2.10. Von Frey Test to Estimate Mechanical Allodynia

To estimate the effects on pain, mechanical stimulation was carried out to measure the pain threshold before CFA injection (Baseline: BL) and 1, 3, 5, and 7 days after the injection of CFA. For a mechanical hypersensitivity test, mice were placed on a metal mesh floor for 0.5 h in a small acrylic box to adapt to the testing environment. Next, mechanical hypersensitivity was measured using von Frey filaments ranging from 0.02 to 6 g. Filaments were applied perpendicularly to plantar surfaces until the mice responded. If mice did not respond to the initially selected filament, thicker filaments were applied. The minimal value which caused licking, foot lifting, and trembling was recorded as the mechanical withdrawal threshold. After measuring the minimum threshold value, the numbers of responses obtained by stimulating the plantar surfaces of mice 10 times with 0.4 g filaments and cotton were recorded as the withdrawal threshold frequency and cotton swab threshold frequency, respectively.