Dexmedetomidine

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.

Human astrocyte 1321N1 cells and rat neuron PC12 cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC, Porton Down, UK). 1321N1 cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) (Gibco; Invitrogen, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin. Some cohort 1321N1 cultures were treated with lipopolysaccharide (LPS) E. coli O111:B4 (100 ng/ml, Sigma-Aldrich, Poole, UK) for 24 h, some cohort cultures were treated with 1 µM dexmedetomidine (Sigma-Aldrich) 30 min before LPS treatment^{22 (link)}, and some cohort cultures were treated with TNF-α (100 ng/ml, Sigma-Aldrich) for 24 h. PC12 cells were maintained in RPMI-1640 medium (Gibco; Invitrogen, USA) containing 10% heat-inactivated horse serum, 5% fetal bovine serum and 100 U/ml penicillin-streptomycin. To induce the differentiation of PC12 cells into neuronal like cells, 50 ng/ml nerve growth factor (NGF) was added into the medium with 1% horse serum and cells were seeded on a Collagen IV coated dish for 24 h. The medium was routinely replaced with fresh medium every 2 days for 5 days until neuronal differentiation had taken place. PC12 cells were treated with calf thymus histones (1000 ng/ml, Sigma-Aldrich) for 24 h. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂.

Bupivacaine hydrochloride (0.5%, Sigma-Aldrich Co., St. Louis, MO, P code: 101524503 B5274-5G), Dexmedetomidine (Sigma-Aldrich Co., St. Louis, MO, P code: 12815 SML0956-10MG) and Yohimbine (Sigma-Aldrich Co., St. Louis, MO, P code: 101509939 Y3125-1G) were used.