IB4(+) nociceptors mediate persistent muscle pain induced by GDNF

Abstract

Skeletal muscle is a well-known source of glial cell line-derived neurotrophic factor (GDNF), which can produce mechanical hyperalgesia. Since some neuromuscular diseases are associated with both increased release of GDNF and intense muscle pain, we explored the role of GDNF as an endogenous mediator in muscle pain. Intramuscularly injected GDNF induced a dose-dependent (0.1-10 ng/20 μl) persistent (up to 3 wk) mechanical hyperalgesia in the rat. Once hyperalgesia subsided, injection of prostaglandin E(2) at the site induced a prolonged mechanical hyperalgesia (>72 h) compared with naïve rats (<4 h; hyperalgesic priming). Selective neurotoxic destruction of IB4(+) nociceptors attenuated both GDNF hyperalgesia and hyperalgesic priming. Ergonomic muscular injury induced by eccentric exercise or mechanical vibration increased muscle GDNF levels at 24 h, a time point where rats also exhibited marked muscle hyperalgesia. Intrathecal antisense oligodeoxynucleotides to mRNA encoding GFRα1, the canonical binding receptor for GDNF, reversibly inhibited eccentric exercise- and mechanical vibration-induced muscle hyperalgesia. Finally, electrophysiological recordings from nociceptors innervating the gastrocnemius muscle in anesthetized rats, revealed significant increase in response to sustained mechanical stimulation after local GDNF injection. In conclusion, these data indicate that GDNF plays a role as an endogenous mediator in acute and induction of chronic muscle pain, an effect likely to be produced by GDNF action at GFRα1 receptors located in IB4(+) nociceptors.

Fig. 1.

Intramuscular injection of glial cell line-derived neurotrophic factor (GDNF) produces acute and chronic pain. A: 1 h after unilateral injection of GDNF (0.1–10 ng/20 μl im; n = 6/dose), a marked mechanical hyperalgesia was observed in the injected (ipsilateral) gastrocnemius muscle (0.1 ng: 2696.2 ± 6.2 vs. 2038.3 ± 38.9 mN, P < 0.001; 1 ng: 2,671.7 ± 4 vs. 1,994 ± 11.5 mN, P < 0.001; 10 ng: 2,677.2 ± 7.9 vs. 1,647.2 ± 43.6 mN, P < 0.001, ANOVA followed by Dunnett's test) but not the contralateral muscle (0.1 ng: 2,697.2 ± 7.8 vs. 2,686.3 ± 16.6 mN, P > 0.05; 1 ng: 2,680.8 ± 2.9 vs. 2,634.3 ± 18.8 mN, P > 0.05; 10 ng: 2,677.5 ± 4.4 vs. 2,600.8 ± 17.2 mN, P > 0.05, ANOVA followed by Dunnett's test, respectively). Data are plotted as %change in mechanical nociceptive threshold of GDNF-injected (ipsilateral) and noninjected (contralateral) hindlimbs compared with baseline and differences are represented by ***P < 0.001. B: GDNF-induced muscle hyperalgesia exhibited dose-dependent amplitude and duration, persisting for >1 wk at 0.1 ng (9th day 2,474.8 ± 19.5 mN, P < 0.001, ANOVA followed by Dunnett's test) and up to 3 wk at 10 ng (21st day 2,419.3 ± 15.5 mN, P < 0.001, ANOVA followed by Dunnett's test) after injection. Data are plotted as %change in mechanical nociceptive threshold compared with baseline (n = 6/group). Solid symbols represent P < 0.05 respect to baseline. C: when nociceptive thresholds returned to pre-GDNF baseline, groups were tested for the presence of hyperalgesic priming. After bilateral PGE₂ administration (1 μg/20 μl im), nociceptive thresholds were tested at 1, 4, 24, and 72 h. One hour after PGE₂ injection, bilateral mechanical hyperalgesia was observed. Regardless the dose of GDNF, such hyperalgesia remained unchanged in hindlimbs ipsilateral to GDNF injection after 72 h (0.1 ng: 1,534.8 ± 28.9 vs. 1,638.8 ± 39.3 mN, P > 0.05; 1 ng: 1,528.3 ± 25.9 vs. 1,603 ± 38.9 mN, P > 0.05; 10 ng: 1,490.3 ± 14.3 vs. 1,600.3 ± 20 mN, P > 0.05, ANOVA followed by Tukey's test, respectively), indicating hyperalgesic priming. However, in hindlimbs contralateral to GDNF injection, mechanical hyperalgesia was significantly decreased 4 h after PGE₂ injection (0.1 ng: 1,635 ± 2.9 vs. 2,470.8 ± 15.4 mN, P < 0.001; 1 ng: 1,569 ± 40.9 vs. 2,384.3 ± 39.6 mN, P < 0.001; 10 ng: 1,560.5 ± 19.5 vs. 2,409.2 ± 77.5 mN, P < 0.001, ANOVA followed by Tukey's test, respectively). Data are plotted as %change in mechanical nociceptive threshold compared with baseline (n = 6/group). Differences in nociceptive responses between readings taken at 1, and 4, 24, and 72 h in hindlimbs ipsi or contralateral to previous GDNF injection are represented by ***P < 0.001.

Fig. 2.

GDNF produces mechanical hyperalgesia and sensitizes muscle nociceptors. A: compared with baseline (2,649.2 ± 2.8 mN) GDNF (n = 6), but not vehicle (n = 6), produced a significant decrease in mechanical muscle threshold at 15 (2,037.3 ± 10.4 mN, P < 0.001, ANOVA followed by Dunnett's test), 30 (1,762.8 ± 18.6 mN, P < 0.001, ANOVA followed by Dunnett's test), and 45 (1,676.3 ± 8.3 mN, P < 0.001, ANOVA followed by Dunnett's test) min. after intramuscular injection. Also, 30 min after injection, GDNF significantly enhanced the nociceptor response to sustained mechanical stimulation, as revealed by increased cumulated number of spikes in recordings obtained during early (first 10 s) and late (last 10 s) parts of the stimulation period (B), as well as total response (60 s) compared with pre-GDNF control values (C). This was also evident in time-course histograms of the nociceptor response, representing recordings obtained before (D) and after (E) GDNF injection (10 ng/5 μl im; n = 16). Comparisons between pre- and post-GDNF treatment were made using one-tail Student's t-test for paired samples; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Ergonomic insults increase GDNF content in affected skeletal muscle. Rats were submitted to a hindlimb vibration or eccentric exercise. Twenty four hours later, a time point where both interventions produce muscle hyperalgesia (Alvarez et al. 2010; Alvarez et al. 2012; Dina et al. 2010), the gastrocnemius muscle was excised and GDNF content (pg/mg protein) determined by means of an ELISA procedure and compared with those exhibited by naïve rats. Both ergonomic interventions produced a significant increase in muscular content of GDNF. Comparisons between GDNF values obtained from naïve rats (white bar, n = 12) and rats submitted to vibration (grey bar, n = 7) or eccentric exercise (black bar, n = 5) were made by using a one-way ANOVA followed by a Dunnett's multiple comparisons test. ***P < 0.001.

Fig. 4.

Intrathecal antisense to GFRα1 inhibits ergonomic insult-induced mechanical hyperalgesia. Rats received a daily intrathecal treatment of antisense (●) or mismatch (○) oligodeoxynucleotide (ODNs) to GFRα1 [40 μg, antisense (AS) or mismatch (MM)] during three consecutive days. They were then submitted to a hindlimb vibration (n = 9/ODN treatment) or eccentric exercise (n = 6/ODN treatment). A: preventive AS, but not MM, treatment inhibited mechanical hyperalgesia after hindlimb vibration on days 1 (AS: 2,376.7 ± 36.3 mN vs. MM: 2,025.1 ± 35.3 mN, P < 0.05), 2 (AS: 2,228.8 ± 37 mN vs. MM: 1,784.9 ± 33.1 mN, P < 0.05), and 3 (AS: 2,108.9 ± 55.7 mN vs. MM: 1,735.3 ± 41.3 mN, P < 0.05) after ergonomic insult. B: preventive AS, but not MM, treatment inhibited mechanical hyperalgesia after hid limb eccentric exercise on days 1 (AS: 2,132.7 ± 0.16 mN vs. MM: 1,680.8 ± 41.8 mN vs. P < 0.05), 2 (AS: 2,319.5 ± 337.5 mN vs. MM: 1,689.7 ± 0.16 mN, P < 0.05), and 3 (AS: 2,296.7 ± 39.2 mN vs. MM: 1,970.8 ± 53.8 mN, P < 0.05) after ergonomic insult. Comparisons to baseline were made using two-way repeated measures ANOVA (time and ODN treatment) followed by Tukey's multiple comparisons test. *P < 0.05.

Fig. 5.

IB4-saporin abrogates acute and chronic mechanical hyperalgesia induced by GDNF intramuscularly. Rats were injected intrathecally with IB4-saporin (n = 6) or kept naïve (control group, n = 5) 10 days before unilateral GDNF injection. A: GDNF injection (10 ng/20 μl im) produced a mechanical hyperalgesia that lasted for at least 3 days in control rats (3rd day: 1,670.7 ± 30.7 mN vs. baseline: 2,674.8 ± 9.2, P < 0.001, ANOVA followed by Tukey's test). In contrast, IB4-saporin-treated rats only exhibited a modest, albeit significant, mechanical hyperalgesia that lasted ∼1 day (2,382.7 ± 11.4 mN vs. baseline: 2,669.8 ± 11.4 mN, P < 0.05, ANOVA followed by Tukey's test). B: 10 days after return to pre-GDNF baseline levels, groups were assessed for hyperalgesic priming by injecting PGE₂ (1 μg/20 μl im) bilaterally. One hour after PGE₂ injection, bilateral mechanical hyperalgesia was observed in naïve (1,497.4 ± 18.8 mN vs. baseline: 2,698.6 ± 7, P < 0.001, ANOVA followed by Tukey's test) and IB4(+)-saporin-treated rats (1,629.7 ± 32.9 mN vs. baseline: 2,658 ± 38.1, P < 0.001, ANOVA followed by Tukey's test). Such hyperalgesia remained unchanged after 24 h in naïve rats (1 h: 1,497.4 ± 18.8 mN vs. 24 h: 1,529.6 ± 16.6 mN, P > 0.05, ANOVA followed by Tukey's test). In contrast, PGE₂-induced mechanical hyperalgesia was significantly decreased in IB4-saporin-treated rats 4 h after PGE₂ injection (1 h: 1,629.7 ± 32.9 mN vs. 4 h: 2,474.2 ± 88.7 mN, P < 0.01, ANOVA followed by Tukey's test), indicating that IB4(+) nociceptors mediate hyperalgesic priming. Data are plotted as %change in mechanical nociceptive threshold compared with baseline. Solid symbols represent P < 0.05 respect to baseline. Differences in nociceptive responses comparing readings taken at 1, 4, and 24 h are represented by **P < 0.01.