Small G-Protein Rheb Gates Mammalian Target of Rapamycin Signaling to Regulate Morphine Tolerance in Mice.

BACKGROUND: Analgesic tolerance due to long-term use of morphine remains a challenge for pain management. Morphine acts on µ-opioid receptors and downstream of the phosphatidylinositol 3-kinase signaling pathway to activate the mammalian target of rapamycin (mTOR) pathway. Rheb is an important regulator of growth and cell-cycle progression in the central nervous system owing to its critical role in the activation of mTOR. The hypothesis was that signaling via the GTP-binding protein Rheb in the dorsal horn of the spinal cord is involved in morphine-induced tolerance. METHODS: Male and female wild-type C57BL/6J mice or transgenic mice (6 to 8 weeks old) were injected intrathecally with saline or morphine twice daily at 12-h intervals for 5 consecutive days to establish a tolerance model. Analgesia was assessed 60 min later using the tail-flick assay. After 5 days, the spine was harvested for Western blot or immunofluorescence analysis. RESULTS: Chronic morphine administration resulted in the upregulation of spinal Rheb by 4.27 ± 0.195-fold (P = 0.0036, n = 6), in turn activating mTOR by targeting rapamycin complex 1 (mTORC1). Genetic overexpression of Rheb impaired morphine analgesia, resulting in a tail-flick latency of 4.65 ± 1.10 s (P < 0.0001, n = 7) in Rheb knock-in mice compared to 10 s in control mice (10 ± 0 s). Additionally, Rheb overexpression in spinal excitatory neurons led to mTORC1 signaling overactivation. Genetic knockout of Rheb or inhibition of mTORC1 signaling by rapamycin potentiated morphine-induced tolerance (maximum possible effect, 52.60 ± 9.56% in the morphine + rapamycin group vs. 16.60 ± 8.54% in the morphine group; P < 0.0001). Moreover, activation of endogenous adenosine 5'-monophosphate-activated protein kinase inhibited Rheb upregulation and retarded the development of morphine-dependent tolerance (maximum possible effect, 39.51 ± 7.40% in morphine + metformin group vs. 15.58 ± 5.79% in morphine group; P < 0.0001). CONCLUSIONS: This study suggests spinal Rheb as a key molecular factor for regulating mammalian target of rapamycin signaling.

Ion Current Rectification Activity Induced by Boron Hydride Nanosheets to Enhance Magnesium Analgesia.

The limited analgesic efficiency of magnesium restricts its application in pain management. Here, we report boron hydride (BH) with ion currents rectification activity that can enhance the analgesic efficiency of magnesium without the risks of drug tolerance or addiction. We synthesize MgB2, comprising hexagonal boron sheets alternating with Mg2+. In pathological environment, Mg2+ is exchanged by H+, forming two-dimensional borophene-analogue BH sheets. BH interacts with the charged cations via cation-pi interaction, leading to dynamic modulation of sodium and potassium ion currents around neurons. Additionally, released Mg2+ competes Ca2+ to inhibit its influx and neuronal excitation. In vitro cultured dorsal root neurons show a remarkable increase in threshold potential from the normal -35.9 mV to -5.9 mV after the addition of MgB2, indicating potent analgesic effect. In three typical pain models, including CFA-induced inflammatory pain, CINP- or CCI-induced neuropathic pain, MgB2 exhibits analgesic efficiency approximately 2.23, 3.20, and 2.0 times higher than clinical MgSO4, respectively, and even about 1.04, 1.66, and 1.95 times higher than morphine, respectively. The development of magnesium based intermetallic compounds holds promise in addressing the non-opioid medical need for pain relief.

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

BACKGROUND AND PURPOSE: The aim of this work was to investigate the role and signal transduction of toll-like receptor 4 (TLR4), TGF-ß-activated kinase 1 (TAK1) and nod-like receptor protein 3 (NLRP3) in microglial in the development of morphine-induced antinociceptive tolerance. METHODS: TLR4 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. RESULTS: Repeated 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. CONCLUSIONS: Microglial 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.

Blockade and reversal of spinal morphine tolerance by P2X3 receptor antagonist.

In recent years, studies have substantiated the view that P2X3 receptors play a part in the generation and transmission of purinergic signals in inflammatory and chronic neuropathic pain. Data have also been presented to suggest that the process of P2X3 receptor antagonism inhibits inflammatory hyperalgesia, involving the spinal opioid system. The aim of this study was to investigate the effect of the selective P2X3 receptor antagonist A-317491 on the development of antinociceptive tolerance to chronic morphine administration in mice. Daily systemic injection of A-317491 attenuated the morphine-induced antinociceptive tolerance to von Frey and thermal stimuli. Repeated morphine injections alone led to a significant rightward shift in the morphine dose-response curve compared with that with A-317491. A single dose of A-317491 also showed a reversal effect in morphine-tolerant mice. In a withdrawal test, co-administration of A-317491 and morphine also reduced the naloxone-induced withdrawal symptoms compared with the morphine-alone group. Thus, we propose that the P2X3 receptor is involved in the process of morphine antinociceptive tolerance and may be a new therapeutic target in the prevention of tolerance to morphine-induced antinociception.

Chronic morphine treatment increased the expression of myeloid differentiation primary response protein 88 in rat spinal cord.

Chronic morphine exposure leads to tolerance, which limits the clinical use of this potent analgesic in the treatment of severe and chronic pain. Compelling evidence suggest that neuro-immune activation (pro-inflammatory cytokines including IL-1ß, IL-6 and TNF) as well as neuro-inflammation have been shown to mediate the development of morphine analgesic tolerance. Toll-like receptors (TLRs), especially TLR-4, have also been reported to contribute to the development of morphine analgesic tolerance. Besides, mitogen-activated protein kinases (MAPKs; especially p38 MAPK and c-Jun N -terminal kinase), as well as nuclear factor-κB (NF-κB) modulate the development of morphine antinociceptive tolerance. Hence, we hypothesis the possible involvement of myeloid differentiation primary response protein 88 (MyD88), a key adaptor protein for the TLR and IL-1R families, in the development of tolerance to morphine-induced analgesia. Our study demonstrated that chronic intrathecal morphine injection led to a robust increase of MyD88 expression in rat spinal cord. Sustained elevation of MyD88 may play a role in modulating the development of morphine antinociceptive tolerance.

Whether ambulatory electroencephalogram and visual tracking system could be the new strategy for pain assessment?

The human pain experience is a complex multi-faceted symptom. Effective pain management begins with a comprehensive assessment. However, a plethora of existing assessment tools for pain assessment focus more on self-report of pain intensity but lack of multi-dimensional impersonal assessment. These unidimensional scales, which capture self-reported levels of pain intensity, not only underestimate the complexity of the pain experience, but also lack stability and objectivity in their own assessments of pain intensity. Therefore, we propose a hypothesis that using scientific and technological means, such as visual tracking and surveillance system, ambulatory electroencephalogram and other techniques, combined with psychological assessment pictures and existing scales, to comprehensively evaluate pain may provide a new method for more effective clinical treatment of pain, especially chronic severe pain.

The role of astrocytes in neuropathic pain.

Neuropathic pain, whose symptoms are characterized by spontaneous and irritation-induced painful sensations, is a condition that poses a global burden. Numerous neurotransmitters and other chemicals play a role in the emergence and maintenance of neuropathic pain, which is strongly correlated with common clinical challenges, such as chronic pain and depression. However, the mechanism underlying its occurrence and development has not yet been fully elucidated, thus rendering the use of traditional painkillers, such as non-steroidal anti-inflammatory medications and opioids, relatively ineffective in its treatment. Astrocytes, which are abundant and occupy the largest volume in the central nervous system, contribute to physiological and pathological situations. In recent years, an increasing number of researchers have claimed that astrocytes contribute indispensably to the occurrence and progression of neuropathic pain. The activation of reactive astrocytes involves a variety of signal transduction mechanisms and molecules. Signal molecules in cells, including intracellular kinases, channels, receptors, and transcription factors, tend to play a role in regulating post-injury pain once they exhibit pathological changes. In addition, astrocytes regulate neuropathic pain by releasing a series of mediators of different molecular weights, actively participating in the regulation of neurons and synapses, which are associated with the onset and general maintenance of neuropathic pain. This review summarizes the progress made in elucidating the mechanism underlying the involvement of astrocytes in neuropathic pain regulation.

What Is New in Classification, Diagnosis and Management of Chronic Musculoskeletal Pain: A Narrative Review.

Chronic musculoskeletal pain (CMP) is the most common type of chronic pain, defined as persistent or recurrent pain condition deriving from musculoskeletal structures such as muscles, joints or bones that lasts for more than 3 months. CMP is multifactorial and severely affects people's quality of life. CMP may be influenced by a number of factors, including contextual factors, the presence of comorbidities, arthritis coping efficacy and access to CMP care. To deepen the comprehensive understanding of CMP, this narrative review provides the latest literature on disease classification, clinical diagnosis, treatment and basic research. In terms of the classification of the disease, here we introduce the 11th edition of the International Classification of Diseases (IDC-11), in which CMP is divided into chronic primary musculoskeletal pain and chronic secondary musculoskeletal pain. In the clinical diagnosis section, the progress of central sensitization in the diagnosis of CMP will also be summarized. In addition, we summarize some recent advances in clinical treatment and basic research.

Efficient processing of top-k frequent spatial keyword queries.

The rapid popularization of high-speed mobile communication technology and the continuous development of mobile network devices have given spatial textual big data (STBD) new dimensions due to their ability to record geographical objects from multiple sources and with complex attributes. Data mining from spatial textual datasets has become a meaningful study. As a popular topic for STBD, the top-k spatial keyword query has been developed in various forms to deal with different retrievals requirements. However, previous research focused mainly on indexing locational attributes and retrievals of few target attributes, and these correlations between large numbers of the textual attributes have not been fully studied and demonstrated. To further explore interrelated-knowledge in the textual attributes, this paper defines the top-k frequent spatial keyword query (tfSKQ) and proposes a novel hybrid index structure, named RCL-tree, based on the concept lattice theory. We also develop the tfSKQ algorithms to retrieve the most frequent and nearest spatial objects in STBD. One existing method and two baseline algorithms are implemented, and a series of experiments are carried out using real datasets to evaluate its performance. Results demonstrated the effectiveness and efficiency of the proposed RCL-tree in tfSKQ with the complex spatial multi keyword query conditions.

mGluR5-Mediated eCB Signaling in the Nucleus Accumbens Controls Vulnerability to Depressive-Like Behaviors and Pain After Chronic Social Defeat Stress.

Stress contributes to major depressive disorder (MDD) and chronic pain, which affect a significant portion of the global population, but researchers have not clearly determined how these conditions are initiated or amplified by stress. The chronic social defeat stress (CSDS) model is a mouse model of psychosocial stress that exhibits depressive-like behavior and chronic pain. We hypothesized that metabotropic glutamate receptor 5 (mGluR5) expressed in the nucleus accumbens (NAc) normalizes the depressive-like behaviors and pain following CSDS. Here, we show that CSDS induced both pain and social avoidance and that the level of mGluR5 decreased in susceptible mice. Overexpression of mGluR5 in the NAc shell and core prevented the development of depressive-like behaviors and pain in susceptible mice, respectively. Conversely, depression-like behaviors and pain were exacerbated in mice with mGluR5 knockdown in the NAc shell and core, respectively, compared to control mice subjected to 3 days of social defeat stress. Furthermore, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), an mGluR5 agonist, reversed the reduction in the level of the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the NAc of susceptible mice, an effect that was blocked by 3-((2-methyl-1, 3-thiazol-4-yl) ethynyl) pyridine hydrochloride (MTEP), an mGluR5 antagonist. In addition, the injection of CHPG into the NAc shell and core normalized depressive-like behaviors and pain, respectively, and these effects were inhibited by AM251, a cannabinoid type 1 receptor (CB1R) antagonist. Based on these results, mGluR5-mediated eCB production in the NAc relieves stress-induced depressive-like behaviors and pain.

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.

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.

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.

Persistent Rheb-induced mTORC1 activation in spinal cord neurons induces hypersensitivity in neuropathic pain.

The small GTPase Ras homolog enriched in the brain (Rheb) can activate mammalian target of rapamycin (mTOR) and regulate the growth and cell cycle progression. We investigated the role of Rheb-mediated mTORC1 signaling in neuropathic pain. A chronic constriction injury (CCI) model was dopted. CCI induced obvious spinal Rheb expression and phosphorylation of mTOR, S6, and 4-E-BP1. Blocking mTORC1 signal with rapamycin alleviated the neuropathic pain and restored morphine efficacy in CCI model. Immunofluoresence showed a neuronal co-localization of CCI-induced Rheb and pS6. Rheb knockin mouse showed a similar behavioral phenotype as CCI. In spinal slice recording, CCI increased the firing frequency of neurons expressing HCN channels; inhibition of mTORC1 with rapamycin could reverse the increased spinal neuronal activity in neuropathic pain. Spinal Rheb is induced in neuropathic pain, which in turn active the mTORC1 signaling in CCI. Spinal Rheb-mTOR signal plays an important role in regulation of spinal sensitization in neuropathic pain, and targeting mTOR may give a new strategy for pain management.

Spinal TLR4/P2X7 Receptor-Dependent NLRP3 Inflammasome Activation Contributes to the Development of Tolerance to Morphine-Induced Antinociception.

BACKGROUND: Long-term use of morphine induces antinociceptive tolerance and limits its clinical efficacy. Neuroinflammation in the spinal cord is thought to play a pivotal role in the development of morphine tolerance. Toll-like receptor 4 (TLR4) and P2X7 receptor (P2X7R) are key modulators of neuroinflammation. Recent studies show that the Nod-like receptor protein 3 (NLRP3) inflammasome play a crucial role in microglia-mediated neuroinflammation. Thus far, the mechanism underlying NLRP3 inflammasome activation during morphine-induced tolerance is not yet fully understood. Therefore, we sought to investigate the mechanisms of NLRP3 inflammasome activation and its role in the development of morphine-induced tolerance. METHODS: Repeated morphine treatment through intrathecal injection (15 µg once daily for 7 days) was given to establish antinociceptive tolerance in mice. Tail-flick latency was used to evaluate morphine-induced antinociception. NLRP3 knockout mice were used to assess the role of NLRP3 inflammasome in morphine tolerance. TLR4 knockout mice and A438079, a P2X7R antagonist, were used to assess the role of TLR4 and P2X7R in chronic morphine-induced NLRP3 inflammasome activation. Western blot and immunofluorescence were used for quantitative comparison. RESULTS: Repeated morphine treatment increased the expression of NLRP3. Knockout of NLRP3 attenuated morphine-induced tolerance and suppressed morphine-induced activation of microglia. Knockout of TLR4 alleviated morphine tolerance and chronic morphine-induced upregulation of spinal NLRP3. Inhibition of spinal P2X7R with A438079 not only prevented the development of morphine-induced tolerance but also inhibited repeated morphine treatment-induced upregulation of spinal NLRP3. Furthermore, spinal NLRP3, TLR4 and P2X7R were collectively colocalized with the microglia marker Iba1. CONCLUSION: This study demonstrates that the NLRP3 inflammasome in microglia plays a crucial role in morphine tolerance and that both TLR4- and P2X7R-dependent pathways are required for NLRP3 inflammasome activation over the course of the development of morphine-induced tolerance. Our results provide a new perspective for the targeted treatment of morphine-induced tolerance.

Disabling phosphorylation at the homer ligand of the metabotropic glutamate receptor 5 alleviates complete Freund's adjuvant-induced inflammatory pain.

Metabotropic glutamate receptor 5 (mGluR5) has been reported to contribute to inflammatory pain. The intracellular C-terminal domain has a Homer-binding motif that can form an mGluR5/Homer complex. Phosphorylation of mGluR5 at the Homer binding domain enhances the mGluR5/Homer interaction and modulates intracellular signal transduction. However, the characteristics of this interaction have not been fully elucidated in inflammatory pain. We aimed to evaluate the effects of CFA-induced phosphorylation of mGluR5 at the Homer binding domain on the mGluR5/Homer interaction. Von-frey filaments and thermal latency were used to monitor the development of inflammatory pain. Spinal mGluR5 phosphorylation at Ser1126 and mGluR5/Homer crosslinking were detected. Mutant mGluR5 that could not be phosphorylated at Thr1123 or Ser1126 was evaluated in inflammatory pain. CFA-induced inflammatory pain resulted in obvious phosphorylation at Ser1126 of mGluR5. Moreover, increased phosphorylation at the Homer-binding motif enhanced crosslinking between mGluR5 and Homer. Mutations at Thr1123 and Ser1126 of mGluR5 blocked the development of CFA-induced inflammatory pain. Overall, our findings showed that disruption of the phosphorylation of mGluR5 Thr1123 and Ser1126 alleviated CFA-induced inflammatory pain.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.

Involvement of neuronal TGF-ß activated kinase 1 in the development of tolerance to morphine-induced antinociception in rat spinal cord.

BACKGROUND AND PURPOSE: 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-ß 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.

Does modulation of spinal N-methyl-D-aspartic acid receptor by pre-activation accelerate the development of morphine tolerance?

Repeated morphine administration usually leads to a number of neuroadaptive processes, including tolerance and sensitization. However, research has shown that the induction and maintenance of central sensitization is dependent on N-methyl-d-aspartic acid receptor (NMDAR) activation. Chronic morphine exposure has been shown to result in spinal sensitization and activation of spinal NMDARs. Chronic morphine treatment and the activation of spinal NMDARs may be synergistic and form a closed loop that may worsen the development of morphine analgesic tolerance and spinal sensitization. Inhibition of NMDARs with an antagonist could effectively alleviate the development of morphine analgesic tolerance. So, what is the effect of modulating spinal NMDAR activation with exogenous agonists or neuropathic input on the development of morphine-induced analgesic tolerance? Our hypothesis is that chronic morphine treatment may worsen the already activation of spinal NMDARs and spinal sensitization after agonist application or neuropathic input to shorten the process of morphine-induced analgesic tolerance.

Inhibitory effect of spinal mGlu(5) receptor antisense oligonucleotide on the up-regulated expression of spinal G protein associated with chronic morphine treatment.

Knockdown of spinal metabotropic glutamate 5 (mGlu5) receptor was shown to inhibit the development of intrathecal morphine antinociceptive tolerance. The present work was designed to evaluate the expression of spinal G-protein during morphine tolerance and knockdown of spinal mGlu5 receptor with antisense oligonucleotide (ODN). Rats were treated with saline, morphine, mGlu5 receptor antisense or mismatch ODN intrathecally. Behavioral tests were employed to test the thermal and mechanical pain thresholds. Five days later, rats were scarified and spinal expression of spinal Gαi, Gαo, Gαq and Gß were detected. Consistent with the previous results, knockdown of spinal mGlu5 receptor could inhibit spinal morphine antinociceptive tolerance in behavioral tests (P<0.05). The mGlu5 receptor antisense ODN produced a significant reduction in mGlu5 receptor protein of about 56.6% compared with the control group (P<0.05). Expression of spinal Gαi, Gαo, Gαq and Gß were up-regulated while morphine tolerance developed (P<0.05). Antisense ODN of spinal mGlu5 receptor, but not mismatched ODN, reduced the spinal dorsal horn levels of Gαi, Gαo, Gαs, Gαq and Gß (P<0.05). We conclude that expression of spinal G (αi, αo, αs, αq and ß) protein may be up-regulated after chronic morphine treatment which could be attenuated by knockdown of spinal mGlu5 receptor with antisense ODN.