Assessing Mechanical, Thermal, and Evaporative Sensitivity in Mice

To assess mechanical sensitivity, calibrated von Frey filaments (0.02–2.0 g, North Coast Medical, USA) were applied to the plantar surfaces of the hindpaws of mice with or without PNI, or peripheral inflammation caused by intraplantar injection of CFA (0.01 mg per 20 μl) and the 50% PWT was determined. The volume of hindpaw oedema was measured by weighing the hindpaw. For the hot-plate test, mice were placed on a metal surface maintained at 45, 49 or 52 °C within a 25-cm-high Plexiglass box (25 × 2 cm). The latency to either lick the hindpaw or jump was recorded as a nocifensive end point54 (link). Noxious heat-evoked tail-flick responses were measured following the application of radiant heat (Ugo Basile, Italy) to the tail. The intensity of the heat stimulus was adjusted to 50 V, and the latency of the tail withdrawal response (seconds) was measured16 (link). In an acetone test, acetone (20 μl) was applied through wire mesh flooring onto the plantar surface of the left hindpaw to produce evaporative cooling, and behaviour was observed during the first 30 s after acetone application and was scored by adding together each of five acetone applications (0, no response; 1, licking or flinching; 2, strong flinching or licking).