The concentration of IL-6 (BioLegend, USA) in the brain supernatant was determined according to the manufacturer’s instruction. IL-6 ELISA (BioLegend ELISA MAX™ Deluxe kit, USA) with sensitivity limit of 4 pg/mL. All the measurements were done at room temperature in accordance with BioLegend instructions using microplate reader with 450 nm filter(Micro READ 1000, Belgium). The concentration of IL-6 from the tissue was extrapolated from the standard curve of IL-6 standards included in the assay kits. The level of IL-6 in the brain was expressed as pg/mg protein.