Figure 4. MOR-EGFR crosstalk in the induction of prolongation of PGE₂ hyperalgesia.

Rats received an intradermal injection of vehicle (5 μL; black bars) or EGFR inhibitor (Tyrphostin AG 1478, 1 μg; dotted bars). The average baseline mechanical nociceptive threshold, before treatments, was 130.1 ± 1.4 g for the vehicle group and 132.0 ± 1.3 g for the EGFR inhibitor group. Ten minutes later, repeated (hourly × 4) intradermal injections of DAMGO (1 μg) were performed on the dorsum of the hind paw. Five days later, when mechanical threshold was not different from the pre-DAMGO baseline (128.8 ± 2.1 g, t₍₅₎ = 0.3162; p = 0.7646, for vehicle-treated group; 130.7 ± 2.0 g, t₍₅₎ = 2.169; p = 0.0822, for EGFR inhibitor-treated group, when the mechanical nociceptive threshold is compared before and 5 days after DAMGO; paired Student's t test), PGE₂ (100 ng) was injected at the same site and mechanical nociceptive threshold evaluated 30 min and 4 hours later. In the group previously treated with EGFR inhibitor, the prolongation of PGE₂ hyperalgesia was almost completely eliminated at the 4^th hour (F_(2,16) = 175.68, *** p < 0.0001, when vehicle-treated group is compared to EGFR inhibitor treated group at the 4^th hour after the injection of PGE₂; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B – C. When PGE₂ (100 ng) was again injected on the dorsum of the hind paw, 15 (B) or 30 (C) days after repeated exposure to DAMGO, the prolongation of PGE₂-induced hyperalgesia was not present at the 4^th hour in the EGFR inhibitor-treated group (F_(2,16) = 98.89, *** p < 0.0001 for 15 days (B) and, F_(2,16) = 120.35, *** p < 0.0001 for 30 days (C), when the vehicle-treated group is compared to EGFR inhibitor-treated group at the 4^th hour after the injection of PGE₂; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These data support a role of crosstalk between mu-opioid and EGF receptors in the induction of the prolongation of PGE₂ hyperalgesia by repeated exposure to DAMGO. Of note, the average baseline mechanical nociceptive threshold, before PGE₂, was 129.7 ± 1.7 g (15 days) and 131.4 ± 1.8 g (30 days) for the vehicle group and 131.1 ± 1.1 g (15 days) and 130.6 ± 1.2 g (30 days) for the EGFR inhibitor group. D. A different group of rats, with average baseline mechanical nociceptive threshold of 130.9 ± 2.0 g, was treated with repeated (hourly × 4) intradermal injections of the EGFR agonist (EGF ligand; 1 μg/5 μL; gray bars) and the mechanical nociceptive threshold evaluated 30 min after each injection. No change in nociceptive threshold was observed after the 1^st (not significant, ns, 130.3 ± 2.2 g, t₍₅₎ = 1.267, p = 0.2610), 2^nd (ns, 130.0 ± 1.6 g, t₍₅₎ = 1.066, p = 0.3352), 3^rd (ns, 128.0 ± 2.1 g, t₍₅₎ = 2.236, p = 0.0756) or 4^th (ns, 128.3 ± 1.9 g, t₍₅₎ = 2.214, p = 0.0778) injection of EGFR agonist (when the mechanical nociceptive threshold before the 1^st injection and 30 min after each injection of EGFR agonist, is compared; paired Student's t test). Five days (5 d) later, when the mechanical threshold was not different from the pre-EGFR agonist baseline (132.3 ± 1.7 g, t₍₅₎ = 2.485; p = 0.0555, when the mechanical nociceptive threshold is compared before and 5 days after repeated injections of EGFR agonist; paired Student's t test), PGE₂ (100 ng) was injected at the same site, on the dorsum of the hind paw, and the mechanical nociceptive threshold evaluated 30 min and 4 hours after injection. PGE₂ induced hyperalgesia 30 min after injection (t₍₅₎ = 10.03, *** p = 0.0002, when mechanical thresholds before and 30 min after PGE₂, were compared; paired Student's t test), but not at the 4^th hour (t₍₅₎ = 0.7895, p = 0.4656, when mechanical thresholds before and 4 hours after PGE₂, were compared; paired Student's t test), indicating that repeated activation of EGF receptor does not induce hyperalgesic priming. n = 6 paws per group.