Sevoflurane

Thirty animals were assigned to 5 dietary groups (n = 6); 1 group was fed AIN-93G as a control diet (20% CN, Oriental Yeast, Tokyo, Japan) (17 (link)), and the other 4 were fed an AIN-93G-based iso-energetic LP diet, containing either 3% CN, 3% CN supplemented with Cyss, 3% WP (Morinaga Milk Industry, Tokyo, Japan), or 3% WG (Wako Pure Chemical, Osaka, Japan). These diets were designated as the control (CT) diet, 3% CN diet, 3% CN + Cyss diet, 3% WP diet, and 3% WG diets, respectively, and their compositions are shown in Table 1. While the CT and 3% CN diet groups were fed each diet ad libitum, the other groups were pair-fed with the 3% CN diet group. These 5 dietary groups were maintained for 4 weeks. Approximately 50–200 μL of blood samples were drawn from the lateral tail vein once a week, using syringes treated with ethylenediaminetetraacetic acid disodium. Blood samples were centrifuged at 1,700 g for 10 min at room temperature (RT), and the upper plasma layers were obtained and stored at −80°C until analysis.
After 4 weeks of the above dietary regimen, animals were euthanized by deep anesthesia with sevoflurane (Mylan, Canonsburg, PA). Blood was drawn from the inferior vena cava, and plasma layers were obtained after centrifugation. Liver samples were also excised and frozen immediately in liquid nitrogen. These samples were stored at −80°C until analysis.

Milk was sampled on days 2, 9, and 16 postpartum according to the method of Keen [16] (link) with slight modifications. Briefly, dams were separated from their litters for 3–4 h before milking and were milked for 30 min between 13:00 and 15:00. Oxytocin (1 I.U., Sigma-Aldrich, St. Louis, MO) was injected intraperitoneally 15 min before milking. The dams were lightly anesthetized with sevoflurane (Mylan Inc., Canonsburg, PA) and were milked by gentle hand stripping. Collected milk samples were immediately incubated at 4°C and were centrifuged (1,200×g, 4°C, 10 min) to remove fat, cells, and large debris. Defatted and cell eliminated supernatants were centrifuged (21,500×g, 4°C; 30 min twice, and 1 h) to remove residual fat and casein, yielding a clear supernatant (whey). Blood samples were obtained at day (d) 21 postpartum (the weaning period in rats) for analyzing serum RNA as representative body fluid RNA. Dams were sacrificed by deep anesthesia with sevoflurane and blood was collected from the inferior vena cava. Blood samples were centrifuged (1,200×g, 25°C, 10 min) to obtain serum. Whey and serum samples were passed through 0.65-µm, 0.45-µm, and 0.22-µm filters to remove residual cell debris and kept at −80°C until analysis.

Rats were anesthetized via a single subcutaneous administration of MMB, followed by intubation and inhalation of 2–3% sevoflurane (Mylan, Canonsburg, PA, USA) using a mechanical ventilator. Surgery was performed on a warming pad to maintain body temperature. For the induction of myocardial infarction, the left anterior chest was incised, the intercostal space was opened with a thoracotomy device to expose the heart, and the middle part of the left anterior descending artery was ligated using 6–0 braided silk (Natsume Seisakusho, Tokyo, Japan). The chest was then closed using 4–0 braided silk (Natsume Seisakusho). Meloxicam (0.2 mg/kg; Metacam, Boehringer Ingelheim, Tokyo, Japan) diluted in saline (Otsuka Pharma) was subcutaneously administered at a dose of 0.05 mL/kg body weight. After intramuscular injection of Atipame, the rats were extubated upon the recovery of spontaneous breathing.