Tgn 020

Male wild-type C57BL/6J mice (6-8weeks old; 20-25 g body weight) were purchased from GemPharmatech Co., Ltd (Nanjing, China), and were housed under controlled environmental condition (12 h light/dark cycles, 24±1°C) with ad libitum access to food and water. Mice were divided into six groups: Sham group, TBI group, TBI+Mino group, TBI+Mino+CYA group, TBI+CYA group and TBI+TGN-020 group. All procedures were performed in accordance with the guidelines from Laboratory Animal Care and Use Committee and Animal Ethics Committee of Wenzhou Medical University. For drug administration, minocycline hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in saline (9 mg/mL) and intraperitoneal (i.p.) injected at the dose of 45 mg/kg 30 min after TBI. CYA (Sangon Biotech, Shanghai, China) and TGN-020 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in normal saline solution and used for the regulation of AQP4. TGN-020 was i.p. injected at the dose of 200 mg/kg 30 min after TBI, while CYA was i.p. injected at the dose of 100 mg/kg 60 min after TBI (CYA needs delayed injection for its combination use with Mino). These three drugs were injected with same doses at 24 h intervals before mice sacrifice. The efficacy and safety of these doses in mice have been demonstrated previously 27 (link), 28 (link). All efforts were made to minimize the pain and prevent infections.

Cerebral cortical astrocytes were prepared from E18 to 19 Sprague-Dawley (SD/Jcl, CLEA Japan) rats. Dissected cortex was treated with 0.25% trypsin (Gibco), triturated in minimum essential medium (MEM, Sigma-Aldrich) containing 10% fetal bovine serum (Cytiva HyClone), and transferred into flasks (Thermo Fisher). Cell cultures were grown to confluence at 37 °C in a humidified 5% CO₂ atmosphere. After 7 to 10 days, flasks were washed with cold Hank’s balanced salt solution (Gibco) and fed with cold MEM before shaking at 115 rpm for 2 days. Remaining adherent cells were dissociated using 0.025% trypsin-EDTA (Gibco) and plated onto coverslips or culture dishes. Cells were used after 4 to 10 days in culture unless specifically stated. For the cellular treatments, following chemicals were used: NH₄Cl (Sigma-Aldrich), ammonium acetate (Sigma-Aldrich), bumetanide (Sigma-Aldrich), TGN-020 (Sigma-Aldrich), L-methionine sulfoximine (Sigma-Aldrich), glutamine (FUJIFILM Wako Pure Chemical Corporation), and sodium hydroxide (FUJIFILM Wako Pure Chemical Corporation).