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. 2017 Jul;158(7):1204-1216.
doi: 10.1097/j.pain.0000000000000898.

Hyperalgesic priming (type II) induced by repeated opioid exposure: maintenance mechanisms

Dioneia Araldi  1 , Luiz F Ferrari, Jon D Levine
Affiliations

Hyperalgesic priming (type II) induced by repeated opioid exposure: maintenance mechanisms

Dioneia Araldi et al. Pain. 2017 Jul.

Abstract

We previously developed a model of opioid-induced neuroplasticity in the peripheral terminal of the nociceptor that could contribute to opioid-induced hyperalgesia, type II hyperalgesic priming. Repeated administration of mu-opioid receptor (MOR) agonists, such as DAMGO, at the peripheral terminal of the nociceptor, induces long-lasting plasticity expressed, prototypically as opioid-induced hyperalgesia and prolongation of prostaglandin E2-induced hyperalgesia. In this study, we evaluated the mechanisms involved in the maintenance of type II priming. Opioid receptor antagonist, naloxone, induced hyperalgesia in DAMGO-primed paws. When repeatedly injected, naloxone-induced hyperalgesia, and hyperalgesic priming, supporting the suggestion that maintenance of priming involves changes in MOR signaling. However, the knockdown of MOR with oligodeoxynucleotide antisense did not reverse priming. Mitogen-activated protein kinase and focal adhesion kinase, which are involved in the Src signaling pathway, previously implicated in type II priming, also inhibited the expression, but not maintenance of priming. However, when Src and mitogen-activated protein kinase inhibitors were coadministered, type II priming was reversed, in male rats. A second model of priming, latent sensitization, induced by complete Freund's adjuvant was also reversed, in males. In females, the inhibitor combination was only able to inhibit the expression and maintenance of DAMGO-induced priming when knockdown of G-protein-coupled estrogen receptor 30 (GPR30) in the nociceptor was performed. These findings demonstrate that the maintenance of DAMGO-induced type II priming, and latent sensitization is mediated by an interaction between, Src and MAP kinases, which in females is GPR30 dependent.

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Conflict of interest statement

Conflict of Interest: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Naloxone induces hyperalgesia in DAMGO-primed paws and after repeated exposure to naloxone, in male rats
A. Male rats received repeated (hourly x4) intradermal injection of vehicle (5 μL) or DAMGO (1 μg) on the dorsum of the hindpaw. One week later (1 w), naloxone (1 μg) was injected at the same site and, the mechanical nociceptive threshold, evaluated. In the group primed by repeated injections of DAMGO, naloxone induced robust mechanical hyperalgesia (black bars), observed 30 min and 1 h after its injection (F1,30; *** p < 0.0001, when DAMGO-primed and the vehicle groups that received naloxone are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that naloxone, an MOR inverse agonist, can reinstate the hyperalgesia in DAMGO-primed paws. (N = 6 paws per group) B. A different group of male rats received repeated (hourly × 4) intradermal injections of vehicle (5 μL) or naloxone (1 μg) on the dorsum of the hindpaw. Thirty minutes after the 4th injection of vehicle or naloxone, the mechanical nociceptive thresholds were evaluated, and significant mechanical hyperalgesia was observed in the naloxone-treated group (F1,10 = 111.76; *** p < 0.0001, when the naloxone- and the vehicle-treated groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). One week later, when PGE2 (100 ng) was injected, at the same site as naloxone or vehicle, mechanical hyperalgesia was observed 30 min after its injection in both groups. In the group that received repeated injections of naloxone, PGE2-induced hyperalgesia was still present at the 4th h after injection (F1,20 = 75.18; *** p < 0.0001, when naloxone- and vehicle-treated groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), suggesting that changes in MOR signaling maintain the nociceptor plasticity. (N = 6 paws per group)
Figure 2
Figure 2. Mu-opioid receptor does not play a role in the expression of type II priming
Male rats received repeated (hourly × 4) intradermal injections of DAMGO (1 μg) on the dorsum of the hindpaw and, 24 hrs after the last injection of DAMGO, treatment with intrathecal injections of MM-ODN (40 μg/20 μL/day; black bars) or AS-ODN (40 μg/20 μL/day; white bars) for mu-opioid receptor mRNA was performed once a day, for 5 consecutive days. On the sixth day, ~17 hrs after the last injection of MM-ODN or AS-ODN, PGE2 (100 ng) was injected on the dorsum of the hindpaw and, the mechanical nociceptive threshold, evaluated 30 min and 4 hrs later. PGE2-induced mechanical hyperalgesia was present at 30 min and 4 hrs, in both MM-ODN- and AS-ODN-treated groups, indicating that mu-opioid receptor does not play a role in the expression of type II priming (F1,22 = 0.22, p = 0.6410, ns, when the MM-ODN and AS-ODN-treated groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). To determine whether the treatment with AS-ODN for mu-opioid receptor affected the maintenance of type II priming, we injected a single dose of DAMGO (1 μg, intradermally) 7 days after the last injection of the ODNs. In both groups DAMGO induced hyperalgesia, indicating the presence of type II priming (F1,22 = 2.91, p = 0.1018, ns, when the MM-ODN and AS-ODN-treated groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). (N = 12 paws per group)
Figure 3
Figure 3. Second messengers involved in the expression of type II priming: MAPK and FAK
A. Male rats received repeated (hourly × 4) intradermal injections of DAMGO (1 μg) on the dorsum of the hindpaw. Seven days later, vehicle (5 μL, black bars) or the MAPK inhibitor U0126 (1 μg; gray bars) was injected at the same site, followed 10 min later by PGE2 (100 ng). In the group treated with U0126, PGE2-induced hyperalgesia was almost completely inhibited at the 4th h (F1,8 = 58.30; *** p < 0.0001, when the vehicle and MAPK inhibitor groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the MAPK plays a role in the expression of type II priming. B. To determine whether the inhibition of type II priming by the MAPK inhibitor was permanent, PGE2 (100 ng) was injected again at the same site, 10 days after the injection of U0126. At this time, PGE2-induced hyperalgesia was present at the 4th h, indicating that MAPK does not play a role in the maintenance of type II priming (F1,16 = 6.34; ns, when the vehicle and MAPK inhibitor groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). C. Male rats received repeated (hourly × 4) intradermal injections of DAMGO (1 μg) on the dorsum of the hindpaw. Twelve days later, vehicle (5 μL, black bars) or the FAK inhibitor (1 μg; white bars) was injected at the same site, followed 10 min later by PGE2 (100 ng). In the group treated with the FAK inhibitor, PGE2-induced hyperalgesia was attenuated at 30 min and 4 hrs (F1,12 = 19.42; *p < 0.05 and *** p < 0.0001, when the vehicle and FAK inhibitor groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating a small participation of FAK in the expression of type II priming. (N = 6 paws per group)
Figure 4
Figure 4. The combination of a Src and MAPK inhibitors reversed DAMGO-induced priming in male rats
A. Rats received repeated (hourly × 4) intradermal injections of DAMGO (1 μg) on the dorsum of the hindpaw. Seven days later, vehicle (5 μL; black bars) or a combination of Src (SU 6656, 1 μg) and MAPK (U0126, 1 μg) inhibitor (gray bars) was injected at the same site. Ten min later, PGE2 (100 ng; intradermally) was injected and the mechanical nociceptive threshold, evaluated 30 min and 4 hrs after injection. In the group treated with the combination of Src and MAPK inhibitors PGE2-induced hyperalgesia, at 30 min, was significantly attenuated and its prolongation, evaluated at the 4th h, completely blocked (F1,20 = 133.49, ** p < 0.01 and ***p < 0.0001, when the vehicle and Src + MAPK inhibitors groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that Src and MAPK play a role in the expression of DAMGO-induced type II priming. B. Fifteen days later, PGE2 (100 ng) was injected again at the same site. Although in all groups PGE2-induced mechanical hyperalgesia was present 30 min after injection, in the Src + MAPK inhibitors-treated group, it was markedly blocked at the 4th h (F1,20 = 73.73, *** p < 0.0001, when the vehicle and Src + MAPK inhibitors groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), supporting the suggestion that both Src and MAPK are required for the maintenance of type II priming. C. To determine whether treatment with the combination of Src and MAPK inhibitors permanently reversed priming, PGE2 was injected again at the same site 30 days later. In the group previously treated with the inhibitors (gray bars), PGE2-induced mechanical hyperalgesia was not observed at the 4th h (F1,20 = 27.98, *** p = 0.0004, when the vehicle and Src + MAPK inhibitors groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), demonstrating that inhibition of Src and MAPK, at the same time, reversed type II priming in male rats. (N = 6 paws per group)
Figure 5
Figure 5. CFA-induced latent sensitization, a model of type II hyperalgesic priming, depends on Src and MAPK in male rats
Mechanical nociceptive threshold was evaluated by Randall-Selitto paw-withdrawal test, in both hindpaws of male rats, before and on days 1, 3, 5, 7, 14, 21 and 28 after an intraplantar injection of CFA (50 μL, non-diluted), in one hindpaw (ipsilateral paw). A. In that paw (black circles), the mechanical nociceptive threshold decreased dramatically already on day 1 (F = 72.47;### p < 0.0001, when days after CFA were compared to baseline [before CFA injection]; one-way repeated measure ANOVA followed by Bonferroni post hoc test. F1,60 = 114.97; *** p < 0.0001 or * p < 0.05, when ipsilateral [black circles] were compared to contralateral [white circles] paws; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), progressively returning to baseline values, days 21 (ns) and 28 (ns). Change in the mechanical nociceptive threshold was not observed in the contralateral paws (A; white circles; F = 1.253; p = 0.3016; one-way repeated measure ANOVA followed by Bonferroni post hoc test). B – C. Twenty-eight days CFA injection, when the mechanical nociceptive threshold returned to baseline values in the ipsilateral paw, rats received an intrathecal injection of naltrexone (NTX; 1 μg/20 μL), and the mechanical nociceptive threshold was evaluated 15, 30, 45, 60, 90 and 120 min after injection. A robust reduction in the mechanical nociceptive threshold was observed in both the contralateral [B; white circles; F = 2.798; *** p < 0.0001, when time (min) after NTX was compared to baseline (before NTX); one-way repeated measure ANOVA followed by Bonferroni post hoc test] and in the ipsilateral [C; black circles; F = 45.21; *** p < 0.0001, when time (min) after NTX was compared to baseline (before NTX); one-way repeated measure ANOVA followed by Bonferroni post hoc test] paws. 120 min after the injection of NTX, the mechanical nociceptive threshold returned to baseline, in both paws (ns; B, white circles; C, black circles). (N = 6 rats). A different group of male rats that received CFA in one hindpaw, 28 days before, was treated with an intrathecal injection of the combination of Src (SU 6656, 10 μg/5 μL) and MAPK (U0126; 10 μg/5 μL) inhibitors, 5 min before of the intrathecal injection of NTX (1 μg/10 μL). At 15, 30, 45, 60, 90 and 120 min after the injection of NTX, we observed a complete inhibition of NTX-induced mechanical hyperalgesia in both paws, contralateral [B; F1,50 = 84.72; *** p < 0.0001, when NTX group (white circles) was compared to Src and MAPK inhibitors followed by NTX group (black square); two-way repeated-measures ANOVA followed by Bonferroni post hoc test; F = 0.9309; p = 0.4873, when time (min) after NTX was compared to baseline (before NTX); one-way repeated measure ANOVA followed by Bonferroni post hoc test] and ipsilateral [C; F1,50 = 149.49; *** p < 0.0001, when NTX group (black circles) was compared to Src and MAPK inhibitors followed by NTX group (white square); two-way repeated-measures ANOVA followed by Bonferroni post hoc test; F = 1.675; p = 0.1616, when time (min) after NTX was compared to baseline (before NTX); one-way repeated measure ANOVA followed by Bonferroni post hoc test]. These findings support the suggestion that the CFA-induced latent sensitization is also dependent on Src and MAPK second messengers. (N = 6 rats)
Figure 6
Figure 6. Expression of type II priming does not depend on Src and MAPK in female rats
Female rats received repeated (hourly × 4) intradermal injections of DAMGO (1 μg) on the dorsum of the hindpaw. A. Seven days later, vehicle (5 μL; black bar) or a combination of Src (SU 6656, 1 μg) and MAPK (U0126; 1 μg) inhibitors (gray bar) was intradermally injected on the dorsum of the hindpaw, followed by injection of PGE2 (100 ng), at the same site. Mechanical nociceptive threshold was evaluated 30 min and 4 hrs after PGE2 injection. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test showed PGE2-induced hyperalgesia at 30 min, which was still present at the 4th h after injection, in both groups, with no significant (ns) difference between the groups (F1,20 = 2.10; p = 0.1783, when vehicle and Src + MAPK inhibitors groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B. Ten days after the injection with Src and MAPK inhibitors, PGE2 (100 ng; intradermally) was again injected. PGE2-induced hyperalgesia was present 30 min and 4 hrs later, in both groups (ns, when vehicle and Src + MAPK inhibitors groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that Src and MAPK do not play a role in the expression or maintenance of priming in female rats, possibly due to sexual dimorphic role of Src and MAPK in this type of neuroplasticity. (N = 6 paws per group).
Figure 7
Figure 7. Knockdown of GPR30 uncovers sensitivity to the combination of Src and MAPK inhibitors in females
Female rats were treated hourly (x 4) with intradermal injections of DAMGO (1 μg) on the dorsum of the hindpaw. Twenty-four hours later, daily spinal intrathecal injection of MM-ODN (40 μg/20 μL/day; black bars) or AS-ODN (40 μg/20 μL/day; white and dotted bars) for ER-α (A), ER-β (B) or GPR30 (C) were performed for 5 consecutive days. On the sixth day, ~ 17 hrs after the last injection of MM-ODN or AS-ODN, rats received intradermal injection of the combination of Src (SU 6656; 1 μg) and MAPK (U0126, 1 μg) inhibitors (black and gray bars) on the dorsum of the hindpaw, followed, 10 min later, by PGE2 (100 ng) at the same site. The mechanical nociceptive threshold was evaluated 30 min and 4 hrs after PGE2 injection. A. PGE2-induced hyperalgesia was present 30 min and 4 hrs later, in all groups (F1,33 = 1.36; p = 0.2523, when the groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the knockdown of ER-α did not affect the inability of the combination of Src and MAPK inhibitors to reverse priming in female rats. B (left panel). PGE2-induced hyperalgesia at 30 min was partially attenuated in the ER-β-treated AS-ODN-treated group, that received the combination of Src and MAPK inhibitors (gray bar; ** p < 0.01, when the MM-ODN- (black bar) and AS-ODN (white bar) groups are compared). At the 4th h, the prolongation of PGE2-induced hyperalgesia was still attenuated in the ER-β AS-ODN-treated group that received Src and MAPK inhibitors (gray bar; F2,33 = 38.54; *** p < 0.001; two-way repeated-measures ANOVA followed by Bonferroni post hoc test); Right panel. To determine whether the reversal of DAMGO-induced priming by treatment with AS-ODN for ER-β in rats that received the combination of Src and MAPK inhibitors, was permanent, PGE2 was injected again at the same site 8 days after the last ODN injection. We observed that PGE2-induced mechanical hyperalgesia was still present at the 4th h, indicating the presence of type II priming (F2,66 = 2.64; p = 0.0862, when the groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). C (left panel). PGE2-induced hyperalgesia was inhibited at 30 min and 4 hrs in the group that had previously been treated with AS-ODN for GPR30 and received the combination of Src and MAPK inhibitors (gray bars; F2,33 = 62.37; ** p < 0.001 and *** p < 0.0001, when AS-ODN, Src and MAPK inhibitors is compared to AS-ODN or MM-ODN, Src and MAPK inhibitors; two-way repeated-measures ANOVA followed by Bonferroni post hoc test); Middle panel. To determine whether the reversal of DAMGO-induced priming by the treatment with AS-ODN for GPR30 that received the combination of Src and MAPK inhibitors was permanent, PGE2 was injected again at the same site 8 days after the last ODN injection. PGE2-induced mechanical hyperalgesia was attenuated at the 4th h in the group previously treated with AS-ODN for GPR30 that received the combination of Src and MAPK inhibitors (gray bar; F2,66 = 31.76; *** p < 0.0001, when AS-ODN, Src and MAPK inhibitors is compared to AS-ODN or MM-ODN, Src and MAPK inhibitors; two-way repeated-measures ANOVA followed by Bonferroni post hoc test); Right panel. Additionally, when PGE2 (100 ng; intradermally) was injected again, 15 days after the last injection of ODNs and 14 days after the injection of the combination of Src and MAPK inhibitors, PGE2-induced mechanical hyperalgesia was attenuated at the 4th h in the group previously treated with AS-ODN for GPR30 that received the combination of Src and MAPK inhibitors (gray bar; F2,66 = 85.64; *** p < 0.0001, when AS-ODN, Src and MAPK inhibitors is compared to the AS-ODN or MM-ODN, Src and MAPK inhibitors; two-way repeated-measures ANOVA followed by Bonferroni post hoc test) indicating that GPR30 regulates the inhibitory effect of the combination of Src and MAPK inhibitors on DAMGO-induced type II priming, in female rats. Of note, the mechanical nociceptive thresholds before the treatment with MM- or AS-ODN antisense were not different when compared to the mechanical nociceptive thresholds ~17 hrs after the last injection of ODNs. In addition, the treatment with AS-ODN alone (white bars) was not able to inhibit the expression and/or maintenance of DAMGO-induced type II priming. (N = 12 paws per group)
Figure 8
Figure 8. Schematic representation of the mechanisms involved in the maintenance of DAMGO-induced type II hyperalgesic priming in the male and female rat
In (A) the induction of type II priming by repeated (hourly × 4) stimulation of mu-opioid receptor in IB4-negative nociceptors is illustrated. The repeated administration of DAMGO (a mu-opioid receptor agonist) stimulates a yet-to-be-determined pathway that produces neuroplastic changes in the nociceptor expressed as prolongation of PGE2-induced hyperalgesia [9]. The maintenance of DAMGO-induced type II hyperalgesic priming is markedly dependent on both Src and MAPK/ERK in male (B), which is regulated through GPR30 in female (C) rats. Abbreviations: EP, prostaglandin E2 receptor; GPCR, G-protein-coupled estrogen receptor 30; MOR, mu-opioid receptor; Gαi, inhibitory G-protein α subunit; IB4, isolectin B4; MAPK/ERK/, mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK); PGE2, prostaglandin E2; Src, Src tyrosine kinase.
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