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Keywords:

  • dorsal root ganglia;
  • DSS colitis;
  • endogenous opioids;
  • IBD

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

Background  Endogenous opioids are implicated in pain-regulation in chronic inflammatory bowel disease (IBD). We sought to examine whether endogenous opioids suppress the excitability of colonic nociceptive dorsal root ganglia (DRG) neurons during chronic IBD, and if so, whether modulation of underlying voltage-gated K+ currents was involved.

Methods  The effects of chronic dextran sulfate sodium (DSS) colitis on afferent signaling in mice was studied using patch clamp recordings. Colonic DRG neurons were identified using Fast Blue retrograde labeling and recordings obtained from small DRG neurons (<40 pF).

Key Results  In current-clamp recordings, the rheobase of neurons was increased 47% (P < 0.01) and action potential discharge at twice rheobase decreased 23% (P < 0.05) following incubation in colonic supernatants from chronic DSS mice. β-endorphin increased 14-fold, and tissue opioid immunoreactivity and expression in CD4+ cells observed by flow cytometry increased in chronic DSS colons. Incubation of naïve neurons in the μ-opioid receptor agonist D-Ala2, N- MePhe4, Gly-ol (DAMGO) (10 nM) partially recapitulated the effects of supernatants from DSS mice on rheobase. Supernatant effects were blocked by the μ-opioid receptor antagonist naloxone. In voltage clamp, chronic DSS supernatants and DAMGO increased IA K+ currents.

Conclusions & Inferences  The release of endogenous opioids during chronic inflammation in mice suppresses the excitability of nociceptive DRG neurons. Targeting immune cells may provide a novel means of modulating IBD pain.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

A flare-up of inflammatory bowel disease (IBD), such as ulcerative colitis (UC), is typically accompanied by abdominal pain, and for many patients this is one of the most distressing symptoms. The release of pro-inflammatory mediators during this acute flare-up leads to activation and sensitization of peripheral nociceptive dorsal root ganglia (DRG) neurons.1 This increased nociceptive signaling has been demonstrated in human studies, where decreased rectal sensory thresholds have been observed during acute flare-ups of colonic IBD.2 In contrast, studies of visceral nociception in chronic IBD patients have revealed normal or increased tolerance to colorectal distension.3 Consistent with these observations, it has also been reported that in the dextran sulfate sodium (DSS) model of IBD in mice, the visceral hyperalgesia of acute inflammation is no longer present during chronic inflammation.4 Evidence is now growing for a dynamic interplay between nociceptive and antinociceptive factors in IBD-related pain, with contributions that vary with the duration and nature of inflammation.

One proposed peripheral mechanism for both anti-inflammatory and antinociceptive actions are endogenous opioids,5–7 although other peptides and cytokines have been implicated.8–10 Exogenous opioids have been shown to exert an anti-inflammatory effect in mouse IBD models, while μ-opioid receptor knockout mice show increased susceptibility to colitis.6 The expression of μ-opioid receptors is upregulated in ileal and colonic enteric neurons, and the immunocytes and mucosa of IBD patients, a process driven in part by inflammatory cytokines.7 In studies of the mouse DSS model, substance P expression decreases while colonic μ-opioid receptors increase during the progression between acute and chronic colitis. In addition, after chronic DSS inflammation there is increased immunoreactivity for β-endorphin in enteric neurons (in comparison with acute DSS or control mice), while the numbers of mucosal CD4+ T cells are also elevated.4 CD4+ T cells are known to be a source of β-endorphin, and infusion of these cells into severe combined immuno-deficient mice alleviates the visceral hyperalgesia of that condition in a naloxone-sensitive manner.11 Thus, release of endogenous opioids during chronic inflammation could have an analgesic action through several mechanisms, including the reduction of pronociceptive cytokine release due to an anti-inflammatory pathway or through opioids acting directly on the intestinal nociceptive DRG neurons.

Here we investigated changes in sensory signaling in DRG neurons innervating the mouse colon in the chronic DSS model of colitis. We found that chronic DSS supernatant from colonic tissue decreased the excitability of nociceptive DRG neurons and examined whether endogenous opioids were involved.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

DSS Colitis

All experiments were performed according to the guidelines of Queen’s University Animal Care Committee and the Canadian Council of Animal Care. Chronic DSS colitis was induced in male C57/BL6 mice by three cycles of 5 days of 2% DSS in drinking water (wt vol−1; MP Biomedicals, Solon, OH), alternating with 5 days of normal water, totaling 30 days. Mice were euthanized on the fifth day of the third water cycle. Acute DSS colitis was evoked by 5 days of 3% DSS, followed by 2 days of water. Mice were euthanized by isoflurane overdose followed by transcardial perfusion of HBSS and cervical transection.

Supernatant generation and use in DRG culture

Colonic tissues were incubated for 24 h in Roswell Park Memorial Institute (RPMI) medium containing gentamycin/amphotericin B, penicillin/streptomycin and 10% FCS, before removal of supernatant and storage at −80 °C. For colonic supernatant production, full circumference segments of 5-mm length were cut, and each segment incubated in 200 μL of RPMI. These supernatants (100 μL) were then combined with F12 medium (900 μL).

Retrograde dye labeling

Briefly, mice were anesthetized with intraperitoneal ketamine/xylazine (Pfizer; New York, NY and Bayer; Etobicoke, ON respectively). A midline laparotomy was then performed to expose the descending colon. Under a dissecting microscope, 17 mg mL−1 Fast Blue (Polysciences Inc., Warrington, PA, USA) was injected (10 μL per injection) into 5–10 sites along the colon wall. The colon was replaced and the wound sutured.

Cell isolation and culture for electrophysiology

The DRG neurons were enzymatically dissociated from T9 to T13 ganglia as previously described.12,13 Dispersed neurons were suspended in DMEM (pH 7.2–7.3, 10% fetal bovine serum, 100 U mL−1 Penicillin, 0.1 mg mL−1 Streptomycin, and 2 mM glutamine), plated on PureCol-coated (60 μL mL−1) (Inamed Biomaterials, Fremont, CA, USA) cover slips and incubated in a humidified incubator at 95% O2 and 5% CO2. Two hours after cells were applied to cover slips, the wellplates were flooded with the supernatant/F12 medium mixture and incubated overnight (16–23 h) until retrieval for electrophysiological studies.

Electrophysiological recordings

Perforated patch clamp experiments were performed in current or voltage clamp modes at room temperature. Fast Blue labeled neurons were identified by their bright blue fluorescence under brief exposure to ultraviolet light, and only these neurons were recorded from. Only neurons ≤40 pF capacitance were studied because these neurons have been shown to display properties associated with nociceptors, including capsaicin sensitivity presence of long duration action potentials, and resistance of the action potential to TTX.12,14–16 Signals were acquired using an Axopatch 200B amplifier and Digidata 1322A A/D converter (Axon Instruments, San Jose, CA, USA), low-pass filtered at 5 kHz and stored at 20 kHz. Capacitive transients were corrected using analogy circuitry. Inclusion criteria for current-clamp analysis were resting membrane potentials more negative than −40 mV and overshooting action potentials with a hump on the falling phase.12 Perforated patch recordings used Amphotericin B (240 μg mL−1) from Sigma (St. Louis, MO, USA). Solutions; (in mM) extracellular solution: 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 D-glucose, pH 7.4. Pipette solution: 110 K-gluconate, 30 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, pH 7.25. The liquid junction potential was calculated to be 12 mV, and corrected for. For K+ current recordings the following solutions were used; Extracellular: 140 NMDG, 4 KCl, 1.8 HEPES, 1 D-glucose, 1 CaCl2, 1 MgCl2, pH 7.4. Pipette solution: 110 K-gluconate, 30 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, pH 7.2. IA and IK currents were separated on the basis of their biophysical properties. Neither current is significantly inactivated when the membrane potential is held at −100 mV, while IA was inactivated when the membrane potential was held at −60 mV. IK was measured from a holding potential of −60 mV, and measured at 400 ms after the onset of the pulse. IA was isolated by subtracting IK from the total K+ current (recorded from a holding potential of −100 mV). Peak IA was measured as the peak of the transient component of this subtracted current. IA inactivation properties were studied using a two pulse protocol, as previously described.13

Flow cytometry

Lamina propria lymphocytes were separated into CD4+ and CD4− groups by magnetic cell sorting, before treatment with 2% paraformaldehyde and single staining by incubation with rabbit anti-β-endorphin antiserum (1 : 250; Millipore, Billerica, MA, USA) followed by goat anti-rabbit phycoerythrin conjugated antiserum (1 : 100; Millipore) and flow cytometry. More details can be found in the supporting infomation.

Immunohistochemistry and ELISAs

For immunohistochemistry, distal colon was dissected and fixed overnight. Rabbit anti-β-endorphin antiserum (1 : 350; Millipore) was applied overnight. Slides were washed and incubated in donkey anti-rabbit Dylight 549 antiserum (1 : 800; Jackson Immuno Research Laboratories, West Grove, PA, USA) for 2 h. Slides were washed and mounted with DAPI mounting media (Vector Laboratories, Burlington, Ontario). β-endorphin ELISAs were performed on the supernatants of homogenized and centrifugated colons, using a rabbit anti-β-endorphin polyclonal antibody (Abcam, Cambridge, MA, USA) with an ELISA kit following the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). Additional details can be found in the supporting information.

Statistical analysis

Results were analyzed with unpaired Student’s t-test, Mann–Whitney tests, one-way or two-way anova (with Dunnett’s or Bonferroni post hoc tests, respectively) with P < 0.05 representing significance. Data are expressed as mean ± SEM. Fitting of patch clamp electrophysiological data was done using the Boltzmann equation fit function in Prism 5.0 (Graphpad, La Jolla, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

Effects of DSS colitis on DRG neuronal excitability

To ascertain whether antinociceptive factors were being produced by inflamed colons, isolated DRG neurons from uninflamed mice were incubated with supernatants from the inflamed colons of chronic DSS mice (Fig. 1). Supernatants from chronic DSS mouse colons reduced the intrinsic excitability of DRG neurons by increasing the rheobase almost 50% compared with the effects of control supernatants (P = 0.0018 by unpaired t-test; controls, n = 34; chronic DSS, n = 24), and reducing the action potential number at twice rheobase (P = 0.023 by Mann–Whitney test). Resting membrane potential was hyperpolarized compared with controls (−69.56 ± 1.00 mV, n = 20 vs−63.57 ± 1.46 mV, n = 21 respectively, P = 0.0018), while input resistance was significantly increased (chronic DSS: 2039 ± 183 MΩ, n = 20; controls: 1322 ± 118 MΩ, n =  21, P = 0.002).

image

Figure 1.  Incubation of naïve dorsal root ganglia (DRG) neurons in supernatant from colons of chronic dextran sulfate sodium (DSS)-treated mice decreases neuronal excitability. Current-clamp recordings of fast blue labeled colonic DRG neurons to assess neuronal excitability. (A) Representative traces from naïve DRG neurons incubated overnight with supernatant from either control or chronic DSS colons. (B) Summary data showing rheobase was increased after overnight incubation with supernatant from chronic DSS supernatant (P = 0.0018). (C) The number of action potentials at twice rheobase was significantly decreased after incubation with supernatant from chronic DSS colons (P = 0.0230, Mann–Whitney test).

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As endogenous opioids may be mediating this reduced excitability,4 their expression levels in colons from chronic DSS colitis animals was compared with those of acute DSS (5 days) and controls. In acute DSS the levels of β-endorphin found in colonic tissue by ELISA was unchanged vs controls (Fig. 2A). However, chronic DSS produced a 14-fold increase in β-endorphin. Immunohistochemical studies of colonic mucosal tissue also revealed a marked increase in β-endorphin immunoreactivity in chronic DSS mice compared with controls (Fig. 2B). Single staining flow cytometry was used to analyze the β-endorphin expression of cell types in the lamina propria of control and chronic DSS-treated mice. CD4+ and CD11b+ cells expressing β-endorphin were increased approximately twofold in the chronic DSS treatment group compared with controls (Fig. 2C).

image

Figure 2.  β-endorphin expression is increased in mouse colons after chronic DSS, but not acute DSS. (A) Concentration of β-endorphin as assayed by ELISA, from colons of control, acute DSS and chronic DSS mice. Chronic DSS colons showed a marked increase in expression (P = 0.0319). (B) Immunohistochemistry confirms increased immunoreactivity for β-endorphin in the mucosa of chronic DSS colons compared with controls. The arrow indicates a group of β-endorphin immunoreactive cells in the lamina propria. (C) Representative histograms show the immunoreactivity for β-endorphin in CD4+ lymphocytes (left panels) obtained from control and chronic DSS-treated mice. β-endorphin expression was quantified using flow cytometry (gray areas under curves in upper panels). β-endorphin was significantly enhanced in CD4+ cells from DSS mice compared with controls (summarized in lower left panel) (P = 0.0354; n = 5 control, n = 7 DSS). Representative histograms of β-endorphin in CD11b+ cells (right panels) from control and DSS-treated mice show a similar increase of β-endorphin in DSS CD11b+ cells (summarized in lower right panel) (P = 0.0175; n = 4 control, n = 5 DSS).

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To investigate whether these endogenous opioids were capable of producing the observed effects of chronic DSS supernatant on DRG neurons, isolated DRG neurons from control mice were incubated overnight in low concentrations (10 nM) of the selective μ-opioid receptor agonist DAMGO, and perforated patch current-clamp measures of intrinsic excitability compared with those of control neurons (Fig. 3A). We focused on the μ-opioid receptor due to previous reports of the importance of this subtype of opioid receptor in colonic inflammation and pain.6,7 The DAMGO-incubated neurons had a significantly increased rheobase vs control (P < 0.0001, n = 28 cells in each group), and were hyperpolarized (DAMGO-treated: 59.20 ± 0.65 mV, n = 28; controls: −56.20 ± 1.06 mV, n = 28; P = 0.019). To test whether opioid receptor activation may underlie the reduction of DRG excitability by chronic DSS supernatants, neurons were co-incubated with supernatant and the μ-receptor antagonist naloxone (10 μM). Naloxone blocked the effect of supernatants from mice with chronic DSS colitis on the excitability of DRG neurons (Fig. 3B).

image

Figure 3.  The effect of chronic DSS colonic supernatants was reproduced by DAMGO and blocked by naloxone. (A) Rheobase was increased by 10 nM DAMGO after overnight incubation (= 0.0002). The number of action potentials at twice rheobase remained unchanged. (B) 10 μM Naloxone decreased the effect of colonic chronic DSS supernatants on the rheobase of DRG neurons (P = 0.0057). Naloxone had no effect by itself on control supernatants incubated cells (P = 0.6032). The number of action potentials at twice rheobase was not significantly changed. (C) DRG neurons from mice that had undergone chronic DSS showed increases in rheobase similar to those produced by DAMGO on naïve DRG neurons (P < 0.01). Subsequent incubation of 10 nM DAMGO with these neurons from DSS mice produced no additional reductions in excitability.

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To examine the effect of chronic exposure to endogenous opioids on neuronal excitability, the intrinsic excitability of colonic DRG neurons isolated from chronic DSS mice was compared with those of control mice. Like DRGs exposed to chronic DSS colonic supernatant or DAMGO, the rheobase was significantly increased (P < 0.001; chronic DSS, n = 19; control, n = 17) (Fig. 3C). Interestingly, incubation of DRG neurons from mice with chronic DSS colitis with 10 nM DAMGO caused no further decrease in excitability (Fig. 3C).

The mechanism of the reduction of neuronal excitability by endogenous opioids was further investigated by using voltage clamp techniques to characterize voltage-gated K+ currents. Fig. 4A shows typical raw-current recordings of step-voltage protocols from different holding potentials to measure whole cell K+ current including and following the inactivation of IA. Compared with control supernatants, incubation of DRG neurons in chronic DSS supernatant significantly enhanced IA current density (P < 0.0001 by two-way anova plus Bonferroni post-test; cDSS, n = 9; control, n = 11), while delayed rectifier (IK) current amplitudes were significantly reduced (P < 0.0001 by two-way anova; cDSS, n = 9; control, n = 11) (Fig. 4B), which was consistent with the −15 mV shift in V50 observed in the IK inactivation curve (P < 0.0001). Overall, the peak total K+ current was significantly enhanced (P = 0.0034 by two-way anova; cDSS, n = 8; control, n = 11). Incubation with 10 nM DAMGO reproduced the enhancement of total and IA currents seen with chronic DSS supernatants (both P < 0.0001 by two-way anova; 10 nM DAMGO, n = 8; control, n = 7; Fig. 4C). However, unlike the effects seen with supernatants, DAMGO incubation did not affect the amplitude of IK.

image

Figure 4.  DAMGO and Chronic DSS supernatants increase the IA voltage-gated potassium current. (A) Representative DRG neuronal currents obtained by manipulating the holding potential (Vh). Total current (left panel, from Vh = −100), non-inactivating sustained IK type current (IK, middle panel, from Vh = −40 mV), subtraction of the sustained from the total current yields the transient, inactivating ‘A’ type current (IA, right panel). (B) Left panels: total DRG K+ currents were significantly increased after incubation with supernatants from chronic DSS mice (P = 0.0034 by two-way anova), however, the component potassium currents underwent differential effects; after chronic DSS supernatants IA currents were increased while IK currents were decreased. (C) Right panels: Incubation of DRG neurons with 10 nM DAMGO increased total K+ currents. IA currents were increased while IK currents remained unchanged. Two-way anova with Bonferroni post tests. *P < 0.05, **P < 0.01, ***P < 0.001.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

There is growing evidence that nociceptive signaling during inflammation is modulated by both pro and antinociceptive factors released from inflamed tissues.4 In previous studies of human UC patients with an acute flare-up of their disease, we have shown that inflammatory mediators secreted by colonic biopsies caused marked hyperexcitability (decreased rheobase and increased action potential discharge) of colonic DRG neurons when compared with biopsies from healthy controls.1 This pro-excitatory effect is consistent with other human studies where testing of pain thresholds to balloon distension in acute UC demonstrated visceral hyperalgesia.2 This visceral hyperalgesia, however, was lost in chronic inflammation in both human studies of chronic colitis3 and mouse chronic DSS colitis.4 In the present study, we have examined how the inflammatory milieu during chronic inflammation of the colon in the mouse DSS model of chronic colitis modulates the intrinsic excitability of colonic DRG neurons. In contrast to our previous studies,1 we found that colonic DRG neurons exposed to these factors exhibited a marked reduction in their excitability. These data suggest that antinociceptive factors can predominate, depending on the duration and state of the inflammatory response.

There are a number of potential antinociceptive mediators released during inflammation, including endocannabinoids and activators of proteinase-activated receptor 4,17–21 but several lines of evidence suggest that endogenous opioids are the key mediators underlying the effects observed in this study. We found a dramatic increase in the levels of endogenous ß-endorphin in colons from chronic DSS mice compared with acute DSS and controls, and increased immunoreactivity of cells in the lamina propria. Flow cytometry demonstrated increased numbers of β-endorphin immunoreactive CD4+ lymphocytes and CD11b+ macrophages. These findings are consistent with studies in the somatic and visceral nervous system4,11,22,23 suggesting that the release of opioids from immunocytes recruited during chronic inflammation could counter the pronociceptive effects of mediators such as TNFα1 during ongoing inflammation. It has been suggested that the enteric nervous system may also be an important source of endogenous opioids in the viscera,4,11 given the increased expression of opioids during chronic inflammation, although this has not been observed in human studies.5,23 To provide direct functional evidence that opioids were involved, we tested whether a μ-opioid receptor antagonist could diminish the antinociceptive effects of supernatants from chronically inflamed colons on DRG neuronal excitability. We found that the changes in excitability were completely reversed in the presence of naloxone. Moreover, the exogenous application of the μ-opioid receptor DAMGO mimicked the effect of the supernatant from the inflamed colon. Together, these data suggest that endogenous opioids are the key mediators of the antinociceptive actions on neuronal excitability and that colonic immunocytes, and possibly enteric neurons, are the primary source.

Our data also provide new insights into the mechanisms by which endogenous opioids exert an antinociceptive action during chronic inflammation. Based on previous studies multiple mechanisms were implied,24–26 including suppression of pronociceptive mediators, modulation of descending inhibition at the level of the spinal cord, or direct effects on the properties of nociceptive neurons such as modulation of TRPV1 channels. We show that the endogenous opioids have a profound effect on the intrinsic excitability of the colonic sensory neurons during chronic inflammation and thus this action could play a significant role in suppression of nociceptive signaling from the inflamed colon. Recent studies of the effects of human peripheral blood mononuclear cell supernatants from healthy controls on mouse colon multi-unit afferent recordings also detected an antinociceptive action of endogenous opioids,27 but how this is manifest in human IBD is unknown.

The opioid-mediated changes in rheobase and action potential discharge suggest altered activity of underlying voltage-gated ion channels that regulate action potential electrogenesis. We found a corresponding increase in IA current in response to both the supernatants and exogenous application of DAMGO (Fig. 4), providing further evidence that endogenous opioids regulate neuronal excitability and that IA is one of the ionic targets. IA plays an important role in determining AP threshold and interspike interval in many neuronal classes.28 Therefore, an increase in IA amplitude is consistent with the observed increase in rheobase. Interestingly, the supernatants, but not DAMGO, also decreased IK currents (Fig. 4) which might be expected to increase neuronal excitability. This opposing action suggests an interplay between pronociceptive and antinociceptive factors on the ionic currents during chronic inflammation. Further studies are needed to determine if opioids may also act to modulate Nav currents, given their role in electrogenesis and known neuroplasticity.13 The hyperpolarization of resting membrane potential produced by supernatants and DAMGO may also contribute to decreased excitability. This, together with the increase in input resistance produced by supernatants, indicates other channels active at rest may also have been affected.

In addition to the antinociceptive effects of inflammatory supernatants on naïve DRG neurons, we also found that the intrinsic excitability of colonic DRG neurons isolated from chronic DSS mice was reduced suggesting that tolerance to chronic endogenous opioid exposure had not occurred. This is consistent with the reported ability of endogenous opioids to sustain μ-receptor sensitivity on sensory nerves during somatic inflammatory models.29 Intriguingly, treatment of these neurons with 10 nM DAMGO did not further decrease their excitability. The endogenous opioids implicated by this study are unlikely to have been present at maximal effective concentrations, but even so, might have be expected to produce tolerance or even paradoxical excitatory effects, possibly via a switch to Gs-coupling of the μ-opioid receptor.30 A possible explanation is that Gi/o mediated effects on intrinsic excitability of low concentrations of endogenous μ-opioid receptor agonists reach a downstream saturation point, and are of a sustained nature. For example, these changes may result from altered levels of expression, changes in K+ channel phosphorylation and/or membrane insertion of potassium channel subunits. Hence, following removal from the chronic opioid environment, it is possible that no additional effects of exogenous agonist would occur.

In summary, we have shown that the release of endogenous opioids from the colon during chronic inflammation in a mouse model causes a significant decrease in nociceptive signaling through activation of μ-opioid receptors. Other studies suggest that κ opioid receptors may also play an antinociceptive role in visceral inflammation but it is unknown whether endogenous opioids also act at these receptors during chronic inflammation.31 We have provided direct evidence for μ-opioid receptor modulation of neuronal excitability and underlying voltage-gated ion channels. Given the dual sensory and local effector role of these neurons i.e. release of substance P and calcitonin gene-related peptide (CGRP) leading to neurogenic inflammation,32 it is also possible that some of the antinociceptive actions of peripheral opioids in colitis6,7 may indirectly result from a reduction of the neurogenic ‘pronociceptive’ component of inflammation, by reducing CGRP and substance P release in the periphery.24–26 Thus, targeting specific immune cells to release opioids and inhibit DRG neurons could result in a dual benefit on pain expression by directly decreasing neuronal excitability and indirectly by reducing the generation of pronociceptive inflammatory mediators.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

This study was funded by the Crohn’s and Colitis Foundation of Canada (SV and AL). MMM was supported by a CAG-CIHR fellowship, RGA was supported by a postdoctoral fellowship CONACYT-144872, and EVM by a postdoctoral fellowship CONACYT-166615.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

EVM, RGA, FOC, JB, IS, and MMM performed the research, AL, DH, and SV designed the research study, EVM, RGA, FOC, JB, IS, and MMM analyzed the data and EVM, RGA, JB, IS, DH, AL, and SV wrote the article.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. References
  11. Supporting Information

Data S1. Methods.

FilenameFormatSizeDescription
NMO_12008_sm_Supplementary-Methods.doc51KSupporting info item

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