4.2.1. Spared Nerve Injury Model of Neuropathic Pain

The SNI model of neuropathic pain was performed as described previously [54,55]. Under isoflurane anesthesia and carprofen analgesia, the common peroneal and tibial nerve branches of the sciatic nerve were ligated with a 6–0 silk suture and approximately 1 mm of the two nerve branches was removed distally, whereas the sural nerve was left intact.

For testing mechanical sensitivity, mice were placed in boxes on an elevated metal mesh floor and habituated for at least 30 min before behavioral testing began. Calibrated von Frey filaments ranging from 0.40 to 39.2 mN (0.02 to 4.0 g; Ugo Basile, Gemonio, Italy) were applied to the lateral part of the hind paw (sural nerve area) until they bowed for 5 s. Only obvious withdrawal responses to the applied stimulus were recorded. The 50% withdrawal thresholds were assessed using the up-down method [56,57] and calculated using an online algorithm (https://bioapps.shinyapps.io/von_frey_app/ [58]).

An automated dynamic weight-bearing (DWB) device (Bioseb, Vitrolles, France) was used to evaluate the dynamic weight distribution in mice after SNI. The system automatically records the average weight (in grams) that each limb exerts on a floor equipped with pressure transducers. A zone was considered valid when the following parameters were detected: ≥ 0.8 g on 1 captor with a minimum of 2 adjacent captors recording ≥ 1.0 g. For testing, the mouse was placed in the chamber and allowed to move freely within the apparatus for 10 min and videotaped. After 5 min of acclimatization, the dynamic weight distribution was recorded for 5 min. In all video sequences, the positions of the paws were manually validated by the observer [59,60]. The results were expressed as percent weight borne by the ipsilateral hind paw of total weight borne by both hind paws [61].