2.2 | Animal preparation
Male rats weighing 400–500 g at the age of 12 weeks received general anesthesia with isoflurane 2% at 2 L/min O2 plus intraperitoneal injection with 25% urethane (5 ml/kg) before the surgical procedures. Anesthetic efficacy was determined by the lack of withdrawal from painful stimulus on the tail. All animals were placed in a supine position with a subcutaneous injection of lidocaine hydrochloride (2%) for local anesthesia. A mid-cervical tracheostomy was performed using a 16-gauge cannula. A polyethylene tube was placed into the femoral artery for the continuous recording of arterial blood pressure and blood sampling. Another catheter was inserted into the femoral vein for intravenous infusion. After these procedures, all animals were paralyzed with an intravenous injection of cisatracurium besylate (0.2 mg/kg), followed by a continuous intravenous infusion of the same agent (0.05 mg/kg/min). The animals were then sedated with a continuous intravenous infusion of propofol (0.7 mg/kg/min). The animals were connected to a volume-controlled animal ventilator (Model 683, Harvard Apparatus, Holliston, MA, USA) to establish conventional ventilation. A tidal volume of 6 mL/kg, an inspiratory versus expiratory ratio of 1:1, a positive end-expiratory pressure (PEEP) of 3 cm H2O, and a fractional FiO2 concentration of 1.0 were maintained throughout the experiments. The original ventilation rate was set at 60 breaths/min, followed by an increment or decrement of 2–3 breaths/min to maintain an arterial blood PCO2 within 40 and 50 torr (5.33–6.67 kPa) and pH > 7.25. The body temperature was maintained at 38 °C–39 °C via a servo-controlled heating blanket with a probe monitoring the anal temperature throughout the experiments.