Mice do not habituate to metabolism cage housing--a three week study of male BALB/c mice

Abstract

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.

Figure 1. Separation failure of the metabolism cages.

The number of samples (frequency of cases) and the average CORT of these samples are subdivided according to the daily creatinine excretion in the corresponding urine samples. Samples in the grey box are excluded due to the high likelihood of (severe) cross-contamination.

Figure 2. Body weights.

Data are presented as mean body weights±SEM.

Figure 3. Feed and water consumption.

Data are presented as means±SEM. The sudden increase in the measured parameters for day 16 is likely to be from badly calibrated scales having been used for weighing the metabolism cage feed tray and water bottles.

Figure 4. Fur scores.

Average fur scores summarized from eight scoring sessions, by two independent blinded observers, spanning three weeks.

Figure 5. Urinary Creatinine Output of the Metabolism Cage Mice.

Data are presented as means±SEM. Data are presented as averages of three days, to compensate for excluded data (refer to Results segment, and Figure 1). A significant elevation is seen over time.

Figure 6. Daily urinary excretion of oxidized nucleic acid metabolites in mice in metabolism cages and correlations between the two markers.

Data are given as means±SEM. Data are presented as averages of three days, to compensate for excluded data (refer to Results segment, and Figure 1). The two markers are highly correlated, but a statistically significant increase over time could only be established in the RNA-based marker (8-oxo-G).

Figure 7. Fecal CORT output.

Immunoreactive corticosterone metabolites (CORT) are quantified as nanogram-equivalents of unmetabolized corticosterone excreted within 24 hours. Data are presented as means±95% confidence intervals of the log-normal distribution. Data are presented as averages of three days, to compensate for excluded data (refer to Results segment, and Figure 1).

Figure 8. Forced swim test (FST) results summarized.

Metabolism cage housed mice (n = 8) and control group (n = 7). The whiskers denote 95% CI. Significant differences were found in the latency to first immobility and the post test serum ACTH levels.

Figure 9. HIH (Hydroxy-dipropylamino-tetralin Induced Hypothermia).

Body temperatures of the mice following an 8-OH-DPAT challenge, a week prior to the experiment and on the last day of the experiment. Data presented as means±SEM; n = 8 for all groups except the control group post-experiment, where n = 7. The pre-test temperature of the metabolism cage housed mice is significantly elevated compared to pre-experiment. The HIH post-experiment is significantly blunted for all mice.

Figure 10. Distribution of subjects in the PCA Space.

Metabolism cage housed mice (n = 8) and control group (n = 7) plotted in the 3D space defined by the extracted components. Factor loadings are presented in Table 2. A perfect separation of the experimental groups can be seen along the axis defined by component 1 (“Stress”).

Figure 11. Correlation between change in body weight and immobility in the FST.

Data from all 15 mice tested in the FST. A linear regression has been fitted to the data (dotted line). The variables are significantly correlated.