*4.4. Paw Withdraw Threshold (PWT)*

PWT was determined by the von Frey behavioral test, which was performed according to the up-down method described by Chaplan et al. [43]. Rats were placed in the individual testing cages for 30 min per day for three continuous days to adapt to the test environment. Before each test, rats were placed into the cage for at least 15 min to acclimate to the environment. The von Frey hairs (Stoelting Co, Thermo, Gilroy, CA, USA) were applied in a consecutive ascending order (0.4, 0.6, 1, 2, 4, 6, 8, 15, and 26 g) to the central surface of the hind paw and sustained for 5 s. The first hair applied corresponded to a force of 4 g. Brisk withdrawal or paw flinching was considered a positive response and marked as "X". A weaker stimulus was then applied. In the case of no responses, "O" was recorded, followed by a stronger stimulus. The interval between each stimulus was not less than 2 min. After the combination of "OX" or "XO" appeared, a series of four stimuli were applied and recorded as above. The 50% PWTs of the rats were calculated by the formula 50% PWTs (g) = 10 ˆ (xf + k \*δ- 4). "xf" is the logarithmic value of the last von Frey hair in the sequence, "k" is the corresponding value of the resulting sequence in the k-value table, and "δ" is the mean difference of each filament strength after logging (0.231 in the current cases). If a positive stimulus appeared five consecutive times, PWT was marked as 26 g. If five "X"s were recorded, PWT was marked as 0.4 g. If the value of 50% PWTs was greater than 26 g, 26 g was used as the maximum. And if the value of 50% PWTs was less than 0.4 g, 0.4 g was used as the minimum. The measuring time was fixed at 9:00–16:00, and the ambient temperature was 23 ± 2 ◦C.
