Fine von frey filament

Mice were placed individually in a plastic cage (11 cm ×18 cm × 15 cm) with a wire mesh bottom and allowed to acclimatize for at least 30 min. Mechanical allodynia in the hind paw was evaluated with a fine von Frey filament (a bending force of 0.69 mN , North Coast Medical Inc., Morgan Hill, CA, USA) [9 (link)]. The von Frey filament was pressed perpendicularly against the central part of the plantar hind paw and was held there for 1–3 s by slight buckling. Responses to the stimulus were scored as follows: no reaction (0), lifting of the hind paw (1), and licking and flinching of the hind paw (2). A stimulation of same intensity was applied three times to each hind paw at intervals of several seconds and the average value of six trials was used as the response score (the maximum score being 2). Cold dysesthesia was evaluated using acetone [11 (link)]. Acetone (10 μl) was applied to the plantar skin. Aversive responses during the 10-s period following acetone stimulation were scored as follows: no response (0), lifting of the hind paw (1), and flinching or licking of the hind paw (2). Naïve mice showed a transient escape response immediately after acetone application, and this response was disregarded. Acetone was applied three times alternately to each hind paw at intervals of more than 20 s, and the average of six trials served as the aversive response score.

Mechanical allodynia of the hind paw was assessed using a fine von Frey filament with a bending force of 0.69 mN (North Coast Medical Inc., Morgan Hill, CA, USA) [12 (link)]. The mice were placed individually in an acrylic cage (11 cm × 18 cm × 15 cm) with a wire mesh bottom. After an acclimation period of at least 30 min, the von Frey filament was pressed perpendicularly against the central part of the plantar hind paw of a freely-moving mouse and was held there for 1–3 s with the filament slightly buckled. Responses to the stimulus were ranked as follows: 0, no reaction; 1, lifting of the hind paw; and 2, licking and flinching of the hind paw. The stimulation of the same intensity was applied to each hind paw three times at intervals of several seconds, and the allodynia score (the total score of six tests) was expressed as a percentage of a maximum score of 12.