For each animal at each PID, percentage relative

to the total number of uses (ipsilateral+contralateral+simultaneous) was calculated for ipsilateral (unimpaired) and contralateral (impaired) uses. An asymmetry score for each animal was calculated at each PID by the following formula: asymmetry score=(% of ipsilateral uses)−(% of contralateral uses). Animals with asymmetry score higher than 15 at PID 0 were discarded for statistical analysis. In the adhesive removal patch test, a small round adhesive paper (13 mm diameter) was placed on the inner portion of each wrist of the animal. One trial consisted in placing the adhesive papers and their subsequent removal by the animal. Four trials were applied at each PID, and trials were always separated Selleck PLX4032 by at least 5 min. Preference was evaluated, and in each trial the first side (ipsilateral Selleck GKT137831 or contralateral to the lesion) of removal was recorded. For each animal at each PID, percentage of contralateral preference relative to the total number of removals (four) was calculated.

Animals with preference to the right forelimb (more than 50% of first removal at pre-ischemic day) suffered focal ischemia in the left hemisphere (see Section 2.2.), and vice-versa. To check for lack of influence of whole experimental procedure in functional loss, untreated sham animals were also evaluated in adhesive test. To evaluate the plasmatic absorption of rutin after an i.p. injection, animals from R50 group were euthanized with CO2 2, 4, 6 or 8 h after the injection. Animals from the control group were also evaluated. Blood was collected by cardiac puncture with heparin and the plasma obtained by centrifugation at 12,000 g for 10 min. Plasma was acidified to pH 4.0 with phosphoric

acid. After acidification, methanol was added (1000 μl: 200 μl of plasma), and the sample was stirred for 1 min and centrifuged at 12,000 g for 10 min. Supernatant was collected, and the organic solvent was evaporated. Pellet was reconstituted with 200 μl of acidified water and analyzed using HPLC (LC-100, Shimadzu®) with reverse-phase column (RP-18, 5 μm, 4.0×250 mm2, Merck®), detector (SPD-M20A, Fenbendazole prominence diode array detector, Shimadzu®), loop injection of 20 μL, pump (LC 20 AT, prominence liquid chromatograph, Shimadzu®), injector (Rheodyne 7725i) and software LC Solution. The eluents were purified water adjusted to pH 3.2 with formic acid (A) and acetonitrile (B). The following solvent gradient was applied: from 100% A and 0% B to 80% A and 20% B within 10 min; from 80% A and 20% B to 75% A and 25% B within 5 min; from 75% A and 25% B to 70% A and 30% B within 10 min; from 70% A and 30% B to 50% A and 50% B within 10 min; and from 50% A and 50% B to 0% A and 100% B within 15 min (total analysis time: 45 min). Flow elution was 1 mL min−1; 20 μL of plasma samples were injected.