ORIGINAL ARTICLE
Reduced antinociceptive responsiveness to hyperbaric oxygen in opioid-tolerant mice Y. Zhang1, P.A. Stolz2, D.Y. Shirachi4, R.M. Quock1,2,3 1 Department of Integrative Physiology and Neuroscience and Graduate Program in Neuroscience, College of Veterinary Medicine, Washington State University, Pullman, USA 2 Department of Psychology, Washington State University, Pullman, USA 3 Translational Addiction Research Center, Washington State University, Pullman, USA 4 Department of Physiology and Pharmacology, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, USA
Correspondence Raymond M. Quock E-mail: [email protected] Funding sources This research was supported by NIH Grant AT-007222 and the Allen I. White Distinguished Professorship at Washington State University. Involvement of funder in study design, data collection, data analysis, manuscript preparation and/or publication decisions: None declared. Conflicts of interest None declared. Accepted for publication 22 November 2013 doi:10.1002/j.1532-2149.2013.00448.x
Abstract Background: Hyperbaric oxygen (HBO2) therapy can produce analgesia in patients experiencing various conditions of chronic pain. Previously, we reported that naloxone antagonized the acute antinociceptive effect of both brief (11 min) and longer (60 min) HBO2 treatments. This implied a possible role for opioid receptors in the antinociceptive effects of HBO2. Objectives: The aim of this study was to determine whether mice previously rendered as tolerant to opioid agonists would exhibit a reduced antinociceptive responsiveness to HBO2. Methods: Male NIH Swiss mice were given repeated injections of the opioid agonists morphine, fentanyl or (-)-U50488H over 4 days to induce tolerance at their respective opioid receptors. Mice receiving saline according to a similar injection schedule served as a vehicle control group. On day 5, 15 h after the last injection, mice received either an antinociceptive challenge dose of the opioid agonists or a 30-min HBO2 exposure at 3.5 atmosphere absolute. The antinociception was then assessed by the 0.6% acetic acid-induced abdominal constriction test. Results: The results showed that mice rendered as tolerant to morphine, fentanyl or (-)-U50488H pretreatment all exhibited reduced antinociceptive responsiveness to themselves and HBO2. Conclusions: These results demonstrated that both μ- and κ-opioid receptors are involved in mediation of the acute antinociceptive response to HBO2.
1. Introduction Hyperbaric oxygen (HBO2) therapy is the intermittent inhalation of 100% oxygen in a hyperbaric chamber at pressures higher than 1.0 atmosphere absolute (ATA). The Food and Drug Administration (FDA) has approved HBO2 therapy for fourteen clinical conditions (Gesell, 2008; Undersea & Hyperbaric Medical Society, 2013). When breathing normobaric air at sea level, the arterial blood oxygen pressure is about 100 mmHg and the tissue oxygen pressure is about 55 mmHg, allowing 3 mL oxygen/L blood to be delivered. However, 100% oxygen at 3.0 ATA can bring © 2014 European Pain Federation - EFIC®
blood oxygen pressure to 2000 mmHg and tissue oxygen pressure to 500 mmHg, allowing 60 mL oxygen/L blood to be delivered. This blood concentration of oxygen is sufficient to provide the amount of oxygen that tissues need without haemoglobin contribution (Tibbles and Edelsberg, 1996; Gill and Bell, 2004). Although it has not yet been approved in the treatment of neuropathic pain, HBO2 has been shown to reduce pain in both experimental animals (Thompson et al., 2010; Li et al., 2011; Gu et al., 2012; Hui et al., 2013) and human subjects (Wilson et al., 1998; Kiralp et al., 2004; Yildiz et al., 2004; Gu et al., 2012). Eur J Pain •• (2014) ••–••
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Reduced HBO2 antinociception in opioid-tolerant mice
What’s already known about this topic? • Our laboratory has demonstrated the involvement of central opioid pathways in HBO2induced antinociception. What does this study add? • Both μ- and κ-opioid receptors are involved in the mechanism of HBO2-induced antinociception. • The reduced antinociceptive effect of HBO2 after repeated administrations of opioids should be taken into consideration for the possible/ expected future application of HBO2 as pain treatment.
However, the precise mechanism of HBO2-induced antinociception remains elusive. There are limited studies on the mechanism of HBO2 in animals, and some researchers have implicated a peripheral antiinflammatory effect of HBO2 that contributes to pain relief (Warren et al., 1979; Wilson et al., 2006, 2007; Hui et al., 2013). Previously, in an acute pain model, the acetic acidinduced abdominal constriction test, we involved opioid receptors in the antinociceptive effect of HBO2 by demonstrating sensitivity of the response to antagonism by intraperitoneal (i.p.) pretreatment with the opioid receptor blocker naltrexone (Zelinski et al., 2009a). A later study showed that intracerebroventricular (i.c.v.) administration of naltrexone reduced HBO2-induced antinociception in mice, thus indicating that the opioid mechanism resided in the central nervous system (Chung et al., 2010). In a rat model of neuropathic pain induced by sciatic nerve crush, we demonstrated that i.c.v. infusion of naltrexone during HBO2 exposure abolished the antiallodynic effect produced by HBO2 (Gibbons et al., 2013). These results support the hypothesis that a central opioid modulating system is responsible for the relief of pain caused by HBO2. Repeated activation of opioid receptors can result in tolerance, in which the responsiveness of the receptors to agonist drugs diminishes over time. Accordingly, human subjects and animals gradually lose their sensitivity to opioid drugs upon repeated drug administration. As a further demonstration of the role of opioid receptors in HBO2-induced antinociception, the present study was designed to ascertain whether mice previously rendered as tolerant to opioid agonists would exhibit a reduced antinociceptive responsiveness to HBO2. 2 Eur J Pain •• (2014) ••–••
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2. Material and methods 2.1 Animals Male NIH Swiss mice, weighing 18–22 g, were purchased from Harlan Laboratories (Indianapolis, IN) and used in this investigation. These experiments were approved by the Washington State University Institutional Animal Care and Use Committee with post-approval review and carried out in accordance with The Guide for the Care and Use of Laboratory Animals, 8th Edition (National Academies Press, Washington, DC, 2010). Mice were housed five per cage in the Animal Resource Unit at Washington State University with access to food and water ad libitum. The facility, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, was maintained on a regular 12-h light:dark cycle (lights on 07:00–19:00 h) under standard conditions of temperature (22 ± 1 °C) and humidity (33%). Mice were kept in the holding room for at least 3 days following arrival in the facility and prior to experimentation.
2.2 Antinociceptive testing Antinociceptive responsiveness was assessed using the abdominal constriction test. Mice were treated i.p. with 0.1 mL per 10 g body weight of 0.6% acetic acid. Exactly 5 min later, the number of abdominal constrictions – lengthwise stretches of the torso with concave arching of the back – in each animal was counted for 6 min. The degree of antinociception (inhibition of abdominal constrictions) produced in various treatment groups of mice was calculated by the following formula:
# constrictions # constrictions − in control mice in treated mice % antinociception = 100 × # constrictions in control mice
2.3 HBO2 treatment Mice were placed in a B-11 research hyperbaric chamber (Reimers Systems, Inc., Lorton, VA). The chamber was ventilated with 100% O2, U.S.P. (A-L Compressed Gases Inc., Spokane, WA) at a flow rate of 20 L/min to minimize N2 and CO2 accumulation. The pressure within the cylindrical clear acrylic chamber (27.9 cm diameter × 55.9 cm L) was increased from 1.0 to 3.5 ATA over 2 min. The mice breathed spontaneously during HBO2 treatment. After completion of the HBO2 treatment, mice were then decompressed over 2–3 min. To investigate the time course of the antinociception produced by a 30-min HBO2 treatment, mice were exposed to HBO2 at 3.5 ATA for 30 min. Then, different groups of mice were assessed for antinociception using the acetic acid constriction assay at different time points (0.25, 0.5, 1.0 and 2.0 h) after the completion of HBO2 treatment.
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In preliminary experiments, treatment with HBO2 failed to produce any observable adverse effects. In another study, even multiple HBO2 treatments failed to produce oxidative stress in mice as determined by measurement of protein carbonyls and malondialdehyde in brain, spinal cord or lung tissue (Liu et al., in press).
2.4 Drugs The following drugs were used in this research: medicalgrade oxygen from A-L Compressed Gases, Inc. (Spokane, WA); morphine sulfate injection, U.S.P. from West-Ward Pharmaceuticals (Eatontown, NJ); fentanyl from SigmaAldrich, Co. (St. Louis, MO); (-)-U50488H from Tocris Bioscience (Bristol, UK). The opioid agonists were prepared in 0.9% sodium chloride solution from Hospira, Inc. (Lake Forest, IL).
2.5 Experimental design 2.5.1 Dose–response curves To produce dose–response curves for the opioid agonists, mice were randomly divided into groups of at least eight mice each and administered different opioid agonists at different doses as follows: morphine sulfate [subcutaneous (s.c.) ], 0.03, 0.1, 0.3, 1 and 3 mg/kg; fentanyl (s.c.), 0.3, 1, 3, 10, 30, 100 μg/kg; and (-)-U50488H (i.p.), 0.1, 0.3, 1, 3 and 10 mg/kg. Twenty-five minutes later, mice received i.p. injections of 0.6% acetic acid and constrictions were counted starting 5 min later. After the calculation of the percent antinociception for each mouse using the formula above, the dose–response curves and the half-maximum effect doses (D50) for each agonist were generated using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA). This determined the appropriate challenge doses of each drug for assessment of opioid tolerance.
2.5.2 Induction and assessment of opioid tolerance Antinociceptive tolerance was induced in mice by repeated injections of morphine sulfate, fentanyl or (-)-U50488H over a 4-day period. For tolerance to morphine, mice were given s.c. injections of morphine sulfate at 08:00 and 16:00 h every day for four consecutive days. The dose of morphine sulfate was 20 mg/kg on the first 3 days and 40 mg/kg on the last day (Liang et al., 2007). For tolerance to fentanyl, a modified method from a previous study was used to develop tolerance (Popik et al., 2000). Briefly, mice were given s.c. injections of 40 μg/kg fentanyl at 08:00, 12:00, 16:00 and 20:00 h every day for 4 days. For tolerance to (-)-U50488H, mice were treated with i.p. injections of 25 mg/kg (-)-U50488H at 08:00 and 16:00 h every day for 4 days (Bhargava et al., 1997). Control mice received repeated injections of saline for 4 days.
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Reduced HBO2 antinociception in opioid-tolerant mice
On day 5, antinociceptive responsiveness was assessed by administration of a challenge dose of the respective opioid agonist: 1.0 mg/kg morphine sulfate; 30 μg/kg fentanyl; or 3.0 mg/kg (-)-U50488H. Opioid agonists were administered 25 min before 0.6% acetic acid injection. Five minutes later, the abdominal constrictions were counted for 6 min. To assess the antinociceptive effect of HBO2 on day 5, mice were exposed to HBO2 at 3.5 ATA for 30 min then returned to room air. One hour later, the antinociceptive responsiveness of the mice was assessed by acetic acid-induced constriction assay.
2.6 Statistical analysis The GraphPad Prism 6 was used to graph dose–response curves and to conduct statistical analysis. The dose–response curves and the D50 values were generated by non-linear regression. The bottom and top limits to calculate D50 values were set as 0 and 100% antinociception. The percent antinociceptive responses between the control and the experimental groups in tolerance assessment were compared using a one-way analysis of variance (ANOVA) with a post hoc Dunnett’s multiple comparisons test.
3. Results 3.1 Dose–response curves of opioid agonists Fig. 1 shows the dose–response curves of the antinociceptive effects of the opioid agonists. The D50 of each agonist are listed in Table 1. For each agonist, the log dose in the dose–response curve that is the next higher than its D50 value was used to assess the tolerance. Therefore, 1.0 mg/kg morphine sulfate (95.1 ± 2.7% antinociception, n = 10), 30 μg/kg fentanyl (71.0 ± 9.1% antinociception, n = 10) and 3.0 mg/kg (-)U50488H (72.8 ± 12.5% antinociception, n = 10) were chosen for later assessment.
3.2 Development of tolerance to opioid agonists by repeated injections In Fig. 2A, 1.0 mg/kg morphine sulfate produced 95.0 ± 2.7% antinociception in naïve mice and 83.3 ± 6.0% antinociception in saline-pretreated mice. There was no significant difference between these two groups. However, morphine-pretreated mice exhibited only a 23.5 ± 7.6% antinociceptive effect, which was significantly lower than the responses in naïve mice (p < 0.0001) and saline-pretreated mice (p < 0.0001), indicating the development of tolerance to morphine sulfate. At a dose of 30 μg/kg, fentanyl produced 71.0 ± 9.1% antinociception in naïve mice and 49.2 ± 13.9% Eur J Pain •• (2014) ••–••
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Figure 1 Dose–response curves of (A) morphine sulfate, (B) fentanyl and (C) (-)-U50488H. Mice were treated with morphine sulfate (0.03, 0.1, 0.3, 1 or 3 mg/kg), fentanyl (0.3, 1, 3, 10, 30 or 100 μg/kg) and (-)-U50488H (0.03, 0.1, 0.3, 1, 3 or 10 mg/kg). Antinociception was assessed 30 min later using the 0.6% acetic acid abdominal constriction assay. Data are presented as the mean ± standard error of the mean (SEM). The doses that produced halfmaximum response (D50) were listed in Table 1.
Table 1 D50 values of morphine sulfate, fentanyl and (-)-U50488H. Opioid agonist
D50 (± SEM) (mg/kg)
Morphine sulfate Fentanyl (-)-U50488H
0.38 (±0.14) 12.79 (±6.49)×10−3 1.03 (±0.27)
SEM, standard error of the mean.
antinociception in saline-pretreated mice, which were not significantly different from each other. Fentanylpretreated mice exhibited an 8.0 ± 12.4% antinociceptive response (Fig. 2B), which was significantly decreased compared with the responses in naïve mice (p < 0.01) and saline-pretreated mice (p < 0.1). Similarly, 3.0 mg/kg (-)-U50488H induced 72.8 ± 12.5%
Figure 2 Antinociceptive tolerance to (A) morphine sulfate, (B) fentanyl and (C) (-)-U50488H. (A) Mice received 4 days of repeated subcutaneous (s.c.) injections of saline (n = 10), morphine sulfate (n = 18) or did not receive any injection in 4 days (naïve group, n = 10). On day 5, mice were challenged with 1.0 mg/kg morphine sulfate. (B) Mice received 4 days of repeated s.c. injections of saline (n = 10), fentanyl (n = 10) or did not receive any injections in 4 days (naïve group, n = 10). On day 5, mice were challenged with 30 μg/kg fentanyl. (C) Mice received 4 days of repeated intraperitoneal (i.p.) injections of saline (n = 10), (-)U50488H (n = 10) or did not receive any injections in 4 days (naïve group, n = 10). On day 5, mice were challenged with 3.0 mg/kg (-)-U50488H. Each column represents the mean percent of antinociceptive response + standard error of the mean (SEM). Significance of difference: **p < 0.01, ****p < 0.0001 compared with naïve group; # p < 0.1, # # p < 0.01, # # # # p < 0.0001 compared with saline group.
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Reduced HBO2 antinociception in opioid-tolerant mice
antinociception in naïve mice and 74.1 ± 7.0% antinociception in saline-pretreated mice, which were not significantly different from one another. (-)U50488H-pretreated mice displayed a 13.0 ± 15.5% antinociceptive response to 3.0 mg/kg to (-)U50488H, significantly lower than that of the naïve mice (p < 0.01) or the saline-pretreated mice (p < 0.01) (Fig. 2C).
3.3 Antinociceptive effect of HBO2 in opioid-tolerant mice According to the 2-h time course shown in Fig. 3A, after a 30-min HBO2 treatment, naïve mice exhibited an antinociceptive response that appeared to be increasing within the first hour and stable for the second hour. However, there was no significant difference in the antinociceptive effect at any two time points. The 30-min HBO2 treatment produced a 74.9 ± 8.4% antinociceptive effect 1 h after the completion of the treatment in naïve mice. This 1-h time point was chosen to assess the antinociceptive effect of HBO2 treatment in opioid-tolerant mice. As shown in Fig. 3B, a 30-min HBO2 treatment produced a 72.2 ± 7.7% antinociception in salinepretreated mice, comparable to the HBO2-induced response in non-pretreated mice. However, mice that were rendered tolerant to morphine, fentanyl or (-)U50488H all exhibited significantly reduced antinociceptive responses to HBO2 treatment. Although HBO2 continued to evoke an antinociceptive effect in morphine-tolerant mice – albeit significantly reduced – under the current pretreatment dose regime (32.9 ± 9.6%, p < 0.1), the antinociceptive effect of HBO2 in mice tolerant to fentanyl (4.7 ± 18.1%, p < 0.0001) or (-)-U50488H (0.4 ± 5.3%, p < 0.0001) was virtually abolished. These results demonstrated that mice tolerant to these μ- and κ-opioid agonists do not respond to HBO2 treatment, indicating that both μ- and κ- opioid receptors play an important role in the mechanism of HBO2-induced antinociception.
4. Discussion Previous findings from our laboratory have demonstrated that a central opioid pathway is involved in the HBO2-induced antinociceptive effect (Zelinski et al., 2009a; Chung et al., 2010). Initial experiments in this study were conducted with morphine, and morphine-tolerant mice were indeed not sensitive to HBO2 treatment, indicating an involvement of μ-opioid receptors. Although it is primarily a μ-opioid receptor agonist, morphine can also © 2014 European Pain Federation - EFIC®
Figure 3 Reduced antinociceptive effect induced by 30-min HBO2 exposure after repeated administrations of different opioid agonists. (A) The 2-h time course of the antinociceptive effect produced by 30-min HBO2 exposure. Mice were treated with HBO2 at 3.5 atmosphere absolute (ATA) for 30 min. Different groups of HBO2-treated mice were assessed for antinociception at different time points (0.25, 0.5, 1 and 2 h, n = 15–19 for each time point). (B) Mice in different groups received 4 days of repeated injections of saline (n = 14), morphine sulfate (n = 10), (-)-U50488H (n = 14) or fentanyl (N = 9), or did not receive any injections (naïve group, n = 15). On day 5, mice in different groups received HBO2 exposure at 3.5 ATA for 30 min and the antinociceptive effect was assessed 60 min after the exposure by 0.6% acetic acid constriction assay. Each column represents the mean percent antinociceptive response + standard error of the mean (SEM) Significance of difference: *p < 0.1, ****p < 0.0001 compared with naïve group; # p < 0.01, # # # # p < 0.0001 compared with saline group.
bind to κ-opioid receptor at higher doses (Pan et al., 1997; Gutstein and Akil, 2006). The high doses of morphine sulfate (20 and 40 mg/kg) used to induce tolerance could plausibly cause activation of κ-opioid receptors, and it is possible that the reduced responsiveness to HBO2 in morphine-tolerant mice might Eur J Pain •• (2014) ••–••
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actually be, at least in part, a κ-opioid receptor phenomenon. To determine whether this might be the case, we subjected additional groups of mice to repeated injections of fentanyl or (-)-U50488H to induce selective tolerance at the μ- and κ-opioid receptors, respectively. Mice tolerant to the κ-selective opioid receptor agonist (-)-U50488H exhibited reduced responsiveness to HBO2 treatment. This is in agreement with the results of an earlier study in which the antinociceptive effect of a 60-min HBO2 treatment was antagonized by i.c.v. administration of a rabbit antiserum against rat dynorphin (Zelinski et al., 2009a). These results are consistent with our hypothesis that HBO2 treatment induces the release of dynorphin and the activation of a κ-opioid receptor pain-relieving pathway. Our results also show that mice tolerant to the μ-selective opioid receptor agonist fentanyl exhibited reduced responsiveness to HBO2 treatment. However, a previous study showed that supraspinal administration of a single dose of β-endorphin antiserum failed to reduce the HBO2 antinociception (Zelinski et al., 2009a). The fact that we did not observe antagonism of HBO2 by β-endorphin antiserum does not mean that it is contradictory to the involvement of μ-opioid receptor. First, as a κ-endogenous opioid peptide, dynorphin is also a weak μ-opioid receptor agonist (Gutstein and Akil, 2006). It is possible that the dynorphin antiserum prevented dynorphin from binding both κ- and μ-opioid receptors. Second, it is possible that the single dose of β-endorphin antiserum was not sufficient to produce a significant effect, which led to the appearance that β-endorphin release was not involved. According to the present study, fentanyl produced a robust reduction in HBO2 antinociceptive effect, indicating that the μ-opioid receptor is largely involved in the antinociception produced by HBO2 treatment. Lastly, we cannot exclude the possibility that HBO2 treatment may cause the release of endomorphins, which are endogenous opioid peptides that are highly selective for μ-opioid receptors but will not react with β-endorphin antiserum (Zadina et al., 1997; Ide et al., 2000; Mizoguchi et al., 2000). It is worthy of mention that the antinociception produced by 30-min HBO2 treatment was almost abolished in mice tolerant to either fentanyl or (-)U50488H, suggesting that the functional loss of either μ- or κ-opioid receptor is able to completely antagonize HBO2 antinociception. It further implies that, in the mechanism of HBO2-induced antinociception, these two receptors are possibly working in a way that the activation of one receptor by HBO2stimulated release of its endogenous opioid peptide 6 Eur J Pain •• (2014) ••–••
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can cause the release of the endogenous opioid peptide of the other receptor in order to activate it, which eventually leads to pain relief. It has been reported that the supraspinal and spinal administration of endomorphin-2 can stimulate μ-opioid receptors, which, in turn, can trigger the release of dynorphin to activate κ-opioid receptors (Mizoguchi et al., 2006; Sakurada et al., 2008). Further investigation is required to understand the exact interaction between μ- and κ-opioid receptors in HBO2 antinociceptive mechanism. One possible mechanism to explain the reduced HBO2 antinociceptive effect in opioid-tolerant mice is cross-tolerance. Cross-tolerance occurs when repeated use of a drug causes the reduction in response not only in that drug but also in other drugs (O’Brien, 2006). It is possible that HBO2 treatment stimulates the release of endogenous opioid peptides. In opioid-tolerant mice, it might be expected that these opioid peptides fail to initiate the downstream events that lead to pain relief. However, we cannot draw such a conclusion without further mechanistic study. We reported several earlier studies in which the exposure of mice to HBO2 at 3.5 ATA produced antinociception in the acetic acid abdominal constriction model (Ohgami et al., 2009; Chung et al., 2010; Quock et al., 2011). The antinociceptive responses were found sensitive not only to opioid antagonism, but also to the inhibition of nitric oxide (NO) function by NO scavenger (Quock et al., 2011), or the inhibition of NO production by nitric oxide synthase (NOS) inhibitors, antisense oligodeoxynucleotides directed against neuronal NOS (nNOS), and nNOS knockout mice (Ohgami et al., 2009). It is not yet known whether there is a direct connection between NO production and endogenous opioid peptides release after HBO2 exposure; however, there is evidence that NO may regulate neuronal release of endogenous opioid peptides in antinociception (Zelinski et al., 2009b; Ohgami et al., 2010). The antinociceptive effects in mice produced by L-arginine, the substrate for NOS and the immediate precursor of NO, and the NO donor 3-morpholinosydnoimine were both dependent on the generation of NO in the brain and mediated by dynorphin (Chung et al., 2006). The anaesthetic gas nitrous oxide (N2O) produces an NO-dependent antinociceptive effect in rats and mice (McDonald et al., 1994). Exposure to N2O resulted in increased levels of β-endorphin and NOx (oxidation products of NO); these increases were blunted by pretreatment with an NOS-inhibitor (Zelinski et al., 2009b). A more direct association between NO and neuronal release of © 2014 European Pain Federation - EFIC®
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β-endorphin was demonstrated by microdialysis experiments in which the increased dialysate levels of β-endorphin and NOx were antagonized by pretreatment with an NOS inhibitor (Ohgami et al., 2010). Whether HBO2-induced antinociception shares the similar mechanism involving NO-dependent release of endogenous opioid peptides remains to be investigated in the future. While we have consistently found that HBO2induced relief of acute and chronic pain is opioid mediated, it must be acknowledged that there is also evidence of a peripheral anti-inflammatory action in HBO2 mechanism. It has been shown that HBO2 treatment suppressed the production of various inflammatory cytokines, including tumour necrosis factor-α, interleukin 1β and interleukin 6, and also reduced chronic constriction injury (CCI)-induced neuropathic pain (Li et al., 2011) or Freund’s adjuvant pain (Hui et al., 2013). It is also important to acknowledge that in many of the FDA-approved clinical indications of HBO2, it is unlikely that there is a common underlying mechanism (Tibbles and Edelsberg, 1996; Gill and Bell, 2004). It would not be surprising that, with the enriched oxygen delivered to blood and tissue, HBO2 can be a benefit for health and recovery by different mechanisms. Beyond the recognition of the antiinflammatory effect of HBO2 treatment in pain relief, we suggest that HBO2 is also capable of activating an opioid-mediated pathway involving both μ- and κopioid receptors in the central nervous system that is capable of modulating the pain response.
5. Conclusion Mice previously rendered as tolerant to μ- and κ-opioid agonists exhibited significantly reduced antinociceptive responsiveness to HBO2 treatment, suggesting that the HBO2-induced antinociceptive effect in mice is mediated by activation of μ- and κ-opioid pain-modulating pathways. Author contributions Y.Z and P.A.S. performed the animal experiments. Y.Z and R.M.Q. designed the experiments, analysed the data and wrote the manuscript. All authors discussed the findings, interpreted the results and commented on the manuscript.
References Bhargava, H.N., Cao, Y.J., Zhao, G.M. (1997). Effect of 7-nitroindazole on tolerance to morphine, U-50,488H and [D-Pen2, D-Pen5] enkephalin in mice. Peptides 18, 797–800.
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Reduced HBO2 antinociception in opioid-tolerant mice
Chung, E., Burke, B., Bieber, A.J., Doss, J.C., Ohgami, Y., Quock, R.M. (2006). Dynorphin-mediated antinociceptive effects of L-arginine and SIN-1 (an NO donor) in mice. Brain Res Bull 70, 245–250. Chung, E., Zelinski, L.M., Ohgami, Y., Shirachi, D.Y., Quock, R.M. (2010). Hyperbaric oxygen treatment induces a 2-phase antinociceptive response of unusually long duration in mice. J Pain 11, 847–853. Gesell, L.B., ed. (2008). The Hyperbaric Oxygen Therapy Committee Report: Indications and Results, 12th edn (Durham: Undersea and Hyperbaric Medical Society). Gibbons, C., Liu, S.L., Zhang, Y., Sayre, C.L., Levitch, B., Moehlmann, S., Shirachi, D.Y., Quock, R.M. (2013). Involvement of opioid receptors in the anti-allodynic effect of hyperbaric oxygen in rats with sciatic nerve crush-induced neuropathic pain. Brain Res 1537, 111–116. Gill, A.L., Bell, C.N.A. (2004). Hyperbaric oxygen: Its uses, mechanisms of action and outcomes. Q J Med 97, 385–395. Gu, N., Niu, J.Y., Liu, W.T., Sun, Y.Y., Liu, S., Lv, Y., Dong, H.L., Song, X.J., Xiong, L.Z. (2012). Hyperbaric oxygen therapy attenuates neuropathic hyperalgesia in rats and idiopathic trigeminal neuralgia in patients. Eur J Pain 16, 1094–1105. Gutstein, H.B., Akil, H. (2006). Chapter 21. Opioid analgesia. In Goodman & Gilman’s the Pharmacological Basis of Therapeutics, 11th edn L.L. Brunton, J.S. Lazo, K.L. Parker, eds. (New York: The McGraw-Hill Companies, Inc.) pp. 547–590. Hui, J., Zhang, Z., Zhang, X., Shen, Y., Gao, Y. (2013). Repetitive hyperbaric oxygen treatment attenuates complete Freund’s adjuvant-induced pain and reduces glia-mediated neuroinflammation in the spinal cord. J Pain 14, 747–758. Ide, S., Sakano, K., Seki, T., Awamura, S., Minami, M., Satoh, M. (2000). Endomorphin-1 discriminates the mu-opioid receptor from the deltaand kappa-opioid receptors by recognizing the difference in multiple regions. Jpn J Pharmacol 83, 306–311. Kiralp, M.Z., Yildiz, S., Vural, D., Keskin, I., Ay, H., Dursun, H. (2004). Effectiveness of hyperbaric oxygen therapy in the treatment of complex regional pain syndrome. J Int Med Res 32, 258–262. Li, F., Fang, L., Huang, S., Yang, Z., Nandi, J., Thomas, S., Chen, C., Camporesi, E. (2011). Hyperbaric oxygenation therapy alleviates chronic constrictive injury-induced neuropathic pain and reduces tumor necrosis factor-alpha production. Anesth Analg 113, 626– 633. Liang, D.Y., Shi, X., Li, X., Li, J., Clark, J.D. (2007). The β2 adrenergic receptor regulates morphine tolerance and physical dependence. Behav Brain Res 181, 118–126. Liu, S., Shirachi, D.Y., Quock, R.M. (in press). The acute antinociceptive effect of hyperbaric oxygen is not accompanied by an increase in markers of oxidative stress. Life Sci. McDonald, C.E., Gagnon, M.J., Ellenberger, E.A., Hodges, B.L., Ream, J.K., Tousman, S.A., Quock, R.M. (1994). Inhibitors of nitric oxide synthesis antagonize nitrous oxide antinociception in mice and rats. J Pharmacol Exp Ther 269, 601–608. Mizoguchi, H., Narita, M., Wu, H., Narita, M., Suzuki, T., Nagase, H., Tseng, L.F. (2000). Differential involvement of μ1-opioid receptors in endomorphin- and β-endorphin-induced G-protein activation in the mouse pons/medulla. Neuroscience 100, 835–839. Mizoguchi, H., Watanabe, H., Hayashi, T., Sakurada, W., Sawai, T., Fujimura, T., Sakurada, T., Sakurada, S. (2006). Possible involvement of dynorphin A-(1-17) release via mu1-opioid receptors in spinal antinociception by endomorphin-2. J Pharmacol Exp Ther 317, 362–368. O’Brien, C.P. (2006). Chapter 23. Drug addiction and drug abuse. In Goodman & Gilman’s the Pharmacological Basis of Therapeutics, 11th edn L.L. Brunton, J.S. Lazo, K.L. Parker, eds. (New York: The McGraw-Hill Companies, Inc.) pp. 607–627. Ohgami, Y., Chung, E., Quock, R.M. (2010). Nitrous oxide-induced NO-dependent neuronal release of β-endorphin from the rat arcuate nucleus and periaqueductal gray. Brain Res 1366, 38–43. Ohgami, Y., Zylstra, C.C., Quock, L.P., Chung, E., Shirachi, D.Y., Quock, R.M. (2009). Nitric oxide in hyperbaric oxygen-induced acute antinociception in mice. Neuroreport 20, 1325–1329. Pan, Z.Z., Tershner, S.A., Fields, H.L. (1997). Cellular mechanism for anti-analgesic action of agonists of the k-opioid receptor. Nature 389, 382–385.
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Popik, P., Kozela, E., Pilc, A. (2000). Selective agonist of group II glutamate metabotropic receptors, LY354740, inhibits tolerance to analgesic effects of morphine in mice. Br J Pharmacol 130, 1425–1431. Quock, L.P., Zhang, Y., Chung, E., Ohgami, Y., Shirachi, D.Y., Quock, R.M. (2011). The acute antinociceptive effect of HBO2 is mediated by a NO-cyclic GMP-PKG-KATP channel pathway in mice. Brain Res 1368, 102–107. Sakurada, S., Sawai, T., Mizoguchi, H., Watanabe, H., Watanabe, C., Yonezawa, A., Morimoto, M., Sata, T., Komatsu, T., Sakurata, T. (2008). Possible involvement of dynorphin A release via μ1 opioid receptor on supraspinal antinociception of endomorphin-2. Peptides 29, 1554– 1560. Thompson, C.D., Uhelski, M.L., Wilson, J.R., Fuchs, P.N. (2010). Hyperbaric oxygen treatment decreases pain in two nerve injury models. Neurosci Res 66, 279–283. Tibbles, P.M., Edelsberg, J.S. (1996). Hyperbaric-oxygen therapy. N Engl J Med 334, 1642–1648. Warren, J., Sacksteder, M.R., Thuning, C.A. (1979). Therapeutic effect of prolonged hyperbaric oxygen in adjuvant arthritis of the rat. Arthritis Rheum 22, 334–339. Wilson, H.D., Toepfer, V.E., Senapati, A.K., Wilson, J.R., Fuchs, F.N. (2007). Hyperbaric oxygen treatment is comparable to acetylsalicylic acid treatment in an animal model of arthritis. J Pain 8, 924– 930.
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Wilson, H.D., Wilson, J.R., Fuchs, P.N. (2006). Hyperbaric oxygen treatment decreases inflammation and mechanical hypersensitivity in an animal model of inflammatory pain. Brain Res 1098, 126–128. Wilson, J.R., Foresman, B.H., Gamber, R.G., Wright, T. (1998). Hyperbaric oxygen in the treatment of migraine with aura. Headache 38, 112–115. Yildiz, S., Kiralp, M.Z., Akin, A., Keskin, I., Ay, H., Dursun, H., Cimsit, M. (2004). A new treatment modality for fibromyalgia syndrome: Hyperbaric oxygen therapy. J Int Med Res 32, 263–267. Zadina, J.E., Hackler, L., Ge, L.J., Kastin, A.J. (1997). A potent and selective endogenous agonist for the mu-opioid receptor. Nature 386, 499–502. Zelinski, L.M., Ohgami, Y., Chung, E., Shirachi, D.Y., Quock, R.M. (2009a). A prolonged nitric oxide-dependent, opioid-mediated antinociceptive effect of hyperbaric oxygen in mice. J Pain 10, 167–172. Zelinski, L.M., Ohgami, Y., Quock, R.M. (2009b). Exposure to nitrous oxide stimulates a nitric oxide-dependent neuronal release of β-endorphin in ventricular cisternally-perfused rats. Brain Res 1300, 37–40.
Web references Undersea & Hyperbaric Medical Society. (2013) Indications for hyperbaric oxygen therapy [WWW Document]. http://membership.uhms.org/ ?page=Indications.
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Reduced antinociceptive responsiveness to hyperbaric oxygen in opioid-tolerant mice Y. Zhang1, P.A. Stolz2, D.Y. Shirachi4, R.M. Quock1,2,3 1 Department of Integrative Physiology and Neuroscience and Graduate Program in Neuroscience, College of Veterinary Medicine, Washington State University, Pullman, USA 2 Department of Psychology, Washington State University, Pullman, USA 3 Translational Addiction Research Center, Washington State University, Pullman, USA 4 Department of Physiology and Pharmacology, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, USA
Correspondence Raymond M. Quock E-mail: [email protected] Funding sources This research was supported by NIH Grant AT-007222 and the Allen I. White Distinguished Professorship at Washington State University. Involvement of funder in study design, data collection, data analysis, manuscript preparation and/or publication decisions: None declared. Conflicts of interest None declared. Accepted for publication 22 November 2013 doi:10.1002/j.1532-2149.2013.00448.x
Abstract Background: Hyperbaric oxygen (HBO2) therapy can produce analgesia in patients experiencing various conditions of chronic pain. Previously, we reported that naloxone antagonized the acute antinociceptive effect of both brief (11 min) and longer (60 min) HBO2 treatments. This implied a possible role for opioid receptors in the antinociceptive effects of HBO2. Objectives: The aim of this study was to determine whether mice previously rendered as tolerant to opioid agonists would exhibit a reduced antinociceptive responsiveness to HBO2. Methods: Male NIH Swiss mice were given repeated injections of the opioid agonists morphine, fentanyl or (-)-U50488H over 4 days to induce tolerance at their respective opioid receptors. Mice receiving saline according to a similar injection schedule served as a vehicle control group. On day 5, 15 h after the last injection, mice received either an antinociceptive challenge dose of the opioid agonists or a 30-min HBO2 exposure at 3.5 atmosphere absolute. The antinociception was then assessed by the 0.6% acetic acid-induced abdominal constriction test. Results: The results showed that mice rendered as tolerant to morphine, fentanyl or (-)-U50488H pretreatment all exhibited reduced antinociceptive responsiveness to themselves and HBO2. Conclusions: These results demonstrated that both μ- and κ-opioid receptors are involved in mediation of the acute antinociceptive response to HBO2.
1. Introduction Hyperbaric oxygen (HBO2) therapy is the intermittent inhalation of 100% oxygen in a hyperbaric chamber at pressures higher than 1.0 atmosphere absolute (ATA). The Food and Drug Administration (FDA) has approved HBO2 therapy for fourteen clinical conditions (Gesell, 2008; Undersea & Hyperbaric Medical Society, 2013). When breathing normobaric air at sea level, the arterial blood oxygen pressure is about 100 mmHg and the tissue oxygen pressure is about 55 mmHg, allowing 3 mL oxygen/L blood to be delivered. However, 100% oxygen at 3.0 ATA can bring © 2014 European Pain Federation - EFIC®
blood oxygen pressure to 2000 mmHg and tissue oxygen pressure to 500 mmHg, allowing 60 mL oxygen/L blood to be delivered. This blood concentration of oxygen is sufficient to provide the amount of oxygen that tissues need without haemoglobin contribution (Tibbles and Edelsberg, 1996; Gill and Bell, 2004). Although it has not yet been approved in the treatment of neuropathic pain, HBO2 has been shown to reduce pain in both experimental animals (Thompson et al., 2010; Li et al., 2011; Gu et al., 2012; Hui et al., 2013) and human subjects (Wilson et al., 1998; Kiralp et al., 2004; Yildiz et al., 2004; Gu et al., 2012). Eur J Pain •• (2014) ••–••
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What’s already known about this topic? • Our laboratory has demonstrated the involvement of central opioid pathways in HBO2induced antinociception. What does this study add? • Both μ- and κ-opioid receptors are involved in the mechanism of HBO2-induced antinociception. • The reduced antinociceptive effect of HBO2 after repeated administrations of opioids should be taken into consideration for the possible/ expected future application of HBO2 as pain treatment.
However, the precise mechanism of HBO2-induced antinociception remains elusive. There are limited studies on the mechanism of HBO2 in animals, and some researchers have implicated a peripheral antiinflammatory effect of HBO2 that contributes to pain relief (Warren et al., 1979; Wilson et al., 2006, 2007; Hui et al., 2013). Previously, in an acute pain model, the acetic acidinduced abdominal constriction test, we involved opioid receptors in the antinociceptive effect of HBO2 by demonstrating sensitivity of the response to antagonism by intraperitoneal (i.p.) pretreatment with the opioid receptor blocker naltrexone (Zelinski et al., 2009a). A later study showed that intracerebroventricular (i.c.v.) administration of naltrexone reduced HBO2-induced antinociception in mice, thus indicating that the opioid mechanism resided in the central nervous system (Chung et al., 2010). In a rat model of neuropathic pain induced by sciatic nerve crush, we demonstrated that i.c.v. infusion of naltrexone during HBO2 exposure abolished the antiallodynic effect produced by HBO2 (Gibbons et al., 2013). These results support the hypothesis that a central opioid modulating system is responsible for the relief of pain caused by HBO2. Repeated activation of opioid receptors can result in tolerance, in which the responsiveness of the receptors to agonist drugs diminishes over time. Accordingly, human subjects and animals gradually lose their sensitivity to opioid drugs upon repeated drug administration. As a further demonstration of the role of opioid receptors in HBO2-induced antinociception, the present study was designed to ascertain whether mice previously rendered as tolerant to opioid agonists would exhibit a reduced antinociceptive responsiveness to HBO2. 2 Eur J Pain •• (2014) ••–••
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2. Material and methods 2.1 Animals Male NIH Swiss mice, weighing 18–22 g, were purchased from Harlan Laboratories (Indianapolis, IN) and used in this investigation. These experiments were approved by the Washington State University Institutional Animal Care and Use Committee with post-approval review and carried out in accordance with The Guide for the Care and Use of Laboratory Animals, 8th Edition (National Academies Press, Washington, DC, 2010). Mice were housed five per cage in the Animal Resource Unit at Washington State University with access to food and water ad libitum. The facility, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, was maintained on a regular 12-h light:dark cycle (lights on 07:00–19:00 h) under standard conditions of temperature (22 ± 1 °C) and humidity (33%). Mice were kept in the holding room for at least 3 days following arrival in the facility and prior to experimentation.
2.2 Antinociceptive testing Antinociceptive responsiveness was assessed using the abdominal constriction test. Mice were treated i.p. with 0.1 mL per 10 g body weight of 0.6% acetic acid. Exactly 5 min later, the number of abdominal constrictions – lengthwise stretches of the torso with concave arching of the back – in each animal was counted for 6 min. The degree of antinociception (inhibition of abdominal constrictions) produced in various treatment groups of mice was calculated by the following formula:
# constrictions # constrictions − in control mice in treated mice % antinociception = 100 × # constrictions in control mice
2.3 HBO2 treatment Mice were placed in a B-11 research hyperbaric chamber (Reimers Systems, Inc., Lorton, VA). The chamber was ventilated with 100% O2, U.S.P. (A-L Compressed Gases Inc., Spokane, WA) at a flow rate of 20 L/min to minimize N2 and CO2 accumulation. The pressure within the cylindrical clear acrylic chamber (27.9 cm diameter × 55.9 cm L) was increased from 1.0 to 3.5 ATA over 2 min. The mice breathed spontaneously during HBO2 treatment. After completion of the HBO2 treatment, mice were then decompressed over 2–3 min. To investigate the time course of the antinociception produced by a 30-min HBO2 treatment, mice were exposed to HBO2 at 3.5 ATA for 30 min. Then, different groups of mice were assessed for antinociception using the acetic acid constriction assay at different time points (0.25, 0.5, 1.0 and 2.0 h) after the completion of HBO2 treatment.
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In preliminary experiments, treatment with HBO2 failed to produce any observable adverse effects. In another study, even multiple HBO2 treatments failed to produce oxidative stress in mice as determined by measurement of protein carbonyls and malondialdehyde in brain, spinal cord or lung tissue (Liu et al., in press).
2.4 Drugs The following drugs were used in this research: medicalgrade oxygen from A-L Compressed Gases, Inc. (Spokane, WA); morphine sulfate injection, U.S.P. from West-Ward Pharmaceuticals (Eatontown, NJ); fentanyl from SigmaAldrich, Co. (St. Louis, MO); (-)-U50488H from Tocris Bioscience (Bristol, UK). The opioid agonists were prepared in 0.9% sodium chloride solution from Hospira, Inc. (Lake Forest, IL).
2.5 Experimental design 2.5.1 Dose–response curves To produce dose–response curves for the opioid agonists, mice were randomly divided into groups of at least eight mice each and administered different opioid agonists at different doses as follows: morphine sulfate [subcutaneous (s.c.) ], 0.03, 0.1, 0.3, 1 and 3 mg/kg; fentanyl (s.c.), 0.3, 1, 3, 10, 30, 100 μg/kg; and (-)-U50488H (i.p.), 0.1, 0.3, 1, 3 and 10 mg/kg. Twenty-five minutes later, mice received i.p. injections of 0.6% acetic acid and constrictions were counted starting 5 min later. After the calculation of the percent antinociception for each mouse using the formula above, the dose–response curves and the half-maximum effect doses (D50) for each agonist were generated using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA). This determined the appropriate challenge doses of each drug for assessment of opioid tolerance.
2.5.2 Induction and assessment of opioid tolerance Antinociceptive tolerance was induced in mice by repeated injections of morphine sulfate, fentanyl or (-)-U50488H over a 4-day period. For tolerance to morphine, mice were given s.c. injections of morphine sulfate at 08:00 and 16:00 h every day for four consecutive days. The dose of morphine sulfate was 20 mg/kg on the first 3 days and 40 mg/kg on the last day (Liang et al., 2007). For tolerance to fentanyl, a modified method from a previous study was used to develop tolerance (Popik et al., 2000). Briefly, mice were given s.c. injections of 40 μg/kg fentanyl at 08:00, 12:00, 16:00 and 20:00 h every day for 4 days. For tolerance to (-)-U50488H, mice were treated with i.p. injections of 25 mg/kg (-)-U50488H at 08:00 and 16:00 h every day for 4 days (Bhargava et al., 1997). Control mice received repeated injections of saline for 4 days.
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Reduced HBO2 antinociception in opioid-tolerant mice
On day 5, antinociceptive responsiveness was assessed by administration of a challenge dose of the respective opioid agonist: 1.0 mg/kg morphine sulfate; 30 μg/kg fentanyl; or 3.0 mg/kg (-)-U50488H. Opioid agonists were administered 25 min before 0.6% acetic acid injection. Five minutes later, the abdominal constrictions were counted for 6 min. To assess the antinociceptive effect of HBO2 on day 5, mice were exposed to HBO2 at 3.5 ATA for 30 min then returned to room air. One hour later, the antinociceptive responsiveness of the mice was assessed by acetic acid-induced constriction assay.
2.6 Statistical analysis The GraphPad Prism 6 was used to graph dose–response curves and to conduct statistical analysis. The dose–response curves and the D50 values were generated by non-linear regression. The bottom and top limits to calculate D50 values were set as 0 and 100% antinociception. The percent antinociceptive responses between the control and the experimental groups in tolerance assessment were compared using a one-way analysis of variance (ANOVA) with a post hoc Dunnett’s multiple comparisons test.
3. Results 3.1 Dose–response curves of opioid agonists Fig. 1 shows the dose–response curves of the antinociceptive effects of the opioid agonists. The D50 of each agonist are listed in Table 1. For each agonist, the log dose in the dose–response curve that is the next higher than its D50 value was used to assess the tolerance. Therefore, 1.0 mg/kg morphine sulfate (95.1 ± 2.7% antinociception, n = 10), 30 μg/kg fentanyl (71.0 ± 9.1% antinociception, n = 10) and 3.0 mg/kg (-)U50488H (72.8 ± 12.5% antinociception, n = 10) were chosen for later assessment.
3.2 Development of tolerance to opioid agonists by repeated injections In Fig. 2A, 1.0 mg/kg morphine sulfate produced 95.0 ± 2.7% antinociception in naïve mice and 83.3 ± 6.0% antinociception in saline-pretreated mice. There was no significant difference between these two groups. However, morphine-pretreated mice exhibited only a 23.5 ± 7.6% antinociceptive effect, which was significantly lower than the responses in naïve mice (p < 0.0001) and saline-pretreated mice (p < 0.0001), indicating the development of tolerance to morphine sulfate. At a dose of 30 μg/kg, fentanyl produced 71.0 ± 9.1% antinociception in naïve mice and 49.2 ± 13.9% Eur J Pain •• (2014) ••–••
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Figure 1 Dose–response curves of (A) morphine sulfate, (B) fentanyl and (C) (-)-U50488H. Mice were treated with morphine sulfate (0.03, 0.1, 0.3, 1 or 3 mg/kg), fentanyl (0.3, 1, 3, 10, 30 or 100 μg/kg) and (-)-U50488H (0.03, 0.1, 0.3, 1, 3 or 10 mg/kg). Antinociception was assessed 30 min later using the 0.6% acetic acid abdominal constriction assay. Data are presented as the mean ± standard error of the mean (SEM). The doses that produced halfmaximum response (D50) were listed in Table 1.
Table 1 D50 values of morphine sulfate, fentanyl and (-)-U50488H. Opioid agonist
D50 (± SEM) (mg/kg)
Morphine sulfate Fentanyl (-)-U50488H
0.38 (±0.14) 12.79 (±6.49)×10−3 1.03 (±0.27)
SEM, standard error of the mean.
antinociception in saline-pretreated mice, which were not significantly different from each other. Fentanylpretreated mice exhibited an 8.0 ± 12.4% antinociceptive response (Fig. 2B), which was significantly decreased compared with the responses in naïve mice (p < 0.01) and saline-pretreated mice (p < 0.1). Similarly, 3.0 mg/kg (-)-U50488H induced 72.8 ± 12.5%
Figure 2 Antinociceptive tolerance to (A) morphine sulfate, (B) fentanyl and (C) (-)-U50488H. (A) Mice received 4 days of repeated subcutaneous (s.c.) injections of saline (n = 10), morphine sulfate (n = 18) or did not receive any injection in 4 days (naïve group, n = 10). On day 5, mice were challenged with 1.0 mg/kg morphine sulfate. (B) Mice received 4 days of repeated s.c. injections of saline (n = 10), fentanyl (n = 10) or did not receive any injections in 4 days (naïve group, n = 10). On day 5, mice were challenged with 30 μg/kg fentanyl. (C) Mice received 4 days of repeated intraperitoneal (i.p.) injections of saline (n = 10), (-)U50488H (n = 10) or did not receive any injections in 4 days (naïve group, n = 10). On day 5, mice were challenged with 3.0 mg/kg (-)-U50488H. Each column represents the mean percent of antinociceptive response + standard error of the mean (SEM). Significance of difference: **p < 0.01, ****p < 0.0001 compared with naïve group; # p < 0.1, # # p < 0.01, # # # # p < 0.0001 compared with saline group.
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antinociception in naïve mice and 74.1 ± 7.0% antinociception in saline-pretreated mice, which were not significantly different from one another. (-)U50488H-pretreated mice displayed a 13.0 ± 15.5% antinociceptive response to 3.0 mg/kg to (-)U50488H, significantly lower than that of the naïve mice (p < 0.01) or the saline-pretreated mice (p < 0.01) (Fig. 2C).
3.3 Antinociceptive effect of HBO2 in opioid-tolerant mice According to the 2-h time course shown in Fig. 3A, after a 30-min HBO2 treatment, naïve mice exhibited an antinociceptive response that appeared to be increasing within the first hour and stable for the second hour. However, there was no significant difference in the antinociceptive effect at any two time points. The 30-min HBO2 treatment produced a 74.9 ± 8.4% antinociceptive effect 1 h after the completion of the treatment in naïve mice. This 1-h time point was chosen to assess the antinociceptive effect of HBO2 treatment in opioid-tolerant mice. As shown in Fig. 3B, a 30-min HBO2 treatment produced a 72.2 ± 7.7% antinociception in salinepretreated mice, comparable to the HBO2-induced response in non-pretreated mice. However, mice that were rendered tolerant to morphine, fentanyl or (-)U50488H all exhibited significantly reduced antinociceptive responses to HBO2 treatment. Although HBO2 continued to evoke an antinociceptive effect in morphine-tolerant mice – albeit significantly reduced – under the current pretreatment dose regime (32.9 ± 9.6%, p < 0.1), the antinociceptive effect of HBO2 in mice tolerant to fentanyl (4.7 ± 18.1%, p < 0.0001) or (-)-U50488H (0.4 ± 5.3%, p < 0.0001) was virtually abolished. These results demonstrated that mice tolerant to these μ- and κ-opioid agonists do not respond to HBO2 treatment, indicating that both μ- and κ- opioid receptors play an important role in the mechanism of HBO2-induced antinociception.
4. Discussion Previous findings from our laboratory have demonstrated that a central opioid pathway is involved in the HBO2-induced antinociceptive effect (Zelinski et al., 2009a; Chung et al., 2010). Initial experiments in this study were conducted with morphine, and morphine-tolerant mice were indeed not sensitive to HBO2 treatment, indicating an involvement of μ-opioid receptors. Although it is primarily a μ-opioid receptor agonist, morphine can also © 2014 European Pain Federation - EFIC®
Figure 3 Reduced antinociceptive effect induced by 30-min HBO2 exposure after repeated administrations of different opioid agonists. (A) The 2-h time course of the antinociceptive effect produced by 30-min HBO2 exposure. Mice were treated with HBO2 at 3.5 atmosphere absolute (ATA) for 30 min. Different groups of HBO2-treated mice were assessed for antinociception at different time points (0.25, 0.5, 1 and 2 h, n = 15–19 for each time point). (B) Mice in different groups received 4 days of repeated injections of saline (n = 14), morphine sulfate (n = 10), (-)-U50488H (n = 14) or fentanyl (N = 9), or did not receive any injections (naïve group, n = 15). On day 5, mice in different groups received HBO2 exposure at 3.5 ATA for 30 min and the antinociceptive effect was assessed 60 min after the exposure by 0.6% acetic acid constriction assay. Each column represents the mean percent antinociceptive response + standard error of the mean (SEM) Significance of difference: *p < 0.1, ****p < 0.0001 compared with naïve group; # p < 0.01, # # # # p < 0.0001 compared with saline group.
bind to κ-opioid receptor at higher doses (Pan et al., 1997; Gutstein and Akil, 2006). The high doses of morphine sulfate (20 and 40 mg/kg) used to induce tolerance could plausibly cause activation of κ-opioid receptors, and it is possible that the reduced responsiveness to HBO2 in morphine-tolerant mice might Eur J Pain •• (2014) ••–••
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actually be, at least in part, a κ-opioid receptor phenomenon. To determine whether this might be the case, we subjected additional groups of mice to repeated injections of fentanyl or (-)-U50488H to induce selective tolerance at the μ- and κ-opioid receptors, respectively. Mice tolerant to the κ-selective opioid receptor agonist (-)-U50488H exhibited reduced responsiveness to HBO2 treatment. This is in agreement with the results of an earlier study in which the antinociceptive effect of a 60-min HBO2 treatment was antagonized by i.c.v. administration of a rabbit antiserum against rat dynorphin (Zelinski et al., 2009a). These results are consistent with our hypothesis that HBO2 treatment induces the release of dynorphin and the activation of a κ-opioid receptor pain-relieving pathway. Our results also show that mice tolerant to the μ-selective opioid receptor agonist fentanyl exhibited reduced responsiveness to HBO2 treatment. However, a previous study showed that supraspinal administration of a single dose of β-endorphin antiserum failed to reduce the HBO2 antinociception (Zelinski et al., 2009a). The fact that we did not observe antagonism of HBO2 by β-endorphin antiserum does not mean that it is contradictory to the involvement of μ-opioid receptor. First, as a κ-endogenous opioid peptide, dynorphin is also a weak μ-opioid receptor agonist (Gutstein and Akil, 2006). It is possible that the dynorphin antiserum prevented dynorphin from binding both κ- and μ-opioid receptors. Second, it is possible that the single dose of β-endorphin antiserum was not sufficient to produce a significant effect, which led to the appearance that β-endorphin release was not involved. According to the present study, fentanyl produced a robust reduction in HBO2 antinociceptive effect, indicating that the μ-opioid receptor is largely involved in the antinociception produced by HBO2 treatment. Lastly, we cannot exclude the possibility that HBO2 treatment may cause the release of endomorphins, which are endogenous opioid peptides that are highly selective for μ-opioid receptors but will not react with β-endorphin antiserum (Zadina et al., 1997; Ide et al., 2000; Mizoguchi et al., 2000). It is worthy of mention that the antinociception produced by 30-min HBO2 treatment was almost abolished in mice tolerant to either fentanyl or (-)U50488H, suggesting that the functional loss of either μ- or κ-opioid receptor is able to completely antagonize HBO2 antinociception. It further implies that, in the mechanism of HBO2-induced antinociception, these two receptors are possibly working in a way that the activation of one receptor by HBO2stimulated release of its endogenous opioid peptide 6 Eur J Pain •• (2014) ••–••
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can cause the release of the endogenous opioid peptide of the other receptor in order to activate it, which eventually leads to pain relief. It has been reported that the supraspinal and spinal administration of endomorphin-2 can stimulate μ-opioid receptors, which, in turn, can trigger the release of dynorphin to activate κ-opioid receptors (Mizoguchi et al., 2006; Sakurada et al., 2008). Further investigation is required to understand the exact interaction between μ- and κ-opioid receptors in HBO2 antinociceptive mechanism. One possible mechanism to explain the reduced HBO2 antinociceptive effect in opioid-tolerant mice is cross-tolerance. Cross-tolerance occurs when repeated use of a drug causes the reduction in response not only in that drug but also in other drugs (O’Brien, 2006). It is possible that HBO2 treatment stimulates the release of endogenous opioid peptides. In opioid-tolerant mice, it might be expected that these opioid peptides fail to initiate the downstream events that lead to pain relief. However, we cannot draw such a conclusion without further mechanistic study. We reported several earlier studies in which the exposure of mice to HBO2 at 3.5 ATA produced antinociception in the acetic acid abdominal constriction model (Ohgami et al., 2009; Chung et al., 2010; Quock et al., 2011). The antinociceptive responses were found sensitive not only to opioid antagonism, but also to the inhibition of nitric oxide (NO) function by NO scavenger (Quock et al., 2011), or the inhibition of NO production by nitric oxide synthase (NOS) inhibitors, antisense oligodeoxynucleotides directed against neuronal NOS (nNOS), and nNOS knockout mice (Ohgami et al., 2009). It is not yet known whether there is a direct connection between NO production and endogenous opioid peptides release after HBO2 exposure; however, there is evidence that NO may regulate neuronal release of endogenous opioid peptides in antinociception (Zelinski et al., 2009b; Ohgami et al., 2010). The antinociceptive effects in mice produced by L-arginine, the substrate for NOS and the immediate precursor of NO, and the NO donor 3-morpholinosydnoimine were both dependent on the generation of NO in the brain and mediated by dynorphin (Chung et al., 2006). The anaesthetic gas nitrous oxide (N2O) produces an NO-dependent antinociceptive effect in rats and mice (McDonald et al., 1994). Exposure to N2O resulted in increased levels of β-endorphin and NOx (oxidation products of NO); these increases were blunted by pretreatment with an NOS-inhibitor (Zelinski et al., 2009b). A more direct association between NO and neuronal release of © 2014 European Pain Federation - EFIC®
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β-endorphin was demonstrated by microdialysis experiments in which the increased dialysate levels of β-endorphin and NOx were antagonized by pretreatment with an NOS inhibitor (Ohgami et al., 2010). Whether HBO2-induced antinociception shares the similar mechanism involving NO-dependent release of endogenous opioid peptides remains to be investigated in the future. While we have consistently found that HBO2induced relief of acute and chronic pain is opioid mediated, it must be acknowledged that there is also evidence of a peripheral anti-inflammatory action in HBO2 mechanism. It has been shown that HBO2 treatment suppressed the production of various inflammatory cytokines, including tumour necrosis factor-α, interleukin 1β and interleukin 6, and also reduced chronic constriction injury (CCI)-induced neuropathic pain (Li et al., 2011) or Freund’s adjuvant pain (Hui et al., 2013). It is also important to acknowledge that in many of the FDA-approved clinical indications of HBO2, it is unlikely that there is a common underlying mechanism (Tibbles and Edelsberg, 1996; Gill and Bell, 2004). It would not be surprising that, with the enriched oxygen delivered to blood and tissue, HBO2 can be a benefit for health and recovery by different mechanisms. Beyond the recognition of the antiinflammatory effect of HBO2 treatment in pain relief, we suggest that HBO2 is also capable of activating an opioid-mediated pathway involving both μ- and κopioid receptors in the central nervous system that is capable of modulating the pain response.
5. Conclusion Mice previously rendered as tolerant to μ- and κ-opioid agonists exhibited significantly reduced antinociceptive responsiveness to HBO2 treatment, suggesting that the HBO2-induced antinociceptive effect in mice is mediated by activation of μ- and κ-opioid pain-modulating pathways. Author contributions Y.Z and P.A.S. performed the animal experiments. Y.Z and R.M.Q. designed the experiments, analysed the data and wrote the manuscript. All authors discussed the findings, interpreted the results and commented on the manuscript.
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