Von Frey filament test

The mechanical withdrawal threshold was measured by the von Frey filament (Stoelting America, Illinois, IL, USA) using an up-down method (Chaplan et al., 1994). Mice were placed on an elevated metal mesh board and under the cover of transparent plastic chambers (9 cm × 9 cm × 4.5 cm). This test was only performed in the light cycle. Before the sequencing tests, the mice should be habituated for 3 days. On the first day of habituation, the mice were put on the board until they fell asleep (about 60–90 min), while on the next 2 days, they were applied two rounds of filament (1 g) probing to the left hind paw (where CFA was injected). During the formal tests, von Frey filaments (bending force ranging from 0.008 to 1.4 g) were applied five times (3 s for each stimulus) to the test site mentioned above. Sudden paw withdrawal and paw licking were considered positive responses. The smallest bending force of the von Frey filament that evokes positive responses with over 50% occurrence frequency is regarded as the mechanical threshold of the mice.