4.4. Allodynia Assessment

Allodynia (a painful response to a non-painful stimulus) was assessed by the measurement of the mechanical nociceptive threshold. Before allodynia assessment, mice were left for 30′ in the experimental room for environmental habituation and in the experimental apparatus for 5′ before testing. The onset of neuropathy was evaluated by measuring the sensitivity of both ipsilateral and contralateral hind paws to normally non-noxious punctuate mechanical stimuli at different time intervals from postoperative day 3 (D3) up to day 60 (D60). The nerve injury-induced mechanical allodynia was tested by using a Dynamic Plantar Aesthesiometer (Ugo Basile, Model 37400, Gemonio VA, Italy), an apparatus that generates a mechanical force linearly increasing with time. The force is applied to the plantar surface of the mouse’s hind paw, and the nociceptive threshold is defined as the force, in grams, at which the mouse withdraws its paw. On each day of testing, the mechanical withdrawal response of ipsilateral and contralateral hind paws was recorded for 3 consecutive trials with at least 10 s between each trial. The withdrawal threshold was taken to be the mean of the 3 trials. The behavioral test was performed in male and female mice (n = 9/10 per group).