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. 2013 Jul;14(7):731-8.
doi: 10.1016/j.jpain.2013.01.779. Epub 2013 May 8.

Peripheral administration of translation inhibitors reverses increased hyperalgesia in a model of chronic pain in the rat

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

Peripheral administration of translation inhibitors reverses increased hyperalgesia in a model of chronic pain in the rat

Luiz F Ferrari et al. J Pain. 2013 Jul.

Abstract

Chronic pain is extremely difficult to manage, in part due to lack of progress in reversing the underlying pathophysiology. Since translation of messenger ribonucleic acids (mRNAs) in the peripheral terminal of the nociceptor plays a role in the transition from acute to chronic pain, we tested the hypothesis that transient inhibition of translation in the peripheral terminal of the nociceptor could reverse hyperalgesic priming, a model of transition from acute to chronic pain. We report that injection of translation inhibitors rapamycin and cordycepin, which inhibit translation by different mechanisms, at the peripheral terminal of the primed nociceptor produces reversal of priming in the rat that outlasted the duration of action of these drugs to prevent the development of priming. These data support the suggestion that interruption of translation in the nociceptor can reverse a preclinical model of at least 1 form of chronic pain.

Perspective: This study provides evidence that ongoing protein translation in the sensory neuron terminals is involved in pain chronification, and local treatment that transiently interrupts this translation may be a useful therapy to chronic pain.

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Conflict of interest statement

Disclosures

This study was funded by the National Institutes of Health (NIH). The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Prevention of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin
Rats received intradermal injection of rapamycin (1 μg) on the dorsum of the hind paw. Evaluation of the mechanical nociceptive threshold, by the Randall-Selitto paw-withdrawal test, before and 30 min after injection, showed no effect of rapamycin by itself [average paw withdrawal thresholds before and 30 min after rapamycin injection were 113.3 ± 1.7 g and 111.6 ± 1.5 g, respectively (paired Student’s t-test showed no significant difference between these values (t5=0.8811, p=0.4186, N=6)]. Carrageenan (CARR, 1%) was then injected at the same site and the mechanical threshold was evaluated daily, until the return to the baseline values (~4th day) [no significant difference on the mechanical nociceptive thresholds was observed immediately before and 4 days after injection of CARR (t5=0.2911, p=0.7827, paired Student’s t-test)]. Daily injections of rapamycin (1 μg) were performed and readings taken before and 30 min after each injection showed no effect of rapamycin on the hyperalgesia induced by CARR (day 1 after CARR: t5=0.5649, p=0.5965; day 2 after CARR: t5=1.976, p=0.1051; day 3 after CARR: t5=1.074, p=0.3318, paired Student’s t-test for each day). On the 4th day after CARR, test for priming was performed: PGE2 (100 ng) was injected at the same site as CARR and rapamycin and the mechanical thresholds were evaluated 30 min and 4 h later. Two-way repeated measures ANOVA followed by Bonferroni post-test showed significant mechanical hyperalgesia 30 min after PGE2 injection (p<0.001), with decrease on the 4th h (p>0.05, when compared to thresholds before PGE2), indicating an inhibitory effect of rapamycin on the prolongation of PGE2 hyperalgesia (black bars, F2,10=196.98, p<0.0001). Of note, in a control group of rats that received CARR 4 days before (gray bars), the PGE2-induced hyperalgesia was still present at that time point [two-way repeated measures ANOVA followed by Bonferroni post-test showed significant difference between both groups (F2,30=55.58, p<0.0001), with decrease in the mechanical hyperalgesia at the 4 h after PGE2 in the rapamycin-treated group, when compared to the control group (***p<0.001); N=6 paws].
Figure 2
Figure 2. Reversal of carrageenan-induced hyperalgesic priming by local injection of the mTOR inhibitor rapamycin
Different groups of rats received intradermal injection of carrageenan (CARR, 1%) on the dorsum of the hind paw. Mechanical nociceptive threshold evaluation, by the Randall-Selitto paw-withdrawal test, showed significant hyperalgesia 3 h after injection (p<0.0001, for both groups when compared to baseline thresholds, paired Student’s t-test, N=6 paws per group) and return to baseline values on the 5th day post CARR injection [average paw withdrawal thresholds before and on the 5th day after CARR injection: 115.0 ± 2.2 g and 116.6 ± 2.1 g, respectively, for the vehicle-treated group (t5=0.4152, p=0.6952, gray bars), and 108.3 ± 3.0 g and 109.3 ± 2.8 g, respectively, for the rapamycin-treated group (t5=0.3437, p=0.7451, black bars) (paired Student’s t-test showed no significant difference between these values). Also, there was no difference between both groups 5 days after CARR injection (t10=2.086, p=0.0635, unpaired Student’s t-test)]. Injection of PGE2 at the same site as CARR induced hyperalgesia that was still present after 4 h, indicating the presence of hyperalgesic priming. Two days later (7 days after CARR injection), vehicle (gray bars) or the mTOR inhibitor rapamycin (black bars) was injected at the same site. No significant change in the mechanical nociceptive threshold was observed 30 min after rapamycin injection (t5=1.126, p=0.3111, paired Student’s t-test). However, when tested for priming with PGE2, no hyperalgesia was observed 4 h later in the rats treated with rapamycin when compared to the rats treated with vehicle (***p<0.001, two-way repeated measures ANOVA followed by Bonferroni post-test), showing inhibition of CARR-induced priming by local injection of rapamycin (N=6 paws per group).
Figure 3
Figure 3. Prevention of carrageenan-induced hyperalgesic priming by local injection of the protein translator inhibitor cordycepin
Different groups of rats received intradermal injection of vehicle (gray bars) or cordycepin (1 μg, black bars) on the dorsum of the hind paw. Evaluation of the mechanical nociceptive thresholds, by the Randall-Selitto paw-withdrawal test, before and 30 min after injection, showed no effect of cordycepin by itself [average paw withdrawal thresholds before and 30 min after injection: 106.6 ± 1.2 g and 105.6 ± 1.6 g, respectively, for the cordycepin group, and 106.6 ± 1.2 g and 106.0 ± 1.8 g, respectively, for the vehicle group (paired Student’s t-test for each group showed no significant difference between these values); also, unpaired Student’s t-test showed no difference between the groups after injections (t10=0.1334, p=0.8965)]. Carrageenan (CARR, 1%) was then injected at the same site and the mechanical thresholds were evaluated daily, until the return to the baseline values (~5th day) [no significant difference on the mechanical nociceptive threshold, for each group, was observed immediately before and 5 days after injection of carrageenan (cordycepin group: t5=0.3071, p=0.7711; vehicle group: t5=0.9325, p=0.3939, paired Student’s t-test)]. Daily injections of cordycepin (1 μg) or vehicle were performed; readings taken 30 min after each injection showed no difference in the hyperalgesia between both groups (F1,60=0.67, p=0.4321, two-way repeated measures ANOVA followed by Bonferroni post-test), i.e., no effect of cordycepin on the hyperalgesia induced by carrageenan. On the 5th day after carrageenan, test for priming was performed: PGE2 (100 ng) was injected at the same site as carrageenan and vehicle/cordycepin and the mechanical thresholds were evaluated 30 min and 4 h later. Two-way repeated measures ANOVA showed no hyperalgesia in the cordycepin-treated group, when compared to the vehicle-treated group, 4 h after PGE2 injection (F2,20=23.51, **p<0.0001, N=6 paws per group).
Figure 4
Figure 4. Reversal of carrageenan-induced hyperalgesic priming by local injection of the protein translator inhibitor cordycepin
Different groups of rats received intradermal injection of carrageenan (CARR, 1%) on the dorsum of the hind paw. Mechanical nociceptive thresholds evaluation, by the Randall-Selitto paw-withdrawal test, showed significant hyperalgesia 3 h after injection and return to baseline values on the 4th day post CARR injection [average paw withdrawal thresholds before and 4 d after injection - cordycepin group: 116.8 ± 1.5 g and 113.3 ± 1.1 g, respectively; control group: 120.0 ± 2.0 g and 117.6 ± 1.7 g, respectively (paired Student’s t-test showed no significant difference between these values for each group: cordycepin: t11=2.116, p=0.0579, N=12 paws; control: t5=1.400, p=0.2204, N=6 paws); unpaired Student’s t-test showed no difference between the groups on the 4th day after carrageenan injection (t16=2.079, p=0.0540)]. No significant difference in the mechanical threshold was observed 30 min after injection of cordycepin, when compared to the control group either (unpaired Student’s t-test, t16=1.038, p=0.3148). Injection of PGE2 at the same site as carrageenan and cordycepin/vehicle induced significant hyperalgesia, evaluated 30 min and 4 h after injection (two-way repeated measures ANOVA followed by Bonferroni post-test, F2,32=205.62, p<0.0001). However, at the 4th h post-PGE2, the magnitude of the hyperalgesia was significantly decreased in the cordycepin-treated group when compared to the control group (***p<0.001), indicating an effect of cordycepin on hyperalgesic priming.
Figure 5
Figure 5. Duration of the effect of the protein translator inhibitor cordycepin on the carrageenan-induced hyperalgesic priming
Panel A: Rats that received intradermal injection of carrageenan (CARR, 1%) on the dorsum of the hind paw were treated with cordycepin (1 μg) on the 4th day (after recovery from CARR-induced hyperalgesia). PGE2 was injected 30 min after cordycepin and the mechanical nociceptive thresholds were evaluated, by the Randall-Selitto paw-withdrawal test, 30 min and 4 h later. Paired Student’s t-test showed significant decrease in the PGE2-induced hyperalgesia on the 4th h after injection, indicating inhibition of hyperalgesic priming by condycepin (t11=8.748, **p<0.0001 when the magnitude of the hyperalgesia on 30 min and 4 h are compared, N=12 paws). When PGE2 was injected, again, 10 and 20 days later, at the same site where carrageenan/cordycepin were previously administered, it failed to induce prolonged hyperalgesia, evaluated at 4 h post injection [Paired Student’s t-test comparing the magnitude of the hyperalgesia on 30 min and 4 h: t11=8.799, **p<0.0001 (10 days after cordycepin injection), t11=10.05, **p<0.0001 (20 days after cordycepin injection)] indicating the long-term reversal of the hyperalgesic priming by cordycepin (N=12 paws); Panel B: Hyperalgesic priming is still present 3 weeks after carrageenan injection: rats that received carrageenan (CARR, 1%) on the dorsum of the hind paws were tested with PGE2 (100 ng) 2 and 3 weeks afterwards. Readings were taken 30 min and 4 h after PGE2 and, as expected, the magnitude of the hyperalgesia was still high at the 4th h, indicating the presence of hyperalgesic priming, induced by previous injection of CARR. This observation is important in order to evaluate the effect of cordycepin seen on Panel A [Paired Student’s t-test comparing the magnitude of the hyperalgesia on 30 min and 4 h: t5=1.101, p=0.3211 (2 weeks after CARR injection), t5=1.849, p=0.1237 (3 weeks after CARR injection)] (N=6 paws).
Figure 6
Figure 6. Duration of the effect of the protein translator inhibitor cordycepin
Rats received intradermal injection of vehicle (white bars) or cordycepin (1 μg, black bars) on the dorsum of the hind paws 20 days before injection of carrageenan (CARR, 1%) at the same site. There was no difference in the carrageenan-induced hyperalgesia, evaluated by the Randall-Selitto paw-withdrawal test, between both groups (data not shown). 5 days later, when the mechanical thresholds were back to pre-carrageenan values (vehicle group: t5=1.464, p=0.2031; cordycepin group: t5=0.4152, p=0.6952, paired Student’s t-test), PGE2 was injected at the same site and the mechanical thresholds were evaluated 30 min and 4 h later. Two-way repeated measures ANOVA followed by Bonferroni post-test showed no significant difference in the PGE2-induced hyperalgesia, both 30 min and 4 h after injection, between the vehicle- and the cordycepin-treated groups (F1,20=0.01, p=0.9067), indicating that, at that time point (20 days after injection), the effect of cordycepin was no longer present (N=6 paws per group).
Figure 7
Figure 7. Local effect of the protein translation inhibitors rapamycin and cordycepin
Rats that had their right hind paw primed with carrageenan (1%) 4 days before received intradermal injection of the protein translation inhibitors rapamycin (1 μg, panel A, white bars) or cordycepin (1 μg, panel B, gray bars) on the dorsum of the contralateral paw. 30 min after injecting the inhibitors, PGE2 was injected in the right hind paw and the mechanical nociceptive threshold was evaluated, by the Randall-Selitto paw-withdrawal test, 30 min and 4 h later (black bars, both panels). Paired Student’s t-test showed no change in the magnitude of PGE2-induced hyperalgesia at 30 min and 4 h (panel A: t5=0.5423, p=0.6109; panel B: t5=0.7006, p=0.5148), indicating lack of effect of rapamycin or cordycepin injected in the contralateral paw on the hyperalgesic priming in the right paws (N=6 paws per group).
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