Inflammatory and neuropathic cold allodynia are selectively mediated by the neurotrophic factor receptor GFRα3

Abstract

Tissue injury prompts the release of a number of proalgesic molecules that induce acute and chronic pain by sensitizing pain-sensing neurons (nociceptors) to heat and mechanical stimuli. In contrast, many proalgesics have no effect on cold sensitivity or can inhibit cold-sensitive neurons and diminish cooling-mediated pain relief (analgesia). Nonetheless, cold pain (allodynia) is prevalent in many inflammatory and neuropathic pain settings, with little known of the mechanisms promoting pain vs. those dampening analgesia. Here, we show that cold allodynia induced by inflammation, nerve injury, and chemotherapeutics is abolished in mice lacking the neurotrophic factor receptor glial cell line-derived neurotrophic factor family of receptors-α3 (GFRα3). Furthermore, established cold allodynia is blocked in animals treated with neutralizing antibodies against the GFRα3 ligand, artemin. In contrast, heat and mechanical pain are unchanged, and results show that, in striking contrast to the redundant mechanisms sensitizing other modalities after an insult, cold allodynia is mediated exclusively by a single molecular pathway, suggesting that artemin-GFRα3 signaling can be targeted to selectively treat cold pain.

Keywords: Gfrα3; allodynia; artemin; cold; pain.

Fig. S1.

Gfrα3^−/− mice display normal acute cold, heat, and mechanosensory behaviors. Withdrawal latencies in response to (A) cold and (C) heat stimuli were similar in naïve adult (8–14 wk of age) Gfrα3^−/− and WT mice (P > 0.05; n = 5–12). (B) Paw withdrawal threshold forces did not differ between genotypes in the electronic von Frey assay (P > 0.05; n = 5–10). (E) Gfrα3^−/− mice injected with artemin (20 μg; P > 0.05 for all time points; n = 7) showed no change in cold sensitivity compared with (D) vehicle-injected mice in the cold plantar assay (n = 7). **P < 0.01; ***P < 0.001. (F) Gfrα3^−/− mice injected with artemin showed no change in cold sensitivity compared with vehicle injection in the evaporative cooling assay (P > 0.05 for all time points; n = 5–6). ARTN, artemin; contr, contralateral; ipsi, ipsilateral; ns, not significant.

Fig. 1.

GFRα3 is required for cold allodynia induced by inflammation, nerve injury, and chemotherapy polyneuropathy. (A) Decreased cold-evoked withdrawal latencies in WT but not Gfrα3^−/− mice 2 d after an intraplantar injection of CFA. Post-CFA latencies for Gfrα3^−/− mice were not statistically different (P > 0.05) than basal. **P < 0.01 (n = 7–9). (B) Cold allodynia observed in the ipsilateral hind paw in WT mice after CCI was absent in Gfrα3^−/− mice with postinjury withdrawal latencies identical to preinjury times (P > 0.05; n = 6–7). **P < 0.01; ***P < 0.001. (C) Oxaliplatin-induced decreases in withdrawal latencies to cold observed in WT controls were absent in Gfrα3^−/− mice, with response times the same as preinjection times for both genotypes (P > 0.05; n = 11–12). contr, Contralateral; ipsi, ipsilateral; ns, not significant; oxal, oxaliplatin. ***P < 0.001.

Fig. S2.

Evaporative cooling assay to assess GFRα3 in cold allodynia induced by inflammation, nerve injury, and chemotherapy polyneuropathy. Increased acetone-cooling evoked response score was observed in WT but not Gfrα3^−/− mice (A) after an intraplantar injection of CFA (n = 8), (B) after CCI of the sciatic nerve (n = 7), and (C) in oxaliplatin polyneuropathy (n = 9). Postinjury scores for Gfrα3^−/− mice were not statistically different (P > 0.05) between ipsilateral and contralateral hind paws (CFA and CCI) or vs. basal (oxalplatin). contr, Contralateral; ipsi, ipsilateral; ns, not significant. **P < 0.01.

Fig. 2.

Heat and mechanical hyperalgesia are not dependent on GFRα3. Both WT and Gfrα3^−/− mice exhibit reduced threshold forces inducing a paw withdrawal (A) 3 d after unilateral CFA injection or (B) 7 d after CCI surgery (ipsilateral vs. contralateral; n = 6–7). Similarly, heat hyperalgesia was observed (C) 3 d after the induction of inflammation or (D) 7 d after nerve injury (ipsilateral vs. contralateral; n = 7–8). contr, Contralateral; ipsi, ipsilateral. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S3.

Oxaliplatin-induced mechanical hyperalgesia is not dependent on GFRα3. Cold allodynia observed 7 d after a single injection of oxaliplatin was robust in both WT and Gfrα3^−/− mice (n = 6–8). ns, Not significant. ***P < 0.001.

Fig. S4.

Expression of genes involved in cold transduction is not dependent on GFRα3. cDNA purified from (A) adult dorsal root and (B) trigeminal ganglia was analyzed by quantitative PCR, showing that transcript levels of a number of genes involved in somatosensory signaling are similar between Gfrα3^−/− and WT tissues (n = 4 mice).

Fig. S5.

Expressions of somatosensory markers are unaltered in the absence of GFRα3. (A–E) Representative images of triple-labeled DRG sections from both adult (Upper) WT and (Lower) Gfrα3^−/− mice. In A–E, TRPM8 (green) and GFRα3 (blue) expressions are compared with a difference marker (red). (A) TRPV1 labeling in DRGs and their overlap with TRPM8 (green) showed no differences in thermosensory TRP channel expression between genotypes. Similarly, expressions of (B) the peptide calcitonin gene-related peptide (CGRP), (C) the nonpeptidergic marker IB4, (D) the A-fiber marker NF200, and (E) the C-fiber marker peripherin (Per) were similar in WT and Gfrα3^−/− mice. Arrowheads indicate triple-labeled neurons (WT) or double-labeled neurons (Gfrα3^−/−), whereas arrows indicate TRPM8 cells that are not positive for either marker. (F) Quantification of immunohistochemical labeling of various sensory neuron makers shows similar proportions in Gfrα3^−/− and WT controls DRGs. Percentages of total DRG neurons that were positive for each marker are similar in Gfrα3^−/− tissue compared with WT tissue (P > 0.05; n = 15–32 sections from at least three mice). ns, Not significant.

Fig. 3.

Artemin neutralization selectively attenuates cold hypersensitivity. (A) Inflammatory cold allodynia was attenuated in WT mice 4 h after s.c. injection of an artemin-neutralizing antibody (P > 0.05, ipsilateral vs. contralateral; n = 6–7) compared with in control mice. **P < 0.01. (B) Chemotherapeutic-induced cold pain was attenuated after antibody injection (P > 0.05, preoxaliplatin vs. postantibody) and significantly different from controls. **P < 0.01. (C) Conversely, inflammatory mechanical hyperalgesia was unaffected (P > 0.05, ipsilateral control vs. ipsilateral antibody; P < 0.001, ipsilateral vs. contralateral for both treatments; n = 5–8). ***P < 0.001. (D) Chemotherapeutic-induced mechanical hyperalgesia was unaffected (P > 0.05, postantibody vs. control; P < 0.01, preoxaliplatin vs. postantibody; n = 5–6). **P < 0.01. (E) Inflammatory thermal hyperalgesia was unaffected (P > 0.05, ipsilateral control vs. ipsilateral antibody; P < 0.001, ipsilateral vs. contralateral for both treatments; n = 6). ARTN, artemin; contr, contralateral; ipsi, ipsilateral; ns, not significant; oxal, oxaliplatin. ***P < 0.001.

Fig. S6.

Artemin neutralization attenuates cold hypersensitivity in the evaporative cooling assay. Inflammation-induced (CFA) cold hypersensitivity was attenuated in WT mice 4 h after s.c. injection of an antiartemin antibody (MAB1085; 10 mg/kg; P > 0.05, ipsilateral vs. contralateral; n = 8) compared with in mice injected with an IgG2A isotype control (10 mg/kg). ARTN, artemin; contr, contralateral; ipsi, ipsilateral; ns, not significant. **P < 0.01.

Fig. 4.

NGF-induced cold allodynia is GFRα3-dependent. (A) WT mice exhibit cold allodynia 1 h but not 3 h after intraplantar NGF injections (P > 0.05; n = 9–11), whereas (B) Gfrα3^−/− mice showed no change in cold sensitivity compared with vehicle-injected mice in the cold plantar assay (P > 0.05; n = 9–11). **P < 0.01. Both (C) WT and (D) Gfrα3^−/− mice displayed robust heat hyperalgesia 1 and 3 h after NGF administration (n = 6). ns, Not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Artemin neutralization blocks NGF-induced cold allodynia. (A) NGF-induced cold allodynia was attenuated in WT mice by artemin neutralization (P > 0.05, pre- vs. post-NGF and vs. vehicle-injected mice; n = 4) 1 h before intraplantar NGF injection. Control mice show robust cold allodynia after NGF injection (pre- vs. post-NGF and vs. vehicle-injected; n = 4). ***P > 0.001. (B) NGF-induced heat hyperalgesia was unaffected by antibody treatment and similar to controls (pre- vs. post-NGF and vs. vehicle-injected; n = 4). **P < 0.01; ***P > 0.001. ARTN, artemin; contr, contralateral; ipsi, ipsilateral; ns, not significant.