Animal care and treatment

Male Sprague-Dawley rats, aged 8 weeks, were purchased from Charles river laboratories Japan, Inc. (Kanagawa, Japan). Rats were acclimatized for 1 week before placement of the gastric catheter. Rats were fasted for 16 hr with free access to water and then an intragastric catheter was placed under the anesthetized condition by isoflurane. To alleviate suffering, intramuscular injection of buprenorphine was conducted after the operation as pain control. The joint between the gastric catheter and the nutrient infusion pump was made free-moving, which reduced restraint stress during administration of the nutrient. The recovery period was 3 days from the day of intragastric catheter. After recovery, SVA (100 mg / kg), LEV (100 mg/kg), and CBZ (50 mg / kg) were orally administered to rats just before RASS via an intragastric catheter. RASS was administered at a dose of 10 g over 30 min (short-term group) or 2 hr (long-term group) based on previous reports [17–19]. In the case of SVA, serial blood samples were obtained from the tail vein 15, 30, and 45 min, and 1, 1.5, 2, and 3 hr after oral administration of the drug. In the case of LEV and CBZ, serial blood samples were obtained from the tail vein 15, 30, and 45 min, and 1, 1.5, 2, 3, 4, and 8 hr after oral administration of these drugs. After the last blood sampling, all rats were euthanized by inhalation of carbon dioxide gas. Blood was centrifuged (2,000 g, 5 min, 4°C) to obtain plasma. For CBZ, a pharmacokinetic study was also conducted in which RASS was started 2 hr after the administration of CBZ. The animal experiment in the present study was carried out in Kamakura Techno Science Co., Ltd., contracted from EN Otsuka Co., Ltd. The experimental protocols and animal care methods used in the present study were approved by the Committee on Animal Research and Ethics at Kamakura Techno Science Co., Ltd.