Impact of short-term isolation stress on urinary stress hormone excretion

Background and objectives: Exposing stressors on mammals, stress hormones are secreted from adrenal glands to blood, consequently excreted into urine. Catecholamines or cortisol (corticosterone in rodents) are produced by the activation of sympathetic adrenomedullary axis (SAM axis) or hypothalamic-pituitary-adrenal axis (HPA axis). By the determination of urinary stress hormone levels, it can estimate the state of stress in the animals. In the previous studies, urine collection from the rodents for measuring stress hormone levels was carried out by a single housing mouse. On the other hand, mouse in single-housing exposes potent social isolation stress, consequently showing depression and anxiety-like behavior. In this study, we compared the levels of urinary stress hormone between mice in single- or pair-housing to verify the validity of the conventional evaluation methods. Methods: A mouse (single-housing group) or two mice (pair-housing group) was housed in metabolic cages for 4 days. Each day during that period, they were weighed, and collected their daily urine into a tube filled with 6N hydrochloric acid. Urinary noradrenaline and adrenaline were determined by HPLC equipped with an electrochemical detector, and their final metabolites, vanillylmandelic acid (VMA) were determined by UPLC-MS/MS. Urinary corticosterone levels were measured using ELISA kit. The level of stress hormone excretion was corrected by urinary creatinine levels. Results: Compared with the pair-housing group, the bodyweight of the single-housing group significantly decreased from day 1, and this change was not recovered during the experimental period. The total amount of catecholamines and corticosterone excreted into urine for 4-days was markedly increased in the single-housing group compared to the pair-housing group. Conclusions: Significantly decreasing body weight and increasing urinary catecholamine and corticosterone in the single-housing group compared with the pair-housing group. It was found that short-term social isolation stress activated the HPA and SAM axis and increased the excretion of stress hormones. Therefore, when verifying the effects of the bioactive compounds on sympathetic nerve activity (e.g. capsaicin), using the urine collected by pair-housing is more appropriate than single-housing.