Bradykinin and nerve growth factor play pivotal roles in muscular mechanical hyperalgesia after exercise (delayed-onset muscle soreness)

Abstract

Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes delayed-onset muscle soreness (DOMS), a kind of muscular mechanical hyperalgesia. The substances that induce this phenomenon are largely unknown. Peculiarly, DOMS is not perceived during and shortly after exercise, but rather is first perceived after approximately 1 d. Using B(2) bradykinin receptor antagonist HOE 140, we show here that bradykinin released during exercise plays a pivotal role in triggering the process that leads to muscular mechanical hyperalgesia. HOE 140 completely suppressed the development of muscular mechanical hyperalgesia when injected before LC, but when injected 2 d after LC failed to reverse mechanical hyperalgesia that had already developed. B(1) antagonist was ineffective, regardless of the timing of its injection. Upregulation of nerve growth factor (NGF) mRNA and protein occurred in exercised muscle over a comparable time course (12 h to 2 d after LC) for muscle mechanical hyperalgesia. Antibodies to NGF injected intramuscularly 2 d after exercise reversed muscle mechanical hyperalgesia. HOE 140 inhibited the upregulation of NGF. In contrast, shortening contraction or stretching induced neither mechanical hyperalgesia nor NGF upregulation. Bradykinin together with shortening contraction, but not bradykinin alone, reproduced lasting mechanical hyperalgesia. We also showed that rat NGF sensitized thin-fiber afferents to mechanical stimulation in the periphery after 10-20 min. Thus, NGF upregulation through activation of B(2) bradykinin receptors is essential (though not satisfactory) to mechanical hyperalgesia after exercise. The present observations explain why DOMS occurs with a delay, and why lengthening contraction but not shortening contraction induces DOMS.

Figure 1.

B₂, but not B₁, antagonist injected before exercise blocked development of muscle mechanical hyperalgesia. A, B, Decrease in the mechanical threshold of exercised muscle observed 1–3 d after LC (saline group, solid circle) was blocked by a B₂ antagonist, HOE 140, administered subcutaneously 30 min before LC (solid triangle in A), but not by injection 30 min before threshold measurement on the second day (open triangle in B). A B₁ antagonist, des-Arg¹⁰-HOE 140, had no effect (open square in A). Arrows and arrowheads, Time points for antagonist injection and LC loading (on day 0), respectively. Mean ± SEM (n = 6 for each group). ⁺p < 0.05, **p < 0.01, ⁺⁺⁺, ***, or ^###p < 0.001 compared with −1 d; ⁺saline, *des-Arg¹⁰-HOE 140, ^#HOE 140. C, Percentage suppression of mechanical hyperalgesia (decrease of the withdrawal threshold), see Materials and Methods for calculation; 100% suppression corresponds to no mechanical hyperalgesia. HOE 140, open column; des-Arg¹⁰-HOE 140, gray column. Not Ex., des-Arg HOE 140 not examined. B₂ antagonist injected before LC almost completely suppressed mechanical hyperalgesia, but neither B₁ nor B₂ antagonist (0.1 mg/kg, s.c.) administered 2 d after LC reversed the mechanical hyperalgesia. B₂ antagonist (0.1 mg/kg, s.c.) administered 30 min after LC had no suppressive effect, either. **p < 0.01, ***p < 0.001, compared with the saline group.

Figure 2.

Upregulation of NGF in the muscle after LC. A, NGF mRNA level in exercised EDL muscle (open column) increased from 12 h to 2 d after LC, whereas no change was seen in the contralateral side (solid column). Representative reverse transcription-PCR profile of NGF mRNA is at the top. n, Number of samples examined. B, NGF protein measured by ELISA increased 12 h to 1 d after LC. *p < 0.05, **p < 0.01 compared with the control (CTL).

Figure 3.

SC induced neither DOMS nor NGF mRNA upregulation in the EDL muscle. A, Muscular mechanical hyperalgesia was induced in rats that underwent LC (solid circle) but not those that underwent SC (open triangle). Vertical axis, Withdrawal threshold (in mN) of the exercised muscle, measured by Randall–Selitto apparatus. Horizontal axis, Days after exercise. Arrowhead, Time point when LC or SC was performed (on day 0). Mean ± SEM (n = 6 for each group). *p < 0.05, **p < 0.01 compared with −1 d. B, There was much less, thus not significant, upregulation of NGF mRNA in the EDL muscle from SC (obliquely hatched column) and stretching (cross-hatched column) than from LC (open column). Relative NGF/GAPDH mRNA abundance in the EDL muscle 12 h after LC or SC. **p < 0.01 compared with the control group (CTL, black column), Dunnett's multiple-comparison test after one-way ANOVA, n = 4 each.

Figure 4.

Time course of changed expression of IL-1β, IL-6, and TNF-α mRNAs in exercised EDL muscle after LC. A–C, Expression of IL-1β (A), IL-6 (B), and TNF-α (C) mRNAs increased biphasically or triphasically after LC. Notably, a marked increase of these mRNAs was observed immediately after LC. n, number of samples examined. *p < 0.05, **p < 0.01 compared with the control group (CTL), Dunnett's multiple-comparison test after one-way ANOVA.

Figure 5.

A–D, Effects of HOE 140 on IL-1β (A), IL-6 (B), and TNF-α (C) mRNAs after LC, their changed expression after SC and stretching, and the effect of anti-IL-6 antibody on mechanical hyperalgesia after LC (D). A–C, HOE 140 had no effect on increase of IL-6 and TNF-α mRNA immediately after LC. Increase of IL-6 mRNA 12 h after LC was suppressed by HOE 140 but not TNF-α mRNA. Shortening contraction also induced upregulation of IL-1β, IL-6, and TNF-α immediately after exercise. TNF-α mRNA also increased 0 and 12 h after stretching, but neither IL-6 nor IL-1β mRNA increased. n = 4 each, except for control group in IL-1β (n = 8) and 0 h samples in stretching group (n = 6). *p < 0.05, **p < 0.01 compared with the control group (CTL). D, Arrow indicates the time of intramuscular injection of anti-IL-6 antibody. ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 compared with −1 d in D. N.S., no significant difference before and after injection, Bonferroni's multiple-comparison test after one-way ANOVA. Intramuscular injection of anti-IL-6 antibody 2 d after LC (D) did not affect muscle mechanical hyperalgesia (n = 6).

Figure 6.

Effects of intramuscular injection of anti-NGF antibody and NGF. A, B, Anti-NGF antibody (10 μg, i.m.) 6 h (A) and (30 μg, i.m.) 2 d (B, right) after LC blocked and reversed, respectively, the mechanical hyperalgesia seen after LC (n = 6 in A; n = 8 in B, right) in the saline (A) and normal goat IgG (B) control groups (solid circle). However, anti-NGF antibody (10 μg, i.m.) injected 2 d after LC failed to reverse the generated hyperalgesia after LC (B, left, n = 6 for each group). ⁺ or *p < 0.05, ⁺⁺ or **p < 0.01, ⁺⁺⁺ or ***p < 0.001 compared with −1 d; ⁺control group, *anti-NGF antibody. C, NGF (20 μl) injected into the gastrocnemius muscle decreased the muscular mechanical nociceptive threshold (n = 8 for each group). ⁺p < 0.05, ⁺⁺ or **p < 0.01, ⁺⁺⁺ or ***p < 0.001 compared with −1 d; ⁺0.2 μm group, *0.8 μm group. Note that the baseline withdrawal threshold was higher than in A and B because a larger probe was used to measure the threshold and that the horizontal axis is not linear.

Figure 7.

Effects of intramuscular injection of bradykinin (BK), and the combination of bradykinin and shortening contraction. A, BK or PBS injected into the EDL muscle tended to decrease the muscular mechanical nociceptive threshold for a short time (n = 6 for bradykinin group, n = 5 for PBS group), but there was no significant interaction between time and treatment (F_(16,112) = 1.09, p > 0.05), and no significant effect in treatment (F_(2,112) = 0.93, p > 0.05, two-way ANOVA with repeated measures). Arrow, Time points for injection (on day 0). B, The combination of bradykinin and SC (BK + SC) induced muscle mechanical hyperalgesia longer than injection of bradykinin alone. ⁺⁺p < 0.01, ***p < 0.001 compared with −1 d; ⁺PBS + SC group; *BK + SC group. Arrow, Time points for injection during shortening contraction (on day 0). C, NGF mRNA level in EDL muscle increased 12 h after BK + SC but not PBS + SC treatment (open columns) compared with the control group (CTL) (no treatment, solid column). **p < 0.01, n = 6 for each group.

Figure 8.

NGF-induced sensitization of C-fiber responses to mechanical stimulation. A, B, Raw recordings of single-muscle C-fiber responses (upper trace, action potentials were retouched because raw trace was too pale to see) to mechanical stimulation (lower trace) before and 120 min after injection of PBS (A) or NGF (B). Right inset shows the receptive field (solid circle) of the recorded fiber and NGF injection site in the EDL muscle. Shadow indicates the tendinous area, and the line at right shows the innervating nerve. Left inset shows the spike form. C, D, Summary of NGF effects on muscle thin-fiber afferent responses to mechanical stimulation. Intramuscularly injected NGF decreased the mechanical threshold (C) and increased the number of discharges (D) responding to the mechanical stimulation (0–196 mN in 10 s). Values are presented as the percentage of the averaged value of PBS group at each time point (for calculation, see Materials and Methods). *p < 0.05, **p < 0.01 compared with PBS group at each time point.