Measurement of malondialdehyde (MDA) level

The concentrations of MDA in the blood and the brain tissue were examined by the MDA assay kit (Abcam). The procedures for measurements were carried out as per manufacturer instructions. Briefly, after the completion of all other examinations, the mice were first terminated by means of cervical vertebral dislocation. Immediately, the blood was collected, and 0.3 gm of brain tissue was sampled. Subsequently, the brain tissue was homogenized in the phosphate-buffered saline, followed by the incubation of the homogenate in a solution composed of 0.37% thiobarbituric acid in 50 nM NaOH and 2.8% trichloroacetic acid for 20 min in the shaken water bath. Furthermore, the sample was centrifuged at a speed of 1,500 rpm (Thermo Fisher Scientific) for 10 min followed by the collection of the supernatant. Eventually, the absorbance value of each sample was determined at 532 nm using a spectrophotometer (UV-Vis Biorad). For the MDA measurement in the blood plasma, the whole blood was centrifuged at 11,000 rpm for 10 min and the plasma was taken using a micropipette (Eppendorf) and processed for the MDA level measurement following the same procedures as previously performed for brain tissue homogenate.