Volume 124, Issue 4, Pages 972-982 (April 2003) Emergence of perianal fistulizing disease in the SAMP1/YitFc mouse, a spontaneous model of chronic ileitis  Jesús Rivera-Nieves, Giorgos Bamias, Alda Vidrich, Marco Marini, Theresa T. Pizarro, Marcia J. McDuffie, Christopher A. Moskaluk, Steven M. Cohn, Fabio Cominelli  Gastroenterology  Volume 124, Issue 4, Pages 972-982 (April 2003) DOI: 10.1053/gast.2003.50148 Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 1 Histologic progression of ileitis in SAMP1/YitFc mice. (A) H&E × 20 original magnification, (B) H&E ×100 original magnification. Histologic ileal sections from a 4-week-old mouse using the Swiss-roll technique. Normal villous/crypt mucosal architecture is seen throughout with a minimal increase in inflammation. There is no thickening of the muscular wall of the intestine. (C) H&E ×20 original magnification, (D) H&E ×100 original magnification. Ten-week-old mouse ileum showed segmental inflammatory disease with loss of normal villous architecture and expansion of the lamina propria with inflammatory cells (neutrophils, lymphocytes, macrophages, and plasma cells) within areas of inflammation. The inflammatory changes are most severe in the distal portion of the ileum (center of roll) and are accompanied by thickening of the muscular wall. (E) H&E ×20 original magnification, (F) H&E ×100 original magnification. Twenty-week-old mouse ileum showed further increases in the extent and degree of inflammatory changes, with pronounced transmural inflammation and further hypertrophy of the muscular layer. (G) H&E ×20 original magnification, (H) H&E ×100 original magnification. In 70-week-old mouse ilea, an increase in the extent of muscular hypertrophy can be appreciated under low power. The high-power image shows an area of ulceration over a pre-existing lymphoid aggregate, similar to the aphthous ulcerations seen in Crohn's disease. In this area, serosal inflammation, indicative of transmural involvement, is particularly pronounced. An area of relatively normal architecture overlying a prominent lymphocyte aggregate and marked inflammation shows the segmental nature of the disease. (Ileal sections representative of the levels of inflammation seen in the different age groups are depicted.) Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 2 Time course of the development of ileal inflammation in SAMP1/YitF Mice. (A) Villus distortion index (flattening or widening of normal mucosal villous architecture). (B) Active inflammatory index (neutrophilic infiltration). (C) Chronic inflammatory index (lymphocytes, plasma cells, and macrophages in the mucosa and submucosa). (D) Total inflammatory score is the sum of the villus, active, and chronic inflammatory indices. Data are presented as mean ± SEM (n = 12, 12, 24, and 18 for 4-, 10-, 40-, and 70-week-old mice, respectively). Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 3 Macroscopic features of ileum, MLNs, and Peyer patches (arrowhead) from SAMP1/YitFc mice with established ileitis. Mice develop chronic inflammation at their terminal ilea (i), which can be identified clearly as it joins the cecum (c). A marked increase in the size and cellularity of the MLN, which coalesces into a single mass at the point of convergence of vessels draining the inflamed ileal segment, was observed in mice older than 10 weeks. (Image representative of MLN seen in 40-week-old SAMP1/YitFc mice.) Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 4 Flow cytometric analysis of MLN cells from 4- and 40-week-old SAMP1/YitFc mice. MLN cells (106 cells per condition) were double labeled with CD4-fluorescein isothiocyanate- and (A) CD45RB-PE- or (B) CD62-phycoerythrin-conjugated monoclonal antibodies, fixed with 1% paraformaldehyde, gated on forward scatter and side scatter, and analyzed by flow cytometry. (Representative dot-plot histograms obtained from 4- and 40-week-old SAMP1/YitFc mice are shown, n = 12-24 mice/age group.) Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 5 Cytokine profiles of MLN cells from 4- and 40-week-old SAMP1/YitFc and AKR mice. MLN cells (1 × 106/mL) were cultured on anti-CD3-coated plates (10 μg/mL) for 48 hours. Secreted (A) IFN-γ, (B) TNF, (C) IL-10, (D) IL-4, and (E) IL-5 from the culture supernatants were measured by specific enzyme-linked immunosorbent assays. Data are presented as mean ± SEM (n = 12-24 mice/age group, *P < 0.001 vs. AKR, †P < 0.01 vs. 4-week-old SAMP1/YitFc mice, ‡P < 0.001 vs. 4-week-old SAMP1/YitFc mice). □, AKR; ▩, SAMP1/YitFc. Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 6 Histologic features of chronic ileitis in SAMP1/YitFc mice. (A) Representative uninflamed ileal section from 4-week-old mice showed normal villous and crypt mucosal architecture, as well as the normal thickness (bracket) of the muscularis propria (H&E ×100 original magnification). (B) Representative inflamed ileal section from 40-week-old mice showed an increased mucosal and submucosal inflammatory infiltrate, with loss of normal villous architecture. The typical hypertrophy of the muscularis propria (bracket) that accompanied the inflammatory lesions is seen clearly (H&E ×100 original magnification). (C) Representative uninflamed ileal section from 4-week-old mice showed the normal complement of collagen fibers (stained blue) within the submucosa (bracket) (Masson trichrome ×200 original magnification). (D) Representative inflamed ileal section from 40-week-old mice showed collagen distribution present primarily within the submucosa (bracket). This stain showed that the increase in width of the muscularis propria is caused primarily by hypertrophy of the muscle fibers (Masson trichrome ×200 original magnification). Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 7 Stricture formation and collagen deposition in SAMP1/YitFc mice. (A) Discrete areas of strictured terminal ileum (arrows) with proximal dilatation (intervening segment) in SAMP1/YitFc mice at 50 weeks of age. (B) Excised terminal ileum segment from AKR mice (top) compared with terminal ileum from SAMP1/YitFc mice (bottom) showed thickened intestinal wall and strictured (arrows)/dilated segments (between arrows). (C) Trichrome-stained strictured segment showed intense focal collagen deposition (blue fibrils) within the hypertrophic muscularis propria. A transmural inflammatory infiltrate is seen within the fibrotic segment (arrow) (Masson trichrome ×20 original magnification). (D) Procollagen α-1 mRNA expression in mouse ilea. Total RNA samples, isolated from ilea and jejuna of three 50-week-old SAMP1/YitFc (S-1, S-2, S-3) and 50-week-old AKR mice (A-1, A-2, A-3) were assayed for pro-α-1 (i) chain of mouse procollagen mRNA by reverse-transcription PCR. Data are presented as mean ± SD of the ratio (inflamed ileum:uninflamed jejunum) of procollagen mRNA for each mouse (n = 3 per mouse strain, *P < 0.05 vs. uninflamed jejunum). is, inflammatory score. ■, Jejunum; ▩, ileum. Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 8 Macroscopic features of perianal disease in SAMP1/YitFc mice. Representative lesions shown are (A) a fissuring mucosal ulceration that extended to the perianal dermis, approximately 2 cm from the anal orifice. (B) Prolapse of the anus and portions of the distal rectum was observed commonly in affected animals. (C) Anocutaneous fistula. (D) Exploration of the anocutaneous fistula with a probe showed a fistulous tract from the anus to the perianal skin. Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 9 Microscopic features of perianal disease in SAMP1/YitFc mice. (A) H&E ×20 original magnification. The anal canal (asterisk) shows extensive mucosal ulceration, accompanied by an inflammatory infiltrate in the surrounding soft tissue. A fistulous tract (arrows) begins in the anal canal and extends laterally through perianal soft tissues. (B) The exit point of the fistulous tract shown in A is in an area of cutaneous ulceration (asterisk) in the perianal skin. The fistulous tract (arrows) travels through an area of inflamed subcutaneous tissue (H&E ×20 original magnification). (C) The margin of the anal ulcer from A is shown at higher magnification. Residual squamous epithelium is seen at the top of the figure, transitioning to denuded mucosa. A submucosal acute and chronic inflammatory infiltrate is shown (H&E ×200 original magnification). (D) The lumen of the subcutaneous fistulous tract from B is shown at higher magnification. The tract is lined by an exudate of fibrin and neutrophils. Fecal material is present within the lumen (H&E ×400 original magnification). Gastroenterology 2003 124, 972-982DOI: (10.1053/gast.2003.50148) Copyright © 2003 American Gastroenterological Association Terms and Conditions