Abstract

Objective

Epigenetic reprogramming may have a possible role in neuropathic pain development; the present study examined the global patterns of lysine histone modification. In this serial study we analyzed the levels of histone 3 lysine 4 monomethylation, histone 3 lysine 4 dimethylation, and histone 3 lysine 9 trimethylation in pertussis toxin (PTX)-induced thermal hyperalgesic rat spinal cords.

Methods

Male Wistar rats implanted with an intrathecal catheter received a single intrathecal PTX (1 μg in 5 μl saline) injection. Four days later, they were randomly assigned to receive either a single injection of saline, or ultra-low-dose naloxone (15 ng in 5 μl saline), followed by morphine (10 μg in 5 μl saline) injection 30 minutes later.

Results

The results showed that PTX injection induced thermal hyperalgesia and significant increase of global histone methylation in the spinal cords. Intrathecal morphine alone did not affect the thermal hyperalgesia and global histone methylation. In contrast, intrathecal administration of ultra-low-dose naloxone plus morphine significantly attenuated the PTX-induced thermal hyperalgesia and down-regulated the global histone methylation.

Conclusion

The results suggest that ultra-low-dose naloxone might be clinical valuable for neuropathic pain management via regulating global histone modification.

Keywords

analgesics, opioid; injections, spinal; methylationpain: neuropathic;

1. Introduction

Neuropathic pain is initiated by a primary lesion or dysfunction of the nervous system; the underlying cellular and molecular mechanisms of neuropathic pain remain controversial.¹ Several possible mechanisms have been proposed, including: (1) down-regulation of μ-opioid receptors, at both pre- and postsynaptic areas after nerve injury²: (2) activation of voltage-gated calcium channel³; (3) increased trafficking of AMPA receptors to the cell surface which enhances glutamate release³; and (4) activation of neuroinflammation cascade.⁴ Moreover, the possibility of epigenetic reprogramming may also play an important role in neuropathic pain development and is worth attention.

Epigenetic modification induces heritable changes in gene expression without changing the DNA sequence; it regulates the transcription and expression of pro- or antinociceptic genes. These mechanisms include alterations in the methylation status of DNA, covalent modification of histone tails, chromatin remodeling, and microRNAs.5, 6 The tails of histones H3 and H4 are methylated at several lysine and arginine residues, and methylation of individual residues is being causally linked to either transcriptional activation or repression.⁷ For example, H3K4 methylation via methyltransferase SET7/9 can affect recruitment of nuclear factor-κB (NF-κB) to proinflammatory genes.⁸ The peripheral mechanisms underpinning chronic inflammatory pain are controlled by the same mediators⁹ and involve action of both glial and neuronal NF-κB,¹⁰ making it likely to process similar epigenetic changes.

Pertussis toxin (PTX) is believed to adenosine diphosphate-ribosylate the α-subunit of G_i/G₀ proteins, thus disrupting the signal transduction of inhibitory G_i/G₀ protein-coupled receptors. Intrathecal injection of PTX not only decreases the antinociceptive effect of opioid agonists, but also produces thermal hyperalgesia and allodynia that appear similar to the symptoms reported in clinical patients who suffer from neuropathic pain.11, 12 The PTX model had been used to study the pathophysiology of neuropathic pain.¹² Certain studies had demonstrated that intrathecal PTX injection could reduce the antinociceptive potency of etorphine, fentanyl,¹³ and selective μ-agonist PL017.¹⁴ Womer et al found that PTX could disrupt the antinociceptive signal transduction of morphine, and therefore reduced morphine's effect in treating central and neuropathic pains than treating acute pain.¹⁵

Opioids are the therapeutic mainstay for alleviation of pain, although are less effective in treating neuropathic pain. Combination with mechanistically distinct analgesics may provide either an additive or synergistic effect to improve the efficacy of morphine at lower dose with fewer side effects, which is superior to the use of single agent or high dose of morphine. Naloxone, an opioid antagonist, at ultra-low nanogram dose, produces not only potentiation of the antinociceptive effect of morphine on neuropathic thermal hyperalgesia, but also attenuation of neuroinflammation and inflammatory pain.¹⁶ We previously had demonstrated that intrathecal ultra-low-dose naloxone could enhance the antinociceptive effect of morphine by inhibiting the activation of microglia and down-regulation of pro-inflammatory cytokine [tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and (IL-6)] and P-p38 MAPK expression, as well as decreasing excitatory amino acids, concentration in the spinal cerebrospinal fluid of PTX-treated rats.17, 18 However, the relationship between histone methylation and PTX-induced neuropathic thermal hyperalgesia is still not clear. The present study not only investigated the H3 methylation change in neuropathic thermal hyperalgesia, but also further examined the underline effect of ultra-low-dose naloxone on regulation of H3 methylation in PTX-treated rats.

2. Materials and methods

Eight-week-old male Wistar rats (320–370 g) were anesthetized by pentobarbital (65 mg/kg, intraperitoneally; Sigma, St Louis, MO, USA) and implanted an intrathecal catheter via the atlanto-occipital membrane down to the lumbar spinal cord segments L5-S3 (Day 0). After catheterization, the rats were returned to their home cages for a 3-dayrecovery before use.Ratsthatdeveloped abnormal behavior (such as poor appetite, decreased stool passage, decreased daily activity, frequent vocalizations, and flaccidity) and motor deficit during the 3-day readjustment were excluded from the study.Three days after catheterization (Day 3), PTX (1 μg in 5 μl of saline; Calbiochem-Novabiochem International, San Diego, CA, USA) was injected intrathecally to the test animals via the implanted catheter to induce thermal hyperalgesia, while the control rats were given 5 μl of saline in the same way. Four days later (Day 7), saline (5 μl) or ultra-low dose naloxone (15 ng in 5 μl of saline; Sigma) was injected intrathecally 30 minutes before saline or morphine (10 μg in 5 μl of saline; Sigma) injection. All drugs, which were purchased from Sigma, were delivered intrathecally in 5 μl and flushed with 5 μl of saline. After drug administration, all rats were subjected to a nociceptive tail-flick test, and then sacrificed for western blot analysis. The use of rats in this study conformed to the Guiding Principles in the Care and Use of Animals of the American Physiology Society and was approved by the Animal Care and Use Committee of our institute.

2.2. Antinociceptive test

The 52 °C hot water tail-flick test was applied to assess the antinociceptive effect as described previously.17, 18 Each test rat was restrained in a cylinder with ventilation holes on the top and in the front. The temperature of the water (52.0 ± 0.2 °C) was maintained by circulating it through an enclosed external chamber within which the circulating water was feedback to a flow-through heater to regulate the temperature. If the test animals failed to flick the noxious stimulus away within 10 seconds, the tail of the animal was removed from the hot water to avoid thermal damage. In the time course of antinociceptive test, we measured the tail-flick latency on Days 2, 4, 6, 8, 10, and 14 at the designated time (10.00 hours) after saline, ultra-low-dose naloxone, or PTX was injected on Day 3 after catheterization.

2.3. Construction of the intrathecal catheter

The intrathecal catheter construction was modified and adapted from our previous study.¹⁹ It was made of an 8 cm polyethylene tube (0.2 mm inner diameter, 3.6 mm outer diameter; Spectranetics, Colorado Springs, CO, USA) and a 3.5 cm silastic tube (Dow Corning, Midland, MI, USA). The silastic tube was inserted onto the polyethylene tube and the joint sealed with epoxy resin and silicon rubber. The dead space of the intrathecal catheter was about 8 μl.

2.4. Spinal cord sample collection

Upon completion of the behavioral test, the rats were sacrificed by exsanguination under isoflurane anesthesia (Abbott Laboratories Ltd., Queenborough, Kent, UK). Laminectomy was performed at the lower edge of the twelfth thoracic vertebra, and the L5-S3 segment of the spinal cord was removed and the dorsal part was used for western blot study. In the time course of methylation analysis, we collected spinal cord samples on Days 2, 4, 6, 8, 10, and 14 after saline, ultra-low-dose naloxone, or PTX injection on Day 3 after catheterization.

2.5. Western blot analysis

The dorsal part of the spinal cord was fractionated into cytosolic, nuclear, and membrane fractions using a cytoplasmic, nuclear, and membrane compartment protein extraction kit as recommended by the manufacturer (Biochain Institute, Inc., Hayward, CA, USA) and stored at −80 °C until subjected to western blotting analysis. Sample loading was checked using mouse anti-rat H3 (Upstate Inc., Lake Placid, NY, USA) antibodies. Samples were separated in 10% SDS-PAGE gel, and electrotransferred to a 0.2 μm PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked for 1 hour at room temperature with 5% albumin from bovine serum (Sigma) in Tris-buffered saline containing 0.1% Tween 20 and incubated overnight at 4 °C with rabbit polyclonal antibodies against rat monomethyl-Histone H3K4, dimethyl-Histone H3K4, or trimethyl-Histone H3K9, and then for 1 hour at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody as appropriate (both from Chemicon, Temecula, CA, USA). After the reaction of the blots with Enhanced Chemiluminescence (ECL) solution (Amersham, Arlington Heights, IL, USA), bound antibody was visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK). Finally, the blots were incubated for 18 min at 56 °C in stripping buffer (62.6 mM Tris-HCl, pH 6.7, 2% SDS, 100 mM mercaptoethanol) and reprobed with a monoclonal mouse anti-H3 antibody as the sample loading control. The density of each band was measured using a computer-assisted imaging analysis system (Gene Tools Match software; Syngene).

2.6. Immunohistochemistry and image analysis

The rats were sacrificed by exsanguination under isoflurane anesthesia and the enlarged lumbar spinal cords (L5 to S3) were immediately removed and embedded in optimal cutting temperature compound (Sakura Finetek Inc., Torrance, CA, USA). Sections (5 μm) were fixed by immersion in ice-cold acetone/methanol (1:1) for 5 min. After washing in ice-cold phosphate-buffered saline, the sections were incubated sequentially with rabbit polyclonal antibodies against rat monomethyl-H3K4, dimethyl-H3K4, or trimethyl-H3K9 antibodies at 4 °C overnight. The sections were then reacted with rhodamine labeled goat anti-rabbit IgG antibody at room temperature for one hour (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The image capture was used an Olympus BX 50 fluorescence microscope (Olympus Optical, Tokyo, Japan) and a Delta Vision disconsolation microscopic system operated by SPOT software (Diagnostic Instruments Inc., Sterling Heights, MI, USA).

2.7. Statistical analysis

All data are presented as mean ± SEM. Statistical analysis was performed using SigmaStat 3.0 software (SYSTAT Software Inc., San Jose, CA, USA). Tail-flick latencies were analyzed using two-way (time and treatment) ANOVA with a posthoc Bonferroni correction followed by subsequent one-way ANOVA (at each time of the experiment). For immunoreactivity data, the intensity of each test band was expressed as the optical density relative to that of the average optical density for the corresponding control band. For statistical analysis, immunoreactivity was analyzed by one-way ANOVA, followed by multiple comparisons with the Student-Newman-Keuls posthoc test. A significant difference was defined as p < 0.05.

3. Results

3.1. PTX induced thermal hyperalgesia and global H3 hypermethylation

Fig. 1 shows the time course of the tail-flick latency test; the average baseline value of control rats was 2.07 ± 0.25 seconds. Intrathecal injection of saline or ultra-low-dose naloxone produced no effect on the normal baseline value. Two days after intrathecal PTX injection, thermal hyperalgesia (0.88 ± 0.12 seconds) was observed; it was associated with an increasing expression of monomethyl-H3K4, dimethyl-H3K4, and trimethyl-H3K9 in spinal dorsal horn nuclei protein extracts (Fig. 2). A significant increase of monomethyl-H3K4, dimethyl-H3K4, and trimethyl-H3K9 expression was observed in PTX-treated rats on Days 2, 4, 6, 8, 10, and 14. At the same time, there was no significant difference in the expression of three types of histone methylation on Days 2, 4, 6, 8, 10, and 14 after saline or ultra-low-dose naloxone injection (data not shown). H3 did not change in the following diverse treatments; therefore total H3 was used as a loading control. The results suggest that the increase of global histone 3 methylation in the spinal cord following PTX administration may play a role in the neuropathic thermal hyperalgesia formation.

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Fig. 1. PTX induced thermal hyperalgesia in rats. Following either PTX (1 μg in 5 μl saline) or saline (5 μl; control) injection, the antinociceptive effect was assessed at Day 2, 4, 6, 8, 10, and 14, expressed by tail-flick latency. #p < 0.05 when compared to the control group. The data are expressed as mean ± SEM and averaged from the indicated number of rats. BL = baseline value; Sal = saline; N = ultra-low-dose naloxone; PTX = pertussis toxin.

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Fig. 2. PTX induced dorsal spinal cord histone methylation in rats. (A) Western blot analysis of monomethyl-H3K4, dimethyl-H3K4, and trimethyl-H3K9 were performed of the nuclear fractions of the spinal cord dorsal horn from PTX (1 μg in 5 μl saline) or saline (5 μl; control) injected rats. Anti-H3 was used as the loading marker for the nuclear fraction. (B) Quantification of the data from Fig. 2A of 5 rats (mean ± SEM). #p < 0.05 compared to the control group.

3.2. Ultra-low-dose naloxone enhances the antinociceptive effect of morphine in PTX-treated rats

As shown in Fig. 3, the maximum antinociceptive effect of morphine (10 μl; intrathecally) was seen 60 minutes after morphine administration in saline control rats and this effect was blocked by the PTX treatment. Similar to our previous studies,17, 18, 19, 20 PTX not only induced thermal hyperalgesia but also reduced the antinociceptive effect of morphine 4 days after PTX injection. Intrathecal morphine alone did not affect the tail-flick latency in PTX-treated rats. However, PTX-induced thermal hyperalgesia was completely blocked by intrathecal ultra-low-dose naloxone when compared PTX-treated rats with PTX + naloxone-treated rats (F = 66.380; p < 0.001); morphine administration further restored morphine's antinociception when compared PTX-treated rats with PTX + naloxone + morphine-treated rats (F = 58.581; p < 0.001).

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Fig. 3. Ultra-low-dose naloxone restores the antinociceptive effect of morphine in PTX-treated rats. On Day 7 after treatment with drugs, rats were placed in plastic restrainers and the environment was kept dim and quiet for antinociception testing. The effect of naloxone pretreatment on the antinociceptive effect of morphine (10 μg in 5 μl saline, intrathecal injection) was examined on Day 4 after intrathecal injection of either PTX or saline. #p < 0.05 when compared to the control group; *p < 0.05 when compared to the PTX group. Data are expressed as mean ± SEM and averaged from the indicated number of rats. P = pertussis toxin, value at 4 days after pertussis toxin injection; Mo = morphine.

3.3. Ultra-low-dose naloxone reverses histone hypermethylation in PTX-treated rats

In western blot analysis, PTX-treated rat spinal cords showed significantly increased expression of monomethyl-H3K4, dimethyl-H3K4, and trimethyl-H3K9 (Fig. 4). Intrathecal administration of morphine or ultra-low-dose naloxone alone did not affect the methylation levels, whereas pretreatment with ultra-low-dose naloxone, 30 minutes prior to morphine injection, significantly down-regulated the methylation levels of H3. In immunohistochemistry analysis, administration of ultra-low-dose naloxone plus morphine administration significantly inhibited the PTX-induced H3 hypermethylation (Fig. 5D–I). These results show that ultra-low-dose naloxone not only enhances the antinociceptive effect of morphine but also down-regulates the hypermethylation of H3 in the spinal cord of PTX-treated rats.

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Fig. 4. Ultra-low-dose naloxone treatment before morphine administration down-regulates histone methylation in PTX-treated rat spinal cords. On Day 7 after drug administration as described above, rats were sacrificed and lumber enlargement (L1-L2) was removed for western blot analysis. (A) Western blot of monomethyl-histone H3K4, dimethyl-histone H3K4, and trimethyl-histone H3K9 was performed on the nuclear fractions of the spinal cord dorsal horn of the indicated treatment groups at 120 minutes after the final morphine injection. (B) Quantification of the data from Fig. 4A of 5 rats (mean ± SEM). Histone 1 was used as the loading control. Data are averaged from 5 rats per group with 20 μg total sample protein of each lane. #p < 0.05 compared to the control group; *p < 0.05 compared to the PTX group.

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Fig. 5. Ultra-low-dose naloxone down-regulates histone methylation in PTX-treated rat spinal cords. On Day 7 after drug administration as described above, rats were sacrificed and lumber enlargement (L1-L2) was removed and cut for immunohistochemistry analysis. Histone methylation staining is shown in red and 4′,6-diamidino-2-phenylindole staining (label for nucleus) is shown in blue. Left panels (A-C), saline controls; center panels (D-F), PTX-treated rats; right panels (G-I), ultra-low-dose naloxone plus morphine intrathecal injection in PTX-treated rats. Scale bar represents 100 μm.

4. Discussion

In the present study, the PTX-induced thermal hyperalgesia was associated with a histone 3 hypermethylation in the dorsal horn of spinal cord of rat, and this was attenuated by the ultra-low-dose naloxone pretreatment with morphine administration. These results show a significant suppressive effect of ultra-low-dose naloxone on the neuropathy in PTX-treated rats.

Histones are DNA-packaging globular proteins that undergo post-translational modifications at specific sites of their N-terminus including acetylation, methylation, phosphorylation, or ubiquitination.²¹ These modifications alter the histone-DNA interaction and therefore the chromatin structure; the consequence can be either transcription activation or repression, depending on the type and site of the modification. Methylation of specific lysine residues within the histone tail is particularly important in defining the histone modification pattern as mutation of the enzymes that catalyze the formation or removal of methyl groups, which affects the physiology of cells. H3 methylation is known to have either positive (H3K4) or negative (H3K9) influence on gene transcription. H3K4 methylation is a marker for transcriptional active genes; methylation of H3K4 occurs in three states: mono-, di- and trimethylated.²² Three methylation levels of H3K4 are differently distributed along with a gene suggesting distinct roles during transcription. H3K4 trimethylation is mainly found around the transcriptional start site for transcription initiation, elongation and RNA processing.23, 24 In contrast, H3K4 dimethylation peaks within the coding region, whereas H3K4 monomethylation accumulates predominantly at the 3′ end of the gene.25, 26 So far, the molecular functions of the different modification states of H3K4 are still not fully understood. H3K9 methylation is involved in gene silencing. Methyl-CpG-binding protein 2, a transcriptional repressor binds to methylated DNA, is associated with the activity of both histone deacetylase and H3K9 methyltransferase. In the present study, we found that intrathecal PTX administration not only induced thermal hyperalgesia but also increased H3K4 mono- and dimethylation, and H3K9 trimethylation expression in the rat spinal dorsal horn. These results indicate that PTX-treatment increases methylation of histone 3 and induces the following epigenetic modification. According to this result, we suggest that PTX treatment may turn on some inflammatory genes and turn off some anti-inflammatory genes. Therefore, we suggest that PTX induces an epigenetic modification that facilitates neuropathic pain formation. Recent literature review further indicates that the importance of inflammatory mediators in the establishment of many pain conditions. There are discourses discussing the influences of epigenetics on the inflammatory processing.²⁷ H3K4 methylation via methyltransferase SET7/9 can affect the recruitment of NF-κB to proinflammatory genes (such as TNF-α).⁸ The peripheral mechanisms underpinning chronic inflammatory pains are controlled by these same mediators⁹ and involve the actions of both glial and neuronal NF-κB transcriptions.¹⁰ Similarly, PTX-treatment significantly increases expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in rat spinal cords¹⁷; we suggest that the epigenetic alterations are likely to play a role for the PTX effect.

Epigenetic techniques such as RNA interference have been employed in pain research to show the contribution of certain proteins to nociception. Certain studies suggest that transcriptional regulation might be a novel treatment for chronic pain; therefore, using epigenetic techniques to inhibit transcription, such as with histone deacetylase (HDAC) inhibitors, of known nociceptive proteins may produce analgesia. HDAC inhibitors have been demonstrated to regulate the expression of opioid receptors.28, 29 Moreover, the analgesic effect of HDAC inhibitors was related to increasing of metabotropic glutamate 2 receptor expression of dorsal root ganglia neurons in the formalin-induced inflammatory pain.³⁰ Wang et al further found that intrathecal 5-azacytidine could inhibit global DNA methylation and MeCP2 expression, which could alleviate the neuropathic pain in injured rats by chronic nerve constriction.³¹ In present study, we found that intrathecal ultra-low-dose naloxone inhibits global histone 3 methylation and attenuates PTX-induced hyperalgesia. According to this result we suggest that ultra-low-dose naloxone may regulate the epigenetic alterations and reverse PTX-induced H3 modification. However, this study has some limitations, in that it only focuses on H3 methylation, and the changes of histone acetylation should be further investigated to complete the whole picture of histone modification during neuropathic pain development and also of their role in morphine tolerance formation.

In comparison with DNA methylation, other epigenetic modification, such as methylation and acetylation of histones, are less well characterized, particularly in neuropathic pain formation. Moreover, histone modification together with DNA methylation seems to have a vital role in organizing nuclear architecture, which in turn involves in regulating transcription and other nuclear processes.³² Methylation of H3 can either be stimulatory or inhibitory to transcription, depending on the particular modified lysine residue.³³ In the case of lysine, it includes formation of mono-, di-, or trimethyl groups, all of which represent a distinct function at the cellular level.

In summary, our results suggest that H3 methylation may play a role in neuropathic pain management. Inhibition of global H3 methylation with drugs such as ultra-low-dose naloxone might be a new therapeutic approach for neuropathic pain management and provide some hints for further studies on the involvement of histone methylation in neuropathic pain.

Disclosure of conflicts of interest

I certify that all my affiliations with or financial involvement in, within the past 5 years and in foreseeable future, any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript are completely disclosed.

Acknowledgments

This study was supported by the grants from the National Science Council (NSC-101-2314-B-281-001-MY3) and the Cathay General Hospital, Taipei, Taiwan (CGH-MR-10113) and was performed at the Neuropathic Pain and Translational Research Laboratory, Cathay Medical Research Institute, Cathay General Hospital, Xizhi, New Taipei City, Taiwan.