Chemically induced mouse models of intestinal inflammation

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1 Chemically induced mouse models of intestinal inflammation Stefan Wirtz, Clemens Neufert, Benno Weigmann & Markus F Neurath Laboratory of Immunology, I Medical Clinic, University of Mainz, Mainz, Germany. Correspondence should be addressed to M.F.N. (neurath@1-med.klinik.uni-mainz.de). Published online 15 March 2007; doi: /nprot PROTOCOL Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and ulcerative colitis, the two major forms of inflammatory bowel disease in humans. Here, we provide protocols for establishing murine 2,4,6- trinitro benzene sulfonic acid (TNBS)-, oxazolone- and both acute and chronic dextran sodium sulfate (DSS) colitis, the most widely used chemically induced models of intestinal inflammation. In the former two models, colitis is induced by intrarectal administration of the covalently reactive reagents TNBS/oxazolone, which are believed to induce a T-cell-mediated response against hapten-modified autologous proteins/luminal antigens. In the DSS model, mice are subjected several days to drinking water supplemented with DSS, which seems to be directly toxic to colonic epithelial cells of the basal crypts. The procedures for the hapten models of colitis and acute DSS colitis can be accomplished in about 2 weeks but the protocol for chronic DSS colitis takes about 2 months. INTRODUCTION Crohn disease (CD) and ulcerative colitis (UC) are the two major forms of chronic inflammatory bowel disease (IBD). Although these two diseases have some characteristics in common, important clinical and genetic differences exist. UC primarily affects the mucosal lining of the colon and rectum, whereas CD usually affects the whole intestinal wall and potentially may extend to any part of the gastrointestinal tract 1. The etiology of both CD and UC still remains largely unclear. However, in recent years, epidemiologic and genetic studies in man and particularly, in IBD-related animal models suggested that a combination of genetic susceptibility factors and altered immune response driven by microbial factors in the enteric environment contributes to the initiation and chronification of these diseases. The clinical appearance of human IBD is heterogeneous, a fact that is also reflected by the steadily increasing number of transgenic or gene-targeted mouse strains displaying IBD-like intestinal alterations 2,3. When chosen appropriately, these models can be used to investigate pathophysiological mechanisms and they are valuable tools to test emerging therapeutic strategies in the preclinical phase. As the onset of inflammation is immediate and the procedure is relatively straightforward, chemically induced models of intestinal inflammation belong to the most commonly used IBD animal models. Although they have, like all other models, limitations, they resemble in some important immunological and histopathological aspects IBDs in humans. TNBS colitis Colitis can be induced in susceptible strains of mice, rats and rabbits by intrarectal instillation of the haptenating substance TNBS in ethanol. Ethanol is required to break the mucosal barrier, whereas TNBS is believed to haptenize colonic autologous or microbiota proteins rendering them immunogenic to the host immune system. As CD4 + T cells have been shown to play a central role in chronic TNBS colitis, this model is useful to study T helper cell-dependent mucosal immune responses. In the mouse, the inflammatory process is highly dependent on the strain, and individual optimization of concentrations of TNBS administered is required, particularly to generate chronic colitis. Murine TNBS colitis has been initially described in SJL/J mice, a mouse strain with high susceptibility that develops chronic TNBS colitis characterized by a predominant Th1-mediated immune response with dense infiltrations of lymphocytes/macrophages and thickening of the colon wall 4,5. However, studies with IFN-g / mice on a Balb/c background showed that in these mice TNBS colitis may be associated with a Th2-mediated colonic patch hypertrophy 6. Oxazolone colitis Rectal administration of the hapten reagent oxazolone dissolved in ethanol induces a severe colitis in rats or mice, characterized by weight reduction, diarrhea and marked loss of goblet cells, leading to high death rates 7,8. In SJL/J mice, the inflammation affects only the distal colon and particularly mucosal layers. Histological features and an elevated production of Th2 cytokines (IL-4, IL-5 and IL-13) of unstimulated and acd3/acd28-stimulated lamina propria T cells are in these mice, in some aspects, similar to characteristics that have been observed in human UC. In contrast to several other murine colitis models, treatment with neutralizing anti-il-4 antibodies or a decoy IL-13Ra2-Fc protein ameliorates disease 8,9. Therefore, this model has been used to study the contribution of the Th2-dependent immune response to intestinal inflammation. As in the TNBS model, oxazolone colitis is straindependent and requires individual optimization. This is exemplified by the fact that oxazolone at lower doses can induce a mixed Th1/Th2-dependent colitis 10. DSS colitis Feeding mice, rats, hamsters or guinea-pigs for several days with DSS polymers in the drinking water induces an acute colitis characterized by bloody diarrhea, ulcerations and infiltrations with granulocytes 11. It is generally believed that DSS is directly toxic to gut epithelial cells of the basal crypts and affects the integrity of the mucosal barrier. As T- and B-cell-deficient C.B-17 scid or Rag1 / mice also develop severe intestinal inflammation, the adaptive immune system obviously does not play a NATURE PROTOCOLS VOL.2 NO

2 major part (at least in the acute phase) in this model 12. Hence, the acute DSS colitis model is particularly useful to study the contribution of innate immune mechanisms of colitis. In susceptible strains, the administration of DSS for several cycles (e.g., 7 days DSS, 14 days water) results in chronic colitis and if combined with a single initial dose of the genotoxic colon carcinogen azoxymethane (AOM), in inflammation-associated colorectal cancer 13. Here, protocols for murine TNBS and oxazolone colitis and both acute and chronic DSS colitis are described. Although the experimental procedures are in principle easy to perform, individual differences in the intestinal microflora between animal facilities and the genetic heterogeneity of mouse inbred strains require pilot experiments to optimize the dosages of TNBS, oxazolone and DSS 14. In addition, one has to consider that there is a substantial variability in different lots of these substances. As covalently MATERIALS REAGENTS week-old sex- and age-matched mice of strain of choice; at least five or more mice per experimental group recommended m CRITICAL All animals must be maintained in accordance with local and national animal care regulations.. TNBS (Sigma, cat. no. p2297) 5% (w/v) in H2 O! CAUTION Harmful. Handle using appropriate safety equipment and measures.. 4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone; Aldrich, cat. no )! CAUTION Harmful. Handle using appropriate safety equipment and measures.. Acetone! CAUTION Flammable. Handle using appropriate safety equipment and measures.. Olive oil. Absolute ethanol! CAUTION Flammable. Handle using appropriate safety equipment and measures.. 50% (v/v) ethanol in distilled water. Ketamine (Ketavest 100 mg ml 1 ; Pfizer). Xylazin (Rompun 2%; Bayer Healthcare). AOM (Sigma)! CAUTION May cause cancer.. Sterile isotonic saline. DSS salt (molecular weight: 36,000 50,000 Da; MP Biomedicals) m CRITICAL It is of critical importance to use DSS of this molecular weight. Other forms of DSS do not induce reproducible colitis or lead to high mortality.. Autoclaved drinking water. Sterile 1 phosphate-buffered saline (PBS) solution without calcium and magnesium (Gibco). Sterile gentamycin sulfate solution (Calbiochem). Sterile cell culture medium: RPMI 1640 medium (PAA) supplemented with 10% heat-inactivated FBS (PAA), 100 U ml 1 penicillin, 100 U ml 1 streptomycin and 2 mm L-glutamine (PAA) EQUIPMENT. Vortexer. Electric razor reactive compounds, TNBS and oxazolone induce intestinal damage. High dosages of these compounds can, therefore be associated with significant death rates owing to colon perforation. The protocols provided below give reproducible results in the common C57BL/6 strain (H2 b ), which shows intermediate to high susceptibility to acute DSS colitis. For this strain, a DSS concentration of 2% (w/v) in the drinking water for 7 days induces strong colitis, but low mortality rates. Some other laboratories use higher DSS concentrations for a shorter time period (e.g., 3.5% for 5 days). Three cycles of 2% (w/v) DSS (7 days DSS, 14 days water) induce in our hands long-lasting chronic DSS colitis in these mice. C57BL/6 mice have been shown to be relatively resistant to TNBS colitis compared with SJL/J(H2 s ) and Balb/c(H2 d ) mice. However, when using the given protocol, which includes a presensitization step, this strain develops significant TNBS colitis.. Small brush. Balance. 3,5 French (F) catheter (B1.2 mm in diameter) or alternatively a blunt-tip steel gavage needle. 1 ml syringe. 3 mm dermal punch (Aesculap Inc.). Cell culture incubator with 5% CO2 humidified air (Thermo Scientific) REAGENT SETUP TNBS presensitization solution Mix acetone and olive oil in a 4:1 volume ratio by rigorous vortexing. Mix 4 volumes of acetone/olive oil with 1 volume of 5% TNBS solution to obtain 1% (w/v) TNBS. Mix by rigorous vortexing.! CAUTION Both acetone and TNBS are harmful. Handle using appropriate safety equipment and measures. TNBS solution Mix 1 volume of 5% (w/v) in H 2 OTNBSsolutionwith 1 volume of absolute ethanol.! CAUTION Flammable. Handle using appropriate safety equipment and measures. Oxazolone presensitization solution Mix acetone and olive oil in a 4:1 volume ratio by rigorous vortexing. Dissolve 60 mg of oxazolone salt in 2 ml of acetone/olive oil to obtain a 3% (w/v) oxazolone solution. Mix by rigorous vortexing.! CAUTION Both acetone and oxazolone are harmful. Handle using appropriate safety equipment and measures. Oxazolone solution Dissolve 20 mg oxazolone salt in 2 ml of 50% ethanol to obtain a 1% (w/v) solution.! CAUTION Flammable. Handle using appropriate safety equipment and measures. Ketamine/xylazine solution Mix 0.6 ml of ketamine (100 mg ml 1 ), 0.4 ml of xylazine (20 mg ml 1 ) and 4 ml of saline. DSS-supplemented drinking water To make 500 ml of a 2% DSS solution, dissolve 10 g DSS powder in 500 ml of autoclaved drinking water. Store until use at 4 1C. AOM solution Dissolve AOM in sterile distilled water to obtain a 10 mg ml 1 stock solution. Aliquot and freeze at 20 1C. m CRITICAL Avoid repeated freeze and thaw cycles. On the day of use, thaw aliquot and dilute 1/10 with sterile saline. PROCEDURE 1 The experimental procedures to induce TNBS- (option A), oxazolone- (option B) and both acute (option C) and chronic (option D) DSS-dependent colitis are described below. Additionally, a protocol to generate colitis-associated colorectal cancer is provided (option E). (A) TNBS colitis (i) On day 1, carefully shave a cm field of the skin of the mouse using an electric razor. To avoid the mouse from licking TNBS (may potentially induce oral tolerance), preferentially select an area on the back between the shoulders. (ii) While holding the mouse with one hand, apply with the other hand, using a 200 ml pipette,150ml of the TNBS presensitization solution to the shaved abdominal skin. The solution is absorbed by the skin quickly. Control mice are treated with presensitization solution without TNBS. Leave the mice until day 8. (iii) On day 8, weigh and mark the mouse. (iv) Anesthetize the mouse by intraperitoneal injection of 80 ml of ketamine/xylazine solution per 10 g body weight. 542 VOL.2 NO NATURE PROTOCOLS

3 (v) Fit a 3.5 F catheter to a 1 ml syringe and fill with TNBS solution. (vi) Insert the catheter into the colon 4 cm proximal to the anus.! CAUTION Proceed carefully to avoid damage or perforation of the colon wall. (vii) Slowly administer 100 ml of TNBS solution into the colon lumen. (viii) Remove the catheter gently from the colon and keep the mouse with the head down in a vertical position for 60 s. m CRITICAL STEP To get reproducible results, it is important to ensure that the TNBS solution remains completely in the colon lumen. (ix) Return the mouse to the cage. (B) Oxazolone colitis (i) On day 1, carefully shave a 1.5 cm 1.5 cm field of the skin of the mouse using an electric razor. To avoid the mouse from licking oxazolone (may potentially induce oral tolerance), preferentially select an area on the back between the shoulders. Control mice are treated with presensitization solution without oxazolone. (ii) While holding the mouse with one hand, apply with the other hand, using a 200 ml pipette,150ml of the oxazolone presensitization solution to the shaved abdominal skin. The solution is absorbed by the skin quickly. (iii) On day 8, weigh and mark the mouse. (iv) Anesthetize the mouse by intraperitoneal injection of 80 ml of ketamine/xylazine solution per 10 g body weight. (v) Fit a 3.5 F catheter to a 1 ml syringe and fill with oxazolone solution. (vi) Insert the catheter into the colon 4 cm proximal to the anus.! CAUTION Proceed very carefully to avoid damage or perforation of the colon wall. (vii) Slowly administer 100 ml of oxazolone solution into the colon lumen. (viii) Remove the catheter gently from the colon and keep the mouse with the head down in a vertical position for 60 s. m CRITICAL STEP To get reproducible results, it is important to ensure that the oxazolone solution remains completely in the colon lumen. (ix) Return the mouse to the cage. (C) Acute DSS colitis (i) On day 1, weigh and mark mice. (ii) Fill the drinking supply of the mouse cages with DSS solution. Calculate 5 ml DSS solution per mouse per day. Control mice receive the same drinking water without DSS. m CRITICAL STEP Mount the bottle lids properly, and ensure that the tips are not congested. (iii) Empty the remaining DSS solution from the cage water bottles at day 3 and refill with DSS solution for another 2 days. (iv) Empty the remaining DSS solution from the bottles at day 5 and refill with DSS solution. (v) Replace the remaining DSS solution by autoclaved water on day 8. (D) Chronic DSS colitis (i) Proceed until Step iv of the acute DSS colitis protocol described in option C. (ii) Replace the remaining DSS solution at day 8 by autoclaved drinking water without DSS for 14 days. (iii) Repeat Steps (ii) (iv) of option C on days ? TROUBLESHOOTING (iv) Replace the remaining DSS solution on day 29 with autoclaved water for 14 days. (v) Repeat Steps (ii) (iv) of option C on days ? TROUBLESHOOTING (vi) Replace the remaining DSS solution on day 50 by autoclaved water. (E) Colitis associated dependent colorectal cancer (i) On day 1, weigh and mark mice. (ii) Inject mice intraperitoneally with 10 mg kg 1 AOM solution! CAUTION AOM may cause cancer. (iii) Proceed with Steps (i) (vi) of the chronic DSS colitis protocol (option D). Sampling of material for evaluation of intestinal inflammation 2 At time point of choice (see ANTICIPATED RESULTS), kill the mouse by cervical dislocation and place suspine on a surgical pad. Using sterile forceps and scissors, carefully open the mouse by a ventral midline incision. Remove the full colon and determine its weight and length. Rinse colon with sterile PBS by using a steel gavage needle attached to a 5 ml syringe to remove feces. 3 Depending on the experimental objectives and equipment available, there are several ways for analyzing chemically induced experimental colitis. Some common options to sample material for subsequent analysis are described here: isolation of mesenteric lymph node (MLN) cells (option A), histological analysis of colitis severity (option B), full-thickness organ culture (option C) and tissue sampling (option D) before protein or mrna analysis. NATURE PROTOCOLS VOL.2 NO

4 (A) Isolation of MLN cells (i) Localize and remove MLNs aseptically. (ii) Make single-cell suspension by pressing through a 40 mm cell strainer positioned on a 50 ml Falcon tube using the plunger of a 1 ml syringe. (iii) Rinse the cell strainer with 10 ml of RPMI cell culture medium. (iv) Pellet cells by centrifugation for 10 min (400g, 20 1C). (v) Discard the supernatant and resuspend cells in 10 ml RPMI cell culture medium. (vi) Pellet cells by centrifugation for 10 min (400g, 20 1C). (vii) Discard the supernatant and resuspend cells in 2 ml RPMI cell culture medium. (viii) Count cells for subsequent fluorescence-activated cell sortingor cytokine-based analysis. (B) Histological evaluation of colitis severity a Body weight (%) c Day Control Ethanol TNBS Oxazolone (i) Cut defined 0.5 cm long colonic fragments from proximal and distal parts of the colon. (ii) Immerse immediately in neutral buffered 10% formalin and incubate overnight at 4 1C. (iii) Prepare 5 mm paraffin cross-sections. (iv) Stain sections with hematoxylin and eosin using appropriate standard procedures. (C) Full-thickness organ culture (i) Open colon longitudinally beginning at the anus with a surgical scissors. (ii) To remove residual intestinal bacteria, wash the colon three times with cold PBS containing 20 mg ml 1 gentamycin. (iii) Using a 3 mm dermal punch biopsy instrument, make 4 6 circular specimens from proximal and distal parts of the colon. (iv) Transfer each biopsy into separate wells of a 48-well plate containing 500 ml of supplemented RPMI1640 culture medium. (v) Incubate for h at 37 1C in a cell culture incubator with 5% CO 2 humidified air. (vi) Collect supernatants and freeze at 20 1C for later cytokine measurements. (D) Tissue sampling for quantitative PCR analysis or protein-based applications (e.g., western blot, myeloperoxidase determination) (i) Cut 0.5 cm long colonic fragments from proximal and distal parts of the colon. (ii) Snap-freeze fragments in liquid nitrogen and store until use at 80 1C. TNBS Figure 1 C57BL/6 mice were subjected to the TNBS or oxazolone colitis protocol as described in the text. (a) Weight analysis after rectal challenge with ethanol only, TNBS or oxazolone. (b) Sample miniendoscopic images 3 days after colitis induction. (c) Photographs of hematoxylin/eosin-stained colon cross-sections 3 days after colitis induction. b Oxa TNBS Oxa a Body weight (%) DSS w/o DSS Day * ** *** *** Figure 2 Acute DSS colitis C57BL/6 mice were subjected to the DSS colitis protocol as described in the text. (a) Weight development after supplementing the drinking water with DSS. (b) Sample miniendoscopic images of day-8 acute DSS colitis and a colonic adenomatous tumor after three cycles of DSS combined with a single initial treatment with AOM. b TIMING TNBS/oxazolone colitis Day 1: presensitization Day 8: induction of colitis Acute DSS colitis Day 1: DSS Day 3: change DSS Day 5: change DSS Day 8: water Chronic DSS colitis Days 1, 22 and 43: DSS Days 3, 24 and 45: change DSS Days 5, 26 and 47: change DSS Days 8, 29 and 50: water 544 VOL.2 NO NATURE PROTOCOLS

5 ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. TABLE 1 Troubleshooting table. Problem Possible reason Solution High incidence of death Mouse strain has low susceptibility for colitis Decrease the dose of colitis-inducing reagent No or only weak colitis Mouse strain has low susceptibility for colitis Increase the dose of colitis-inducing reagent High variability within experimental groups ANTICIPATED RESULTS Colitis in the models described here is usually strongly associated with wasting disease (Figs. 1 and 2). Determination of weight every day or every other day is, therefore, important to get a rough idea of colitis severity and is often indicative of differences in colitis development between experimental groups. In addition, inflammation-associated rectal bleeding can be determined by examination of blood in the stool, for example, by fecal occult blood test (Table 2). Another, more sophisticated method to monitor colitis severity without the need to kill the mice is high-resolution miniature endoscopy (Figs. 1 and 2). As intestinal inflammation is usually strongest then, most investigators kill mice at days 3 5 after intrarectal challenge with TNBS or oxazolone. In the DSS model, a good time point for termination of the experiment with high damage scores and epithelial repair activity is days After killing of the mice, colitis development can be characterized using different methods. Standard methods include histological analysis of hematoxylin/eosin-stained colon sections. Several systems to quantify/ evaluate acute and chronic colitis-associated histological alterations in the colon mucosa have been developed 18,19. One scoring system, for example, to assess the hapten-based models, is provided in Table 3. The determination of myeloperoxidase activity in the gut tissue is an important indicator of neutrophil infiltration in the intestinal mucosa 20. It is important to obtain information about immunological features of the inflammatory response in the gut. This can be achieved by isolation and characterization of lamina propria mononuclear cells and immune cells obtained from MLNs. In addition, fresh full-thickness colon fragments of a defined size can be incubated in culture media to analyze cytokine responses. DSS colitis: bottle lids are not tight or are congested TNBS/oxazolone colitis: colon is physically damaged, TNBS/oxazolone solution does not remain completely in the colon TABLE 2 Scoring system for the comparative analysis of intestinal bleeding. Score Stool consistency Blood 0 Normal Negative hemoccult 1 Soft but still formed Positive hemoccult 2 Very soft Blood traces in stool visible 3 Diarrhea Rectal bleeding Check bottle lids every day Carefully execute intrarectal application step; do not damage or puncture the gut; hold the mice in a vertical position (heads down) after removing the catheter TABLE 3 Scoring system for inflammation-associated histological changes in the colon. Score Histologic changes 0 No evidence of inflammation 1 Low level of inflammation with scattered infiltrating mononuclear cells (1 2 foci) 2 Moderate inflammation with multiple foci 3 High level of inflammation with increased vascular density and marked wall thickening 4 Maximal severity of inflammation with transmural leukocyte infiltration and loss of goblet cells COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at Reprints and permissions information is available online at reprintsandpermissions 1. Podolsky, D.K. Inflammatory bowel disease. N. Engl. J. Med. 347, (2002). 2. Wirtz, S. & Neurath, M.F. Animal models of intestinal inflammation: new insights into the molecular pathogenesis and immunotherapy of inflammatory bowel disease. Int. J. Colorectal Dis. 15, (2000). 3. Elson, C.O. et al. Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota. Immunol. Rev. 206, (2005). 4. Neurath, M.F., Fuss, I., Kelsall, B.L., Stuber, E. & Strober, W. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J. Exp. Med. 182, (1995). 5. Neurath, M.F., Pettersson, S., Meyer zum Buschenfelde, K.H. & Strober, W. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice. Nat. Med. 2, (1996). 6. Dohi, T. et al. Hapten-induced colitis is associated with colonic patch hypertrophy and T helper cell 2-type responses. J. Exp. Med. 189, (1999). 7. Ekstrom, G.M. Oxazolone-induced colitis in rats: effects of budesonide, cyclosporin A, and 5-aminosalicylic acid. Scand. J. Gastroenterol. 33, (1998). 8. Boirivant, M., Fuss, I.J., Chu, A. & Strober, W. Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4. J. Exp. Med. 188, (1998). NATURE PROTOCOLS VOL.2 NO

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