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BACKGROUND: Pain is reported as the leading cause of disability in the common forms of inflammatory arthritis conditions. Acting as a key player in nociceptive processing, neuroinflammation, and neuron-glia communication, the chemokine CCL2/CCR2 axis holds great promise for controlling chronic painful arthritis. Here, we investigated how the CCL2/CCR2 system in the dorsal root ganglion (DRG) contributes to the peripheral inflammatory pain sensitization. METHODS: Repeated intrathecal (i.t.) administration of the CCR2 antagonist, INCB3344 was tested for its ability to reverse the nociceptive-related behaviors in the tonic formalin and complete Freund's adjuvant (CFA) inflammatory models. We further determined by qPCR the expression of CCL2/CCR2, SP and CGRP in DRG neurons from CFA-treated rats. Using DRG explants, acutely dissociated primary sensory neurons and calcium mobilization assay, we also assessed the release of CCL2 and sensitization of nociceptors. Finally, we examined by immunohistochemistry following nerve ligation the axonal transport of CCL2, SP, and CGRP from the sciatic nerve of CFA-treated rats. RESULTS: We first found that CFA-induced paw edema provoked an increase in CCL2/CCR2 and SP expression in ipsilateral DRGs, which was decreased after INCB3344 treatment. This upregulation in pronociceptive neuromodulators was accompanied by an enhanced nociceptive neuron excitability on days 3 and 10 post-CFA, as revealed by the CCR2-dependent increase in intracellular calcium mobilization following CCL2 stimulation. In DRG explants, we further demonstrated that the release of CCL2 was increased following peripheral inflammation. Finally, the excitation of nociceptors following peripheral inflammation stimulated the anterograde transport of SP at their peripheral nerve terminals. Importantly, blockade of CCR2 reduced sensory neuron excitability by limiting the calcium mobilization and subsequently decreased peripheral transport of SP towards the periphery. Finally, pharmacological inhibition of CCR2 reversed the pronociceptive action of CCL2 in rats receiving formalin injection and significantly reduced the neurogenic inflammation as well as the stimuli-evoked and movement-evoked nociceptive behaviors in CFA-treated rats. CONCLUSIONS: Our results provide significant mechanistic insights into the role of CCL2/CCR2 within the DRG in the development of peripheral inflammation, nociceptor sensitization, and pain hypersensitivity. We further unveil the therapeutic potential of targeting CCR2 for the treatment of painful inflammatory disorders.
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Quimiocina CCL2/metabolismo , Ganglios Espinales/metabolismo , Hiperalgesia/metabolismo , Dolor/metabolismo , Receptores CCR2/antagonistas & inhibidores , Receptores CCR2/metabolismo , Animales , Células Cultivadas , Adyuvante de Freund/toxicidad , Ganglios Espinales/efectos de los fármacos , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inyecciones Espinales , Masculino , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Pirrolidinas/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
Following publication of this article, the authors noticed an error in the abstract, where they incorrectly stated that: "Direct application of IL-1ß to ex vivo hippocampal slices induced non-synaptic depolarisation and irreversible loss of membrane potential in CA1 neurons from diseased animals and systemic LPS increased apoptosis in the degenerating brain, in an IL-1RI-/--dependent fashion". This has now been corrected to: "Direct application of IL-1ß to ex vivo hippocampal slices induced non-synaptic depolarisation and irreversible loss of membrane potential in CA1 neurons from diseased animals and systemic LPS increased apoptosis in the degenerating brain, in an IL-1RI-dependent fashion". The authors would like to apologise for this error. This has been corrected in both the PDF and HTML versions of the article.
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Systemic inflammation can impair cognition with relevance to dementia, delirium and post-operative cognitive dysfunction. Episodes of delirium also contribute to rates of long-term cognitive decline, implying that these acute events induce injury. Whether systemic inflammation-induced acute dysfunction and acute brain injury occur by overlapping or discrete mechanisms remains unexplored. Here we show that systemic inflammation, induced by bacterial LPS, produces both working-memory deficits and acute brain injury in the degenerating brain and that these occur by dissociable IL-1-dependent processes. In normal C57BL/6 mice, LPS (100 µg/kg) did not affect working memory but impaired long-term memory consolidation. However prior hippocampal synaptic loss left mice selectively vulnerable to LPS-induced working memory deficits. Systemically administered IL-1 receptor antagonist (IL-1RA) was protective against, and systemic IL-1ß replicated, these working memory deficits. Dexamethasone abolished systemic cytokine synthesis and was protective against working memory deficits, without blocking brain IL-1ß synthesis. Direct application of IL-1ß to ex vivo hippocampal slices induced non-synaptic depolarisation and irreversible loss of membrane potential in CA1 neurons from diseased animals and systemic LPS increased apoptosis in the degenerating brain, in an IL-1RI-dependent fashion. The data suggest that LPS induces working memory dysfunction via circulating IL-1ß but direct hippocampal action of IL-1ß causes neuronal dysfunction and may drive neuronal death. The data suggest that acute systemic inflammation produces both reversible cognitive deficits, resembling delirium, and acute brain injury contributing to long-term cognitive impairment but that these events are mechanistically dissociable. These data have significant implications for management of cognitive dysfunction during acute illness.
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Lesiones Encefálicas/inmunología , Disfunción Cognitiva/inmunología , Interleucina-1/metabolismo , Animales , Encéfalo/metabolismo , Cognición/fisiología , Trastornos del Conocimiento/inmunología , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/metabolismo , Citocinas/metabolismo , Demencia/inmunología , Femenino , Hipocampo/metabolismo , Inflamación/complicaciones , Inflamación/metabolismo , Interleucina-1/inmunología , Lipopolisacáridos/farmacología , Trastornos de la Memoria/inmunología , Memoria a Corto Plazo/fisiología , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismoRESUMEN
BACKGROUND: Accumulating evidence suggests that the C-C chemokine ligand 2 (CCL2, or monocyte chemoattractant protein 1) acts as a neuromodulator in the central nervous system through its binding to the C-C chemokine receptor 2 (CCR2). Notably, it is well established that the CCL2/CCR2 axis plays a key role in neuron-glia communication as well as in spinal nociceptive transmission. Gene silencing through RNA interference has recently emerged as a promising avenue in research and drug development, including therapeutic management of chronic pain. In the present study, we used 27-mer Dicer-substrate small interfering RNA (DsiRNA) targeting CCR2 and assessed their ability to reverse the nociceptive behaviors induced by spinal CCL2 injection or following intraplantar injection of complete Freund's adjuvant. RESULTS: To this end, we first developed high-potency DsiRNAs designed to target different sequences distributed across the rat CCR2 (rCCR2) messenger RNA. For optimization, methyl groups were added to the two most potent DsiRNA candidates (Evader and M7 2'-O-methyl modified duplexes) in order to improve in vivo duplex stability and to reduce potential immunostimulatory activity. Our results demonstrated that all modified candidates formulated with the cell-penetrating peptide reagent Transductin showed strong RNAi activity following intrathecal delivery, exhibiting >50% rCCR2 knockdown in lumbar dorsal root ganglia. Accordingly, we found that these DsiRNA duplexes were able to reduce spinal microglia activation and were effective at blocking CCL2-induced mechanical hypersensitivity. Along with similar reductions of rCCR2 messenger RNA, both sequences and methylation patterns were similarly effective in inhibiting the CCL2 nociceptive action for the whole seven days testing period, compared to mismatch DsiRNA. DsiRNAs against CCR2 also reversed the hypernociceptive responses observed in the complete Freund's adjuvant-induced inflammatory chronic pain model. CONCLUSION: Altogether, these results validate CCR2 as a an appropriate molecular target for pain control and demonstrate that RNAi-based gene therapy represent an highly specific alternative to classical pharmacological approaches to treat central pathologies such as chronic pain.
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Dolor/metabolismo , Dolor/prevención & control , ARN Interferente Pequeño/metabolismo , Receptores CCR2/antagonistas & inhibidores , Ribonucleasa III/metabolismo , Animales , Forma de la Célula , Fluorescencia , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Hiperalgesia/complicaciones , Hiperalgesia/metabolismo , Inflamación/complicaciones , Inflamación/patología , Masculino , Neuroglía/metabolismo , Dolor/complicaciones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Receptores CCR2/genética , Reproducibilidad de los Resultados , Médula Espinal/metabolismo , Especificidad por SustratoRESUMEN
BACKGROUND: Functional alterations in the properties of Aß afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Na(v)1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Na(v)1.8 in controlling Aß-fiber excitability following persistent inflammation. METHODS: Distribution and expression of Na(v)1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund's adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Na(v)1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Na(v)1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats. RESULTS: Our findings revealed that Na(v)1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Na(v)1.8 peak current densities are enhanced in inflamed large myelinated Aß-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aß-fiber neuron excitability by shifting the voltage-dependent activation of Na(v)1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Na(v)1.8 currents in Aß-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Na(v)1.8 regulation in Aß-fibers contributes to inflammatory pain. CONCLUSIONS: Collectively, these findings support a key role for Na(v)1.8 in controlling the excitability of Aß-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.
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Ganglios Espinales/citología , Regulación de la Expresión Génica/fisiología , Inflamación/patología , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Neuronas/metabolismo , Nervio Ciático/metabolismo , Ambroxol/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Adyuvante de Freund , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/complicaciones , Masculino , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
CCL2 chemokine and its receptor CCR2 may contribute to neuropathic pain development. We tested the hypothesis that injury to peripheral nerves triggers CCL2 release from afferents in the dorsal horn spinal cord (DHSC), leading to pronociceptive effects, involving the production of proinflammatory factors, in particular. Consistent with the release of CCL2 from primary afferents, electron microscopy showed the CCL2 immunoreactivity in glomerular boutons and secretory vesicles in the DHSC of naive rats. Through the ex vivo superfusion of DHSC slices, we demonstrated that the rate of CCL2 secretion was much lower in neonatal capsaicin-treated rats than in controls. Thus, much of the CCL2 released in the DHSC originates from nociceptive fibers bearing TRPV1 (transient receptor potential vanilloid 1). In contrast, high levels of CCL2 released from the DHSC were observed in neuropathic pain animal model induced by chronic constriction of the sciatic nerve (SN-CCI). The upregulated expression of proinflammatory markers and extracellular signal-regulated kinase (ERK) 1/2 pathway activation (ERK1/2 phosphorylation) in the DHSC of SN-CCI animals were reversed by intrathecal administration of the CCR2 antagonist INCB3344 (N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide). These pathological pain-associated changes in the DHSC were mimicked by the intrathecal injection of exogenous CCL2 in naive rats and were prevented by the administration of INCB3344 or ERK inhibitor (PD98059). Finally, mechanical allodynia, which was fully developed 2 weeks after SN-CCI in rats, was attenuated by the intrathecal injection of INCB3344. Our data demonstrate that CCL2 has the typical characteristics of a neuronal mediator involved in nociceptive signal processing and that antagonists of its receptor are promising agents from treating neuropathic pain.
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Quimiocina CCL2/metabolismo , Inflamación/patología , Neuralgia/patología , Neuronas/metabolismo , Traumatismos de los Nervios Periféricos , Médula Espinal/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/farmacología , Enfermedad Crónica , Constricción Patológica , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Técnica del Anticuerpo Fluorescente , Hiperalgesia/patología , Inmunohistoquímica , Masculino , Microscopía Electrónica , Inhibidores de Proteínas Quinasas/farmacología , Pirrolidinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores CCR2/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nervio Ciático/lesiones , Nervio Ciático/patología , Médula Espinal/citologíaRESUMEN
Changes in function of voltage-gated sodium channels in nociceptive primary sensory neurons participate in the development of peripheral hyperexcitability that occurs in neuropathic and inflammatory chronic pain conditions. Among them, the tetrodotoxin-resistant (TTX-R) sodium channel Na(v)1.8, primarily expressed by small- and medium-sized dorsal root ganglion (DRG) neurons, substantially contributes to the upstroke of action potential in these neurons. Compelling evidence also revealed that the chemokine CCL2 plays a critical role in chronic pain facilitation via its binding to CCR2 receptors. In this study, we therefore investigated the effects of CCL2 on the density and kinetic properties of TTX-R Na(v)1.8 currents in acutely small/medium dissociated lumbar DRG neurons from naive adult rats. Whole-cell patch-clamp recordings demonstrated that CCL2 concentration-dependently increased TTX-resistant Na(v)1.8 current densities in both small- and medium-diameter sensory neurons. Incubation with CCL2 also shifted the activation and steady-state inactivation curves of Na(v)1.8 in a hyperpolarizing direction in small sensory neurons. No change in the activation and inactivation kinetics was, however, observed in medium-sized nociceptive neurons. Our electrophysiological recordings also demonstrated that the selective CCR2 antagonist INCB3344 [N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide] blocks the potentiation of Na(v)1.8 currents by CCL2 in a concentration-dependent manner. Furthermore, the enhancement in Na(v)1.8 currents was prevented by pretreatment with pertussis toxin (PTX) or gallein (a Gßγ inhibitor), indicating the involvement of Gßγ released from PTX-sensitive G(i/o)-proteins in the cross talk between CCR2 and Na(v)1.8. Together, our data clearly demonstrate that CCL2 may excite primary sensory neurons by acting on the biophysical properties of Na(v)1.8 currents via a CCR2/Gßγ-dependent mechanism.
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Quimiocina CCL2/farmacología , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Células Receptoras Sensoriales/metabolismo , Canales de Sodio/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Quimiocina CCL2/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Canal de Sodio Activado por Voltaje NAV1.8 , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
Recent observations suggest that besides their role in the immune system, chemokines have important functions in the brain. There is a great line of evidence to suggest that chemokines are a unique class of neurotransmitters/neuromodulators, which regulate many biological aspects as diverse as neurodevelopment, neuroinflammation and synaptic transmission. In physiopathological conditions, many chemokines are synthesized in activated astrocytes and microglial cells, suggesting their involvement in brain defense mechanisms. However, when evoking chemokine functions in the nervous system, it is important to make a distinction between resting conditions and various pathological states including inflammatory diseases, autoimmune or neurodegenerative disorders in which chemokine functions have been extensively studied. We illustrate here the emergent concept of the neuromodulatory/neurotransmitter activities of neurochemokines and their potential role as a regulatory alarm system and as a group of messenger molecules for the crosstalk between neurons and cells from their surrounding microenvironment. In this deliberately challenging review, we provide novel hypotheses on the role of these subtle messenger molecules in brain functions leading to the evidence that previous dogmas concerning chemokines should be reconsidered.
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Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Quimiocinas/fisiología , Animales , Humanos , Modelos Biológicos , Enfermedades del Sistema Nervioso/metabolismo , Neurotransmisores/fisiologíaRESUMEN
Voltage-gated ion channels play a key role in the action potential (AP) initiation and its propagation in sensory neurons. Modulation of their activity during chronic inflammation creates a persistent pain state. In this study, we sought to determine how peripheral inflammation caused by complete Freund's adjuvant (CFA) alters the fast sodium (INa ), L-type calcium (ICaL ), and potassium (IK ) currents in primary afferent fibers to increase nociception. In our model, intraplantar administration of CFA induced mechanical allodynia and thermal hyperalgesia at day 14 post-injection. Using whole-cell patch-clamp recording in dissociated small (C), medium (Aδ), and large-sized (Aß) rat dorsal root ganglion (DRG) neurons, we found that CFA prolonged the AP duration and increased the amplitude of the tetrodotoxin-resistant (TTX-r) INa in Aß fibers. In addition, CFA accelerated the recovery of INa from inactivation in C and Aδ nociceptive fibers but enhanced the late sodium current (INaL ) only in Aδ and Aß neurons. Inflammation similarly reduced the amplitude of ICaL in each neuronal cell type. Fourteen days after injection, CFA reduced both components of IK (IKdr and IA ) in Aδ fibers. We also found that IA was significantly larger in C and Aδ neurons in normal conditions and during chronic inflammation. Our data, therefore, suggest that targeting the transient potassium current IA represents an efficient way to shift the balance toward antinociception during inflammation, since its activation will selectively decrease the AP duration in nociceptive fibers. Altogether, our data indicate that complex interactions between IK , INa , and ICaL reduce pain threshold by concomitantly enhancing the activity of nociceptive neurons and reducing the inhibitory action of Aß fibers during chronic inflammation.
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Potenciales de Acción , Neuronas Aferentes/metabolismo , Dolor Nociceptivo/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiología , Masculino , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Nocicepción , Dolor Nociceptivo/fisiopatología , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
BACKGROUND: Central neurotensin (NT) administration results in a naloxone-insensitive antinociceptive response in animal models of acute and persistent pain. Both NTS1 and NTS2 receptors were shown to be required for different aspects of NT-induced analgesia. We recently demonstrated that NTS2 receptors were extensively associated with ascending nociceptive pathways, both at the level of the dorsal root ganglia and of the spinal dorsal horn. Then, we found that spinally administered NTS2-selective agonists induced dose-dependent antinociceptive responses in the acute tail-flick test. In the present study, we therefore investigated whether activation of spinal NTS2 receptors suppressed the persistent inflammatory pain symptoms observed after intraplantar injection of formalin. RESULTS: We first demonstrated that spinally administered NT and NT69L agonists, which bind to both NTS1 and NTS2 receptors, significantly reduced pain-evoked responses during the inflammatory phase of the formalin test. Accordingly, pretreatment with the NTS2-selective analogs JMV-431 and levocabastine was effective in inhibiting the aversive behaviors induced by formalin. With resolution at the single-cell level, we also found that activation of spinal NTS2 receptors reduced formalin-induced c-fos expression in dorsal horn neurons. However, our results also suggest that NTS2-selective agonists and NTS1/NTS2 mixed compounds differently modulated the early (21-39 min) and late (40-60 min) tonic phase 2 and recruited endogenous pain inhibitory mechanisms integrated at different levels of the central nervous system. Indeed, while non-selective drugs suppressed pain-related behaviors activity in both part of phase 2, intrathecal injection of NTS2-selective agonists was only efficient in reducing pain during the late phase 2. Furthermore, assessment of the stereotypic pain behaviors of lifting, shaking, licking and biting to formalin also revealed that unlike non-discriminative NTS1/NTS2 analogs reversing all nociceptive endpoint behaviors, pure NTS2 agonists specifically inhibited paw lifting, supporting a role of NTS2 in spinal modulation of persistent nociception. CONCLUSION: The present study provides the first demonstration that activation of NTS2 receptors produces analgesia in the persistent inflammatory pain model of formalin. The dichotomy between these two classes of compounds also indicates that both NTS1 and NTS2 receptors are involved in tonic pain inhibition and implies that these two NT receptors modulate the pain-induced behavioral responses by acting on distinct spinal and/or supraspinal neural circuits. In conclusion, development of NT agonists targeting both NTS1 and NTS2 receptors could be useful for chronic pain management.
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Dolor/etiología , Receptores de Neurotensina/fisiología , Animales , Formaldehído/administración & dosificación , Formaldehído/farmacología , Antagonistas de los Receptores Histamínicos H1 no Sedantes/farmacología , Inflamación , Masculino , Neurotensina/análogos & derivados , Neurotensina/farmacología , Dolor/tratamiento farmacológico , Dolor/prevención & control , Fragmentos de Péptidos/farmacología , Piperidinas/farmacología , Células del Asta Posterior/patología , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Neurotensina/agonistas , Nervios Espinales , Factores de TiempoRESUMEN
RNA interference (RNAi) is gaining acceptance as a potential therapeutic strategy against peripheral disease, and several clinical trials are already underway with 21-mer small-interfering RNA (siRNA) as the active pharmaceutical agent. However, for central affliction like pain, such innovating therapies are limited but nevertheless crucial to improve pain research and management. We demonstrate here the proof-of-concept of the use of 27-mer Dicer-substrate siRNA (DsiRNA) for silencing targets related to CNS disorders such as pain states. Indeed, low dose DsiRNA (0.005 mg/kg) was highly efficient in reducing the expression of the neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, our results show a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3-4 days after administration of DsiRNA. In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), we also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor. Altogether, DsiRNAi represents a potent tool for dissecting nociceptive pathways and could further lead to a new class of central active drugs.
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Dolor/tratamiento farmacológico , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Receptores de Neurotensina/antagonistas & inhibidores , Ribonucleasa III/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Ganglios Espinales/metabolismo , Masculino , Oligonucleótidos/administración & dosificación , Oligonucleótidos/uso terapéutico , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Central administration of the neuropeptide neurotensin (NT) was shown to induce antinociceptive responses both spinally and supraspinally. Although NTS2 receptors play an important role in modulating the activity of spinal neurons, we have recently implicated NTS1 receptors in NT's analgesic effects in acute spinal pain paradigms. The current experiments were thus designed to examine the antinociceptive effects of intrathecal administration of NTS1 agonists in formalin-induced tonic pain in rats. We first established, using immunoblotting and immunohistochemical approaches, that NTS1 receptors were present in small- and medium-sized dorsal root ganglion cells and localized in the superficial layers of the dorsal horn of the spinal cord. We then examined the effects of intrathecal injection of NT (1-15 microg/kg) or NTS1 preferring agonists on the nocifensive response to intraplantar formalin. Both NTS1-agonists, PD149163 (10-120 microg/kg) and NT69L (1-100 microg/kg), dose-dependently attenuated the formalin-induced behaviors. Accordingly, NTS1 agonists markedly suppressed pain-evoked c-fos expression in the superficial, nucleus proprius and neck regions of the spinal dorsal horn. The concomitant administration of PD149163 with the NTS1 antagonist SR48692 (3 microg/kg) significantly reversed PD149163-induced antinociception, confirming the implication of NTS1 in tonic pain. In contrast, NT69L's analgesic effects were partly abolished by co-administration of SR48692, indicating that NT69L-induced effects may also be exerted through interaction with NTS2. These results demonstrate that NTS1 receptors play a key role in the mediation of the analgesic effects of NT in persistent pain and suggest that NTS1-selective agonists may represent a new line of analgesic compounds.
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Dimensión del Dolor/métodos , Dolor/fisiopatología , Receptores de Neurotensina/fisiología , Transducción de Señal/fisiología , Animales , Modelos Animales de Enfermedad , Formaldehído , Masculino , Neurotensina/fisiología , Neurotensina/uso terapéutico , Dolor/prevención & control , Ratas , Ratas Sprague-Dawley , Receptores de Neurotensina/agonistasRESUMEN
A better understanding of the mechanisms linked to chemokine pronociceptive effects is essential for the development of new strategies to better prevent and treat chronic pain. Among chemokines, MCP-1/CCL2 involvement in neuropathic pain processing is now established. However, the mechanisms by which MCP-1/CCL2 exerts its pronociceptive effects are still poorly understood. In the present study, we demonstrate that MCP-1/CCL2 can alter pain neurotransmission in healthy rats. Using immunohistochemical studies, we first show that CCL2 is constitutively expressed by primary afferent neurons and their processes in the dorsal horn of the spinal cord. We also observe that CCL2 is co-localized with pain-related peptides (SP and CGRP) and capsaicin receptor (VR1). Accordingly, using in vitro superfusion system of lumbar dorsal root ganglion and spinal cord explants of healthy rats, we show that potassium or capsaicin evoke calcium-dependent release of CCL2. In vivo, we demonstrate that intrathecal administration of CCL2 to healthy rats produces both thermal hyperalgesia and sustained mechanical allodynia (up to four consecutive days). These pronociceptive effects of CCL2 are completely prevented by the selective CCR2 antagonist (INCB3344), indicating that CCL2-induced pain facilitation is elicited via direct spinal activation of CCR2 receptor. Therefore, preventing the activation of CCR2 might provide a fruitful strategy for treating pain.
Asunto(s)
Quimiocina CCL2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hiperalgesia/fisiopatología , Neuronas Aferentes/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Pirrolidinas/farmacología , Receptores CCR2/antagonistas & inhibidores , Médula Espinal/citología , Análisis de Varianza , Animales , Conducta Animal , Péptido Relacionado con Gen de Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcio/metabolismo , Quimiocina CCL2/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Ganglios Espinales/citología , Masculino , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Sustancia P/genética , Sustancia P/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
BACKGROUND: Lack of validity in osteoarthritis pain models and assessment methods is suspected. Our goal was to 1) assess the repeatability and reproducibility of measurement and the influence of environment, and acclimatization, to different pain assessment outcomes in normal rats, and 2) test the concurrent validity of the most reliable methods in relation to the expression of different spinal neuropeptides in a chemical model of osteoarthritic pain. METHODS: Repeatability and inter-rater reliability of reflexive nociceptive mechanical thresholds, spontaneous static weight-bearing, treadmill, rotarod, and operant place escape/avoidance paradigm (PEAP) were assessed by the intraclass correlation coefficient (ICC). The most reliable acclimatization protocol was determined by comparing coefficients of variation. In a pilot comparative study, the sensitivity and responsiveness to treatment of the most reliable methods were tested in the monosodium iodoacetate (MIA) model over 21 days. Two MIA (2 mg) groups (including one lidocaine treatment group) and one sham group (0.9 % saline) received an intra-articular (50 µL) injection. RESULTS: No effect of environment (observer, inverted circadian cycle, or exercise) was observed; all tested methods except mechanical sensitivity (ICC <0.3), offered good repeatability (ICC ≥0.7). The most reliable acclimatization protocol included five assessments over two weeks. MIA-related osteoarthritic change in pain was demonstrated with static weight-bearing, punctate tactile allodynia evaluation, treadmill exercise and operant PEAP, the latter being the most responsive to analgesic intra-articular lidocaine. Substance P and calcitonin gene-related peptide were higher in MIA groups compared to naive (adjusted P (adj-P) = 0.016) or sham-treated (adj-P = 0.029) rats. Repeated post-MIA lidocaine injection resulted in 34 times lower downregulation for spinal substance P compared to MIA alone (adj-P = 0.029), with a concomitant increase of 17 % in time spent on the PEAP dark side (indicative of increased comfort). CONCLUSION: This study of normal rats and rats with pain established the most reliable and sensitive pain assessment methods and an optimized acclimatization protocol. Operant PEAP testing was more responsive to lidocaine analgesia than other tests used, while neuropeptide spinal concentration is an objective quantification method attractive to support and validate different centralized pain functional assessment methods.
Asunto(s)
Artritis Experimental , Neuropéptidos/análisis , Osteoartritis , Dimensión del Dolor/métodos , Proteómica/métodos , Aclimatación/fisiología , Animales , Artritis Experimental/complicaciones , Artritis Experimental/metabolismo , Cromatografía Líquida de Alta Presión , Condicionamiento Operante , Femenino , Ácido Yodoacético/toxicidad , Osteoartritis/complicaciones , Osteoartritis/metabolismo , Dolor/etiología , Umbral del Dolor , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
It is increasingly clear that systemic inflammation has both adaptive and deleterious effects on the brain. However, detailed comparisons of brain effects of systemic challenges with different pro-inflammatory cytokines are lacking. In the present study, we challenged female C57BL/6 mice intraperitoneally with LPS (100 µg/kg), IL-1ß (15 or 50 µg/kg), TNF-α (50 or 250 µg/kg) or IL-6 (50 or 125 µg/kg). We investigated effects on core body temperature, open field activity and plasma levels of inflammatory markers at 2 hours post injection. We also examined levels of hepatic, hypothalamic and hippocampal inflammatory cytokine transcripts. Hypothermia and locomotor hypoactivity were induced by LPS>IL-1ß>TNF-α>>IL-6. Systemic LPS, IL-1ß and TNF-α challenges induced robust and broadly similar systemic and central inflammation compared to IL-6, which showed limited effects, but did induce a hepatic acute phase response. Important exceptions included IFNß, which could only be induced by LPS. Systemic IL-1ß could not induce significant blood TNF-α, but induced CNS TNF-α mRNA, while systemic TNF-α could induce IL-1ß in blood and brain. Differences between IL-1ß and TNF-α-induced hippocampal profiles, specifically for IL-6 and CXCL1 prompted a temporal analysis of systemic and central responses at 1, 2, 4, 8 and 24 hours, which revealed that IL-1ß and TNF-α both induced the chemokines CXCL1 and CCL2 but only IL-1ß induced the pentraxin PTX3. Expression of COX-2, CXCL1 and CCL2, with nuclear localisation of the p65 subunit of NFκB, in the cerebrovasculature was demonstrated by immunohistochemistry. Furthermore, we used cFOS immunohistochemistry to show that LPS, IL-1ß and to a lesser degree, TNF-α activated the central nucleus of the amygdala. Given the increasing attention in the clinical literautre on correlating specific systemic inflammatory mediators with neurological or neuropsychiatric conditions and complications, these data will provide a useful resource on the likely CNS inflammatory profiles resulting from systemic elevation of particular cytokines.
Asunto(s)
Encéfalo/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Lipopolisacáridos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Temperatura Corporal , Encéfalo/metabolismo , Citocinas/sangre , Citocinas/inmunología , Femenino , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/inmunología , Interleucina-1beta/administración & dosificación , Interleucina-6/administración & dosificación , Lipopolisacáridos/administración & dosificación , Hígado/inmunología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Actividad Motora , ARN Mensajero/genética , ARN Mensajero/inmunología , Activación Transcripcional , Factor de Necrosis Tumoral alfa/administración & dosificaciónRESUMEN
Preclinical pain assessment remains a key step for the development of new and potent painkillers. Significant progress in pain evaluation has been achieved with the development of non-reflexive tools. Seeking efficient and clinically relevant devices for pain-related quality of life assessment, we evaluated a new Dynamic Weight Bearing (DWB) device based on pressure captors in three different preclinical chronic pain models. Inflammatory (CFA), neuropathic (CCI) and bone cancer pain (femoral tumor) models were evaluated in Sprague Dawley rats for mechanical allodynia using dynamic von Frey for pain-related behaviors and DWB for discomfort. We observed similar impairment patterns in all of the models for both von Frey (allodynia) and DWB (weight balance) during the complete observation period, starting at day 3 in CCI- and CFA-affected limbs and at day 14 in bone cancer-afflicted rats, indicating that the DWB could be a useful tool for supporting pain assessment. Interestingly, we demonstrated that the main compensation, when animals experienced pain, was seen in the forepaws, ranging from 46% to 69% of increased load compared to normal. Other pain-related coping behaviors were also measured, such as the time spent on each paw and the contact surface. Our results revealed that CFA, CCI and cancerous rats decreased the use of their ipsilateral hind paws by 30% and showed a 50% reduction in paw surface pressed against the floor. In conclusion, this new device improves methods for preclinical evaluation of discomfort and quality of life proxies and could be helpful in screening putative analgesics.