2.2. Sleep Deprivation Procedure

The previously described disc-on-water (DOW) method was applied [23,33,37,38]. An apparatus was used to induce sleep deprivation in experimental rats without excessive physical exertion. The yoked control group (ESC group) allowed to sleep although confined to the same apparatus, as the apparatus was turned off for a designated period of time [39]. Briefly, the apparatus was comprised of two rectangular clear plastic chambers (60 × 20 × 60 cm each) placed side by side. Beneath the disk floor were 5 cm deep trays that contained a small amount of water. Before the experiment, a rat to be sleep-deprived and its yoked control were placed in the apparatus for at least 3 days for environmental adaptation. During this period, the chambers were fitted with a solid mat in place of water. Sleep deprivation depended on rats’ hatred of water, as rats rarely enter water spontaneously. The disc was rotated slowly at a moderate speed of 3.5 rpm by computerized monitoring system. The rats had to keep pace to avoid stepping into the water. Previous studies have shown that the validated DOW setup causes experimental rats to be effectively deprived of sleep [38,39]. The animals were closely observed, including via camera surveillance, to ensure their active and safe movement on the platform or in the water. Food and water were provided ad libitum in the device and the physical condition and tolerance of the experimental rats were closely monitored. All sleep deprivation procedures were approved by the Laboratory Animal Center of Chung Shan Medical University.