Intraplantar injection of 10, 25, or 60 μg of substance P (SP) into the hindpaw of a normal rat dose-dependently induced mechanical allodynia to von Frey testing. (A) Allodynia developed slowly, peaking at 6 h post-injection, and gradually resolving over 48 h. (B) There were no changes in hindpaw temperature after injecting any dose of SP. (C) Even the lowest dose of SP (10 μg) evoked hindpaw edema within 5 minutes, peaking at 1 h, and then gradually resolving over 24 h. Measurements for (A), (B), and (C) represent the difference between the injected side and the contralateral paw; thus, a positive value represents an increase in temperature or paw thickness on the injected side, and a negative value represents a decrease in mechanical nociceptive thresholds on the injected side. Data were analyzed to compare SP injection vs. vehicle injection cohorts at various time points by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001, SP- vs. vehicle-injected rats at indicated time points (n = 6 for each injection group). (B) There was no significant difference in hindpaw temperature between SP-injected and vehicle-injected rats at any time point. (C) Injection of SP at all concentrations (10, 25, and 60 μg) significantly evoked edema at all three time points of 1 h, 3 h and 6 h (***P < 0.001 SP- vs. vehicle-injected cohorts).