A chronic combinatory stress model that activates the HPA axis and avoids habituation in BALB/C mice.

A detailed protocol is described to induce chronic stress in BALB/c mice, which affects the hypothalamic-pituitary-adrenal (HPA) axis. The protocol is based on a combination of two mild physical stressors: restraint stress and forced swimming. Physical stressors were applied on an alternative schedule: one day restraint, next day swimming, during a 28-day period. Mice were sacrificed at days 7, 14, 21 and 28 and plasma was obtained. Optimized chromatographic system with electrochemical detection and a commercially available enzyme immunoassay kit were used to measure catecholamines [representative of the activation of the autonomic nervous system (ANS)] and corticosterone (representative of the activation of the HPA axis). Corticosterone levels increased in mice under stress and remained significantly higher in stressed mice compared to control animals throughout the experimental procedure, indicating that mice did not show habituation to the combined stress. In our experimental conditions, catecholamine levels were not useful as an index of stress. The stress model applied here provoked a steady activation of the HPA axis resulting in the constant secretion of corticosterone from the adrenal gland, which may prevent the activation of the ANS axis. The simple and economic stress model presented here affected the HPA axis but not the ANS of BALB/c mice and is useful for the study of metabolic stress-related gastric pathology and stress hormone secretion in mice.