Inflammatory cytokine analysis

The brain tissue was homogenised in phosphate-buffered saline (PBS; pH 7.4, 5% weight/volume), and the resultant homogenates were centrifuged at 10,000 × g for 5 min at 4 °C. Post-mitochondrial supernatants were obtained after an additional centrifugation step at 10,000 × g for 20 min at 4 °C and used to perform the enzyme-linked immunosorbent assay (ELISA) (Abcam, Cambridge, MA, USA). The levels of interleukin-1 beta (IL-1β) and tumour necrosis factor-α (TNF-α) in the brain tissue were measured with ELISA using a commercially available kit according to the manufacturer’s instructions. The detection limit of the assay was 0.1 mg/mL. The absorbance of the reaction products was measured at 450 nm using a microplate reader.