibiotics, were analyzed with a paired 2-tailed t test. All other data (black bars: wild-type; 129/B6 [129/B6], mice with abnormal leukocyte recruitment; [FTVII−/−], mice lacking mature T and B cells [Rag 2−/−]) were analyzed using a 1-way ANOVA followed by a Dunnett's multiple comparisons post test. For all comparisons, n ≥ 4/group, expressed as mean ± SEM. *P < 0.05, **P < 0.01. Gastroenterology 2000 119, 699-705DOI: (10.1053/gast.2000.16497) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Indomethacin-induced macroscopic gastric damage. Gastric mucosal lesions were assessed 24 hours following indomethacin (5 mg/kg) and 1 hour following bethanechol (2.5 mg/kg). Most lesions were linear, thus data are expressed as average linear length per animal per group. All mice were of 129/B6 background (129/B6) except for the gp91phox−/−, CGD mice, which were C57/B6. Paired data, gray bars; 129/B6 mice treated with control antibody and antibiotics (129/B6+Abx) vs. 129/B6 mice depleted of neutrophils with Ly6G antibody and antibiotics (anti-Ly6G), and open bars; C57/B6 mice treated with antibiotics (C57/B6+Abx) vs. gp91phox−/−, CGD mice treated with antibiotics, were analyzed with a paired 2-tailed t test. All other data (black bars: wild type; 129/B6 [129/B6], mice with abnormal leukocyte recruitment; [FTVII−/−], mice lacking mature T and B cells [Rag 2−/−]) were analyzed using a 1-way ANOVA followed by a Dunnett's multiple comparisons post test. For all comparisons, n ≥ 4/group, expressed as mean ± SEM. *P < 0.05. Gastroenterology 2000 119, 699-705DOI: (10.1053/gast.2000.16497) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Histologic assessment of (A) indomethacin-induced gastric and (B) small bowel damage. Indomethacin-induced gastric (A) and small bowel (B) damage and inflammation were assessed 24 hours following indomethacin (5 mg/kg intraperitoneally) and 1 hour following bethanechol (2.5 mg/kg intraperitoneally). Data are presented as the mean ± SEM from the review of a minimum of 2 sections per animal and 4 animals per group. Paired data, 129/B6 mice treated with control antibody and antibiotics (129/B6+Abx) vs. 129/B6 mice depleted of neutrophils with Ly6G antibody and antibiotics (anti-Ly6G), and C57/B6 mice treated with antibiotics (C57/B6+Abx) vs. gp61phox−/−, CGD mice (CGD) treated with antibiotics, were analyzed with a Mann–Whitney U test for nonparametric data. All other data (wild type; 129/B6 [129/B6], mice with abnormal leukocyte recruitment; [FTVII−/−], mice lacking mature T and B cells [Rag 2−/−]) were analyzed using a Kruskal–Wallis test (nonparametric ANOVA) followed by a Dunn's multiple comparisons post test. For all comparisons; n ≥ 4/group, ≥2 sections per animal, expressed as mean ± SEM, *P < 0.05, ***P < 0.001. Histologic evaluation: damage score; 0, normal; 1, edema and/or vacuolation, minimal changes in crypt architecture; 2, superficial epithelial disruption; 3, disruption extending to the muscularis mucosae. Inflammation score: 0, normal; 1, minimal inflammatory cells; 2, moderate number of inflammatory cells; 3, severe increase in inflammatory cells. Gastroenterology 2000 119, 699-705DOI: (10.1053/gast.2000.16497) Copyright © 2000 American Gastroenterological Association Terms and Conditions