Distribution and time course of corticosterone excretion in faeces and urine of female mice with varying systemic concentrations
Introduction
In striving towards more accurate and continued ethically defensible in vivo biomedical research it is of great value to be able to assess stress levels, non-invasively, in laboratory animals. The ultimate aim with stress level assessments is the complete elimination of avoidable stress and pain in animal experimentation. The most popular animal species in biomedical research in Europe is, by far, the laboratory mouse (Commission to the Council and the European Parliament, 2007), and this is probably also the case worldwide (Taylor et al., 2008). The most common biomarker used to evaluate acute stress response in mice is serum corticosterone. Corticosterone (CORT) is an effector hormone of the hypothalamic–pituitary–adrenal (HPA) axis, and is released as the HPA axis is activated in response to a stressor. Blood sampling, and the associated physical restraint of the animal, is however a stressful procedure and serum CORT is therefore affected (Abelson et al., 2005, Gartner et al., 1980, Vachon and Moreau, 2001). Furthermore, serum CORT only provides a snapshot of a parameter which varies greatly over time and between individuals (Atkinson et al., 2006). For these reasons, measuring corticosterone and corticosterone metabolites (CM) in excreta has become a widely accepted alternative (Bamberg et al., 2001, Möstl and Palme, 2002), providing a non-invasive method for assessing stress levels. By measuring CM in faeces or a pooled sample of urine an average CORT turnover over a longer time period can be established (Palme et al., 2005).
An implicit assumption in comparing excreted CM levels to determine relative stress levels in animals is that the time course and distribution of CM in the two terminal compartments, urine and faeces, remain unaffected by varying concentrations of circulating CORT. This is an assumption that has never been tested. Considering that both time course and terminal distribution – i.e. excretion in faeces and urine – of a number of endogenous (e.g. folate (Gregory et al., 1998)), as well as exogenous (e.g. mercury (Morcillo and Santamaria, 1995)), compounds are highly variable, and affected by the systemic concentration, it is vital to test whether this is also the case for corticosterone.
The aim of the present study was to investigate the time course and terminal distribution of CM following tail vein injection of mice with varying doses of CORT, where a subset had been radioactively labelled. This provides valuable information on whether faecal and urinary CM can be used as independent, but equally applicable, measures of preceding serum CORT concentrations.
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Animals
Thirty-six female, 7–9 weeks old, BALB/c mice (Taconic, Ry, Denmark) with a FELASA approved health status, each weighing approximately 20 g, were group housed and acclimatised for 2 weeks prior to the experiment. The animals were subjected to standard housing conditions throughout the experiment: Diurnal rhythm was regulated through a 12 h light/12 h dark cycle (lights on from 6:00), temperature was kept at 20–22 °C, with a relative humidity of 30–60%. Food pellets (Altromin 1319; Brogaarden,
Time course
The recovered 3H activity resulted in corrected doses covering a range of 0–51 μg/kg bw. No two experimental units received the same dose. In two of the experimental units, there was cross-contamination between two of the urine samples (from the same experimental unit) prior to the scintillation analysis. No mean residence time could therefore be calculated for these units, but a CM distribution between the terminal compartments could still be established.
Fig. 1a and b present the mean residence
Discussion
The concentration–time profiles for excreted 3H-CORT, as well as the final measurements, supported our and others’ (Touma et al., 2003) previous findings, where all of the injected CORT is excreted within 26 h. For female BALB/c mice the present results demonstrate that 60% of any injected CORT is excreted in the urine with a delay of, on average, 5 h. The remaining 40% is excreted in the faeces nearly 9 h after entering the blood stream. The faecal/urinary ratio of CM coincided well with previous
Acknowledgments
This study received generous funding from the Danish Research Council. The authors thank Trine Marie Nielsen, Niels Grunnet and Lis Frandsen for expert technical assistance.
References (21)
- et al.
Partly ordered synthesis and degradation of glycogen in cultured rat myotubes
J. Biol. Chem.
(2002) - et al.
Urinary excretion of [2H4] folate by nonpregnant women following a single oral dose of [2H4]folic acid is a functional index of folate nutritional status
J. Nutr.
(1998) - et al.
Hormones as indicators of stress
Domest. Anim. Endocrinol.
(2002) - et al.
Anxiety responses, plasma corticosterone and central monoamine variations elicited by stressors in reactive and nonreactive mice and their reciprocal F1 hybrids
Behav. Brain Res.
(2007) - et al.
Acute and chronic stress exert opposing effects on antibody responses associated with changes in stress hormone regulation of T-lymphocyte reactivity
J. Neuroimmunol.
(2003) - et al.
Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones
Horm. Behav.
(2004) - et al.
Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice
Gen. Comp. Endocrinol.
(2003) - et al.
High plasma corticosterone levels persist during frequent automatic blood sampling in rats
In Vivo
(2005) - et al.
Distribution of [3H]-corticosterone in urine, feces and blood of male Sprague-Dawley rats after tail vein and jugular vein injections
In Vivo
(2009) - et al.
Diurnal variation in the responsiveness of the hypothalamic–pituitary–adrenal axis of the male rat to noise stress
J. Neuroendocrinol.
(2006)
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