Nitric Oxide Measurement in Rat Brain

The NO production was assessed by the accumulation of nitrites (NO₂⁻) by the modified technique of Griess as described previously [69 (link)]. The cerebral cortex and hippocampus were obtained 1 h after the memory test (n = 5 rats in each group), mechanically homogenized in phosphate-buffered saline solution pH 7.4 (PBS), and centrifuged at 12,500 rpm for 30 min at 4°C by using a Z216MK microcentrifuge (HERMLE Labortechnik; Wehingen, Germany). Briefly, the NO concentration was measured in 10 μL of supernatant after the addition of 10 μL of Griess reagent (composed of equal volumes of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride and 1.32% sulfanilamide in 60% acetic acid). In addition, the optical density in a 2 μL sample was measured with a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Bancroft Building, Wilmington, USA) at 540 nm, and the optical density values were interpolated from the standard curve of NaNO₂ (1 to 10 μM) to obtain NO concentration.