Feb 2, 1994 - 0 1994 by John Wiley & Sons, Ltd. available species with well-defined inbred ...... Grisham MB. (1993). Mucosal injury and inflam- mation in a ...
MICROBIAL ECOLOGY IN HEALTH AND DISEASE
VOL.
7: 207-2 15 (1994)
Experimental Colitis Induced by Dextran Sulphate in Normal and Germfree Mice A.-C. BYLUND-FELLENIUS?, E. LANDSTROMI, L.-G. AXELSSONtf* and T. MIDTVEDTg
?Department of Pharmacology, Kabi Pharmacia Therapeutics, S-751 82 Uppsala, Sweden; $Department of Zoophysiology, Uppsala University, S-751 22 Uppsala. Sweden and §Department of Medical Microbial Ecology, Karolinska Institute, S-104 01 Stockholm, Sweden Received 2 February 1994; revised 21 March 1994
Experimental colitis in mice, induced by administration of 2.5 or 5 per cent dextran sodium sulphate (DSS; Mwt=40 kDa, S=16 per cent) in the drinking water, was characterised and optimised. The degree of colitis was evaluated after 7-10 d of acute exposure ( 5 per cent), after continuous supply for 22, 27 and 35 d (2.5 per cent), and after two and three 7 d cycles with DSS (5 per cent) in water. The colitis was manifested by typical clinical symptoms such as weight loss, rectal bleeding and diarrhoea. Macroscopically a shortening of the colon was observed and histologically epithelial cell damage, crypt loss and infiltration of mononuclear cells was verified. A stable subacute colitis with maintained diarrhoea (wet/dry weight of the faeces) and shortening of the colon, but no mortality or gross rectal bleeding was induced within 3 wk either by continuous supply of 2.5 per cent or by two 7 d periods of exposure to 5 per cent DSS. The development of colitis in germfree animals excludes a critical role of the intestinal microbiota for the induction of colitis by DSS. KEY
woms-Inflammatory
bowel diseases; Colitis, ulcerative; Intestinal microorganisms.
INTRODUCTION A relevant animal model for ulcerative colitis would be a powerful tool for future pathophysiological and pharmacological studies, related to this disease. S ontaneously developing colitis is rare in animals,1a723 and inbred strains with inflammatory bowel disease are not yet available. Experimental studies are therefore dependent on artificially induced colitis models, and the relative merits and disadvantages of these are currently disc ~ s s e d . ~A , ' ~number , ~ ~ of the existing models are based on local administration of irritants in the colon and reflect primarily the early stages of acute inflammation and ulceration, while immunologically induced models that reflect the chronic aspects of the disease are rare. Important criteria for a relevant model are that it resembles the human disease in terms of clinical manifestations and pathogenesis, and responds to established therapy. A useful model should also be easy to induce, have a reasonable time-course and give reproducible results. Ideally the model should also involve an *Author to whom correspondence should be addressed CCC 0891-060X/94/040207-9 0 1994 by John Wiley & Sons, Ltd.
available species with well-defined inbred genetic strains, such as the mouse. For these reasons we have further evaluated and optimised the dextran sodium sulphate (DSS) induced colitis model in mice, recently described by Okayasu et a l l 9 In this study we have evaluated shorter protocols of exposure to DSS and demonstrate a stable colitis already present after three weeks. In addition to the clinical symptoms of weight loss and rectal bleeding, we also report an increase in the wet/dry weight ratio of the faeces as an estimation of the diarrhoea. Furthermore, we have evaluated the effect of DSS in germfree animals to address the role of the intestinal microbiota for the induction of colitis. METHODS Mice Specific pathogen-free BALB/c female mice (Charles River Wiga GmbH, Sulzfeld, Germany), 8-9 wk of age, weighing about 17 g, were used. The animals were kept under standard laboratory conditions with free
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access to animal chow (R34, Lactamin AB, Vadstena, Sweden). Five to ten animals were kept in the same cage. Drinking water with or without DSS was provided ad libitum. Germfree and conventional mice of the NMRUKI strain were also used. The strain originates from the Institute fur Versuchstierzucht, Hannover, Germany, and has been inbred for 28 generations at the Department of Medical Microbial Ecology, Karolinska Institute, Stockholm, Sweden. The germfree mice were reared as described in detail previously.’ They had free access to autoclaved R34 diet and sterile drinking water with or without DSS. The conventional NMRI/KI mice were kept under standard laboratory conditions and received the same autoclaved diet and had free access to the same drinking solutions as their germfree counterparts. Preparation and administration of dextran sodium sulphate DSS was provided as a 2.5 or 5 per cent solution in the drinking water. DSS was dissolved in distilled water, and the pH was adjusted to 8.5 and sterile filtered. The daily water consumption was measured initially, and the water intake was not affected by addition of DSS. The daily oral dose achieved by free access to 2.5 and 5 per cent DSS corresponds to approximately 3.7 and 7.4 g per kg per day. DSS with an average molecular weight of 40 kDa and a sulphur content of 15-17 per cent was obtained from TdB Consultancy (NAntunavagen 36F, S-757 57 Uppsala, Sweden). The DSS was synthesised from dextran (B512F) by standard sulphation techniques using chlorosulphonic acid. The purity with regard to N-impurities and free sulphate was controlled. The molecular weight distribution for the batch used, determined by size exclusion chromatography,’ is given in Table 1. To determine the stability of DSS in solution, the following tests were performed: molecular weight distribution, pH, free sulphate and optical rotation. The changes in these parameters were insignificant after 3 wk of storage of a 2.5 per cent DSS solution at room temperature. The stability of the DSS solution remaining in the animals’ drinking bottles after a 3 wk experiment was also analysed to evaluate if the bacterial contamination that normally occurs would alter the chemical composition of DSS. There was no difference compared with freshly made solutions, and it was concluded that DSS is stable during these conditions. As a
Table 1. Molecular weight distribution of DSS with a mean molecular weight of 40 kDa, determined by size exclusion chromatography Molecular weight
Percentage distribution
80 000
15 20 25 25 15
routine the animals’ drinking bottles were changed and sterilised once a week, and the DSS solution was replenished every day with sterile filtered solution. Experimental protocols A total of 186 mice was randomised into different groups, according to the following treatment regimens: (1) BALB/c mice received either water alone or 2.5 or 5 per cent DSS according to the following administration protocols: (A) 5 per cent DSS for 7, 8, 9 or 10 d; (B) 5 per cent DSS for 9 d followed by water for 11 d; ( C ) 5 per cent DSS 7 d, water 7 d and 5 per cent DSS 7 d; (D) 5 per cent DSS 7d, water 7d, 5 per cent DSS 7d, water 7 d and 5 per cent DSS 7 d; (E) 2.5 per cent DSS for 22, 27 or 35 d. (2) 2-5 and 5 per cent DSS were also administered to germfree and conventional mice of the NMRUKI strain for up to 12 d. These experiments were approved by the local animal welfare committee. Observation of colitis The animals were inspected daily for indications of colitis. The body weight was measured and rectal bleeding was observed. Rectal bleeding was defined as the presence of blood clots around the anus, apparent by eye inspection. At the end of the experimental period the animals were anaesthetised with Methofane (Pitman-Moore, Mundelein, IL, USA) and killed by cervical dislocation. The abdomen was opened and the colon was resected. Care was taken to make the distal cut of the colon as close to the anus as possible, and the proximal cut was performed at the junction with the caecum. The length of the colon (in a relaxed position without stretching) was measured in a blind
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EXPERIMENTAL COLITIS IN MICE
Table 2. Induction of acute colitis by administration of 5 per cent DSS in mice: Protocol A Groups
Length of colon (cm)
Control, day 7 DSS, day 7 DSS, day 8 DSS, day 10
7.9 0.5 5.6 f 0.8** 6.0 f 0.4** 5.7 ?c 1.3**
*
Wetldry weight of faeces
Rectal bleeding"
Spleen weight (mg)
Body weight (g)
2.90 f 0.65 4.14 f 0.71 ** 5.00 f 0.58** 7.41 3,60**
0110 0110 6110** 415 * *
95.3 f 10.4 90.4 f 12.2 82.9 f 9.2* 60.9 f 27.8**
17.8 0.7 16.3 f 1.5* 17.9 ?c 1.1 15.7 f 1.8**
*
*
Mortalityb 0110 0110 0110 217
Balb/c-mice were given distilled water containing 0 per cent (control) or 5 per cent DSS ad libitum "Number of mice with rectal bleeding/surviving mice. bNumber of dead miceltotal. *P
