RESUMO
A multiplex analysis for profiling the expression of candidate genes along with epigenetic modification may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we found that partial sciatic nerve ligation most remarkably increased the expression of monocyte chemotactic protein 3 (MCP-3, known as CCL7) a total of 33 541 genes in the spinal cord, which lasted for 4 weeks. This increase in MCP-3 gene transcription was accompanied by the decreased trimethylation of histone H3 at Lys27 at the MCP-3 promoter. The increased MCP-3 expression associated with its epigenetic modification observed in the spinal cord was almost abolished in interleukin 6 knockout mice with partial sciatic nerve ligation. Consistent with these findings, a single intrathecal injection of recombinant proteins of interleukin 6 significantly increased MCP-3 messenger RNA with a decrease in the level of Lys27 trimethylation of histone H3 at the MCP-3 promoter in the spinal cord of mice. Furthermore, deletion of the C-C chemokine receptor type 2 (CCR2) gene, which encodes a receptor for MCP-3, failed to affect the acceleration of MCP-3 expression in the spinal cord after partial sciatic nerve ligation. A robust increase in MCP-3 protein, which lasted for up to 2 weeks after surgery, in the dorsal horn of the spinal cord of mice with partial sciatic nerve ligation was seen mostly in astrocytes, but not microglia or neurons. On the other hand, the increases in both microglia and astrocytes in the spinal cord by partial sciatic nerve ligation were mostly abolished in interleukin 6 knockout mice. Moreover, this increase in microglia was almost abolished by CCR2 gene deletion, whereas the increase in astrocytes was not affected in nerve-ligated mice that lacked the CCR2 gene. We also found that either in vivo or in vitro treatment with MCP-3 caused robust microglia activation. Under these conditions, intrathecal administration of MCP-3 antibody suppressed the increase in microglia within the mouse spinal cord and neuropathic pain-like behaviours after nerve injury. With the use of a functional magnetic resonance imaging analysis, we demonstrated that a single intrathecal injection of MCP-3 induced dramatic increases in signal intensity in pain-related brain regions. These findings suggest that increased MCP-3 expression associated with interleukin 6 dependent epigenetic modification at the MCP-3 promoter after nerve injury, mostly in spinal astrocytes, may serve to facilitate astrocyte-microglia interaction in the spinal cord and could play a critical role in the neuropathic pain-like state.
Assuntos
Comunicação Celular/fisiologia , Quimiocina CCL7/biossíntese , Epigênese Genética/fisiologia , Interleucina-6/metabolismo , Neuralgia/fisiopatologia , Ativação Transcricional/fisiologia , Animais , Astrócitos/metabolismo , Axotomia , Western Blotting , Quimiocina CCL7/genética , Imunoprecipitação da Cromatina , Dor Crônica/genética , Dor Crônica/metabolismo , Dor Crônica/fisiopatologia , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Imuno-Histoquímica , Interleucina-6/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Análise em Microsséries , Microglia/metabolismo , Neuralgia/genética , Neuralgia/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nervo Isquiático/lesões , Medula Espinal/metabolismo , Medula Espinal/fisiopatologiaRESUMO
BACKGROUND: Evidence obtained in humans and rodents indicates that beta-endorphin (encoded by the proopiomelanocortin [POMC] gene) is critical in the regulation of alcohol drinking behavior. However, the alcohol effect on POMC gene expression has not been studied in rodent mesolimbic regions, such as the nucleus accumbens (NAc). METHODS: In this study, we first utilized POMC-enhanced green fluorescent protein (EGFP) transgenic mice to visualize POMC neurons and found that POMC-EGFP cells were modestly distributed throughout the NAc shell and core, in addition to the hypothalamic arcuate nucleus. POMC mRNA expression in the NAc of mice and rats was confirmed using reverse transcriptase-polymerase chain reaction and solution hybridization assays. We then investigated whether there are genetically determined differences in basal mRNA levels of POMC and mu opioid receptor (MOP-r) between selectively bred Sardinian alcohol-preferring (sP) and nonpreferring (sNP) rats, and whether these mRNA levels are altered in sP rats after alcohol drinking (10%, unlimited access) for 17 days. RESULTS: Alcohol-naïve sP rats had higher basal POMC mRNA levels than sNP rats only in hypothalamus. Alcohol drinking increased POMC mRNA levels in both the NAc shell (by 100%) and the hypothalamus (by 50%) of sP rats. Although sP rats had lower basal levels of MOP-r mRNA and GTPγS binding in NAc shell than sNP rats, voluntary alcohol consumption had no effect on MOP-r mRNA levels in the NAc shell. CONCLUSIONS: Our results define the distribution of POMC-expressing neurons in the NAc of mice and rats. Higher POMC expression at basal levels in sP rats (genetically determined), along with increases after drinking (alcohol-induced) in the NAc shell and hypothalamus, suggests that the POMC systems play a role in high alcohol preference and consumption.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Hipotálamo/metabolismo , Núcleo Accumbens/metabolismo , Pró-Opiomelanocortina/biossíntese , Pró-Opiomelanocortina/genética , Regulação para Cima/genética , Consumo de Bebidas Alcoólicas/psicologia , Animais , Etanol/administração & dosagem , Hipotálamo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Núcleo Accumbens/efeitos dos fármacos , Ratos , Ratos Mutantes , Regulação para Cima/efeitos dos fármacosRESUMO
Environmental enrichment is an experimental paradigm that increases brain-derived neurotrophic factor (BDNF) gene expression accompanied by neurogenesis in the hippocampus of rodents. In the present study, we investigated whether an enriched environment could cause epigenetic modification at the BDNF gene in the hippocampus of mice. Exposure to an enriched environment for 3-4 weeks caused a dramatic increase in the mRNA expression of BDNF, but not platelet-derived growth factor A (PDGF-A), PDGF-B, vascular endothelial growth factor (VEGF), nerve growth factor (NGF), epidermal growth factor (EGF), or glial fibrillary acidic protein (GFAP), in the hippocampus of mice. Under these conditions, exposure to an enriched environment induced a significant increase in histone H3 lysine 4 (H3K4) trimethylation at the BDNF P3 and P6 promoters, in contrast to significant decreases in histone H3 lysine 9 (H3K9) trimethylation at the BDNF P4 promoter and histone H3 lysine 27 (H3K27) trimethylation at the BDNF P3 and P4 promoters without any changes in the expression of their associated histone methylases and demethylases in the hippocampus. The expression levels of several microRNAs in the hippocampus were not changed by an enriched environment. These results suggest that an enriched environment increases BDNF mRNA expression via sustained epigenetic modification in the mouse hippocampus.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Epigênese Genética , Hipocampo/metabolismo , Animais , Meio Ambiente , Fator de Crescimento Epidérmico/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteína Glial Fibrilar Ácida , Hipocampo/crescimento & desenvolvimento , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Fator de Crescimento Derivado de Plaquetas/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-sis/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Neuropathic pain is the most difficult pain to manage in the pain clinic, and sleep problems are common among patients with chronic pain including neuropathic pain. In the present study, we tried to visualize the intensity of pain by assessing neuronal activity and investigated sleep disturbance under a neuropathic pain-like state in mice using functional magnetic resonance imaging (fMRI) and electroencephalogram (EEG)/electromyogram (EMG), respectively. Furthermore, we investigated the effect of gabapentin (GBP) on these phenomena. In a model of neuropathic pain, sciatic nerve ligation caused a marked decrease in the latency of paw withdrawal in response to a thermal stimulus only on the ipsilateral side. Under this condition, fMRI showed that sciatic nerve ligation produced a significant increase in the blood oxygenation level-dependent (BOLD) signal intensity in the pain matrix, which was significantly decreased 2 h after the i.p. injection of GBP. Based on the results of an EEG/EMG analysis, sciatic nerve-ligated animals showed a statistically significant increase in wakefulness and a decrease in non-rapid eye movement (NREM) sleep during the light phase, and the sleep disturbance was almost completely alleviated by a higher dose of GBP in nerve-ligated mice. These findings suggest that neuropathic pain associated with sleep disturbance can be objectively assessed by fMRI and EEG/EMG analysis in animal models. Furthermore, GBP may improve the quality of sleep as well as control pain in patients with neuropathic pain.
Assuntos
Aminas/farmacologia , Analgésicos/farmacologia , Mapeamento Encefálico/métodos , Encéfalo/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos/farmacologia , Neuralgia/complicações , Transtornos do Sono-Vigília/fisiopatologia , Ácido gama-Aminobutírico/farmacologia , Animais , Axotomia , Ondas Encefálicas/efeitos dos fármacos , Gabapentina , Imageamento por Ressonância Magnética , Magnetoencefalografia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/tratamento farmacológico , Medição da Dor/métodos , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Transtornos do Sono-Vigília/etiologiaRESUMO
Pain is evoked by noxious body stimulation or through negative emotional events and memories. There are several caveats to the simple proposition that pain and emotion are linked in the cingulate cortex (CG). In this study, we investigated whether mild noxious heat stimuli could affect the neuronal activity in the CG of rats with sciatic nerve ligation. We produced a partial sciatic nerve injury by tying a tight ligature in rats. Seven days after sciatic nerve ligation, rats received mild noxious heat stimuli. Mild noxious heat stimuli produced flinching behaviors in sciatic nerve-ligated rats, but not sham-operated rats. In addition, the mild noxious heat stimuli caused a significant increase in the release of glutamate in the CG of nerve-ligated rats compared with that of sham-operated rats. Furthermore, phosphorylated-NR1-positive cells in this area significantly increased after mild noxious heat stimuli under a neuropathic pain. Under this condition, there were no significant changes in the levels of immediate-early genes such as c-fos, c-jun, JunB, and Fra1 in the CG between nerve-ligated and sham-operated rats. However, mild noxious heat stimuli under a neuropathic pain-like state produced a marked increase in the phosphorylated-c-jun (p-c-jun) immunoreactivity, which is commonly used to map neurons in the brain that can be activated after N-methyl-D-aspartate receptor activation. These findings raise the possibility that mild noxious heat stimuli under a peripheral nerve injury may increase the release of glutamate and promote its related postneuronal activity in the CG.
Assuntos
Ácido Glutâmico/metabolismo , Giro do Cíngulo/metabolismo , Giro do Cíngulo/fisiopatologia , Hiperalgesia/metabolismo , Estimulação Física/efeitos adversos , Ciática/patologia , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Hiperalgesia/etiologia , Proteínas Imediatamente Precoces/metabolismo , Masculino , Medição da Dor , Limiar da Dor/fisiologia , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Ciática/fisiopatologiaRESUMO
Recent research has suggested that epigenetic mechanisms, which exert lasting control over gene expression without altering the genetic code, could mediate stable changes in brain function. A growing body of evidence supports the idea that epigenetic changes play a role in the etiology of aging and its associated brain dysfunction. The present study was undertaken to evaluate the age-related changes in the expression of doublecortin, which is a marker for neuronal precursors, along with epigenetic modification in the hippocampus of aged mice. In the present study, the doublecortin-positive cells were almost completely absent from the dentate gyrus of the hippocampus of 28-month-old mice. Furthermore, the expression level of doublecortin mRNA was significantly decreased in the hippocampus of aged mice. Under these conditions, a significant decrease in H3K4 trimethylation and a significant increase in H3K27 trimethylation at doublecortin promoters were observed with aging without any changes in the expression of their associated histone methylases and demethylases in the hippocampus. These findings suggest that aging produces a dramatic decrease in the expression of doublecortin along with epigenetic modifications in the hippocampus.
Assuntos
Envelhecimento/genética , Epigênese Genética/fisiologia , Hipocampo/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Animais , Metilação de DNA/genética , Giro Denteado/citologia , Giro Denteado/metabolismo , Proteínas do Domínio Duplacortina , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos/genética , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
A variety of mechanisms that contribute to the accumulation of age-related damage and the resulting brain dysfunction have been identified. Recently, decreased neurogenesis in the hippocampus has been recognized as one of the mechanisms of age-related brain dysfunction. However, the molecular mechanism of decreased neurogenesis with aging is still unclear. In the present study, we investigated whether aging decreases neurogenesis accompanied by the activation of microglia and astrocytes, which increases the expression of IL-1beta in the hippocampus, and whether in vitro treatment with IL-1beta in neural stem cells directly impairs neurogenesis. Ionized calcium-binding adaptor molecule 1 (Iba1)-positive microglia and glial fibrillary acidic protein (GFAP)-positive astrocytes were increased in the dentate gyrus of the hippocampus of 28-month-old mice. Furthermore, the mRNA level of IL-1beta was significantly increased without related histone modifications. Moreover, a significant increase in lysine 9 on histone H3 (H3K9) trimethylation at the promoter of NeuroD (a neural progenitor cell marker) was observed in the hippocampus of aged mice. In vitro treatment with IL-1beta in neural stem cells prepared from whole brain of E14.5 mice significantly increased H3K9 trimethylation at the NeuroD promoter. These findings suggest that aging may decrease hippocampal neurogenesis via epigenetic modifications accompanied by the activation of microglia and astrocytes with the increased expression of IL-1beta in the hippocampus.
Assuntos
Envelhecimento/fisiologia , Hipocampo/fisiologia , Interleucina-1beta/biossíntese , Neurogênese/fisiologia , Neuroglia/fisiologia , Animais , Astrócitos/fisiologia , Diferenciação Celular/fisiologia , DNA/biossíntese , DNA/genética , Feminino , Imunofluorescência , Hipocampo/citologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Análise Serial de Proteínas , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/fisiologiaRESUMO
BACKGROUND: Dynorphin A in the spinal cord is considered to contribute to nociceptive stimuli. However, it has not yet been determined whether activation of the spinal dynorphinergic system under nociceptive stimuli plays a role in direct acceleration of the ascending nociceptive pathway. In this study, the authors investigated the role of spinal dynorphinergic transmission in ongoing brain activation under noxious stimuli in mice. METHODS: The changes in prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity in the mouse spinal cord were determined after the intraplantar injection of complete Freund's adjuvant in mice. The signal intensity in different brain regions after the intraplantar injection of complete Freund's adjuvant or intrathecal injection of dynorphin A (1-17) was measured by a pharmacological functional magnetic resonance imaging analysis. RESULTS: Complete Freund's adjuvant injection produced pain-associated behaviors and induced a dramatic increase in signal intensity in the mouse cingulate cortex, somatosensory cortex, insular cortex, and thalamic nuclei. These effects were not seen in prodynorphin knockout mice. Prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity on the ipsilateral side of the spinal cord were markedly increased in complete Freund's adjuvant-injected mice. Furthermore, intrathecal injection of dynorphin A (1-17) at relatively high doses caused pain-associated behaviors and a remarkable increase in the activities of the cingulate cortex, somatosensory cortex, insular cortex, and medial and lateral thalamic nuclei in mice. CONCLUSIONS: These findings indicate that spinally released dynorphin A (1-17) by noxious stimuli leads to the direct activation of ascending pain transmission.
Assuntos
Encéfalo/fisiologia , Dinorfinas/fisiologia , Inflamação/fisiopatologia , Medula Espinal/fisiologia , Animais , Comportamento Animal , Química Encefálica/genética , Dinorfinas/administração & dosagem , Dinorfinas/farmacologia , Encefalinas/genética , Adjuvante de Freund , Imuno-Histoquímica , Inflamação/induzido quimicamente , Injeções Espinhais , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dor/psicologia , Precursores de Proteínas/genética , RNA/genética , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transmissão Sináptica/fisiologiaRESUMO
Despite the importance of prefrontal cortical dopamine in modulating reward, little is known about the implication of the specific subregion of prefrontal cortex in opioid reward. We investigated the role of neurons projecting from the ventral tegmental area (VTA) to the anterior cingulate cortex (ACG) in opioid reward. Microinjection of the retrograde tracer fluorogold (FG) into the ACG revealed several retrogradely labelled cells in the VTA. The FG-positive reactions were noted in both tyrosine hydroxylase (TH)-positive and -negative VTA neurons. The released levels of dopamine and its major metabolites in the ACG were increased by either the electrical stimulation of VTA neurons or microinjection of a selective µ-opioid receptor (MOR) agonist, (D-Ala²,N-MePhe4,Gly-ol5) enkephalin (DAMGO), into the VTA. MOR-like immunoreactivity was seen in both TH-positive and -negative VTA neurons projecting to the ACG. The conditioned place preference induced by intra-VTA injection of DAMGO was significantly attenuated by chemical lesion of dopaminergic terminals in the ACG. The depletion of dopamine in the ACG induced early extinction of µ-opioid-induced place preference. The levels of phosphorylated DARPP32 (Thr34) and phosphorylated CREB (Ser133) were increased in the ACG of rats that had maintained the morphine-induced place preference, whereas the increases of these levels induced by morphine were blocked by pre-treatment of a selective dopamine D1 receptor antagonist SCH23390. These findings suggest that VTA-ACG transmission may play a crucial role in the acquisition and maintenance of µ-opioid-induced place preference. The activation of DARPP32 and CREB through dopamine D1 receptors in the ACG could be implicated in the maintenance of µ-opioid-induced place preference.
Assuntos
Condicionamento Operante/efeitos dos fármacos , Dopamina/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Giro do Cíngulo/efeitos dos fármacos , Motivação/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Receptores Opioides mu/agonistas , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Aprendizagem por Associação/efeitos dos fármacos , Benzazepinas/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Masculino , Rememoração Mental/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D1/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
Morphine is a prototypical mu-opioid receptor (MOR) agonist, and can directly inhibit pain transmission at both spinal and supraspinal levels. In the present study, we investigated the properties of thalamic neurons in an opioid-sensitive pain-modulating circuit. Application of morphine to cultured thalamic neurons evoked a potentiation of glutamate-induced peak currents, which was blocked by the MOR antagonist. Application of the protein kinase C inhibitor chelerythrine significantly inhibited the morphine-evoked enhancement of glutamate-induced currents. Immunoreactivity for MOR was observed with high density in the habenular nucleus (Hb) of the thalamus in rats, which was clearly co-localized with NMDA receptor subunit 1 (NRI). In this study, we show that microinjection of morphine into the Hb produced a dose-dependent increase in the tail-flick latency and enhanced the antinociceptive effect induced by the intra-Hb injection of glutamate. When fluoro-gold (FG) was used as a retrograde tracer, we found that FG-labeled neurons in the Hb after the microinjection of FG into the periaqueductal gray expressed both MOR and NR1. The present data suggest that the stimulation of MOR in the Hb may be involved in activation of the descending antinociceptive pathway through glutamatergic neurotransmission via the NMDA receptor.
Assuntos
Morfina/farmacologia , Entorpecentes/farmacologia , Neurônios/citologia , Dor/tratamento farmacológico , Transmissão Sináptica/efeitos dos fármacos , Tálamo/citologia , Animais , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Embrião de Mamíferos , Ácido Glutâmico/farmacologia , Injeções Espinhais , Masculino , Microinjeções , Neuroglia , Dor/induzido quimicamente , Dor/fisiopatologia , Medição da Dor/métodos , Técnicas de Patch-Clamp/métodos , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Substância Cinzenta Periaquedutal/fisiopatologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/metabolismo , Estilbamidinas/metabolismoRESUMO
Clinically, it is well known that chronic pain induces depression, anxiety, and a reduced quality of life. There have been many reports on the relationship between pain and emotion. We previously reported that chronic pain induced anxiety with changes in opioidergic function in the central nervous system. In this study, we evaluated the anxiolytic-like effects of several types of antidepressants under a chronic neuropathic pain-like state and searched for the brain site of action where antidepressants show anxiolytic or antinociceptive effects. Sciatic nerve-ligated mice exhibited thermal hyperalgesia and tactile allodynia from days 7 to 28 after nerve ligation. At 4 weeks after ligation, these mice showed a significant anxiety-related behavior in the light-dark test and the elevated plus-maze test. Under these conditions, repeated administration of antidepressants, including the tricyclic antidepressant (TCA) imipramine, the serotonin noradrenaline reuptake inhibitor (SNRI) milnacipran, and the selective serotonin reuptake inhibitor (SSRI) paroxetine, significantly prevented the anxiety-related behaviors induced by chronic neuropathic pain. These antidepressants also produced a significant reduction in thermal hyperalgesia and tactile allodynia. Moreover, the microinjection of paroxetine into the basolateral amygdala or cingulate cortex reduced anxiety-related behavior, and microinjection into the primary somatosensory cortex significantly attenuated thermal hyperalgesia. These findings suggest that serotonergic antidepressants are effective for treating anxiety associated with chronic neuropathic pain and may be useful for treating neuropathic pain with emotional dysfunction such as anxiety. Furthermore, SSRIs show anxiolytic and antinociceptive effects by acting on different brain regions.
Assuntos
Analgésicos , Ansiolíticos , Antidepressivos/farmacologia , Ansiedade/tratamento farmacológico , Ansiedade/etiologia , Encéfalo/efeitos dos fármacos , Dor/tratamento farmacológico , Dor/etiologia , Doenças do Sistema Nervoso Periférico/complicações , Animais , Antidepressivos/administração & dosagem , Antidepressivos Tricíclicos/administração & dosagem , Antidepressivos Tricíclicos/farmacologia , Escuridão , Temperatura Alta , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Dor/complicações , Medição da Dor/efeitos dos fármacos , Estimulação Física , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/complicações , Neuropatia Ciática/patologia , Neuropatia Ciática/psicologia , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
It has often been proposed that opioid addiction does not arise as a consequence of opioid treatment for pain. Recently, we demonstrated that activated protein kinase C (PKC) in the spinal cord associated with chronic pain-like hyperalgesia suppressed the morphine-induced rewarding effect in mice. In the present study, we investigated whether a gene deletion for an endogenous mu-opioid peptide beta-endorphin could affect pain-like behavior and the suppression of the morphine-induced rewarding effect by the direct activation of PKC in the spinal cord. We found that activation of spinal PKC by intrathecal (i.t.) treatment with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused thermal hyperalgesia, pain-like behaviors and suppression of the morphine-induced rewarding effect. This suppression of morphine reward was eliminated in mice that lacked beta-endorphin. In contrast, thermal hyperalgesia and pain-like behaviors were not affected in beta-endorphin knockout mice. These results suggest that the activation of PKC in the spinal cord may play an essential role in the suppression of the morphine-induced rewarding effect in mice with neuropathic pain through the constant release of beta-endorphin.
Assuntos
Encéfalo/efeitos dos fármacos , Dependência de Morfina/enzimologia , Dor/enzimologia , Proteína Quinase C/metabolismo , Medula Espinal/efeitos dos fármacos , beta-Endorfina/genética , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Ativação Enzimática/genética , Ativadores de Enzimas/farmacologia , Feminino , Hiperalgesia/induzido quimicamente , Hiperalgesia/enzimologia , Hiperalgesia/genética , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Dependência de Morfina/genética , Dependência de Morfina/fisiopatologia , Entorpecentes/farmacologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Dor/genética , Dor/fisiopatologia , Recompensa , Medula Espinal/enzimologiaRESUMO
The aim of the present study was to investigate whether direct activation of protein kinase C (PKC) in the spinal cord could change brain activation using a functional magnetic resonance imaging (fMRI) analysis in mice that lack the PKCgamma gene. The activation of spinal PKC by intrathecal (i.t.) injection with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused a time-dependent decrease in paw-withdrawal latency to the heat thermal stimulus. In contrast, i.t. injection of PDBu failed to cause thermal hyperalgesia in mice which lacked the PKCgamma gene. We found that the i.t. injection with PDBu caused a remarkable increase in the activity of several brain regions in wild-type mice compared with vehicle injection. In the somatosensory cortex and lateral and medial thalamus, i.t. injection of PDBu produced a dramatic and time-dependent increase in signal intensity at 1-6h after i.t. PDBu injection. In contrast, i.t. injection of PDBu produced a delayed but significant increase in signal intensity at 3-6h in the cingulate cortex, at 4-6h in the nucleus accumbens and at 3-6h in the ventral tegmental area. In addition, all effects of PDBu were abolished in mice that lacked the PKCgamma gene. These results suggest that the activation of spinal PKCgamma associated with the activation of ascending pain transmission may be an important factor in chronic pain-like hyperalgesia with changes in emotionality.
Assuntos
Vias Aferentes/enzimologia , Encéfalo/enzimologia , Emoções/fisiologia , Dor/enzimologia , Proteína Quinase C/metabolismo , Medula Espinal/enzimologia , Vias Aferentes/efeitos dos fármacos , Vias Aferentes/fisiopatologia , Animais , Encéfalo/fisiopatologia , Mapeamento Encefálico , Emoções/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/enzimologia , Hiperalgesia/genética , Injeções Espinhais , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/enzimologia , Nociceptores/efeitos dos fármacos , Nociceptores/enzimologia , Dor/induzido quimicamente , Dor/genética , Medição da Dor/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Dibutirato de 12,13-Forbol/farmacologia , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/genética , Medula Espinal/efeitos dos fármacos , Fatores de TempoRESUMO
Recent clinical studies have demonstrated that when opioids are used to control pain, psychological dependence is not a major problem. In this study, we further investigated the mechanisms that underlie the suppression of opioid reward under neuropathic pain in rodents. Sciatic nerve ligation suppressed a place preference induced by the selective mu-opioid receptor agonist [d-Ala(2), N-MePhe(4), Gly-ol(5)] enkephalin (DAMGO) and reduced both the increase in the level of extracellular dopamine by s.c. morphine in the nucleus accumbens and guanosine-5'-o-(3-[(35)S]thio) triphosphate ([(35)S]GTPgammaS) binding to membranes of the ventral tegmental area (VTA) induced by DAMGO. These effects were eliminated in mice that lacked the beta-endorphin gene. Furthermore, intra-VTA injection of a specific antibody to the endogenous mu-opioid peptide beta-endorphin reversed the suppression of the DAMGO-induced rewarding effect by sciatic nerve ligation in rats. These results provide molecular evidence that nerve injury results in the continuous release of endogenous beta-endorphin to cause the dysfunction of mu-opioid receptors in the VTA. This phenomenon could explain the mechanism that underlies the suppression of opioid reward under a neuropathic pain-like state.
Assuntos
Morfina/administração & dosagem , Entorpecentes/administração & dosagem , Recompensa , Ciática/tratamento farmacológico , Ciática/psicologia , beta-Endorfina/fisiologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Condicionamento Operante/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/uso terapêutico , Feminino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Masculino , Camundongos , Camundongos Knockout , Medição da Dor , Ligação Proteica/efeitos dos fármacos , Tempo de Reação/efeitos dos fármacos , Ciática/genética , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismo , beta-Endorfina/deficiênciaRESUMO
Pain pathways terminate in discrete brain areas that monitor the sensory and affective qualities of the initiating stimulus and show remarkable plasticity. Here, we found that chronic pain by sciatic nerve ligation caused a dramatic increase in glial fibrillary acidic protein (GFAP)-like immunoreactivity (IR), which is located in the dendritic astrocytes, with its expanding distribution in the cingulate cortex (CG) of mice. The branched GFAP-like IR in the CG of nerve-ligated mice was overlapped with S100beta-like IR, which is highly limited to the cell body of astrocytes, whereas there was no difference of S100beta-like IR between sham-operated and nerve-ligated mice. The number of BrdU-positive cells on the CG was not changed by sciatic nerve ligation. Furthermore, subventricular zone (SVZ)-derived neural stem cells marked by pEGFP-C1 did not migrate toward the CG after sciatic nerve ligation. In the behavioral assay, the thermal hyperalgesia observed on the ipsirateral side in nerve-ligated mice was significantly suppressed by a single pre-microinjection of a glial-modulating agent propentofylline into the CG 24 h before nerve ligation. These results suggest that chronic painful stimuli induces astrocyte activation in the CG, whereas they do not affect the cell proliferation/differentiation from neural stem cells in the CG and the migration of neural stem cells from the SVZ area. The astrocyte activation in the CG may, at least in part, contribute to the development of a chronic pain-like state following sciatic nerve ligation in mice.
Assuntos
Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Gliose/fisiopatologia , Giro do Cíngulo/fisiopatologia , Dor Intratável/fisiopatologia , Células-Tronco/fisiologia , Animais , Astrócitos/citologia , Biomarcadores , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Doença Crônica , Denervação , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/etiologia , Gliose/metabolismo , Giro do Cíngulo/citologia , Giro do Cíngulo/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Dor Intratável/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Neuropatia Ciática/complicações , Neuropatia Ciática/fisiopatologia , Transplante de Células-Tronco , Células-Tronco/citologiaRESUMO
In the previous study, we reported that exposure to bisphenol-A induced the potentiation of dopamine receptor functions in the mouse limbic area, resulting in supersensitivity to methamphetamine-induced pharmacological actions. The present study was undertaken to investigate whether prenatal exposure to bisphenol-A could produce morphological change in dopaminergic neuron and the pattern of expression of genes regulating the dopaminergic neuron development. Here we found that prenatal and neonatal exposures to bisphenol-A increased the tyrosine hydroxylase- and dopamine transporter-like immunoreactivities in the adult mouse limbic area. The present molecular biological study shows that chronic bisphenol-A treatment produced a significant decrease in the dopaminergic neuron development factors, sonic hedgehog and glial cell line-derived neurotrophic factor, which were also decreased by prenatal exposure to bisphenol-A. These results suggest that chronic exposure to bisphenol-A could disrupt the dopaminergic neurotransmission in the process of dopaminergic neuron development.
Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Proteínas Hedgehog/biossíntese , Fenóis/farmacologia , Animais , Compostos Benzidrílicos , Regulação para Baixo , Feminino , Masculino , Exposição Materna , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Receptores de Dopamina D3/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Recently, it has been reported that both thrombin-sensitive protease-activated receptor 1 (PAR-1) and platelet-derived growth factor (PDGF) are present not only in platelets, but also in the CNS, which indicates that they have various physiological functions. In this study, we evaluated whether PAR-1/PDGF in the spinal cord could contribute to the development of a neuropathic pain-like state in mice. Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were significantly suppressed by repeated intrathecal injection of hirudin, which is characterized as a specific and potent thrombin inhibitor. Furthermore, a single intrathecal injection of thrombin produced long-lasting hyperalgesia and allodynia, and these effects were also inhibited by hirudin in normal mice. In nerveligated mice, the increase in the binding of [35S]GTPgammaS to membranes of the spinal cord induced by thrombin and PAR-1-like immunoreactivity (IR) in the spinal cord were each greater than those in sham-operated mice. Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were also suppressed by repeated intrathecal injection of either the PDGF alpha receptor (PDGFRalpha)/Fc chimera protein or the PDGFR-dependent tyrosine kinase inhibitor AG17 [(3,5-di-tert-butyl-4-hydroxybenzylidene)-malononitrile]. Moreover, thermal hyperalgesia and tactile allodynia induced by thrombin in normal mice were virtually eliminated by intrathecal pretreatment with PDGFRalpha/Fc. In immunohistochemical studies, PAR-1-like IR-positive cells in the spinal dorsal horn were mostly colocated on PDGF-like IR-positive neuronal cells. These data provide novel evidence that PAR-1 and PDGF-A-mediated signaling pathway within spinal cord neurons may be directly implicated in neuropathic pain after nerve injury in mice.
Assuntos
Neuralgia/metabolismo , Neurônios/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor PAR-1/metabolismo , Neuropatia Ciática/metabolismo , Medula Espinal/citologia , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Lateralidade Funcional , Terapia com Hirudina/métodos , Hirudinas/farmacologia , Imuno-Histoquímica/métodos , Masculino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/farmacologia , Neuralgia/etiologia , Neurônios/efeitos dos fármacos , Medição da Dor/métodos , Estimulação Física , Fator de Crescimento Derivado de Plaquetas/uso terapêutico , Neuropatia Ciática/complicações , Neuropatia Ciática/tratamento farmacológico , Trombina/uso terapêutico , Fatores de TempoRESUMO
It is well known that prolonged exposure to morphine results in tolerance to morphine-induced antinociception. In the present study, we found that mice that were tolerant to morphine-induced antinociception exhibited an increase in immunoreactivity for the neural cell adhesion molecule in the dorsal horn of the spinal cord, which was highly overlapped with immunoreactivity for the increased metabotropic glutamate receptor 5 induced by morphine. These findings support the idea that repeated stimulation of mu-opioid receptors increases the expression of neural cell adhesion molecule and metabotropic glutamate receptor 5. This phenomenon leads to the enhanced excitatory synaptic transmission in the dorsal horn of the spinal cord, and in turn suppresses the morphine-induced antinociception.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Morfina/administração & dosagem , Entorpecentes/administração & dosagem , Moléculas de Adesão de Célula Nervosa/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Medula Espinal/citologia , Animais , Western Blotting/métodos , Relação Dose-Resposta a Droga , Esquema de Medicação , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/metabolismoRESUMO
According to the World Health Organization (WHO) guidelines for patients with moderate or severe pain, morphine has been used as a "gold standard" treatment for cancer pain. Recent clinical experiences have demonstrated that when morphine is used to control pain in cancer patients, psychological dependence is not a major concern. However, undue anxiety about psychological dependence on morphine in cancer patients has caused physicians and patients to use inadequate doses of opioids. In basic research, we reported that the morphine-induced rewarding effects can be dramatically suppressed under a neuropathic pain-like state induced by sciatic nerve ligation and an inflammatory pain-like state produced by intraplantar injection of formalin or carrageenan in rodents. The use of morphine for the treatment of pain is sometimes accompanied with side effects such as emesis, constipation and drowsiness. We show that the lower doses of morphine produce emesis, whereas antinociceptive doses of morphine show no emetic responses in ferrets. These findings provide further evidence that an adequate dose of morphine is useful and safe in a clinical setting.
Assuntos
Analgésicos Opioides/uso terapêutico , Morfina/uso terapêutico , Neoplasias/fisiopatologia , Dor/prevenção & controle , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Animais , Constipação Intestinal/induzido quimicamente , Relação Dose-Resposta a Droga , Fentanila/efeitos adversos , Fentanila/uso terapêutico , Humanos , Morfina/administração & dosagem , Morfina/efeitos adversos , Oxicodona/efeitos adversos , Oxicodona/uso terapêutico , Dor/tratamento farmacológico , Dor/psicologia , Medição da Dor , Vômito/induzido quimicamente , Organização Mundial da SaúdeRESUMO
UNLABELLED: : Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of â¼50-100 cells for passaging, cell counting is time-consuming. In the present study, using a time-lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase-contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time-lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase-contrast images. This method provided real-time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC-based therapy. SIGNIFICANCE: This is the first study to use a noninvasive method using images to systemically determine the growth of human pluripotent stem cells (hPSCs) without damaging or wasting cells. This method would be useful for quality control during cell culture of clinical hPSCs.