Atipamezole

The experimental procedures were approved by the Peking University Biomedical Ethics Committee experimental animal ethics branch (Approval No. J201807) and performed in accordance with the institutional guidelines. Adult male Sprague-Dawley rats (250–300 g) were purchased from the Experimental Animal Center of Peking University Health Center (Beijing, China). Rats were housed in standardized conditions with room temperature 24 ± 2 °C, controlled humidity 55 ± 5%, 12 h light/12 h dark cycle and access to water ad libitum. Rats were injected intraperitoneally with 10 mg/kg LPS (Sigma-Aldrich), dissolved and diluted in sterile normal saline. Dexmedetomidine (Sigma-Aldrich) was then intraperitoneally injected at a dose of 25 µg/kg every 2 h for three times immediately after LPS administration^{22 (link)}. One cohort was injected intraperitoneally with 500 µg/kg α₂-adrenoceptor antagonist atipamezole (Sigma-Aldrich) every 2 h in three doses prior to the administration of Dexmedetomidine^{22 (link)}. Controls received comparable volume injections of saline. At 24 h after the first treatment, animals were sacrificed with pentobarbitone (100 mg/kg, i.p.). Their hippocampus was quickly dissected and snap frozen in liquid nitrogen from the half of the animals from each group. The remaining animals were perfused with 4% paraformaldehyde and their brains were collected.

Male Sprague-Dawley rats (8–10 weeks old and 220–250 g) were purchased from the Medical Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine. Fifty-six rats were randomized into seven groups (n = 8 per group). Rats in the sham-operated group (S) did not undergo OALT. Rats in the model group (M) were intraperitoneally injected with saline 30 min before OALT. Rats in the D1 and D2 groups were intraperitoneally injected with 10 μg/kg (drug/body weight) and 50 μg/kg Dex (Hengrui Pharmaceutical Co., Ltd., Jiangsu, China) 30 min before OALT, respectively. Rats in the B1, B2, and B3 groups were intraperitoneally injected with 500 g/kg atipamezole (a nonspecific α_2A-AR siRNA blocker, Sigma-Aldrich, USA), 50 g/kg ARC239 (a specific α_2B/C-AR blocker, Santa Cruz Biotechnology, Inc., USA), and 1.5 mg/kg BRL-44408 (a specific α_2A-AR siRNA blocker, Sigma-Aldrich, USA) 40 min before receiving 50 μg/kg Dex prior to OALT, respectively. This study was performed with the approval of the Institutional Animal Care and Use Committee of Sun Yat-sen University in Guangzhou, China, and in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals.

Mice were pretreated with dexmedetomidine (50 μg/kg, i.p.) or vehicle at 30 min prior to 4-hour exposure to 1.3% isoflurane (in humidified 30% oxygen carrier gas) in a chamber partially submerged in a 37°C water bath [6 (link),8 (link)]. Isoflurane was delivered using a vaporizer (Aerrane, Baxter Healthcare, Deerfield, IL, USA). Concentrations of oxygen, carbon dioxide and isoflurane in the chamber were monitored using a monitor from Detex-Ohmeda (Louisville, KY, USA). A group of mice not exposed to isoflurane was included as an additional control. Core body temperature was measured with a rectal probe and maintained at 37 ± 0.5°C using a heating blanket.
In some experiments, animals were pretreated with the α₂ adrenoreceptor antagonist atipamezole (250 μg/kg, i.p.), the JAK2 inhibitor AG490 (15 mg/kg i.p; Sigma-Aldrich, St Louis, MO, USA) or the STAT3 inhibitor WP1066 (40 mg/kg i.p.; Sigma-Aldrich) at 30 min before dexmedetomidine injection.
Mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p., n = 16) at the end of the 4-hour isoflurane exposure and blood samples were obtained from the femoral artery of animals for measurement of arterial blood gases and blood glucose. Animals were then allowed to recover from anesthesia. Cognition was assessed using a Morris water maze test at 2 w after isoflurane exposure.

Dexmedetomidine (Hengrui, Jiangsu, China), atipamezole (Sigma-Aldrich, USA), BRL44408 (Sigma-Aldrich, USA), and ARC239 (Santa Cruz, USA) were dissolved in 0.9% saline at 2.5 μg/ml, 25 μg/ml, 50 μg/ml and 2.5 μg/ml concentrations, respectively. Heparin (Chen Xin Pharmaceutical Co., Ltd., Shandong, China) was diluted in 0.9% saline or acetic acid Ringer’s solution at 25 U/ml; and 12.5 U/ml of protamine sulfate (KaiYue Pharmaceutical Co. Ltd., Beijing, China) was diluted in saline at 0.05% concentration.