Gas monitor

SD rats of postnatal day 7 (P7; weight, 16–17 g) were randomly allocated into 4 groups: An air-treated control group and 0.3, 1.3 and 2.3% sevoflurane (Abbott Laboratories, Abott Park, IL, USA) treatment groups, with 21 rats in each treatment group. Rats in the sevoflurane treatment groups were placed in a plastic chamber and exposed to 0.3, 1.3 or 2.3% sevoflurane for 6 h with air as a carrier at a gas flow of 2 l/min. During sevoflurane exposure, the chamber was heated to 38°C using a warming device (NPS-A3; Midea Group Co. Ltd., Foshan, China). sevoflurane, oxygen and carbon dioxide levels in the chamber were calibrated by a gas monitor (Datex-Ohmeda, Inc., Madison, WI, USA). After 6 h, sevoflurane delivery ceased, and the animals were exposed to air again. When these rats moved freely, they were placed back into the maternal cage. During anesthesia, the respiratory frequency and skin color of the rats were monitored by an investigator. If signs of apnea or hypoxia were observed, the rat was exposed to air immediately and excluded from the experiment. Rats in the control group were placed into the same container as those in the sevoflurane groups but exposed to air for 6 h.

Male Sprague-Dawley rats (n=144, 20 months of age and weighing 500–600 g) were purchased from the Dongchuang Laboratory Animal Center (Changsha, Hunan, China) and housed in standard barrier facilities. They were maintained under a 12-h light/dark cycle (lights on at 07:00) with food and water ad libitum. The animals had a recovery period of at least 7 days to adapt to their new environment before experiments began.
isoflurane exposure was performed as previously described (2 (link)). In a transparent anesthetic chamber, rats were exposed to 1.5% isoflurane (Baxter Healthcare, Deerfield, IL, USA) with 2 l/min of 100% oxygen (Beijing Millennium City Gas Sales Center, China) as the carrying gas, or to vehicle (2 l/min of 100% oxygen) for 4 h. At the outlet of the chamber, gas composition (the concentrations of isoflurane, oxygen, and carbon dioxide) within the chamber was continuously analyzed by a gas monitor (Datex-Ohmeda, Inc., Louisville, CO, USA). After anesthesia, the rats received 100% oxygen until they recovered complete consciousness. In a previous study, it has been shown that this anesthesia protocol does not cause significant changes in glucose or blood gas (1 (link)).

The animals were randomly exposed to isoflurane (Baxter Healthcare of Puerto Rico, Guayama, Puerto Rico). isoflurane (1.5%) with 2 l/min 100% oxygen as carrying gas (n=30) or vehicle gas (Beijing Millennium City Gas Sales Center (Beijing, China) was employed at 2 l/min 100% oxygen (n=30) for 4 h. To examine the role of GluN2B in anesthetic-induced changes of spatial learning and memory, an additional two groups (n=30, respectively) were administered the specific GluN2B antagonist, Ro25-6981 (3 mg/kg, 2 ml/kg, i.p.; Sigma-Aldrich, St. Louis, MO, USA) daily for 6 days, 24 h after exposure to isoflurane or vehicle gas.
The animals were maintained in an anesthesia chamber during isoflurane or vehicle gas exposure. At the outlet of the chamber, the concentrations of isoflurane, oxygen, and carbon dioxide in the chamber were continuously analyzed with a gas monitor (Datex-Ohmeda, Inc., Louisville, CO, USA). In a previous study, it was shown that this anesthesia protocol did not cause significant changes in blood gas or glucose (1 (link),2 (link)).