Role of substance P signaling in enhanced nociceptive sensitization and local cytokine production after incision

Abstract

Substance P (SP) signaling facilitates nociceptive sensitization in various inflammatory and chronic pain models and we postulated that SP signaling might also contribute to the development of post-incisional hyperalgesia. These studies used mice with a deletion of the pre-protachykinin A gene (ppt-A(-/-)) which codes for SP to determine the role of SP signaling in post-incisional pain and in the increased cytokine and nerve growth factor (NGF) expression observed in the incised skin. SP deficient ppt-A(-/-) mice displayed reduced mechanical allodynia and heat hyperalgesia compared to the wild-type (wt) mice at all post-incision time points, despite similar baseline values (p<0.001). Furthermore, the NK-1 receptor antagonist LY303870 attenuated mechanical allodynia produced by incision in the wt mice (p<0.001). Incision also up-regulated IL-6, TNF-alpha and KC levels but not IL-1beta after 2h in the wt mice skin. However, ppt-A(-/-) mice had more skin NGF levels 2h post-incision. Subcutaneous hind paw SP injection produced acute and transient elevations of IL-1beta, IL-6, and KC but modest elevations in TNF-alpha levels in the wt mice. Systemic LY303870 reversed the SP-induced elevations of these cytokines. Hind paw injection of IL-6 and NGF dose dependently produced less mechanical allodynia in the ppt-A(-/-) compared to wt mice. Additionally, SP produced mechanical allodynia in a dose-dependent fashion in wt mice. Therefore, SP supports nociceptive sensitization after hind paw incision and potentially participates directly in modulating the intensity of inflammatory response in peri-incisional tissue.

Figure 1

Assessment of mechanical allodynia and heat hyperalgesia after hind paw incision. A-Mechanical allodynia was measured in the wild type (n=8) and ppt-A(−/−) (n=8) mice using calibrated von Frey filaments before and at different time points after incision. B- Paw withdrawal latencies to heat stimuli was measured in the wild type (n=12) and ppt-A(−/−) (n=12) mice using the Hargreaves method. Data are presented as Mean ± S.E.M and were analyzed by two way ANOVA with post hoc Bonferroni tests comparing strains at corresponding time points. *p<0.05, **p<0.01, ***p<0.001.

Figure 2

Assessment of selective NK-1 receptor antagonist effects on incision induced mechanical allodynia. Mechanical hypersensitivity was measured in two different groups of wild type mice: vehicle pretreatment/incision, LY303870 (40 mg/kg. i.p.) pretreatment/incision (n=8 each group). Mean ± S.E.M values of each group were analyzed by two way ANOVA with post hoc Bonferroni tests comparing treatment groups at each time point. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Effects of hind paw incision on peripheral cytokines and NGF levels. The levels of cytokines and NGF were measured at baseline and at the 2–48 hours time points after incision. The selected time points were based on the behavior data presented in Figure 1. Different groups of mice of both wild type and ppt-A(−/−) were used for each time point (n=6 per group). Data are presented as Mean ± S.E.M and were analyzed by two way ANOVA with post hoc Bonferroni tests comparing strains at corresponding time points. *p<0.05, **p<0.01, ***p<0.001.

Figure 4

Effects of intra-plantar substance P (SP:1 µg/15 µL), LY303870 (40 mg/kg. i.p.) pretreatment/ intra-plantar substance P (SP:1 µg/15 µL) and vehicle (15 µL Saline) on peripheral cytokines and NGF levels. The levels of cytokines and NGF were measured 2–48 hours time points after SP administration. Different groups of mice of both wild type and ppt-A(−/−) were used for each time point (n=4–6 per group). Data are presented as Mean ± S.E.M and were analyzed by two way ANOVA with post hoc Bonferroni tests comparing strains at corresponding time points. Significant difference from vehicle:*p<0.05, **p<0.01, ***p<0.001. Significant difference from SP: ^#p<0.05, ^##p<0.01, ^###p<0.001.

Figure 5

Effects of peripheral administration of substance-P, IL-6 and NGF on mechanical sensetivity. A: Intra-plantar substance P and vehicle were given after baseline mechanical paw withdrawal threshold determination. B–C: Intra-plantar IL-6, NGF and vehicle given after baseline measurements of paw withdrawal threshed. Data of the effect of different doses of SP, IL-6 and NGF are presented as mean percent effect, with baseline values as being zero effect. Higher doses of NGF produced ongoing mechanical hyperalgesia for several days (Data not shown). D–E: Comparison of the paw withdrawal thresholds of wild type and ppt-A(−/−) mice at various time points given 1ng/15 µL of IL-6 and 50ng/15 µL of NGF by intra-plantar injection. Data are presented as Mean ± S.E.M and were analyzed by two way ANOVA with post hoc Bonferroni tests comparing strains at corresponding time points. *p<0.05, **p<0.01, ***p<0.001.