RESUMEN
Neuropathic pain (NP) is associated with profound gene expression alterations within the nociceptive system. DNA mechanisms, such as epigenetic remodeling and repair pathways have been implicated in NP. Here we have used a rat model of peripheral nerve injury to study the effect of a recently developed RARß agonist, C286, currently under clinical research, in NP. A 4-week treatment initiated 2 days after the injury normalized pain sensation. Genome-wide and pathway enrichment analysis showed that multiple mechanisms persistently altered in the spinal cord were restored to preinjury levels by the agonist. Concomitant upregulation of DNA repair proteins, ATM and BRCA1, the latter being required for C286-mediated pain modulation, suggests that early DNA repair may be important to prevent phenotypic epigenetic imprints in NP. Thus, C286 is a promising drug candidate for neuropathic pain and DNA repair mechanisms may be useful therapeutic targets to explore.
RESUMEN
Oxadiazole replacement of an amide linkage in an RARα agonist template 1, followed by lead optimisation, has produced a highly potent and selective RARß agonist 4-(5-(4,7-dimethylbenzofuran-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (10) with good oral bioavailability in the rat and dog. This molecule increases neurite outgrowth in vitro and induces sensory axon regrowth in vivo in a rodent model of avulsion and crush injury, and thus has the potential for the treatment of nerve injury.
Asunto(s)
Oxadiazoles/química , Receptores de Ácido Retinoico/agonistas , Administración Oral , Animales , Perros , Evaluación Preclínica de Medicamentos , Semivida , Locomoción/efectos de los fármacos , Células de Riñón Canino Madin Darby , Proyección Neuronal/efectos de los fármacos , Traumatismos del Nervio Óptico/tratamiento farmacológico , Oxadiazoles/farmacocinética , Oxadiazoles/farmacología , Ratas , Receptores de Ácido Retinoico/metabolismo , Relación Estructura-ActividadRESUMEN
In the CNS, oligodendrocytes are responsible for myelin formation and maintenance. Following spinal cord injury, oligodendrocyte loss and an inhibitory milieu compromise remyelination and recovery. Here, we explored the role of retinoic acid receptor-beta (RARß) signaling in remyelination. Using a male Sprague Dawley rat model of PNS-CNS injury, we show that oral treatment with a novel drug like RARß agonist, C286, induces neuronal expression of the proteoglycan decorin and promotes myelination and differentiation of oligodendrocyte precursor cells (NG2+ cells) in a decorin-mediated neuron-glia cross talk. Decorin promoted the activation of RARα in NG2+ cells by increasing the availability of the endogenous ligand RA. NG2+ cells synthesize RA, which is released in association with exosomes. We found that decorin prevents this secretion through regulation of the EGFR-calcium pathway. Using functional and pharmacological studies, we further show that RARα signaling is both required and sufficient for oligodendrocyte differentiation. These findings illustrate that RARß and RARα are important regulators of oligodendrocyte differentiation, providing new targets for myelination.SIGNIFICANCE STATEMENT This study identifies novel therapeutic targets for remyelination after PNS-CNS injury. Pharmacological and knock-down experiments show that the retinoic acid (RA) signaling promotes differentiation of oligodendrocyte precursor cells (OPCs) and remyelination in a cross talk between neuronal RA receptor-beta (RARß) and RARα in NG2+ cells. We show that stimulation of RARα is required for the differentiation of OPCs and we describe for the first time how oral treatment with a RARß agonist (C286, currently being tested in a Phase 1 trial, ISRCTN12424734) leads to the endogenous synthesis of RA through retinaldehyde dehydrogenase 2 (Raldh2) in NG2 cells and controls exosome-associated-RA intracellular levels through a decorin-Ca2+ pathway. Although RARß has been implicated in distinct aspects of CNS regeneration, this study identifies a novel function for both RARß and RARα in remyelination.
Asunto(s)
Exosomas/metabolismo , Vaina de Mielina/metabolismo , Regeneración Nerviosa/efectos de los fármacos , Receptores de Ácido Retinoico/agonistas , Traumatismos de la Médula Espinal/tratamiento farmacológico , Tretinoina/metabolismo , Animales , Decorina/metabolismo , Receptores ErbB/metabolismo , Vaina de Mielina/efectos de los fármacos , Regeneración Nerviosa/fisiología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Traumatismos de la Médula Espinal/metabolismoRESUMEN
Stimulation of retinoic acid (RA) mediated signalling pathways following neural injury leads to regeneration in the adult nervous system and numerous studies have shown that the specific activation of the retinoic acid receptor ß (RARß) is required for this process. Here we identify a novel mechanism by which neuronal RARß activation results in the endogenous synthesis of RA which is released in association with exosomes and acts as a positive cue to axonal/neurite outgrowth. Using an established rodent model of RARß induced axonal regeneration, we show that neuronal RARß activation upregulates the enzymes involved in RA synthesis in a cell specific manner; alcohol dehydrogenase7 (ADH7) in neurons and aldehyde dehydrogenase 2 (Raldh2) in NG2 expressing cells (NG2+ cells). These release RA in association with exosomes providing a permissive substrate to neurite outgrowth. Conversely, deletion of Raldh2 in the NG2+ cells in our in vivo regeneration model is sufficient to compromise axonal outgrowth. This hitherto unidentified RA paracrine signalling is required for axonal/neurite outgrowth and is initiated by the activation of neuronal RARß signalling.
Asunto(s)
Antígenos/metabolismo , Exosomas/metabolismo , Regeneración Nerviosa/fisiología , Neuroglía/metabolismo , Proyección Neuronal/fisiología , Proteoglicanos/metabolismo , Tretinoina/metabolismo , Aldehído Oxidorreductasas/metabolismo , Animales , Transporte Biológico/fisiología , Células Cultivadas , Médula Cervical/metabolismo , Médula Cervical/patología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Exosomas/patología , Masculino , Ratones , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Ratas Sprague-Dawley , Receptores de Ácido Retinoico/metabolismo , Retinal-Deshidrogenasa/metabolismo , Raíces Nerviosas Espinales/lesiones , Raíces Nerviosas Espinales/metabolismo , Raíces Nerviosas Espinales/patologíaRESUMEN
Following peripheral axon injury, dysregulation of non-coding microRNAs (miRs) occurs in dorsal root ganglia (DRG) sensory neurons. Here we show that DRG neuron cell bodies release extracellular vesicles, including exosomes containing miRs, upon activity. We demonstrate that miR-21-5p is released in the exosomal fraction of cultured DRG following capsaicin activation of TRPV1 receptors. Pure sensory neuron-derived exosomes released by capsaicin are readily phagocytosed by macrophages in which an increase in miR-21-5p expression promotes a pro-inflammatory phenotype. After nerve injury in mice, miR-21-5p is upregulated in DRG neurons and both intrathecal delivery of a miR-21-5p antagomir and conditional deletion of miR-21 in sensory neurons reduce neuropathic hypersensitivity as well as the extent of inflammatory macrophage recruitment in the DRG. We suggest that upregulation and release of miR-21 contribute to sensory neuron-macrophage communication after damage to the peripheral nerve.
Asunto(s)
Exosomas/metabolismo , Ganglios Espinales/metabolismo , Macrófagos/inmunología , MicroARNs/metabolismo , Neuralgia/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Axones/metabolismo , Exosomas/genética , Ganglios Espinales/citología , Ganglios Espinales/lesiones , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Neuralgia/genética , Neuralgia/inmunología , Fagocitosis , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) patients frequently show weak correlations between the magnitude of pain and inflammation suggesting that mechanisms other than overt peripheral inflammation contribute to pain in RA. We assessed changes in microglial reactivity and spinal excitability and their contribution to pain-like behaviour in the early stages of collagen-induced arthritis (CIA) model. METHODS: Mechanically evoked hypersensitivity, spinal nociceptive withdrawal reflexes (NWRs) and hind paw swelling were evaluated in female Lewis rats before and until 13 days following collagen immunization. In the spinal dorsal horn, microgliosis was assayed using immunohistochemistry (Iba-1/p-p38) and cyto(chemo)kine levels in the cerebrospinal fluid (CSF). Intrathecal administration of microglia-targeting drugs A-438079 (P2X7 antagonist) and LHVS (cathepsin S inhibitor) were examined upon hypersensitivity, NWRs, microgliosis and cyto(chemo)kine levels in the early phase of CIA. RESULTS: The early phase of CIA was associated with mechanical allodynia and exaggerated mechanically evoked spinal NWRs, evident before hind paw swelling, and exacerbated with the development of swelling. Concomitant with the development of hypersensitivity was the presence of reactive spinal microgliosis and an increase of IL-1ß levels in CSF (just detectable in plasma). Prolonged intrathecal administration of microglial inhibitors attenuated the development of mechanical allodynia, reduced microgliosis and attenuated IL-1ß increments. Acute spinal application of either microglial inhibitor significantly diminished the sensitization of the spinal NWRs. CONCLUSIONS: Mechanical hypersensitivity in the early phase of CIA is associated with central sensitization that is dependent upon microglial-mediated release of IL-1ß in the spinal cord. Blockade of these spinal events may provide pain relief in RA patients.
Asunto(s)
Artritis Experimental/fisiopatología , Artritis Reumatoide/fisiopatología , Sensibilización del Sistema Nervioso Central/fisiología , Hiperalgesia/fisiopatología , Neuronas/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Electromiografía , Femenino , Gliosis/inmunología , Gliosis/metabolismo , Gliosis/fisiopatología , Hiperalgesia/inmunología , Hiperalgesia/metabolismo , Inmunohistoquímica , Interleucina-1beta/metabolismo , Microglía/inmunología , Microglía/metabolismo , Microglía/patología , Ratas , Ratas Endogámicas Lew , Médula Espinal/inmunología , Médula Espinal/fisiopatologíaRESUMEN
Failure of axonal regeneration in the central nervous system (CNS) is mainly attributed to a lack of intrinsic neuronal growth programs and an inhibitory environment from a glial scar. Phosphatase and tensin homolog (PTEN) is a major negative regulator of neuronal regeneration and, as such, inhibiting its activity has been considered a therapeutic target for spinal cord (SC) injuries (SCIs). Using a novel model of rat cervical avulsion, we show that treatment with a retinoic acid receptor ß (RARß) agonist results in locomotor and sensory recovery. Axonal regeneration from the severed roots into the SC could be seen by biotinylated dextran amine labeling. Light micrographs of the dorsal root entry zone show the peripheral nervous system (PNS)-CNS transition of regrown axons. RARß agonist treatment also resulted in the absence of scar formation. Mechanism studies revealed that, in RARß-agonist-treated neurons, PTEN activity is decreased by cytoplasmic phosphorylation and increased secretion in exosomes. These are taken up by astrocytes, resulting in hampered proliferation and causing them to arrange in a normal-appearing scaffold around the regenerating axons. Attribution of the glial modulation to neuronal PTEN in exosomes was demonstrated by the use of an exosome inhibitor in vivo and PTEN siRNA in vitro assays. The dual effect of RARß signaling, both neuronal and neuronal-glial, results in axonal regeneration into the SC after dorsal root neurotmesis. Targeting this pathway may open new avenues for the treatment of SCIs. SIGNIFICANCE STATEMENT: Spinal cord injuries (SCIs) often result in permanent damage in the adult due to the very limited capacity of axonal regeneration. Intrinsic neuronal programs and the formation of a glial scar are the main obstacles. Here, we identify a single target, neuronal retinoic acid receptor ß (RARß), which modulates these two aspects of the postinjury physiological response. Activation of RARß in the neuron inactivates phosphatase and tensin homolog and induces its transfer into the astrocytes in small vesicles, where it prevents scar formation. This may open new therapeutic avenues for SCIs.
Asunto(s)
Astrocitos/metabolismo , Cicatriz/metabolismo , Exosomas/metabolismo , Neuroglía/metabolismo , Fosfohidrolasa PTEN/metabolismo , Receptores de Ácido Retinoico/fisiología , Regeneración de la Medula Espinal/fisiología , Animales , Células Cultivadas , Cicatriz/prevención & control , Masculino , Ratones , Neuroglía/patología , Neuronas/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/fisiologíaRESUMEN
G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiological function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mechanical or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter molecule 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array analysis revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addition, the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1ß, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger associated with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired.
Asunto(s)
Regulación de la Expresión Génica/genética , Umbral del Dolor/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Ciática/metabolismo , Ciática/fisiopatología , Animales , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Hipersensibilidad/etiología , Hipersensibilidad/genética , Inflamación/etiología , Inflamación/genética , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Dimensión del Dolor , Estimulación Física/efectos adversos , Receptores Acoplados a Proteínas G/genética , Ciática/patología , Médula Espinal/metabolismo , Temperatura , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: To evaluate the contribution of sensory neurons in ankle joints and adjacent tissue to the development of pain in collagen-induced arthritis (CIA), and to determine the relationship between pain and the appearance of clinical signs. METHODS: Mechanical and heat hypersensitivity and hind paw swelling were assessed in Lewis rats before and until 18 days following collagen immunization. We examined the effect of intrathecal administration of a calcitonin gene-related peptide (CGRP) antagonist (CGRP(8-37) ) from day 11 to day 18 postimmunization on CIA-induced hypersensitivity. During CIA development, CGRP and p-ERK immunoreactivity was quantified in lumbar dorsal root ganglia in which sensory neurons innervating the ankle joint were identified by retrograde labeling with Fluoro-Gold. Microgliosis in the lumbar dorsal horn was assessed by immunohistochemistry, and release of CGRP evoked by activity of primary afferent fibers was measured using a preparation of isolated dorsal horn with dorsal roots attached. RESULTS: CIA was associated with mechanical hypersensitivity that was evident before hind paw swelling and that was exacerbated with the development of swelling. Heat hyperalgesia developed along with swelling. Concomitant with the development of mechanical hypersensitivity, joint innervating neurons exhibited enhanced CGRP expression and an activated phenotype (increased p-ERK expression), and significant microgliosis became evident in the dorsal horn; these peripheral and central changes were augmented further with disease progression. CGRP release evoked by dorsal root stimulation was higher in the dorsal horn on day 18 in rats with CIA compared to control rats. Prolonged intrathecal administration of CGRP(8-37) attenuated established mechanical hypersensitivity and reduced spinal microgliosis. CONCLUSION: Sensory neuron-derived CGRP sustains mechanical hypersensitivity and spinal microglial reactivity in CIA, suggesting that central mechanisms play critical roles in chronic inflammatory pain. Blockade of these central events may provide pain relief in rheumatoid arthritis patients.
Asunto(s)
Articulación del Tobillo/metabolismo , Artralgia/metabolismo , Artritis Experimental/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Hiperalgesia/metabolismo , Microglía/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Artralgia/etiología , Artritis Experimental/complicaciones , Péptido Relacionado con Gen de Calcitonina/antagonistas & inhibidores , Péptido Relacionado con Gen de Calcitonina/farmacología , Hiperalgesia/etiología , Inyecciones Espinales , Fragmentos de Péptidos/farmacología , Ratas , Ratas Endogámicas Lew , Médula Espinal/citologíaRESUMEN
A major dose-limiting side effect associated with cancer-treating antineoplastic drugs is the development of neuropathic pain, which is not readily relieved by available analgesics. A better understanding of the mechanisms that underlie pain generation has potential to provide targets for prophylactic management of chemotherapy pain. Here, we delineate a pathway for pain that is induced by the chemotherapeutic drug vincristine sulfate (VCR). In a murine model of chemotherapy-induced allodynia, VCR treatment induced upregulation of endothelial cell adhesion properties, resulting in the infiltration of circulating CX3CR1⺠monocytes into the sciatic nerve. At the endothelial-nerve interface, CX3CR1⺠monocytes were activated by the chemokine CX3CL1 (also known as fractalkine [FKN]), which promoted production of reactive oxygen species that in turn activated the receptor TRPA1 in sensory neurons and evoked the pain response. Furthermore, mice lacking CX3CR1 exhibited a delay in the development of allodynia following VCR administration. Together, our data suggest that CX3CR1 antagonists and inhibition of FKN proteolytic shedding, possibly by targeting ADAM10/17 and/or cathepsin S, have potential as peripheral approaches for the prophylactic treatment of chemotherapy-induced pain.
Asunto(s)
Antineoplásicos Fitogénicos/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Monocitos/metabolismo , Dolor/metabolismo , Receptores de Quimiocina/metabolismo , Vincristina/efectos adversos , Animales , Antineoplásicos Fitogénicos/farmacología , Receptor 1 de Quimiocinas CX3C , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Regulación de la Expresión Génica/genética , Hiperalgesia/inducido químicamente , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/patología , Ratones , Ratones Noqueados , Monocitos/patología , Dolor/inducido químicamente , Dolor/genética , Dolor/patología , Receptores de Quimiocina/genética , Vincristina/farmacologíaRESUMEN
BACKGROUND: The past decade has seen an abundance of transcriptional profiling studies of preclinical models of persistent pain, predominantly employing microarray technology. In this study we directly compare exon microarrays to RNA-seq and investigate the ability of both platforms to detect differentially expressed genes following nerve injury using the L5 spinal nerve transection model of neuropathic pain. We also investigate the effects of increasing RNA-seq sequencing depth. Finally we take advantage of the "agnostic" approach of RNA-seq to discover areas of expression outside of annotated exons that show marked changes in expression following nerve injury. RESULTS: RNA-seq and microarrays largely agree in terms of the genes called as differentially expressed. However, RNA-seq is able to interrogate a much larger proportion of the genome. It can also detect a greater number of differentially expressed genes than microarrays, across a wider range of fold changes and is able to assign a larger range of expression values to the genes it measures. The number of differentially expressed genes detected increases with sequencing depth. RNA-seq also allows the discovery of a number of genes displaying unusual and interesting patterns of non-exonic expression following nerve injury, an effect that cannot be detected using microarrays. CONCLUSION: We recommend the use of RNA-seq for future high-throughput transcriptomic experiments in pain studies. RNA-seq allowed the identification of a larger number of putative candidate pain genes than microarrays and can also detect a wider range of expression values in a neuropathic pain model. In addition, RNA-seq can interrogate the whole genome regardless of prior annotations, being able to detect transcription from areas of the genome not currently annotated as exons. Some of these areas are differentially expressed following nerve injury, and may represent novel genes or isoforms. We also recommend the use of a high sequencing depth in order to detect differential expression for genes with low levels of expression.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Neuralgia/metabolismo , Neuralgia/patología , Células Receptoras Sensoriales/metabolismo , Análisis de Secuencia de ARN , Transcripción Genética/fisiología , Animales , Mapeo Cromosómico , Modelos Animales de Enfermedad , Ganglios Espinales/patología , Perfilación de la Expresión Génica , Genoma/fisiología , Masculino , Análisis por Micromatrices/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Wistar , Médula Espinal/patología , Nervios Espinales/lesionesRESUMEN
Peripheral nerve injuries caused by trauma are associated with increased sensory neuron excitability and debilitating chronic pain symptoms. Axotomy-induced alterations in the function of ion channels are thought to largely underlie the pathophysiology of these phenotypes. Here, we characterise the mRNA distribution of Kv2 family members in rat dorsal root ganglia (DRG) and describe a link between Kv2 function and modulation of sensory neuron excitability. Kv2.1 and Kv2.2 were amply expressed in cells of all sizes, being particularly abundant in medium-large neurons also immunoreactive for neurofilament-200. Peripheral axotomy led to a rapid, robust and long-lasting transcriptional Kv2 downregulation in the DRG, correlated with the onset of mechanical and thermal hypersensitivity. The consequences of Kv2 loss-of-function were subsequently investigated in myelinated neurons using intracellular recordings on ex vivo DRG preparations. In naïve neurons, pharmacological Kv2.1/Kv2.2 inhibition by stromatoxin-1 (ScTx) resulted in shortening of action potential (AP) after-hyperpolarization (AHP). In contrast, ScTx application on axotomized neurons did not alter AHP duration, consistent with the injury-induced Kv2 downregulation. In accordance with a shortened AHP, ScTx treatment also reduced the refractory period and improved AP conduction to the cell soma during high frequency stimulation. These results suggest that Kv2 downregulation following traumatic nerve lesion facilitates greater fidelity of repetitive firing during prolonged input and thus normal Kv2 function is postulated to limit neuronal excitability. In summary, we have profiled Kv2 expression in sensory neurons and provide evidence for the contribution of Kv2 dysfunction in the generation of hyperexcitable phenotypes encountered in chronic pain states.
Asunto(s)
Enfermedades del Sistema Nervioso Periférico/metabolismo , Células Receptoras Sensoriales/metabolismo , Canales de Potasio Shab/metabolismo , Animales , Axotomía/métodos , Péptido Relacionado con Gen de Calcitonina/metabolismo , Ganglios Espinales/citología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas del Tejido Nervioso/metabolismo , Dolor/etiología , Dolor/metabolismo , Dimensión del Dolor , Enfermedades del Sistema Nervioso Periférico/complicaciones , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Células Receptoras Sensoriales/fisiología , Canales de Potasio Shab/genéticaRESUMEN
Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug-induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.
Asunto(s)
Inhibidores de Histona Desacetilasas/uso terapéutico , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Neuralgia/complicaciones , Animales , Antirretrovirales/efectos adversos , Benzamidas/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Neuralgia/inducido químicamente , Dimensión del Dolor , Piridinas/uso terapéutico , Pirimidinas/uso terapéutico , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.
Asunto(s)
Regulación hacia Abajo , Neuralgia/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Axotomía , Conducta Animal/fisiología , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Masculino , Fibras Nerviosas Mielínicas/metabolismo , Neuralgia/etiología , Neuralgia/fisiopatología , Dimensión del Dolor , Traumatismos de los Nervios Periféricos/complicaciones , Traumatismos de los Nervios Periféricos/fisiopatología , Canales de Potasio con Entrada de Voltaje/genética , ARN Interferente Pequeño , Ratas , Ratas WistarRESUMEN
OBJECTIVE: The induction of rheumatoid arthritis (RA) by active and passive immunization of mice results in the development of pain at the same time as the swelling and inflammation, with both peripheral and central sensitization contributing to joint pain. The purpose of this study was to examine the development of pain in the rat model of collagen-induced arthritis (CIA) and to evaluate the contribution of neuroimmune interactions to established arthritis pain. METHODS: Mechanical hypersensitivity was assessed in female Lewis rats before and up to 18 days after induction of CIA by immunization with type II collagen. The effect of selective inhibitors of microglia were then evaluated by prolonged intrathecal delivery of a cathepsin S (CatS) inhibitor and a fractalkine (FKN) neutralizing antibody, from day 11 to day 18 following immunization. RESULTS: Rats with CIA developed significant mechanical hypersensitivity, which started on day 9, before the onset of clinical signs of arthritis. Mechanical hypersensitivity peaked with the severity of the disease, when significant microglial and astrocytic responses, alongside T cell infiltration, were observed in the spinal cord. Intrathecal delivery of microglial inhibitors, a CatS inhibitor, or an FKN neutralizing antibody attenuated mechanical hypersensitivity and spinal microglial response in rats with CIA. CONCLUSION: The inhibition of microglial targets by centrally penetrant CatS inhibitors and CX(3) CR1 receptor antagonists represents a potential therapeutic avenue for the treatment of pain in RA.
Asunto(s)
Artritis Experimental/metabolismo , Catepsinas/metabolismo , Quimiocina CX3CL1/metabolismo , Dolor Crónico/metabolismo , Hiperalgesia/metabolismo , Microglía/metabolismo , Médula Espinal/metabolismo , Animales , Anticuerpos Neutralizantes/farmacología , Catepsinas/antagonistas & inhibidores , Quimiocina CX3CL1/antagonistas & inhibidores , Dipéptidos/farmacología , Femenino , Microglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Endogámicas Lew , Médula Espinal/efectos de los fármacos , Sulfonas/farmacologíaRESUMEN
In the majority of spinal cord injuries (SCIs), some axonal projections remain intact. We examined the functional status of these surviving axons since they represent a prime therapeutic target. Using a novel electrophysiological preparation, adapted from techniques used to study primary demyelination, we quantified conduction failure across a SCI and studied conduction changes over time in adult rats with a moderate severity spinal contusion (150 kdyn; Infinite Horizon impactor). By recording antidromically activated single units from teased dorsal root filaments, we demonstrate complete conduction block in ascending dorsal column axons acutely (1-7 d) after injury, followed by a period of restored conduction over the subacute phase (2-4 weeks), with no further improvements in conduction at chronic stages (3-6 months). By cooling the lesion site, additional conducting fibers could be recruited, thus revealing a population of axons that are viable but unable to conduct under normal physiological conditions. Importantly, this phenomenon is still apparent at the most chronic (6 month) time point. The time course of conduction changes corresponded with changes in behavioral function, and ultrastructural analysis of dorsal column axons revealed extensive demyelination during the period of conduction block, followed by progressive remyelination. A proportion of dorsal column axons remained chronically demyelinated, suggesting that these are the axons recruited with the cooling paradigm. Thus, using a clinically relevant SCI model, we have identified a population of axons present at chronic injury stages that are intact but fail to conduct and are therefore a prime target for therapeutic strategies to restore function.
Asunto(s)
Axones/fisiología , Conducción Nerviosa/fisiología , Neuronas/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/fisiopatología , Potenciales de Acción/fisiología , Animales , Femenino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Following peripheral nerve injury microglia accumulate within the spinal cord and adopt a proinflammatory phenotype a process which contributes to the development of neuropathic pain. We have recently shown that neuregulin-1, a growth factor released following nerve injury, activates erbB 2, 3, and 4 receptors on microglia and stimulates proliferation, survival and chemotaxis of these cells. Here we studied the intracellular signaling pathways downstream of neuregulin-1-erbB activation in microglial cells. We found that neuregulin-1 in vitro induced phosphorylation of ERK1/2 and Akt without activating p38MAPK. Using specific kinase inhibitors we found that the mitogenic effect of neuregulin-1 on microglia was dependant on MEK/ERK1/2 pathway, the chemotactic effect was dependant on PI3K/Akt signaling and survival was dependant on both pathways. Intrathecal treatment with neuregulin-1 was associated with microgliosis and development of mechanical and cold pain related hypersensitivity which was dependant on ERK1/2 phosphorylation in microglia. Spinal nerve ligation results in a robust microgliosis and sustained ERK1/2 phosphorylation within these cells. This pathway is downstream of neuregulin-1/erbB signaling since its blockade resulted in a significant reduction in microglial ERK1/2 phosphorylation. Inhibition of the MEK/ERK1/2 pathway resulted in decreased spinal microgliosis and in reduced mechanical and cold hypersensitivity after peripheral nerve damage. We conclude that neuregulin-1 released after nerve injury activates microglial erbB receptors which consequently stimulates the MEK/ERK1/2 pathway that drives microglial proliferation and contributes to the development of neuropathic pain.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Microglía/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuralgia/metabolismo , Neurregulina-1/metabolismo , Traumatismos de los Nervios Periféricos , Análisis de Varianza , Animales , Western Blotting , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Microglía/efectos de los fármacos , Neuralgia/fisiopatología , Neurregulina-1/administración & dosificación , Nervios Periféricos/efectos de los fármacos , Nervios Periféricos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas WistarRESUMEN
Immune-neuronal interactions contribute to neuropathic pain. Thus, immune-competent cells such as microglia may provide targets for pain relief, as may infiltrating lymphocytes. We evaluated the nature of the lymphocyte response in the spinal cord in association with the maintenance of neuropathic allodynia. We assessed T cell contribution to pain processing by targeting these cells with Glatiramer acetate (GA) which when administered systemically reversed neuropathic allodynia, inhibited microglia response and increased IL-10 and IL-4 expressing T cells in neuropathic dorsal horns. These studies advance understanding of lymphocyte contribution to chronic pain and reveal a new mechanism of T cell intervention.