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. 2013 Jul 3;33(27):11002-11.
doi: 10.1523/JNEUROSCI.1785-13.2013.

Role of nociceptor αCaMKII in transition from acute to chronic pain (hyperalgesic priming) in male and female rats

Luiz F Ferrari  1 , Oliver Bogen, Jon D Levine
Affiliations

Role of nociceptor αCaMKII in transition from acute to chronic pain (hyperalgesic priming) in male and female rats

Luiz F Ferrari et al. J Neurosci. .

Abstract

We have previously shown that activation of protein kinase Cε (PKCε) in male rats induces a chronic, long-lasting change in nociceptors such that a subsequent exposure to proinflammatory mediators produces markedly prolonged mechanical hyperalgesia. This neuroplastic change, hyperalgesic priming, is dependent on activation of cytoplasmic polyadenylation element-binding protein (CPEB), downstream of PKCε, and consequent translation of mRNAs in the peripheral terminal of the nociceptor. Since α calmodulin-dependent protein kinase II (αCaMKII), a molecule implicated in neuroplasticity, is a target of CPEB and can also affect CPEB function, we investigated its role in the transition from acute to chronic pain. Priming induced by direct activation of PKCε can be prevented by inhibition of αCaMKII. In addition, direct activation of αCaMKII induces priming, which was not prevented by pretreatment with PKCε antisense, suggesting that αCaMKII is downstream of PKCε in the induction of priming. Activation of ryanodine receptors (RyRs), which can lead to activation of αCaMKII, also induced priming, in a calcium- and αCaMKII-dependent manner. Similarly, inhibition of the RyR and a calcium buffer prevented induction of priming by PKCε. Unlike activation of PKCε, ryanodine and αCaMKII induced priming in female as well as male rats. Our results demonstrate a contribution of αCaMKII to induction of hyperalgesic priming, a phenomenon implicated in the transition from acute to chronic pain.

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Figures

Figure 1.
Figure 1.
PKCε-induced hyperalgesic priming is αCaMKII dependent. Rats were treated with daily intrathecal injections of ODN AS (filled bars) or mismatch (MM; open bars) for αCaMKII mRNA for 3 consecutive days. In addition, the CaMKII inhibitor (1 μg, filled bars) or vehicle (open bars) was injected on the dorsum of the hindpaw daily. On the third day of ODN AS plus inhibitor or MM plus vehicle treatment, ψεRACK (1 μg) was administered at the same site as the inhibitor or vehicle. Mechanical nociceptive thresholds were then evaluated by the Randall-Sellitto paw-withdrawal test 30 min and 4, 24, 48, and 96 h after ψεRACK administration. The ODN plus inhibitor or vehicle treatments were continued until the return of the mechanical threshold to baseline values (on the seventh day). Repeated-measures ANOVA followed by Bonferroni's post hoc test showed significant mechanical hyperalgesia induced by injection of ψεRACK in both groups (F(5,50) = 31.28; p < 0.0001 compared with baseline thresholds). However, when both groups were compared, ψεRACK-induced mechanical hyperalgesia was significantly attenuated in the AS plus inhibitor group (F(1,50) = 237.8; p < 0.0001). Seven days after the last treatment with ODN AS plus inhibitor or ODN MM plus vehicle (11 d after ψεRACK injection), we tested for hyperalgesic priming by intradermal injection of PGE2 (100 ng) in the same site as ψεRACK and the CaMKII inhibitor (or vehicle). Two-way repeated-measures ANOVA followed by Bonferroni's post hoc test showed that, although the mechanical hyperalgesia was not significantly different in both groups 30 min after PGE2 (p > 0.05), at the fourth hour a significant attenuation in the group previously treated with ODN AS plus inhibitor was observed, when compared with the MM plus vehicle-treated group (***p < 0.001). n = 6 paws per group.
Figure 2.
Figure 2.
αCaMKII activation induces mechanical hyperalgesia and hyperalgesic priming. A, αCaMKII was activated in vitro in the presence (black bars) or absence (white bars) of the CaMKII inhibitor CaM2INtide. Intradermal injections, on the dorsum of the hindpaws, were performed in different groups of rats (25 ng of αCaMKII). A control group (gray bars) received injection of the vehicles instead of αCaMKII. Mechanical nociceptive threshold was evaluated 30 min, 4 h, 24 h, 96 h, 7 d, and 10 d after injections. Repeated-measures ANOVA followed by Bonferroni's post hoc test showed significant mechanical hyperalgesia induced by αCaMKII activated in the absence of the inhibitor (F(12,78) = 11.04; ***p < 0.0001 when compared with the groups treated with αCaMKII plus inhibitor or vehicles) that was still significant 7 d after the injection (**p < 0.01). Comparison of the groups that received the αCaMKII plus its inhibitor or the vehicles showed no significant statistical difference (F(6.48) = 1.70; p = 0.1412). n = 6 paws per group. B, Rats received intradermal injection of activated αCaMKII (25 ng, black bars) or its vehicle (white bars). No significant difference was observed between the mechanical thresholds before and 10 d after injection of αCaMKII or vehicle, i.e., immediately before PGE2 injection (data not shown). PGE2 (100 ng) was then injected at the same site as αCaMKII or vehicle, and the mechanical nociceptive thresholds were evaluated 30 min and 4 h later. Repeated-measures ANOVA followed by Bonferroni's post hoc test showed that PGE2-induced hyperalgesia was still significant at the fourth hour in the paws pretreated with αCaMKII, whereas in the vehicle-treated paws, the mechanical threshold had already returned to baseline at that time point (***p < 0.001, when comparing both groups at the fourth hour). n = 10 paws in the αCaMKII group; n = 6 paws in the vehicle group.
Figure 3.
Figure 3.
Prolongation of PGE2-induced mechanical hyperalgesia in αCaMKII primed rats is PKCε dependent. Activated αCaMKII (25 ng) was injected intradermally in the rat hindpaw. Two weeks later, when the mechanical thresholds had returned to baseline (paired Student's t test showed no difference in the mechanical thresholds between both groups; t(10) = 1.340, p = 0.2099; data not shown), the PKCε-specific translocation inhibitor peptide PKCεV1–2 (PKCε-I, 1 μg, filled bars), or its vehicle (control, open bars), was administered in the same site. Five minutes later, PGE2 (100 ng) was injected, and the mechanical nociceptive thresholds were evaluated 30 min and 4 h later. Significant attenuation of the prolonged PGE2-induced hyperalgesia, measured at 4 h, was observed in the paws treated with PKCε-I (***p < 0.001), when compared with the vehicle-treated group, without affecting hyperalgesia at the 30 min time point (two-way repeated-measures ANOVA followed by Bonferroni's post hoc test; F(1,10) = 27.36, p = 0.0004, both groups compared at 30 min). n = 6 paws per group.
Figure 4.
Figure 4.
Prolongation of PGE2-induced mechanical hyperalgesia in αCaMKII primed rats is dependent on local protein translation. Activated αCaMKII (25 ng) was injected intradermally on the dorsum of the hindpaw of different groups of rats. Mechanical nociceptive threshold evaluation showed significant hyperalgesia 30 min and 4 h after injection and mechanical thresholds similar to baseline values on the day of the test with PGE2 (2 weeks after αCaMKII injection; data not shown). A, B, Injection of PGE2 (100 ng) at the same site as αCaMKII was preceded (30 min before) by injection of cordycepin (A, 1 μg, filled bars) or rapamycin (B, 1 μg, filled bars). Control groups received vehicle in the PGE2 injection site (open bars). Repeated-measures ANOVA followed by Bonferroni's post hoc test showed significant hyperalgesia, evaluated 30 min and 4 h after injection in the groups treated with vehicle; however, in the groups treated with cordycepin or rapamycin, the magnitude of the PGE2-induced hyperalgesia was significantly decreased at the fourth hour (A, F(1,10) = 44.42, ***p < 0.0001; B, F(1,10) = 73,25, ***p < 0.0001; when the protein translation inhibitor groups are compared with the vehicle groups), indicating an effect of protein translation inhibitors on the prolongation of the PGE2-induced hyperalgesia in paws primed with activated αCaMKII. n = 6 paws per group.
Figure 5.
Figure 5.
Induction of hyperalgesic priming by αCaMKII is not PKCε dependent. Rats were treated with ODN AS (filled bars) or MM (open bars) for PKCε mRNA for 10 consecutive days. Activated αCaMKII (25 ng) was injected on the dorsum of the hind paw on the third day of ODN AS or MM treatment. Three days after the last ODN treatment (on the 13th day), PGE2 (100 ng) was injected at the same site as activated αCaMKII (paired Student's t test showed no significant difference between the mechanical thresholds in both groups before αCaMKII administration and immediately before PGE2 injection: t(5) = 0.8660, p = 0.4261 for the AS group and t(5) = 0.7559, p = 0.4838 for the MM group, respectively). Two-way repeated-measures ANOVA followed by Bonferroni's post hoc test showed that in both the AS and the MM groups the PGE2-induced mechanical hyperalgesia was still present at the fourth hour, indicating that PKCε is not necessary for the induction of priming by αCaMKII (F(1,10) = 2.10; p = 0.1784, when comparing both groups; NS). n = 6 paws per group.
Figure 6.
Figure 6.
PKCε-induced hyperalgesic priming is dependent on the RyR. Rats received intradermal injection of vehicle (white bars), dantrolene (1 μg, gray bars), or TMB-8 (1 μg, black bars) on the dorsum of the hindpaw. Ten minutes later, the PKCε activator ψεRACK (1 μg) was injected at the same site and the mechanical nociceptive thresholds were evaluated (data not shown). Daily injections of vehicle, dantrolene, or TMB-8 continued for 4 d, until the mechanical thresholds had returned to baseline. Two days after the last injection (6 d after ψεRACK), PGE2 (100 ng) was administered at the same site, and the mechanical thresholds were evaluated 30 min and 4 h later. Two-way repeated-measures ANOVA followed by Bonferroni's post hoc test showed significant mechanical hyperalgesia induced by PGE2 in all groups (F(2,30) = 24.01; p < 0.0001 when compared with baseline mechanical thresholds). However, in the groups that received dantrolene or TMB-8, the PGE2-induced hyperalgesia was significantly attenuated at the 4 h time point (***p < 0.001 for both groups when comparing 30 min and 4 h time points), in contrast with the vehicle-treated group (NS; p > 0.05). n = 6 paws per group.
Figure 7.
Figure 7.
Ryanodine induces hyperalgesic priming. A, B, Rats received intradermal injection of ryanodine (A, 1 μg, filled bars) or vehicle (B, open bars) on the dorsum of the hindpaw, and the mechanical nociceptive thresholds were evaluated 30 min later. Repeated-measures ANOVA showed significant mechanical hyperalgesia induced by ryanodine, but not by vehicle (F(1,10) = 46.08; p < 0.0001 when the ryanodine group is compared with the vehicle group). Testing for priming with intradermal injection of PGE2 (100 ng) was performed 7 d later, when the mechanical nociceptive threshold had returned to pre-ryanodine baseline (paired Student's t test showed no significant difference between the mechanical thresholds before and 7 d after ryanodine or vehicle administration; t(5) = 0.1784, p = 0.1048 for the ryanodine group; t(5) = 0.4385, p = 0.6793 for the vehicle group). Repeated-measures ANOVA followed by Bonferroni's post hoc test showed significant hyperalgesia induced by PGE2 injected at the same site as ryanodine or vehicle at 30 min. And, in contrast to the vehicle group, in which the hyperalgesia was no longer present 4 h after PGE2 injection, in the paws that received ryanodine 1 week before, the PGE2-induced hyperalgesia was unattenuated (***p < 0.001, when compared with the vehicle group). n = 6 paws per group.
Figure 8.
Figure 8.
Ryanodine-induced priming is calcium dependent. A, B, Rats received intradermal injection of the calcium buffer TMB-8 (A, 1 μg, filled bars) or its vehicle (B, open bars) on the dorsum of the hindpaw. Ten minutes later, ryanodine (1 μg) was injected at the same site and the mechanical nociceptive thresholds were evaluated after 30 min. Two-way repeated-measures ANOVA followed by Bonferroni's post hoc test showed significant mechanical hyperalgesia induced by ryanodine in the control group, as opposed to the group treated with TMB-8, in which the hyperalgesia was significantly attenuated (F(1,20) = 15.67; ***p < 0.001 when both groups are compared 30 min after ryanodine injection). Seven days later, when the mechanical thresholds were back to baseline (paired Student's t test showed no significant difference between the mechanical thresholds before and 7 d after ryanodine plus TMB-8 or ryanodine plus vehicle administration; t(5) = 0.6547, p = 0.5416 and t(5) = 0.7565, p = 0.3701, respectively), testing for priming was performed by intradermal injection of PGE2 (100 ng) at the same site as ryanodine. Repeated-measures ANOVA followed by Bonferroni's post hoc test showed that the group treated with TMB-8, but not with vehicle, before ryanodine injection had a significant decrease in the PGE2-induced hyperalgesia at the fourth hour (F(1,10) = 92.90; ***p < 0.001 when the TMB-8-treated group is compared with the vehicle-treated group at the fourth hour). n = 6 paws per group.
Figure 9.
Figure 9.
αCaMKII is downstream of the RyR in the induction of hyperalgesic priming. Rats were treated with ODN AS (A, filled bars) or MM (B, open bars) for αCaMKII for 6 consecutive days. On the third day of ODN AS or MM treatment, ryanodine (1 μg) was coinjected with the CaMKII inhibitor CaM2INtide (A, 1 μg, filled bars), or vehicle (B, open bars), on the dorsum of the hindpaws. Mechanical thresholds were evaluated 30 min later. Ryanodine induced mechanical hyperalgesia in both groups, without significant statistical difference between the groups (two-way repeated-measures ANOVA followed by Bonferroni's post hoc test; F(1,20) = 2.04; p = 0.1841). The daily treatment with ODN AS plus CaMKII inhibitor or MM plus vehicle continued for 3 more days. PGE2 (100 ng) was injected, 4 d after the ODN AS or MM treatment was discontinued, at the same site as the injection of ryanodine plus CaMKII inhibitor or vehicle. Repeated-measures ANOVA showed that the treatment with αCaMKII AS plus CaMKII inhibitor prevented the induction of priming by ryanodine, since the PGE2-induced hyperalgesia, although similar in both groups at the 30 min time point, was significantly attenuated in the AS plus inhibitor group but not on the MM plus vehicle group (***p < 0.001, when both groups are compared at the fourth hour). n = 6 paws per group.
Figure 10.
Figure 10.
αCaMKII and ryanodine induce priming in female rats. A, Female rats received intradermal injection of activated αCaMKII (25 ng, gray bars) or its vehicle (white bars). Mechanical nociceptive threshold was evaluated before and 30 min, 4 h, 24 h, and 10 d after their injection. Two-way repeated-measures ANOVA followed by Bonferroni's post hoc test showed significant hyperalgesia in the αCaMKII-treated group but not in the vehicle-treated group (F(1,40) = 162.26; ***p < 0.001), with no significant (NS) difference between both groups on the 10th day. Test for priming was then performed with intradermal injection of PGE2 (100 ng) on day 10. Repeated-measures ANOVA showed that PGE2-induced hyperalgesia was still significant at the fourth hour in the αCaMKII-treated paws, whereas in the vehicle-treated group, the mechanical threshold had already returned to baseline values at that time point (F(1,10) = 26.61; ***p < 0.001 when both groups are compared at the fourth hour). n = 6 paws per group. B, Ryanodine (1 μg, gray bars) or vehicle (white bars) was injected intradermally on the dorsum of the hindpaw of female rats, and the mechanical thresholds were evaluated 30 min later. Significant hyperalgesia was observed in the ryanodine-treated group but not in the vehicle-treated paws 30 min after injection (***p < 0.001, when ryanodine group is compared with the vehicle group). Seven days after ryanodine or vehicle injection, PGE2 (100 ng) was injected at the same site (paired Student's t test showed no significant difference between the mechanical thresholds before and 7 d after ryanodine or vehicle administration: t(5) = 1.225, p = 0.2752 and t(5) = 1.035, p = 0.3481, respectively). Two-way repeated-measures ANOVA followed by Bonferroni's post hoc test showed that the PGE2-induced mechanical hyperalgesia was still present 4 h after injection in the rats that previously received ryanodine, whereas in the group that received vehicle, the nociceptive thresholds had returned to baseline at that time point (F(1,10) = 55.24; ***p < 0.001). n = 6 paws per group.
Figure 11.
Figure 11.
Schematic diagram of the proposed mechanisms underlying the induction of hyperalgesic priming in male and female rats. In male but not female rats, activation of TNFα and IL-6 receptors or direct activation of PKCε in the peripheral terminal of the primary afferent nociceptor triggers a cascade of events that culminate in hyperalgesic priming. CPEB, a downstream target of PKCε, activates αCaMKII in the induction of priming. In the PKCε-induced priming, αCaMKII is downstream of CPEB. However, since αCaMKII is also able to affect CPEB activity (dashed arrow) (Atkins et al., 2004), the maintenance of the primed state is hypothesized to be a loop in which αCaMKII is both upstream and downstream of CPEB. Importantly, activation of αCaMKII induces priming in male and female rats (events below the dotted line are involved in priming in both sexes). Direct activation of RyR also produces priming in male and female rats by releasing calcium (Ehrlich et al., 1994; MacKrill, 1999, 2012) and activating αCaMKII (Shakiryanova et al., 2007, 2011; Wong et al., 2009). In italics are shown the inhibitors of the mediators involved in the induction of priming, with the respective steps on the pathway where they act: PKCε AS, CPEB AS, αCaMKII AS and the CaMKII inhibitor CaMINtide; the RyR inhibitor dantrolene; the calcium chelator TMB-8; and the protein translation inhibitors cordycepin and rapamycin. In addition, estrogen is the critical mediator that prevents the development of hyperalgesic priming in female rats, acting at the level of PKCε (Joseph et al., 2003).
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