2.10. von Frey Test for Determining Mechanical Allodynia

Pain reactions were evaluated using the von Frey tests, which were carried out according to a previously described method for mice [14] using an electronic von Frey apparatus (IITC Inc., CA, USA). Briefly, we placed the mice individually in a transparent acrylic box (9 × 9 × 15 cm) with a wire mesh bottom. We allowed them to acclimate to their environment for at least 30 minutes. A mechanical stimulus was then applied from underneath to the plantar aspect of the hind limb, with a gradual increase in pressure. The end point was removal of the paw, followed by clear flinching movements. After paw withdrawal, the intensity of the pressure was automatically recorded. Each test was repeated three times at intervals of 5 minutes to gain an average value. The area under the time-effect curve (AUC) was calculated for each animal according to the following formula [14]: ([paw withdrawal pressure at each time point] − [paw withdrawal pressure at preoperative baseline value]) × time (days).