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. 2008 May 28;28(22):5721-30.
doi: 10.1523/JNEUROSCI.0256-08.2008.

Stress induces a switch of intracellular signaling in sensory neurons in a model of generalized pain

Sachia G Khasar  1 , Jennifer Burkham, Olayinka A Dina, Adrienne S Brown, Oliver Bogen, Nicole Alessandri-Haber, Paul G Green, David B Reichling, Jon D Levine
Affiliations

Stress induces a switch of intracellular signaling in sensory neurons in a model of generalized pain

Sachia G Khasar et al. J Neurosci. .

Abstract

Stress dramatically exacerbates pain in diseases such as fibromyalgia and rheumatoid arthritis, but the underlying mechanisms are unknown. We tested the hypothesis that stress causes generalized hyperalgesia by enhancing pronociceptive effects of immune mediators. Rats exposed to nonhabituating sound stress exhibited no change in mechanical nociceptive threshold, but showed a marked increase in hyperalgesia evoked by local injections of prostaglandin E(2) or epinephrine. This enhancement, which developed more than a week after exposure to stress, required concerted action of glucocorticoids and catecholamines at receptors located in the periphery on sensory afferents. The altered response to pronociceptive mediators involved a switch in coupling of their receptors from predominantly stimulatory to inhibitory G-proteins (G(s) to G(i)), and for prostaglandin E(2), emergence of novel dependence on protein kinase C epsilon. Thus, an important mechanism in generalized pain syndromes may be stress-induced coactivation of the hypothalamo-pituitary-adrenal and sympathoadrenal axes, causing a long-lasting alteration in intracellular signaling pathways, enabling normally innocuous levels of immune mediators to produce chronic hyperalgesia.

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Figures

Figure 1.
Figure 1.
Sound stress enhances hyperalgesia induced by prostaglandin E2 and epinephrine. A, A 4 d cycle of sound stress consists of 30 min of intermittent exposure to sound stress (s) administered on days 1, 3, and 4. Pain threshold measurements (M) were taken on poststress days 1, 7, 14, and 21. B, C, Dose–response relationships for PGE2- and epinephrine-induced hyperalgesia were determined after exposure to sound stress or sham (exposure to the sound stress device without sound). Logarithmically increasing doses of PGE2 or epinephrine were administered cumulatively as indicated. B, Changes in PGE2 hyperalgesia produced by sound stress exposure. There was a significant main effect of group for sound stress on PGE2 hyperalgesia (F(4,261) = 29.70; p < 0.001). Post hoc analysis showed that PGE2 hyperalgesia in the group of rats that was tested 14 d (n = 24) or 21 d (n = 12) after sound stress was significantly enhanced (p < 0.001 each) compared with the sham sound-stressed group (n = 20). PGE2 hyperalgesia in the rest of the groups was not significantly different from the sham-stressed group (p > 0.05). C, Effect of stress on epinephrine hyperalgesia. There was a significant main effect of sound stress on epinephrine hyperalgesia compared with the sham sound stress group (F(4,207) = 110.70; p < 0.001). Post hoc analysis showed that epinephrine hyperalgesia in the group of rats that was tested 24 h after sound stress (n = 22) was significantly less (p < 0.001) than in the sham-stressed group (n = 12). However, 14 d (n = 12) or 21 d (n = 12) after sound stress, epinephrine hyperalgesia was significantly enhanced compared with the sham sound-stressed group (p < 0.001). D, Dose–response relationship for PGE2 in the gastrocnemius muscle in naive rats and 14 d after sound stress. Repeated-measures ANOVA showed a significant difference in the response of gastrocnemius muscle to PGE2 between naive and sound-stressed rats (F(1,55) = 53.2; p < 0.001; n = 12 each). Error bars indicate SEM.
Figure 2.
Figure 2.
The effect of stress is mediated by glucocorticoid receptors in sensory neurons. A, Dose–response relationship for epinephrine hyperalgesia shows that systemic administration of the glucocorticoid receptor antagonist mifepristone (RU 38486), starting at the same time as initiation of sound stress and continuing for 13 d after, prevented the stress-induced enhancement of epinephrine hyperalgesia. All measurements were taken 14 d after the final exposure to sound stress. There was a significant main effect of mifepristone treatment on epinephrine hyperalgesia between the groups (F(3,132) = 23.5; p < 0.001). Post hoc analysis showed that subcutaneous injection of mifepristone (n = 12), but not its vehicle (n = 4), prevented sound-stress-induced enhancement of epinephrine hyperalgesia (p = 0.001). Epinephrine hyperalgesia in the mifepristone-treated group was not significantly different from that in the sham sound-stressed group (p > 0.05). B, Immunohistochemical analysis of the GCR expression in rat lumbar dorsal root ganglia. Anti-GCR immunoreactivity (ir) was detected in sensory neurons. As expected, anti-GCR-ir shows a predominantly nuclear distribution. A, Negative control, immunohistochemistry in the absence of the anti-GCR antibody. B, Immunohistochemistry in the presence of the anti-GCR antibody. Scale bars, 100 μm. C, Expression of glucocorticoid receptor was decreased in peripheral nerves after intrathecal antisense treatment. A 90 kDa band corresponding to the glucocorticoid receptor was detected by Western blotting of saphenous nerve. There was a 33 ± 8% decrease in protein expression in antisense compared with mismatch ODN-treated rats (p = 0.004, unpaired Student's t test; n = 11 for both antisense- and mismatch-treated rats). D, Glucocorticoid receptor antisense inhibits sound-stress-induced enhancement of epinephrine hyperalgesia. Rats were injected intrathecally daily with antisense (AS) or mismatch control (MM) oligodeoxynucleotides to the GCR. These rats were exposed to sound stress, and epinephrine hyperalgesia was measured at 14 d postexposure. The antisense, but not mismatch, ODN eliminated the enhancement of epinephrine hyperalgesia seen 14 d after sound stress. Overall ANOVA showed significant main effect of ODN treatment on epinephrine hyperalgesia between the groups (F(3,156) = 30.3; p < 0.001). Glucocorticoid receptor antisense treatment (n = 12) significantly inhibited stress-induced enhancement (n = 12) of epinephrine hyperalgesia [GCR AS compared with GCR MM or sound stress (14 d); p = 0.001]. Error bars indicate SEM.
Figure 3.
Figure 3.
Corticosterone mimics the ability of stress to enhance epinephrine hyperalgesia. A, Dose–response relationships for hyperalgesia induced by intradermal injection of epinephrine show that pretreatment with intradermal corticosterone (1 μg/2.5 μl) enhanced responses to epinephrine, measured 24 h later. Corticosterone was injected in one paw (Cort), while vehicle was injected in the contralateral paw (vehicle). No effect was observed in untreated contralateral paws. There was a significant main effect of corticosterone-treatment on epinephrine hyperalgesia between the groups (F(3,84) = 8.32; p < 0.001). Post hoc analysis showed significant enhancement of epinephrine hyperalgesia 24 h (n = 10) after corticosterone treatment compared with the vehicle-treated paws (n = 10) during the same time period (p = 0.004). Epinephrine hyperalgesia remained significantly enhanced in the corticosterone-treated paws 7 d after corticosterone injection (n = 6), whereas the vehicle-treated paws (n = 6) remained unaffected (p < 0.005). B, Intradermal corticosterone did not significantly increase sound stress enhancement of epinephrine hyperalgesia compatible with an occlusion of overlapping mechanisms. There was a significant main effect of corticosterone treatment on epinephrine hyperalgesia (F(3,144) = 32.4; p < 0.001). Post hoc analysis showed that corticosterone treatment did not further enhance epinephrine hyperalgesia 14 d after sound stress (n = 10) compared with sound stress with (p > 0.05; n = 10) or without (p > 0.05; n = 20) corticosterone vehicle. Error bars indicate SEM.
Figure 4.
Figure 4.
Corticosterone-enhanced hyperalgesia is mediated by glucocorticoid receptors on sensory neurons. A, Intrathecally administered glucocorticoid receptor antisense (AS), but not mismatch (MM), ODN treatment abolished enhancement of PGE2 hyperalgesia after intradermal glucocorticoid treatment. Overall ANOVA showed significant main effect of ODN treatment group on PGE2 hyperalgesia (F(3,60) = 7.2; p = 0.002). Post hoc analysis showed that glucocorticoid receptor antisense ODN treatment (n = 6) significantly reversed corticosterone-induced enhancement (n = 6) of PGE2 hyperalgesia (p = 0.030, GCR AS compared with GCR MM or corticosterone). B, Rats received intrathecal injections of glucocorticoid receptor antisense and mismatch ODN and were then treated with intradermal corticosterone 24 h before measurement of epinephrine hyperalgesia. Antisense but not mismatch glucocorticoid receptor treatment abolished enhancement of epinephrine hyperalgesia that is normally seen after intradermal glucocorticoid treatment. There were significant main effects of ODN treatment on epinephrine hyperalgesia between the antisense and mismatch groups (F(3,84) = 8.0; p < 0.001). Post hoc analysis showed that glucocorticoid antisense treatment (n = 6) significantly reversed corticosterone-induced enhancement (n = 6) of epinephrine hyperalgesia (p = 0.035, GCR AS compared with GCR MM or corticosterone). Epinephrine-induced hyperalgesia in the vehicle-treated paw of control rats was not different from that of the paws in the antisense ODN-treated group (p > 0.05). Error bars indicate SEM.
Figure 5.
Figure 5.
Epinephrine augments the glucocorticoid-mediated enhancement of epinephrine hyperalgesia. A, Dose–response curves for epinephrine show that adrenal medullectomy prevented sound-stress-induced enhancement of epinephrine hyperalgesia. There were differences between the groups (F(3,89) = 10.2; p < 0.001). Post hoc analysis showed that, whereas epinephrine hyperalgesia in naive control (n = 30), AMedx (n = 12), and AMedx sound stress (n = 20) groups was not significantly different (p > 0.05), it was significantly enhanced in the sound stress (n = 20) group (p < 0.001). B, Adrenalectomized rats (n = 6) were reconstituted with both corticosterone (100 mg) pellets and epinephrine (5.4 μg/h) on the day of surgery. Three days after the implants, three rats were given bolus injection of corticosterone (25 mg/kg, i.p.) on days 1, 3, and 4 (similar to a sound stress protocol), the other three rats received injection of vehicle. Fourteen days after the last injection, intradermal injection of epinephrine (1–1000 ng) produced enhanced hyperalgesia compared with the naive control or vehicle-treated groups. Overall ANOVA showed significant main effect of reconstitution group on epinephrine hyperalgesia (F(3,189) = 20.3; p < 0.001). Post hoc analysis showed a significant enhancement of epinephrine hyperalgesia (p < 0.001; n = 8) in adrenalectomized rats that were given a bolus injection of corticosterone, compared with naive controls (n = 31). Error bars indicate SEM.
Figure 6.
Figure 6.
Adrenal medulla is required for stress-induced enhancement of hyperalgesia and pertussis toxin sensitivity. A, The Gi/o inhibitor pertussis toxin (PTx) (100 ng/2.5 μl) partially inhibited epinephrine hyperalgesia (shown here as a decrease in the nociceptive threshold). PTx sensitivity was enhanced after sound stress. One-way ANOVA showed a significant main effect of sound stress group on epinephrine hyperalgesia (F(3,40) = 95.8; p < 0.001). Post hoc analysis showed epinephrine hyperalgesia after sound stress (n = 16) was significantly more inhibited by PTx than epinephrine hyperalgesia in the nonstressed group (sham sound; p = 0.013; n = 8). B, Adrenal medullectomy prevented the inhibition of epinephrine hyperalgesia by PTx in sham sound-stressed rats. Adrenal medullectomy also prevented sound-stress-induced enhancement as well as inhibition of epinephrine hyperalgesia by PTx. One-way ANOVA showed no significant main effect of adrenal medullectomy on epinephrine hyperalgesia between the sham sound-stressed and sound-stressed groups, whether PTx-treated or not (F(3,44) = 0.26; p > 0.05). C, Whereas PTx treatment enhanced PGE2 hyperalgesia in sham sound-stressed rats, it inhibited it in sound-stress-induced enhancement of PGE2 hyperalgesia. One-way ANOVA showed a significant main effect of PTx treatment on PGE2 hyperalgesia between the sham sound-stressed and sound-stressed groups (F(3,24) = 8.2; p < 0.001). Post hoc analysis showed that, whereas PTx significantly enhanced PGE2 hyperalgesia in sham sound-stressed rats [p = 0.002, sham sound stress (n = 6) compared with sham sound stress plus PTx (n = 6)], PTx inhibited sound-stress-induced enhancement of PGE2 hyperalgesia [p = 0.002, sound stress (n = 8) compared with sound stress plus PTx (n = 8)]. D, PKCεI (1 μg/2.5 μl) peptide had no effect on PGE2-evoked hyperalgesia in sham-stressed rats, but it abolished the enhancement of PGE2 hyperalgesia caused by sound stress. The scrambled peptide [PKCεI (-ve)] had no effect on stress-induced enhancement of PGE2 hyperalgesia. The dose of epinephrine used in A and B was 100 ng/2.5 μl, whereas the dose of PGE2 used in both C and D was 1 ng/2.5 μl. Error bars indicate SEM.
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