RESUMO
Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the N-Methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here, we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt (LSOS) or the D-serine degrading enzyme, D-amino acid oxidase (DAAO) on post-operative days 0-3 significantly reduced the CCI-induced increase in nitric oxide (NO) levels and nicotinamide adenine dinucleotide phosphate-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and protein kinase C (PKC)-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, 3-morpholinosydnonimine hydrochloride (SIN-1). In naïve mice, exogenous D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechanical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively, we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1 and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.
Assuntos
Astrócitos/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Serina/farmacologia , Animais , Western Blotting , D-Aminoácido Oxidase/metabolismo , Hiperalgesia/etiologia , Masculino , Camundongos , Molsidomina/análogos & derivados , Molsidomina/farmacologia , N-Metilaspartato/metabolismo , Neuralgia/etiologia , Fosforilação/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/análogos & derivados , Serina/metabolismoRESUMO
Despite the relatively high prevalence of migraine or headache, the pathophysiological mechanisms triggering headache-associated peripheral hypersensitivities, are unknown. Since nitric oxide (NO) is well known as a causative factor in the pathogenesis of migraine or migraine-associated hypersensitivities, a mouse model has been established using systemic administration of the NO donor, nitroglycerin (NTG). Here we tried to investigate the time course development of facial or hindpaw hypersensitivity after repetitive NTG injection. NTG (10 mg/kg) was administrated to mice every other day for nine days. Two hours post-injection, NTG produced acute mechanical and heat hypersensitivity in the hind paws. By contrast, cold allodynia, but not mechanical hypersensitivity, occurred in the facial region. Moreover, this hindpaws mechanical hypersensitivity and the facial cold allodynia was progressive and long-lasting. We subsequently examined whether the depletion of capsaicin-sensitive primary afferents (CSPAs) with resiniferatoxin (RTX, 0.02 mg/kg) altered these peripheral hypersensitivities in NTG-treated mice. RTX pretreatment did not affect the NTG-induced mechanical allodynia in the hind paws nor the cold allodynia in the facial region, but it did inhibit the development of hind paw heat hyperalgesia. Similarly, NTG injection produced significant hindpaw mechanical allodynia or facial cold allodynia, but not heat hyperalgesia in transient receptor potential type V1 (TRPV1) knockout mice. These findings demonstrate that different peripheral hypersensitivities develop in the face versus hindpaw regions in a mouse model of repetitive NTG-induced migraine, and that these hindpaw mechanical hypersensitivity and facial cold allodynia are not mediated by the activation of CSPAs.
Assuntos
Doenças do Nervo Facial/fisiopatologia , Hiperalgesia/fisiopatologia , Transtornos de Enxaqueca/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios Aferentes/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/fisiopatologia , Canais de Cátion TRPV/metabolismo , Animais , Capsaicina/farmacologia , Temperatura Baixa/efeitos adversos , Diterpenos/toxicidade , Resistência a Medicamentos , Doenças do Nervo Facial/induzido quimicamente , Doenças do Nervo Facial/metabolismo , Doenças do Nervo Facial/patologia , Membro Posterior , Temperatura Alta/efeitos adversos , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/patologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Neurônios Aferentes/metabolismo , Neurônios Aferentes/patologia , Neurotoxinas/toxicidade , Doadores de Óxido Nítrico/toxicidade , Nitroglicerina/toxicidade , Especificidade de Órgãos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genéticaRESUMO
Background Self-injurious behaviors (SIBs) are devastating traits in autism spectrum disorder (ASD). Although deficits in pain sensation might be one of the contributing factors underlying the development of SIBs, the mechanisms have yet to be addressed. Recently, the Shank2 synaptic protein has been considered to be a key component in ASD, and mutations of SHANK2 gene induce the dysfunction of N-methyl-D-aspartate (NMDA) receptors, suggesting a link between Shank2 and NMDA receptors in ASD. Given that spinal NMDA receptors play a pivotal role in pain hypersensitivity, we investigated the possible role of Shank2 in nociceptive hypersensitivity by examining changes in spontaneous pain following intrathecal NMDA injection in S hank2-/- ( Shank2 knock-out, KO) mice. Results Intrathecal NMDA injection evoked spontaneous nociceptive behaviors. These NMDA-induced nociceptive responses were significantly reduced in Shank2 KO mice. We also observed a significant decrease of NMDA currents in the spinal dorsal horn of Shank2 KO mice. Subsequently, we examined whether mitogen-activated protein kinase or AKT signaling is involved in this reduced pain behavior in Shank2 KO mice because the NMDA receptor is closely related to these signaling molecules. Western blotting and immunohistochemistry revealed that spinally administered NMDA increased the expression of a phosphorylated form of extracellular signal-regulated kinase (p-ERK) which was significantly reduced in Shank2 KO mice. However, p38, JNK, or AKT were not changed by NMDA administration. The ERK inhibitor, PD98059, decreased NMDA-induced spontaneous pain behaviors in a dose-dependent manner in wild-type mice. Moreover, it was found that the NMDA-induced increase in p-ERK was primarily colocalized with Shank2 proteins in the spinal cord dorsal horn. Conclusion Shank2 protein is involved in spinal NMDA receptor-mediated pain, and mutations of Shank2 may suppress NMDA-ERK signaling in spinal pain transmission. This study provides new clues into the mechanisms underlying pain deficits associated with SIB and deserves further study in patients with ASD.
Assuntos
Hiperalgesia/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Nociceptividade/efeitos dos fármacos , Dor/patologia , Medula Espinal/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Modelos Animais de Doenças , Agonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Flavonoides/farmacologia , Hiperalgesia/induzido quimicamente , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Metilaspartato/toxicidade , Proteínas do Tecido Nervoso/genética , Dor/induzido quimicamente , Medição da Dor , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Medula Espinal/efeitos dos fármacosRESUMO
Cancer chemotherapy with platinum-based antineoplastic agents including oxaliplatin frequently results in a debilitating and painful peripheral neuropathy. We evaluated the antinociceptive effects of the alpha-2 adrenoceptor agonist, clonidine on oxaliplatin-induced neuropathic pain. Specifically, we determined if (i) the intraperitoneal (i.p.) injection of clonidine reduces mechanical allodynia in mice with an oxaliplatin-induced neuropathy and (ii) concurrent inhibition of p38 mitogen-activated protein kinase (MAPK) activity by the p38 MAPK inhibitor SB203580 enhances clonidine's antiallodynic effect. Clonidine (0.01-0.1 mg kg(-1), i.p.), with or without SB203580(1-10 nmol, intrathecal) was administered two weeks after oxaliplatin injection(10 mg kg(-1), i.p.) to mice. Mechanical withdrawal threshold, motor coordination and blood pressure were measured. Postmortem expression of p38 MAPK and ERK as well as their phosphorylated forms(p-p38 and p-ERK) were quantified 30 min or 4 hr after drug injection in the spinal cord dorsal horn of treated and control mice. Clonidine dose-dependently reduced oxaliplatin-induced mechanical allodynia and spinal p-p38 MAPK expression, but not p-ERK. At 0.1 mg kg(-1), clonidine also impaired motor coordination and decreased blood pressure. A 10 nmol dose of SB203580 alone significantly reduced mechanical allodynia and p-p38 MAPK expression, while a subeffective dose(3 nmol) potentiated the antiallodynic effect of 0.03 mg kg(-1) clonidine and reduced the increased p-p38 MAPK. Coadministration of SB203580 and 0.03 mg kg(-1) clonidine decreased allodynia similar to that of 0.10 mg kg(-1) clonidine, but without significant motor or vascular effects. These findings demonstrate that clonidine treatment reduces oxaliplatin-induced mechanical allodynia. The concurrent administration of SB203580 reduces the dosage requirements for clonidine, thereby alleviating allodynia without producing undesirable motor or cardiovascular effects.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Antineoplásicos/efeitos adversos , Clonidina/farmacologia , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Compostos Organoplatínicos/efeitos adversos , Animais , Pressão Sanguínea/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hiperalgesia/diagnóstico , Hiperalgesia/tratamento farmacológico , Imidazóis/farmacologia , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Oxaliplatina , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/efeitos dos fármacos , Corno Dorsal da Medula Espinal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We recently demonstrated that activation of spinal sigma-1 receptors (Sig-1Rs) induces pain hypersensitivity via the activation of neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2). However, the potential direct interaction between nNOS-derived nitric oxide (NO) and Nox2-derived reactive oxygen species (ROS) is poorly understood, particularly with respect to the potentiation of N-methyl-D-aspartate (NMDA) receptor activity in the spinal cord associated with the development of central sensitization. Thus, the main purpose of this study was to investigate whether Sig-1R-induced and nNOS-derived NO modulates spinal Nox2 activation leading to an increase in ROS production and ultimately to the potentiation of NMDA receptor activity and pain hypersensitivity. Intrathecal pretreatment with the nNOS inhibitor, 7-nitroindazole or with the Nox inhibitor, apocynin significantly inhibited the mechanical and thermal hypersensitivity induced by intrathecal administration of the Sig-1R agonist, 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride (PRE084). Conversely, pretreatment with 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron(III) (FeTPPS; a scavenger of peroxynitrite, a toxic reaction product of NO and superoxide) had no effect on the PRE084-induced pain hypersensitivity. Pretreatment with 7-nitroindazole significantly reduced the PRE084-induced increase in Nox2 activity and concomitant ROS production in the lumbar spinal cord dorsal horn, whereas apocynin did not alter the PRE084-induced changes in nNOS phosphorylation. On the other hand pretreatment with apocynin suppressed the PRE084-induced increase in the protein kinase C (PKC)-dependent phosphorylation of NMDA receptor GluN1 subunit (pGluN1) at Ser896 site in the dorsal horn. These findings demonstrate that spinal Sig-1R-induced pain hypersensitivity is mediated by nNOS activation, which leads to an increase in Nox2 activity ultimately resulting in a ROS-induced increase in PKC-dependent pGluN1 expression.
Assuntos
Hiperalgesia/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores sigma/metabolismo , Animais , Temperatura Alta , Masculino , Camundongos Endogâmicos ICR , NADPH Oxidase 2 , Óxido Nítrico/metabolismo , Dor/metabolismo , Estimulação Física , Corno Dorsal da Medula Espinal/metabolismo , Receptor Sigma-1RESUMO
We have previously demonstrated that activation of the spinal sigma-1 receptor (Sig-1R) plays an important role in the development of mechanical allodynia (MA) via secondary activation of the N-methyl-d-aspartate (NMDA) receptor. Sig-1Rs have been shown to localize to astrocytes, and blockade of Sig-1Rs inhibits the pathologic activation of astrocytes in neuropathic mice. However, the mechanism by which Sig-1R activation in astrocytes modulates NMDA receptors in neurons is currently unknown. d-serine, synthesized from l-serine by serine racemase (Srr) in astrocytes, is an endogenous co-agonist for the NMDA receptor glycine site and can control NMDA receptor activity. Here, we investigated the role of d-serine in the development of MA induced by spinal Sig-1R activation in chronic constriction injury (CCI) mice. The production of d-serine and Srr expression were both significantly increased in the spinal cord dorsal horn post-CCI surgery. Srr and d-serine were only localized to astrocytes in the superficial dorsal horn, while d-serine was also localized to neurons in the deep dorsal horn. Moreover, we found that Srr exists in astrocytes that express Sig-1Rs. The CCI-induced increase in the levels of d-serine and Srr was attenuated by sustained intrathecal treatment with the Sig-1R antagonist, BD-1047 during the induction phase of neuropathic pain. In behavioral experiments, degradation of endogenous d-serine with DAAO, or selective blockade of Srr by LSOS, effectively reduced the development of MA, but not thermal hyperalgesia in CCI mice. Finally, BD-1047 administration inhibited the development of MA and this inhibition was reversed by intrathecal treatment with exogenous d-serine. These findings demonstrate for the first time that the activation of Sig-1Rs increases the expression of Srr and d-serine in astrocytes. The increased production of d-serine induced by CCI ultimately affects dorsal horn neurons that are involved in the development of MA in neuropathic mice.
Assuntos
Astrócitos/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Receptores sigma/metabolismo , Serina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Modelos Animais de Doenças , Etilenodiaminas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Células do Corno Posterior/metabolismo , Racemases e Epimerases/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Receptor Sigma-1RESUMO
BACKGROUND: We previously developed a thrombus-induced ischemic pain (TIIP) animal model, which was characterized by chronic bilateral mechanical allodynia without thermal hyperalgesia (TH). On the other hand we had shown that intraplantar injection of acidic saline facilitated ATP-induced pain, which did result in the induction of TH in normal rats. Because acidic pH and increased ATP are closely associated with ischemic conditions, this study is designed to: (1) examine whether acidic saline injection into the hind paw causes the development of TH in TIIP, but not control, animals; and (2) determine which peripheral mechanisms are involved in the development of this TH. RESULTS: Repeated intraplantar injection of pH 4.0 saline, but not pH 5.5 and 7.0 saline, for 3 days following TIIP surgery resulted in the development of TH. After pH 4.0 saline injections, protein levels of hypoxia inducible factor-1α (HIF-1α) and carbonic anhydrase II (CA II) were elevated in the plantar muscle indicating that acidic stimulation intensified ischemic insults with decreased tissue acidity. At the same time point, there were no changes in the expression of TRPV1 in hind paw skin, whereas a significant increase in TRPV1 phosphorylation (pTRPV1) was shown in acidic saline (pH 4.0) injected TIIP (AS-TIIP) animals. Moreover, intraplantar injection of chelerythrine (a PKC inhibitor) and AMG9810 (a TRPV1 antagonist) effectively alleviated the established TH. In order to investigate which proton- or ATP-sensing receptors contributed to the development of TH, amiloride (an ASICs blocker), AMG9810, TNP-ATP (a P2Xs antagonist) or MRS2179 (a P2Y1 antagonist) were pre-injected before the pH 4.0 saline. Only MRS2179 significantly prevented the induction of TH, and the increased pTRPV1 ratio was also blocked in MRS2179 injected animals. CONCLUSION: Collectively these data show that maintenance of an acidic environment in the ischemic hind paw of TIIP rats results in the phosphorylation of TRPV1 receptors via a PKC-dependent pathway, which leads to the development of TH mimicking what occurs in chronic ischemic patients with severe acidosis. More importantly, peripheral P2Y1 receptors play a pivotal role in this process, suggesting a novel peripheral mechanism underlying the development of TH in these patients.
Assuntos
Membro Posterior/irrigação sanguínea , Hiperalgesia/complicações , Isquemia/etiologia , Dor/etiologia , Receptores Purinérgicos P2Y1/metabolismo , Canais de Cátion TRPV/metabolismo , Trombose/complicações , Ácidos , Acrilamidas/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Benzofenantridinas/farmacologia , Western Blotting , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Modelos Animais de Doenças , Diterpenos/farmacologia , Membro Posterior/patologia , Temperatura Alta , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Hipóxia/etiologia , Hipóxia/metabolismo , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Injeções , Canais Iônicos/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Dor/metabolismo , Dor/patologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio/administração & dosagem , Cloreto de Sódio/farmacologia , Trombose/metabolismo , Trombose/patologia , Extratos de TecidosRESUMO
Neuroactive steroids (NASs) directly affect neuronal excitability. Despite their role in the nervous system is intimately linked to pain control, knowledge is currently limited. This study investigates the peripheral involvement of NASs in chronic ischemic pain by targeting the cytochrome P450 side-chain cleavage enzyme (P450scc). Using a rat model of hind limb thrombus-induced ischemic pain (TIIP), we observed an increase in P450scc expression in the ischemic hind paw skin. Inhibiting P450scc with intraplantar aminoglutethimide (AMG) administration from post-operative day 0 to 3 significantly reduced the development of mechanical allodynia. However, AMG administration from post-operative day 3 to 6 did not affect established mechanical allodynia. In addition, we explored the role of the peripheral sigma-1 receptor (Sig-1R) by co-administering PRE-084 (PRE), a Sig-1R agonist, with AMG. PRE reversed the analgesic effects of AMG during the induction phase. These findings indicate that inhibiting steroidogenesis with AMG alleviates peripheral ischemic pain during the induction phase via Sig-1Rs.
Assuntos
Modelos Animais de Doenças , Hiperalgesia , Isquemia , Ratos Sprague-Dawley , Receptores sigma , Animais , Hiperalgesia/tratamento farmacológico , Hiperalgesia/patologia , Hiperalgesia/complicações , Masculino , Isquemia/complicações , Isquemia/patologia , Receptores sigma/antagonistas & inibidores , Receptores sigma/metabolismo , Receptor Sigma-1 , Dor/tratamento farmacológico , Dor/complicações , Dor/etiologia , Dor/patologia , Membro Posterior/efeitos dos fármacos , Ratos , Sistema Enzimático do Citocromo P-450/metabolismoRESUMO
We have recently demonstrated that spinal sigma-1 receptors (Sig-1Rs) mediate pain hypersensitivity in mice and neuropathic pain in rats. In this study, we examine the role of NADPH oxidase 2 (Nox2)-induced reactive oxygen species (ROS) on Sig-1R-induced pain hypersensitivity and the induction of chronic neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in rats. Mechanical allodynia and thermal hyperalgesia were evaluated in mice and CCI-rats. Western blotting and dihydroethidium (DHE) staining were performed to assess the changes in Nox2 activation and ROS production in spinal cord, respectively. Direct activation of spinal Sig-1Rs with the Sig-1R agonist, PRE084 induced mechanical allodynia and thermal hyperalgesia, which were dose-dependently attenuated by pretreatment with the ROS scavenger, NAC or the Nox inhibitor, apocynin. PRE084 also induced an increase in Nox2 activation and ROS production, which were attenuated by pretreatment with the Sig-1R antagonist, BD1047 or apocynin. CCI-induced nerve injury produced an increase in Nox2 activation and ROS production in the spinal cord, all of which were attenuated by intrathecal administration with BD1047 during the induction phase of neuropathic pain. Furthermore, administration with BD1047 or apocynin reversed CCI-induced mechanical allodynia during the induction phase, but not the maintenance phase. These findings demonstrate that spinal Sig-1Rs modulate Nox2 activation and ROS production in the spinal cord, and ultimately contribute to the Sig-1R-induced pain hypersensitivity and the peripheral nerve injury-induced induction of chronic neuropathic pain.
Assuntos
Hiperalgesia/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neuralgia/metabolismo , Receptores sigma/metabolismo , Animais , Etilenodiaminas/farmacologia , Temperatura Alta , Masculino , Camundongos , Camundongos Endogâmicos ICR , Morfolinas/farmacologia , NADPH Oxidase 2 , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores sigma/agonistas , Receptores sigma/antagonistas & inibidores , Medula Espinal/metabolismo , Tato , Receptor Sigma-1RESUMO
BACKGROUND INFORMATION: Although previous reports have examined the function of prostaglandin E2 (PGE2) on gap junctions and undifferentiated stem cells, its effects on the reciprocal action of connexin (Cx) isoforms and undifferentiation in embryonic stem cells (ESCs) are unclear. Therefore, we investigated the role of PGE2 on Cx isoforms and maintenance of mouse ESC undifferentiated state. RESULTS: We have analysed 10 Cx genes, but found nine of them. PGE2 (50 µM) stimulated Cx31, Cx32, Cx40, Cx43 and Cx45 mRNA expression. Amongst them, PGE2 maximally stimulated the Cx43 mRNA expression and gap junction inter-cellular coupling. Therefore, we investigated the effect of PGE2 on Cx43 expression. PGE2 activated cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation. In addition, treatments of adenylate cyclase activators increased Cx43 expression, but not PI3K/Akt phosphorylation. PGE2 also inactivated GSK-3ß and stimulated active-ß-catenin. Furthermore, a ChiP assay demonstrated the association of ß-catenin with the Cx26 (as control) and Cx43 promoter. Finally, down-regulation of PGE2-induced Cx isoforms by AH 6809, Cx31-, Cx43-, Cx45 small interfering (si)RNA and 18α-glycyrrhetinic acid decreased levels of undifferentiated markers of ESCs, including Oct4, FoxD3, Sox2 and SSEA-1, but Nanog did not be down-regulated by Cx43 siRNA. CONCLUSIONS: PGE2 stimulates Cx isoforms via GSK-3ß/ß-catenin via EP2-receptor-dependent cAMP/PKA and PI3K/Akt in mouse ESCs, thereby partially contributing to the maintenance of their undifferentiated state.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Conexinas/metabolismo , Dinoprostona/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , beta Catenina/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Conexina 26 , Conexina 43/genética , Conexina 43/metabolismo , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Glicogênio Sintase Quinase 3 beta , Camundongos , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Peripheral or central nerve injury often leads to neuropathic pain. Although ketamine and pregabalin are first line options for the treatment of neuropathic pain, their clinical application is limited due to side effects such as sedation, dizziness and somnolence. We designed this study to determine whether the intrathecal (i.t.) co-treatment with ketamine and pregabalin at sub-effective low doses would elicit a sufficient pain relief without producing side effect in a neuropathic pain mouse model. At day 7 after chronic constriction injury (CCI) of sciatic nerve, dose dependent effects of i.t. ketamine (3, 10, 30, 100 µg) or i.t. pregabalin (10, 30, 100 µg) on mechanical allodynia and thermal hyperalgesia were measured. For combination treatment, 3 or 10 µg of ketamine and 30 µg of pregabalin were selected because these doses of drugs were not effective on neuropathic pain. Interestingly, combined i.t. treatment groups (ketamine 3 µg+pregabalin 30 µg and ketamine 10 µg+pregabalin 30 µg) produced strong analgesia on neuropathic pain although these doses of ketamine and pregabalin alone are not effective. Moreover, rota rod test revealed that normal motor function was not affected by combined treatment while i.t. ketamine at doses above 10 µg showed a significant motor dysfunction. Results of this study suggested that i.t. co-treatment with ketamine and pregabalin at sub-effect low doses may be a useful therapeutic method for the treatment of neuropathic pain patients.
Assuntos
Analgésicos/administração & dosagem , Hiperalgesia/tratamento farmacológico , Ketamina/administração & dosagem , Neuralgia/tratamento farmacológico , Ácido gama-Aminobutírico/análogos & derivados , Animais , Modelos Animais de Doenças , Sinergismo Farmacológico , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Destreza Motora/efeitos dos fármacos , Pregabalina , Ácido gama-Aminobutírico/administração & dosagemRESUMO
Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1), also known as growth differentiation factor-15 (GDF-15), is associated with cancer, diabetes, and inflammation, while there is limited understanding of the role of NAG-1 in nociception. Here, we examined the nociceptive behaviors of NAG-1 transgenic (TG) mice and wild-type (WT) littermates. Mechanical sensitivity was evaluated by using the von Frey filament test, and thermal sensitivity was assessed by the hot-plate, Hargreaves, and acetone tests. c-Fos, glial fibrillary acidic protein (GFAP), and ionized calcium binding adaptor molecule-1 (Iba-1) immunoreactivity was examined in the spinal cord following observation of the formalin-induced nociceptive behaviors. There was no difference in mechanical or thermal sensitivity for NAG-1 TG and WT mice. Intraplantar formalin injection induced nociceptive behaviors in both male and female NAG-1 TG and WT mice. The peak period in the second phase was delayed in NAG-1 TG female mice compared with that of WT female mice, while there was no difference in the cumulative time of nociceptive behaviors between the two groups of mice. Formalin increased spinal c-Fos immunoreactivity in both TG and WT female mice. Neither GFAP nor Iba-1 immunoreactivity was increased in the spinal cord of TG and WT female mice. These findings indicate that NAG-1 TG mice have comparable baseline sensitivity to mechanical and thermal stimulation as WT mice and that NAG-1 in female mice may have an inhibitory effect on the second phase of inflammatory pain. Therefore, it could be a novel target to inhibit central nervous system response in pain.
RESUMO
Progesterone has been shown to have neuroprotective capabilities against a wide range of nervous system injuries, however there are negative clinical studies that have failed to demonstrate positive effects of progesterone therapy. Specifically, we looked into whether progesterone receptors or its metabolizing enzymes, cytochrome P450c17 and 5α-reductase, are involved in the effects of progesterone on neuropathic pain after chronic constriction injury (CCI) of the sciatic nerve in mice. Intrathecal progesterone administration during the induction phase of chronic pain enhanced mechanical allodynia development and spinal glial fibrillary acidic protein (GFAP) expression, and this enhancement was inhibited by administration of ketoconazole, a P450c17 inhibitor, but not finasteride, a 5α-reductase inhibitor. Furthermore, phospho-serine levels of P450c17 in the spinal cord were elevated on day 1 after CCI operation, but not on day 17. In contrast, intrathecal progesterone administration during the maintenance phase of chronic pain decreased the acquired pain and elevated GFAP expression; this inhibition was restored by finasteride administration, but not by ketoconazole. The modification of mechanical allodynia brought on by progesterone in CCI mice was unaffected by the administration of mifepristone, a progesterone receptor antagonist. Collectively, these findings imply that progesterone suppresses spinal astrocyte activation via 5α-reductase activity during the maintenance phase of chronic pain and has an analgesic impact on the mechanical allodynia associated with the growing neuropathy. Progesterone, however, stimulates spinal astrocytes during the induction stage of peripheral neuropathy and boosts the allodynic impact caused by CCI through early spinal P450c17 activation.
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Prostaglandin E(2) (PGE(2)) is well known to regulate cell functions through cAMP; however, the role of exchange protein directly activated by cAMP (Epac1) and protein kinase A (PKA) in modulating such functions is unknown in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). Therefore, we investigated the relationship between Epac1 and PKA during PGE(2)-induced hUCB-MSC proliferation and its related signaling pathways. PGE(2) increased cell proliferation, and E-type prostaglandin (EP) 2 receptor mRNA expression level and activated cAMP generation, which were blocked by EP2 receptor selective antagonist AH 6809. PGE(2) increased Epac1 expression, Ras-related protein 1 (Rap1) activation level, and Akt phosphorylation, which were inhibited by AH 6809, adenylyl cyclase inhibitor SQ 22536, and Epac1/Rap1-specific siRNA. Also, PGE(2) increased PKA activity, which was inhibited by AH 6809, SQ 22536, and PKA inhibitor PKI. HUCB-MSCs were incubated with the Epac agonist 8-pCPT-cAMP or the PKA agonist 6-phe-cAMP to examine whether Epac1/Rap1/Akt activation was independent of PKA activation. 8-pCPT-cAMP increased Akt phosphorylation but not PKA activity. 6-Phe-cAMP increased PKA activity, but not Akt phosphorylation. Additionally, an Akt inhibitor or PKA inhibitor (PKI) did not block the PGE(2) -induced increase in PKA activity or Akt phosphorylation, respectively. Moreover, PGE(2) increased glycogen synthase kinase (GSK)-3ß phosphorylation and nuclear translocation of active-ß-catenin, which were inhibited by Akt inhibitor or/and PKI. PGE(2) increased c-Myc and vascular endothelial growth factor (VEGF) expression levels, which were blocked by ß-catenin siRNA. In conclusion, PGE(2) stimulated hUCB-MSC proliferation through ß-catenin-mediated c-Myc and VEGF expression via Epac/Rap1/Akt and PKA cooperation.
Assuntos
Dinoprostona/farmacologia , Sangue Fetal/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Complexo Shelterina , Transdução de Sinais , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: It has been reported that the selective blockade of Nav1.8 sodium channels could be a possible target for the development of analgesics without unwanted side effects. However, the precise role of spinal Nav1.8 in the induction and maintenance of persistent pain, e.g., mechanical allodynia (MA) and thermal hyperalgesia (TH), is not clear. We designed this study to investigate whether spinal Nav1.8 contributes to capsaicin-induced and peripheral ischemia-induced MA and TH. METHODS: The Nav1.8 blockers, A-803467 or ambroxol, were injected intrathecally either before or after intraplantar capsaicin injection. To evaluate capsaicin-induced neuronal activation in the spinal cord, we quantified the number of Fos-immunoreactive cells in the dorsal horn. In the thrombus-induced ischemic pain model, we determined the differential effect of A-803467 on the induction phase or maintenance phase of MA. RESULTS: Intrathecal injection of A-803467 (10, 30, 100 nmol) or ambroxol (241, 724, 2410 nmol) before intraplantar injection of capsaicin dose dependently prevented the induction of both MA and TH. However, posttreatment with A-803467 (100 nmol) and ambroxol (2410 nmol) did not reduce the MA that had already developed, but did significantly suppress capsaicin-induced TH. Moreover, the capsaicin-induced increase of spinal Fos-immunoreactive cells was significantly diminished by pretreatment, but not posttreatment with Nav1.8 blockers. In thrombus-induced ischemic pain rats, repetitive treatments of A-803467 during the induction period also prevented the development of MA, whereas A-803467 treatments during the maintenance period were ineffective in preventing or reducing MA. CONCLUSIONS: These results demonstrate that spinal activation of Nav1.8 mediates the early induction of MA, but not the maintenance of MA. However, both the induction and maintenance of TH are modulated by the intrathecal injection of Nav1.8 blockers. These findings suggest that early treatment with a Nav1.8 blocker can be an important factor in the clinical management of chronic MA associated with inflammatory and ischemic pain.
Assuntos
Ambroxol/administração & dosagem , Compostos de Anilina/administração & dosagem , Capsaicina , Furanos/administração & dosagem , Hiperalgesia/etiologia , Isquemia/complicações , Dor/etiologia , Bloqueadores dos Canais de Sódio/administração & dosagem , Canais de Sódio/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Temperatura Alta , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Injeções Espinhais , Isquemia/metabolismo , Isquemia/fisiopatologia , Masculino , Canal de Sódio Disparado por Voltagem NAV1.8 , Dor/induzido quimicamente , Dor/metabolismo , Dor/fisiopatologia , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Estimulação Física , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Sódio/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Fatores de TempoRESUMO
Cold allodynia is an important distinctive feature of neuropathic pain. The present study examined whether single or repetitive treatment of diluted bee venom (DBV) reduced cold allodynia in sciatic nerve chronic constriction injury (CCI) rats and whether these effects were mediated by spinal adrenergic receptors. Single injection of DBV (0.25 or 2.5 mg/kg) was performed into Zusanli acupoint 2 weeks post CCI, and repetitive DBV (0.25 mg/kg) was injected for 2 weeks beginning on day 15 after CCI surgery. Single treatment of DBV at a low dose (0.25 mg/kg) did not produce any anticold allodynic effect, while a high dose of DBV (2.5 mg/kg) significantly reduced cold allodynia. Moreover, this effect of high-dose DBV was completely blocked by intrathecal pretreatment of idazoxan (α2-adrenoceptor antagonist), but not prazosin (α1-adrenoceptor antagonist) or propranolol (nonselective ß-adrenoceptor antagonist). In addition, coadministration of low-dose DBV (0.25 mg/kg) and intrathecal clonidine (α2-adrenoceptor agonist) synergically reduced cold allodynia. On the other hand, repetitive treatments of low-dose DBV showing no motor deficit remarkably suppressed cold allodynia from 7 days after DBV treatment. This effect was also reversed by intrathecal idazoxan injection. These findings demonstrated that single or repetitive stimulation of DBV could alleviate CCI-induced cold allodynia via activation of spinal α2-adrenoceptor.
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In this study, we examined the antinociceptive effect of Cyperi rhizoma (CR) and Corydalis tuber (CT) extracts using a chronic constriction injury-induced neuropathic pain rat model. After the ligation of sciatic nerve, neuropathic pain behavior such as mechanical allodynia and thermal hyperalgesia were rapidly induced and maintained for 1 month. Repeated treatment of CR or CT (per oral, 10 or 30 mg/kg, twice a day) was performed either in induction (day 0~5) or maintenance (day 14~19) period of neuropathic pain state. Treatment of CR or CT at doses of 30 mg/kg in the induction and maintenance periods significantly decreased the nerve injury-induced mechanical allodynia. In addition, CR and CT at doses of 10 or 30 mg/kg alleviated thermal heat hyperalgesia when they were treated in the maintenance period. Finally, CR or CT (30 mg/kg) treated during the induction period remarkably reduced the nerve injury-induced phosphorylation of NMDA receptor NR1 subunit (pNR1) in the spinal dorsal horn. Results of this study suggest that extracts from CR and CT may be useful to alleviate neuropathic pain.
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Recent studies have revealed that glial sigma-1 receptor (Sig-1R) in the spinal cord may be a critical factor to mediate sensory function. However, the functional role of Sig-1R in astrocyte has not been clearly elucidated. Here, we determined whether Sig-1Rs modulate calcium responses in primary cultured astrocytes and pathological changes in spinal astrocytes, and whether they contribute to pain hypersensitivity in naïve mice and neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve in mice. Sig-1R was expressed in glial fibrillary acidic protein (GFAP)-positive cultured astrocytes. Treatment with the Sig-1R agonist, PRE-084 or neurosteroid dehydroepiandrosterone (DHEA) increased intracellular calcium responses in cultured astrocytes, and this increase was blocked by the pretreatment with the Sig-1R antagonist, BD-1047 or neurosteroid progesterone. Intrathecal administration of PRE-084 or DHEA for 10 days induced mechanical and thermal hypersensitivity and increased the number of Sig-1R-immunostained GFAP-positive cells in the superficial dorsal horn (SDH) region of the spinal cord in naïve mice, and these changes were inhibited by administration with BD-1047 or progesterone. In CCI mice, intrathecal administration of BD-1047 or progesterone at post-operative day 14 suppressed the developed mechanical allodynia and the number of Sig-1R-immunostained GFAP-positive cells that were increased in the SDH region of the spinal cord following CCI of the sciatic nerve. These results demonstrate that Sig-1Rs play an important role in the modulation of intracellular calcium responses in cultured astrocytes and pathological changes in spinal astrocytes and that administration of BD-1047 or progesterone alleviates the Sig-1R-induced pain hypersensitivity and the peripheral nerve injury-induced mechanical allodynia.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Neuroesteroides/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Receptores sigma/metabolismo , Medula Espinal/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Camundongos , Neuralgia/tratamento farmacológico , Neuralgia/fisiopatologia , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Traumatismos dos Nervos Periféricos/fisiopatologia , Progesterona/farmacologia , Receptores sigma/antagonistas & inibidores , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia , Receptor Sigma-1RESUMO
The sigma-1 receptor (Sig-1R) plays an important role in spinal pain transmission by increasing phosphorylation of the N-methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). As a result Sig-1R has been suggested as a novel therapeutic target for prevention of chronic pain. Here we investigated whether interleukin-1ß (IL-1ß) modulates the expression of the Sig-1R in spinal astrocytes during the early phase of nerve injury, and whether this modulation affects spinal pGluN1 expression and the development of neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve. Repeated intrathecal (i.t.) administration of IL-1ß from days 0-3 post-surgery significantly reduced the increased pGluN1 expression at the Ser896 and Ser897 sites in the ipsilateral spinal cord, as well as, the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw of CCI mice, which were restored by co-administration of IL-1 receptor antagonist with IL-1ß. Sciatic nerve injury increased the expression of Sig-1R in astrocytes of the ipsilateral spinal cord, and this increase was suppressed by i.t. administration of IL-1ß. Agonistic stimulation of the Sig-1R with PRE084 restored pGluN1 expression and the development of mechanical allodynia that were originally suppressed by IL-1ß in CCI mice. Collectively these results demonstrate that IL-1ß administration during the induction phase of neuropathic pain produces an analgesic effect on neuropathic pain development by controlling the expression of Sig-1R in spinal astrocytes.
Assuntos
Analgésicos/administração & dosagem , Astrócitos/efeitos dos fármacos , Hiperalgesia/prevenção & controle , Interleucina-1beta/administração & dosagem , Neuralgia/prevenção & controle , Limiar da Dor/efeitos dos fármacos , Receptores sigma/metabolismo , Medula Espinal/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Injeções Espinhais , Masculino , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/metabolismo , Neuralgia/fisiopatologia , Fosforilação , Receptores de N-Metil-D-Aspartato/metabolismo , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Receptor Sigma-1RESUMO
BACKGROUND: Scalding burn injuries can occur in everyday life but occur more frequently in young children. Therefore, it is important to develop more effective burn treatments. OBJECTIVES: This study examined the effects of bee venom (BV) stimulation on scalding burn injury-induced nociception in mice as a new treatment for burn pain. METHODS: To develop a burn injury model, the right hind paw was immersed temporarily in hot water (65°C, 3 seconds). Immediately after the burn, BV (0.01, 0.02, or 0.1 mg/kg) was injected subcutaneously into the ipsilateral knee area once daily for 14 days. A von Frey test was performed to assess the nociceptive response, and the altered walking parameters were evaluated using an automated gait analysis system. In addition, the peripheral and central expression changes in substance P (Sub P) were measured in the dorsal root ganglion and spinal cord by immunofluorescence. RESULTS: Repeated BV treatment at the 2 higher doses used in this study (0.02 and 0.1 mg/kg) alleviated the pain responses remarkably and recovered the gait performances to the level of acetaminophen (200 mg/kg, intraperitoneal, once daily), which used as the positive control group. Moreover, BV stimulation had an inhibitory effect on the increased expression of Sub P in the peripheral and central nervous systems by a burn injury. CONCLUSIONS: These results suggest that a peripheral BV treatment may have positive potency in treating burn-induced pain.