The Mongolian gerbil as a model for inflammatory bowel disease

Abstract

Mongolian gerbils are used as biomedical research models for a variety of diseases and are in some cases suited better than other rodents for basic research and therapeutic studies. The aim of this study was to establish and characterize a dextran sulphate sodium (DSS)-induced model in gerbils for the human inflammatory bowel disease (IBD) and to utilize them for a therapeutic study in vivo. Four concentrations (0.5%, 1%, 2% and 4%) of DSS were administered via drinking water for 7 days; based on these results, a concentration of 3% DSS was given for 9 days in a second approach. Fluid uptake and general clinical condition were assessed daily using a clinical score. Caecum and colon were scored histologically. Fluid uptake was affected by addition of DSS to the drinking water. First clinical symptoms were observed at day 4 of DSS treatment with a considerable increase in clinical score parameters only in gerbils receiving 2% or 4% DSS. Histologically, ulceration and inflammation were observed predominantly in the caecum of gerbils treated with at least 1% DSS; reproducible inflammation in the colon required at least 2% DSS. Using 3% DSS for 9 days, considerably more inflammation was induced in the colon, comparable with lesions usually observed in the mouse model. Using an optimized protocol, DSS treatment induces reproducibly typhlocolitis in Mongolian gerbils, rendering them as a useful model for IBD.

Introduction

Inflammatory bowel disease (IBD) comprise a group of inflammatory conditions of the intestine with two major forms, Crohn’s disease and ulcerative colitis, that differ in anatomic location and pathological lesions. Crohn’s disease affects any part of the intestinal tract from mouth to anus with the terminal ileum being a major site. Ulcerative colitis is restricted to colon and anus. Microscopically, ulcerative colitis is limited to the mucosa whereas Crohn’s disease is characterized by transmural inflammation. Extraintestinal manifestations of IBD include inflammatory lesions of the skin, joints, liver and eyes (Das 1999).

To increase knowledge about aetiology, diagnosis and treatment of IBD, various animal models were developed, e.g. the dextran sulphate sodium (DSS)-induced model of colitis in mice (Okayasu et al. 1990). DSS (30–50 kDA) is usually given via drinking water for several days in concentrations up to 10% and likely leads to disruption of the mucosa by a toxic effect on epithelial cells and subsequently to acute typhlocolitis, which is manifested by bloody diarrhoea, weight loss, shortening of the colon, mucosal ulceration and neutrophilic infiltration; repeated cycles of DSS treatment induce a chronic form of disease (Elson 1999).

For pharmacological studies, it was necessary to establish a colitis model in the Mongolian gerbil (Meriones unguiculatus) that serves as an animal model for a wide range of diseases, e.g. for the investigation of Helicobacter pylori-induced gastritis (Hirayama et al. 1996, Watanabe et al. 1998, Bleich et al. 2005). Because of its probable applicability in a variety of species, the DSS-induced model was chosen. However, gerbils are adapted to their original habitat, semi-deserts and steppes, and are therefore able to reduce water uptake (National Research Council 1995), which might interfere with the application of DSS via drinking water. Furthermore, gerbils are very sensitive to intestinal (and systemic) disease induced by clostridial species (e.g. Clostridium piliforme or Clostridium difficile) (Bergin et al. 2005), possibly rendering them very susceptible to intestinal lesions induced by a chemical and sequential inflammation. Therefore, the aim of this study was to investigate whether DSS-treated Mongolian gerbils serve as a suitable animal model of IBD.

Materials and methods

Animals

Eight- to 10-week-old male Mongolian gerbils obtained by Charles River, Sulzfeld, Germany, weighting 64.71 ± 8.86 g were used in this study. All gerbils were maintained in a controlled environment (21 ± 2 °C, 55 ± 5% relative humidity, 12 h light/dark cycle, 12–14 changes of air/h) in groups of two animals per cage on bedding of dust-free soft-wood fibres and autoclaved hay at the Central Animal Facility, Hannover Medical School. The gerbils had free access to a pellet diet (Altromin 1314; Altromin, Lage, Germany) and tap water or DSS solution, respectively, supplied in bottles. Personnel entering the room were required to wear a gown, cap, surgical mask, overshoes and gloves.

Routine microbiological monitoring according to modified FELASA recommendations (Rehbinder et al. 1996; Glage et al. 2007) did not reveal any evidence of infections with common pathogens.

This study was conducted in accordance with the German animal protection law and with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. All experiments were approved by the Local Institutional Animal Care and Research Advisory Committee and permitted by the local government.

Measurement of water uptake

Water uptake was measured in three autonomous groups. The bottles were weighed once daily at a particular time and the uptake was calculated for each animal.

Colitis induction

Acute colitis was induced in groups of four gerbils each by giving four different concentrations (0.5%, 1%, 2% and 4%) or no DSS (n = 3) for 7 days. In a second experiment, four gerbils were treated with 3% DSS for 9 days.

Evaluation of clinical signs and colitis

For each animal, weight loss, consistency of stool and general clinical condition were assessed and graded to quantify the severity of disease on a daily base (Table 1). Weight loss was graded in per cent compared with pretreatment weights, stool was evaluated for consistency and appearance of blood, and the general clinical condition was determined as being not, mildly, moderately or severely changed compared with control animals. For each animal, all scores were added and an overall disease score was calculated ranging from 0 to 23.

Table 1.

Parameters used for clinical scoring

Clinical parameters	Score
Weight
0–3% weight loss or weight gain	0
4–10% weight loss	1
11–20% weight loss	2
>20% weight loss	3
Stool consistency (normal, soft, soft stool with blood)	0–2
General clinical parameters
Posture of the animal (normal to hunched)	0–3
Spontaneous behaviour (normal to no activity of the animals before disturbing)	0–3
Provoked behaviour (normal to no activity of the animals after disturbing)	0–3
Evaluation of the eyes (clearness, openness)	0–3
Evaluation of the fur (cleanliness, gloss, smoothness)	0–3
General appearance (not, mildly, moderately, severely disturbed)	0–3
Total (not, mildly, moderately, or severely affected)	0–23

Histological evaluation of colitis

Gerbils were killed by CO₂ asphyxiation and exsanguination. Small intestine, caecum and colon were removed and the length of the colon from the colocaecal junction to the anus was measured. The small intestine was divided in two equal parts, and both parts as well as the colon were prepared as a ‘Swiss Roll’ (Moolenbeek & Ruitenberg 1981) without being opened before rolling, the caecum was bisected with a razor blade after overnight fixation (Bleich et al. 2004).

All parts were processed routinely for histological examination, and stained with haematoxylin and eosin. Histological lesions were graded separately for the small intestine, caecum, and proximal, middle and distal colon as follows: epithelial changes (crypts unchanged: 0, loss of basal 1/3: 1, basal 2/3: 2, complete: 3, loss of crypts plus ulceration: 4), cellular infiltration (none: 0, infiltrates in lamina propria: 1, in lamina propria plus oedema formation: 2, in lamina propria and submucosa: 3), area involved (none: 0, 10–30%: 1, 40–60%: 2, >60%: 3; for each parameter individually applied), yielding scores from 0–13 for each segment (de Buhr et al. 2009).

Results

Water uptake

The average water uptake per day of untreated gerbils housed in the Central Animal Facility was 7.25 ± 1.4 ml. DSS addition to the drinking water affected the fluid uptake. Compared with pretreatment water uptake, water consumption was decreased on day 1 of treatment (which was statistically significant in the 2% and 4% group) except in the 0.5% group, increased and remained stable over the next days, and dropped again at day 5 (2% and 4% group) or day six (1% group). Compared with pretreated gerbils, water uptake was significantly reduced on day 6 (1%, 2%, and 4% groups) and day 7 (0.5% and 4% group) (Figure 1a). However, reduced fluid intake did not negatively affect colitis development (see below).

Figure 1.

Water uptake (relative to pretreatment values), clinical and histological scores for gerbils treated with different concentrations of dextran sulphate sodium (DSS) for 7 days or controls (0%). (a) Water uptake of gerbils treated with 0.5%, 1%, 2%, or 4% DSS at day 1–7 of and before DSS treatment (pre-treatment, P). A decreased water uptake was observed in gerbils treated with 1%, 2% and 4% DSS on day 1 compared with pretreatment values (mean water uptake of 3 days before DSS treatment started). Water intake started to decrease again at day 5 (2% and 4% groups) or day 6 (1% group) and was significantly reduced on day 6 (1%, 2% and 4% groups) and day 7 (0.5% and 4% group) compared with water consumption in pretreated animals.*P < 0.05; **P < 0.01 (Dunnett’s test) (b) Clinical scores from day 0 (pretreatment values) to day 7 for the different treatment groups. Groups marked with ‘A’ showed statistically significant differences to groups denoted with ‘B’ as determined using Newman–Keuls test; in particular: day 4 P < 0.01 (1%vs. 4%), P < 0.05 (0% and 0.5%vs. 4%); day 5 P < 0.01 (0%, 0.5%, and 1%vs. 4%), P < 0.05 (2%vs. 4%); day 6 P < 0.001 (0% and 0.5%vs. 2% and 4%; 1%vs. 4%), P < 0.01 (1%vs. 2%); day 7 P < 0.001 (0% and 0.5%vs. 4%), P < 0.01 (0%, 0.5%, and 1%vs. 2%; 1%vs. 4%). (c) Histological scores for gerbils treated with different concentrations of DSS for 7 days. Caecum P < 0.001 (0% and 0.5%vs. 4%), P < 0.01 (0% and 0.5%vs. 1% and 2%); middle colon P < 0.001 (0% and 0.5%vs. 2% and 4%), P < 0.01 (1%vs. 2% and 4%) (Newman–Keuls test).

Clinical symptoms and histology

Clinical scoring of gerbils using the parameters described in Table 1 revealed first symptoms on day 4 of treatment. A considerable increase in symptoms (in particular weight loss, data not shown) was observed only in the 2% and 4% group (Figure 1b). Stool of gerbils remained to be formed but was considerably softer than normal. Blood attached to faecal pellets or after was noted in one animal of the 4% and one gerbil of the 2% group on day 7.

Histology revealed inflammation in all parts of the large intestine in the 2% and 4% group, predominantely in the caecum and middle colon (Figure 1c); no inflammatory lesions were detected in the small intestine. Compared with gerbils receiving 4% DSS, more variation in histological scores of the caecum was observed in gerbils treated with 2% DSS. Notably, none or only mild inflammation was observed in the proximal part of the colon.

As clinical signs developed rather late during treatment, a further experiment was conducted for 9 days. Based on the first experiment revealing an optimal DSS concentration of 2–4%, a DSS concentration of 3% was chosen. Indeed, gerbils showed a significant weight loss on day 8 and 9 and a significant increase in the clinical parameter score on day 5 of treatment (compared with pretreatment values on day 0). Faeces showed a notably soft consistency between day 5 and 9 in all gerbils, and blood was detected on faeces during this time for at least 1 day. With an average total clinical score of 13 (maximum score: 23) on day 9, no gerbil had to be killed prematurely. As determined by histology (Figures 2c and 3), all gerbils developed a well reproducible disease with caecal lesions being similar to those of gerbils that received 4% DSS over a period of 7 days. Interestingly, inflammation increased in the distal colon compared with gerbils treated for 7 days, which makes this model more comparable to the acute DSS model in mice.

Figure 2.

Weight reduction, clinical score and histological score for gerbils treated with 3% dextran sulphate sodium (DSS) for 9 days. A statistically significant weight reduction (a) was observed at day 8 and 9 of treatment and a significant increase in clinical parameters (b) was observed from day 5 on compared with pretreatment values as determined using Dunnett’s test. Histological scores for caecum, proximal (Prox), middle (Mid) and distal (Dist) colon as well as the total colon scores (Total) are shown (c). *P < 0.05, **P < 0.01.

Histological lesions induced by dextran sulphate sodium (DSS)-treatment (3%) for 9 days. (a) Scan of a longitudinally cut caecum including the caeco-colonic junction, both halves placed on one slide, showing a considerably thickening of the caecal wall, massive ulceration and reactive mucosal hyperplasia. (b) Higher magnification of the boxed area in (a) showing ulceration and transmural inflammation characterized by a mixed cellular infiltration in the muscular layer (c), submucosa (d), and mucosa (d, e), ulceration (e) and massive exudation of granulocytes (f). (g) Longitudinal section of a colon prepared as a roll with the proximal part (with its folds) being placed in the middle and the distal part on the outside of the roll. While the proximal part appears almost normal, the middle and distal colon show ulceration, thickening of the mucosa and submucosa with oedema and reactive epithelial hyperplasia, and mixed cellular infiltration as well as crypt shortening and distortion (h, j) as typical DSS-induced lesions. Original magnifications: a, g (1×); b, h (4×); c–f, j (40×).

Discussion

Induction of typhlocolitis in gerbils was achieved by oral administration of DSS for 7 or 9 days. Optimal results were obtained by treatment of gerbils with 3% DSS for 9 days, indicating a similar course of disease as in mice (Okayasu et al. 1990). Therefore, DSS-induced colitis in gerbils serves as an adequate model for IBD; based on histological findings, in particular multi-focal transmural inflammatory lesions, crypt loss, and large areas of ulceration, this model shows similarities to both ulcerative colitis and Crohn’s disease. Inflammatory lesions were limited to the large intestine; the small intestine remained unaffected. Most severe inflammation was detected in the caecum of gerbils. Therefore, it is important to include the caecum in histological scoring, which also applies for mice, especially as severity of inflammation in caecum and colon depends on the genetic background (Mähler et al. 1998). Changes in stool consistency during DSS administration were more subtle in gerbils than what was expected from the previous studies in mice. The absence of diarrhoea in gerbils probably reflects their adaptation to their natural habitat (semi-deserts and steppes), enabling a more efficient resorption of water from the intestine (Snipes 1982). During necropsy, a considerably shortening of the caecum and colon was noted in DSS-treated gerbils which was not evaluated systematically; however, this parameter likely serves as a useful macroscopic marker for inflammation in prospective studies like it has been described in mice (Elson 1999; Obermeier et al. 2005).

The reduction in fluid uptake upon DSS addition to drinking water did not negatively affect typhlocolitis development. As an alternative to DSS application via drinking water, application via oral gavage was tested, which showed equally potency in induction of intestinal inflammation in a preliminary study (data not shown). However, as a result of considerably high volumes and repeated application over at least 1 week, we considered this approach as inappropriate, especially as gerbils are known to react very sensitive to stressors, e.g. with genetically predisposed epileptiform seizures (Donnelly & Quimby 2002).

Gerbils that were treated for 9 days received autoclaved hay that was used as nesting material by the animals. However, small amounts were also fed on by gerbils but did not change fluid intake or outcome of disease. Therefore, we decided to provide hay in subsequent experiments.

The clinical scoring sheet developed for this study enabled a fast and simple scheme to evaluate the general condition of DSS-treated gerbils. This scoring provides useful data to follow the induction and to evaluate the severity of disease as it corresponds quite well to the severity of histological lesions observed by histology. This score sheet also provides a good basis for the determination of humane endpoints for these experiments.

In summary, DSS treatment allows to induce a well reproducible typhlocolitis in gerbils with a moderate increase in clinical parameters. The course of disease should be monitored daily using an adequate scoring sheet.