Epigenetic regulation of spinal cord gene expression controls opioid-induced hyperalgesia

Abstract

Background: The long term use of opioids for the treatment of pain leads to a group of maladaptations which includes opioid-induced hyperalgesia (OIH). OIH typically resolves within few days after cessation of morphine treatment in mice but is prolonged for weeks if histone deacetylase (HDAC) activity is inhibited during opioid treatment. The present work seeks to identify gene targets supporting the epigenetic effects responsible for OIH prolongation.

Results: Mice were treated with morphine according to an ascending dose protocol. Some mice also received the selective HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) additionally. Chronic morphine treatment with simultaneous HDAC inhibition enhanced OIH, and several spinal cord genes were up-regulated. The expression of Bdnf (Brain-derived neurotrophic factor) and Pdyn (Prodynorphin) were most closely related to the observed behavioral changes. ChIP (Chromatin immuoprecipation) assays demonstrated that promoter regions of Pdyn and Bdnf were strongly associated with aceH3K9 (Acetylated histone H3 Lysine9) after morphine and SAHA treatment. Furthermore, morphine treatment caused an increase in spinal BDNF and dynorphin levels, and these levels were further increased in SAHA treated mice. The selective TrkB (tropomyosin-receptor-kinase) antagonist ANA-12 reduced OIH when given one or seven days after cessation of morphine. Treatment with the selective kappa opioid receptor antagonist nor-BNI also reduced established OIH. The co-administration of either receptor antagonist agent daily with morphine resulted in attenuation of hyperalgesia present one day after cessation of treatment. Additionally, repeated morphine exposure induced a rise in BDNF expression that was associated with an increased number of BDNF+ cells in the spinal cord dorsal horn, showing strong co-localization with aceH3K9 in neuronal cells. Lastly, spinal application of low dose BDNF or Dynorphin A after resolution of OIH produced mechanical hypersensitivity, with no effect in controls.

Conclusions: The present study identified two genes whose expression is regulated by epigenetic mechanisms during morphine exposure. Treatments aimed at preventing the acetylation of histones or blocking BDNF and dynorphin signaling may reduce OIH and improve long-term pain using opioids.

Figure 1

Epigenetic effects of chronic morphine treatment on spinal cord gene expression. (A) Schematic representation of experimental timeline showing the daily dosing schedule of morphine treatment. (B-C) Epigenetic effects of escalating dose morphine treatment on spinal cord gene expression changes after 7 days of washout with/without co-administration of SAHA 50 mg/kg daily. Concomitant HDAC inhibition leads to significant up-regulation in Bdnf and Pdyn mRNA expression 7 days after cessation of morphine treatment (B). The promoter regions of Pdyn and Bdnf exon-IV genes were more strongly associated with acetylated histone H3: aceH3K9 (C). Error bars: SEM, n = 6-8/group, ^*p < 0.05, **p < 0.01 and ***p < 0.001 for comparison with controls. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 for comparison with morphine group. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc tests.

Figure 2

HDAC inhibitor treatment increases spinal cord BDNF and dynorphin protein levels after morphine treatment. SAHA treatment resulted in significantly increased expression of BDNF after 1 or 7 days since cessation of morphine treatment compared to vehicle treatment (A). Levels of dynorphin were similarly increased on day 5 in SAHA and vehicle groups and it was significantly up-regulated 7 days after morphine and SAHA treatment only (B). HDAC inhibition was carried out for the duration of escalating dose morphine administration. Error bars: SEM, n = 5-6/group; *p < 0.05, **p < 0.01, ***p < 0.001 for comparison with vehicle and ^###p < 0.001 for comparison with morphine treatment. Data were analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons within each timepoint.

Figure 3

Selective acute inhibition of BDNF and prodynorphin signaling attenuates opioid induced hyperalgesia. Effects of single dose ANA-12 (TrkB receptor antagonist, 1 mg/kg) or nor-BNI (KOR antagonist, 1 mg/kg) in the setting of OIH was examined. Selective antagonism of TrkB (A) receptor and KOR (B) produces a reversal of mechanical hypersensitivity, whether given once at day 5 (day 1 after completion of morphine protocol) or day 12 (day 7 after completion of morphine). However, acute ANA-12 but not nor-BNI administration reversed the sensitization present in the morphine + SAHA group. Error bars: SEM, n = 6/group, *p < 0.05, **p < 0.01 and ***p < 0.001 for comparison between treatments for each timepoint. Data for each timepoint were analyzed by one-way ANOVA followed by Sidak multiple comparisons tests.

Figure 4

Selective inhibition of BDNF and prodynorphin signaling during morphine treatment attenuates opioid induced hyperalgesia. ANA-12 (TrkB receptor antagonist, 0.5 mg/kg) or nor-BNI (KOR antagonist, 0.5 mg/kg) administration once daily with morphine during the 4 days of escalating morphine treatment resulted in attenuation of sensitization on day 5 (A-B). However, only ANA-12 was associated with less sensitization in the morphine + SAHA group on day 12 (one week after completion of drug administration). Error bars: SEM, n = 6/group, ns: p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001 for comparison between treatments for each timepoint. Data for each timepoint were analyzed by one-way ANOVA followed by Sidak multiple comparisons tests.

Figure 5

Chronic morphine with SAHA treatment promotes histone acetylation dependent increase in BDNF expression in spinal cord tissue. (A-B) Micrographs of representative spinal cord dorsal horn sections (Scale bar = 100 micron) showing increased number of BDNF⁺ cells in the dorsal horn 7 days after cessation of morphine and SAHA (M + S) treatment. (C) Quantification of BDNF positive cells in 10–15 randomly (2 slices per mouse) selected high-power fields (HPF, 400X) of spinal cord dorsal horn per animal. (D) BDNF and acetylated H3K9 co-staining in the M + S treatment group, demonstrating strong association between both. Error bars: SEM, n = 4/treatment group. Data analyzed by unpaired two tailed student t-tests, **p < 0.01 for differences between treatment conditions.

Figure 6

Chronic morphine with SAHA treatment increases the number of BDNF ⁺ expressing spinal cord dorsal horn neurons. (A-C) Double staining for BDNF and NeuN markers demonstrates strong association with BDNF in neuronal cells (Scale bar = 20 micron). (B-D) Quantification of double staining for BDNF and NeuN markers reveals increased percentage of neurons expressing BDNF in lumbar spinal cord dorsal horn tissue (lamina I-II and III-VI) following M + S treatment. (E) Double staining for BDNF and GFAP markers shows less abundant BDNF expression by astrocytes. Error bars: SEM, n = 4/ treatment group with the numbers of NeuN positive cells (neurons) and BDNF positive neurons were counted in 10–15 randomly selected (2 slices per mouse) high-power fields (HPF, 400X) of spinal cord dorsal horn per animal. Data analyzed by unpaired two tailed student t-tests, **p < 0.01 and ***p < 0.001 for differences between treatment conditions.

Figure 7

Spinal application of low dose BDNF or dynorphin A produces hyperalgesia in previously morphine treated mice. Effects of single intrathecal dose of (A) saline (5uL), (B) BDNF 1 ng/5uL or (C) Dynorphin A 1 nmol (5uL) in mice 7 days after cessation of morphine/saline treatment OIH were examined. The drug doses selected fail to produce mechanical hypersensitivity in animals previously treated with saline. However, in the setting of resolved OIH both agents produce robust mechanical allodynia. Error bars: SEM, n = 6/group, ^**p < 0.01 and ^***p < 0.001 for comparison between treatments for each timepoint. Data for each timepoint were analyzed by two-way ANOVA followed by Sidak multiple comparisons tests.