RESUMO
ABSTRACT: Secreted microRNAs (miRNAs) have been detected in various body fluids including the cerebrospinal fluid, yet their direct role in regulating synaptic transmission remains uncertain. We found that intrathecal injection of low dose of let-7b (1 µg) induced short-term (<24 hours) mechanical allodynia and heat hyperalgesia, a response that is compromised in Tlr7-/- or Trpa1-/- mice. Ex vivo and in vivo calcium imaging in GCaMP6-report mice revealed increased calcium signal in spinal cord afferent terminals and doral root ganglion/dorsal root ganglia neurons following spinal perfusion and intraplantar injection of let-7b. Patch-clamp recordings also demonstrated enhanced excitatory synaptic transmission (miniature excitatory postsynaptic currents [EPSCs]) in spinal nociceptive neurons following let-7b perfusion or optogenetic activation of axonal terminals. The elevation in spinal calcium signaling and EPSCs was dependent on the presence of toll-like receptor-7 (TLR7) and transient receptor potential ion channel subtype A1 (TRPA1). In addition, endogenous let-7b is enriched in spinal cord synaptosome, and peripheral inflammation increased let-7b in doral root ganglion/dorsal root ganglia neurons, spinal cord tissue, and the cerebrospinal fluid. Notably, let-7b antagomir inhibited inflammatory pain and inflammation-induced synaptic plasticity (EPSC increase), suggesting an endogenous role of let-7b in regulating pain and synaptic transmission. Furthermore, intrathecal injection of let-7b, at a higher dose (10 µg), induced persistent mechanical allodynia for >2 weeks, which was abolished in Tlr7-/- mice. The high dose of let-7b also induced microgliosis in the spinal cord. Of interest, intrathecal minocycline only inhibited let-7b-induced mechanical allodynia in male but not female mice. Our findings indicate that the secreted microRNA let-7b has the capacity to provoke pain through both neuronal and glial signaling, thereby establishing miRNA as an emerging neuromodulator.
Assuntos
MicroRNAs , Microglia , Medula Espinal , Transmissão Sináptica , Animais , Masculino , Camundongos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Gânglios Espinais/metabolismo , Hiperalgesia/fisiopatologia , Hiperalgesia/metabolismo , Glicoproteínas de Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Nociceptores/metabolismo , Nociceptores/fisiologia , Transdução de Sinais/fisiologia , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Transmissão Sináptica/fisiologia , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/genética , Canal de Cátion TRPA1/metabolismo , Canal de Cátion TRPA1/genéticaRESUMO
Inhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride concentration in neurons, maintained by KCC2, a neuroprotective ion transporter that extrudes intracellular neuronal chloride. To identify Kcc2 gene expressionenhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain-like behavior in mouse models of nerve injury and bone cancer. In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via KAISO transcription factor. Transient spinal over-expression of delta-catenin mimicked KP analgesia. Our findings of a newly repurposed compound and a novel, genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming.
Assuntos
Analgésicos/uso terapêutico , Benzazepinas/uso terapêutico , Reposicionamento de Medicamentos , Indóis/uso terapêutico , Transmissão Sináptica/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Analgésicos/farmacologia , Animais , Benzazepinas/farmacologia , Dor do Câncer/tratamento farmacológico , Cateninas/genética , Cateninas/metabolismo , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Humanos , Indóis/farmacologia , Camundongos , Neuralgia/tratamento farmacológico , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Corno Dorsal da Medula Espinal/efeitos dos fármacos , Corno Dorsal da Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/patologia , Simportadores/genética , Simportadores/metabolismo , Fatores de Transcrição/metabolismo , Ácido gama-Aminobutírico/metabolismo , delta CateninaRESUMO
Mechanisms of pain resolution are largely unclear. Increasing evidence suggests that specialized pro-resolving mediators (SPMs), derived from fish oil docosahexaenoic acid (DHA), promote the resolution of acute inflammation and potently inhibit inflammatory and neuropathic pain. In this study, we examined the analgesic impact of DHA and DHA-derived SPMs in a mouse model of post-operative pain induced by tibial bone fracture (fPOP). Intravenous perioperative treatment with DHA (500 µg), resolvin D1 (RvD1, 500 ng) and maresin 1 (MaR1, 500 ng), 10 min and 24 h after the surgery, delayed the development of fPOP (mechanical allodynia and cold allodynia). In contrast, post-operative intrathecal (IT) administration of DHA (500 µg) 2 weeks after the surgery had no effects on established mechanical and cold allodynia. However, by direct comparison, IT post-operative treatment (500 ng) with neuroprotectin D1 (NPD1), MaR1, and D-resolvins, RvD1 and RvD5, but not RvD3 and RvD4, effectively reduced mechanical and cold allodynia. ELISA analysis showed that perioperative DHA treatment increased RvD1 levels in serum and spinal cord samples after bone fracture. Interestingly, sham surgery resulted in transient allodynia and increased RvD1 levels, suggesting a correlation of enhanced SPM levels with acute pain resolution after sham surgery. Our findings suggest that (1) perioperative treatment with DHA is effective in preventing and delaying the development of fPOP and (2) post-treatment with some SPMs can attenuate established fPOP. Our data also indicate that orthopedic surgery impairs SPM production. Thus, DHA and DHA-derived SPMs should be differentially supplemented for treating fPOP and improving recovery.
RESUMO
Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.
Assuntos
Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Tálamo/metabolismo , Animais , Células COS , Chlorocebus aethiops , Dominância Ocular , Humanos , Camundongos , Camundongos Knockout , Doenças do Sistema Nervoso/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Sinapses/metabolismoRESUMO
Accumulating evidence suggests that activation of spinal microglia contributes to the development of inflammatory and neuropathic pain. However, the role of spinal microglia in the maintenance of chronic pain remains controversial. Bone cancer pain shares features of inflammatory and neuropathic pain, but the temporal activation of microglia and astrocytes in this model is not well defined. Here, we report an unconventional role of spinal microglia in the maintenance of advanced-phase bone cancer pain in a female rat model. Bone cancer elicited delayed and persistent microglial activation in the spinal dorsal horn on days 14 and 21, but not on day 7. In contrast, bone cancer induced rapid and persistent astrocytic activation on days 7-21. Spinal inhibition of microglia by minocycline at 14 d effectively reduced bone cancer-induced allodynia and hyperalgesia. However, pretreatment of minocycline in the first week did not affect the development of cancer pain. Bone cancer increased ATP levels in CSF, and upregulated P2X7 receptor, phosphorylated p38, and IL-18 in spinal microglia. Spinal inhibition of P2X7/p-38/IL-18 pathway reduced advanced-phase bone cancer pain and suppressed hyperactivity of spinal wide dynamic range (WDR) neurons. IL-18 induced allodynia and hyperalgesia after intrathecal injection, elicited mechanical hyperactivity of WDR neurons in vivo, and increased the frequency of mEPSCs in spinal lamina IIo nociceptive synapses in spinal cord slices. Together, our findings demonstrate a novel role of microglia in maintaining advanced phase cancer pain in females via producing the proinflammatory cytokine IL-18 to enhance synaptic transmission of spinal cord nociceptive neurons.
Assuntos
Interleucina-18/metabolismo , Microglia/metabolismo , Neuralgia/fisiopatologia , Células do Corno Posterior/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/líquido cefalorraquidiano , Animais , Neoplasias Ósseas/complicações , Potenciais Pós-Sinápticos Excitadores , Feminino , Interleucina-18/genética , Microglia/fisiologia , Potenciais Pós-Sinápticos em Miniatura , Minociclina/farmacologia , Minociclina/uso terapêutico , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Neuralgia/metabolismo , Células do Corno Posterior/fisiologia , Ratos , Ratos Wistar , Receptores Purinérgicos P2X7/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Inflammatory pain such as arthritic pain is typically treated with opioids and cyclo-oxygenase-2 inhibitors with well known side effects. Transient receptor potential subtype vanilloid 1 (TRPV1) and TRP ankyryn 1 (TRPA1) contribute importantly to the genesis of inflammatory pain via both peripheral mechanisms (peripheral sensitization) and spinal cord mechanisms (central sensitization). Although these TRP channels have been intensively studied, little is known about their endogenous inhibitors. Recent studies have demonstrated that the endogenous lipid mediators resolvins (RvE1 and RvD1), derived from ω-3 unsaturated fatty acids, are potent inhibitors for inflammatory pain, without noticeable side effects. However, the molecular mechanisms underlying resolvins' distinct analgesic actions in mice are unclear. RvD2 is a novel family member of resolvins. Here we report that RvD2 is a remarkably potent inhibitor of TRPV1 (IC(50) = 0.1 nm) and TRPA1 (IC(50) = 2 nm) in primary sensory neurons, whereas RvE1 and RvD1 selectively inhibited TRPV1 (IC(50) = 1 nm) and TRPA1 (IC(50) = 9 nm), respectively. Accordingly, RvD2, RvE1, and RvD1 differentially regulated TRPV1 and TRPA1 agonist-elicited acute pain and spinal cord synaptic plasticity [spontaneous EPSC (sEPSC) frequency increase]. RvD2 also abolished inflammation-induced sEPSC increases (frequency and amplitude), without affecting basal synaptic transmission. Intrathecal administration of RvD2 at very low doses (0.01-1 ng) prevented formalin-induced spontaneous pain. Intrathecal RvD2 also reversed adjuvant-induced inflammatory pain without altering baseline pain and motor function. Finally, intrathecal RvD2 reversed C-fiber stimulation-evoked long-term potentiation in the spinal cord. Our findings suggest distinct roles of resolvins in regulating TRP channels and identify RvD2 as a potent endogenous inhibitor for TRPV1/TRPA1 and inflammatory pain.
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Inflamação/metabolismo , Plasticidade Neuronal/fisiologia , Dor/metabolismo , Medula Espinal/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/metabolismo , Ácido Eicosapentaenoico/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Masculino , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Medula Espinal/efeitos dos fármacosRESUMO
Mechanisms of inflammatory pain are not fully understood. We investigated the role of TRPV1 (transient receptor potential subtype V1) and TNF-α, two critical mediators for inflammatory pain, in regulating spinal cord synaptic transmission. We found in mice lacking Trpv1 the frequency but not the amplitude of spontaneous EPSCs (sEPSCs) in lamina II neurons of spinal cord slices is reduced. Further, C-fiber-induced spinal long-term potentiation (LTP) in vivo is abolished in Trpv1 knock-out mice. TNF-α also increases sEPSC frequency but not amplitude in spinal outer lamina II (lamina IIo) neurons, and this increase is abolished in Trpv1 knock-out mice. Single-cell PCR analysis revealed that TNF-α-responding neurons in lamina IIo are exclusively excitatory (vGluT2(+)) neurons. Notably, neuroprotectin-1 (NPD1), an anti-inflammatory lipid mediator derived from ω-3 polyunsaturated fatty acid (docosahexaenoic acid), blocks TNF-α- and capsaicin-evoked sEPSC frequency increases but has no effect on basal synaptic transmission. Strikingly, NPD1 potently inhibits capsaicin-induced TRPV1 current (IC(50) = 0.4 nm) in dissociated dorsal root ganglion neurons, and this IC(50) is ≈ 500 times lower than that of AMG9810, a commonly used TRPV1 antagonist. NPD1 inhibition of TRPV1 is mediated by GPCRs, since the effects were blocked by pertussis toxin. In contrast, NPD1 had no effect on mustard oil-induced TRPA1 currents. Spinal injection of NPD1, at very low doses (0.1-10 ng), blocks spinal LTP and reduces TRPV1-dependent inflammatory pain, without affecting baseline pain. NPD1 also reduces TRPV1-independent but TNF-α-dependent pain hypersensitivity. Our findings demonstrate a novel role of NPD1 in regulating TRPV1/TNF-α-mediated spinal synaptic plasticity and identify NPD1 as a novel analgesic for treating inflammatory pain.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Neurônios/efeitos dos fármacos , Dor/tratamento farmacológico , Medula Espinal/patologia , Canais de Cátion TRPV/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Acrilamidas , Análise de Variância , Animais , Compostos Bicíclicos Heterocíclicos com Pontes , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Adjuvante de Freund/efeitos adversos , Gânglios Espinais/citologia , Técnicas In Vitro , Inflamação/induzido quimicamente , Inflamação/complicações , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Dor/etiologia , Dor/patologia , Medição da Dor , Técnicas de Patch-Clamp , Receptores do Fator de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/deficiênciaRESUMO
Arthritis-associated inflammatory pain represents a serious medical problem worldwide. Current treatments for arthritic pain are not very effective and cause unwanted side effects. Resolvins, including the resolvin D and resolvin E series, are a family of novel endogenous lipid mediators derived from ω-3 polyunsaturated fatty acids and display potent anti-inflammatory and pro-resolution actions in animal models of inflammation. Emerging evidence also points to a potent anti-hyperalgesic role of resolvins in animal models of inflammatory pain. The study by Lima-Garcia et al. in this issue of the BJP demonstrated that systemic treatment with 17(R)-hydroxy-docosahexaenoic acid (17(R)HDoHE), the precursor of resolvin D series and its product, aspirin-triggered resolvin D1 (AT-RvD1), at very low doses (1 µg·kg(-1)), reduced inflammatory pain in an adjuvant-induced arthritis model. Particularly 17(R)HDoHE reduced joint stiffness but not paw and joint oedema. Given their potency and safety profile, resolvins may represent a new class of analgesics well suited to treating inflammatory pain associated with arthritis. LINKED ARTICLE This article is a commentary on Lima-Garcia et al., pp. 278-293 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2011.01345.x.
Assuntos
Analgésicos/metabolismo , Analgésicos/uso terapêutico , Artrite/complicações , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/uso terapêutico , Dor/tratamento farmacológico , Analgésicos/química , Ácidos Docosa-Hexaenoicos/química , Humanos , Estrutura Molecular , Dor/etiologiaRESUMO
Inflammatory pain, such as arthritis pain, is a growing health problem. Inflammatory pain is generally treated with opioids and cyclooxygenase (COX) inhibitors, but both are limited by side effects. Recently, resolvins, a unique family of lipid mediators, including RvE1 and RvD1 derived from omega-3 polyunsaturated fatty acid, have shown marked potency in treating disease conditions associated with inflammation. Here we report that peripheral (intraplantar) or spinal (intrathecal) administration of RvE1 or RvD1 in mice potently reduces inflammatory pain behaviors induced by intraplantar injection of formalin, carrageenan or complete Freund's adjuvant (CFA), without affecting basal pain perception. Intrathecal RvE1 injection also inhibits spontaneous pain and heat and mechanical hypersensitivity evoked by intrathecal capsaicin and tumor necrosis factor-alpha (TNF-alpha). RvE1 has anti-inflammatory activity by reducing neutrophil infiltration, paw edema and proinflammatory cytokine expression. RvE1 also abolishes transient receptor potential vanilloid subtype-1 (TRPV1)- and TNF-alpha-induced excitatory postsynaptic current increases and TNF-alpha-evoked N-methyl-D-aspartic acid (NMDA) receptor hyperactivity in spinal dorsal horn neurons via inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. Thus, we show a previously unknown role for resolvins in normalizing the spinal synaptic plasticity that has been implicated in generating pain hypersensitivity. Given the potency of resolvins and the well-known side effects of opioids and COX inhibitors, resolvins may represent new analgesics for treating inflammatory pain.