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Venlafaxine prevents morphine antinociceptive tolerance: The role of neuroinflammation and the l -arginine-nitric oxide pathway
Experimental Neurology 303 (2018) 134–141
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Research paper
Venlafaxine prevents morphine antinociceptive tolerance: The role of neuroinflammation and the L-arginine-nitric oxide pathway
T
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Mohammad Taghi Mansouria,b, , Bahareh Naghizadehc, Behnam Ghorbanzadehd, ⁎ Soheila Alboghobeishc, Neda Amirgholamic, Gholamreza Houshmande, Omar Caulif, a
Department of Pharmacology, School of Pharmacy, Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Neuroanesthesia Laboratory, Department of Anesthesiology, Emory University School of Medicine, 3B South, Emory University Hospital, 1364 Clifton Road, NE Atlanta, GA 30322, USA c Department of Pharmacology, School of Pharmacy, Toxicology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran d Department of Pharmacology, School of Medicine, Dezful University of Medical Sciences, Dezful, Iran e Department of Pharmacology, School of Medicine, Mazandaran University of Medical Sciences, Mazandaran, Iran f Department of Nursing, University of Valencia, Valencia, Spain b
A R T I C L E I N F O
A B S T R A C T
Keywords: Morphine tolerance Venlafaxine Cytokines L-arg-NO pathway Neuroinflammation Oxidative stress Brain-derived neurotrophic factor
Opioid-induced neuroinflammation and the nitric oxide (NO) signal-transduction pathway are involved in the development of opioid analgesic tolerance. The antidepressant venlafaxine (VLF) modulates NO in nervous tissues, and so we investigated its effect on induced tolerance to morphine, neuroinflammation, and oxidative stress in mice. Tolerance to the analgesic effects of morphine were induced by injecting mice with morphine (50 mg/kg) once a day for three consecutive days; the effect of co-administration of VLF (5 or 40 mg/kg) with morphine was similarly tested in a separate group. To determine if the NO precursor L-arginine hydrochloride (Larg) or NO are involved in the effects rendered by VLF, animals were pre-treated with L-arg (200 mg/kg), or the NO synthesis inhibitors N(ω)-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg) or aminoguanidine hydrochloride (AG; 100 mg/kg), along with VLF (40 mg/kg) for three days before receiving morphine for another three days. Nociception was assessed with a hot-plate test on the fourth day, and the concentration of tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-10, brain-derived neurotrophic factor, NO, and oxidative stress factors such as total thiol, malondialdehyde content, and glutathione peroxidase (GPx) activity in the brain was also determined. Co-administration of VLF with morphine attenuated morphine-induced analgesic tolerance and prevented the upregulation of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), NO, and malondialdehyde in brains of mice with induced morphine tolerance; chronic VLF administration inhibited this decrease in brain-derived neurotrophic factor, total thiol, and GPx levels. Moreover, repeated administration of L-arg before receipt of VLF antagonized the effects induced by VLF, while L-NAME and AG potentiated these effects. VLF attenuates morphine-induced analgesic tolerance, at least partly because of its anti-inflammatory and antioxidative properties. VLF also appears to suppress the development of morphine-induced analgesic tolerance through an L-arg–NO-mediated mechanism.
1. Introduction The administration of narcotics is the most effective means of attenuating severe pain in a wide range of conditions. Morphine is a widely used analgesic drug, however, multiple preclinical and clinical studies have shown that its chronic administration is associated with the development of analgesic tolerance (Hutchinson et al., 2011). A growing body of evidence has shown that opioid-induced tolerance is a complex phenomenon involving multiple behavioral and cellular adaptations, including alterations in neuronal plasticity at the cellular, ⁎
synaptic, and network levels in the central nervous system (Ueda et al., 2003; Von Zastrow, 2004). The role of cytokines in the development of morphine tolerance has been extensively studied (Kapasi et al., 2000; Pacifici et al., 2000). Indeed, chronic morphine treatment causes both neuronal and non-neuronal cells to release of several inflammatory mediators involved in the development of tolerance, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). There are also reports that suppressing neuroinflammation by inhibiting microglial activation and proinflammatory cytokines, TNF-α, IL-1β, and IL-6, can enhance the antinociceptive effect of morphine and
Corresponding authors. E-mail addresses: [email protected], [email protected] (M.T. Mansouri), [email protected] (O. Cauli).
https://doi.org/10.1016/j.expneurol.2018.02.009 Received 12 October 2017; Received in revised form 13 January 2018; Accepted 14 February 2018 Available online 15 February 2018 0014-4886/ © 2018 Elsevier Inc. All rights reserved.
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USA). Morphine sulfate and venlafaxine hydrochloride were donated by the Temad Pharmaceuticals (Tehran, Iran) and Darupakhsh Pharmaceuticals (Tehran, Iran), respectively. All the drugs were dissolved or suspended in 0.9% NaCl and buffered to pH 7.3. The drug dilutions were freshly prepared so that the required dose could be injected in a volume of 5 mL/kg of body weight; the respective animal controls received only the vehicle. Morphine was administered subcutaneously (s.c.) and all the other drugs were administered intraperitoneally (i.p.). DTNB (2,20-dinitro-5,50-dithiodibenzoic acid), TBA (2-thiobarbituric acid), n-butanol, tris base, ethylenediaminetetraacetic acid disodium, sodium acetate, glacial acetic acid, phosphoric acid, potassium chloride, and tetramethoxypropane were obtained from Merck (Darmstadt, Germany).
inhibit the formation of morphine tolerance (Song and Zhao, 2001). Brain-derived neurotrophic factor (BDNF) has trophic effects on the structures and functional plasticity of central synapses in the mammalian brain (Park and Poo, 2013). It has a well-documented pronociceptive role in inflammatory pain responses, acting at the descending brain-stem pain pathways (Merighi et al., 2008). Moreover, BDNF is essential for neuronal system adaptations in response to chronic opiate exposure, including cyclic AMP pathway superactivation, tyrosine hydroxylase upregulation, and restoration of neuronal firing (Akbarian et al., 2002). In contrast, chronic morphine administration induces oxidative stress in the brain (Ozmen et al., 2007) and furthermore, overactivation of the glutamatergic system is accompanied by increased formation of reactive oxygen species (Alekseenko et al., 2009). The action of glutamate, mediated through N-methyl-D-aspartate (NMDA) receptors, subsequently activates nitric oxide (NO) synthase (NOS) and the formation of NO (Kone et al., 2003). In addition, several free radical scavengers and NOS inhibitors suppress the development of morphine tolerance and dependence. Thus, oxidative stress and NO play a role in morphine tolerance (Mori et al., 2007). Venlafaxine (VLF), a bicyclic phenylethylamine derivative and serotonin and norepinephrine reuptake inhibitor, is widely used to treat major depression (Fournier et al., 2010). Like other antidepressants, it can relieve several pain conditions, including neuropathic pain; it also has anti-inflammatory (Hajhashemi et al., 2015) and antioxidant (Di Napoli et al., 2001) effects and regulates neurotrophic factors (Wu et al., 2008). VLF has been reported to inhibit NOS activity in the brain, causing subsequent NO release inhibition (Dhir and Kulkarni, 2007; Krass et al., 2011). A NO mechanism is thought to be involved in VLF's protective effect against acute immobilization and stress-induced behavioral and biochemical alterations (Kumar et al., 2009), and this mechanism has been implicated in some of the drug's most important functions in the nervous system (Dhir and Kulkarni, 2007; Uzbay, 2008; Krass et al., 2011; Gaur and Kumar, 2010). Polypharmacy is one option for improving the side-effect profile of opioid analgesics. Therefore, the aim of this work was to evaluate the potential role of VLF in preventing morphine antinociceptive tolerance by altering BDNF levels, oxidative stress, and neuroinflammation in mouse brains; we also assessed the involvement of the L-arg–NO pathway in mediating these effects.
2.3. Induction and assessment of morphine tolerance The animals were rendered tolerant to morphine using a previously published method (Zarrindast et al., 2002). After a morphine challenge test-dose on day one, tolerance was induced by administering morphine (50 mg/kg; s.c.) once daily at 9.00 A.M. for three consecutive days. On day four, the animals were assessed for tolerance as previously described (Way et al., 1969); the loss of the antinociceptive effects of morphine were tested using a hot-plate sensitivity test 15, 30, 60, and 90 min after a challenge-dose injection of 50 mg/kg morphine (Way et al., 1969; Zarrindast et al., 2002). Each animal was placed on a 52 ± 1 °C hot plate which was surrounded by a clear acrylic cage, and the latency time(s) to either hind paw licking or jumping (whichever came first) was recorded. To avoid tissue damage, the cut-off time was 15 s (Eddy and Leimbach, 1953). 2.4. Experimental groups To assess the effect of VLF in the development of morphine analgesic tolerance, we administered VLF (5 or 40 mg/kg, i.p.) once a day for three consecutive days, 30 min prior to the injection of morphine (50 mg/kg, s.c.); hot-plate tests were performed on the fourth day. In another experiment, to investigate the role of NO in VLF-mediated tolerance modulation, L-arg (200 mg/kg), L-NAME (30 mg/kg), AG (100 mg/kg), or saline (5 mL/kg) were injected with each dose of VLF (Fig. 1); these doses were chosen based on previous studies (Homayoun et al., 2002a; Mansouri et al., 2014b, 2013; Schreiber et al., 1999; Way et al., 1969; Zarrindast et al., 2002; Homayoun et al., 2002b).
2. Materials and methods 2.1. Animals
2.5. Brain sample collection Adult male Swiss mice (weighing 25–30 g) obtained from the central animal house of the Ahvaz Jundishapur University of Medical Sciences (Iran) were used in this study; they were housed at 22 ± 2 °C with 12 h light/dark cycles (light from 7:00 to 19:00) with free access to food and water. The animals were randomly divided into groups of 6–8, acclimatized to the laboratory environment for at least one week before commencing the experiments, and used only once. All the animal experiments were carried out in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animals. The Institutional Animal Ethics Committee at the Ahvaz Jundishapur University of Medical Sciences, formed under the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Reg. No. APRC), approved the experimental protocols. All the behavioral tests were performed by an investigator who was blinded to the interventions.
At the end of experiments, the animals were decapitated and the brain tissue was quickly removed, rinsed with saline, and frozen at −80 °C until their use. The tissues were homogenized in 0.1 M phosphate buffer (pH 7.4) to give a 10% homogenate suspension. The homogenate was centrifuged at 10,000g for 15 min, and aliquots of the supernatant were separated and used for biochemical experiments. 2.6. Antioxidant assays 2.6.1. Lipid peroxidation assay The level of thiobarbituric acid-reactive substances (TBARS) produced by free radicals, an index of lipid peroxidation, was measured. Malondialdehyde (MDA) reacts with TBA to produce a red complex with a peak absorbance at 532 nm. Briefly, 3 mL phosphoric acid (1%) and 1 mL TBA (0.6%) were added to 0.5 mL of homogenate in a centrifuge tube and the mixture was heated for 45 min in a boiling water bath. After cooling, 4 mL n-butanol was added and the mixture was vortexed for 1 min followed by centrifugation at 2000g for 20 min. The colored layer was transferred to a fresh tube and its absorbance was measured at 532 nm. The levels of TBARS were determined using 1,1,3,3-tetramethoxypropane as a standard; the standard curve of MDA
2.2. Drugs The nitric oxide precursor, L-arginine hydrochloride (L-arg), NO synthase inhibitor, N(ω)-nitro-L-arginine methyl ester hydrochloride (LNAME), and selective inducible NOS inhibitor, aminoguanidine hydrochloride (AG) were purchased from Sigma-Aldrich (St. Louis, MO, 135
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Fig. 1. Timeline of the experimental schedule in the hot-plate test (HP). (A) Acute antinociceptive effect of venlafaxine and role of L-arg/NO/cGMP pathway. (B) Effects of venlafaxine on the development of morphine tolerance and role of L-arg/NO/cGMP pathway.
was constructed over a 0–20 μM concentration range (Naghizadeh et al., 2013). MDA concentration was expressed as nmol per gram of wet tissue.
sulfanilamide, and 2.5% phosphoric acid] as previously described (Green et al., 1982). Equal volumes of supernatant and Griess reagent were mixed; the mixture was incubated for 10 min at room temperature in the dark and the absorbance was determined with spectrophotometer at 540 nm. The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and expressed as a percentage of the control. Nitrite level was expressed as μM per gram of wet tissue.
2.6.2. Glutathione peroxidase assay The concentration of glutathione (GSH) peroxidase (GPx) was measured with a GPx kit (Randox Labs, Crumlin, UK). GPx concentration was expressed as unit per gram of wet tissue. 2.6.3. Total sulfhydryl groups assay Total sulfhydryl (–SH) groups were measured using DTNB; this reagent reacts with these groups to produce a yellow complex with a peak absorbance at 412 nm (Ellman, 1959). Briefly, 1 mL Tris-EDTA buffer (pH 8.6) was added to 50 μL homogenate in 2 mL cuvettes and the sample absorbance was read at 412 nm against Tris-EDTA buffer alone (A1). Then, 20 μL DTNB reagent (10 mM in methanol) was added to the mixture and, after 15 min at room temperature, the sample absorbance was read again (A2). The absorbance of the DTNB reagent was also read as a blank (B). The total thiol concentration (mM) was calculated from the following equation:
2.7.3. Brain-derived neurotrophic factor measurement BDNF was measured with an enzyme-linked immunosorbent assay kit (Emax™ ImmunoAssay Systems, Promega, Madison, Wisconsin, USA) following the manufacturer's instructions. Briefly, the BDNF standard or brain samples were distributed in 96-well immunoassay plates pre-coated with monoclonal anti-mouse BDNF antibody (100 μL/ well) and were incubated for 20 h at room temperature. After washing, the plates were incubated with an anti-human BDNF antibody for 2 h at room temperature. The plates were washed again and then incubated with an anti-IgG horseradish peroxidase for 1 h at room temperature. Tetramethylbenzidine/peroxidase substrate solution was added to the wells to produce a colorimetric reaction which was measured at 450 nm with a microplate reader (Dynatech MR 5000, Dynatech Laboratories, Chantilly, Virginia, USA). BDNF concentrations were determined from the regression line for the BDNF standard incubated under similar conditions in each assay. BDNF level was expressed as nanogram per gram of wet tissue The sensitivity of the assay was about 15 pg/mg and the cross-reactivity with other related neurotrophic factors (nerve growth factor, neurotrophin-3 and -4) is considered nil (Aloe et al., 1999).
Total thiol concentration (mM) = (A2−A1 − B) × 1.07/0.05 × 13.6 The sulfhydryl content is inversely correlated to protein oxidative damage. 2.7. Biochemical assays 2.7.1. Proinflammatory cytokine measurement Brain levels of TNF-α, IL-6, IL1β, and IL-10 were determined by enzyme immunoassay kits, as previously described (de Waal Malefyt et al., 1991) and according to the manufacturer's guidelines (eBioscience, Vienna, Austria). The absorbance of the produced color was measured at 450 nm using microplate reader. Cytokines levels were expressed as picogram per gram of wet tissue.
2.8. Statistical analysis To evaluate the global analgesic effect and compare the different treatment groups, the area under the curve (AUC; 15–90 min) of the hot-plate latency was calculated using the trapezoidal rule, based on the observed values (Ghavimi et al., 2014). The results are presented as means ± the standard error of the mean (SEM). One-way analysis of variance (ANOVA) and Tukey tests were used to compare: the effects of (1) repeated VLF administrations compared to baseline data at the first
2.7.2. Nitrite measurement The accumulation of nitrite in the supernatant, an indicator of the production of NO, was determined with a colorimetric assay with Griess reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% 136
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Fig. 3. Effect of L-arginine (L-Arg), L-NAME and aminoguanidine (AG) on antinociceptive effect venlafaxine (VLF) in combination with morphine (MPH) in morphine tolerant mice in the mouse hot-plate test. Data are expressed as the under the curve of the time course curves (AUC). Bars are the means ± S.E.M. for 6–8 animals. *P < 0.05 compared to animals treated with normal saline (NS) in morphine (MPH)-dependent mice, #P < 0.05 and ##P < 0.01 compared to NS + VLF, as determined by two-way ANOVA followed by Bonferroni's test.
3.2. Effect of L-arginine, Nω-nitro-L-arginine methyl ester, or aminoguanidine on venlafaxine-induced inhibition of antinociceptive tolerance in morphine-dependent mice Concurrent i.p. administration of L-NAME and VLF (40 mg/kg each, i.p.) 30 min before each morphine injection for three consecutive days enhanced the inhibitory effect of VLF on the development of morphine tolerance (Fig. 3); this inhibitory effect was potentiated by concurrent i.p. administration of AG and antagonized by concurrent i.p. administration of L-arg [F(5.33) = 57.05, P < 0.01]. 3.3. Effect of venlafaxine in combination with L-arginine, Nω-nitro-Larginine methyl ester, or aminoguanidine on brain malondialdehyde, total thiol, nitrite levels, and glutathione peroxidase activity in morphinedependent mice
Fig. 2. Effects of venlafaxine (VLF) on nociceptive latency responses (A) and the area under the curve (AUC) of the time course curves (B) in morphine tolerant mice in the mouse hot-plate test. The latency to nociceptive responses was measured 15, 30, 60 and 90 min after challenge dose of morphine (5 mg/kg). Data are expressed as the means ± S.E.M. Each group consisted of 8 mice. **P < 0.01 compared with the normal saline (NS) at day 1, ##P < 0.01 compared with the normal saline at day 4 (one-way ANOVA with Tukey's test).
Repeated s.c. administration of 50 mg/kg morphine for three consecutive days resulted in a progressive increase in brain MDA and nitrite levels as well as a decrease in total brain thiol levels and GPx activity. Moreover, administration of VLF 30 min before each morphine injection for three consecutive days significantly inhibited morphineinduced increases in brain MDA and nitrite levels (Fig. 4A and D) and the decreases in thiol levels and GPx activity were inhibited by pretreatment with VLF (Fig. 4B and C). The inhibitory effect of VLF (40 mg/kg) on brain MDA [F(5.24) = 73.00, P < 0.01] and serum nitrite levels [F(5.30) = 26.27, P < 0.01] in morphine-dependent mice was increased by concurrent i.p. administration of L-NAME or AG. In addition, co-administration of these agents with VLF enhanced its effect of increasing total brain thiol levels [F(5.24) = 25.57, P < 0.01] and GPx activity [F(5.24) = 24.31, P < 0.01] which was inhibited in morphine-dependent mice.
injection and (2) co-administration of VLF with other agents and morphine, and (3) the overall combined effects of these drugs at each administration (Mansouri et al., 2014a). P-values < 0.05 were considered statistically significant. All calculations and statistical analyses were done with GraphPad Prism v6 (GraphPad Software Inc., San Diego, CA, USA).
3. Results 3.1. Effects of venlafaxine on the tolerance to morphine analgesia Receipt of a morphine injection for three consecutive days caused a decline in its antinociceptive effects, indicating analgesic tolerance [F (4.65) = 5.32, P < 0.01; Fig. 2A]. In contrast, co-administration of VLF (5 or 40 mg/kg, i.p.) with morphine for three consecutive days in morphine-tolerant mice prevented the induction of antinociceptive tolerance to high doses of morphine [F(3.23) = 55.9, P < 0.01; Fig. 2B].
3.4. Effect of venlafaxine in combination with L-arginine, Nω-nitro-Larginine methyl ester, or aminoguanidine on brain TNF-α, IL-6, IL1β, and IL-10 levels in morphine-dependent mice Repeated s.c. administration of morphine to mice for three consecutive days resulted in a progressive increase in TNF-α [F (5.30) = 24.71, P < 0.01], IL-6 [F(5.30) = 39.10, P < 0.01], IL-1β [F (5.30) = 48.14, P < 0.01], and IL-10 [F(5.30) = 4.28, P < 0.05] in the brains of morphine-dependent mice. However, concurrent 137
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Fig. 4. Effect of venlafaxine (VLF) and VLF in combination with, L-arginine (L-arg), L-NAME or aminoguanidine (AG) in brain malondialdehyde (MDA) (A), glutathione peroxidase (GPx) activity (B), total thiol level (C), and nitrite level (D) in morphine (MPH)-dependent mice. Bars are the means ± S.E.M. for 6–8 animals. *P < 0.05 and **P < 0.01 compared to animals treated with normal saline (NS) in morphine (MPH)-dependent mice, #P < 0.05 compared to NS + VLF, as determined by two-way ANOVA followed by Bonferroni's test.
Fig. 5. Effect of venlafaxine (VLF) and VLF in combination with, L-arginine (L-arg), L-NAME or aminoguanidine (AG) in brain TNF-α (A), IL-1β (B), IL-10 (C) and IL-6 (D) level in morphine (MPH)-dependent mice. Bars are the means ± S.E.M. for 6–8 animals. *P < 0.05 and **P < 0.01 compared to animals treated with normal saline (NS) in morphine (MPH)dependent mice, #P < 0.05 compared to NS + VLF, as determined by two-way ANOVA followed by Bonferroni's test.
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pharmacokinetics (Luo and Cizkova, 2000). The biological effects of NO may be mediated through direct interaction with its targets or the activation of soluble guanylate cyclase and subsequent production of cyclic GMP (cGMP) which then activates downstream targets including cGMP-dependent protein kinase, ion channels, and receptors. However, animal studies have suggested that other pathways are also involved in NO-induced hyperalgesia (Ichinose et al., 1998; Inoue et al., 1998). Here we showed that pre-treatment with L-arg reversed the antinociceptive effect of VLF in morphine-dependent mice, while L-NAME and AG potentiated it. Similarly, NG-nitro-L-arginine treatment prevents morphine tolerance and L-arg accelerates it (Babey et al., 1994). Moreover, AG, a selective inhibitor of inducible NO synthase, inhibits the development of morphine tolerance (Abdel-Zaher et al., 2006) and co-administration of morphine with VLF attenuates analgesic tolerance to morphine (Ozdemir et al., 2012). NO is a neurotoxin which is elevated in morphine-tolerance and dependence states (Dambisya and Lee, 1996). Moreover, it is well established that NO/GC/cGMP pathway contributes to neuronal adaptations in response to repeated exposure and that NOS inhibition ameliorates morphine-induced analgesia tolerance in different animal models (Cao et al., 2005; Machelska et al., 1997). In this study we observed that repeated administration of morphine for three consecutive days produced an increase in NO production in mouse brains, but that pretreatment with VLF significantly attenuated this effect. Furthermore, pretreatment with L-arginine reversed the effect of VLF in morphine-dependent mice but in contrast, L-NAME and AG potentiated its effect on NO production. These findings agree with previous results indicating that repeated administration of morphine over several consecutive days produces a progressive increase in NO production (AbdelZaher et al., 2013). It has been reported that repeated administration of morphine produced tolerance and a progressive increase in glutamate levels which induced oxidative stress in rat brain cortex (Yang et al., 1998). Activation of NMDA receptors by glutamate was also found to induce oxidative stress in rat brain synaptosomes (Alekseenko et al., 2009), and similarly, morphine administration decreased intracellular GSH levels in rat brains (Guzmán et al., 2006). In our experiments, administration of morphine to mice for three consecutive days produced oxidative stress in brain tissues, as indicated by an increase in brain MDA levels and a decrease in the non-enzymatic brain antioxidant, intracellular total thiol and in the enzymatic antioxidant, GPx. Our results suggest that repeated co-administration of VLF and morphine to mice attenuated the increase in brain MDA levels and the decrease in intracellular –SH levels and GPx activity induced by repeated morphine administration. This fits with a previous report that the antioxidant alpha-lipoic acid can suppress oxidative stress in the brains of morphine-dependent mice (Abdel-Zaher et al., 2013). To further investigate how VLF reduces morphine tolerance, we evaluated the levels of neuro-inflammatory proteins in the brain tissue of morphine-tolerant mice. Non-neuronal cells, especially microglia, are crucial in the pathogenesis of morphine tolerance (Mika, 2008). After chronic exposure to morphine, activated microglia express more proinflammatory cytokines, including IL-1β, IL-6, TNF-α, and chemokines (Johnston et al., 2004; Finley et al., 2008) and these changes contribute to morphine analgesic tolerance. Furthermore, both central and peripheral administration of proinflammatory cytokines TNF-α, IL-1β, and IL-6 facilitate pain transmission (Reeve et al., 2000). Here, we found that chronic morphine administration increased the levels of IL-1β, IL6, and TNF-α in mouse brains, but that VLF inhibited morphine-induced production of these cytokines. Hence, our data suggest that VLF may help to reduce microglial activation and morphine tolerance. In line with these findings, Pan et al. (2016) suggested that metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation (Pan et al., 2016) and both pro- and anti-inflammatory cytokines are involved in the development and maintenance of morphine tolerance (Johnston et al., 2004). IL-10 is a
Fig. 6. Effect of venlafaxine (VLF) and VLF in combination with, L-arginine (L-arg), LNAME or aminoguanidine (AG) in brain BDNF level in morphine (MPH)-dependent mice. Bars are the means ± S.E.M. for 6–8 animals. *P < 0.05 compared to animals treated with normal saline (NS) in morphine (MPH)-dependent mice, #P < 0.05 compared to NS + VLF, as determined by two-way ANOVA followed by Bonferroni's test.
administration of VLF (40 mg/kg) 30 min before each morphine injection for three consecutive days in morphine-dependent mice significantly inhibited this increase; this inhibitory effect was increased by concurrent i.p. administration of 30 mg/kg L-NAME or 100 mg/kg AG, but these inhibitory effects were reversed by concurrent i.p. administration of 300 mg/kg L-arg. The progressive increase in brain IL-10 levels induced by repeated administration of morphine was not changed by co-administration of VLF (Fig. 5A–D). 3.5. Effect of venlafaxine in combination with L-arginine, Nω-nitro-Larginine methyl ester, or aminoguanidine on brain-derived neurotrophic factor levels in morphine-dependent mice Subcutaneous administration of 50 mg/kg morphine to mice for three consecutive days resulted in a decrease in BDNF in the brains of morphine-dependent mice. However, co-administration of VLF (40 mg/ kg) 30 min before each morphine injection for three consecutive days significantly inhibited this decline in morphine-dependent mice [F (5.30) = 6.05; P < 0.01]; this inhibitory effect was increased by concurrent i.p. administration of L-NAME or AG but was antagonized by concurrent i.p. administration of L-arg (Fig. 6). 4. Discussion In this study, first we observed that chronic administration of VLF blocks pre-existing morphine analgesic tolerance through the L-arg-NO pathway. Moreover, further investigation indicated that the NO system mediates alterations in the neuro-inflammation and oxidation produced by repeated morphine administration. Opioids are effective drugs for moderating severe pain but the rapid development of analgesic tolerance to them remains as a problem (Ren et al., 2015). There are many hypotheses regarding how opioid tolerance might develop, including receptor desensitization, opioid receptor phosphorylation, internalization and/or downregulation of μ-opioid receptors, upregulation of the cyclic AMP and protein kinase C pathways, neuro-inflammation, glutamate homeostasis, NO production, oxidative stress, a modulatory role for ion channels or neurotrophic factors, or glial cell activation (AbdelZaher et al., 2013). The L-arg–NO axis plays an important role in modulating opioid analgesia and morphine-sensitive nociceptive processes. However, the role of NO in pain is contradictory because it can have both pro- and anti-nociceptive effects, depending on the specific experimental conditions (Ghorbanzadeh et al., 2014; Miclescu and Gordh, 2009); this inconsistency might be due to drug specificity, distribution, and 139
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powerful anti-inflammatory cytokine with a wide spectrum of biological effects (Bao et al., 2014), which may be involved in regulating morphine receptors and implicated mediating morphine tolerance (Zaringhalam et al., 2014). We found that morphine increased IL-10 levels in mouse brains but that VLF did not alter IL-10 levels in morphine-dependent mice. Interestingly, gabapentin increases the antinociceptive effect of morphine by activating IL-10 expression and its downstream signaling pathway which suppresses proinflammatory cytokine expression (Bao et al., 2014). Neurotrophic factors such as BDNF, which is involved in neuronal survival, differentiation, synaptogenesis, and maintenance (Carrasco et al., 2007; Mizuno et al., 1994), may also be important in opioidinduced neuronal degeneration. BDNF is anterogradely transported to the central terminals of the spinal dorsal horn where pain signaling from different pain stimuli is transduced and modulated. BDNF expression increases in the primary sensory neurons after peripheral inflammation (Obata and Noguchi, 2006); here we found that morphine decreased the levels of BDNF in mouse brains but that VLF inhibited this decline. In this regard, most antidepressants, including VLF and mirtazapine (Cooke et al., 2009; Rogoz et al., 2005), increase the hippocampal expression of BDNF, while others such as fluoxetine do not (Dias et al., 2003). Although we did not determine the source of BDNF after systemic VLF administration, there is evidence that cerebral and peripheral BDNF are related (Sartorius et al., 2009), and BDNF can cross the blood-brain barrier via a high-capacity and saturable transport system (Pan et al., 1998). However, whether serum BDNF reflects or contributes to brain BDNF levels remains unknown. Furthermore, we observed that pretreatment with L-arg reversed the effect of VLF in morphine-dependent mice and conversely, that L-NAME and AG potentiated the effect of VLF on NO production in mouse brains. NO influences gene expression by altering the activity of transcription factors through the cGMP/protein kinase G cascade and by S-nitrosylation (Contestabile, 2008). Moreover, NO regulates BDNF expression in conditions of brain ischemia (Chen et al., 2005), and may be able to regulate BDNF signaling in morphine-dependent animals (Peregud et al., 2016).
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5. Conclusions Chronic morphine treatment produces progressive analgesic tolerance and impedes clinical efficacy and neuro-inflammation and oxidative stress plays an important role in the development of morphine tolerance. Our data suggest that the L-arg–NO signaling pathway contributes to the VLF's ability to inhibit neuro-inflammation and oxidative stress in the brains of morphine-tolerant mice. Accordingly, our results also show that VLF-mediated inhibition of morphine-tolerance was also associated with the interaction of VLF with inflammatory cytokine cascades and oxidative factors. Hence, although further studies are required to clarify the mechanisms underlying VLF's effects on morphineinduced tolerance, this drug could be used alongside morphine to restore its analgesic properties. References Abdel-Zaher, A.O., Hamdy, M.M., Aly, S.A., Abdel-Hady, R.H., Abdel-Rahman, S., 2006. Attenuation of morphine tolerance and dependence by aminoguanidine in mice. Eur. J. Pharmacol. 540, 60–66. Abdel-Zaher, A.O., Mostafa, M.G., Farghaly, H.S., Hamdy, M.M., Abdel-Hady, R.H., 2013. Role of oxidative stress and inducible nitric oxide synthase in morphine-induced tolerance and dependence in mice. Effect of alpha-lipoic acid. Behav. Brain Res. 247, 17–26. Akbarian, S., Rios, M., Liu, R.J., Gold, S.J., Fong, H.F., Zeiler, S., Coppola, V., Tessarollo, L., Jones, K.R., Nestler, E.J., Aghajanian, G.K., Jaenisch, R., 2002. Brain-derived neurotrophic factor is essential for opiate-induced plasticity of noradrenergic neurons. J. Neurosci. 15, 4153–4162. Alekseenko, A.V., Kolos, V.A., Vasim, T.V., Fedorovich, S.V., 2009. Glutamate induces the formation of free radicals in rat brain synaptosomes. Biofizika 54, 876–880. Aloe, L., Properzi, F., Probert, L., Akassoglou, K., Kassiotis, G., Micera, A., Fiore, M., 1999. Learning abilities, NGF and BDNF brain levels in two lines of TNF-alpha transgenic
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Research paper
Venlafaxine prevents morphine antinociceptive tolerance: The role of neuroinflammation and the L-arginine-nitric oxide pathway
T
⁎
Mohammad Taghi Mansouria,b, , Bahareh Naghizadehc, Behnam Ghorbanzadehd, ⁎ Soheila Alboghobeishc, Neda Amirgholamic, Gholamreza Houshmande, Omar Caulif, a
Department of Pharmacology, School of Pharmacy, Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Neuroanesthesia Laboratory, Department of Anesthesiology, Emory University School of Medicine, 3B South, Emory University Hospital, 1364 Clifton Road, NE Atlanta, GA 30322, USA c Department of Pharmacology, School of Pharmacy, Toxicology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran d Department of Pharmacology, School of Medicine, Dezful University of Medical Sciences, Dezful, Iran e Department of Pharmacology, School of Medicine, Mazandaran University of Medical Sciences, Mazandaran, Iran f Department of Nursing, University of Valencia, Valencia, Spain b
A R T I C L E I N F O
A B S T R A C T
Keywords: Morphine tolerance Venlafaxine Cytokines L-arg-NO pathway Neuroinflammation Oxidative stress Brain-derived neurotrophic factor
Opioid-induced neuroinflammation and the nitric oxide (NO) signal-transduction pathway are involved in the development of opioid analgesic tolerance. The antidepressant venlafaxine (VLF) modulates NO in nervous tissues, and so we investigated its effect on induced tolerance to morphine, neuroinflammation, and oxidative stress in mice. Tolerance to the analgesic effects of morphine were induced by injecting mice with morphine (50 mg/kg) once a day for three consecutive days; the effect of co-administration of VLF (5 or 40 mg/kg) with morphine was similarly tested in a separate group. To determine if the NO precursor L-arginine hydrochloride (Larg) or NO are involved in the effects rendered by VLF, animals were pre-treated with L-arg (200 mg/kg), or the NO synthesis inhibitors N(ω)-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg) or aminoguanidine hydrochloride (AG; 100 mg/kg), along with VLF (40 mg/kg) for three days before receiving morphine for another three days. Nociception was assessed with a hot-plate test on the fourth day, and the concentration of tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-10, brain-derived neurotrophic factor, NO, and oxidative stress factors such as total thiol, malondialdehyde content, and glutathione peroxidase (GPx) activity in the brain was also determined. Co-administration of VLF with morphine attenuated morphine-induced analgesic tolerance and prevented the upregulation of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), NO, and malondialdehyde in brains of mice with induced morphine tolerance; chronic VLF administration inhibited this decrease in brain-derived neurotrophic factor, total thiol, and GPx levels. Moreover, repeated administration of L-arg before receipt of VLF antagonized the effects induced by VLF, while L-NAME and AG potentiated these effects. VLF attenuates morphine-induced analgesic tolerance, at least partly because of its anti-inflammatory and antioxidative properties. VLF also appears to suppress the development of morphine-induced analgesic tolerance through an L-arg–NO-mediated mechanism.
1. Introduction The administration of narcotics is the most effective means of attenuating severe pain in a wide range of conditions. Morphine is a widely used analgesic drug, however, multiple preclinical and clinical studies have shown that its chronic administration is associated with the development of analgesic tolerance (Hutchinson et al., 2011). A growing body of evidence has shown that opioid-induced tolerance is a complex phenomenon involving multiple behavioral and cellular adaptations, including alterations in neuronal plasticity at the cellular, ⁎
synaptic, and network levels in the central nervous system (Ueda et al., 2003; Von Zastrow, 2004). The role of cytokines in the development of morphine tolerance has been extensively studied (Kapasi et al., 2000; Pacifici et al., 2000). Indeed, chronic morphine treatment causes both neuronal and non-neuronal cells to release of several inflammatory mediators involved in the development of tolerance, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). There are also reports that suppressing neuroinflammation by inhibiting microglial activation and proinflammatory cytokines, TNF-α, IL-1β, and IL-6, can enhance the antinociceptive effect of morphine and
Corresponding authors. E-mail addresses: [email protected], [email protected] (M.T. Mansouri), [email protected] (O. Cauli).
https://doi.org/10.1016/j.expneurol.2018.02.009 Received 12 October 2017; Received in revised form 13 January 2018; Accepted 14 February 2018 Available online 15 February 2018 0014-4886/ © 2018 Elsevier Inc. All rights reserved.
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USA). Morphine sulfate and venlafaxine hydrochloride were donated by the Temad Pharmaceuticals (Tehran, Iran) and Darupakhsh Pharmaceuticals (Tehran, Iran), respectively. All the drugs were dissolved or suspended in 0.9% NaCl and buffered to pH 7.3. The drug dilutions were freshly prepared so that the required dose could be injected in a volume of 5 mL/kg of body weight; the respective animal controls received only the vehicle. Morphine was administered subcutaneously (s.c.) and all the other drugs were administered intraperitoneally (i.p.). DTNB (2,20-dinitro-5,50-dithiodibenzoic acid), TBA (2-thiobarbituric acid), n-butanol, tris base, ethylenediaminetetraacetic acid disodium, sodium acetate, glacial acetic acid, phosphoric acid, potassium chloride, and tetramethoxypropane were obtained from Merck (Darmstadt, Germany).
inhibit the formation of morphine tolerance (Song and Zhao, 2001). Brain-derived neurotrophic factor (BDNF) has trophic effects on the structures and functional plasticity of central synapses in the mammalian brain (Park and Poo, 2013). It has a well-documented pronociceptive role in inflammatory pain responses, acting at the descending brain-stem pain pathways (Merighi et al., 2008). Moreover, BDNF is essential for neuronal system adaptations in response to chronic opiate exposure, including cyclic AMP pathway superactivation, tyrosine hydroxylase upregulation, and restoration of neuronal firing (Akbarian et al., 2002). In contrast, chronic morphine administration induces oxidative stress in the brain (Ozmen et al., 2007) and furthermore, overactivation of the glutamatergic system is accompanied by increased formation of reactive oxygen species (Alekseenko et al., 2009). The action of glutamate, mediated through N-methyl-D-aspartate (NMDA) receptors, subsequently activates nitric oxide (NO) synthase (NOS) and the formation of NO (Kone et al., 2003). In addition, several free radical scavengers and NOS inhibitors suppress the development of morphine tolerance and dependence. Thus, oxidative stress and NO play a role in morphine tolerance (Mori et al., 2007). Venlafaxine (VLF), a bicyclic phenylethylamine derivative and serotonin and norepinephrine reuptake inhibitor, is widely used to treat major depression (Fournier et al., 2010). Like other antidepressants, it can relieve several pain conditions, including neuropathic pain; it also has anti-inflammatory (Hajhashemi et al., 2015) and antioxidant (Di Napoli et al., 2001) effects and regulates neurotrophic factors (Wu et al., 2008). VLF has been reported to inhibit NOS activity in the brain, causing subsequent NO release inhibition (Dhir and Kulkarni, 2007; Krass et al., 2011). A NO mechanism is thought to be involved in VLF's protective effect against acute immobilization and stress-induced behavioral and biochemical alterations (Kumar et al., 2009), and this mechanism has been implicated in some of the drug's most important functions in the nervous system (Dhir and Kulkarni, 2007; Uzbay, 2008; Krass et al., 2011; Gaur and Kumar, 2010). Polypharmacy is one option for improving the side-effect profile of opioid analgesics. Therefore, the aim of this work was to evaluate the potential role of VLF in preventing morphine antinociceptive tolerance by altering BDNF levels, oxidative stress, and neuroinflammation in mouse brains; we also assessed the involvement of the L-arg–NO pathway in mediating these effects.
2.3. Induction and assessment of morphine tolerance The animals were rendered tolerant to morphine using a previously published method (Zarrindast et al., 2002). After a morphine challenge test-dose on day one, tolerance was induced by administering morphine (50 mg/kg; s.c.) once daily at 9.00 A.M. for three consecutive days. On day four, the animals were assessed for tolerance as previously described (Way et al., 1969); the loss of the antinociceptive effects of morphine were tested using a hot-plate sensitivity test 15, 30, 60, and 90 min after a challenge-dose injection of 50 mg/kg morphine (Way et al., 1969; Zarrindast et al., 2002). Each animal was placed on a 52 ± 1 °C hot plate which was surrounded by a clear acrylic cage, and the latency time(s) to either hind paw licking or jumping (whichever came first) was recorded. To avoid tissue damage, the cut-off time was 15 s (Eddy and Leimbach, 1953). 2.4. Experimental groups To assess the effect of VLF in the development of morphine analgesic tolerance, we administered VLF (5 or 40 mg/kg, i.p.) once a day for three consecutive days, 30 min prior to the injection of morphine (50 mg/kg, s.c.); hot-plate tests were performed on the fourth day. In another experiment, to investigate the role of NO in VLF-mediated tolerance modulation, L-arg (200 mg/kg), L-NAME (30 mg/kg), AG (100 mg/kg), or saline (5 mL/kg) were injected with each dose of VLF (Fig. 1); these doses were chosen based on previous studies (Homayoun et al., 2002a; Mansouri et al., 2014b, 2013; Schreiber et al., 1999; Way et al., 1969; Zarrindast et al., 2002; Homayoun et al., 2002b).
2. Materials and methods 2.1. Animals
2.5. Brain sample collection Adult male Swiss mice (weighing 25–30 g) obtained from the central animal house of the Ahvaz Jundishapur University of Medical Sciences (Iran) were used in this study; they were housed at 22 ± 2 °C with 12 h light/dark cycles (light from 7:00 to 19:00) with free access to food and water. The animals were randomly divided into groups of 6–8, acclimatized to the laboratory environment for at least one week before commencing the experiments, and used only once. All the animal experiments were carried out in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animals. The Institutional Animal Ethics Committee at the Ahvaz Jundishapur University of Medical Sciences, formed under the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Reg. No. APRC), approved the experimental protocols. All the behavioral tests were performed by an investigator who was blinded to the interventions.
At the end of experiments, the animals were decapitated and the brain tissue was quickly removed, rinsed with saline, and frozen at −80 °C until their use. The tissues were homogenized in 0.1 M phosphate buffer (pH 7.4) to give a 10% homogenate suspension. The homogenate was centrifuged at 10,000g for 15 min, and aliquots of the supernatant were separated and used for biochemical experiments. 2.6. Antioxidant assays 2.6.1. Lipid peroxidation assay The level of thiobarbituric acid-reactive substances (TBARS) produced by free radicals, an index of lipid peroxidation, was measured. Malondialdehyde (MDA) reacts with TBA to produce a red complex with a peak absorbance at 532 nm. Briefly, 3 mL phosphoric acid (1%) and 1 mL TBA (0.6%) were added to 0.5 mL of homogenate in a centrifuge tube and the mixture was heated for 45 min in a boiling water bath. After cooling, 4 mL n-butanol was added and the mixture was vortexed for 1 min followed by centrifugation at 2000g for 20 min. The colored layer was transferred to a fresh tube and its absorbance was measured at 532 nm. The levels of TBARS were determined using 1,1,3,3-tetramethoxypropane as a standard; the standard curve of MDA
2.2. Drugs The nitric oxide precursor, L-arginine hydrochloride (L-arg), NO synthase inhibitor, N(ω)-nitro-L-arginine methyl ester hydrochloride (LNAME), and selective inducible NOS inhibitor, aminoguanidine hydrochloride (AG) were purchased from Sigma-Aldrich (St. Louis, MO, 135
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Fig. 1. Timeline of the experimental schedule in the hot-plate test (HP). (A) Acute antinociceptive effect of venlafaxine and role of L-arg/NO/cGMP pathway. (B) Effects of venlafaxine on the development of morphine tolerance and role of L-arg/NO/cGMP pathway.
was constructed over a 0–20 μM concentration range (Naghizadeh et al., 2013). MDA concentration was expressed as nmol per gram of wet tissue.
sulfanilamide, and 2.5% phosphoric acid] as previously described (Green et al., 1982). Equal volumes of supernatant and Griess reagent were mixed; the mixture was incubated for 10 min at room temperature in the dark and the absorbance was determined with spectrophotometer at 540 nm. The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and expressed as a percentage of the control. Nitrite level was expressed as μM per gram of wet tissue.
2.6.2. Glutathione peroxidase assay The concentration of glutathione (GSH) peroxidase (GPx) was measured with a GPx kit (Randox Labs, Crumlin, UK). GPx concentration was expressed as unit per gram of wet tissue. 2.6.3. Total sulfhydryl groups assay Total sulfhydryl (–SH) groups were measured using DTNB; this reagent reacts with these groups to produce a yellow complex with a peak absorbance at 412 nm (Ellman, 1959). Briefly, 1 mL Tris-EDTA buffer (pH 8.6) was added to 50 μL homogenate in 2 mL cuvettes and the sample absorbance was read at 412 nm against Tris-EDTA buffer alone (A1). Then, 20 μL DTNB reagent (10 mM in methanol) was added to the mixture and, after 15 min at room temperature, the sample absorbance was read again (A2). The absorbance of the DTNB reagent was also read as a blank (B). The total thiol concentration (mM) was calculated from the following equation:
2.7.3. Brain-derived neurotrophic factor measurement BDNF was measured with an enzyme-linked immunosorbent assay kit (Emax™ ImmunoAssay Systems, Promega, Madison, Wisconsin, USA) following the manufacturer's instructions. Briefly, the BDNF standard or brain samples were distributed in 96-well immunoassay plates pre-coated with monoclonal anti-mouse BDNF antibody (100 μL/ well) and were incubated for 20 h at room temperature. After washing, the plates were incubated with an anti-human BDNF antibody for 2 h at room temperature. The plates were washed again and then incubated with an anti-IgG horseradish peroxidase for 1 h at room temperature. Tetramethylbenzidine/peroxidase substrate solution was added to the wells to produce a colorimetric reaction which was measured at 450 nm with a microplate reader (Dynatech MR 5000, Dynatech Laboratories, Chantilly, Virginia, USA). BDNF concentrations were determined from the regression line for the BDNF standard incubated under similar conditions in each assay. BDNF level was expressed as nanogram per gram of wet tissue The sensitivity of the assay was about 15 pg/mg and the cross-reactivity with other related neurotrophic factors (nerve growth factor, neurotrophin-3 and -4) is considered nil (Aloe et al., 1999).
Total thiol concentration (mM) = (A2−A1 − B) × 1.07/0.05 × 13.6 The sulfhydryl content is inversely correlated to protein oxidative damage. 2.7. Biochemical assays 2.7.1. Proinflammatory cytokine measurement Brain levels of TNF-α, IL-6, IL1β, and IL-10 were determined by enzyme immunoassay kits, as previously described (de Waal Malefyt et al., 1991) and according to the manufacturer's guidelines (eBioscience, Vienna, Austria). The absorbance of the produced color was measured at 450 nm using microplate reader. Cytokines levels were expressed as picogram per gram of wet tissue.
2.8. Statistical analysis To evaluate the global analgesic effect and compare the different treatment groups, the area under the curve (AUC; 15–90 min) of the hot-plate latency was calculated using the trapezoidal rule, based on the observed values (Ghavimi et al., 2014). The results are presented as means ± the standard error of the mean (SEM). One-way analysis of variance (ANOVA) and Tukey tests were used to compare: the effects of (1) repeated VLF administrations compared to baseline data at the first
2.7.2. Nitrite measurement The accumulation of nitrite in the supernatant, an indicator of the production of NO, was determined with a colorimetric assay with Griess reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% 136
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Fig. 3. Effect of L-arginine (L-Arg), L-NAME and aminoguanidine (AG) on antinociceptive effect venlafaxine (VLF) in combination with morphine (MPH) in morphine tolerant mice in the mouse hot-plate test. Data are expressed as the under the curve of the time course curves (AUC). Bars are the means ± S.E.M. for 6–8 animals. *P < 0.05 compared to animals treated with normal saline (NS) in morphine (MPH)-dependent mice, #P < 0.05 and ##P < 0.01 compared to NS + VLF, as determined by two-way ANOVA followed by Bonferroni's test.
3.2. Effect of L-arginine, Nω-nitro-L-arginine methyl ester, or aminoguanidine on venlafaxine-induced inhibition of antinociceptive tolerance in morphine-dependent mice Concurrent i.p. administration of L-NAME and VLF (40 mg/kg each, i.p.) 30 min before each morphine injection for three consecutive days enhanced the inhibitory effect of VLF on the development of morphine tolerance (Fig. 3); this inhibitory effect was potentiated by concurrent i.p. administration of AG and antagonized by concurrent i.p. administration of L-arg [F(5.33) = 57.05, P < 0.01]. 3.3. Effect of venlafaxine in combination with L-arginine, Nω-nitro-Larginine methyl ester, or aminoguanidine on brain malondialdehyde, total thiol, nitrite levels, and glutathione peroxidase activity in morphinedependent mice
Fig. 2. Effects of venlafaxine (VLF) on nociceptive latency responses (A) and the area under the curve (AUC) of the time course curves (B) in morphine tolerant mice in the mouse hot-plate test. The latency to nociceptive responses was measured 15, 30, 60 and 90 min after challenge dose of morphine (5 mg/kg). Data are expressed as the means ± S.E.M. Each group consisted of 8 mice. **P < 0.01 compared with the normal saline (NS) at day 1, ##P < 0.01 compared with the normal saline at day 4 (one-way ANOVA with Tukey's test).
Repeated s.c. administration of 50 mg/kg morphine for three consecutive days resulted in a progressive increase in brain MDA and nitrite levels as well as a decrease in total brain thiol levels and GPx activity. Moreover, administration of VLF 30 min before each morphine injection for three consecutive days significantly inhibited morphineinduced increases in brain MDA and nitrite levels (Fig. 4A and D) and the decreases in thiol levels and GPx activity were inhibited by pretreatment with VLF (Fig. 4B and C). The inhibitory effect of VLF (40 mg/kg) on brain MDA [F(5.24) = 73.00, P < 0.01] and serum nitrite levels [F(5.30) = 26.27, P < 0.01] in morphine-dependent mice was increased by concurrent i.p. administration of L-NAME or AG. In addition, co-administration of these agents with VLF enhanced its effect of increasing total brain thiol levels [F(5.24) = 25.57, P < 0.01] and GPx activity [F(5.24) = 24.31, P < 0.01] which was inhibited in morphine-dependent mice.
injection and (2) co-administration of VLF with other agents and morphine, and (3) the overall combined effects of these drugs at each administration (Mansouri et al., 2014a). P-values < 0.05 were considered statistically significant. All calculations and statistical analyses were done with GraphPad Prism v6 (GraphPad Software Inc., San Diego, CA, USA).
3. Results 3.1. Effects of venlafaxine on the tolerance to morphine analgesia Receipt of a morphine injection for three consecutive days caused a decline in its antinociceptive effects, indicating analgesic tolerance [F (4.65) = 5.32, P < 0.01; Fig. 2A]. In contrast, co-administration of VLF (5 or 40 mg/kg, i.p.) with morphine for three consecutive days in morphine-tolerant mice prevented the induction of antinociceptive tolerance to high doses of morphine [F(3.23) = 55.9, P < 0.01; Fig. 2B].
3.4. Effect of venlafaxine in combination with L-arginine, Nω-nitro-Larginine methyl ester, or aminoguanidine on brain TNF-α, IL-6, IL1β, and IL-10 levels in morphine-dependent mice Repeated s.c. administration of morphine to mice for three consecutive days resulted in a progressive increase in TNF-α [F (5.30) = 24.71, P < 0.01], IL-6 [F(5.30) = 39.10, P < 0.01], IL-1β [F (5.30) = 48.14, P < 0.01], and IL-10 [F(5.30) = 4.28, P < 0.05] in the brains of morphine-dependent mice. However, concurrent 137
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Fig. 4. Effect of venlafaxine (VLF) and VLF in combination with, L-arginine (L-arg), L-NAME or aminoguanidine (AG) in brain malondialdehyde (MDA) (A), glutathione peroxidase (GPx) activity (B), total thiol level (C), and nitrite level (D) in morphine (MPH)-dependent mice. Bars are the means ± S.E.M. for 6–8 animals. *P < 0.05 and **P < 0.01 compared to animals treated with normal saline (NS) in morphine (MPH)-dependent mice, #P < 0.05 compared to NS + VLF, as determined by two-way ANOVA followed by Bonferroni's test.
Fig. 5. Effect of venlafaxine (VLF) and VLF in combination with, L-arginine (L-arg), L-NAME or aminoguanidine (AG) in brain TNF-α (A), IL-1β (B), IL-10 (C) and IL-6 (D) level in morphine (MPH)-dependent mice. Bars are the means ± S.E.M. for 6–8 animals. *P < 0.05 and **P < 0.01 compared to animals treated with normal saline (NS) in morphine (MPH)dependent mice, #P < 0.05 compared to NS + VLF, as determined by two-way ANOVA followed by Bonferroni's test.
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pharmacokinetics (Luo and Cizkova, 2000). The biological effects of NO may be mediated through direct interaction with its targets or the activation of soluble guanylate cyclase and subsequent production of cyclic GMP (cGMP) which then activates downstream targets including cGMP-dependent protein kinase, ion channels, and receptors. However, animal studies have suggested that other pathways are also involved in NO-induced hyperalgesia (Ichinose et al., 1998; Inoue et al., 1998). Here we showed that pre-treatment with L-arg reversed the antinociceptive effect of VLF in morphine-dependent mice, while L-NAME and AG potentiated it. Similarly, NG-nitro-L-arginine treatment prevents morphine tolerance and L-arg accelerates it (Babey et al., 1994). Moreover, AG, a selective inhibitor of inducible NO synthase, inhibits the development of morphine tolerance (Abdel-Zaher et al., 2006) and co-administration of morphine with VLF attenuates analgesic tolerance to morphine (Ozdemir et al., 2012). NO is a neurotoxin which is elevated in morphine-tolerance and dependence states (Dambisya and Lee, 1996). Moreover, it is well established that NO/GC/cGMP pathway contributes to neuronal adaptations in response to repeated exposure and that NOS inhibition ameliorates morphine-induced analgesia tolerance in different animal models (Cao et al., 2005; Machelska et al., 1997). In this study we observed that repeated administration of morphine for three consecutive days produced an increase in NO production in mouse brains, but that pretreatment with VLF significantly attenuated this effect. Furthermore, pretreatment with L-arginine reversed the effect of VLF in morphine-dependent mice but in contrast, L-NAME and AG potentiated its effect on NO production. These findings agree with previous results indicating that repeated administration of morphine over several consecutive days produces a progressive increase in NO production (AbdelZaher et al., 2013). It has been reported that repeated administration of morphine produced tolerance and a progressive increase in glutamate levels which induced oxidative stress in rat brain cortex (Yang et al., 1998). Activation of NMDA receptors by glutamate was also found to induce oxidative stress in rat brain synaptosomes (Alekseenko et al., 2009), and similarly, morphine administration decreased intracellular GSH levels in rat brains (Guzmán et al., 2006). In our experiments, administration of morphine to mice for three consecutive days produced oxidative stress in brain tissues, as indicated by an increase in brain MDA levels and a decrease in the non-enzymatic brain antioxidant, intracellular total thiol and in the enzymatic antioxidant, GPx. Our results suggest that repeated co-administration of VLF and morphine to mice attenuated the increase in brain MDA levels and the decrease in intracellular –SH levels and GPx activity induced by repeated morphine administration. This fits with a previous report that the antioxidant alpha-lipoic acid can suppress oxidative stress in the brains of morphine-dependent mice (Abdel-Zaher et al., 2013). To further investigate how VLF reduces morphine tolerance, we evaluated the levels of neuro-inflammatory proteins in the brain tissue of morphine-tolerant mice. Non-neuronal cells, especially microglia, are crucial in the pathogenesis of morphine tolerance (Mika, 2008). After chronic exposure to morphine, activated microglia express more proinflammatory cytokines, including IL-1β, IL-6, TNF-α, and chemokines (Johnston et al., 2004; Finley et al., 2008) and these changes contribute to morphine analgesic tolerance. Furthermore, both central and peripheral administration of proinflammatory cytokines TNF-α, IL-1β, and IL-6 facilitate pain transmission (Reeve et al., 2000). Here, we found that chronic morphine administration increased the levels of IL-1β, IL6, and TNF-α in mouse brains, but that VLF inhibited morphine-induced production of these cytokines. Hence, our data suggest that VLF may help to reduce microglial activation and morphine tolerance. In line with these findings, Pan et al. (2016) suggested that metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation (Pan et al., 2016) and both pro- and anti-inflammatory cytokines are involved in the development and maintenance of morphine tolerance (Johnston et al., 2004). IL-10 is a
Fig. 6. Effect of venlafaxine (VLF) and VLF in combination with, L-arginine (L-arg), LNAME or aminoguanidine (AG) in brain BDNF level in morphine (MPH)-dependent mice. Bars are the means ± S.E.M. for 6–8 animals. *P < 0.05 compared to animals treated with normal saline (NS) in morphine (MPH)-dependent mice, #P < 0.05 compared to NS + VLF, as determined by two-way ANOVA followed by Bonferroni's test.
administration of VLF (40 mg/kg) 30 min before each morphine injection for three consecutive days in morphine-dependent mice significantly inhibited this increase; this inhibitory effect was increased by concurrent i.p. administration of 30 mg/kg L-NAME or 100 mg/kg AG, but these inhibitory effects were reversed by concurrent i.p. administration of 300 mg/kg L-arg. The progressive increase in brain IL-10 levels induced by repeated administration of morphine was not changed by co-administration of VLF (Fig. 5A–D). 3.5. Effect of venlafaxine in combination with L-arginine, Nω-nitro-Larginine methyl ester, or aminoguanidine on brain-derived neurotrophic factor levels in morphine-dependent mice Subcutaneous administration of 50 mg/kg morphine to mice for three consecutive days resulted in a decrease in BDNF in the brains of morphine-dependent mice. However, co-administration of VLF (40 mg/ kg) 30 min before each morphine injection for three consecutive days significantly inhibited this decline in morphine-dependent mice [F (5.30) = 6.05; P < 0.01]; this inhibitory effect was increased by concurrent i.p. administration of L-NAME or AG but was antagonized by concurrent i.p. administration of L-arg (Fig. 6). 4. Discussion In this study, first we observed that chronic administration of VLF blocks pre-existing morphine analgesic tolerance through the L-arg-NO pathway. Moreover, further investigation indicated that the NO system mediates alterations in the neuro-inflammation and oxidation produced by repeated morphine administration. Opioids are effective drugs for moderating severe pain but the rapid development of analgesic tolerance to them remains as a problem (Ren et al., 2015). There are many hypotheses regarding how opioid tolerance might develop, including receptor desensitization, opioid receptor phosphorylation, internalization and/or downregulation of μ-opioid receptors, upregulation of the cyclic AMP and protein kinase C pathways, neuro-inflammation, glutamate homeostasis, NO production, oxidative stress, a modulatory role for ion channels or neurotrophic factors, or glial cell activation (AbdelZaher et al., 2013). The L-arg–NO axis plays an important role in modulating opioid analgesia and morphine-sensitive nociceptive processes. However, the role of NO in pain is contradictory because it can have both pro- and anti-nociceptive effects, depending on the specific experimental conditions (Ghorbanzadeh et al., 2014; Miclescu and Gordh, 2009); this inconsistency might be due to drug specificity, distribution, and 139
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powerful anti-inflammatory cytokine with a wide spectrum of biological effects (Bao et al., 2014), which may be involved in regulating morphine receptors and implicated mediating morphine tolerance (Zaringhalam et al., 2014). We found that morphine increased IL-10 levels in mouse brains but that VLF did not alter IL-10 levels in morphine-dependent mice. Interestingly, gabapentin increases the antinociceptive effect of morphine by activating IL-10 expression and its downstream signaling pathway which suppresses proinflammatory cytokine expression (Bao et al., 2014). Neurotrophic factors such as BDNF, which is involved in neuronal survival, differentiation, synaptogenesis, and maintenance (Carrasco et al., 2007; Mizuno et al., 1994), may also be important in opioidinduced neuronal degeneration. BDNF is anterogradely transported to the central terminals of the spinal dorsal horn where pain signaling from different pain stimuli is transduced and modulated. BDNF expression increases in the primary sensory neurons after peripheral inflammation (Obata and Noguchi, 2006); here we found that morphine decreased the levels of BDNF in mouse brains but that VLF inhibited this decline. In this regard, most antidepressants, including VLF and mirtazapine (Cooke et al., 2009; Rogoz et al., 2005), increase the hippocampal expression of BDNF, while others such as fluoxetine do not (Dias et al., 2003). Although we did not determine the source of BDNF after systemic VLF administration, there is evidence that cerebral and peripheral BDNF are related (Sartorius et al., 2009), and BDNF can cross the blood-brain barrier via a high-capacity and saturable transport system (Pan et al., 1998). However, whether serum BDNF reflects or contributes to brain BDNF levels remains unknown. Furthermore, we observed that pretreatment with L-arg reversed the effect of VLF in morphine-dependent mice and conversely, that L-NAME and AG potentiated the effect of VLF on NO production in mouse brains. NO influences gene expression by altering the activity of transcription factors through the cGMP/protein kinase G cascade and by S-nitrosylation (Contestabile, 2008). Moreover, NO regulates BDNF expression in conditions of brain ischemia (Chen et al., 2005), and may be able to regulate BDNF signaling in morphine-dependent animals (Peregud et al., 2016).
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5. Conclusions Chronic morphine treatment produces progressive analgesic tolerance and impedes clinical efficacy and neuro-inflammation and oxidative stress plays an important role in the development of morphine tolerance. Our data suggest that the L-arg–NO signaling pathway contributes to the VLF's ability to inhibit neuro-inflammation and oxidative stress in the brains of morphine-tolerant mice. Accordingly, our results also show that VLF-mediated inhibition of morphine-tolerance was also associated with the interaction of VLF with inflammatory cytokine cascades and oxidative factors. Hence, although further studies are required to clarify the mechanisms underlying VLF's effects on morphineinduced tolerance, this drug could be used alongside morphine to restore its analgesic properties. References Abdel-Zaher, A.O., Hamdy, M.M., Aly, S.A., Abdel-Hady, R.H., Abdel-Rahman, S., 2006. Attenuation of morphine tolerance and dependence by aminoguanidine in mice. Eur. J. Pharmacol. 540, 60–66. Abdel-Zaher, A.O., Mostafa, M.G., Farghaly, H.S., Hamdy, M.M., Abdel-Hady, R.H., 2013. Role of oxidative stress and inducible nitric oxide synthase in morphine-induced tolerance and dependence in mice. Effect of alpha-lipoic acid. Behav. Brain Res. 247, 17–26. Akbarian, S., Rios, M., Liu, R.J., Gold, S.J., Fong, H.F., Zeiler, S., Coppola, V., Tessarollo, L., Jones, K.R., Nestler, E.J., Aghajanian, G.K., Jaenisch, R., 2002. Brain-derived neurotrophic factor is essential for opiate-induced plasticity of noradrenergic neurons. J. Neurosci. 15, 4153–4162. Alekseenko, A.V., Kolos, V.A., Vasim, T.V., Fedorovich, S.V., 2009. Glutamate induces the formation of free radicals in rat brain synaptosomes. Biofizika 54, 876–880. Aloe, L., Properzi, F., Probert, L., Akassoglou, K., Kassiotis, G., Micera, A., Fiore, M., 1999. Learning abilities, NGF and BDNF brain levels in two lines of TNF-alpha transgenic
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