Epigenetic regulation of spinal cord gene expression contributes to enhanced postoperative pain and analgesic tolerance subsequent to continuous opioid exposure

Abstract

Background: Opioids have become the mainstay for treatment of moderate to severe pain and are commonly used to treat surgical pain. While opioid administration has been shown to cause opioid-induced hyperalgesia and tolerance, interactions between opioid administration and surgery with respect to these problematic adaptations have scarcely been addressed. Accumulating evidence suggests opioids and nociceptive signaling may converge on epigenetic mechanisms in spinal cord to enhance or prolong neuroplastic changes. Epigenetic regulation of Bdnf (brain-derived neurotrophic factor) and Pdyn (prodynorphin) genes may be involved.

Results: Four days of ascending doses of morphine treatment caused opioid-induced hyperalgesia and reduced opioid analgesic efficacy in mice. Both opioid-induced hyperalgesia and the reduced opioid analgesic efficacy were enhanced in mice that received hindpaw incisions. The expression of Bdnf and Pdyn (qPCR) was increased after morphine treatment and incision. Chromatin immunoprecipitation assays demonstrated that the Pdyn and Bdnf promoters were more strongly associated with acetylated H3K9 after morphine plus incision than in the morphine or incision alone groups. Selective tropomyosin-related kinase B (ANA-12) and κ-opioid receptor (nor-binaltorphimine) antagonists were administered intrathecally, both reduced hyperalgesia one or three days after surgery. Administration of ANA-12 or nor-binaltorphimine attenuated the decreased morphine analgesic efficacy on day 1, but only nor-binaltorphimine was effective on day 3 after incision in opioid-exposed group. Coadministration of histone acetyltransferase inhibitor anacardic acid daily with morphine blocked the development of opioid-induced hyperalgesia and attenuated incision-enhanced hyperalgesia in morphine-treated mice. Anacardic acid had similar effects on analgesic tolerance, showing the involvement of histone acetylation in the interactions detected.

Conclusions: Spinal epigenetic changes involving Bdnf and Pdyn may contribute to the enhanced postoperative nociceptive sensitization and analgesic tolerance observed after continuous opioid exposure. Treatments blocking the epigenetically mediated up-regulation of these genes or administration of TrkB or κ-opioid receptor antagonists may improve the clinical utility of opioids, particularly after surgery.

Keywords: BDNF and dynorphin; Epigenetics; histone acetylation; incision; opioid-induced hyperalgesia.

Figure 1.

Effects of morphine treatment on mechanical hypersensitivity after incision. (a) Schematic representation of experimental timeline showing the daily dosing schedule of morphine treatment and procedures carried out. (b) Morphine treatment or incision alone resulted in increased mechanical hypersensitivity. Mice that had the incision after morphine treatment had significantly lower mechanical thresholds than groups which received either morphine or incision alone. Data were analyzed by multiple t tests using the Holm-Sidak method to correct for multiple comparisons. * indicates significant difference in comparison with morphine, and ^# indicates significant difference in comparison with incision. Error bars: SEM, n = 8/group.

Figure 2.

Effects of morphine treatment on opioid analgesic efficacy after incision. Mice that had the incision after morphine treatment displayed decreased analgesic efficacy to morphine on days 3 and 6 after surgery compared to morphine treatment alone. Data were analyzed by one-way ANOVA followed by Sidak post hoc test for multiple comparisons for each dose. **p < 0.01, ***p < 0.001 for comparison with morphine and ^##p < 0.01 for comparison with controls. Error bars: SEM, n = 6/group.

Figure 3.

Effects of morphine treatment, incision and the combination on spinal cord gene expression. Expression patterns of spinal Bdnf and Pdyn on (a) day 1 and (b) day 3 after incision. Expression of Bdnf was higher in morphine-treated plus incision animals one day after surgery. Increased expression changes for Pdyn were observed at both one and three days after incision for all groups, and greater enhancement of expression in the morphine plus incision group at both time points. Error bars: SEM, n = 5/group; *p < 0.05, ***p < 0.001 for comparison with controls and ^#p < 0.05 for within groups comparisons. Data were analyzed by two-way analysis of variance (ANOVA) followed by Fisher post hoc test for multiple comparisons within each time point.

Figure 4.

Epigenetic effects of morphine treatment, incision and the combination on spinal Bdnf and Pdyn expression. Chromatin immunoprecipitation (ChIP) assays done on lumbar spinal tissue (a) day 1 and (b) day 3 after incision. ChIP assays showed that promoter regions of Bdnf and Pdyn were more strongly associated with aceH3K9 on day 1 after incision in morphine-treated group compared with morphine or incision alone groups. Error bars: SEM, n = 5–6/group; *p < 0.05, ***p < 0.001 for comparison with controls and ^#p < 0.05, ^##p < 0.01 for within groups comparisons. Data were analyzed by two-way analysis of variance (ANOVA) followed by Fisher post hoc test for multiple comparisons within each time point.

Figure 5.

Effects of chronic morphine treatment and incision on spinal cord BDNF and dynorphin protein levels. Enzyme immunoassay (ELISA) for BDNF and dynorphin protein levels in lumbar spinal cord segments were done to determine the time course of the elevated proteins after incision in mice previously exposed to morphine. (a) BDNF levels were elevated on days 1 and 3 but return to baseline values by day 6 after incision. (b) Dynorphin levels remain persistently elevated up to day 6 after incision. Error bars: SEM, n = 6/group, *p < 0.05 and **p < 0.01 for comparison with baseline. Data were analyzed by one-way ANOVA followed by Fisher post hoc test multiple comparisons tests.

Figure 6.

Effects of selective inhibition of spinal BDNF and dynorphin signaling. (a) Effects of selective antagonists of the tropomyosin-receptor-kinase (TrkB) and κ-opioid receptors, ANA-12 and nor-BNI, respectively. Both drugs reduced hyperalgesia given on day 1 or 3 after surgery compared to vehicle treatment. (b) The administration of ANA-12 or nor-BNI attenuated the reduced morphine analgesic efficacy on day 1, but only nor-BNI was effective on day 3 after surgery in opioid-exposed group. Error bars: SEM, n = 6/group, *p < 0.05, **p < 0.01 and ***p < 0.001 for comparison between treatments for each time point. Data for each time point were analyzed by one-way ANOVA followed by Sidak multiple comparisons tests.

Figure 7.

Effects of HAT inhibition on enhanced hyperalgesia and tolerance after incision following morphine treatment. (a) Coadministration of anacardic acid daily with morphine prevented opioid-induced hyperalgesia from occurring. Anancardic acid also attenuated enhanced hyperalgesia seen on days 1, 3, and 6 after incision. (b) Coadministration of anacardic acid daily with morphine attenuated the reduced opioid analgesic efficacy on day 3 after incision, having no effect on morphine analgesic efficacy in the control group. Error bars: SEM, n = 6/group, ns: p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.00. Data were analyzed by one-way ANOVA followed by Sidak multiple comparisons tests.