Mouse models of IBD and how to avoid them

Choosing the right mouse model is a major issue in studies of experimental colitis. In previous years some quite extensive overviews of the different models available have been published,^3,6–8 but most authors refrain from giving advice on the best model to choose. Indeed, if any advice is given it is very limited. For example, Goyal et al.⁷ concluded that the “…currently available animal models are relevant to human IBD if they are chosen carefully (chronic, immune mediated)” and DeVoss and Diehl⁶ indicated that a successful approach requires “…careful utilization of pathway models to query specific scientific or efficacy questions.”

There are a number of chemically induced acute colitis models that are easy to use, rapid and of low cost and therefore widely used. However, these models may not be the best models to study IBD, because chemical damage to the gut epithelium results in self-limiting inflammation rather than chronic inflammation. Comparative analysis of colonic gene expression in the 2,4,6-trinitrobenzenesulfonic acid (TNBS), dextran sulfate sodium (DSS) and T-cell transfer models with human IBD revealed that the pattern of gene expression in the T-cell transfer model most closely reflects altered gene expression in IBD.⁹ Chemically induced models should only be used if the intention is to study the physiology of epithelial regeneration or intestinal wound healing.

In general, the different mouse models of colitis may reflect human IBD subtypes as described in table 1.  However, there is not one single experimental colitis model that resembles all aspects of human IBD. Making the choice of which model to use should combine the research question and the IBD subtype to achieve the best outcome. 

In 2006, a critical appraisal of experimental colitis induction using TNBS revealed that the protocol followed differed in each of the studies included.¹⁰ Indeed, the mouse strains used, mouse age, dosing and times of TNBS administration, percentage of ethanol used and the duration of the experiment all varied. In 2007 Wirtz et al.¹¹ published experimental protocols for the chemical induction of colitis in mouse models, thereby setting the gold standard for this methodology. Unfortunately, since then not many studies using these models seem to have followed the procedure described in this protocol.⁵ For the T-cell transfer model an excellent protocol, including critical parameters and troubleshooting, has been published in Current Protocols in Immunology¹² and by Ostanin et al.¹³

The lack of consistency in the experimental protocols used for the chemical induction of colitis in mouse models hampers reproducibility, which is fundamental for any scientific experiment.^14,15 In addition, standardization of environmental factors, such as circadian rhythms, nutrition, age, sex and strain are important confounders that have to be identified and acknowledged.^16,17 To ensure consistency and reproducibility, the same protocol and environmental circumstances should be secured in every experiment and preferably shared by several laboratories.

Randomly allocating animals to groups is a relevant issue when studying intestinal inflammation, because it ensures that subtle differences between the animals are unlikely to influence the experimental outcome. Usually, the body weight (or body weight change) of the animal, besides sex and age, is the most important parameter to account for in the randomization process. As the composition of the microbiome has a great influence on the development of intestinal inflammation,¹⁸ and this can differ between cages, the animals in each experimental group should be co-housed, so that representatives of the different experimental groups are within a single cage, and the groups are replicated across a series of cages.¹⁹ This way the differences between the groups and the cages can be measured independently.

In some cases it may be impossible to mix the experimental groups in one cage. For example, mice may not be mixed due to gender differences. Also, it is impossible to mix DSS animals and control animals (i.e. non-DSS-treated mice) in one cage. If this is the case it is recommended to randomly allocate the cages within the same room. In addition, when the animals are sacrificed the sequence should be randomized to avoid the introduction of bias. There are several ways to randomly allocate the animals used in an experiment. A random sequence generator for randomization of animals and the random integer set generator for randomization of an intervention can be found at www.random.org.

Another important consideration when setting up an accurate animal experiment is that it should be blinded at several levels. The division between experimental and control groups should be blinded to avoid selection bias. The person who is responsible for the daily care of the animals should be unaware of the intervention(s) to avoid performance bias. Moreover, the people involved in determining the outcome parameters should not be aware of the intervention(s) to avoid detection bias. In general, blinding can be realised by having an independent outsider give each animal an individual mark/number coupled to the intervention(s) and only disclosing the mark/number at the end of the experiment. In general, the same care and quality control should be incorporated in animal experimentation as is customary in human clinical trials. Hooijmans et al. recently described a tool that can be used to assess the risk of bias for animal studies.²⁰

For most autoimmune diseases there is a clear difference in susceptibility between the sexes, with females more frequently affected than males.⁴¹ In experimental models of colitis sex-specific effects have also been described. For DSS colitis greater male susceptibility has been observed,^30,42 and for TNBS the wasting disease has been shown to have a greater effect on female mice.³³

Most experiments are performed with either male or female mice. However, in incidental experiments in which both sexes have been used,^43–45 or a comparison was made between experiments,⁵ differences can be observed. In the T-cell transfer model both male¹³ and female ³⁵ mice are used. In general, DeVoss et al.⁶ recommend using female animals if possible. They indicate that male animals are more prone to display aggressive behaviour resulting in fighting, with the resulting stress and wounds potentially having a negative impact on a study. This finding hampers the random allocation of the mice because non-littermates cannot be housed together. However, single housing of male animals also has an effect on wellbeing⁴⁶ and is expensive. In a study in which several aspects of the current usage of experimental colitis models was analysed, the predominant use of male animals was observed.⁵

Experimental colitis models are frequently used for preclinical drug evaluation. The pharmacological approach is an important topic, and several aspects and considerations have been reviewed by Koboziev et al.⁴⁹ Here, we focus on some of the main issues.

Of great importance is being aware that in chemical models of colitis the administered compound can potentially interfere with the DSS or TNBS and result in a reduced colitis induction. Also, in DSS colitis it must be confirmed that the treatment regimen does not influence the water consumption. So, this should be carefully monitored. Drugs can also have an effect on the microbiota composition and in this way affect disease development.

In experimental colitis models in which the induction of the colitis is fixed on a specific time point, as is the case in the chemically induced models, it is calculated that 78% of the treatments are applied before or within 24 h after the induction of colitis.⁵ In this situation it can be questioned whether a positive effect is due to actual treatment or interference with induction of colitis. It is actually essential to treat established disease. Koboziev et al⁴⁹ indicate that “…one of the best predictors of clinical efficacy of a drug is its ability to reverse established disease in at least two different animal models of chronic intestinal inflammation.” This idea is also advocated in a recent commentary on reproducibility.¹⁷ With the introduction of endoscopy,⁵⁰ researchers are able to investigate the effect on established disease more efficiently, enabling the comparison of disease characteristics (semi-quantitative score of endoscopy) before and after treatment for each individual animal (and do paired statistical analysis).