Scoring spontaneous pain. Wild-type mice (C57BL/6J; n = 5/group) were subjected to CCI, placed in plexiglass cylinders (19 × 31 cm), and allowed to habituate. Baseline response measurements were determined for each animal prior to testing. One animal at a time was continuously observed for 2 min. This was repeated 2 more times within the next 2 h. Various positions of the injured hind paw were continuously observed and rated according to the following numerical scoring system: 0, paw placed normally on the floor; 1, paw placed lightly on the floor and toes ventroflexed; 2, only internal edge of the paw contacted the floor; 3, contact restricted to the heel; 4, total paw elevation. During each 2-minute observation period, scoring was done at 5-s intervals by an investigator who was blinded to the experimental groupings. The spontaneous pain index for each observation period was calculated by multiplying the interval number by the interval score and dividing by the total number of intervals (25 in all/session) (80). The overall pain index for each mouse on each day was obtained by averaging the scores obtained during the 3 observation periods. In general, the 3 scores obtained on a single day with 1 mouse varied by less than 20% relative to the mean. Results at each day are presented as the mean ± SD for the cohort.