Figure 1: Location of incision in the hind leg of Göttingen minipigs

Figure 2: Effect of incision on mechanical nociception in saline-dosed animals
Results are reported as mean±SD. A low withdrawal force (g) [log10] was detected for ≥144 hours (6 days).
*p<0.001 vs. baseline, ‡p<0.05 vs. 1-hour post-incision.
SD: standard deviation.

Figure 3:Effect of incision on mechanical nociception (pre-dosing and 1-hour post-dosing)
Results are shown as mean±SD 1hour post-dosing (Day 0 for saline and ropivacaine 1^st dose PN, and Day 1 for saline and morphine 1^st and 2^nd doses IM).
*p<0.001 vs. saline.
‡p<0.05 vs. pre-dosing.
IM: intramuscular; PN: perineural; SD: standard deviation

Figure 4: Results of the Human Approach Test (HAT)
Results are shown as mean±SD. HAT cut off during the 6-day acclimitisation period was 120 seconds. Incision was performed on Day 0.
*p<0.001 vs. Day -6 (first day of acclimitisation, 6 days pre-incision).
#p<0.05 vs. saline-dosed animals.
†p<0.05 vs. Day -1 (pre-incision).
‡p<0.001 vs. Day -1 (pre-incision).
HAT: human approach test; SD: standard deviation.

Figure 5a-c: Results of the Open Field Test
(a) Saline-dosed animals. Note: the saline-dosed animals on Day 2 were previously dosed with morphine (0.5 mg/kg and 1.0 mg/kg IM) on Day 1.
(b) Morphine-dosed animals. Note: animals were dosed with 0.5 mg/kg and 1.0 mg/kg morphine (IM) on Day 1, and 2.0 mg/kg and 4.0 mg/kg morphine (IM) on Day 2 (1st and 2nd doses, respectively).
(c) Ropivacaine-dosed animals. Note: animals dosed with 25 mg/pig were dosed immediately after incision on Day 0 (1st dose) whereas the animals dosed with 35 mg/kg were dosed on Day 1 post-incision (2nd dose).
Results are shown as mean±SD.
*p<0.05 vs. pre-incision.
‡p<0.01 vs. pre-incision.
†p<0.01 vs. saline-dosed animals.
IM: intramuscular; PN: perineural; SD: standard deviation.

Figure 6a-c: Effect of systemic treatment with morphine on locomotor function
(a) Walking pattern post-incision (0.5 hours post IM injection); (b) time spent next to wall A or D (saline dosed IM group); and (c) time spent next to wall A or D (morphine dosed IM group)
Results are reported as mean±SD.
*p<0.05 vs. saline dosed animals.
SD: standard deviation.

Figure 7a-c: Results of the intraepidermal nerve fibre (IENF) density analysis
(a) Representative image showing immunodetection of the PGP 9.5 antigen in flank, (b) representative image showing immunodetection of the PGP 9.5 antigen in hind leg, and (c) mean±SD IENF recorded in hind leg vs. flank region. Yellow arrows indicate PGP9.5 positive intraepidermal nerve fibres.
*p<0.05 IENF in the hind leg vs. flank region.
IENF: intraepidermal nerve fibre.