Chronic restraint stress protocol

Mice were randomly divided into two groups: A control and a chronic restraint stress group. Mice in the control group were housed in the individual cages and were left undisturbed. Mice in the chronic restraint stress group were subjected to restraint stress using a ventilated plastic 50 ml tube that allowed for a close fit to mice. Subsequently, mice were submitted to immobilization stress for 2 h per day for period of 14 consecutive days using a self-made restraint device (a 50 ml centrifugal tube with a number of ventilation holes ~5 mm in diameter and a small hole for the tail) (17,18). During the stress period, mice were not allowed access to food and water. Following chronic restrained stress, mice were maintained in individual cages and allowed free access to food and water. Body weight and food intake were monitored every two days during the stress period.