Morphine-induced Antinociceptive Tolerance Research Articles

Experimental Study: Interleukin-31 Augments Morphine-Induced Antinociceptive Activity and Suppress Tolerance Development in Mice.

Morphine-induced antinociception is partially reduced in interleukin-31 (IL-31) receptor A (IL-31RA)-deficient mice, indicating that IL-31RA is crucial for morphine-induced peripheral antinociception. Herein, we examined the combined effects of IL-31 and morphine on the antinociceptive activity and itch-associated scratching behavior (LLS) in mice and elucidated the regulatory mechanisms. A hot-plate test was used to assess antinociception. LLS was automatically detected and recorded via a computer. IL-31RA mRNA expression was assessed using real-time polymerase chain reaction. Repeated pre-treatment with IL-31 resulted in significant antinociceptive activity. Repeated administration of morphine decreased the morphine-induced antinociceptive activity, LLS counts, and regular dose and inhibited IL-31-induced LLS. These results suggested that the repeated administration of morphine depleted inter-neuronal IL-31RA levels, preventing morphine-induced antinociception. Therefore, IL-31 may be helpful as an adjunct analgesic to morphine. To explore the benefits of IL-31, its influence on morphine-induced antinociceptive tolerance in mice was examined. An IL-31 and morphine combination increased the analgesic action, which increased the expression of DRG neuronal IL-31RA, elucidating the site of peripheral antinociception of morphine. This site may induce exocytosis of IL-31RA in the sensory nervous system. Collectively, the suppressive effect of IL-31 on morphine-induced antinociceptive tolerance may result from IL-31RA supplementation in sensory nerves.

Involvement of the nociceptin opioid peptide receptor in morphine-induced antinociception, tolerance and physical dependence in female mice.

Nociceptin opioid peptide (NOP) receptor modulates pain transmission and is considered a prospective target for pain management. Under acute pain conditions in rodents, however, no definitive conclusions about effects of systemically intervening NOP receptors on nociception, classical opioid-induced antinociception, tolerance and physical dependence have been drawn. Given that opioid analgesia has sex differences, and females experience greater pain and consume more opioids, clarifying these issues in females will help develop novel analgesics. To clarify the role of NOP receptors on the pharmacological profiles of µ-opioid receptor agonists, in this study, a selective agonist (SCH221510) and antagonist (SB612111) of the NOP receptor were subcutaneously administered in female mice in multiple animal models. In hot-plate test, neither SCH221510 (3 and 10mg/kg, sc) nor SB612111 (10mg/kg, sc) produced significant antinociception. SCH221510 (3mg/kg, sc) attenuated but SB612111 (10mg/kg, sc) enhanced morphine-induced antinociception, with rightward and leftward shift of morphine dose-response curves, respectively. SCH221510 (3mg/kg, sc) combined with morphine (10mg/kg, sc) accelerated the development of morphine antinociceptive tolerance. Conversely, SB612111 (10mg/kg, sc) delayed morphine tolerance development. Neither SCH221510 (3mg/kg, sc) nor SB612111 (10mg/kg, sc) statistically significantly altered the development of morphine-induced physical dependence. Therefore, systemic activation of NOP receptors attenuated morphine antinociception to acute thermal stimuli, facilitated morphine-induced antinociceptive tolerance but did not robustly alter physical dependence in female mice. Systemic blockade of NOP receptors produced opposite actions. These findings demonstrate that N/OFQ-NOP receptor system plays diverse roles in modulating pharmacological profiles of µ-opioid receptor agonists.

Sphingosine-1-phosphate Receptor Subtype 1 Antagonists may be the Unmet Medical Need for Morphine-induced Hyperalgesia and Antinociceptive Tolerance

Opioids such as morphine are frequently used for chronic pain management despite their many adverse effects. Ongoing research aims at either finding new treatments to replace opioids or reducing its heavy adverse effects due to long-term use: opioid-induced hyperalgesia and antinociceptive tolerance. In a recent study, Doyle et al. (2020) demonstrate that the activation of sphingosine-1-phosphate receptor subtype 1 (S1PR1) in the central nervous system contributes to morphine-induced hyperalgesia and antinociceptive tolerance in a rodent model of chronic pain. By targeting S1PR1 with molecules with functional antagonistic properties (some of which are FDA-approved for multiple sclerosis treatment), hyperalgesia and tolerance were significantly reduced without modifying morphine pharmacokinetics or efficacy.

Involvement of TCF7L2 in generation of morphine-induced antinociceptive tolerance and hyperalgesia by modulating TLR4/ NF-κB/NLRP3 in microglia

Morphine is an opioid agonist and a nonselective mu, kappa and delta receptor agonist. It is a commonly used analgesic drug for the treatment of acute and chronic pain as well as cancer pain. Morphine is particularly important to address the problem of morphine tolerance. Tcf7l2, known as a risk gene for schizophrenia and autism, encodes a member of the LEF1/TCF transcription factor family. TCF7L2 is an important transcription factor that is upregulated in neuropathic pain models. However, the relationship between TCF7L2 and morphine tolerance has not been reported. In this study, we found that morphine tolerance led to the upregulation of TCF7L2 in the spinal cord, and also led to the upregulation of TCF7L2 expression in glial cells, which promoted inflammation related signal, and activated TLR4 / NF-κB/NLRP3 pathway. In addition, TCF7L2 regulated microglial cell activation induced by chronic morphine treatment. Mechanically, we found that TCF7L2 transcriptionally regulated TLR4 expression, and the depletion of TCF7L2 alleviated morphine tolerance induced by chronic morphine treatment, and further alleviated pain hypersensitivity induced by chronic morphine treatment. We therefore suggested that TCF7L2 regulates the activation of TLR4/ NF-κB/NLRP3 pathway in microglia, and is involved in the formation of morphine tolerance. Our results provide a new idea for the regulation mechanism of morphine tolerance.

Microglial TLR4-induced TAK1 phosphorylation and NLRP3 activation mediates neuroinflammation and contributes to chronic morphine-induced antinociceptive tolerance

Background and purposeThe aim of this work was to investigate the role and signal transduction of toll-like receptor 4 (TLR4), TGF-β-activated kinase 1 (TAK1) and nod-like receptor protein 3 (NLRP3) in microglial in the development of morphine-induced antinociceptive tolerance. MethodsTLR4 and NLRP3 knockout mice and 5Z-7-oxozeaeno (a selective inhibitor against TAK1 activity) were used to observe their effect on the development of morphine tolerance. Intrathecal injections of morphine (0.75 mg/kg once daily for 7 days) were used to establish anti-nociceptive tolerance, which was measured by the tail-flick test. Spinal TLR4, TAK1, and NLRP3 expression levels and phosphorylation of TAK1 were evaluated by Western blotting and immunofluorescence. ResultsRepeated treatment with morphine increased total expression of spinal TLR4, TAK1, and NLRP3 and phosphorylation of TAK1 in wild-type mice. TLR4 knockout attenuated morphine-induced tolerance and inhibited the chronic morphine-induced increase in NLRP3 and phosphorylation of TAK1. Compared with controls, mice that received 5Z-7-oxozeaenol showed decreased development of morphine tolerance and inhibition on repeated morphine-induced increase of NLRP3 but not TLR4. NLRP3 knockout mice showed resistance to morphine-induced analgesic tolerance with no effect on chronic morphine-induced expression of TLR4 and TAK1. TLR4, TAK1, and NLRP3 were collectively co-localized together and with the microglia marker Iba1. ConclusionsMicroglial TLR4 regulates TAK1 expression and phosphorylation and results in NLRP3 activation contributes to the development of morphine tolerance through regulating neuroinflammation. Targeting TLR4-TAK1-NLRP3 signaling to regulate neuro-inflammation will be alternative therapeutics and strategies for chronic morphine-induced antinociceptive tolerance.

Simvastatin prevents morphine antinociceptive tolerance and withdrawal symptoms through antioxidative effect and nitric oxide pathway in mice

Oxidative stress and the nitric oxide (NO) pathway are involved in the development of opioid analgesic tolerance and dependence. Simvastatin modulates NO and oxidative stress, so the present study aimed to investigate its effect on the development and expression of morphine analgesic tolerance and withdrawal signs in mice. Morphine tolerance and dependence were induced by twice daily morphine injection (10 mg/kg, s.c.) for 5 consecutive days. Tolerance was assessed by the hot-plate test and dependence by naloxone challenge, on the sixth day. To determine if the NO is involved in the effects of simvastatin, mice were pre-treated with l-arginine (200 mg/kg) or the NO synthesis inhibitors (L-NAME; 30 mg/kg) along with simvastatin (300 mg/kg). The results showed that acute and chronic administration of simvastatin reversed the antinociceptive tolerance of morphine and attenuated withdrawal signs in morphine-dependent mice, and this effect is reversed by l-arginine and augmented by l-NAME. Also, the concentration of NO and oxidative stress factors such as malondialdehyde content, total thiol, and glutathione peroxidase (GPx) activity in brain tissues was evaluated. Chronic administration of simvastatin reduced NO and malondialdehyde, and increased total thiol and GPx levels in the cerebral cortex and hippocampus of morphine-dependent mice which were antagonized by l-arginine, and augmented by l-NAME. In summary, simvastatin attenuates morphine-induced antinociceptive tolerance and withdrawal symptoms, at least partly, through antioxidative properties and nitric oxide pathway.

Chronic morphine induces cyclic adenosine monophosphate formation and hyperpolarization-activated cyclic nucleotide-gated channel expression in the spinal cord of mice

Chronic morphine exposure persistently activates Gαi/o protein-coupled receptors and enhances adenylyl cyclase (AC) activity, which can increase cyclic adenosine monophosphate (cAMP) production. Direct binding of cAMP to the cytoplasmic site on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels increases the probability of channel opening. HCN channels play a prominent role in chronic pain the disease that shares some common mechanisms with opioid tolerance. This compensatory AC activation may be responsible for the induction of morphine-induced analgesic tolerance. We investigated spinal cAMP formation and expression of HCN2 in the spinal cord, and observed the effect of AC inhibition on the induction of morphine analgesic tolerance. We found that chronic morphine-induced antinociceptive tolerance increased spinal cAMP formation and the expression of spinal HCN2. Inhibition of spinal AC partially blocked chronic morphine-induced cAMP formation and prevented the induction of morphine-induced analgesic tolerance. Inhibition of HCN2 also showed a partial preventive effect on morphine-induced tolerance, hypothermia tolerance and also the right-shift of the dose-response curve. We conclude that repeated morphine treatment increases AC activity and cAMP formation, and also spinal HCN2 expression, blockade of AC or HCN2 can prevent the development of morphine-induced analgesic tolerance.

DAMGO-induced μ opioid receptor internalization and recycling restore morphine sensitivity in tolerant rat

This study investigated the effect of DAMGO-induced μ opioid receptor (MOR) internalization on morphine tolerance. Male Sprague-Dawley rats (200–250 g) aged 6–8 weeks were administered morphine via intrathecal (i.t.) injection (15 μg/10 μl twice daily for 6 days) to induce antinociceptive tolerance, which was evaluated using the tail-flick and paw-withdrawal tests. Response latency was calculated as the percentage of maximum possible effect (%MPE). A bolus of DAMGO was administered by i.t. injection on day 6, and the tail-flick and paw-withdrawal tests were carried out 24, 48, and 72 h later. Membrane and cytosolic MOR expression was assessed by western blotting. HEK293 cells were transfected with MOR-FLAG plasmid and after 6 days of morphine treatment (10 μM), the cells were treated with 1 μM DAMGO, and MOR localization was examined by immunofluorescence analysis 30 and 60 min later. Repeated morphine treatment induced tolerance after 5 days; however, i.t. DAMGO administration restored morphine sensitivity and enhanced acute morphine-induced antinociception after 24, 48, and 72 h. In HEK293 cells, DAMGO treatment stimulated MOR internalization after 30 min and MOR recycling to the membrane after 1 h. Membrane and cytoplasmic MOR expression in vivo was unchanged 24, 48, and 72 h after i.t. DAMGO injection. Morphine does not cause significant MOR internalization or downregulation, and can readily induce tolerance. DAMGO counters this effect by enhancing receptor endocytosis, thereby reversing morphine-induced antinociceptive tolerance and restoring its analgesic efficacy.

Blood-borne and brain-derived microparticles in morphine-induced anti-nociceptive tolerance

We hypothesized that elevations of microparticles (MPs) would occur with morphine administration to mice. Repetitive dosing to induce anti-nociceptive tolerance increases blood-borne MPs by 8-fold, and by 10-fold in deep cervical lymph nodes draining brain glymphatics. MPs express proteins specific to cells including neutrophils, microglia, astrocytes, neurons and oligodendrocytes. Interleukin (IL)-1β content of MPs increases 68-fold. IL-1β antagonist administration diminishes blood-borne and cervical lymph node MPs, and abrogates tolerance induction. Intravenous polyethylene glycol Telomer B, a surfactant that lyses MPs, and intraperitoneal methylnaltrexone also inhibit MPs elevations and tolerance. Critically, neutropenic mice do not develop anti-nociceptive tolerance, elevations of blood-borne or cervical node MPs. Immunohistochemical evidence for microglial activation by morphine does not correlated with the MPs response pattern. Neutrophil-derived MPs appear to be required for morphine-induced anti-nociceptive tolerance. Further, patients entering treatment for opioid use disorder exhibit similar MPs elevations as do tolerant mice.

Pellino1 Contributes to Morphine Tolerance by Microglia Activation via MAPK Signaling in the Spinal Cord of Mice.

Chronic morphine-induced antinociceptive tolerance is a major unresolved issue in clinical practices, which is associated with microglia activation in the spinal cord. E3 ubiquitin ligase Pellino1 (Peli1) is known to be an important microglia-specific regulator. However, it is unclear whether Peli1 is involved in morphine tolerance. Here, we found that Peli1 levels in the spinal cord were significantly elevated in morphine tolerance mouse model. Notably, Peli1 was expressed in a great majority of microglia in the spinal dorsal horn, while downregulation of spinal Peli1 attenuated the development of morphine tolerance and associated hyperalgesia. Our biochemical data revealed that morphine tolerance-induced increase in Peli1 was accompanied by spinal microglia activation, activation of mitogen-activated protein kinase (MAPK) signaling, and production of proinflammatory cytokines. Peli1 additionally was found to promote K63-linked ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) in the spinal cord after repeated morphine treatment. Furthermore, knocking down Peli1 in cultured BV2 microglial cells significantly attenuated inflammatory reactions in response to morphine challenge. Therefore, we conclude that the upregulation of Peli1 in the spinal cord plays a curial role in the development of morphine tolerance via Peli1-dependent mobilization of spinal microglia, activation of MAPK signaling, and production of proinflammatory cytokines. Modulation of Peli1 may be a potential strategy for the prevention of morphine tolerance.

Reversal of the Development of Antinociceptive Effects to Chronic Morphine in Mice by Fecal Microbiota Transplantation (FMT)

Morphine remains one of the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. However, the long‐term use of this excellent pain reliever in man is limited by side‐effects that include analgesic tolerance, dependence and constipation. We have previously shown that depletion of gut bacteria by oral vancomycin results in the loss of antinociceptive tolerance, decreases epithelial permeability and reduces colonic inflammation by chronic morphine. The aim of this study was to investigate the effect of colonizing the gut with non‐opioid commensal microbiome on reversal of morphine‐induced antinociceptive tolerance. Gut bacteria were collected from 5 day placebo and morphine pelleted mice from fecal pellets designated as donor mice groups (PP‐FMT and MP‐FMT). The microbiome was then orally gavaged (1×109 cells/dose) daily for 5 days to placebo (PP) and morphine (MP) pelleted (75 mg pellet, 5 days) recipient mice groups, and the presence of antinociceptive tolerance was determined by morphine challenge (10 mg/kg, s.c.) in the tail immersion and hot plate tests. Daily gavage of the microbiome from morphine pelleted mice to the morphine‐pelleted recipient mice (MP+MP‐FMT) resulted in antinociceptive tolerance to morphine challenge. However, gavage of the microbiome from placebo pelleted donor mice to the morphine pelleted recipient mice (PP+MP‐FMT), prevented the development of antinociceptive tolerance. We further investigated whether an exogenous microbiome could reverse chronic morphine induced antinociceptive tolerance development. In these studies, oral gavage of gut microbiome was commenced after development of tolerance to morphine. Similar to the induction of tolerance, oral gavage of placebo microbiome to morphine tolerant mice (MP+PP‐FMT) reversed antinociceptive tolerance. In order to determine if transplantation of the gut microbiome also prevents the loss of epithelial integrity by chronic morphine treatment, intestinal permeability was measured by oral administration of FITC (Fluorescein isothiocyanate)‐dextran (44 mg/kg, p.o.) in placebo or morphine pelleted mice for 5 days. FITC levels were significantly higher in morphine pelleted mice (MP) treated with the microbiome from morphine donors (MP‐FMT) than placebo pelleted controls (PP, p<0.0001). However, the epithelial integrity was maintained in morphine pelleted mice (MP) treated with the commensal microbiome from placebo donors (PP‐FMT). These studies define a novel microbiome‐gut‐brain interaction in the development of opioid tolerance.Support or Funding InformationNIH R01 DA036975 and NIH T32 DA007027This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of glucosamine against morphine-induced antinociceptive tolerance and dependence in mice

BackgroundThe most important limitations of morphine in pain therapy are its tolerance and dependence. In this study, we evaluated the protective effect of glucosamine against morphine-induced tolerance and dependence in mice.MethodsMice received twice daily morphine (20 mg/kg, s.c.) alone, or along with orally administered glucosamine (500, 1000 and 2000 mg/kg), for 9 continuous days. To assess antinociceptive effect of morphine, percentage of maximal possible effect (%MPE) of animals exposed to thermal stimulus was measured in the hot plate test, 30 min after morphine administration. Test was performed on days 1, 3, 5, 7 and 9. The effect of glucosamine on the naloxone (5 mg/kg, i.p.)-precipitated morphine withdrawal, was also evaluated. Changes in brain gene expression levels of induced nitric oxide synthase (iNOS), enzyme responsible for nitric oxide generation, as well as pro-inflammatory mediator, tumor necrosis alpha (TNF-α) were measured in morphine tolerated animals, as well as after withdrawal by real-time polymerase chain reaction (RT-PCR). Protein content of TNF-α was evaluated via ELISA assay.ResultsTolerance to antinociceptive effect of morphine was developed after 7 days of morphine treatment. The concurrent administration of glucosamine (500, 1000 and 2000 mg/kg) with morphine, significantly inhibited tolerance development, on days 7 and 9. In addition, glucosamine ameliorated the naloxone-precipitated opioid withdrawal symptoms (tremor, jumping, teeth chattering, grooming). However, diarrhea was significantly improved only with the dose of 500 mg/kg. Increased mRNA expression of iNOS as well as TNF-α mRNA expression and protein, after both morphine tolerance and withdrawal, were considerably reduced by glucosamine (1000 mg/kg) in the morphine withdrawal animals.ConclusionThese data support the utility of glucosamine in attenuating both tolerance to nociceptive effects of morphine as well as withdrawal-induced behavioral profile. Anti-oxidant and anti-inflammatory effects are responsible, at least in part, for the protective effects of this drug.

Metabotropic glutamate receptor 5 signalling induced NMDA receptor subunits alterations during the development of morphine-induced antinociceptive tolerance in mouse cortex

Background and purposeThe aim of this study was to investigate the interactions between mGluR5 and NMDAR during the development of tolerance to the analgesic effect of chronic morphine following disruption of the link between mGluR5 and NMDAR. MethodsFor these experiments, mGluR5 knockout (KO) mice, Shank3 ΔC mutant mice and their littermate controls were used. Repeated morphine treatment through intrathecal injections (i.t. 10 μg/10 μl twice daily for 5 days) was used to induce antinociceptive tolerance and thermal hyperalgesia, which was determined by tail-flick latency and calculated as a percentage of the maximum possible effect (%MPE). Expression of NMDAR subunits (NR1, NR2A and NR2B) and phosphorylation of NR2B in cortex were evaluated by Western blot. ResultsChronic morphine treatment increased the total expression of mGluR5 and NR2A but not NR1 and NR2B in the cortex and enhanced the phosphorylation of NR2B in wild-type mice after tolerance developed. mGluR5 KOs had attenuated morphine-induced tolerance and thermal hyperalgesia and reduced chronic morphine-induced phosphorylation of NR2B but not total NR2A expression. Shank3 ΔC mice were used to imitate the loss of the physiological connection between mGluR5 and NMDAR, and the mice showed a similar analgesic effect to morphine as their littermate controls, and there were no differential effects on NMDAR subunits compared with controls during the development of morphine-induced tolerance. ConclusionsIt may be mGluR5-mediated PKC signalling that plays the most significant role in the mechanisms underlying morphine-induced antinociceptive tolerance rather than the physiological connection between mGluR5 and NMDAR.

Cathepsin S in the spinal microglia facilitates morphine-induced antinociceptive tolerance in rats

Microglia-derived lysosomal cysteine protease cathepsin S (CatS) is increasingly recognized as important mediators to exaggerate nociceptive signaling. However, the patterns and functional roles of CatS in morphine tolerance have never been investigated. Here, we showed that mature form of CatS was exclusively upregulated in the spinal microglia following chronic morphine treatment. Pharmacological blockade of CatS before each morphine treatment prolonged the efficacy of morphine analgesia. Correspondingly, inhibition of CatS suppressed activation of spinal microglia and phosphorylated p38 MAPK. Finally, intrathecal injection of selective microglia inhibitor minocycline reduced upregulation of mature CatS induced by chronic morphine treatment. Our data provide novel insight into the cellular mechanisms underlying morphine antinociceptive tolerance and highlight CatS as a therapeutic target for preventing morphine tolerance.

Sumatriptan effects on morphine-induced antinociceptive tolerance and physical dependence: The role of nitric oxide

Sumatriptan, a 5HT (5-hydroxytryptamine)1B/1D receptor agonist, showed neuroprotection in different studies. The aim of the present study was to investigate the effect of sumatriptan on morphine-induced antinociceptive tolerance and physical dependence. We also investigated the possible role of nitric oxide (NO) on sumatriptan effects.Tolerance was induced by morphine injection (50, 50, 75 mg/kg) three times daily for five days. Antinociceptive latency after acute and chronic treatment with sumatriptan (0.001, 0.01, 0.1 and 1 mg/kg) was measured by hot plate test in morphine-dependent animals. To investigate the possible involvement of NO, different isoforms of nitric oxide synthase (NOS) inhibitors including L-NAME, aminoguanidine and 7-nitroindazole were co-administered with sumatriptan. Nitrite level in mice hippocampus was quantified by Griess method. To examine the role of sumatriptan on physical dependence, three parameters of withdrawal signs were recorded after injection of naloxone (4 mg/kg).Acute treatment with sumatriptan (0.01, 0.1 and 1 mg/kg) attenuated the antinociceptive tolerance (P < 0.001). Chronic injection of sumatriptan (0.001, 0.01 and 0.1 mg/kg), as well, decreased the antinociceptive tolerance (P < 0.001). Moreover, co-administration of NOS inhibitors prevented the effects of sumatriptan. Sumatriptan significantly increased the level of nitrite only after chronic administration. Sumatriptan administration showed no alteration in naloxone-precipitated withdrawal signs.Acute and chronic administration of sumatriptan attenuated morphine antinociceptive tolerance; at least in chronic phase via nitrergic pathway. Our data did not support beneficial effects of sumatriptan on morphine-induced physical dependence in mice.

The Effect of Colonic Inflammation on Morphine Induced Antinociceptive Tolerance

Opioids are one of the most widely prescribed classes of drugs for the treatment of moderate to severe pain. Opioids are prescribed for abdominal pain, a major symptom associated with Crohn's Disease. Recent studies have reported that continued opioid use in patients with Crohn's Disease is associated with increased mortality rate, duration and severity of disease, as well as infection. However, it is not clear if this association is causal. We have previously demonstrated that chronic use of morphine, in addition to the development of antinociceptive tolerance, also causes the disruption of the intestinal barrier function resulting in bacterial translocation from the gut lumen into the peritoneal organs, thus inducing an inflammatory response in the colon. Therefore, we tested if inflammation in the colon, as in Crohn's disease, affects the rate of antinociceptive tolerance development to morphine. To test this hypothesis, we induced colonic inflammation in Swiss Webster male mice using 2,4,6‐trinitro‐benzene sulfonic acid (TNBS) or vehicle intrarectally and then surgically implanted 25 mg, 50 mg (2 × 25 mg), or 75 mg slow release morphine pellets or placebo pellets subcutaneously on the dorsum. Tail immersion assay was performed on day 3, 4, and 5 post treatment and colons were extracted from the animals to measure mRNA expression of Interleukin 1 beta (IL‐1β) and tumor necrosis factor alpha (TNF‐α) by real‐time polymerase chain reaction (RT‐PCR). A morphine (10 mg/kg) challenge was used to test for the development of antinociceptive tolerance at various times in morphine pelleted with (n= 7) or without TNBS (n= 5). In TNBS + morphine pelleted mice, tolerance developed by day 5 in 25 mg morphine pelleted mice, by day 4 in 50 mg (2 × 25 mg) morphine pelleted mice, and as early as day 2 in 75 mg morphine pelleted mice. In the absence of TNBS‐induced inflammation, tolerance to morphine did not develop to either 25 mg or 50 mg morphine pellets up to day 5, and at day 4 in 75 mg morphine pellets. Furthermore, IL‐1β production was significantly increased in the morphine and placebo pelleted treated animals with TNBS from day 1 compared to the vehicle treated groups. In summary, chronic morphine administration in the presence of colonic inflammation leads to an increased rate and extent of antinociceptive tolerance development.Support or Funding InformationAgency: NIDA Agency Number: 5R01DA036975‐04This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spinal TRPC6 channels contributes to morphine-induced antinociceptive tolerance and hyperalgesia in rats

The chronic administration of opioids results in the development of morphine analgesic tolerance and withdrawl-induced hyperalgesia, which limits their clinical utility in pain treatment. However, the cellular mechanisms underlying opioid-induced tolerance and hyperalgesia are not fully understood. The transient receptor potential canonical channel TRPC6 is important for brain development and function, as it regulates cytosolic, endoplasmic reticulum, and mitochondrial Ca2+ levels in neural cells. Here, we report that TRPC6 expression in the spinal cord was up-regulated after chronic morphine treatment. Furthermore, inhibition of TRPC6 in the spinal cord blocked the induction of morphine tolerance and hyperalgesia without affecting basal pain perception. These effects were attributed to the attenuation of morphine-induced neuroimmune activation and increased levels of CaMKIIα and nNOS in the spinal cord. This data suggests that specific TRPC6 inhibitors could be utilized for the prevention of morphine-induced antinociceptive tolerance and hyperalgesia in chronic pain management.

Blockade of neuronal dopamine D2 receptor attenuates morphine tolerance in mice spinal cord.

Tolerance induced by morphine remains a major unresolved problem and significantly limits its clinical use. Recent evidences have indicated that dopamine D2 receptor (D2DR) is likely to be involved in morphine-induced antinociceptive tolerance. However, its exact effect and molecular mechanism remain unknown. In this study we examined the effect of D2DR on morphine antinociceptive tolerance in mice spinal cord. Chronic morphine treatment significantly increased levels of D2DR in mice spinal dorsal horn. And the immunoreactivity of D2DR was newly expressed in neurons rather than astrocytes or microglia both in vivo and in vitro. Blockade of D2DR with its antagonist (sulpiride and L-741,626, i.t.) attenuated morphine antinociceptive tolerance without affecting basal pain perception. Sulpiride (i.t.) also down-regulated the expression of phosphorylation of NR1, PKC, MAPKs and suppressed the activation of astrocytes and microglia induced by chronic morphine administration. Particularly, D2DR was found to interact with μ opioid receptor (MOR) in neurons, and chronic morphine treatment enhanced the MOR/D2DR interactions. Sulpiride (i.t.) could disrupt the MOR/D2DR interactions and attenuate morphine tolerance, indicating that neuronal D2DR in the spinal cord may be involved in morphine tolerance possibly by interacting with MOR. These results may present new opportunities for the treatment and management of morphine-induced antinociceptive tolerance which often observed in clinic.

Thalidomide attenuates the development and expression of antinociceptive tolerance to μ-opioid agonist morphine through l-arginine-iNOS and nitric oxide pathway

Morphine is a μ-opioid analgesic drug which is used in the treatment and management of chronic pain. However, due to development of antinociceptive tolerance its clinical use is limited. Thalidomide is an old glutamic acid derivative which recently reemerged because of its potential to counteract a number of disorders including neurodegenerative disorders. The potential underlying mechanisms and effects of thalidomide on morphine-induced antinociceptive tolerance is still elusive. Hence, the present study was designed to explore the effect of thalidomide on the development and expression of morphine antinociceptive tolerance targeting l-arginine-nitric oxide (NO) pathway in mice and T98G human glioblastoma cell line. When thalidomide was administered in a dose of 17.5mg/kg before each dose of morphine chronically for 5days it prevented the development of antinociceptive tolerance. Also, a single dose of thalidomide 20mg/kg attenuated the expression phase of antinociceptive tolerance. The protective effect of thalidomide was augmented in development phase when co-administration with NOS inhibitors like L-NAME (non- selective NOS inhibitor; 2mg/kg) or aminoguanidine (selective inducible NOS inhibitor; 50mg/kg). Also, the reversal effect of thalidomide in expression phase was potentiated when concomitantly administrated with L-NAME (5mg/kg) or aminoguanidine (100mg/kg). Co-administration of ODQ (a guanylyl cyclase inhibitor) 10mg/kg in developmental phase or 20mg/kg in expression phase also progressively increased the pain threshold. In addition, thalidomide (20μM) also significantly inhibited the overexpression of iNOS gene induced by morphine (2.5μM) in T98G cell line. Hence, our findings suggest that thalidomide has protective effect both in the development and expression phases of morphine antinociceptive tolerance. It is also evident that this effect of thalidomide is induced by the inhibition of NOS enzyme predominantly iNOS.

Atorvastatin attenuates the antinociceptive tolerance of morphine via nitric oxide dependent pathway in male mice.

The development of morphine-induced antinociceptive tolerance limits its therapeutic efficacy in pain management. Atorvastatin, or competitive inhibitor of 3-hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase, is mainstay agent in hypercholesterolemia treatment. Beyond the cholesterol-lowering activity, exploration of neuroprotective properties of this statin indicates its potential benefit in central nervous disorders. The aim of the present study was to assess the effects of atorvastatin in development and expression of morphine-induced analgesic tolerance in male mice and probable involvement of nitric oxide. Chronic and acute treatment with atorvastatin 10 and 20mg/kg, respectively, could alleviate morphine tolerance in development and expression phases. Chronic co-administration of nitric oxide synthase (NOS) inhibitors including L-NAME (non selective NOS inhibitor; 2mg/kg), aminoguanidine (selective inducible NOS inhibitor; 50mg/kg) and 7-NI (selective neuronal NOS inhibitor; 15mg/kg) with atorvastatin blocked the protective effect of atorvastatin in tolerance reversal. Moreover, reversing the atorvastatin effect was also observed in acute simultaneous treatment of L-NAME (5mg/kg) and aminoguanidine (100mg/kg) with atorvastatin. Co-treatment of guanylyl cyclase inhibitor, ODQ (chronic dose: 10mg/kg and acute dose: 20mg/kg) was associated with prevention of atorvastatin anti-tolerance properties. Our results revealed that the atorvastatin modulating role in morphine antinociceptive tolerance is mediated at least in part via nitric oxide in animal pain models of hot plate and tail flick.