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. 2019 Sep 4;39(36):7061-7073.
doi: 10.1523/JNEUROSCI.1191-19.2019. Epub 2019 Jul 12.

In Vitro Nociceptor Neuroplasticity Associated with In Vivo Opioid-Induced Hyperalgesia

Eugen V Khomula  1 , Dioneia Araldi  1 , Jon D Levine  2
Affiliations

In Vitro Nociceptor Neuroplasticity Associated with In Vivo Opioid-Induced Hyperalgesia

Eugen V Khomula et al. J Neurosci. .

Abstract

Opioid-induced hyperalgesia (OIH) is a serious adverse event produced by opioid analgesics. Lack of an in vitro model has hindered study of its underlying mechanisms. Recent evidence has implicated a role of nociceptors in OIH. To investigate the cellular and molecular mechanisms of OIH in nociceptors, in vitro, subcutaneous administration of an analgesic dose of fentanyl (30 μg/kg, s.c.) was performed in vivo in male rats. Two days later, when fentanyl was administered intradermally (1 μg, i.d.), in the vicinity of peripheral nociceptor terminals, it produced mechanical hyperalgesia (OIH). Additionally, 2 d after systemic fentanyl, rats had also developed hyperalgesic priming (opioid-primed rats), long-lasting nociceptor neuroplasticity manifested as prolongation of prostaglandin E2 (PGE2) hyperalgesia. OIH was reversed, in vivo, by intrathecal administration of cordycepin, a protein translation inhibitor that reverses priming. When fentanyl (0.5 nm) was applied to dorsal root ganglion (DRG) neurons, cultured from opioid-primed rats, it induced a μ-opioid receptor (MOR)-dependent increase in [Ca2+]i in 26% of small-diameter neurons and significantly sensitized (decreased action potential rheobase) weakly IB4+ and IB4- neurons. This sensitizing effect of fentanyl was reversed in weakly IB4+ DRG neurons cultured from opioid-primed rats after in vivo treatment with cordycepin, to reverse of OIH. Thus, in vivo administration of fentanyl induces nociceptor neuroplasticity, which persists in culture, providing evidence for the role of nociceptor MOR-mediated calcium signaling and peripheral protein translation, in the weakly IB4-binding population of nociceptors, in OIH.SIGNIFICANCE STATEMENT Clinically used μ-opioid receptor agonists such as fentanyl can produce hyperalgesia and hyperalgesic priming. We report on an in vitro model of nociceptor neuroplasticity mediating this opioid-induced hyperalgesia (OIH) and priming induced by fentanyl. Using this model, we have found qualitative and quantitative differences between cultured nociceptors from opioid-naive and opioid-primed animals, and provide evidence for the important role of nociceptor μ-opioid receptor-mediated calcium signaling and peripheral protein translation in the weakly IB4-binding population of nociceptors in OIH. These findings provide information useful for the design of therapeutic strategies to alleviate OIH, a serious adverse event of opioid analgesics.

Keywords: calcium; excitability; fentanyl; hyperalgesic priming; mu-opioid receptor (MOR); opioid-induced hyperalgesia (OIH).

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Figures

Figure 1.
Figure 1.
Effect of fentanyl on mechanical nociceptive threshold. A, Male rats were treated systemically (subcutaneous, s.c.) with an analgesic dose of fentanyl (30 μg/kg, s.c.) or vehicle (saline, 100 μl/100 g body weight, s.c.). Mechanical nociceptive threshold was evaluated 1, 2, 3, and 48 h after each treatment. Systemic fentanyl induced analgesia measured 1 h after administration (F(1,10) = 23, ***p = 0.0007, when the mechanical nociceptive threshold was compared between vehicle and fentanyl, 1 h after systemic administration; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). By 2 h after systemic fentanyl no significant difference in nociceptive threshold was observed between vehicle- and fentanyl-treated rats. (n = 6 paws per group). B, A different group of rats was treated systemically with fentanyl (30 μg/kg, s.c.) or vehicle (saline, s.c.). Two days later, fentanyl was injected intradermally (1 μg/5 μl), in both groups, to evaluate for the presence of OIH; the mechanical nociceptive threshold was evaluated 30 min after intradermal fentanyl. Nociceptive threshold before intradermal fentanyl was not different from systemic pretreatment baselines (t5 = 0.41; p = 0.70, for the vehicle-treated group and, t5 = 0.17; p = 0.87, for the fentanyl-treated group; paired Student's t test). In the group previously treated with systemic vehicle, intradermal fentanyl did not induce changes in the mechanical nociceptive threshold. However, intradermal fentanyl-induced hyperalgesia (OIH) in the systemic fentanyl-treated group (t10 = 14, ****p < 0.0001, when the hyperalgesia in the groups was compared, 30 min after intradermal fentanyl; unpaired Student's t test; n = 6 paws per group).
Figure 2.
Figure 2.
Systemic fentanyl induces hyperalgesic priming. Rats received vehicle (saline, s.c.) or an analgesic dose of fentanyl (30 μg/kg, s.c.). Forty-eight hours later, PGE2 (100 ng/5 μl) was injected intradermally on the dorsum of the hindpaw and mechanical nociceptive threshold evaluated 30 min and 4 h later. Nociceptive threshold before intradermal injection of PGE2 was not different from presystemic fentanyl baseline (t5 = 1.6; p = 0.17, for the vehicle-treated group and, t5 = 0.70; p = 0.52, for the fentanyl-treated group; paired Student's t test). PGE2 induced hyperalgesia, 30 min after injection, in both vehicle- and fentanyl-treated groups. However, prolongation of PGE2-induced hyperalgesia, at the fourth hour, was present only in the group previously treated with fentanyl (F(1,10) = 113, ****p < 0.0001; when the hyperalgesia in the vehicle- and fentanyl-treated groups is compared at the fourth hour after intradermal PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). Thus, systemic administration of a single analgesic dose of fentanyl (30 μg/kg, s.c.) induces hyperalgesic priming, as shown by subsequent prolongation of PGE2 hyperalgesia. (n = 6 paws per group).
Figure 3.
Figure 3.
OIH is protein translation dependent. A, Rats were treated with fentanyl (30 μg/kg, s.c.) subcutaneously. Forty-eight hours later, vehicle (20 μl), the combination of SU6656 (10 μg/10 μl) + U0126 (10 μg/10 μl) or cordycepin (4 μg/20 μl) was injected intrathecally, followed 10 min later by fentanyl (1 μg/5 μl), injected intradermally, on the dorsum of the hindpaw. Mechanical nociceptive threshold was evaluated 30 min after intradermal fentanyl. In both the group treated with cordycepin and the group treated with the combination of SU6656 + U0126, intradermal fentanyl-induced hyperalgesia was blocked (F(2,15) = 57, ****p < 0.0001; when the hyperalgesia in the vehicle-, in the SU6656 + U0126- and in the cordycepin-treated groups is compared 30 min after intradermal fentanyl; one-way ANOVA followed by Bonferroni's post hoc test). B, One month after intrathecal treatment with vehicle, SU6656 + U0126, or cordycepin, fentanyl (1 μg/5 μl) was again injected intradermally and mechanical nociceptive threshold evaluated 30 min later. Although hyperalgesia induced by intradermal fentanyl was still reversed in the group treated with cordycepin (F(2,15) = 66, ****p < 0.0001; when the hyperalgesia in the vehicle-, SU6656 + U0126-, and cordycepin-treated groups was compared 30 min after intradermal fentanyl; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), in the groups treated with vehicle or the combination of SU6656 + U10126, intradermal fentanyl-induced hyperalgesia was present. These findings support the suggestion that intradermal fentanyl-induced hyperalgesia (OIH) in fentanyl-primed rats is dependent on protein translation in the nociceptor, sharing a mechanism in common with type I priming (n = 6 paws per group).
Figure 4.
Figure 4.
Fentanyl-induced Ca2+ signals in sensory neurons. Rats were primed in vivo by systemic administration of fentanyl (30 μg/kg, s.c.) 3 d before the preparation of DRG neuron cultures. In vitro recordings were made after 24 h in culture. Only small DRG neurons (soma diameter <30 μm; putative nociceptors) were considered for this analysis. A, Illustrative traces for time course of [Ca2+]i from each group, obtained by ratiometric calcium imaging with Fura-2 dye and reported as F340/F380 fluorescence ratio. The vertical gray arrow depicts the start of fentanyl (0.5 nm) (or vehicle for vehicle group) administration (bath application). Fentanyl remained in the perfusion chamber during recordings. Gray horizontal bar above the trace for the Primed CTOP group indicates presence of selective MOR antagonist CTOP (1 μm) in the recording chamber, starting 10 min before and continuing during fentanyl administration. B, Bars and dots show pooled magnitudes, mean and individual responses correspondingly, of maximum increase of the “F340/F380” ratio during the 5 min administration of fentanyl (0.5 nm) (or vehicle for “Primed, vehicle” group) compared with preadministration baseline. The distribution of the response magnitudes of cells from primed animals (n = 34) did not pass testing for normality and therefore was subdivided into two groups, responders (Primed resp, 9 of 34 neurons in the group, 26%) and nonresponders (Primed no resp, 25 of 34 neurons in the group, 74%), based on the magnitude of their response. A response magnitude of 0.032 was set as the threshold, based on the [mean+2 × SD] in the control group (shown as dash line). Both groups demonstrated normality after splitting into responders and nonresponders. Enhanced Ca2+ responses in cultured neurons from primed animals were dependent on activation of MOR as revealed by significant attenuation when CTOP (1 μm) was coadministered with fentanyl (Primed CTOP bar). One-way ANOVA with Dunnet's post hoc test revealed a significant difference between primed responders and all other groups (****p < 0.0001; F(4,88) = 33). Number of cells in experimental groups: control, n = 28; primed vehicle, n = 15; primed CTOP, n = 16; primed responders, n = 9; primed nonresponders, n = 25.
Figure 5.
Figure 5.
Fentanyl-induced sensitization of primed sensory neurons. Rats were primed by the systemic administration of fentanyl (30 μg/kg, s.c.) 3 d before preparing neuronal cultures; recordings were made after 24 h in culture. Bars show pooled magnitudes of decrease in rheobase, relative to preadministration baseline, 5 min after fentanyl (0.5 nm) was added to the perfusion chamber. Small DRG neurons from fentanyl-primed and control (opioid-naive) rats, depicted by the white and gray bars, correspondingly, were separated by IB4-binding intensity into strongly IB4+, weakly IB4+ and IB4− classes. In sensory neurons from opioid-naive animals, a significant fentanyl-induced decrease of rheobase was detected in strongly IB4+, but not in weakly IB4+ and IB4− neurons. In contrast, in sensory neurons from primed rats, fentanyl decreased rheobase in all three classes of nociceptors, with no significant difference in magnitude between classes. Two-way ANOVA confirmed a statistically significant effect of group (i.e., priming in vivo versus naive animals; F(1,56) = 17, p = 0.0001), but not IB4-binding class (F(2,56) = 1.8, p = 0.17) or their interaction (F(2,56) = 2.3, p = 0.11). Sidak's post hoc test demonstrated a statistically significant difference between primed and control groups for IB4− (t56 = 2.8, * adjusted p = 0.02) and weakly IB4+ (t56 = 3.6, ** adjusted p = 0.002), but not for strongly IB4+ neurons (t56 = 0.74, adjusted p = 0.85). Number of cells in experimental groups: strongly IB4+ in control, n = 12; the other 5 bars, n = 10. n.s., statistically not significant (p > 0.05).
Figure 6.
Figure 6.
Fentanyl-induced decrease in rheobase in primed sensory neurons. A, Bars and dots show pooled magnitudes, mean and individual responses, respectively, of decrease in rheobase during the 5 min administration of fentanyl (0.5 nm) relative to preadministration baseline in all sensory neurons (regardless of IB4-binding status) from primed animals. Neuronal responses were split into nonresponders and responders based on the magnitude of the fentanyl-induced decrease in rheobase with threshold selected at 20% (based on the visual gap in the distribution shown in B). Nonresponders (below the threshold; noted as NonResp and depicted by white bar with gray squares) constituted 40% of neurons (n = 12 of 30), had average sensitization of 8 ± 2%, ranging from −10 to +18%. Responders (above the threshold; noted as Resp and depicted by gray bar with white circles) constituted 60% of neurons (n = 18 of 30), had average sensitization of 38 ± 3%, ranging from 23 to 64%. Responders and nonresponders were found in all three IB4-binding classes of neurons cultured from primed animals.
Figure 7.
Figure 7.
Effect of fentanyl on [Ca2+]i and rheobase in sensory neurons from cordycepin-treated primed rats. Rats were treated with systemic fentanyl (30 μg/kg, s.c.) and, 2 d later, cordycepin (4 μg/20 μl) was injected intrathecally (in the same way as described in Fig. 3). As shown in behavioral experiments, this protocol eliminates the ability of intradermal fentanyl to induce hyperalgesia (OIH). The next day neuronal cultures were prepared; rats used for culture preparation did not receive intradermal fentanyl. The effect of fentanyl (0.5 nm) on calcium signaling (A) and electrical excitability (B) was examined in the same way as described in Figures 4 and 5, correspondingly, starting 1 d after preparation of neuronal culture. These results constitute the reversal groups in A and B. A, Bars and dots show pooled magnitudes, mean and individual responses correspondingly, in small DRG neurons from the reversal group (most right group) along with data for control and primed responders and nonresponders, which were repeated from Figure 4B for the purpose of comparison. The same threshold (0.032 a.u. as in Fig. 4B; shown as dash line) was used to split neurons from the reversal group into responders (n = 2) and nonresponders (n = 31). Fractions of responders were significantly different between groups (χ2 test for 3 groups: p = 0.0025, χ2 = 12, df = 2). Effect of OIH reversal protocol was significant, compared with the primed group (two-sided Fisher's exact test, p = 0.044), supporting the suggestion that reversal of OIH in vivo significantly diminishes changes in calcium signaling produced by in vitro fentanyl. B, Bars show pooled magnitudes of decrease in rheobase, relative to preadministration baseline, 5 min after fentanyl (0.5 nm) was added to the perfusion chamber. Only weakly IB4+ and IB4− neurons were considered for this analysis, as those were affected by fentanyl-induced priming (Fig. 5). Neurons from primed and control (opioid-naive) rats, depicted by the white and gray bars, correspondingly, were transferred from Figure 5 for the purpose of comparison. Sensitization effect of in vitro fentanyl was abolished only in weakly IB4+ neurons from the reversal group, whereas decrease in rheobase in IB4− neurons from the reversal group remained unattenuated, compared with the primed group (two-way ANOVA: F(2,46) = 9.9, p = 0.0003 for the effect of primed vs reversal condition; Holm-Sidak's post hoc test: comparison within weakly IB4+ neurons: t46 = 3.5, ** adjusted p = 0.003 for control vs primed; t46 = 0.70, adjusted p = 0.49 for control vs reversal; t46 = 2.3, * adjusted p = 0.047 for primed vs reversal; comparison within IB4− neurons: t46 = 2.8, # adjusted p = 0.025 for control vs primed; t46 = 2.6, # adjusted p = 0.025 for control vs reversal; t46 = 0.22, adjusted p = 0.83 for primed vs reversal). Thus, cordycepin-induced reversal of OIH in vivo eliminated in vitro electrophysiological changes attributed to neuroplasticity induced by systemic fentanyl, only in weakly IB4+. Number of cells in experimental groups in A: control, n = 28; primed responders, n = 9 and primed nonresponders n = 25, reversal n = 33; in B: control and primed groups, n = 10 per IB4-binding class; reversal groups, n = 6 per IB4-binding class. n.s., statistically not significant (p > 0.05).
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