Analgesic Potency Of Morphine Research Articles

Blockade of peripheral nociceptive signal input relieves the formation of spinal central sensitization and retains morphine efficacy in a neuropathic pain rat model

Neural plasticity, especially central sensitization, is essential for developing and maintaining neuropathic pain. Unfortunately, the analgesic potency of morphine is greatly reduced in animal models and patients with neuropathic pain. We hypothesized that pre-activation of spinal N-methyl-d-aspartate receptors (NMDARs) by agonist or neuropathic pain facilitated the development of morphine-induced analgesic tolerance. We therefore investigated the effects of spinal NMDAR activation, induced by neuropathic pain, on the development of morphine-induced analgesic tolerance in male Sprague-Dawley rats. Four days of chronic constriction injury (CCI) induced upregulation of spinal NR1. Once established, spinal central sensitization accelerated the development of morphine-induced analgesic tolerance. Continuous ropivacaine infusion prevented CCI-induced increases in spinal Substance P (SP), NR1, and TRPV1. Blockade of peripheral nociceptive inputs prevented chronic morphine-induced increases in spinal SP, NR1, and TRPV1 and a rightward shift of the morphine dose-response curve in the CCI model. These findings suggest that pre-activation of spinal NMDARs contributes to central sensitization and potentiates the development of morphine-induced analgesic tolerance. Interruption of the peripheral nociceptive inputs during the induction phase could prevent spinal central sensitization and retain morphine efficacy, thereby delaying the development of morphine-induced tolerance in patients with neuropathic conditions.

Exchange factor directly activated by cAMP-PKCε signalling mediates chronic morphine-induced expression of purine P2X3 receptor in rat dorsal root ganglia.

The P2X3 receptor is a major receptor in the processing of nociceptive information in dorsal root ganglia. We investigated the role of the P2X3 receptor and the detailed mechanisms underlying chronic morphine-induced analgesic tolerance in rats. Repeated i.t. morphine treatment was used to induce anti-nociceptive tolerance. The expression of spinal P2X3 receptor, phosphorylated PKCε and exchange factor directly activated by cAMP (Epac) were evaluated. Effects of A-317491 (P2X3 antagonist), ε-V1-2 (PKCε inhibitor) and ESI-09 (Epac inhibitor) on mechanical pain thresholds and tail-flick latency after chronic morphine treatment were determined. Co-localization of P2X3 receptor with NeuNs (marker of neuron), IB4 (marker of small DRG neurons), peripherin, PKCε and Epac were performed by double immunofluorescence staining. Chronic morphine time-dependently increased the expression of P2X3 receptor, phosphorylated PKCε and Epac in DRGs. ε-V1-2 prevented chronic morphine-induced expression of P2X3 receptor. ESI-09 decreased the phosphorylation of PKCε and up-regulated expression of Epac after chronic morphine exposure. Mechanical pain thresholds and tail-flick latency showed that A317491, ε-V1-2 and ESI-09 significantly attenuated the loss of morphine's analgesic potency. Morphine-induced P2X3 receptor expression mainly occurred in neurons staining for IB4 and peripherin. Co-localization of P2X3 receptor with PKCε and Epac was demonstrated in the same neurons. Chronic morphine exposure increased the expression of P2X3 receptor, and i.t. P2X3 receptor antagonists attenuated the loss of morphine's analgesic effect. Inhibiting Epac/PKCε signalling was shown to play a significant inhibitory role in chronic morphine-induced P2X3 receptor expression and attenuate morphine-induced tolerance.

Reversal of morphine-induced respiratory depression by doxapram in anesthetized rats

The present study was undertaken to investigate whether doxapram, a blocker of tandem pore K+ (TASK-1/−3) channels, is a useful tool for recovery from morphine-induced ventilatory disturbances. Spontaneous ventilation and the hind leg withdrawal response against noxious thermal stimulation were recorded simultaneously in anesthetized rats. Morphine (1.0mg/kg, i.v.) decreased the minute volume resulting from depression of the ventilatory rate and tracheal airflow. Concomitantly, it prolonged the latency of withdrawal response against the thermal stimulation. Subsequent intravenous injection of doxapram recovered the morphine-induced ventilatory depression. This effect of doxapram declined rapidly after a single injection (1.0–3.0mg/kg, i.v.) but persisted with a continuous infusion (0.33mg/kg/min). Neither single injection nor continuous infusion of doxapram had any detectable effect on the analgesic potency of morphine. The central respiratory activity was recorded from the phrenic nerve in anesthetized, vagotomized, paralyzed and artificially ventilated rats. Morphine (3.0mg/kg, i.v.) induced respiratory depression, characterized by a prolonged plateau-like inspiratory discharge (apneustic discharge) in the phrenic nerve. Doxapram (10mg/kg, i.v.) restored the morphine-induced apneustic discharge to an augmenting inspiratory discharge. This study demonstrated that doxapram counteracted morphine-induced respiratory depression by stimulating the central respiratory network without compromising morphine antinociception. These results support the clinical use of doxapram for amelioration of ventilatory disturbances in patients treated with opioids.

Contribution of adrenomedullin to the switch of G protein-coupled μ-opioid receptors from Gi to Gs in the spinal dorsal horn following chronic morphine exposure in rats.

Chronic exposure to morphine increases spinal adrenomedullin (AM) bioactivity resulting in the development and maintenance of morphine tolerance. This study investigated the possible involvement of AM in morphine-evoked alteration in μ-opioid receptor-coupled G proteins. Agents were administered intrathecally (i.t.) in rats. Nociceptive behaviours and cumulative dose-response of morphine analgesia were assessed. Neurochemicals in the spinal dorsal horn were assayed by immunoprecipitation, Western blot analysis and ELISA. Intrathecal injection of AM (8μg) for 9days decreased and increased the levels of μ receptor-coupled Gi and Gs proteins respectively. Morphine stimulation (5μg) after chronic treatment with AM also induced an increase in cAMP production in the spinal dorsal horn. Co-administration of the selective AM receptor antagonist AM22-52 inhibited chronic morphine-evoked switch of G protein-coupled μ receptor from Gi to Gs. Chronic exposure to AM increased the phosphorylation of cAMP-responsive element-binding protein (CREB) and ERK. Co-administration of the PKA inhibitor H-89 (5μg) or MEK1 inhibitor PD98059 (1μg) reversed the AM-induced thermal/mechanical hypersensitivity, decline in morphine analgesic potency, switch of G protein-coupled μ receptor and increase in cAMP. The present study supports the hypothesis that an increase in AM activity in the spinal dorsal horn contributes to the switch of the μ receptor-coupled G protein from Gi to Gs protein via the activation of cAMP/PKA/CREB and ERK signalling pathways in chronic morphine use.

(334) Differences in morphine analgesic potency between sensory and affective components of formalin-evoked pain in male and female rats

(334) Differences in morphine analgesic potency between sensory and affective components of formalin-evoked pain in male and female rats

Involvement of neuronal TGF‐β activated kinase 1 in the development of tolerance to morphine‐induced antinociception in rat spinal cord

Tolerance induced by morphine and other opiates remains a major unresolved problem in the clinical management of pain. There is now good evidence for the importance of MAPKs in morphine-induced antinociceptive tolerance. A member of the MAPK kinase kinase family, TGF-β activated kinase 1 (TAK1) is the common upstream kinase of MAPKs. Here, we have assessed the involvement of TAK1 in the development of tolerance to morphine-induced analgesia. The effects of an antagonist of TAK1 on morphine tolerance were investigated in vivo using the Randall-Selitto test, and the mechanism was investigated using Western blot and immunohistochemistry. The expression of TAK1 after chronic morphine exposure was also evaluated in vitro by immunohistochemistry. Chronic intrathecal morphine exposure up-regulated protein levels and phosphorylation of spinal TAK1. TAK1 immunoreactivity was co-localized with the neuronal marker NeuN. Intrathecal administration of 5Z-7-oxozeaenol (OZ), a selective TAK1 inhibitor, attenuated the loss of morphine analgesic potency and morphine-induced TAK1 up-regulation. Furthermore, OZ decreased the up-regulated expression of spinal p38 and JNK after repeated morphine exposure. In vitro studies demonstrated that sustained morphine treatment induced TAK1 up-regulation, which was reversed by co-administration of OZ. A bolus injection of OZ showed some reversal of established morphine antinociceptive tolerance. TAK1 played a pivotal role in the development of morphine-induced antinociceptive tolerance. Modulation of TAK1 activation by the selective inhibitor OZ in the lumbar spinal cord may prove to be an attractive adjuvant therapy to attenuate such tolerance.

Melanocortin 4 Receptor Is Involved in the Development of Morphine Tolerance

Background: Prolonged morphine treatment usually results in the development of analgesic tolerance. Melanocortin 4 receptor (MC4R) is involved in the development of morphine tolerance. The aim of this study was to examine the effects of an MC4R antagonist, HS014, on MC4R-mediated hyperalgesia and on microglia and cytokine expression in the spinal cord of rat during morphine tolerance.Methods: Thirty rats were assigned randomly to the N group, M group, HM group, NM group and HN group (N=6 in each group). Rats received 5-day treatment with saline (N group) or morphine (M group). Rats were given an intrathecal injection (i.t.) of HS014 (HM group) or saline (NM group) at 15 minutes prior to the morphine challenge. In the HN group, rats were injected with HS014 at 15 minutes prior to the saline. To observe the effect of HS014 on the development of morphine tolerance, morphine-tolerant rats were assigned to M1 group, M2 group, M3 group and N group on day 6 (N=6 in each group). The morphine-tolerant rats in M1 group, M2 group and M3 group received morphine (10 mg/kg, i.p.), HS014 (5 μg, i.t.) and HS014 (5 μg, i.t.) followed 15 minutes later by injection of morphine (10 mg/kg, i.p.), respectively. Control rats (N group, N=6) were injected with saline under identical conditions.Results: Morphine treatment (10 mg/kg, i.p. twice daily) over 5 days induced tolerance as reflected by a significant reduction of withdrawal latency from 29.67 ± 1.81 s to 8.67 ± 1.70 seconds in the hot-plate test. Repeated HS014 injection prior to morphine administration inhibited the development of morphine tolerance. Furthermore, a single administration of HS014 restored morphine analgesic potency in morphine-tolerant rats. Immunohistochemical staining showed that the administration of HS014 during the induction of morphine tolerance inhibited the activation of microglia, reduced the expression of proinflammatory cytokines, such as interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α, and increased the expression of anti-inflammatory cytokines (IL-10) at the L5 lumbar spinal cord.Conclusions: HS014 attenuates the development of antinociceptive tolerance following chronic administration of morphine by inhibiting microglial activation and reducing the expression of IL-1, IL-6 and TNF-α. Citation: Hong-Mei Xu, Ze-Jun Niu, Hai-Chen Chu, Xue-Feng Zhang. Melanocortin 4 receptor is involved in the development of morphine tolerance. J Anesth Perioper Med 2014; 1: 90-6. doi: 10.24015/JAPM.2014.0014This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Platelet-derived growth factor receptor-β antagonism restores morphine analgesic potency against neuropathic pain.

BackgroundChronic, intractable pain is a problem of pandemic proportions. Pain caused by nerve injuries (neuropathic pain) is extremely difficult to treat. For centuries, opiates such as morphine have been the first-line treatment for severe chronic pain. However, opiates are often ineffective against neuropathic pain, leaving few options for suffering patients. We previously demonstrated that platelet-derived growth factor- β (PDGFR-β) inhibition completely eliminated morphine tolerance. In these studies, we determined whether PDGFR-β inhibition could improve the effectiveness of morphine for neuropathic pain treatment.Results and FindingsSpinal nerve ligation was performed in male Sprague-Dawley rats. The clinically used PDGFR antagonist imatinib did not relieve mechanical pain in a nerve injury model as determined by Von Frey assay. Surprisingly, combining imatinib with a previously ineffective dose of morphine led to complete pain relief. Scavenging released PDGF-B also markedly augmented the analgesic effect of morphine.ConclusionsThese findings suggest the novel hypothesis that PDGF-B released by injured nerves renders animals resistant to morphine, implying that PDGFR-β inhibition could potentially eliminate the tremendous suffering caused by neuropathic pain.

Opioid Antagonists

Opioid Antagonists

Activation of Mas oncogene-related gene (Mrg) C receptors enhances morphine-induced analgesia through modulation of coupling of μ-opioid receptor to Gi-protein in rat spinal dorsal horn

Mas oncogene-related gene (Mrg) G protein-coupled receptors are exclusively expressed in small-sized neurons in trigeminal and dorsal root ganglia (DRG) in mammals. The present study investigated the effect of MrgC receptor activation on morphine analgesic potency and addressed its possible mechanisms. Intrathecal (i.t.) administration of the specific MrgC receptor agonist bovine adrenal medulla 8-22 (BAM8-22, 3nmol) increased morphine-induced analgesia and shifted the morphine dose–response curve to the left in rats. Acute morphine (5μg) reduced the coupling of μ-opioid receptors (MORs) to Gi-, but not Gs-, protein in the spinal dorsal horn. The i.t. BAM8-22 (3nmol) prevented this change of G-protein repertoire while the inactive MrgC receptor agonist BAM8-18 (3nmol, i.t.) failed to do so. A double labeling study showed the co-localization of MrgC and MORs in DRG neurons. The i.t. BAM8-22 also increased the coupling of MORs to Gi-protein and recruited Gi-protein from cytoplasm to the cell membrane in the spinal dorsal horn. Application of BAM8-22 (10nM) in the cultured ganglion explants for 30min increased Gi-protein mRNA, but not Gs-protein mRNA. The present study demonstrated that acute administration of morphine inhibited the repertoire of MOR/Gi-protein coupling in the spinal dorsal horn in vivo. The findings highlight a novel mechanism by which the activation of MrgC receptors can modulate the coupling of MORs with Gi-protein to enhance morphine-induced analgesia. Hence, adjunct treatment of MrgC agonist BAM8-22 may be of therapeutic value to relieve pain.

Experimental Pain and Opioid Analgesia in Volunteers at High Risk for Obstructive Sleep Apnea

BackgroundObstructive sleep apnea (OSA) is characterized by recurrent nocturnal hypoxia and sleep disruption. Sleep fragmentation caused hyperalgesia in volunteers, while nocturnal hypoxemia enhanced morphine analgesic potency in children with OSA. This evidence directly relates to surgical OSA patients who are at risk for airway compromise due to postoperative use of opioids. Using accepted experimental pain models, we characterized pain processing and opioid analgesia in male volunteers recruited based on their risk for OSA.MethodsAfter approval from the Intitutional Review Board and informed consent, we assessed heat and cold pain thresholds and tolerances in volunteers after overnight polysomnography (PSG). Three pro-inflammatory and 3 hypoxia markers were determined in the serum. Pain tests were performed at baseline, placebo, and two effect site concentrations of remifentanil (1 and 2 µg/ml), an μ-opioid agonist. Linear mixed effects regression models were employed to evaluate the association of 3 PSG descriptors [wake after sleep onset, number of sleep stage shifts, and lowest oxyhemoglobin saturation (SaO2) during sleep] and all serum markers with pain thresholds and tolerances at baseline, as well as their changes under remifentanil.ResultsForty-three volunteers (12 normal and 31 with a PSG-based diagnosis of OSA) were included in the analysis. The lower nadir SaO2 and higher insulin growth factor binding protein-1 (IGFBP-1) were associated with higher analgesic sensitivity to remifentanil (SaO2, P = 0.0440; IGFBP-1, P = 0.0013). Other pro-inflammatory mediators like interleukin-1β and tumor necrosis factor-α (TNF-α) were associated with an enhanced sensitivity to the opioid analgesic effect (IL-1β, P = 0.0218; TNF-α, P = 0.0276).ConclusionsNocturnal hypoxemia in subjects at high risk for OSA was associated with an increased potency of opioid analgesia. A serum hypoxia marker (IGFBP-1) was associated with hypoalgesia and increased potency to opioid analgesia; other pro-inflammatory mediators also predicted an enhanced opioid potency.Trial Registration: ClinicalTrials.gov NCT00672737.

Analgesic Tolerance of Opioid Agonists in Mutant Mu-Opioid Receptors Expressed in Sensory Neurons following Intrathecal Plasmid Gene Delivery

BackgroundPhosphorylation sites in the C-terminus of mu-opioid receptors (MORs) are known to play critical roles in the receptor functions. Our understanding of their participation in opioid analgesia is mostly based on studies of opioid effects on mutant receptors expressed in in vitro preparations, including cell lines, isolated neurons and brain slices. The behavioral consequences of the mutation have not been fully explored due to the complexity in studies of mutant receptors in vivo. To facilitate the determination of the contribution of phosphorylation sites in MOR to opioid-induced analgesic behaviors, we expressed mutant and wild-type human MORs (hMORs) in sensory dorsal root ganglion (DRG) neurons, a major site for nociceptive (pain) signaling and determined morphine- and the full MOR agonist, DAMGO,-induced effects on heat-induced hyperalgesic behaviors and potassium current (IK) desensitization in these rats.FindingsA mutant hMOR DNA with the putative phosphorylation threonine site at position 394 replaced by an alanine (T394A), i.e., hMOR-T, or a plasmid containing wild type hMOR (as a positive control) was intrathecally delivered. The plasmid containing GFP or saline was used as the negative control. To limit the expression of exogenous DNA to neurons of DRGs, a neuron-specific promoter was included in the plasmid. Following a plasmid injection, hMOR-T or hMOR receptors were expressed in small and medium DRG neurons. Compared with saline or GFP rats, the analgesic potency of morphine was increased to a similar extent in hMOR-T and hMOR rats. Morphine induced minimum IK desensitization in both rat groups. In contrast, DAMGO increased analgesic potency and elicited IK desensitization to a significantly less extent in hMOR-T than in hMOR rats. The development and extent of acute and chronic tolerance induced by repeated morphine or DAMGO applications were not altered by the T394A mutation.ConclusionsThese results indicate that phosphorylation of T394 plays a critical role in determining the potency of DAMGO-induced analgesia and IK desensitization, but has limited effect on morphine-induced responses. On the other hand, the mutation contributes minimally to both DAMGO- and morphine-induced behavioral tolerance. Furthermore, the study shows that plasmid gene delivery of mutant receptors to DRG neurons is a useful strategy to explore nociceptive behavioral consequences of the mutation.

Increased central and peripheral inflammation and inflammatory hyperalgesia in Zucker rat model of leptin receptor deficiency and genetic obesity

This study investigated whether sensitivity to nociceptive stimuli is altered in obese rats using established models of inflammatory pain, and using real-time PCR, profiled alterations in expression of key adipokine and inflammatory mediator mRNA (adiponectin, tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, inducible nitric oxide synthase (iNOS)) in spinal cord with obesity. Responses to thermal and mechanical stimulation of the hindpaw and paw oedema were assessed in adult male Zucker fatty rats (fa/fa) and their lean littermates (fa/-; n = 6-9 per group) in the absence of inflammation (acute nociception), then in response to intradermal hindpaw injection of carrageenan (3%; 50 μl) or capsaicin (10 μg; 50 μl) or hindpaw incision. The analgesic potency of morphine (1, 2.5 or 5 mg kg(-1) or vehicle; s.c.) was also assessed. Acute nociception was unaltered in obese animals, but following carrageenan-induced inflammation the obese rats were significantly more sensitive to mechanical and thermal stimulation of the inflamed paw, and displayed greater paw oedema. No difference in the capsaicin- or paw-incision-induced pain sensitivity or in the analgesic potency of morphine was observed between groups. Levels of adiponectin and inducible nitric oxide synthase mRNA were downregulated in spinal cord from obese rats, whereas tumour necrosis factor-α mRNA was upregulated; interleukin-1β and cyclo-oxygenase were unchanged. The increased pain sensitivity and inflammatory response together with changes in spinal adipokine expression in obese rats fit well with the hypothesis that obesity is a chronic low-grade inflammatory disorder, producing a state where responses to subsequent inflammatory challenge are potentiated.

Melanocortin 4 receptor antagonists attenuates morphine antinociceptive tolerance, astroglial activation and cytokines expression in the spinal cord of rat

Chronic use of morphine is accompanied by the development of morphine tolerance, which is one of the major problems associated with opiate treatment. Experimental evidence indicates that melanocortin 4 receptor (MC4R) is involved in development of morphine tolerance. Therefore, we investigated the influence of repeated intrathecal injection of a MC4R antagonist (HS014) on the development of morphine tolerance as measured by hot-plate test. It was also examined whether a single i.t. HS014 administration could counteract the loss of analgesic potency of morphine in morphine tolerant rats. We examined also the influence of i.t. HS014 administration on astrocytes activation and cytokines expression in the spinal cord of rat during morphine tolerance. Morphine treatment (10mg/kg, i.p. twice daily) over 5 days induced tolerance as reflected by a significant reduction of withdrawal latency from 29.67±1.81s to 8.67±1.70s in the hot-plate test. Repeated coadministration of HS014 and morphine, significantly prevented the development of morphine tolerance. A single administration of an MC4R antagonist restored morphine analgesic potency in morphine tolerant rats. Using immunohistochemical staining, we demonstrated the administration of MC4R during the induction of morphine tolerance inhibited the activation of astrocytes; reduced the expression of proinflammatory cytokines interleukin-1β, IL-6, and tumor necrosis factor-α; upregulated the expression of anti-inflammatory cytokines IL-10 at the L5 lumbar spinal cord. These results suggest that MC4R may be involved in the mechanisms of morphine tolerance and antagonists of this receptor may be a possible new target in the search for strategies preventing the development of morphine tolerance.

Different effects of morphine and oxycodone in experimentally evoked hyperalgesia: a human translational study

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT * Previous studies using short-lasting experimental pain stimulations in healthy volunteers have shown differences in opioid effects regarding visceral pain stimulations. However, these differences can be more pronounced in patients due to a sensitized pain system. Therefore, the aim of the present study was to mimic the clinical situation by investigating opioid effects on experimental pain in healthy volunteers after experimentally evoked hyperalgesia. WHAT THIS STUDY ADDS? * We now know that morphine and oxycodone exerts different effects in the sensitized pain system as we found a greater analgesic effect of oxycodone in response to skin, muscle and oesophageal pain stimulation. This supports clinicians' experiences that oxycodone can be superior to morphine in the treatment of some pain conditions. The evoked hyperalgesia bridged findings from studies in healthy volunteers to patients, and new fundamental knowledge on different analgesic effects in hyperalgesia was found. AIM Similar analgesics may have different analgesic potencies especially in patients in whom the pain system is sensitized. The aim was to investigate different opioid effects on experimental pain after the sensitized pain system was mimicked evoking hyperalgesia in healthy volunteers. METHODS Twenty-four healthy volunteers were randomized to treatment with morphine (30 mg orally) and oxycodone (15 mg orally) or placebo in a double-blind crossover study. Hyperalgesia was induced by oesophageal perfusion with acid and capsaicin. Several exploratory endpoints were studied using skin heat, muscle pressure and oesophageal mechanical, heat and electrical stimulation. Effects on pain from deeper structures were considered most important. RESULTS Different analgesic potencies were found. Oxycodone had a greater analgesic effect than morphine attenuating pain from: (i) heat stimulation of skin (P= 0.016); difference between the means of 0.39 degrees C, 95% CI 0.22, 2.09. (ii) muscle pressure (P < 0.001); difference between the means of 11.93kPa, 95% CI 5.4, 18.5. (iii) oesophageal heat stimulation (P < 0.001); difference between the means of 38.54 cm(2), 95% CI 15.37, 61.71 and (iv) oesophageal electrical stimulation (P= 0.016); difference between the means of 6.69mA, 95% CI 1.23, 12.13. CONCLUSION After sensitization of the pain system different analgesic potencies of morphine and oxycodone were found in response to skin, muscle and oesophageal pain stimulation, in which oxycodone had a greater effect. As similar differential analgesic potencies of the two opioids have been found in patients with chronic pain, the experimental hyperalgesia model bridged findings from studies in healthy volunteers to patients.

Involvement of Spinal Microglial P2X7 Receptor in Generation of Tolerance to Morphine Analgesia in Rats

Morphine loses analgesic potency after repeated administration. The underlying mechanism is not fully understood. Glia are thought to be involved in morphine tolerance, and P2X(7) purinergic receptor (P2X(7)R) has been implicated in neuron-glia communication and chronic pain. The present study demonstrated that P2X(7)R immunoreactivity was colocalized with the microglial marker OX42, but not the astrocytic marker GFAP, in the spinal cord. The protein level of spinal P2X(7)R was upregulated after chronic exposure to morphine. Intrathecal administration of Brilliant Blue G (BBG), a selective P2X(7)R inhibitor, significantly attenuated the loss of morphine analgesic potency, P2X(7)R upregulation, and microglial activation. Furthermore, RNA interference targeting the spinal P2X(7)R exhibited a similar tolerance-attenuating effect. Once morphine analgesic tolerance is established, it was no longer affected by intrathecal BBG. Together, our results suggest that spinal P2X(7)R is involved in the induction but not maintenance of morphine tolerance.

Changes in the gene expression of specific G-protein subunits correlate with morphine insensitivity in streptozotocin-induced diabetic rats

Several animal and human studies have shown a decreased analgesic potency of morphine in diabetic subjects. Since G-protein subunits have an important role in morphine effects at the cellular level and the exact mechanism(s) of diabetes-induced morphine insensitivity has not been fully clarified yet, the present study was designed to determine the changes in the levels of G(alphai), G(alphas), G(beta) mRNAs and proteins involved in this phenomenon. All experiments were carried out on male Wistar rats. The tail-flick test was used to assess the nociceptive threshold. Diabetes was induced by injection of 50 mg/kg (i.p.) streptozotocin. Four weeks after diabetes induction, the dorsal half of the lumbar spinal cord was assayed for the expression of G-protein subunits using semiquantitative RT-PCR and immunoblotting. The antinociceptive effect of intrathecal morphine (5, 10 and 15 microg i.t.) was significantly reduced in diabetic rats and these effects were reversed with insulin replacement. In diabetic animals, a significant increase in the mRNA levels of G(alphai) (23.5%) was observed in the dorsal portion of the lumbar spinal cord. The mRNA level of G(alphas) and G(beta) did not change. Following diabetes a significant decrease in the protein levels of G(alphai) was induced. In contrast, no significant changes were observed in the protein level of G(alphas) and G(beta). In diabetic animals that received insulin, levels of G(alphai) mRNA and protein were close to those in control rats. In conclusion, our results demonstrate that the expression pattern of the cellular components involved in morphine analgesia changes in diabetic animals. This may be, at least partly, responsible for diabetes-induced morphine insensitivity.

Increased analgesic tolerance to acute morphine in fosB knock-out mice: A gender study

The proteins of Fos family are a potential candidate to link molecular mechanisms of morphine action with behavioural effects such as morphine-induced reward, dependence and tolerance. We used both male and female mice lacking fosB gene to study its contribution to morphine effects. Morphine analgesia (tail-flick test) and hypothermia were studied using morphine at cumulative doses in morphine-naïve and morphine-tolerant (tolerance induced by 24 h prior 100 mg/kg morphine administration) mice. FosB −/− mice, as compared to fosB +/+ mice, developed enhanced tolerance to morphine-induced analgesia. No effects of genotype or gender on tolerance to morphine-induced hypothermia were observed. These results suggest that fosB may be involved in the development of tolerance to morphine analgesia but not hypothermia. The gender study implicates that lack of FosB proteins in female fosB −/− mice enhanced morphine analgesic potency. In conclusion, we show that fosB gene is important to analgesia but not hypothermia phenotype indicating its role in morphine effects.

Maternal Separation Leads to Persistent Reductions in Pain Sensitivity in Female Rats

We determined responses to noxious thermal stimuli, before and after morphine, and mu-opioid receptor binding in brain regions involved in nociception in maternally separated (MS), neonatally handled (H) and nonhandled (NH) female rats. Long-Evans dams were randomly assigned to either 180-minute (MS) or 15-minute (H) minute daily separations from their litters or left undisturbed (NH). At 120 days of age, paw lick latency (50 degrees C hot plate) was determined in offspring during diestrous. Rats were then given 1, 2, 5, or 10 mg/kg morphine and paw lick latency was measured. Rats were killed during diestrous and mu-opioid receptor binding was determined in discrete brain regions, using [(3)H]DAMGO autoradiography. MS rats had significantly longer (P < .05) paw lick latencies compared with H rats. The percent maximal possible effect of morphine was significantly (P < .05) lower in MS compared with H rats for the 5 mg/kg dose. Mu-Opioid receptor binding capacity was significantly greater (P < .05) in MS rats compared with H rats in the medial preoptic nucleus. In conclusion, MS and H treatments led to antipodal differences in pain sensitivity in female rats and differential mu-opioid receptor binding in the medial preoptic nucleus. This article describes the persistent impact of early life adversity on pain sensitivity and the analgesic potency of morphine. Clinically, early life history may play an important role in pain symptoms and responses to opioid analgesics.

Involvement of cannabinoid (CB 1)-receptors in the development and maintenance of opioid tolerance

Sustained exposure to opioid agonists such as morphine increases levels of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn, a response implicated in the development of opioid tolerance and physical dependence. Recent evidence suggests that both the opioid-induced increase in CGRP and the development of opioid physical dependence are suppressed by blockade of spinal cannabinoid (CB 1)-receptors. The present study examined whether CB 1-receptor activity also has a role in the development of opioid tolerance. In rats implanted with spinal catheters, repeated acute injections of morphine (15 μg) delivered over 4 h resulted in a rapid decline of thermal and mechanical antinociception and a significant loss of analgesic potency, reflecting development of acute opioid tolerance. In another set of experiments, chronic administration of spinal morphine (15 μg) once daily for 5 days produced a similar loss of analgesic effect and a marked increase in CGRP-immunoreactivity in the superficial laminae of the dorsal horn. Consistent with the in vivo findings, primary cultures of adult dorsal root ganglion (DRG) neurons exposed to morphine for 5 days showed a significant increase in the number of CGRP-immunoreactive neurons. Co-administration of acute or chronic morphine with a CB 1-receptor antagonist/inverse agonist, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM-251), inhibited the development of both acute and chronic analgesic tolerance. In animals already exhibiting tolerance to morphine, intervention with AM-251 restored morphine analgesic potency. Co-administration with AM-251 attenuated the morphine-induced increase in CGRP-immunoreactivity in the spinal cord and in DRG cultured neurons. Collectively, the results of this study suggest that activity of endocannabinoids, mediated via CB 1-receptors, contributes to both the development and maintenance of opioid tolerance by influencing the opioid-induced increase in spinal CGRP.