Generating a Stress-Reducing Environment
Items that were placed in enhanced cages included a cardboard nest box, shredded paper nesting material, and a polycarbonate mouse tube (). Additionally, certain cages contained a silent running wheel (). Eight-week-old BALB/c mice were used in these studies. Although females are known to be less territorial and aggressive, we used male mice to circumvent estrous cycle variability and its effects on hormonal regulation. Mice were randomly assigned to one of four caging groups (n = 10 per group): Cntl, Calm, Cntl Ex or Calm Ex. Cntl animals were group housed (five animals per cage) in standard caging without enhancements (). Calm animals were housed in larger caging containing a cardboard nest box for shelter, paper nesting material to promote nesting behavior, and a polycarbonate tube to mimic tunneling (). Animals in Cntl Ex were housed in standard caging equipped with a running wheel (). Lastly, Calm Ex caging included the same enrichments as Calm with the addition of a running wheel (). See Materials and Methods for further details.
Before being placed in their respective experimental caging environments, all mice were kept in Cntl caging for a period of 14 d to allow them to acclimatize to the animal husbandry facility (). After acclimatization, animals were housed in experimental caging, according to their randomized group assignments, until the end of the experiment. On d 42 all animals were subjected to assessment of anxiety-related behavior on an EPM. At the end of the experiment, on d 70, animals were killed for blood collection, in vitro immunologic studies, and assessment of body composition. At regular intervals fecal samples were collected from each animal and analyzed for corticosterone metabolites.
Calming and Exercise Increased Body Mass
Body mass was monitored weekly in all groups. We found that both calming and exercise resulted in significantly (time*Calm, P < 0.0001; time*exercise, P < 0.0001) increased body mass (). After 7 d of experimental caging, Calm animals exhibited a significantly greater mean body mass compared with Cntl (7d, difference in means [Md] 0.94 g, 95% confidence interval [CI] 0.04–1.84, P = 0.041), and this difference increased (70 d, Md 2.50 g, CI 1.23–3.77, P = 0.0006) over the course of the experiment (). Animals housed in Cntl Ex caging also had a greater mean body mass than Cntl animals (35d, Md 1.23 g, CI 0.16–2.37, P = 0.027) (). Similar to Calm, Calm Ex animals showed a greater mean body mass than Cntl animals (7 d, Md 1.05 g, CI 0.10–2.00, P = 0.032), and increased the difference over the duration of the experiment (70 d, Md 3.26 g, CI 2.17–4.35, P < 0.0001). Collectively, these findings suggest that both Calm caging and voluntary exercise can induce rapid gains in body mass in growing mice.
Energy Intake Was Affected by Exercise
To further characterize the differences in body mass described above, we investigated changes in energy intake, body composition and peptide hormone levels in experimental and control animals (). Dry food consumption was monitored and recorded weekly by cage for the length of the experiment. These data are expressed as weekly cage-total energy intake per mean cage body mass (kcal/g) at the end of each week to account for longitudinal increases in body mass. Remarkably, although we observed similar proportional weekly energy intake between the Calm and Cntl groups (), Calm animals exhibited significantly greater body mass than Cntl animals (). We also observed that Cntl Ex and Calm Ex animals consumed greater quantities of food per week than Cntl and Calm animals (). In comparing the two exercise groups, we noted that for time points 14, 21, 28 and 35 d, Cntl Ex animals displayed greater energy intake () but less body mass () than Calm Ex animals, although these differences did not reach statistical significance.
Calm Caging Had Little Effect on Body Composition, whereas Exercise Reduced Adiposity
To determine whether the increased body mass in experimental groups was attributable to differences in body composition, we carried out an MRI protocol (). Animals were imaged by using a three-dimensional fast-spin–echo sequence that emphasized the lipid signal contrast (, red arrows), facilitating subsequent adipose tissue segmentation and quantification (see Materials and Methods).
Cntl and Calm animals had similar quantities of lipid, whereas Cntl Ex and Calm Ex animals had less visible lipid than Cntl animals (). Quantification of the proportion of adipose tissue was expressed as total lipid volume per total body mass. These data revealed that Cntl and Calm animals had comparable adiposity (Md 1.56 mm3/g, CI −6.16 to 9.29) and similar body composition (). Cntl Ex and Calm Ex animals, however, exhibited a significantly smaller proportion of adipose tissue compared with Cntl (Cntl Ex, Md −10.15 mm3/g, CI −18.87 to −1.41, P = 0.025; Calm Ex, Md −11.11 mm3/g, CI −21.37 to −0.85, P = 0.035), but were similar to each other (). Together, these findings suggest that increased body mass in Cntl Ex and Calm Ex animals may be partially attributable to increased lean muscle mass as a result of voluntary wheel running. However, Cntl and Calm animals appeared to have similar body composition, a finding that provided little explanation of how Calm mice gained significantly more body mass while maintaining the same proportional energy intake as Cntl animals.
Effects of Calming on Peptide Hormones
To determine whether Calm-associated body mass differences were related to circulating hormone concentrations, we collected serum samples at the 70-d endpoint and assayed them for IGF-1 and insulin (). IGF-1, a focal mediator of growth hormone, is produced by the liver and can stimulate growth in most cell types, including muscle, bone and connective tissue. Previous studies of enriched environments have reported upregulation of IGF-1 in the rodent central nervous system (39
), and thus, we hypothesized that serum IGF-1 might be upregulated as a product of Calm caging or exercise. We found, however, that compared with Cntl animals, serum levels of IGF-1 were lower in Cntl Ex (Md
−278 ng/mL, CI −897 to 342) and Calm Ex (Md
−489 ng/mL, CI −1032 to 54) and significantly lower (Md
−459 ng/mL, CI −832 to 86, P
= 0.019) in Calm animals (). We also measured serum levels of insulin, the principal hormone responsible for regulating glucose metabolism. We found that both Calm and Calm Ex animals exhibited trends toward higher levels of serum insulin compared with Cntl animals (Calm, Md
0.42 ng/mL, CI −0.06 to 1.46; Calm Ex, Md
0.28, CI −0.10 to 0.65), although the results lacked statistical significance (). Given the functional role that insulin plays in metabolism and energy homeostasis, we reasoned that higher levels of insulin might be associated with enhanced ability to derive energy from digested dry food. To examine this relationship, we looked at the association between serum insulin level and final body mass within each group of our study. We found that a linear regression for Cntl animals revealed a negative relationship between serum insulin level at the end of the experiment and final body mass (Cntl insulin effect −0.98 g per ng/mL of insulin, CI −0.40 to −1.56, P
= 0.0018) (). However, Calm, Cntl Ex and Calm Ex regressions had positive effects, indicating that higher insulin levels were associated with greater body mass (Calm insulin effect 0.99 g per ng/mL of insulin, CI 0.58 to 1.41, P
< 0.0001; Cntl Ex insulin effect 0.26 g per ng/mL of insulin, CI −1.04 to 1.56, P
= 0.69; Calm Ex insulin effect 0.67 g per ng/mL of insulin, CI −0.43 to 1.78, P
= 0.22) (). The overall insulin by caging group interaction was statistically significant (P
< 0.0001). These results suggest that caging-induced changes in body mass were related to altered responses to serum insulin level, although further work is needed to confirm this association.
Both Calming and Exercise Reduced Corticosterone Production
The most widely used biomarker of rodent stress is the production of corticosterone. Collecting blood from an animal to measure circulating corticosterone levels, however, may elicit a substantial stress response. For the purposes of assessing corticosterone production in the present study, we employed a less stressful, noninvasive technique in which we measured CMs found in fecal pellets from each animal (). CM measures are well correlated with a graded average of corticosterone production from the preceding 10 h, and these results have been extensively validated in laboratory mice and other species (35
). Fecal pellets were collected from individual mice and were later assayed for CM as described in Materials and Methods. We found that on d 0, before animals were placed in their respective experimental caging environments, the mean levels of CMs were similar in all groups (). After mice were housed in Calm, Cntl Ex or Calm Ex caging for 14 d, they exhibited significantly lower fecal CM levels (Calm, Md
15.1 ng, CI 1.9–28.2, P
= 0.027; Cntl Ex, Md
20.8 ng, CI 4.8–36.8, P
= 0.014; Calm Ex, Md
26.4, CI 11.4–41.4, P
= 0.0017) compared with Cntl animals (). On d 49, mean fecal CM levels from experimental animals were even lower (Calm, Md
20.5 ng, CI 11.7–29.4, P
< 0.0001; Cntl Ex, Md
22.5 ng, CI 13.4–31.5, P
< 0.0001; Calm Ex, Md
21.8, CI 11.7–32.0, P
= 0.0003) and this effect persisted through d 70 (Calm, Md
23.1 ng, CI 1.3–44.9, P
= 0.039; Cntl Ex, Md
28.4 ng, CI 13.2–43.5, P
= 0.0010; Calm Ex, Md
23.9 ng, CI 7.4–40.4, P
= 0.0070) the last day of the experiment (). These findings suggest that both Calm caging and caging equipped with a running wheel can effectively reduce stress levels as evidenced by lasting reductions in CM production.
Immunologic Changes in Mice Housed in Calm and Exercise Caging
To characterize the effects of Calm and exercise caging on murine immune function, we examined spleens and carried out functional in vitro studies using splenocytes (). At the experimental endpoint animals were sacrificed and spleens were aseptically removed and massed. We found that both calming and exercise generated a significant increase (Calm, P = 0.0086; exercise, P = 0.049) in spleen mass ().
Chronic social stress has been shown to affect innate and adaptive immunity in humans and animals. For example, studies of chronic stress have revealed increased production of IL-6 after lipopolysaccharide (LPS) stimulation of splenocytes (41
), and both reduced lymphocyte proliferation and suppressed IL-2 production (5
). In this work, to assess whether there were changes in innate or adaptive immune function we stimulated splenocyte cultures with LPS (1 μg/mL) or anti-CD3 plus anti-CD28 (CD3/CD28) Dynabeads (1 bead/cell), respectively. Some stimulated splenocyte cultures were treated with different concentrations of corticosterone, to assess leukocyte sensitivity to corticosterone-mediated suppression of cytokine production. All cells were cultured in splenocyte medium containing 5% charcoal-stripped FBS, which minimizes background hormone levels commonly found in serum and reduces unspecific binding of the glucocorticoid receptor. Because murine immune cells usually function in the presence of a small concentration of corticosterone (43
), the results displayed are from cultures containing 0.005 μmol/L corticosterone, the most physiologically relevant condition tested ().
LPS-stimulated splenocyte cultures from Calm and Cntl Ex animals exhibited comparable levels of IL-6 to those of Cntl animals (). Cultures from Calm Ex animals displayed a slight trend toward greater production of IL-6 after stimulation with LPS, but these data were not statistically significant (). Although splenocyte production of IL-2 in response to CD3/CD28 bead stimulation was similar among Cntl, Cntl Ex and Calm Ex groups, cultures from Calm animals had slightly higher (Md 138.0 pg/mL, CI −18.8 to 296.1, P = 0.081) mean levels of IL-2 ().
Acute Stress and Immunomodulation
Stress is known to be immunomodulatory and may enhance or suppress immune responses depending on the type of stressor, acute or chronic, and the cell populations being studied, innate or adaptive (9
). For example, human studies have shown that acute psychological stress results in increased production of IL-6 in LPS-stimulated peripheral blood mononuclear cells (46
). In the context of animal studies, the handling of mice before sacrifice is a substantial acute stressor, resulting in increased serum levels of stress mediators such as corticosterone and epinephrine (47
). To assess the level of acute stress at the end of the experiment we measured the quantity of corticosterone found in serum samples taken from mice at the time of sacrifice. We observed a distinct pattern, in that within individual cages, the animals killed first often had lower levels of acute stress than subsequent animals, as indicated by serum corticosterone concentration (Supplementary Figure 1). Thus, in each group there was a broad range of serum corticosterone levels at the time of sacrifice (Supplementary Figures 1A–D). The within-group variation in serum corticosterone provided an opportunity to investigate the effects of varying levels of acute stress on functional immunity in animals housed in each of the four caging conditions.
To determine whether the level of acute stress at the time of sacrifice had any effect on cytokine production, we carried out linear regression analyses to examine the relationship between serum corticosterone at the time of death and in vitro stimulation of splenocyte cytokine production (). We found that all four groups displayed a positive association between serum corticosterone and LPS-stimulated production of splenocyte IL-6, but the correlation was a strong fit and statistically significant (P = 0.0029) only for Calm Ex animals (). In contrast, the overall trend for serum corticosterone and IL-2 production after CD3/CD28 bead stimulation was a negative correlation (). Similar to the results of IL-6 analysis, we found that the strongest and most significant (P = 0.0074) relationship between serum corticosterone and IL-2 production was within the Calm Ex group ().
To investigate whether the above observations were specific to the presence of 0.005 μmol/L corticosterone in the splenocyte cultures, we applied similar regression analyses to cultures that contained other concentrations of exogenous corticosterone, including: 0, 0.025, 0.05, and 0.075 μmol/L (Supplementary Table 1). These analyses showed that the positive association between serum corticosterone and splenocyte IL-6 production remained intact at several other concentrations of in vitro corticosterone and that the strongest relationships were found in Calm Ex animals (Supplementary Table 1). Similarly, the negative correlations observed between serum corticosterone and IL-2 production were found at other concentrations of in vitro corticosterone, and again, the Calm Ex group displayed highly significant correlations (Supplementary Table 1).
In summary, animals housed in Calm, Cntl Ex and Calm Ex caging had greater mean spleen mass than Cntl, and our regression analyses suggest that the acute stress of handling had an enhancing effect on innate immune function and a suppressive effect on adaptive immune function as determined by changes in cytokine production. Finally, Calm Ex animals exhibited the greatest sensitivity to acute stress-mediated alteration of immune function.
Caging Environment and EPM Behavior
To assess the behavioral effects of housing animals in enhanced caging, we applied the EPM, a widely used test of rodent anxiety (). The EPM is a raised, plus-shaped apparatus consisting of two open arms and two arms with a walled enclosure, or closed arms. The principle of the assay relies on the innate aversion that rodents have to open elevated alleys. Interpretation of EPM behavior is derived from original work showing that rodents given anxiolytic agents spent a greater proportion of time in open arms of the maze (48
). In pilot studies of the Calm model, we found that biomarkers of stress decreased to the lowest levels after 6 wks, and we hypothesized that behavioral anxiety might be reduced on a similar temporal scale. In the present work, at 42 d each animal was placed in the EPM and behavior was digitally recorded for 5 min and later analyzed by a blinded observer (see Materials and Methods).
We found that Calm animals spent a smaller proportion of time in open arms of the EPM than Cntl animals, although none of the between-groups comparisons reached statistical significance (). Similarly, the proportion of time spent in the closed arms of the EPM was not statistically significantly different between groups nor were the number of open- or closed-arm entries (, data not shown). Lastly, we measured the total distance traveled by each animal, an indicator of exploration, and found that Cntl Ex animals traveled the greatest distance during the course of the observation period, whereas Cntl, Calm, and Calm Ex mice traversed the maze comparably ().