Mechanical nociceptive von Frey test

Mechanical nociception, reflected by hindpaw withdrawal thresholds, was determined by an observer who was blind to the experimental groups using von Frey filaments that ranged from 3.63 to 281.838 g as previously reported (Dixon, 1965; Kononoff et al., 2018). A test session began after 10 min of habituation to the testing environment immediately following the subcutaneous injection of naloxone (WD, WD+ABX, and SAL groups) or saline (OXY and OXY+ABX groups). A series of von Frey filaments were applied from below the wire mesh to the central region of the plantar surface of the left and right hindpaws in ascending order, beginning with the smallest filament (3.63 g). The filament was applied until buckling occurred, and it remained in place for ∼2 s. A sharp withdrawal of the hindpaw indicated a positive response. The stimulus was incrementally increased until a positive response was observed and then decreased until a negative response was observed according to the statistical up-down method of Dixon (1965). The 50% paw withdrawal threshold was determined by the formula Xf + kδ, where Xf is the last von Frey filament applied, k is the Dixon value that corresponded to the response pattern, and δ is the mean difference between stimuli. Once the threshold was determined for the left hindpaw, the same testing procedure was repeated for the right hindpaw after 5 min. Paw withdrawal thresholds were recorded at baseline, during intoxication, and during precipitated withdrawal.