Abstract
Stress associated with transport and change of environment may have widespread effects on physiological parameters in laboratory animals. To investigate the time needed for mice to acclimatize to a new environment, based on fecal IgA and corticosterone excretion, eightweek-old BALB/c mice of both genders were housed either in groups of eight in different cage types in open conventional cages, in Individual Ventilated Cages (IVC), in open conventional cages inside a plastic isolator, or in different group sizes (8, 4, 8, 10 or 12 mice in each group) in open conventional cages. Feces were collected from each cage on routine cage changing. There was no significant difference in corticosterone excretion in feces between animals housed in the different cage types or between animals housed in different group sizes. IgA excretion for both males and females was found to be affected by transfer of mice into a novel cage, and it was found that it takes at least four weeks for the mice to acclimatize to a new environment with respect to this parameter.
Stress associated with transportation and translocation between facilities has widespread effects on physiological parameters in laboratory animals, including changes in the cardiovascular, endocrine, immune, central nervous, and reproductive systems, and even the phenotype may change following the move of a model from one facility to another (1-4). Although short-lived, these changes may confound research if animals are utilized before homeostasis is restored and physiological measures return to a new baseline. Therefore, a period of acclimatization following transportation is generally practiced to restore homeostasis (4-7). The following two questions should be considered to determine an adequate period for acclimatization: May physiological changes confound the research to be conducted? And, if no: what is the length of the time necessary for confounding physiological changes to normalize? Most literature on the physiological impact of translocation of laboratory animals has focused on acclimatization to changes in ambient temperature. It is documented that many physiological parameters, including stress-associated hormones, such as glucocorticoids, return to baseline within the first week following transportation (8-10). Blood pressure seems to return to normal within approximately three weeks (4, 6, 7, 11). Other measurements, such as circadian rhythm, immunological parameters and reproductive performance may take several weeks to months to normalize (12, 4).
The aim of the present study was to investigate the time needed for mice to acclimatize with respect to the stress-sensitive biomarker fecal corticosterone and the immunological parameter fecal IgA, and whether the acclimatization was influenced by cage type and social grouping.
Secretory IgA and corticosterone are both recognized as stress-sensitive molecules in rodents (3, 13-15). Corticosterone is the major glucocorticoid in mice, and since it is excreted into urine and feces, this makes the collection of its metabolites through fecal samples very easy and non-invasive (3, 16, 17). When animals are confronted with a physical or emotional stressor, they rely on different biological systems to cope with the situation and this can lead to different behavioral, autonomic, neuroendocrine or immune responses (16, 17). Environmental, as well as psychological, stressors will activate the hypothalamo pituitary adrenocortical (HPA) axis, and glucocorticoid, of which corticosterone is the active hormone in rodents, is secreted from the adrenal cortex to the blood stream to their target tissues. The glucocorticoids are thereafter metabolized in the liver and are excreted as conjugates (sulfates or glucoronids) through urine and bile in feces (16, 17). Although steroids in the gut are subjected to some extent to an enterohepatic circulation, i.e. reabsorption into the blood stream, and are metabolized by the microbial flora, the steane skeletal structure of these steroids are not degraded. Therefore specific steroid metabolites can be detected in the feces (17). Since there is no need to capture, handle or restrain the animals when collecting fecal samples, it is a good way to carry out repeated sampling without affecting the animal's behavior or endocrine status. In addition circulating hormone levels in the feces are integrated over a certain period. Hence, rather than the actual steroid concentration in the circulation, fecal hormone metabolite levels reflect the production rate, that is, the cumulative secretion and elimination of hormones over several hours (17). Therefore, fecal samples are less affected by episodic fluctuations or pulsatility of hormone secretion.
It has been shown that glucocorticoid levels in body fluids are useful indicators of acute stress, but not necessarily of chronic stress, since the elevated levels may return to normal even if the stressor persists. It is, on the other hand, well-documented that stress of long duration results in immunosuppression. In the immune system, glucocorticoid receptors can be found on all ajor subsets of leucocytes at different densities. Total serum levels of IgA, IgG and IgM are reduced after the administration of high doses of glucocorticoids, but treatment with lower doses may instead increase the level of IgA, IgG and IgM production (14).
Secretory immunoglobulin A (sIgA) is one of five classes of immunoglobulins found in serum and secretory fluids, including saliva, breast milk and nasal, gastrointestinal and bronchial secretions. IgA-producing plasma cells are primarily situated within the lamina propria of the mucosa but are also present in the bone marrow and the secondary lymphoid organs (18). The presence of IgA antibodies on the gastrointestinal mucosal surfaces makes it a major effector of host defense against microorganisms (19). Fecal IgA seems to be usable as a marker of stress of longer duration in rats (14). IgA levels have been shown to be reduced in the presence of chronic stress and high workload situations in humans and animals (19-21). Although it is still widely agreed that chronic stress reduces immune functioning, there is growing evidence that acute stress can actually lead to an increase in several measures of immunity in laboratory settings (19-21). There appears to be a stress-activated pathway from the central nervous system to the mucosal immune cells, however, the mechanism by which stress induces immunomodulation is still not sufficiently understood (22, 23). It has been shown that even a short period of relatively mild stress, as for instance restraint stress, enhances intestinal epithelial permeability to macromolecules (22, 24). A result of this barrier dysfunction is that the permeability of the intestines is increased, and this will be followed by an increase in antigen invasion, which will probably influence the intestinal immunity to a considerable extent (22, 24, 25). There also seems to be a sex difference, and differences dependent on the time of day; female rats had consistently higher IgA levels in fecal samples than did males, and that more IgA was excreted in the feces in the evening than in the morning (14).
Materials and Methods
Animals and housing conditions. For the study, housing the mice in different population densities, 105 eight-week-old female and 105 eight-week-old male BALB/c mice were used. The mice were randomly allocated into groups of 1, 4, 8, 10 or 12 male or female mice in each cage and three experimental units in each group. The animals were studied for six weeks.
For the study, housing the mice in different cage types, 72 eight-week-old female BALB/c mice were used. The mice were randomly allocated into groups of eight mice and three experimental units in each group, housed in either conventional open type III cages, type III Individual Ventilated Cages (IVC), or in type III conventional cages inside an isolator from Ehret flexible plastic isolator, Germany. All cages were from Tecniplast, Varese, Italy with a floor area of 756 cm2. All mice were from Taconic, Ry, Denmark and experimentally-naïve.
The animals were housed in conventional animal rooms, with a temperature between 19° and 23°C under a 12:12 hour light:dark cycle, with lights on at 6.00. All animals were provided with standard cage enrichment, cardboard shelter, wooden stick and nesting materials. The animals were fed fortified Altromin 1319 (Brogaarden, Denmark) and acidified (citric acid) tap water (pH=3) ad libitum.
Throughout the study, the animals were handled like other mice in the facility. The cage changing regimen was the same, as what would normally be used, since it has previously been shown that human interference, such as changing cages and handling, may have marked effects on the animals and their activity (26).
To avoid any bias of social hierarchy development, the animals were put into new groups one week before the study. It is common practice to allow laboratory animals to adapt to the new environment after arrival from the breeder prior to experimentation (27).
Sampling. Feces were collected once a week from each cage just after routine cage changing, but once every two weeks for animals caged in IVC cages, since this is the regimen of cage changing for IVC cages in our facility. The fecal pellets were stored in plastic test tubes at −20°C before analysis. The samples were dried in a drying oven at 95°C for 1h for water to evaporate from the sample.
Extraction method. To extract corticosterone, 0.9 g of dried feces were mixed with 6.75 ml of 96% ethanol. The sample was placed on a shaking table at room temperature overnight, then centrifuged (2500 ×g, 10 min) and 4.8 ml of the supernatant was placed in a water bath and left to evaporate to dryness. Then 300 μl Phosphate Buffered Saline (PBS) were added to re-suspend the sample. To extract the IgA, 0.8 g of dried feces were homogenized with 20 ml PBS (pH 7.2). The sample was centrifuged and the supernatant was collected.
Quantification of corticosterone metabolites and IgA. The corticosterone metabolite concentrations were analyzed using a commercial ELISA corticosterone assay from DRG Diagnostic (Marburg, Germany), using the manufacturer's instruction manual. All samples were analyzed in duplicate. The IgA concentrations were analyzed according to a previously described ELISA assay (14). Detection limits were estimated to 0,3 ng/ml extraction medium for Corticosterone and 20 ng/ml extraction medium for IgA. All samples were analyzed in duplicate.
Concentration of corticosterone (cort) in feces of mice housed in different cage types.
Statistical testing. The effects of sex, group size and time – allowing for interactions – on the excretion of both corticosterone metabolites and IgA were tested using analysis of variance. All statistical testings were carried out in PASW Statistics v.18 (IBM corp., Armonk, NY, USA).
Results
Cage type. The effect of different caging systems on the fecal corticosterone metabolite excretion is shown in Figure 1 and those of the fecal IgA concentration in Figure 2. There were no significant differences in the concentration of fecal corticosterone metabolite excretion or fecal IgA excretion in animals housed in open cages, IVCs or open cages in flexible plastic isolators.
Social grouping. There was no significant difference in corticosterone metabolite excretion in feces between animals housed in group sizes of 1, 4, 8, 10 or 12 animals per cage (Figure 3). Females had consistently higher corticosterone levels in fecal samples than did males (F1, 14=49.4, p<0.01). No interaction between sex and group size was, however, found (F1, 14=1.20, p=0.313).
IgA excretion for both males and females was found to adhere to a bimodal distribution consisting of two log-normal distributions, with minimal overlap (Figure 4). A cut-off point was set at 2,2 μg/g feces, as this was found to be a good approximation of the geometric mean bisection of the inter-modal distance and both sub-populations were found to agree well with log-normal distributions using Q-Q plots and the Shapiro-Wilk test of normality. Over time, the animals were found to move from the population of high excreters to low excreters (Figure 5), and after six weeks, most animals had moved from the high-excreting to the low-excreting group, but not all; four individuals still excreted IgA levels outside the 95% CI for the low excreting population. Thus, nearly all mice may be considered as having transferred from the high-excreting population to the low-excreting population during the course of the study, notably without producing any in-between states.
Concentration of IgA in feces of mice housed in different cage types.
Corticosterone (cort) concentration in feces for the different population densities. Error bars represent±SEM.
Apart from the change over time, analysis of variance revealed an additional effect as the three-way interaction term sex×time×group size was found to be significant (F49, 59=2.10, p<0.01). That is to say, although the trend for a decrease in excreted IgA is present in nearly all mice, certain groups were quicker than others in adopting a low-excreting profile. Three out of the four high excreters found in week six were single-housed females – i.e. none of the single-housed females reduced their IgA excretion during the experiment, whereas all male mice housed in groups of ten were found to be in the low-excreting population, as early as week two. Between these two extremes, no clear trend could be found with increasing/decreasing group sizes or between sexes (Figure 6).
Histograms of fecal IgA concentrations. The frequency refers to the total number of observations during the six-week period. The grand population – whether sub-divided by sex (inset graphs), or not – adhere to a bimodal distribution consisting of two log-normal populations. The high-excreting population is here defined as excreting an excess of 100 μg IgA/g feces.
Discussion
The present study demonstrated that the animals had stable fecal excretion of corticosterone metabolites, already after one week's habituation to the new cage environment when fecal sampling commenced. This is in agreement with studies demonstrating that corticosterone responses to stressors return to baseline within a couple of days (1, 9, 27, 29, 30). Neither cage type nor social group size had any significant quantitative impact on fecal excretion of corticosterone metabolites. The recorded gender difference in excretion of corticosterone metabolites confirms previous findings (1, 3, 23).
Many immunological parameters have been documented to be highly susceptible to stress, and studies of many different species, including man, have shown that secretory IgA is sensitive to psychological variables and stress (15, 19, 31, 32). A transient stressor, such as being moved to a novel environment – which results in a corticosterone response of short duration, has been shown to result in an immediate immune response, up-regulating B-cell-mediated immune functioning, whereas prolonged stress has been reported to inhibit immune system functioning (20, 21, 33). Yamamoto and collaborators found that daily foot shocks for two weeks led to significantly elevated levels of IgA in lymph nodes proximal to the gut mucosa of rats (22). This is in agreement with the findings of the present study in which a gradual decline in fecal IgA excretion from an implied elevated initial level was recorded. After four weeks, the fecal excretion of IgA seemed to stabilize at baseline value, suggesting that the immune system of the animals takes much longer to habituate and acclimatize to a new environment, than do many other physiological parameters. On the other hand, we do not know if the levels seen after a four-week period are really a baseline level. It could also be a lowered level of IgA excretion due to persistent stress. Studies in other species (12, 34-36) have documented that a variety of immunological parameters are disturbed by translocation to a new environment and that individuals adapt surprisingly slowly with respect to immune function to environmental changes. We do not fully-understand the mechanisms by which stress induces immunomodulation.
IgA in feces was analyzed using a commercial ELISA assay. This assay does not distinguish between sIgA and serum IgA excreted in feces. IgA is also present in serum where it functions as an inflammatory antibody through interactions with the IgA Fc receptor (FcαR) on immune effector cells. Mice display a relatively high rate of biliary transport of plasma IgA, it was reported that 22-28% of injected 125I-labeled IgA was led into the duodenal fluid through the liver, which is 38% of the eliminated intravascular IgA from mouse plasma (37). The rest of the serum IgA is cleared by internal catabolism (37). In order to distinguish between sIgA and the IgA excreted from serum to the intestines via the liver, measurements should be made on part of sIgA that is distinguishable from serum IgA, for instance the secretory component. The measurement made in this study is the total amount of IgA in the feces. Since acute stress will lead to a general elevation in the IgA level, both in serum and in mucus surfaces, this would be the correct way of measuring a rise or fall of total IgA.
Change in IgA excretion over time is illustrated as transference of number of animals placed in the high excreting population moving to the low excreting population over a 6 weeks period. Frequency refers to the number of observations in both populations obtained for each week. Total number of experimental units (i.e. cages) is 30.
It has been shown that there are gender differences in the behavior induced when mice are housed individually, and it seems that females are more sensitive than males to single-housing (38-42). In the present study, the single-housed females did not decrease in any IgA excretion during the experiment, and three out of the four high excreters found in week six were single-housed females. Whether this means that females have a higher than normal level of IgA or they actually have normal levels and it is all the others that have lowered levels of IgA is not certain. Studies in other species (12, 34-36) have documented that a variety of immunological parameters are disturbed by translocation to a new environment and that individuals adapt surprisingly slowly with respect to immune function to such environmental changes.
In conclusion, the present study demonstrates that fecal excretion of IgA is affected by translocation of mice into a novel cage, suggesting that it takes up to four weeks for mice to acclimatize to a new environment, with respect to this parameter; this possibly may be even longer for single-housed female mice. This response was uniform, regardless of social group size and whether the cage was conventional, IVC, or located in a flexible plastic isolator.
The concentration of IgA in feces of male and female mice housed in group sizes of 1, 4, 8, 10 or 12 animals in a 6-week period.
- Received June 15, 2012.
- Revision received August 10, 2012.
- Accepted August 13, 2012.
- Copyright © 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
