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. 2015 Apr 15;35(15):6107-16.
doi: 10.1523/JNEUROSCI.5085-14.2015.

Distinct terminal and cell body mechanisms in the nociceptor mediate hyperalgesic priming

Luiz F Ferrari  1 , Dioneia Araldi  1 , Jon D Levine  2
Affiliations

Distinct terminal and cell body mechanisms in the nociceptor mediate hyperalgesic priming

Luiz F Ferrari et al. J Neurosci. .

Abstract

Hyperalgesic priming, a form of neuroplasticity in nociceptors, is a model of the transition from acute to chronic pain in the rat, which involves signaling from the site of an acute tissue insult in the vicinity of the peripheral terminal of a nociceptor to its cell body that, in turn, induces a signal that travels back to the terminal to mediate a marked prolongation of prostaglandin E2-induced hyperalgesia. In the present experiments, we studied the underlying mechanisms in the cell body and compared them to the mechanisms in the nerve terminal. Injection of a cell-permeant cAMP analog, 8-bromo cAMP, into the dorsal root ganglion induced mechanical hyperalgesia and priming with an onset more rapid than when induced at the peripheral terminal. Priming induced by intraganglion 8-bromo cAMP was prevented by an oligodeoxynucleotide antisense to mRNA for a transcription factor, cAMP response element-binding protein (CREB), and by an inhibitor of importin, which is required for activated CREB to get into the nucleus. While peripheral administration of 8-bromo cAMP also produced hyperalgesia, it did not produce priming. Conversely, interventions administered in the vicinity of the peripheral terminal of the nociceptor that induces priming-PKCε activator, NGF, and TNF-α-when injected into the ganglion produce hyperalgesia but not priming. The protein translation inhibitor cordycepin, injected at the peripheral terminal but not into the ganglion, reverses priming induced at either the ganglion or peripheral terminal of the nociceptor. These data implicate different mechanisms in the soma and terminal in the transition to chronic pain.

Keywords: dorsal root ganglion; hyperalgesic priming; mechanical hyperalgesia; nociceptor; rat.

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Figures

Figure 1.
Figure 1.
Induction of hyperalgesic priming by intrathecal (i.t.) injection of MCP-1 is dependent on gene transcription. Rats received three daily intraganglion (i.gl.) injections of vehicle (white bars) or the protein transcription inhibitor actinomycin D (10 μg, black bars). Thirty minutes after the first injection of actinomycin D, the central terminal of these nociceptors was stimulated with MCP-1 (20 ng/μl, 20 μl), injected intrathecally. No effect of actinomycin D on MCP-1-induced hyperalgesia was observed (data not shown). The injections of vehicle or actinomycin D continued, once a day, for 3 d. On the fourth day, PGE2 (100 ng) was injected intradermally into the dorsum of the hindpaw at the site of nociceptive testing to evaluate for the presence of priming. The average paw-withdrawal thresholds before injections of vehicle/MCP-1 and immediately before the injection of PGE2 (4 d later) were 110.0 ± 3.6 and 110.0 ± 2.7 g, respectively; for the groups that received actinomycin D/MCP-1, 113.3 ± 3.3 and 113.5 ± 2.2 g, respectively (t(5) = 0.0000; p = 1.0000, NS in both cases; paired Student's t test). Mechanical hyperalgesia was then evaluated by the Randall–Sellitto paw-withdrawal test 30 min and 4 h after PGE2 injection. We observed that, in the paws ipsilateral to the DRGs that received vehicle, the magnitude of hyperalgesia was still significant at the fourth hour after PGE2 injection, indicating the presence of hyperalgesic priming, while in the paws ipsilateral to the DRGs that received actinomycin D, the PGE2-induced hyperalgesia was significantly attenuated at this time point, which is compatible with the prevention of priming (***p < 0.001, when both groups are compared at the fourth hour; two-way repeated-measures ANOVA followed by Bonferroni post-test, N = 6 paws per group). The results of this experiment support the suggestion that the inhibition of transcription in the cell body prevented the induction of priming by intrathecal MCP-1.
Figure 2.
Figure 2.
Intraganglion, but not intradermal, injection of 8-bromo cAMP induces hyperalgesic priming. Rats received injection of 8-bromo cAMP in the DRG (10 μg, black bars) or intradermally in the dorsum of the hindpaw (10 μg, white bars). Robust mechanical hyperalgesia, evaluated 30 min later, by the Randall–Sellitto paw-withdrawal test, was observed in both groups (p > 0.05 when compared). One week later, a time point when the mechanical thresholds were not significantly different from pre-8-bromo cAMP baseline thresholds (data not shown), PGE2 (100 ng) was injected intradermally into the dorsum of the hindpaw. The average paw-withdrawal thresholds before the injection of 8-bromo cAMP and immediately before the injection of PGE2 (1 week later) were as follows: 120.6 ± 1.6 and 116.3 ± 2.0 g, respectively, for the DRG group (t(5) = 1.904; p = 0.1152, NS); 122.3 ± 3.0 and 116.3 ± 2.3 g, respectively, for the paw group (t(5) = 1.861; p = 0.1219, NS); no significant difference (NS) between these two pairs of values was observed (paired Student's t test). Although the mechanical hyperalgesia induced by PGE2 at the 30 min time point was not different between the groups, 4 h after injection, the PGE2 hyperalgesia was still significant in the group previously treated with intraganglion 8-bromo cAMP, but not in the group that received an injection of 8-bromo cAMP in the hindpaw (***p < 0.001, when both groups are compared at the fourth hour; two-way repeated-measures ANOVA followed by Bonferroni post-test, N = 6 paws per group), indicating that 8-bromo cAMP induces priming by acting at the level of the cell body of the nociceptor in the DRG.
Figure 3.
Figure 3.
Time course for the development of hyperalgesic priming induced by intraganglion injection of 8-bromo cAMP. Groups of rats received intraganglion (i.gl.) injection of 8-bromo cAMP (10 μg, black bars) or vehicle. A–C, PGE2 (100 ng) was then injected intradermally into the dorsum of the hindpaw 6 (A), 12 (B), or 24 (C) hours later. Mechanical nociceptive paw-withdrawal thresholds were evaluated before, and 30 min and 4 h after the intradermal injection of PGE2 by the Randall–Selitto paw-withdrawal test. Average paw-withdrawal thresholds measured before, and 6, 12, and 24 h after i.gl. injection of 8-bromo cAMP were as follows: 112.6 ± 0.8 and 116.6 ± 1.7 g, respectively (6 h group); 129.0 ± 3.0 and 128.3 ± 4.0 g, respectively (12 h group); and 126.6 ± 3.9 and 122.6 ± 3.7 g, respectively (24 h group); no significant difference (NS) among these values was observed: after 6 h: t(5) = 2.582; p = 0.4930; NS; after 12 h: t(5) = 0.1147; p = 0.9131, NS; after 24 h: t(5) = 1.054; p = 0.3401, NS; paired Student's t test. For the groups that received vehicle, the average paw-withdrawal thresholds measured before, and 6, 12, and 24 h after i.gl. injection were as follows: 115.6 ± 3.2 and 117.0 ± 2.1 g, respectively (6 h group); 128.3 ± 1.8 and 126.6 ± 3.3 g, respectively (12 h group); and 128.0 ± 4.1 and 118.0 ± 1.2 g, respectively (24 h group). No significant differences among these values were observed: after 6 h: t(5) = 0.7559; p = 0.4838; NS; after 12 h: t(5) = 0.5207; p = 0.6248, NS; after 24 h: t(5) = 2.348; p = 0.0657, NS; paired Student's t test. A, In the group treated with i.gl. injection of 8-bromo cAMP, followed by PGE2 injection into the hindpaw 6 h later, the PGE2-induced hyperalgesia was no longer present when evaluated 4 h after injection (no significant difference was observed when compared with the i.gl.-vehicle group; p > 0.05, two-way repeated-measures ANOVA followed by Bonferroni post-test, N = 6 paws per group), which is compatible with the absence of priming. B, C, However, when PGE2 was injected 12 h (B) or 24 h (C) after i.gl. injection of 8-bromo cAMP, the hyperalgesia observed was still present at the 4 h time point, indicating that hyperalgesic priming was established (***p < 0.001 when compared the vehicle-treated group at the fourth hour; two-way repeated-measures ANOVA followed by Bonferroni post-test, N = 6 paws per group).
Figure 4.
Figure 4.
CREB antisense prevents (A), but does not reverse (B), hyperalgesic priming induced by intraganglion (i.gl.) injection of 8-bromo cAMP. A, Rats were treated with daily spinal intrathecal injections of ODN AS (black bars) for CREB mRNA, for 3 consecutive days, which decreases its levels in the sensory neuron, and prevents its activation by the priming inducer 8-bromo cAMP (10 μg, injected into the DRG, i.gl.), injected on the fourth day. Control animals were treated, following the same protocol, with ODN MM (white bars). To prevent the possibility that prolonged activation of a signaling pathway might produce priming following the administration of 8-bromo cAMP, the ODN treatment continued through day 5, when the mechanical nociceptive paw-withdrawal thresholds were not significantly (NS) different from pre 8-bromo cAMP baseline values (t(5) = 2.445; p = 0.0583, NS, for the MM group; t(5) = 0.1117; p = 0.9154, NS, for the AS group; paired Student's t test). The presence of hyperalgesic priming was assessed by intradermal injection of PGE2 (100 ng) into the dorsum of the hindpaw. Mechanical hyperalgesia was evaluated 30 min and 4 h later, by the Randall–Sellitto paw-withdrawal test. Average paw-withdrawal thresholds before the injection of 8-bromo cAMP and before the injection of PGE2 (1 d later) were as follows: 119.0 ± 2.7 and 114.3 ± 2.0 g, respectively, for the CREB MM-treated group; and 118.0 ± 2.0 and 118.3 ± 2.0 g, respectively, for the AS-treated group. Two-way repeated-measures ANOVA followed by Bonferroni post-test showed significant mechanical hyperalgesia induced by PGE2, measured 30 min after injection, in both groups. However, while in the MM-treated group the magnitude of PGE2 hyperalgesia was still significant at the fourth hour, in the AS-treated group it was strongly attenuated (***p < 0.001 when the hyperalgesia in those groups was compared at that time point). When tested again for priming with PGE2 1 week after the last treatment with ODN AS or MM, the prolongation of PGE2-induced hyperalgesia was still attenuated (at the 4 h time point) in the ODN AS-treated group, but not in the ODN MM-treated group, indicating a role of CREB in the induction of hyperalgesic priming by i.gl. injection of 8-bromo cAMP (***p < 0.001 when the MS- and the AS-treated groups are compared; N = 6 paws per group). Of note, no difference in the mechanical thresholds was observed at this time point, when compared with prepriming stimuli baseline thresholds: 119.0 ± 2.7 and 116.3 ± 3.1 g, respectively, for the CREB MM-treated group (t(5) = 2.169; p = 0.0822, NS), and 118.0 ± 2.0 and 115.3 ± 2.2 g, respectively, for the AS-treated group (t(5) = 1.019; p = 0.3548, NS; paired Student's t test). B, Rats that were treated with i.gl. injection of 8-bromo cAMP (10 μg) 5 d before were treated with ODN AS (black bars) for CREB mRNA for 3 consecutive days, to decrease the levels of CREB in the nociceptor. Control animals were treated, following the same protocol, with MM (white bars). On the fourth day, PGE2 (100 ng) was injected intradermally into the dorsum of the hindpaw, and the mechanical threshold was evaluated 30 min and 4 h later. The average paw-withdrawal thresholds before the injection of 8-bromo cAMP and before the injection of PGE2 were 118.3 ± 3.0 and 120.3 ± 3.6 g, respectively, for the CREB MM-treated group (t(5) = 0. 0000; p = 1.0000, NS), and 123.3 ± 3.6 and 122.6 ± 2.1 g, respectively, for the AS-treated group (t(5) = 0.6742; p = 0.5301, NS). Paired Student's t test showed no significant (NS) difference between these two values. Two-way repeated-measures ANOVA followed by Bonferroni post-test showed no difference in the magnitude of the PGE2-induced hyperalgesia at 30 min and 4 h between the ODN AS- and MM-treated groups (p > 0.05, NS), indicating that CREB does not play a role in the maintenance of hyperalgesic priming induced by 8-bromo cAMP (N = 6 paws per group).
Figure 5.
Figure 5.
Hyperalgesic agents that also induce priming when injected into the paw produce hyperalgesia, but not priming, when injected in the DRG. A–C, Groups of rats received intraganglion (i.gl.) injection of the hyperalgesic mediators ψεRACK (10 μg; A), NGF (1 μg; B), or TNF-α (10 μg; C). The mechanical threshold was evaluated 60 min and 24 h later. A–C, The paws ipsilateral to the DRGs that received injection of these mediators showed significant mechanical hyperalgesia when compared with the control paws (ipsilateral to the DRGs treated with vehicles; A, **p < 0.01; B, ***p < 0.001, **p < 0.01; C, p < 0.001, when the change in mechanical threshold induced by the i.gl. priming inducers or control is compared over 24 h; two-way repeated-measures ANOVA followed by Bonferroni post-test). One week later, when the mechanical thresholds were not significantly (NS) different from the baseline nociceptive thresholds evaluated before the injection of the mediators (A: ψεRACK group, t(5) = 0.8402, p = 0.4391; control group, t(5) = 1.164, p = 0.2968; B: NGF group, t(5) = 0.9543, p = 0.3837; control group, t(5) = 0.4740, p = 0.6554; C: TNF-α group, t(5) = 0.7559, p = 0.4838; control group, t(5) = 0.9764, p = 0.3737; paired Student's t test), PGE2 (100 ng) was injected intradermally into the dorsum of the hindpaw, and mechanical hyperalgesia was evaluated 30 min and 4 h later. Average paw-withdrawal thresholds before the injection of the mediators and before the injection of PGE2 (1 week later) were as follows: 123.0 ± 2.3 and 119.0 ± 2.7 g, respectively, for the ψεRACK-treated group, and 117.6 ± 1.8 and 115.0 ± 1.0 g, for the control-treated group; 118.6 ± 1.8 and 116.3 ± 1.7 g, respectively, for the NGF-treated group, and 117.6 ± 2.9 and 119.0 ± 1.9 g, for the control-treated group; and 118.3 ± 1.8 and 121.0 ± 2.5 g, respectively, for the TNF-α-treated group; and 117.3 ± 2.1 and 119.6 ± 2.0 g, for the control-treated group. Evaluation of the mechanical hyperalgesia induced by PGE2 showed significant hyperalgesia at 30 min after injection, which was no longer present at the fourth hour time point, in all groups. Two-way repeated-measures ANOVA followed by Bonferroni post-test showed no difference between the controls and the groups treated with the mediators (NS), indicating that i.gl. injection of ψεRACK, NGF, or TNF-α did not produce hyperalgesic priming (N = 6 paws per group).
Figure 6.
Figure 6.
Transport into the nucleus plays a role in the induction of hyperalgesic priming. Rats received intraganglion (i.gl.) injection of vehicle (white bars) or ivermectin (10 μg, black bars), an inhibitor of the nuclear transporter importin. Thirty minutes later, MCP-1 [20 ng/μl, 20 μl, injected intrathecally (i.t.) into the spinal cord, A] or 8-bromo cAMP [10 μg, injected into the DRG, B] was administered. No effect of ivermectin on MCP-1- or 8-bromo cAMP-induced hyperalgesia was observed (data not shown). The injection of vehicle or ivermectin continued daily for 3 d. On the fourth day, PGE2 (100 ng) was injected intradermally into the dorsum of the hindpaw. A, Average paw-withdrawal thresholds before injection of vehicle/MCP-1 and immediately before the injection of PGE2 (4 d later) were 121.3 ± 2.1 and 118.6 ± 1.6 g, respectively (t(5) = 0.9325; p = 0.3939, NS); for the groups that received ivermectin/MCP-1, they were 123.0 ± 2.5 and 123.5 ± 1.6 g, respectively (t(5) = 0.0000; p = 1.0000, NS); no significant (NS) difference between these two values was observed (paired Student's t test). B, Average paw-withdrawal thresholds before injection of vehicle/8-bromo cAMP and immediately before the injection of PGE2 (4 d later) were 117.3 ± 2.1 and 114.6 ± 1.8 g, respectively (t(5) = 0.9035; p = 0.4077, NS); for the groups that received ivermectin/8-bromo cAMP, they were 122.3 ± 1.6 and 122. 5 ± 2.6 g, respectively (t(5) = 0.0000; p = 1.0000, NS); no significant (NS) difference between these two values was observed (paired Student's t test). Mechanical hyperalgesia was then evaluated 30 min and 4 h after PGE2 injection using the Randall–Sellitto paw-withdrawal test. In both A and B, in the paws ipsilateral to the DRGs that received vehicle, the magnitude of hyperalgesia was still significant at the fourth hour after PGE2 injection. However, in the paws ipsilateral to the DRGs that received ivermectin, the prolongation of PGE2-induced hyperalgesia was significantly attenuated (***p < 0.001, A and B, when vehicle and ivermectin groups are compared at the fourth hour; two-way repeated-measures ANOVA followed by Bonferroni post-test), indicating that the induction of priming by i.t. MCP-1 or i.gl. 8-bromo cAMP requires transport into the nucleus. When tested again with PGE2 1 week later; attenuation of the hyperalgesia induced by PGE2 at the fourth hour was still observed (***p < 0.001, A and B, when vehicle and ivermectin groups are compared at the fourth hour), indicating that ivermectin prevented the development of hyperalgesic priming (N = 6 paws per group).
Figure 7.
Figure 7.
Expression of hyperalgesic priming induced by intraganglion injection of 8-bromo cAMP is dependent on PKCε and αCaMKII. Rats primed with intraganglion (i.gl.) injection of 8-bromo cAMP (10 μg) 1 week prior received intradermal injection of PGE2 (100 ng) into the dorsum of the hindpaw in the presence or absence of inhibitors of the second messengers, PKCε (PKCε-I; 1 μg/5 μl, gray bars) and αCaMKII (CaMINtide; 1 μg/5 μl, black bars). Of note, in order to achieve a stronger inhibition of αCaMKII, rats were treated, for 3 consecutive days, prior to injection of the inhibitor plus PGE2, with ODN AS for αCaMKII. Mechanical nociceptive thresholds were evaluated 30 min and 4 h after PGE2, injected 5 min after the inhibitors, by the Randall–Selitto paw-withdrawal test. We observed, in both cases, significant attenuation of the hyperalgesia induced by PGE2 at the fourth hour (***p < 0.001) in the groups treated with the PKCε inhibitor or αCaMKII ODN AS plus CaM2INtide when compared with the vehicle groups (PKCε-I: F(1,10) = 29.55, p = 0.0003; αCaMKII ODN AS plus CaM2INtide: F(1,10) = 54.61, p < 0.0001, two-way repeated-measures ANOVA followed by Bonferroni post-test, N = 6 paws per group), indicating a role of PKCε and αCaMKII in the prolongation of PGE2 hyperalgesia in the i.gl. 8-bromo cAMP-induced hyperalgesic priming (A). B shows that, when PGE2 was injected again at the same site 1 week later, it produced prolonged hyperalgesia, evaluated at the fourth hour, indicating that the reversal of hyperalgesic priming induced by i.gl. 8-bromo cAMP by the inhibition of PKCε or αCaMKII is not permanent (PKCε-I: t(5) = 3.072, p = 0.2770; αCaMKII ODN AS + CaM2INtide: t(5) = 0.6532, p = 0.5424, paired Student's t test, no difference when the 30 min and 4 h time points are compared in both cases; N = 6 paws per group).
Figure 8.
Figure 8.
Protein translation inhibitor reverses priming when it is administered at the terminal of the nociceptor, but not at its cell body. The effect of the protein translation inhibitor cordycepin on priming was evaluated in rats that had received intraganglion (i.gl.) injection of 8-bromo cAMP (10 μg) 2 weeks before. The average paw-withdrawal thresholds before and 2 weeks after 8-bromo cAMP (when the experiments were performed) were 121.6 ± 1.4 and 120.0 ± 1.4 g, respectively; no significant (NS) difference between these two values was observed (t(17) = 1.062, p = 0.3032, NS; paired Student's t test, data not shown). Rats were then divided into a control group (white bars) and two experimental groups, one that received cordycepin intradermally on the dorsum of the hindpaw (1 μg, gray bars) and the other that received i.gl. injection to the DRG (10 μg, black bars). Fifteen minutes later, PGE2 (100 ng) was injected into the dorsum of the hindpaw, and the paw-withdrawal threshold was evaluated 30 min and 4 h later. We observed that, while PGE2-induced hyperalgesia was still significant at the fourth hour in the control group and the experimental group that received cordycepin in the DRG (p > 0.05, when both groups are compared), in the group that received cordycepin in the paw it was significantly attenuated at the fourth hour (***p < 0.001, when compared with the control group), indicating a role of protein translation in the terminal of the nociceptor, but not in the DRG, in hyperalgesic priming (two-way repeated-measures ANOVA followed by Bonferroni post-test; N = 6 paws per group).
Figure 9.
Figure 9.
Hyperalgesic priming mechanisms in the nociceptor terminals and cell body. A schematic of the nociceptor with the central terminal in the spinal cord, and the peripheral terminal in the rat hindpaw, is shown. The blue arrows represent the message triggered by stimulation in either of the terminals and directed toward the cell body, and the red arrows represent the message coming from the cell body to the terminals, where the maintenance of the primed state will take place. On the top is described the induction phase of hyperalgesic priming, separated by the events occurring in the nociceptor terminals (1) and in the cell body (2): inflammatory stimuli such as TNF-α, IL-6, or MCP-1, or the administration of neurotrophins (NGF or GDNF) in the terminals of the nociceptor (1) induce the events that lead to the development of hyperalgesic priming. Subsequent activation of PKCε stimulates CPEB, αCaMKII, or the release of calcium (Ca2+; Aley et al., 2000; Reichling and Levine, 2009; Bogen et al., 2012; Ferrari et al., 2013b), which will produce the message directed toward the cell body (Ferrari et al., 2015). a–c, There (2), activation of cAMP (a) activates the transcription factor CREB (b; pCREB), which will be transported into the nucleus by importin, where gene transcription will take place (c). d, The resulting protein or mRNA will then be transported to the terminals of the nociceptor. On the bottom, the maintenance phase of hyperalgesic priming is shown: at the terminals, ongoing protein translation, triggered by the message originated in the cell body (Bogen et al., 2012; Ferrari et al., 2013c, 2015), is responsible for the maintenance of the neuroplasticity observed in the primed state.
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