Figure 2. Neonatal animals display pain-like behaviors following a muscle incision that can be prevented by local GH administration. A, One day after a reverse neonatal muscle incision, animals guard the injured hindpaw demonstrating an overall effect of day (F(1,34) = 23.16, p < 0.001), injury (F(2,34) = 12.74, p < 0.001), and an interaction (F(2,34) = 12.64, p < 0.001). Tukey's post hoc test showed that animals that received a muscle incision guard at day 1 (*p < 0.001 vs BL), while those that receive a skin incision only (p = 0.309) or sham (p = 0.575) do not. Also, muscle incised animals guard significantly more compared with both controls (̂p < 0.001 vs controls). B, Mechanical withdrawal thresholds in the same animals demonstrated an overall effect of day (F(1,34) = 5.2, p = 0.029), injury (F(2,34) = 7.28, p = 0.002), and an interaction (F(2,34) = 4.52, p = 0.018). Only animals that received a muscle incision displayed lower squeezing withdrawal thresholds compared with BL (*p < 0.001 vs BL); n = 10–15/group, two-way RM ANOVA, Tukey's post hoc test. C, Representative image and analysis of muscle GH levels using Western blotting. Following incision, GH levels are reduced in incised animals compared with naive animals and this is restored with exogenous GH treatment. F(2,6) = 9.96, *p = 0.012 versus naive; n = 3/group, one-way ANOVA, Tukey's post hoc test. D, Spontaneous paw guarding is increased following incision in animals treated with vehicle but not in GH-treated animals at 1 d. By 3 d, neither group is different from their BL or each other. Overall effect of day (F(1,16) = 13.79, p = 0.002), injury (F(1,16) = 7.89, p = 0.013), and interaction (F(1,16) = 9.37, p = 0.007). Tukey's post hoc test indicated that vehicle-injected animals guard at day 1 (*p < 0.001 vs BL), while those injected with 1.5 mg/kg of GH do not (p = 0.668). There was also a significant difference between groups at day 1 (̂p < 0.001 vs controls); n = 8–10/group, two-way RM ANOVA. The shades of the data points indicate the age range of the animals (also true for E, G). Light gray = ≤P7. Medium gray = P8 and P9. Dark gray = P10, P11. Black = ≥P12. E, Muscle withdrawal thresholds are reduced in incised animals with vehicle injection at 1 d, but this is inhibited in GH-treated animals. By 3 d, both groups have increased withdrawal thresholds compared with BL. Overall effect of day (F(1,16) = 8.16, p = 0.011). Multiple comparisons indicate that vehicle-injected animals have reduced thresholds compared with BL (*p = 0.015 vs BL) but GH-injected animals do not (p = 0.186); n = 8–10/group, two-way ANOVA, Tukey's post hoc test. F, One day after an injury, animals dosed with GH at 0, 0.1, 0.5, and 1.0 mg/kg guarding showed an overall effect of day (F(1,37) = 53.64, p < 0.001), a modest effect of injection (F(4,37) = 2.199, p = 0.088) and an interaction (F(4,37) = 3.71, p = 0.012). Tukey's correction indicated that all groups significantly guarded (*p < 0.05 vs BL) except animals injected with 1.5 mg/kg of GH (p = 0.629). Also, animals injected with 1.5 mg/kg had significantly lower guarding scores compared with vehicle-injected animals at 1 d (̂p < 0.001 vs controls). G, When GH treatment is delayed 8 h following the injury an overall effect of day (F(1,17) = 42.89, p < 0.001) and injection (F(1,17) = 5.35, p = 0.033) with a moderate interaction (F(1,17) = 4.01, p = 0.062) was detected. Tukey's correction demonstrated adjusted p values indicating a difference in each group from BL (*p = 0.004 within GH vs BL; *p < 0.001 within vehicle vs BL). Also, within day 1, there is a significant reduction in the amount of guarding in GH-injected animals (̂p = 0.005 vs controls); n = 10/group, two-way RM ANOVA, Tukey's post hoc test. H, Representative images of Western blottings for GH in naive, incised, and GH-treated incised adolescent animals. Quantification indicates no differences between groups. F(2,9) = 0.063, p = 0.94; n = 4/group, one-way ANOVA. I, Adolescent animals with vehicle+incision or GH+incision guard have a main effect of day (F(1,21) = 95.35, p < 0.001), injection (or naive; F(2,21) = 20.2, p < 0.001), and interaction (F(2,21) = 19.01, p < 0.001). Tukey's post hoc test indicate that both incised groups demonstrating guarding (*p < 0.001 vs BL) and do not differ from one another (p = 1). J, For muscle mechanical withdrawal thresholds, a main effect of day (F(1,21) = 138.97, p < 0.001), injection (or naive; F(2,21) = 3.55, p = 0.047), and interaction (F(2,21) = 17.92, p < 0.001) was detected. After correction, we found that incised groups had reduced threshold 1 d after injury (*p < 0.001 vs BL and naive) and the incised groups do not differ from one another (p = 0.874); n = 8/group, two-way RM ANOVA, Tukey's post hoc test. Data shown as the mean ± SEM.