Rats sacrificed by decapitation without anaesthesia had their bra

Rats sacrificed by decapitation without anaesthesia had their brain rapidly excised on a Petri dish placed on ice. The olfactory bulbs, pons, medulla, cerebellum, hippocampus and striatum were discarded, and the cerebral cortex was dissected, weighed and homogenized in 10 volumes (w/v) of 20 mM sodium phosphate buffer, pH 7.4, containing 140 mM KCl. Homogenates were centrifuged at 750g for 10 min at 4 °C to discard nuclei and cell debris ( Evelson et al., 2001). The pellet was discarded and the

supernatant, a suspension of mixed and preserved organelles, including mitochondria, was pre-incubated at 37 °C for 1 h with Prist at concentrations of 1, 10, 100 Ruxolitinib clinical trial or 200 μM. Controls did not contain this fatty acid in the incubation medium. Immediately after incubation, aliquots were taken to measure TBA-RS, carbonyl formation, sulfhydryl content and GSH levels. In some experiments, antioxidants were co-incubated with supernatants at the following final concentrations: 10 μM Trolox (TRO, soluble α-tocoferol), 1000 μM GSH, 1000 μM N-acetylcysteine (NAC),

combination of 20 mU/mL superoxide dismutase (SOD) plus 20 mU/mL catalase (CAT), 750 μM Nω-nitro-L-arginine methyl ester (L-NAME) and 1000 μM melatonin (MEL). The chosen concentrations of the antioxidants were those capable to efficiently scavenge free radicals ( Halliwell and Gutteridge, 2007). TBA-RS was determined according to the method of Esterbauer and Cheeseman (1990). Briefly, Uroporphyrinogen III synthase 300 µL of cold 10% trichloroacetic acid were added Metformin mouse to 150 µL of pre-incubated cerebral cortex supernatants and centrifuged at 3000g for 10 min. Three hundred microliters

of the pre-incubated supernatants were transferred to a pyrex tube and incubated with 300 μL of 0.67% TBA in 7.1% sodium sulphate on a boiling water bath for 25 min. The tubes containing the mixture were allowed to cool on running tap water for 5 min. The resulting pink-stained TBA-RS was determined in a spectrophotometer at 532 nm. A calibration curve was performed using 1,1,3,3-tetramethoxypropane, and each curve point was subjected to the same treatment as supernatants. TBA-RS values were calculated as nmol/mg protein and represented as percentage of control. Protein carbonyl formation, a marker of protein oxidative damage, was measured spectrophotometrically according to Reznick and Packer (1994). Two hundred microliters of the aliquots from the pre-treated supernatants were treated with 400 µL of 10 mM 2,4-dinitrophenylhidrazine (DNPH) dissolved in 2.5 N HCl or with 2.5 N HCl (blank) and left in the dark for 1 h. Samples were then precipitated with 600 μL 20% trichloroacetic acid and centrifuged for 5 min at 9000g. The pellet was then washed with 1 mL ethanol:ethyl acetate (1:1, V/V) and dissolved in 550 μL 6 M guanidine prepared in 2.5 N HCl at 37 ºC for 5 min. The difference between the DNPH-treated and HCl-treated samples (blank) was used to calculate the carbonyl content determined at 365 nm.

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