Assessment of mechanical allodynia

Mechanical allodynia was assessed by measuring 50% paw withdrawal threshold (PWT) as described in our previous studies.^5,44 The PWT in response to a series of von Frey filament (Stoelting, Wood Dale, US) was determined by the up-down method. ⁴⁵ Briefly, the rat was placed on a metal mesh floor covered with a clear plastic cage (18 × 8 × 8cm) and allowed a habituation period for 20 minutes. Eight von Frey filaments with approximately equal logarithmic incremental bending forces (0.224) were chosen (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10g). Each test started with a 2.00g von Frey filament which was delivered perpendicularly to the plantar surface of the left hindpaw for 2 to 3 seconds. Abrupt withdrawal of the foot during stimulation or immediately after the removal of the filament was regarded as a positive response. Whenever there was a positive or negative response, the next weaker or stronger filament was applied, respectively. This procedure was completed until 6 stimuli after the first change in response had been observed. The 50% PWT was calculated using the following formula: 50% PWT (g) = (10^[X _f ^+κδ]), where X _f is the value of the final von Frey filament used (in log units), κ is a value acquired from the pattern of positive/negative response, and δ is the average interval (0.224, in log units) between the von Frey filaments. ⁴⁶ An allodynia rat is defined as whose 50% PWT is less than 4.0g (i.e., withdrawal in response to non-noxious tactile stimulus). ⁴⁷