Volume 131, Issue 6, Pages 1844-1855 (December 2006) A Mouse Model of Hereditary Pancreatitis Generated by Transgenic Expression of R122H Trypsinogen  Herbert Archer, Natalia Jura, James Keller, Matthew Jacobson, Dafna Bar–Sagi  Gastroenterology  Volume 131, Issue 6, Pages 1844-1855 (December 2006) DOI: 10.1053/j.gastro.2006.09.049 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 Transgene construct and expression analysis. (A) Schematic representation of the R122H_mPRSS1 transgene construct. Ela, elastase promoter; Tr, trypsinogen; UTR, untranslated region. (B) Immunohistochemical detection of acinar-specific transgene expression using anti-FLAG antibody (original magnification 100× and 400×). (Inset, bottom right panel in B) The granular localization of FLAG-trypsin in R122H_mPRSS1 mice. (C) Real-time RT-PCR analysis of the relative expression levels of the transgene in R122H_mPRSS1 animals of different ages. The average value obtained for 4 wild-type animals was subtracted from expression level obtained for each transgenic animal. Gastroenterology 2006 131, 1844-1855DOI: (10.1053/j.gastro.2006.09.049) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 Acinar cell necrosis is an early manifestation of the phenotype in R122H_mPRSS1 transgenic mice. (A) H&E image of the wild-type pancreas showing normal acinar (ac) and duct (d) structures (original magnification 100×). (B) Representative H&E image from a 7-week-old R122H_mPRSS1 animal indicates increased vacuolization of the acinar cells (original magnification 200×). (C) Immunohistochemical staining for apoptotic cells with anti-cleaved caspase-3 antibody in the pancreata of R122H_mPRSS1 mice (original magnification 200×). (D and E) Immunohistochemical staining for necrotic cells with anti-HMG1 antibody in (D) wild-type and (E) R122H_mPRSS1 mice (original magnification 200×). Arrows point to the nuclear accumulation of HMGB-1 in the wild-type animal and its cytosolic translocation in the transgenic mouse. Gastroenterology 2006 131, 1844-1855DOI: (10.1053/j.gastro.2006.09.049) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 R122H_mPRSS1 transgenic mice develop inflammatory lesions. (A) Intra-acinar inflammation (arrow) in the pancreata of 16-week-old mice (original magnification 200×). (B) Immunohistochemical analysis showing positive Flag staining (arrows) in association with inflammation (asterisk) (original magnification 200×). (C) A fibrotic region in 1-year-old R122H_mPRSS1 mice (original magnification 50×). (D) Immunohistochemical staining for α–smooth muscle actin in a fibrotic lesion from a 1-year-old R122H_mPRSS1 mouse (original magnification 100×). (E) Trichrome staining (blue) indicates deposition of collagen in the areas of fibrosis in a 1-year-old R122H_mPRSS1 mouse (original magnification 200×). Insets represent higher magnifications of selected areas in the corresponding images. (F) Quantitative analysis of the inflammatory phenotype observed in R122H_mPRSS1 animals. Data are presented as percent of the animals that display fibrotic lesions in the indicated age group. Wild-type mice, gray bars; R122H_mPRSS1 mice, black bars; n, number of animals within the age group. Wild-type animals used in this experiment had the same genetic background as transgenic animals. The statistical significance of the differences was calculated using the Fisher exact test (*P < .05). Gastroenterology 2006 131, 1844-1855DOI: (10.1053/j.gastro.2006.09.049) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 Characterization of the inflammatory infiltrates in R122H_mPRSS1 mice. (A) A representative H&E image of an inflammatory infiltrate from an R122H_mPRSS1 mouse (original magnification 100×). Immunohistochemical staining of R122H_mPRSS1 inflammatory infiltrates for (B) the common leukocyte antigen CD-45, (C) the T-cell specific marker CD-3, (D) the B-cell specific marker B220, (E) the macrophage specific marker F4-80, and (F) a proinflammatory cytokine, TNF-α. Insets represent higher magnifications of selected areas in the corresponding images. Gastroenterology 2006 131, 1844-1855DOI: (10.1053/j.gastro.2006.09.049) Copyright © 2006 AGA Institute Terms and Conditions

Figure 5 The effect of cerulein administration on R122H_mPRSS1 mice. Representative H&E images of sections obtained from (A and C) wild-type and (B and D) R122H_mPRSS1 mice at (A and B) 1 day and (C and D) 1 week after cessation of cerulein treatment (original magnification 100×). (E and F) Assessment of fibrotic reaction in (E) wild-type and (F) R122H_mPRSS1 mice by trichrome staining for collagen (blue) (original magnification 200×). (G) Morphometric quantitation of fibrosis in the wild-type (light gray bars) and R122H_mPRSS1 mice (dark gray bars) at 1 day and 1 week after cerulein injection. Error bars represent SEM; differences between experiments were examined using Student t test (*P < .05). Gastroenterology 2006 131, 1844-1855DOI: (10.1053/j.gastro.2006.09.049) Copyright © 2006 AGA Institute Terms and Conditions

Figure 6 Inflammation induces compartment-specific activation of signaling pathways in pancreata of R122H_mPRSS1 mice. Evaluation of the extent of activation of JNK, NF-κB, and ERK signaling pathways by immunohistochemical analysis of (A–C) c-jun (original magnification 400×), (D–F) p65 (original magnification 400×), and (G–I) pERK (original magnification 100×) expression pattern in pancreata of 6–12-month-old R122H_mPRSS1 animals and age-matched controls. Gastroenterology 2006 131, 1844-1855DOI: (10.1053/j.gastro.2006.09.049) Copyright © 2006 AGA Institute Terms and Conditions

Figure 7 Chronic inflammatory lesions in R122H_mPRSS1 mice. (A) A representative H&E image of intralobular inflammation and fibrosis in a 1-year-old R122H_mPRSS1 animal (original magnification 100×). (B) Immunohistochemical assessment of acinar cell proliferation in a fibrotic area from the R122H_mPRSS1 mouse using proliferating cell nuclear antigen as a marker (original magnification 400×). Note the cytosolic staining of proliferating cell nuclear antigen, which represents cells that entered mitosis (arrow). (C and D) Selected areas from A are shown at higher magnification (original magnification 400×). Note the decreased apical cell height (arrowhead) and the enlargement of the acinar lumen (arrow). (E) Immunohistochemical staining for the acinar cell marker amylase (original magnification 400×). (F) Immunohistochemical staining for the ductal cell marker cytokeratin 19 (original magnification 200×). Arrows point to the cytokeratin 19–negative tubular structures. Gastroenterology 2006 131, 1844-1855DOI: (10.1053/j.gastro.2006.09.049) Copyright © 2006 AGA Institute Terms and Conditions