5 bromo 2 deoxyuridine brdu

Rapamycin (Rap) obtained from Meilun Biotechnology Co., Ltd., China was dissolved in 100% ethanol and prepared into 10 mg/ml stock solution, which was kept at −20°C. The stock solution is dissolved with 5% tween-80 and 5% PEG-400 to desired concentration and then used for injection. Alcohol and tween-80 are used to promote drug dissolution. PEG-400 is used to promote drug dissolution and maintain drug stability. The desired concentration of rapamycin is 4 mg/kg. The 5-bromo-2′-deoxyuridine (BrdU, Beijing Solarbio technology Co.,Ltd., China) of 100 mg/kg was injected into the animal 2 h before the sample was collected. The stock concentration of BrdU was 10 mg/ml and kept away from light.

Mice were anesthetized with isoflurane (induced at 3%, and maintained at 1.2–1.6%) and positioned in a stereotaxic frame. Corpus callosum demyelination was induced by stereotaxic injection of 2 μL (1 μL for each point) of 1% lysophosphatidylcholine (LPC; Sigma, St. Louis, MO), which was an endogenous lysophospholipid that disrupts myelin-associated lipids leading to focal demyelination, in 0.9% NaCl solution at the rate of 0.5 μL/min using 1 μL microsyringe at two points of corpus callosum. Mice of sham group were injected with equal volume of saline in double-point injection: (1) left: 1.0 mm lateral to the bregma, 1.1 mm anterior, and 2.4 mm deep; (2) right: 1.0 mm lateral to the bregma, 0.6 mm anterior, and 2.1 mm deep. After injection, the needle was kept in the place for an additional 5 min to prevent backflow. The day of injection was regarded as day 0 (0 dpi). For all groups, the mice were daily administered 5-bromo-2’-deoxyuridine (BrdU, 50 mg/kg; Solarbio, Beijing, China) at 2, 3, and 4 dpi by intraperitoneal (i.p.) injection. A specific Kv1.3 blocker 5-(4-phenoxybutoxy) psoralen (PAP, 6 mg/kg; Santa Cruz Biotechnology, Santa Cruz, CA) was applied by i.p. injection for the PAP and PAP + LPC groups at 2, 3, and 4 dpi. The brain tissues were taken at 5 dpi for cryostat section or Western blot analysis.

NBP (C₁₂H₁₄O₂) was a gift from Shijiazhuang Pharmaceutical Company (Shijiazhuang, China). The NBP stock solution was diluted with corn oil for gavage. 5-Bromo-2′-deoxyuridine (BrdU) was purchased from Solarbio (Beijing, China). Antibodies against the following proteins were used in this study: myelin basic protein (MBP; cat. no. ab40390), SIRT1 (cat. no. ab189494) and BrdU (cat. no. ab8152; all from Abcam, Cambridge, UK); glial fibrillary acidic protein (GFAP; cat. no. 16825-1-AP), oligodendrocyte transcription factor (Olig)2 (cat. no. 13999-1-AP), NF-κB (cat. no. 10745-1-AP), tumor necrosis factor (TNF)-α (cat. no. 17590-1-AP), and β-actin (cat. no. 20536-1-AP; all from Proteintech Biotechnology, Wuhan, China); phosphorylated (p-)STAT3 (cat. no. YP0250) and p-AMPKα1/2 (cat. no. YP0575; both from ImmunoWay, Plano, TX, USA).