Solutions for injection were prepared immediately before the experiments
by adding isotonic NaCl. The volume of subcutaneous (s.c.) injection selleck inhibitor into the dorsum was 4 ml/kg. The volume of s.c. injection into the dorsum of the right hind paw was 20 μl. Formaldehyde (0.92% v/v in isotonic saline; 20 μl) was injected into the dorsum of the right hind paw of mice. Each mouse was placed under a transparent glass funnel (18 cm diameter, 15 cm-high) and the amount of time the animal licked the injected paw was determined between 0 and 5 min (first phase) and 15 and 30 min (second phase) after the injection of formaldehyde. To evaluate the effects induced by AMV, F<10, melittin, melittin-free AMV, venom of T. serrulatus or venom of B. jararaca on the nociceptive response induced by formaldehyde, the substances were previously (30 min) injected s.c. into the dorsum of the animals. AMV (50 or 100 pg), F<10 (50 or 100 pg),
melittin (25 or 50 pg), T. serrulatus (1 pg; Nascimento et al., 2005) or B. jararaca venom (1 pg; Carneiro et al., 2002 and Olivo et al., 2007), in a volume of 20 μl, were injected s.c. into the dorsum of the right Cytoskeletal Signaling inhibitor hind paw of mice. Each mouse was placed under a transparent glass funnel (18 cm diameter, 15 cm-high) and the amount of time the animal licked the injected paw was determined between 0 and 30 min after injection. In one protocol, the effect induced by previous (30 min) s.c. injection of the AMV into the dorsum of mice on the nociceptive response induced by the injection of AMV into the right hind paw was investigated. Paw oedema was measured with a plethysmometer (Model 7140, Ugo Basile, Comerio, Italy). The basal volume of the right hind paw was determined before administration of any drug. After determination of the basal volume, the animals were divided in the experimental groups in such a way that the mean volumes of the different groups were similar. AMV, F<10, melittin or dexamethasone were administered 30 min Axenfeld syndrome before s.c. injection of formaldehyde (0.92%, 20 μl) into the dorsum of the right hind paw. The paw volume was measured
at 30 and 60 min after injection of formaldehyde. The results were presented as the paw volume changes in relation to the baseline. Thirty minutes after treatment with AMV, F<10, melittin or morphine, the animals were placed on a heated (54 °C) metal plate (20 × 20 cm with 18 cm-high walls). The latency to lick one of the hind paws or to jump off the plate was determined. Mice were removed from the hot-plate immediately after the response. The cut off time was 30 s to avoid tissue damage. The motor activity of the animals was evaluated in a rota-rod apparatus. The day before the experiment, the animals were trained in the apparatus. On the testing day, the animals were placed on a rotating rod (20 rpm) and the time they spent on the apparatus was measured. The cut off time was 2 min (Miyamoto, 2006).