RESUMO
In view of postsynaptic density 95kDA (PSD95) tethers neuronal NO synthase (nNOS) to N-methyl-d-aspartate receptor (NMDAR), the PSD95-nNOS complex represents a therapeutic target of neuropathic pain. This study therefore sought to explore the ability of PCC-0105002, a novel PSD95-nNOS small molecule inhibitor, to alter pain sensitivity in rodent neuropathic pain models. Firstly, the IC50 of PCC-0105002 for PSD95 and NOS1 binding activity was determined using an Alpha Screen assay kit. Then, we examined the effects of PCC-0105002 in the mouse formalin test and in the rat spinal nerve ligation (SNL) model, and explored the ability of PCC-0105002 to mediate analgesia and to effect motor coordination in a rota-rod test. Moreover, the mechanisms whereby PCC-0105002 mediates analgesia was explored via western blotting, Golgi staining, and co-immunoprecipitation experiments in dorsal horn. The outcomes indicated that PCC-0105002 exhibited dose-dependent attenuation of phase II pain-associated behaviors in the formalin test. The result indicated that PCC-0105002 disrupted the PSD95-nNOS interaction with IC50 of 1.408 µM. In the SNL model, PCC-0105002 suppressed mechanical allodynia, thermal hyperalgesia, and abnormal dorsal horn wide dynamic range neuron discharge. PCC-0105002 mediated an analgesic effect comparable to that of MK-801, while it was better able to enhance motor coordination as compared with MK-801. Moreover, PCC-0105002 altered signaling downstream of NMDAR and thus functionally and structurally attenuating synaptic plasticity through respective regulation of the NR2B/GluR1/CaMKIIα and Rac1/RhoA pathways. These findings suggest that the novel PSD95-nNOS inhibitor PCC-0105002 is an effective agent for alleviating neuropathic pain, and that it produces fewer motor coordination-associated side effects than do NMDAR antagonists.
Assuntos
Aminobenzoatos/uso terapêutico , Analgésicos/farmacologia , Proteína 4 Homóloga a Disks-Large/metabolismo , Ésteres/uso terapêutico , Atividade Motora/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Óxido Nítrico Sintase Tipo I/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Nervos Espinhais/efeitos dos fármacos , Aminobenzoatos/farmacologia , Analgésicos/toxicidade , Animais , Modelos Animais de Doenças , Ésteres/farmacologia , Masculino , Camundongos , Neuralgia/enzimologia , Neuralgia/fisiopatologia , Plasticidade Neuronal/efeitos dos fármacos , Células do Corno Posterior/enzimologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos Sprague-Dawley , Teste de Desempenho do Rota-Rod , Transdução de Sinais , Nervos Espinhais/enzimologia , Nervos Espinhais/fisiopatologiaRESUMO
It is well known that remifentanil, a widely used intravenous anesthesia drug, can paradoxically induce hyperalgesia. The underlying mechanisms are still not clear despite the wide investigations. The present study demonstrated that withdrawal from spinal application of remifentanil could dose-dependently induce long term potentiation (LTP) of C-fiber evoked field potentials. Remifentanil withdrawal could activate Src family kinases (SFKs) in microglia, and upregulate the expression of tumor necrosis factor alpha (TNFα) in spinal dorsal horn. Furthermore, pretreatment with either microglia inhibitor Minocycline, SFKs inhibitor PP2 or TNF αneutralization antibody could block remifentanil withdrawal induced spinal LTP, whereas supplement of recombinant rat TNFα to the spinal cord could reverse the inhibitory effect of Minocycline or PP2 on remifentanil withdrawal induced LTP. Our results suggested that TNFαrelease following SFKs activation in microglia is involved in the induction of LTP induced by remifentanil withdrawal.
Assuntos
Potenciação de Longa Duração/fisiologia , Microglia/enzimologia , Fibras Nervosas Amielínicas/fisiologia , Piperidinas/administração & dosagem , Células do Corno Posterior/enzimologia , Quinases da Família src/metabolismo , Analgésicos Opioides/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Microglia/efeitos dos fármacos , Fibras Nervosas Amielínicas/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Remifentanil , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologiaRESUMO
Plasma membrane calcium ATPases (PMCAs) are ion pumps that expel Ca2+ from cells and maintain Ca2+ homeostasis. Four isoforms and multiple splice variants play important and non-overlapping roles in cellular function and integrity and have been implicated in diseases including disorders of the central nervous system (CNS). In particular, one of these isoforms, PMCA2, is critical for spinal cord (SC) neuronal function. PMCA2 expression is decreased in SC neurons at onset of symptoms in animal models of multiple sclerosis. Decreased PMCA2 expression affects the function and viability of SC neurons, with motor neurons being the most vulnerable population. Recent studies have also shown that PMCA2 could be an important contributor to pain processing in the dorsal horn (DH) of the SC. Pain sensitivity was altered in female, but not male, PMCA2+/- mice compared to PMCA2+/+ littermates in a modality-dependent manner. Changes in pain responsiveness in the female PMCA2+/- mice were paralleled by female-specific alterations in the expression of effectors, which have been implicated in the excitability of DH neurons, in mechanisms governing nociception and in the transmission of pain signals. Other PMCA isoforms and in particular, PMCA4, also contribute to the excitability of neurons in the dorsal root ganglia (DRG), which contain the first-order sensory neurons that convey nociceptive information from the periphery to the DH. These findings suggest that specific PMCA isoforms play specialized functions in neurons that mediate pain processing. Further investigations are necessary to unravel the precise contribution of PMCAs to mechanisms governing pathological pain in models of injury and disease.
Assuntos
Neurônios Motores/enzimologia , Doenças Neurodegenerativas/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Células do Corno Posterior/enzimologia , Medula Espinal/enzimologia , Animais , Humanos , Neurônios Motores/patologia , Doenças Neurodegenerativas/patologia , Percepção da Dor/fisiologia , Células do Corno Posterior/patologia , Medula Espinal/patologiaRESUMO
OBJECTIVE: To investigate whether analgesic effect of electroacupuncture (EA) is affected by p38 mitogen-activated protein kinase (p38 MAPK) on microglia. METHODS: There were two experiments. The experiment 1: 40 male Sprague-Dawley (SD) rats were randomly divided into the normal, surgery, EA and sham EA groups, and the L5 spinal nerve ligation (SNL) on the right side was used to establish neuropathic pain model. EA was applied to bilateral Zusanli (ST36) and Kunlun (BL60) at 24, 48 and 72 h after SNL for 30 min, once per day. The paw withdrawal thresholds (PWTs) were measured before surgery (as base) and at 24, 25, 49 and 73 h after surgery. Phospho-p38 MAPK (p-p38 MAPK), oxycocin-42 (OX-42, marker of microglia), and glial fibrillary acidic protein (GFAP, marker of astrocyte) in bilateral spinal cord dorsal horn (SCDH) were detected by immunofluorescence, respectively. The experiment 2: 40 male SD rats were cannulated for SNL-induced neuropathic pain, and then were randomly divided into the dimethyl sulfoxide (DMSO), EA plus DMSO, 4-(4-fluorophenyl)-2-(4-methylsulfonylpheny)-5-(4-pyridyl)-1H-imidazole (SB203580) and EA plus SB203580 groups. SB203580 (30 nmol/L) was administered 5 min prior to EA treatment. The PWTs and OX-42 in bilateral SCDH were measured as mentioned above. RESULTS: SNL-induced neuropathic pain reduced PWTs and increased the expression of p-p38 MAPK and OX-42 in bilateral lumbar SCDH of rats (P<0.01). Spinal p-p38 MAPK was only co-localized with OX-42 in our study. EA treatment significantly alleviated SNL-mediated mechanical hyperalgesia, and suppressed the expression of p-p38 MAPK and OX-42 in lumbar SCDH (P<0.05 or P<0.01). Intrathecal injection of low dose SB203580 had no influence on PWTs (P>0.05), but significantly inhibited the expression of OX-42 positive cells in bilateral SCDH (P<0.01 or P<0.05). EA plus SB203580 synergistically increased PWTs, and reduced the expression of bilateral spinal OX-42 (P<0.01 or P<0.05). CONCLUSIONS: The central mechanism of EA-induced anti-hyperalgesia may be partially associated with the reduced expression of p-p38 MAPK, and subsequently reducing the activation of OX-42 in neuropathic pain. Therefore, EA may be a new complementary and alternative therapy for neuropathic pain.
Assuntos
Eletroacupuntura , Microglia/enzimologia , Microglia/patologia , Nervos Espinhais/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Imunofluorescência , Hiperalgesia/patologia , Hiperalgesia/terapia , Imidazóis/farmacologia , Ligadura , Masculino , Microglia/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Fosforilação/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/enzimologia , Células do Corno Posterior/patologia , Piridinas/farmacologia , Ratos Sprague-Dawley , Nervos Espinhais/efeitos dos fármacosRESUMO
Tranexamic acid (TXA) is an antifibrinolytic agent widely used to reduce blood loss during surgery. However, a serious adverse effect of TXA is seizure due to inhibition of γ-aminobutyric acid (GABA) and glycine receptors in cortical neurons. These receptors are also present in the spinal cord, and antagonism of these receptors in spinal dorsal horn neurons produces pain-related phenomena, such as allodynia and hyperalgesia, in experimental animals. Moreover, some patients who are injected intrathecally with TXA develop severe back pain. However, the effect of TXA on spinal dorsal horn neurons remain poorly understood. Here, we investigated the effects of TXA by using behavioral measures in rats and found that TXA produces behaviors indicative of spontaneous pain and mechanical allodynia. We then performed whole-cell patch-clamp experiments that showed that TXA inhibits GABAA and glycine receptors in spinal dorsal horn neurons. Finally, we also showed that TXA facilitates activation of the extracellular signal-regulated kinase in the spinal cord. These results indicated that TXA produces pain by inhibiting GABAA and glycine receptors in the spinal dorsal horn.
Assuntos
Dor/induzido quimicamente , Dor/fisiopatologia , Células do Corno Posterior/patologia , Ácido Tranexâmico/efeitos adversos , Animais , Comportamento Animal , Biomarcadores/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/patologia , Glicina/farmacologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Potenciais Pós-Sinápticos Inibidores , Injeções Intraperitoneais , Injeções Espinhais , Masculino , Modelos Biológicos , Nociceptividade/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/enzimologia , Ratos Wistar , Ácido Tranexâmico/administração & dosagem , Ácido gama-Aminobutírico/farmacologiaRESUMO
AIMS: To explore the possible relationship between protein kinase C gamma (PKCγ) and phosphorylated forms of extracellular signal-regulated kinases 1/2 (pERK1/2) in the rat medullary dorsal horn and the facial hypersensitivity indicative of dynamic mechanical allodynia (DMA) following chronic constriction of the infraorbital nerve (CCI-IoN). METHODS: A well-established rat model of trigeminal neuropathic pain involving CCI-IoN was used. Facial mechanical hypersensitivity was tested with non-noxious dynamic mechanical stimulation (air-puff), and the medullary dorsal horn was examined immunohistochemically using PKCγ and pERK1/2 as pain markers. Statistical analysis was performed using Student t test or one-way analysis of variance (ANOVA). RESULTS: Increased PKCγ and pERK1/2 expressions within the medullary dorsal horn were associated with DMA following CCI-IoN. A segmental network composed of PKCγ-positive cells located in medullary dorsal horn laminae II/III, contacting more superficially located pERK1/2-expressing cells, was identified. Ultrastructural analysis confirmed the presence of PKCγ to pERK1/2-positive cells. Moreover, intracisternal administration of the selective PKCγ inhibitor KIG31-I blocked both the DMA and pERK1/2 expression in a dose-dependent manner. Although the number of pERK1/2-positive cells was significantly elevated with air-puff stimulation, DMA rats not receiving air-puff stimulation showed significant pERK1/2 expression, suggesting they were experiencing spontaneous pain. CONCLUSION: PKCγ cells in the medullary dorsal horn may be involved in DMA following CCI-IoN through the activation of pERK1/2-expressing cells, which then may relay non-nociceptive information to lamina I cells in the medullary dorsal horn.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Nociceptividade/fisiologia , Células do Corno Posterior/enzimologia , Proteína Quinase C/fisiologia , Tato/fisiologia , Neuralgia do Trigêmeo/fisiopatologia , Animais , Biomarcadores/análise , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/análise , Dor Facial/enzimologia , Dor Facial/fisiopatologia , Hiperalgesia/enzimologia , Hiperalgesia/fisiopatologia , Imuno-Histoquímica , Isoenzimas/análise , Isoenzimas/antagonistas & inibidores , Isoenzimas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Vias Neurais/enzimologia , Vias Neurais/fisiopatologia , Vias Neurais/ultraestrutura , Órbita/inervação , Fosforilação , Células do Corno Posterior/fisiologia , Células do Corno Posterior/ultraestrutura , Proteína Quinase C/análise , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Neuralgia do Trigêmeo/enzimologia , Neuralgia do Trigêmeo/patologiaRESUMO
The aim of this research was to reveal the changes in the NADPH-d reactivity in the lumbal spinal cord (L6/L7) of cats with unilateral acute myositis of the mm. gastrocnemius-soleus after intramuscular injections of carrageenan. The effect of unilateral muscle inflammation was expressed in a significant increase in the number of NADPH-d-reactive neurons in ipsilateral and contralateral intermediate (lamina VII; 17.62 ± 2.7 and 20.67 ± 13.3) and medial (lamina VIII; 7.3 ± 1.9 and 6.0 ± 2.1 respectively) zones of the ventral horns. However, a clear decline of the reactive cells was recorded on the ipsilateral side within the area around the central canal (lamina X). An increase in the NADPH-d reactivity within the ventral horns on both sides on the spinal cord and the induction of such reactivity (contralaterally) in large multipolar neurons localized in the dorsal part of the intermediate zone were revealed in cats with unilateral acute muscle inflammation. It is hypothesized, that during acute myositis, plastic changes in different layers of the dorsal and ventral horns activate the processes of disinhibition due to an increase in the number of NOS-containing/NADPH-d-reactive neurons in the spinal gray matter.
Assuntos
Células do Corno Anterior/enzimologia , Substância Cinzenta/enzimologia , Músculo Esquelético/enzimologia , Miosite/enzimologia , NADPH Desidrogenase/metabolismo , Células do Corno Posterior/enzimologia , Animais , Células do Corno Anterior/efeitos dos fármacos , Células do Corno Anterior/patologia , Carragenina , Gatos , Expressão Gênica , Substância Cinzenta/efeitos dos fármacos , Substância Cinzenta/patologia , Substância Cinzenta/fisiopatologia , Histocitoquímica , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Miosite/induzido quimicamente , Miosite/patologia , Miosite/fisiopatologia , NADPH Desidrogenase/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/patologiaRESUMO
Inhibitory interneurons are an important component of dorsal horn circuitry where they serve to modulate spinal nociception. There is now considerable evidence indicating that reduced inhibition in the spinal dorsal horn contributes to neuropathic pain. A loss of these inhibitory neurons after nerve injury is one of the mechanisms being proposed to account for reduced inhibition; however, this remains controversial. This is in part because previous studies have focused on global measurements of inhibitory neurons without assessing the number of inhibitory synapses. To address this, we conducted a quantitative analysis of the spatial and temporal changes in the number of inhibitory terminals, as detected by glutamic acid decarboxylase 65 (GAD65) immunoreactivity, in the superficial dorsal horn of the spinal cord following a chronic constriction injury (CCI) to the sciatic nerve in rats. Isolectin B4 (IB4) labelling was used to define the location within the dorsal horn directly affected by the injury to the peripheral nerve. The density of GAD65 inhibitory terminals was reduced in lamina I (LI) and lamina II (LII) of the spinal cord after injury. The loss of GAD65 terminals was greatest in LII with the highest drop occurring around 3-4 weeks and a partial recovery by 56 days. The time course of changes in the number of GAD65 terminals correlated well with both the loss of IB4 labeling and with the altered thresholds to mechanical and thermal stimuli. Our detailed analysis of GAD65+ inhibitory terminals clearly revealed that nerve injury induced a transient loss of GAD65 immunoreactive terminals and suggests a potential involvement for these alterations in the development and amelioration of pain behaviour.
Assuntos
Glutamato Descarboxilase/metabolismo , Inibição Neural/fisiologia , Células do Corno Posterior/enzimologia , Neuropatia Ciática/patologia , Corno Dorsal da Medula Espinal/patologia , Análise de Variância , Animais , Modelos Animais de Doenças , Lateralidade Funcional/fisiologia , Hiperalgesia/etiologia , Lectinas/metabolismo , Masculino , Ratos , Ratos Wistar , Neuropatia Ciática/complicações , Fatores de TempoRESUMO
The activation of calcium/calmodulin-dependent protein kinase II (CaMKII) has been proposed as a key factor in chronic pain development. This study therefore aimed to investigate the expression of CaMKII in the dorsal horn in a rat model of early phase diabetes mellitus (DM) types 1 and 2. Sprague-Dawley rats were used. DM1 was induced using streptozotocin (STZ) (55mg/kg injected intraperitoneally (i.p.)). DM2 was induced using a combination of a high fat diet (HFD) and STZ (35mg/kg i.p.). Controls received an i.p. injection of pure citrate buffer solution. DM2 animals and their controls also received HFD 2 weeks prior to the i.p. injection. Rats were sacrificed 2 weeks and 2 months after diabetes induction. The expression of tCaMKII, pCaMKIIα and IB4 in the dorsal horns was quantified using immunohistochemistry. Increased expression of tCaMKII and pCaMKIIα was seen in the dorsal horns of DM1 animals 2 weeks and 2 months after diabetes induction. In DM2 animals, similar changes in the expression of tCaMKII and pCaMKIIα were observed after 2 weeks, but not after 2 months. The expression of pCaMKIIα was most pronounced in laminae I-III. No difference in IB4 expression was observed between the groups. These results suggest a potential role for CaMKII in diabetic neuropathy development. Inhibition of CaMKII signaling pathways should be further explored as a potential treatment target in painful diabetic neuropathy.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/biossíntese , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Células do Corno Posterior/enzimologia , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/enzimologia , Dieta Hiperlipídica , Regulação Enzimológica da Expressão Gênica/genética , Masculino , Vias Neurais/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To observe the effect of different intensities of manual acupuncture (MA) stimulation on mechanical pain thresholds (PTs) and the expression of phosphorylated extracellular signal-regulated kinases (p-ERK) in lumbar spinal dorsal horn regions in rats with neuropathic mirror-image pain, so as to explore its mechanisms underlying analgesia. METHODS: Forty male SD rats were equally and randomly divided into control, spinal nerve ligation (SNL) model, mild MA-stimulation, and strong MA-stimulation groups. Neuropathological pain model was established by ligature of the spinal nerve (L 5). Three days after the SNL, bilateral "Huantiao" (GB 30) were stimulated by rotating the thin (0.22 mm x 13 mm) or thick (0.3 mm x 13 mm) filiform needles at a frequencies of 60 times/min or 180 times/min and at an angle of 180 degrees or 360 degrees for 2 min for rats in the mild and strong MA-stimulation groups, respectively, followed by remaining the needle in place for 30 min. The mechanical PTs were measured before and after SNL. The expression of p-ERK protein in bilateral dorsal horn regions of the lumbar spinal cord (L4- L 6) was detected by Western blot. RESULTS: In comparison with the control group, the mechanical PTs were significantly decreased beginning from the 3rd day on after SNL on the affected side and from the 7th day on after SNL on the healthy hindpaw (P < 0.05), simultaneously, p-ERK protein expression levels of dorsal horn regions on both sides of the spinal cord were considerably up-regulated on the 12th day (P < 0.05). Compared with the model group, the PTs of the affected hindpaw and the healthy hindpaw were significantly increased on the 7th and 12th day in the strong MA-stimulation group (P < 0.05, P < 0.01), whereas pERK expression levels in the bilateral spinal dorsal horn regions were obviously down-regulated in the strong MA-stimulation group (P < 0.05). No significant differences were found between the model and mild MA-stimulation groups in the PTs of bilateral hindpaws and p-ERK expression levels of the bilateral spinal dorsal horn regions (P > 0.05) except the PTs of the healthy hindpaw on 7th day (P < 0.05). CONCLUSION: Strong MA-stimulation can alleviate neuropathic mirror-image pain in SNL rats, which is closely related to its effect in down-regulating the expression of p-ERK in the bilateral spinal dorsal horn regions.
Assuntos
Pontos de Acupuntura , Terapia por Acupuntura/métodos , Dor nas Costas/terapia , MAP Quinases Reguladas por Sinal Extracelular/genética , Neuralgia/terapia , Células do Corno Posterior/enzimologia , Terapia por Acupuntura/instrumentação , Animais , Dor nas Costas/enzimologia , Dor nas Costas/genética , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Masculino , Neuralgia/enzimologia , Neuralgia/genética , Limiar da Dor , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Phosphodiesterase 2A (PDE2A) is an evolutionarily conserved enzyme that catalyzes the degradation of the cyclic nucleotides, cyclic adenosine monophosphate, and/or cyclic guanosine monophosphate. Recent studies reported the expression of PDE2A in the dorsal horn of the spinal cord, pointing to a potential contribution to the processing of pain. However, the functions of PDE2A in spinal pain processing in vivo remained elusive. METHODS: Immunohistochemistry, laser microdissection, and quantitative real-time reverse transcription polymerase chain reaction experiments were performed to characterize the localization and regulation of PDE2A protein and messenger RNA in the mouse spinal cord. Effects of the selective PDE2A inhibitor, BAY 60-7550 (Cayman Chemical, Ann Arbor, MI), in animal models of inflammatory pain (n = 6 to 10), neuropathic pain (n = 5 to 6), and after intrathecal injection of cyclic nucleotides (n = 6 to 8) were examined. Also, cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in spinal cord tissues were measured by liquid chromatography tandem mass spectrometry. RESULTS: The authors here demonstrate that PDE2A is distinctly expressed in neurons of the superficial dorsal horn of the spinal cord, and that its spinal expression is upregulated in response to hind paw inflammation. Administration of the selective PDE2A inhibitor, BAY 60-7550, increased the nociceptive behavior of mice in animal models of inflammatory pain. Moreover, BAY 60-7550 increased the pain hypersensitivity induced by intrathecal delivery of cyclic adenosine monophosphate, but not of cyclic guanosine monophosphate, and it increased the cyclic adenosine monophosphate levels in spinal cord tissues. CONCLUSION: Our findings indicate that PDE2A contributes to the processing of inflammatory pain in the spinal cord.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/fisiologia , Inflamação/enzimologia , Inflamação/fisiopatologia , Dor/enzimologia , Dor/fisiopatologia , Medula Espinal/enzimologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/biossíntese , Hipersensibilidade a Drogas/fisiopatologia , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Imuno-Histoquímica , Inflamação/complicações , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microdissecção , Neuralgia/enzimologia , Neuralgia/fisiopatologia , Neuralgia/psicologia , Dor/etiologia , Medição da Dor , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Fosfodiesterase/farmacologia , Células do Corno Posterior/enzimologia , Reação em Cadeia da Polimerase em Tempo Real , Triazinas/administração & dosagem , Triazinas/farmacologia , Regulação para Cima/genética , Regulação para Cima/fisiologia , ZimosanRESUMO
BACKGROUND: Itch, chronic itch in particular, can have a significant negative impact on an individual's quality of life. However, the molecular mechanisms underlying itch processing in the central nervous system remain largely unknown. RESULTS: We report here that activation of ERK signaling in the spinal cord is required for itch sensation. ERK activation, as revealed by anti-phosphorylated ERK1/2 immunostaining, is observed in the spinal dorsal horn of mice treated with intradermal injections of histamine and compound 48/80 but not chloroquine or SLIGRL-NH2, indicating that ERK activation only occurs in histamine-dependent acute itch. In addition, ERK activation is also observed in 2, 4-dinitrofluorobenzene (DNFB)-induced itch. Consistently, intrathecal administration of the ERK phosphorylation inhibitor U0126 dramatically reduces the scratching behaviors induced by histamine and DNFB, but not by chloroquine. Furthermore, administration of the histamine receptor H1 antagonist chlorpheniramine decreases the scratching behaviors and ERK activation induced by histamine, but has no effect on DNFB-induced itch responses. Finally, the patch-clamp recording shows that in histamine-, chloroquine- and DNFB-treated mice the spontaneous excitatory postsynaptic current (sEPSC) of dorsal horn neurons is increased, and the decrease of action potential threshold is largely prevented by bathing of U0126 in histamine- and DNFB-treated mice but not those treated with chloroquine. CONCLUSION: Our results demonstrate a critical role for ERK activation in itch sensation at the spinal level.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Prurido/enzimologia , Prurido/patologia , Sensação , Medula Espinal/enzimologia , Medula Espinal/patologia , Animais , Butadienos/farmacologia , Cloroquina , Dinitrofluorbenzeno , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Histamina , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neuroglia/efeitos dos fármacos , Neuroglia/enzimologia , Nitrilas/farmacologia , Oligopeptídeos , Fosforilação/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/enzimologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Prurido/fisiopatologia , Receptores Histamínicos H1/metabolismo , Sensação/efeitos dos fármacos , Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (IIi ) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina IIi PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A- and C-fibers low threshold mechanoreceptors (LTMRs) terminate in lamina IIi raise the general issue of synaptic inputs to lamina IIi PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ-immunoreactivity is mostly restricted to interneurons in lamina IIi of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ-immunoreactive interneurons are asymmetric (likely excitatory). PKCγ-immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3-immunoreactive bouton, specific to C-LTMRs, on PKCγ-immunoreactive interneurons. PKCγ-immunoreactive interneurons contain GABAA ergic and glycinergic receptors. At the subcellular level, PKCγ-immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ-immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia.
Assuntos
Hiperalgesia/metabolismo , Interneurônios/enzimologia , Mecanorreceptores/enzimologia , Proteína Quinase C/metabolismo , Sinapses/ultraestrutura , Animais , Western Blotting , Imuno-Histoquímica , Interneurônios/ultraestrutura , Masculino , Mecanorreceptores/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/ultraestrutura , Nociceptores/enzimologia , Nociceptores/ultraestrutura , Células do Corno Posterior/enzimologia , Células do Corno Posterior/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapses/enzimologiaRESUMO
The aim of this study was to investigate the expression of total calcium/calmodulin-dependent protein kinase II (CaMKII) and its phosphorylated α isoform in the dorsal horn of the spinal cord in an animal model of long-term diabetes. Diabetes was induced in Sprague-Dawley rats using 55 mg/kg streptozotocin, and expression of total CaMKII, the phosphorylated α-CaMKII isoform, and isolectin B4 was analyzed by immunohistochemical analysis in the dorsal horn of the spinal cord 6 and 12 months after diabetes induction. Results were compared with those for control rats of the same age. Increased expression of total CaMKII and its activated α isoform was seen in the dorsal horn of diabetic rats 6 months after diabetes induction. The increase in CaMKII fluorescence was restored to control values after 12 months. The expression of activated α-CaMKII 12 months after diabetes induction was most pronounced in laminae I-VI of the dorsal horn, not corresponding with the highest expression of isolectin B4 in laminae I-III. Increased expression of CaMKII in the dorsal horn during long-term diabetes could be involved in the development of neuropathic symptoms in diabetes. The expression pattern of CaMKII during long-term diabetes indicates that it affects the entire sensory input.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Diabetes Mellitus Experimental/enzimologia , Células do Corno Posterior/enzimologia , Animais , Ratos , Ratos Sprague-DawleyRESUMO
Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered an important enzyme contributing to the pathogenesis of persistent pain. The aim of this study was to test whether intrathecal injection of CaMKII inhibitors may reduce pain-related behavior in diabetic rats. Male Sprague-Dawley rats were used. Diabetes was induced with intraperitoneal injection of 55mg/kg streptozotocin. Two weeks after diabetes induction, CaMKII inhibitor myristoil-AIP or KN-93 was injected intrathecally. Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2h and 24h after the intrathecal injection. The expression of total CaMKII and its alpha isoform in dorsal horn was quantified using immunohistochemistry. Intrathecal injection of mAIP and KN-93 resulted in significant decrease in expression of total CaMKII and CaMKII alpha isoform activity. Also, mAIP and KN93 injection significantly increased sensitivity to a mechanical stimulus 24h after i.t. injection. Intrathecal inhibition of CaMKII reduced the expression of total CaMKII and its CaMKII alpha isoform activity in diabetic dorsal horn, which was accompanied with an increase in pain-related behavior. Further studies about the intrathecal inhibition of CaMKII should elucidate its role in nociceptive processes of diabetic neuropathy.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas/fisiopatologia , Dor/fisiopatologia , Animais , Benzilaminas/administração & dosagem , Benzilaminas/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/enzimologia , Neuropatias Diabéticas/enzimologia , Neuropatias Diabéticas/etiologia , Injeções Espinhais , Masculino , Dor/enzimologia , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Estimulação Física , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/enzimologia , Ratos , Ratos Sprague-Dawley , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , TemperaturaRESUMO
Paclitaxel (taxol) is a first-line chemotherapy-drug used to treat many types of cancers. Neuropathic pain and sensory dysfunction are the major toxicities, which are dose-limiting and significantly reduce the quality of life in patients. Two known critical spinal mechanisms underlying taxol-induced neuropathic pain are an increased production of pro-inflammatory cytokines including interleukin-1ß (IL-1ß) and suppressed glial glutamate transporter activities. In this study, we uncovered that increased activation of glycogen synthase kinase 3beta (GSK3ß) in the spinal dorsal horn was concurrently associated with increased protein expressions of GFAP, IL-1ß and a decreased protein expression of glial glutamate transporter 1 (GLT-1), as well as the development and maintenance of taxol-induced neuropathic pain. The enhanced GSK3ß activities were supported by the concurrently decreased AKT and mTOR activities. The changes of all these biomarkers were basically prevented when animals received pre-emptive lithium (a GSK3ß inhibitor) treatment, which also prevented the development of taxol-induced neuropathic pain. Further, chronic lithium treatment, which began on day 11 after the first taxol injection, reversed the existing mechanical and thermal allodynia induced by taxol. The taxol-induced increased GSK3ß activities and decreased AKT and mTOR activities in the spinal dorsal horn were also reversed by lithium. Meanwhile, protein expressions of GLT-1, GFAP and IL-1ß in the spinal dorsal horn were improved. Hence, suppression of spinal GSK3ß activities is a key mechanism used by lithium to reduce taxol-induced neuropathic pain, and targeting spinal GSK3ß is an effective approach to ameliorate GLT-1 expression and suppress the activation of astrocytes and IL-1ß over-production in the spinal dorsal horn.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Lítio/farmacologia , Neuralgia/enzimologia , Neuralgia/prevenção & controle , Paclitaxel/toxicidade , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Glicogênio Sintase Quinase 3 beta , Lítio/uso terapêutico , Masculino , Neuralgia/induzido quimicamente , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
Nerve injury and inflammation can both induce neuropathic pain via the production of pro-inflammatory cytokines. In the process, G protein-coupled receptors (GPCRs) were involved in pain signal transduction. GPCR kinase (GRK) 6 is a member of the GRK family that regulates agonist-induced desensitization and signaling of GPCRs. However, its expression and function in neuropathic pain have not been reported. In this study, we performed a chronic constriction injury (CCI) model in adult male rats and investigated the dynamic change of GRK6 expression in spinal cord. GRK6 was predominantly expressed in the superficial layers of the lumbar spinal cord dorsal horn neurons and its expression was decreased bilaterally following induction of CCI. The changes of GRK6 were mainly in IB4 and P substrate positive areas in spinal cord dorsal horn. And over-expression of GRK6 in spinal cord by lentivirus intrathecal injection attenuated the pain response induced by CCI. In addition, the level of TNF-α underwent the negative pattern of GRK6 in spinal cord. And neutralized TNF-α by antibody intrathecal injection up-regulated GRK6 expression and attenuated the mechanical allodynia and heat hyperalgesia in CCI model. All the data indicated that down-regulation of neuronal GRK6 expression induced by cytokine may be a potential mechanism that contributes to increasing neuronal signaling in neuropathic pain.
Assuntos
Quinases de Receptores Acoplados a Proteína G/fisiologia , Neuralgia/enzimologia , Traumatismos da Medula Espinal/enzimologia , Animais , Constrição , Quinases de Receptores Acoplados a Proteína G/biossíntese , Hiperalgesia/fisiopatologia , Masculino , Neuralgia/fisiopatologia , Células do Corno Posterior/enzimologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
OBJECTIVE: To investigate the effects of chronic morphine administration on pain behaviors in young rats and to explore the mechanisms involved. METHODS: Sixteen SD young rats of 3-4 weeks were randomly divided into control and morphine administration groups. Young rats received saline (1 mL/kg) or morphine (10 mg/kg) subcutaneously. Each regimen was given once daily for 14 days. Pain behaviors were examined on day 1, 3, 5, 7, and 14 before the daily drug administration. Western blot was used to examine the expression of glutamate decarboxylase 65 (GAD65) in the spinal cord dorsal horn on day 14 after the last drug administration. RESULTS: Following 14 days of morphine administration, mechanical hyperalgesia was induced in young rats. Compared with control group, the mechanical withdrawal threshold of morphine group significantly decreased on days 3, 5,7 and 14. Chronic administration of morphine downregulated the expression of GAD65 in the spinal cord dorsal horn of young rats. CONCLUSION: Chronic morphine administration could induce mechanical hyperalgesia in young rats, and the downregulation of GAD65 in the spinal cord dorsal horn might play a critical role in the molecular mechanisms of morphine-induced hyperalgesia.
Assuntos
Glutamato Descarboxilase/metabolismo , Hiperalgesia/induzido quimicamente , Morfina/administração & dosagem , Morfina/efeitos adversos , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Animais , Regulação para Baixo , Masculino , Células do Corno Posterior/enzimologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/enzimologia , Fatores de TempoRESUMO
Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.
Assuntos
Amidoidrolases/antagonistas & inibidores , Analgesia , Ácidos Araquidônicos/metabolismo , Benzamidas/farmacologia , Carbamatos/farmacologia , Endocanabinoides/metabolismo , Neuralgia/tratamento farmacológico , Alcamidas Poli-Insaturadas/metabolismo , Células do Corno Posterior/enzimologia , Canais de Cátion TRPV/metabolismo , Amidas , Animais , Araquidonato 15-Lipoxigenase/metabolismo , Benzamidas/administração & dosagem , Sinalização do Cálcio , Carbamatos/administração & dosagem , Diterpenos/farmacologia , Etanolaminas/metabolismo , Flavanonas/farmacologia , Glicerídeos/metabolismo , Células HEK293 , Humanos , Hiperalgesia/tratamento farmacológico , Injeções Espinhais , Inibidores de Lipoxigenase/farmacologia , Masculino , Ácidos Oleicos , Ácidos Palmíticos/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Ratos , Ratos Wistar , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologia , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
It has been reported that the expression of GTP cyclohydrolase I (GCH1) and neuronal apoptosis in dorsal root ganglion (DRG) is related to the generation of neuropathic pain. In this study, we hypothesize that GCH1 protein and neuronal apoptosis in rat spinal dorsal horn may also increase after chronic sciatic nerve injury. To establish the neuropathic pain model, we slightly ligated the right sciatic nerve of experimental rats. Mechanical allodynia was observed in CCI group on the 3rd day postoperatively determined by the Von Frey test, and it lasted until the 14th day after operation. No matter which method we used, western blotting or immunohistochemistry analysis, they all showed the enhancement of GCH1 protein in spinal dorsal horn on the 3rd, 7th, and 14th days after operation due to the sciatic nerve injury (P < 0.05). The apoptosis of nerve cells also increased evidently compared with sham group detected by TUNEL staining (P < 0.05). Therefore, the data suggested that along with mechanical allodynia caused by peripheral nerve injury, both GCH1 level and apoptosis index of nerve cells in spinal dorsal horn was elevated, which might also contribute to the generation of neuropathic pain.