The flavonoid quantification was carried out using calibration graph with nine data points. Calibration graph for HPLC was recorded with rutin amounts ranging from 0.156 to 50.0 μg/mL. The relationship between peak areas (detector responses) and amount of rutin was linear (r2=0.9953). To evaluate the repeatability of the injection integration, the rutin standard solution and all samples were injected three times

and the relative standard deviation values were calculated. Identification was performed comparing the retention time (tR) and UV spectrum of peaks in the samples of plasma with standard rutin: tR=18.6 min (97.5% purity). To quantify the extension this website of lesion, animals from control and R50 groups suffered two injections, one just after the end of surgical procedure and other 24 h after ischemia. They were euthanized with CO2 48 h after ischemia. Untreated sham animals were also evaluated to check for lack of cortical injury. Brains were rapidly removed

from the skull and sectioned in the coronal plane at 2 mm thickness using a rat brain blocker/slicer (Insight Ltda.). The slices (five for each animal) were immersed for 30 min into 2% 2,3,5-triphenyl tetrazolium chloride (TTC) solution at 37 °C. Digital images from reacted slices were captured under conventional light illumination using a Nikon digital camera (Nikon Co., Tokyo, Japan) coupled to a dissecting microscope and a PC computer. Oligomycin A in vivo Lesion areas of slices were measured from digital images using the ImageJ software (NIH). The lesion area of each slice was multiplied by its thickness (2 mm), obtaining the volume (mm3). For each animal, the total lesion volume was calculated by summing the lesion volumes of its slices. To analyze the effect of treatment with rutin in neurodegeneration, animals from control and R50

groups suffered three injections, one just after the end of surgical procedure, one 24 h and one 48 h after ischemia. They were euthanized with CO2 72 h after ischemia and intracardially perfused with cold 0.9% NaCl solution followed by a solution of 4% paraformaldehyde, in 100 mM phosphate Tideglusib buffer (pH 7.4). Brains were removed and immersed in 100 mM phosphate buffer containing 20% sucrose for 24 h at 10 °C. Brains were sectioned in the coronal plane at 30 μm thickness at 20 °C on a CM 1850 cryostat (Leica Instruments GmbH, Heidelberg, Baden-Wurttemberg, Germany). Sections were subjected to FJC staining, in accordance with the manufacturer’s instructions (Schmued et al., 2005). Briefly, they were immersed in a solution of 1% sodium hydroxide in 80% alcohol for 5 min, 70% alcohol for 2 min, distilled water for 2 min, and 0.06% potassium permanganate for 15 min. They were immersed into a solution of 0.0005% FJC (Histo-Chem Inc., Jefferson, AR, USA) in 0.