Inhibitory Effects of Mas-Related Gene C Receptor on Chronic Morphine-Induced Spinal Glial Activation in Rats.

Glial activation plays a pivotal role in morphine tolerance. This study investigated effects of Mas-related gene (Mrg) C receptor on morphine-induced activation of microglia and astrocytes in the spinal cord and its underlying mechanisms. Intrathecal administration of morphine (20 µg, daily) for 6 days induced a great decline in morphine antinociception and increased expression of glial fibrillary acidic protein and OX-42 in the spinal dorsal horn. These changes were greatly attenuated by the intermittent coinjection of bovine adrenal medulla 8-22 (BAM8-22, 1 nmol), a specific agonist of MrgC receptor. These modulatory effects were accompanied by the reduction of P2X4 and interleukin-1ß expressions in the spinal dorsal horn. Chronic morphine increased the expression of fractalkine in medium- and small-sized neurons of dorsal root ganglia (DRG). Treatment with BAM8-22 inhibited these changes as well as an increase in Toll-like receptor 4 (TLR4) protein in DRG. Chronic treatment of DRG explant cultures with morphine (3.3 µM, 5 days) increased the levels of fractalkine mRNA. Application of BAM8-22 (10 nM) for 60 minutes completely blocked the increase of fractalkine mRNA induced by morphine. Our findings indicate that the inhibition of morphine tolerance by MrgC receptor was associated with the modulation of astrocytes and microglia in the spinal dorsal horn and fractalkine and TLR4 expressions in DRG. As MrgC receptor is exclusively located in DRG, intermittent combination of MrgC receptor agonist could be a promising adjunct with limited side effects for chronic use of opiates.

Intrathecal administration of adrenomedullin induces mechanical allodynia and neurochemical changes in spinal cord and DRG.

This study investigated the effect of adrenomedullin (AM) on mechanical pain sensitivity and its possible mechanisms. Intrathecal injection of AM receptor agonist AM1-50 (20 µg) once per day briefly reduced mechanical pain threshold on days 1 and 2 but induced prolonged mechanical allodynia on day 3. However, AM1-50 did not change mechanical pain sensation when the AM receptor antagonist AM22-52 (20 µg) was intrathecally co-administered. Daily administration of AM1-50 (20 µg) for 3 days increased expression of phosphorylated extracellular signal-regulated protein kinase (pERK) and neuronal nitric oxide synthase (nNOS) in the spinal dorsal horn. The AM-induced increase in pERK and nNOS was inhibited by the co-administration of AM22-52. The chronic administration of AM1-50 also increased expression of microglial maker Iba1 and astrocytic marker GFAP (glial fibrillary acidic protein) in the spinal dorsal horn in an AM22-52-sensitive manner. Furthermore, the application of AM1-50 (10 nM, 3 h) to dorsal root ganglion (DRG) explant cultures induced an increase in the expression of transient receptor potential vanilloid 1 (TRPV1). The treatment with AM1-50 did not change TRPV1 expression in DRG in the presence of AM22-52 (2 µM). These results suggest that the increased AM bioactivity induced mechanical allodynia and may contribute to the mechanical pain hypersensitivity under pathological conditions. The mechanisms may involve the activation of ERK signaling pathway and spinal glia as well as the recruitment of nNOS and TRPV1 in the spinal dorsal horn or DRG. The present study indicates that inhibition of the activation AM receptor might provide a fruitful strategy to relieving chronic pain.

Inhibition of morphine tolerance by MrgC receptor via modulation of interleukin-1ß and matrix metalloproteinase 9 in dorsal root ganglia in rats.

Opiate tolerance is a critical issue in pain management. Previous studies show that activation of Mas-related gene (Mrg) C receptor can modulate the development of morphine tolerance. This study was designed to investigate the underlying mechanism(s). Intrathecal (i.t.) administration of morphine (20µg) increased the expression of interleukin-1ß (IL-1ß) and matrix metalloproteinase-9 (MMP-9) in small- and medium-sized neurons in dorsal root ganglia (DRG). Co-administration of bovine adrenal medulla 8-22 (BAM8-22), a selective MrgC receptor agonist, via i.t. route inhibited the increase of IL-1ß and MMP-9 in the DRG. Exposure of DRG cultures to morphine (3.3µM) for 3 or 5 days, but not for 1 day, induced an increase in MMP-9 mRNA expression. The treatment with BAM8-22 (10nM) for 20, 40 or 60min abolished chronic (5 days) morphine-induced increase of MMP-9 mRNA in the cultured DRG. The treatment with BAM8-22 for 1h inhibited chronic morphine-induced increase of MMP-9 and IL-1ß mRNA in DRG but these effects were abolished by MrgC receptor antibody. The treatment with BAM8-22 for 24 and 72h respectively inhibited and enhanced morphine-induced expression of MMP-9 and IL-1ß mRNA in the cultured DRG. The BAM8-22-induced inhibition and enhancement were abolished by MrgC receptor antibody. The results suggest that the inhibition of IL-1ß and MMP-9 expressions in DRG underlain the modulation of morphine tolerance by the acute activation of MrgC receptors. The chronic activation of MrgC receptors can facilitate morphine-induced increase of MMP-9 and IL-1ß expressions in DRG.

[Effects of intrathecal administration of AM22-52 on mechanical allodynia and CCL2 expression in DRG in bone cancer rats].

The pain peptide adrenomedullin (AM) plays a pivotal role in pathological pain. The present study was designed to investigate the effect of blockade of AM receptor on bone cancer pain (BCP) and its mechanism. BCP was developed by inoculation of Walker 256 mammary gland carcinoma cells in the tibia medullary cavity of Sprague Dawley rats. The selective AM receptor antagonist AM22-52 was administered intrathecally on 15 d after the inoculation. Quantitative real-time PCR was used to detect mRNA level of CC chemokine ligand 2 (CCL2) in dorsal root ganglion (DRG). Double immunofluorescence staining was used to analyze the localizations of CCL2 and AM in DRG of normal rats. The results showed that, from 6 to15 d after the inoculation, the animals showed significant reduction in the mechanical pain threshold in the ipsilateral hindpaw, companied by the decline in bone density of tibia bone. The expression of CCL2 mRNA in DRG of BCP rats was increased by 3 folds (P < 0.001 vs saline group). Intrathecal administration of AM22-52 abolished bone cancer-induced mechanical allodynia and increase of CCL2 mRNA level (P < 0.001). In normal rats, CCL2 was co-localized with AM in DRG neurons. These results suggest that AM may play a role in the pathogenesis of BCP. The increased AM bioactivity up-regulates CCL2 expression in DRG, which may contribute to the induction of pain hypersensitivity in bone cancer.

[Inhibition of prostaglandins synthesis in the inflamed site results in opioid-mediated hypoalgesia in rats].

This study was designed to investigate the contribution of prostaglandins to the maintenance of inflammatory pain. Inflammation was induced by intraplantar (i.pl.) injection of carrageenan in right hindpaw in rats. Indomethacin (non-selective COX inhibitor) was administered i.pl. 1 h after the carrageenan injection, and paw withdrawal latency (PWL) responding to noxious heat was measured. ß-endorphin (ß-END) and µ-opioid receptor (MOR) expressed in the inflamed site were examined by using immunocytochemistry, ELISA and RT-PCR techniques. The results showed that indomethacin dose-dependently increased PWL to the levels that were above the baseline on the day 2 and 3, referred to as hypoalgesia. The hypoalgesia was abolished by a local injection of the non-selective opioid receptor inhibitor naloxone methiodide. The number of ß-END-positive cells, the content of ß-END and the expression of MOR mRNA in the inflammatory site of inflammation model rats were all significantly increased by indomethacin. These results reveal a novel mechanism of prostaglandins for the inhibition of inflammation-induced endogenous opioid activity. This study provides further evidence that inhibition of prostaglandins in inflamed site could be a promising therapy for inflammatory pain.

Adrenomedullin mediates tumor necrosis factor-α-induced responses in dorsal root ganglia in rats.

Adrenomedullin (AM), a member of the calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pain peptide. This study investigated the possible involvement of AM in tumor necrosis factor-alpha (TNF-α)-induced responses contributing to neuronal plasticity in the dorsal root ganglia (DRG). Exposure of the DRG explant cultures to TNF-α (5nM) for 48h upregulated the expression of AM mRNA. The treatment with TNF-α also increased the level of CGRP, CCL-2 and MMP-9 mRNA in the cultured DRG. This increase was attenuated by the co-treatment with the selective AM receptor antagonist AM22-52 (2µM). The blockade of AM receptors inhibited TNF-α-induced increase of the glial fibrillary acidic protein (GFAP), interleukin-1ß (IL-1ß), phosphorylated cAMP response element binding protein (pCREB) and nuclear factor kappa B (pNF-κB) proteins. On the other hand, the treatment with the AM receptor agonist AM1-50 (10nM) for 96h induced an increase in the level of GFAP, IL-1ß, pCREB and pNF-κB proteins. The inhibition of AM activity did not change TNF-α-induced phosphorylation of extracellular signal-related kinase (pERK) while the treatment with AM1-50 still increased the level of pERK in the cultured DRG. Immunofluorescence assay showed the colocalization of AM-like immunoreactivity (IR) with TNF-α-IR in DRG neurons. The present study suggests that the increased AM receptor signaling mediated the many, but not all, TNF-α-induced activities, contributing to peripheral sensitization in neuropathic pain.

Blockade of 5-HT2A receptors at the site of inflammation inhibits activation of spinal dorsal horn neurons in rats.

Repeated inflammation in the periphery is a major cause of chronic inflammatory pain. We have showed that blockade of 5-HT2A receptors in the periphery inhibits repeated inflammation-induced pain hypersensitivity. The present study investigated whether inhibition of 5-HT2A receptor signaling at the site of inflammation could inhibit repeated inflammation-induced neurochemical changes in spinal dorsal horn neurons. Treatment with the 5-HT2A receptor antagonist ketanserin (20µg) in the hindpaw following intraplantar injection of carrageenan inhibited the increase in nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity, a marker of nitric oxide synthase (NOS), in the spinal dorsal horn. Administration of formalin (1%) in the hindpaw following the carrageenan injection evoked a great increase in NADPH-d reactivity in spinal dorsal horn neurons on the ipsilateral and contralateral sides. These changes were abolished by the pretreatment of ketanserin (20µg). The ketanserin treatment also reduced the repeated inflammation-induced expression of protein kinase gamma (PKCγ) in the membrane of spinal dorsal horn neurons and increased PKCγ protein level in the cytosol. Following the treatment with the opioid receptor antagonist naloxone methiodide (5mg/kg, s.c.), the administration of ketanserin failed to inhibit the repeated inflammation-induced increase in NADPH-d reactivity and c-Fos expression in the spinal dorsal horn. These results suggest that 5-HT2A receptor bioactivity in the inflammatory site plays an important role in repeated inflammation-induced central sensitization. The present study supports the notion that targeting 5-HT2A receptors in the periphery can be a promising therapy for relieving chronic inflammatory pain.

Reversal of neurochemical alterations in the spinal dorsal horn and dorsal root ganglia by Mas-related gene (Mrg) receptors in a rat model of spinal nerve injury.

The rodent Mas-related gene (Mrg) receptor subtype C has been demonstrated to inhibit pathological pain. This study investigated the mechanisms underlying the reversal of pain hypersensitivity by the selective MrgC receptor agonist bovine adrenal medulla 8-22 (BAM8-22) in a rat model of L5 spinal nerve ligation (SNL). Intrathecal (i.t.) administration of BAM8-22 (0.1-10nmol) attenuated mechanical allodynia in a dose-dependent manner on day 10 after SNL. The antiallodynia effect of BAM8-22 was abolished by MrgC receptor antibody, but not by naloxone. I.t. BAM8-22 (10nmol) inhibited SNL-induced upregulation of neuronal nitric oxide synthesis (nNOS) and phosphorylation of cyclic AMP response element-binding protein (p-CREB) in the spinal dorsal horn. The BAM8-22 treatment reversed the SNL-induced astrocyte activation, increase of interleukin-1ß (IL-1ß) expression and phosphorylation of extracellular signal-regulated kinase (p-ERK) in the spinal cord. BAM8-22 also reversed the upregulation of fractalkine and IL-1ß in small- and medium-sized dorsal root ganglion (DRG) neurons. Furthermore, the BAM8-22 exposure suppressed the lipopolysaccharide (LPS)-induced increase of nNOS and IL-1ß in the DRG explant cultures and the BAM8-22-induced suppression disappeared in the presence of MrgC receptor antibody. The present study provides evidence that activation of MrgC receptors inhibits nerve injury-induced increase of pronociceptive molecules in DRG neurons, suppressing astrocyte activation, the upregulation of excitatory mediators and phosphorylation of transcription factors in the spinal dorsal horn. As MrgC receptors are unequally expressed in the dorsal root and trigeminal ganglia, this study suggests that targeting MrgC receptors could be a new therapy for neuropathic pain with limited unwanted effects.

Mas-related gene (Mrg) C receptors inhibit mechanical allodynia and spinal microglia activation in the early phase of neuropathic pain in rats.

Mas-related gene (Mrg) C receptors are exclusively expressed in the trigeminal and dorsal root ganglia (DRG). However, their functional roles are poorly understood. This study was aimed to determine the effect of MrgC receptors on pain hypersensitivity in the early phase of neuropathic pain and its underlying mechanisms. Intrathecal (i.t.) administration of the selective MrgC receptor agonist bovine adrenal medulla 8-22 (BAM8-22) at 1 or 10nmol attenuated mechanical allodynia one day after L5 spinal nerve ligation (SNL) surgery. I.t. BAM8-22 (10 nmol) inhibited SNL-induced microglia activation in the spinal dorsal horn on day 2 post-SNL. The BAM8-22 treatment also abolished SNL-induced upregulation of neuronal nitric oxide synthesis (nNOS) in the dorsal root ganglia (DRG). On the other hand, SNL, but not sham, surgery reduced the expression of MrgC receptor mRNA in the injured L5 DRG without changing thier levels in the adjacent uninjured L4 or L6 DRG on day 2 following the surgery. These results suggest that the activation of MrgC receptors can relieve pain hypersensitivity by the inhibition of nNOS increase in DRG neurons and microglia activation in the spinal dorsal horn in the early time following peripheral nerve injury. This study provides evidence that MrgC receptors could be targeted as a novel therapy for neuropathic pain with limited unwanted effects.

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.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: Intrathecal injection of AM (8 µg) for 9 days decreased and increased the levels of µ receptor-coupled Gi and Gs proteins respectively. Morphine stimulation (5 µg) 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 (5 µg) or MEK1 inhibitor PD98059 (1 µg) reversed the AM-induced thermal/mechanical hypersensitivity, decline in morphine analgesic potency, switch of G protein-coupled µ receptor and increase in cAMP. CONCLUSIONS AND IMPLICATIONS: 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.

[Effects of blockade of 5-HT2A receptors in inflammatory site on complete Freund's adjuvant-induced chronic hyperalgesia and neuropeptide Y expression in the spinal dorsal horn in rats].

5-hydroxytryptamine (5-HT) released in inflammatory tissues plays a pivotal role in pain hypersensitivity. However, it is not clear whether 5-HT2A receptors in the inflamed tissues mediate this effect. The present study investigated the contribution of 5-HT2A receptors in the periphery to chronic inflammatory pain. Complete Freund's adjuvant (CFA) was injected subcutaneously in the hindpaw of rats. The selective 5-HT2A receptor antagonist ketanserin was given in the inflamed site. Paw withdrawal latency responding to heat or mechanical stimuli was measured. Expression of neuropeptide Y (NPY) in the spinal dorsal horn and dorsal root ganglia (DRG) was assayed using immunohistochemistry technique. The results showed that ketanserin administered in the inflamed site inhibited thermal hyperalgesia in a dose-dependent manner (20, 40 and 80 µg) induced by the intraplantar injection of CFA. Ketanserin given once per day at a dose of 80 µg abolished heat hyperalgesia and also attenuated mechanical allodynia on the third day. CFA injection increased the expression of NPY in superficial laminae of the spinal cord, but not in the DRG. The local treatment of ketanserin completely inhibited CFA-induced increase in NPY expression in superficial laminae of the spinal cord. These results indicated that activation of 5-HT2A receptors in the inflamed tissues was involved in the pathogenesis of inflammatory pain and the blockade of 5-HT2A receptors in the periphery could relieve pain hypersensitivity and normalize the cellular disorder in the spinal dorsal horn associated with pathological pain. The present study suggests that the peripheral 5-HT2A receptors can be a promising target for pharmaceutical therapy to treat chronic inflammatory pain without central nervous system side effects.

[Activation of spinal MrgC receptors inhibits hyperalgesia in rats].

This study was aimed to investigate the mechanisms of the modulation effect of activation of spinal Mas-related gene C (MrgC) receptors on hyperalgesia induced by intraplantar (i.pl.) injection of (Tyr6)-γ2-MSH-6-12 (MSH) or complete Freund's adjuvant (CFA). Paw withdrawal latency test and immunohistochemistry were used to observe the effect of intrathecal (i.t.) administration of MSH or BAM8-22, two selective agonists of MrgC receptor, in hyperalgesia in rats. The results showed that i.t. administration of MSH inhibited acute hyperalgesic response induced by i.pl. application of MSH, while did not change thermal nociceptive threshold in naïve rats. The i.t. administration of MSH also attenuated CFA-induced inflammatory hyperalgesia. However, i.t. administration of the µ-opioid receptor (MOR) antagonist CTAP blocked the induction of delayed anti-hyperalgesia by MSH. The i.t. injection of BAM8-22 at a dose of 30 nmol evidently reduced the number of CFA-evoked nitric oxide synthase (NOS)-positive neurons and the expression of calcitonin gene-related peptide (CGRP)-immunoreactivity positive nerve fibers at L3-L5 segments of the spinal cord. These results suggest that the activation of MrgC receptor in CFA-induced inflammation reduces inflammatory hyperalgesia through inactivation of NOS neurons and down-regulation of CGRP expressions, and generates delayed but long-lasting anti-nociception through the endogenous activation of MOR via indirect mechanisms. Agonists for MrgC receptors may, therefore, represent a new class of antihyperalgesics for treating inflammatory pain because of the highly specific expression of their targets.

[Involvement of adrenomedullin in the pathogenesis of inflammatory pain and morphine tolerance].

The increase of pronociceptive mediators in the dorsal root ganglia (DRG) and spinal dorsal horn is an important mechanism in the pathogenesis of inflammatory pain and opioid tolerance. Adrenomedullin (AM) belongs to calcitonin gene-related peptide (CGRP) family and has been recently demonstrated to be a pain-related peptide. It has also been shown that the expression and release of AM are increased in the DRG and spinal dorsal horn during inflammation and repeated use of morphine. Intrathecal administration of the selective AM receptor antagonist AM22-52 abolishes inflammatory pain and morphine tolerance, suggesting that enhanced AM receptor signaling in the DRG and spinal dorsal horn contributes to the induction of inflammatory pain and morphine tolerance. The present review highlights the recent developments regarding the involvement of AM in these two disorders. The neurological mechanisms of AM's actions are also discussed.

Activation of Mas Oncogene-Related G Protein-Coupled Receptors Inhibits Neurochemical Alterations in the Spinal Dorsal Horn and Dorsal Root Ganglia Associated with Inflammatory Pain in Rats.

Mas oncogene-related G protein-coupled receptor C (MrgC) is unequally expressed in sensory ganglia and has been shown to modulate pathologic pain. This study investigated the mechanism underlying the effect of MrgC receptors on inflammatory pain. Intrathecal administration of the selective MrgC receptor agonist bovine adrenal medulla 8-22 (BAM8-22) (30 nmol) inhibited complete Freund's adjuvant-evoked hyperalgesia. This was associated with the inhibition of protein kinase C-γ and phosphorylated extracellular signal-regulated protein kinase in the spinal cord and/or dorsal root ganglia (DRG). The complete Freund's adjuvant injection in the hindpaw induced an increase in Gq, but not Gi and Gs, protein in the spinal dorsal horn. This increase was inhibited by the intrathecal administration of BAM8-22. The exposure of DRG cultures to bradykinin (10 µM) and prostaglandin E2 (1 µM) increased the expression of calcitonin gene-related peptide (CGRP) and neuronal nitric oxide synthase in small- and medium-sized neurons as well as the levels of CGRP, aspartate, and glutamate in the cultured medium. The bradykinin/prostaglandin E2-induced alterations were absent in the presence of BAM8-22 (10 nM). These results suggest that the activation of MrgC receptors can modulate the increase in the expression of CGRP and neuronal nitric oxide synthase as well as the release of CGRP and excitatory amino acids in DRG associated with inflammatory pain. This modulation results in the inhibition of pain hypersensitivity by suppressing the expression of Gq protein and protein kinase C-γ and extracellular signal-regulated protein kinase signaling pathways in the spinal cord and/or DRG. The present study suggests that MrgC receptors may be a novel target for relieving inflammatory pain.

[MrgC receptor activation reverses chronic morphine-evoked alterations of glutamate transporters and nNOS in rats].

This study was aimed to investigate the mechanisms underlying the modulation effect of Mas-related gene (Mrg) C receptors (MrgC) on morphine tolerance. Saline, morphine (20 µg), morphine plus bovine adrenal medulla 8-22 (BAM8-22, 1 nmol) or (Tyr(6))-2-MSH-6-12 (MSH, 5 nmol) were administered intrathecally in rats for 6 days. Pain-related molecules in the spinal cord and dorsal root ganglion (DRG) were examined using Western blot, immunocytochemistry and RT-PCR techniques. The results showed that intrathecal administration of the selective MrgC receptor agonists (BAM8-22 or MSH) remarkably attenuated or abolished chronic morphine-evoked reduction in glutamate transporters (GLAST, GLT-1 and EAAC1) in the spinal cord and increase in neuronal nitric oxide synthase (nNOS) in the spinal cord as well as DRG. In addition, MrgC receptor-like immunoreactivity (IR) was detected in superficial laminae of the spinal cord. Chronic morphine induced significant increases in MrgC receptor-IR in the spinal cord and MrgC receptor mRNA levels in DRG. These results suggest that the modulation of pro-nociceptive mediators in the spinal cord and DRG underlies the inhibition of morphine tolerance by MrgC receptor activation.

Activated microglia in the spinal cord underlies diabetic neuropathic pain.

Diabetes mellitus is an increasingly common chronic medical condition. Approximately 30% of diabetic patients develop neuropathic pain, manifested as spontaneous pain, hyperalgesia and allodynia. Hyperglycemia induces metabolic changes in peripheral tissues and enhances oxidative stress in nerve fibers. The damages and subsequent reactive inflammation affect structural properties of Schwann cells and axons leading to the release of neuropoietic mediators, such as pro-inflammatory cytokines and pro-nociceptive mediators. Therefore, diabetic neuropathic pain (DNP) shares some histological features and underlying mechanisms with traumatic neuropathy. DNP displays, however, other distinct features; for instance, sensory input to the spinal cord decreases rather than increasing in diabetic patients. Consequently, development of central sensitization in DNP involves mechanisms that are distinct from traumatic neuropathic pain. In DNP, the contribution of spinal cord microglia activation to central sensitization and pain processes is emerging as a new concept. Besides inflammation in the periphery, hyperglycemia and the resulting production of reactive oxygen species affect the local microenvironment in the spinal cord. All these alterations could trigger resting and sessile microglia to the activated phenotype. In turn, microglia synthesize and release pro-inflammatory cytokines and neuroactive molecules capable of inducing hyperactivity of spinal nociceptive neurons. Hence, it is imperative to elucidate glial mechanisms underlying DNP for the development of effective therapeutic agents. The present review highlights the recent developments regarding the contribution of spinal microglia as compelling target for the treatment of DNP.

Involvement of adrenomedullin in the attenuation of acute morphine-induced analgesia in rats.

Adrenomedullin (AM) is a member of calcitonin gene-related peptide (CGRP) family and a pain-related peptide. We have shown that chronic administration of morphine (20 µg) upregulates AM activity contributing to morphine tolerance. The present study investigated if AM is involved in acute morphine-induced analgesia. Single intrathecal (i.t.) injection of morphine at a dose of 5 µg increased the tail-flick latency (TFL). This analgesic effect was potentiated by the co-administration of the AM receptor antagonist AM22-52 (5 and 10 nmol). Exposure of sensory ganglion culture to morphine increased AM content in the ganglia in concentration (0.33-10 µM)- and time (10-240 min)-dependent manners. However, treatment with morphine (3.3 µM) for 30-240 min did not alter AM mRNA levels in the cultured ganglia. Furthermore, exposure of ganglion cultures to morphine (3.3 µM) for 30-240, but not 10 min induced an increase in AM content in the culture medium. These results reveal that a single morphine treatment potentiates post-translational change and the release of AM in sensory ganglia masking morphine-induced analgesia. Thus, targeting AM and its receptors should be considered as a novel approach to improve the analgesic potency of opiates during their acute use.

Effects of a combination of ketanserin and propranolol on inflammatory hyperalgesia in rats.

Pain management is still challenging in clinic as current analgesics either are not very effective or produce serious adverse effects. This study aimed to examine if old drugs could display the new use and to develop a novel therapy for inflammatory pain. Injection of carrageenan in hindpaw evoked hyperalgesia detected by noxious heat stimulation. Intraplantar (i.pl.) injection of the 5-HT1A receptor antagonist WAY-100635 increased paw withdrawal latency (PWL) above normal level (hypoalgesia) during the late phase of carrageenan-evoked inflammation. The hypoalgesia was completely abolished by systemic injection of naloxone chloride and naloxone methiodide. Moreover, i.pl. injection of a combination of WAY-100635 and ketanserin, a 5-HT2A receptor antagonist, at their minimal doses attenuated hyperalgesia in the late phase of carrageenan-evoked inflammation. Subcutaneous (s.c.) injection of both ketanserin and propranolol dose-dependently inhibited carrageenan-evoked hyperalgesia. The treatment with a combination of ketanserin and propranolol by s.c. injection abolished carrageenan-evoked hyperalgesia at the doses, at which the drugs failed to alter the hypersensitivity when they were given alone. Furthermore, the combination of ketanserin and propranolol was also effective in relieving arthritic hyperalgesia and muscle pain at a minimal dose. The present study suggests that the activation of 5-HT1A receptors suppressed naloxone-reversible antinociception contributing to the maintenance of inflammatory pain, and that the concomitant blockade of 5-HT1A and 5-HT2A receptors in the periphery produced synergistic effects on inflammatory hyperalgesia. It is proposed that the combination of ketanserin and propranolol injected s.c. could be a promising therapy for relieving inflammatory pain with minimal side effects.

Effect of Mas-related gene (Mrg) receptors on hyperalgesia in rats with CFA-induced inflammation via direct and indirect mechanisms.

BACKGROUND AND PURPOSE: Mas oncogene-related gene (Mrg) receptors are exclusively distributed in small-sized neurons in trigeminal and dorsal root ganglia (DRG). We investigated the effects of MrgC receptor activation on inflammatory hyperalgesia and its mechanisms. EXPERIMENTAL APPROACH: A selective MrgC receptor agonist, bovine adrenal medulla peptide 8-22 (BAM8-22) or melanocyte-stimulating hormone (MSH) or the µ-opioid receptor (MOR) antagonist CTAP was administered intrathecally (i.t.) in rats injected with complete Freund's adjuvant (CFA) in one hindpaw. Thermal and mechanical nociceptive responses were assessed. Neurochemicals were measured by immunocytochemistry, Western blot, ELISA and RT-PCR. KEY RESULTS: CFA injection increased mRNA for MrgC receptors in lumbar DRG. BAM8-22 or MSH, given i.t., generated instant short and delayed long-lasting attenuations of CFA-induced thermal hyperalgesia, but not mechanical allodynia. These effects were associated with decreased up-regulation of neuronal NOS (nNOS), CGRP and c-Fos expression in the spinal dorsal horn and/or DRG. However, i.t. administration of CTAP blocked the induction by BAM8-22 of delayed anti-hyperalgesia and inhibition of nNOS and CGRP expression in DRG. BAM8-22 also increased mRNA for MORs and pro-opiomelanocortin, along with ß-endorphin content in the lumbar spinal cord and/or DRG. MrgC receptors and nNOS were co-localized in DRG neurons. CONCLUSIONS AND IMPLICATIONS: Activation of MrgC receptors suppressed up-regulation of pronociceptive mediators and consequently inhibited inflammatory pain, because of the activation of up-regulated MrgC receptors and subsequent endogenous activity at MORs. The uniquely distributed MrgC receptors could be a novel target for relieving inflammatory pain.

[Roles of calcitonin gene-related peptide family in pain and opioid tolerance].

The calcitonin gene-related peptide (CGRP) family mainly includes CGRPα, CGRPß, adrenomedullin, calcitonin and amylin. The members of CGRP family and their receptors are widely distributed in the central and peripheral nervous systems. Studies show that members of CGRP family such as CGRP and adrenomedullin play important roles in the transmission of nociceptive information. At spinal level, CGRP promotes the transmission of nociceptive information, spinal morphine tolerance, migraine, inflammatory pain and neuropathic pain. At superspinal level, CGRP suppresses the transmission of nociceptive information. Adrenomedullin is a pain-related neuropeptide which has recently been demonstrated. It facilitates the transmission of nociceptive information and is involved in the development and maintenance of opioid tolerance. The involvement of amylin and calcitonin in pain is not clear yet.