Mice Do Not Habituate to Metabolism Cage Housing–A Three Week Study of Male BALB/c Mice

et al. (2013) Mice Do Not Habituate to Metabolism Cage Housing-A Three Week Study of Male BALB/c Mice. PLoS ONE 8(3): e58460. doi:10.1371/journal.pone.0058460 Mice Do Not Habituate to Metabolism Cage Housing-A Three Week Study of Male BALB/c Mice Otto Kalliokoski 0 Kirsten R. Jacobsen 0 Huda S. Darusman 0 Trine Henriksen 0 Allan Weimann 0 Henrik E. Poulsen 0 Jann Hau 0 Klas S. P. Abelson 0 Hemachandra Reddy, Oregon Health & Science University, United States of America 0 1 Department of Experimental Medicine, University of Copenhagen and University Hospital , Copenhagen , Denmark , 2 Department of Anatomy, Physiology and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural University , Bogor , Indonesia , 3 Laboratory of Clinical Pharmacology , Rigshospitalet, Copenhagen , Denmark , 4 Department of Clinical Pharmacology, Bispebjerg Hospital , Copenhagen , Denmark The metabolism cage is a barren, non-enriched, environment, combining a number of recognized environmental stressors. We investigated the ability of male BALB/c mice to acclimatize to this form of housing. For three weeks markers of acute and oxidative stress, as well as clinical signs of abnormality were monitored. Forced swim tests were conducted to determine whether the animals experienced behavioral despair and the serotonergic integrity was tested using an 8-OH-DPAT challenge. The metabolism cage housed mice excreted approximately tenfold higher amounts of corticosterone metabolites in feces throughout the study when compared to controls. Urinary biomarkers confirmed that these mice suffered from elevated levels of oxidative stress, and increased creatinine excretions indicated increased muscle catabolism. Changes in the core body temperature (stress-induced hyperthermia) and the fur state of the mice also indicated impaired well-being in the metabolism cage housed mice. However, monitoring body weight and feed intake was found misleading in assessing the wellbeing of mice over a longer time course, and the forced swim test was found poorly suited for studying chronic stress in mice in the present setup. In conclusion, the mice were found not to acclimatize to the metabolism cages whereby concern for animal welfare would dictate that mice should be housed in this way for as short periods as possible. The elevated degree of HPA axis activity, oxidative stress, and increased overall metabolism warrant caution when interpreting data obtained from metabolism cage housed mice, as their condition cannot be considered representative of a normal physiology. - Metabolism cage housing of laboratory rodents provides researchers with unique possibilities of investigating particular biological events and their progression, and it is widely used in pharmacokinetic and pharmacodynamic studies. However, it constitutes a form of single housing on wire mesh without bedding, often without enrichment, comprising of a smaller living area with no shelter. All of the aforementioned factors have been associated with induction of stress or discomfort in laboratory mice and rats, as observed in e.g. Zhu et al. [1] on enrichment, Ishida et al. [2] on the effect of living area, Bartolomucci et al. [3] on single housing, and Manser et al. [4,5] on the effect of being housed on wire mesh. Considering that it is generally accepted that prolonged stress has profound consequences on many of the factors the metabolism cages have been designed specifically to studye.g. the eponymous metabolism [6,7], pharmacokinetics [8], kidney function [9], and intestinal function [10]there are remarkably few studies on the effect of this type of housing on the wellbeing of the test subjects [11]. Whereas it has been suggested that rats may be able to adjust to metabolism cages [1214], mice are considered to be less malleable and more sensitive to low-level stressors [15,16]. Indeed some guidelines suggest that mice should be kept in metabolism cages for the shortest possible time [17]. Others are vaguer in the matter. According to the current EU directive [18], metabolism cage housing for more than 24 hours should be classed as a moderate to severe procedure with respect to pain, suffering and/or distress. On the other hand, it is common best practice to let animals habituate and acclimatize to changes in location and caging in order to obtain reliable results from subsequent experimentation [19]. In mice transferred to metabolism cages, food and water intake, urinary output, and body weight do not seem to stabilize until 34 days after the novel housing [20]. The stabilization of these parameters is sometimes interpreted as adjustment to the new environment. This thus provides a counterpoint, as reliable measures cannot be obtained until perhaps a week after housing mice in metabolism cages. Consequently, other guidelines recommend an acclimatization period to be included in studies in metabolism cages [21]. Unfortunately, a great majority of laboratory animal guidelines and handbooks do not address the conflict between the metabolism cages being considered stressful and the scientific need to let the animals acclimatize prior to experimentation. The central question, which remains unanswered, is whether mice are at all capable of adjusting to the extreme environmental condition which is metabolism cage housing. We hypothesized that the mouse physiology may find a new equilibrium after a few days housing in metabolism cages, but that the stressfulness of the condition persists. This in turn means that endogenous measures obtained from laboratory mice housed in this fashion need not, and in most cases will not, be reflective of a normal physiology. Acknowledging that emotional stress is a multi-dimensional construct, also in rodents [22], we used a combination of biochemical, clinical, and behavioral measures to assess the state of mice housed in metabolism cages for three weeks. We chose BALB/c mice for this study as they are the most numerous strain in our facilities, and male mice as they have been shown to better cope with single housing [23]a condition we would obviously also impose on our control group. Traditionally measures such as body weight, feed and water consumption, defecation, and level of activity have been used to great extent to evaluate the effect of environmental stressors on laboratory mice in the past. Due to the radically different construction of the metabolism cages when compared to standard open cages, and the difference in available enrichment, activity levels are not meaningful to compare between the two cage types. Defecation is, in itself, not a useful comparative measure as the bedding material in standard cages desiccates the fecal matter greatly, reducing its mass. In the present study we opted to measure body weight, feed and water intake while also assessing fur quality, as this has been used convincingly in studies of the effect of low-intensity stressors [24]. Forcing a mouse into an inescapable (...truncated)