All animals were then promptly treated with oxytetracycline hydrochloride (400 mg/kg) and the experiments were performed 1 week later. After each experiment, the animal was anesthetized as before, and the location of the cannula track was histologically find more verified. Animals which showed cannula misplacement, blockage upon injection or abnormal weight gain patterns were excluded from the study. A different group of rats was used for each experiment, i.e., each animal was used only once. In a first set of experiments, rats were treated intraperitoneally (i.p.)

with either the NK1 receptor antagonist SR140333B (0.3, 1 or 3 mg/kg dissolved in saline plus Tween 80 1%) or vehicle (2 ml/kg, control), 30 min

prior to injection of E. coli LPS (30 μg/kg, i.p.) or sterile saline (2 ml/kg, i.p., control). To confirm the effectiveness of the peripheral treatment, another group of animals was treated with SR140333B (1 mg/kg) and after 30 min, under pentobarbital anesthesia (50 mg/kg, i.p.), they received an Caspase pathway injection of Evans Blue dye (50 mg/kg, i.v.) followed by 40 ng of SP (50 μl) or the same volume of saline in the skin. After 15 min, animals were killed, the dorsal skin was immediately excised and the blue-stained area at each injection site was removed for dye extraction ( Rattmann et al., 2008). The plasma leakage was measured as described previously ( Brain and Williams, 1985). In another set of experiments, rats were treated intracerebroventricularly (i.c.v.) with either the NK1 receptor antagonist SR140333B (0.3, 1 or 3 μg dissolved in 2 μl saline plus Tween 80 0.3%) or vehicle (2 μl, control), 30 min prior to injection of E. coli LPS (30 μg/kg, i.p.) or sterile saline (2 ml/kg, i.p., control). In the following set of experiments animals were treated with SR140333B (3 μg/ 2 μl, i.c.v.) or the Selleckchem Sirolimus respective vehicle (Tween 80 0.3%) 30 min prior to injection of the angiotensin converting-enzyme inhibitor captopril (5 μg/ 2 μl, i.c.v.) or the same volume of vehicle (sterile saline), followed by the injection of SP (250, 500 or 750 ng, i.c.v) or saline (2 μl) 30 min later. In

the final set of experiments, rats were treated with the same dose of SR140333B or the vehicle 30 min before injecting either IL-1β (3.1 ng/ 2 μl, i.c.v.) or CCL3/MIP-1α (500 pg) or sterile saline. Pyrogenic stimuli were always injected between 10:00 and 11:00 h. Doses of each pyrogenic stimulus were based on previous studies and do not represent doses that cause maximal responses ( Fraga et al., 2008, Melo Soares et al., 2006, Werner et al., 2006 and Zampronio et al., 2000). The following drugs were employed: LPS from E. coli 0111:B4, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gli-Leu-Met-NH2) and captopril (Sigma Chem Co., St. Louis, U.S.A.), rat IL-1β and rat CCL3/MIP-1α (R&D Systems Inc., Minneapolis, U.S.A.