RESUMEN
Investigation of neural circuits underlying visceral pain is hampered by the difficulty in achieving selective manipulations of individual circuit components. In this study, we adapted a dual AAV approach, used for projection-specific transgene expression in the CNS, to explore the potential for targeted delivery of transgenes to primary afferent neurons innervating visceral organs. Focusing on the extrinsic sensory innervation of the mouse colon, we first characterized the extent of dual transduction following intrathecal delivery of one AAV9 vector and intracolonic delivery of a second AAV9 vector. We found that if the two AAV9 vectors were delivered one week apart, dorsal root ganglion (DRG) neuron transduction by the second vector was greatly diminished. Following delivery of the two viruses on the same day, we observed colocalization of the transgenes in DRG neurons, indicating dual transduction. Next, we delivered intrathecally an AAV9 vector encoding the inhibitory chemogenetic actuator hM4D(Gi) in a Cre-recombinase dependent manner, and on the same day injected an AAV9 vector carrying Cre-recombinase in the colon. DRG expression of hM4D(Gi) was demonstrated at the mRNA and protein level. However, we were unable to demonstrate selective inhibition of visceral nociception following hM4D(Gi) activation. Taken together, these results establish a foundation for development of strategies for targeted transduction of primary afferent neurons for neuromodulation of peripheral neural circuits.
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Chronic pain remains a significant burden worldwide, and treatments are often limited by safety or efficacy. The decarboxylated form of L-arginine, agmatine, antagonizes N-methyl-d-aspartate receptors, inhibits nitric oxide synthase, and reverses behavioral neuroplasticity. We hypothesized that expressing the proposed synthetic enzyme for agmatine in the sensory pathway could reduce chronic pain without motor deficits. Intrathecal delivery of an adeno-associated viral (AAV) vector carrying the gene for arginine decarboxylase (ADC) prevented the development of chronic neuropathic pain as induced by spared nerve injury in mice and rats and persistently reversed established hypersensitivity 266 days post-injury. Spinal long-term potentiation was inhibited by both exogenous agmatine and AAV-human ADC (hADC) vector pre-treatment but was enhanced in rats treated with anti-agmatine immunoneutralizing antibodies. These data suggest that endogenous agmatine modulates the neuroplasticity associated with chronic pain. Development of approaches to access this inhibitory control of neuroplasticity associated with chronic pain may yield important non-opioid pain-relieving options.
Asunto(s)
Agmatina , Dolor Crónico , Humanos , Ratas , Ratones , Animales , Dolor Crónico/terapia , Roedores/metabolismo , Agmatina/farmacología , Receptores de N-Metil-D-AspartatoRESUMEN
Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products.
Asunto(s)
Dependovirus , Vectores Genéticos , Animales , Dependovirus/genética , Dependovirus/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/genética , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Transducción GenéticaRESUMEN
The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.
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Dependovirus , Vectores Genéticos/farmacocinética , Animales , ADN Viral/análisis , ADN Viral/sangre , Femenino , Vectores Genéticos/administración & dosificación , Inyecciones Espinales , Masculino , Ratones , Ratones Endogámicos ICR , Reacción en Cadena en Tiempo Real de la Polimerasa , Médula Espinal/química , Distribución Tisular , Transducción Genética/métodosRESUMEN
Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). The two current treatments [hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT)], are insufficiently effective in addressing neurologic disease, in part due to the inability of lysosomal enzyme to cross the blood brain barrier. With a goal to more effectively treat neurologic disease, we have investigated the effectiveness of AAV-mediated IDUA gene delivery to the brain using several different routes of administration. Animals were treated by either direct intracerebroventricular (ICV) injection, by intrathecal (IT) infusion into the cerebrospinal fluid, or by intranasal (IN) instillation of AAV9-IDUA vector. AAV9-IDUA was administered to IDUA-deficient mice that were either immunosuppressed with cyclophosphamide (CP), or immunotolerized at birth by weekly injections of human iduronidase. In animals treated by ICV or IT administration, levels of IDUA enzyme ranged from 3- to 1000-fold that of wild type levels in all parts of the microdissected brain. In animals administered vector intranasally, enzyme levels were 100-fold that of wild type in the olfactory bulb, but enzyme expression was close to wild type levels in other parts of the brain. Glycosaminoglycan levels were reduced to normal in ICV and IT treated mice, and in IN treated mice they were normalized in the olfactory bulb, or reduced in other parts of the brain. Immunohistochemical analysis showed extensive IDUA expression in all parts of the brain of ICV treated mice, while IT treated animals showed transduction that was primarily restricted to the hind brain with some sporadic labeling seen in the mid- and fore brain. At 6 months of age, animals were tested for spatial navigation, memory, and neurocognitive function in the Barnes maze; all treated animals were indistinguishable from normal heterozygous control animals, while untreated IDUA deficient animals exhibited significant learning and spatial navigation deficits. We conclude that IT and IN routes are acceptable and alternate routes of administration, respectively, of AAV vector delivery to the brain with effective IDUA expression, while all three routes of administration prevent the emergence of neurocognitive deficiency in a mouse MPS I model.
RESUMEN
The enteric nervous system of the lower gastrointestinal tract comprises intrinsic neural circuits as well as extrinsic afferent and efferent innervation. The development of strategies for neuronal gene transfer has created new opportunities for functional analysis, circuit mapping, and neuromodulation in the enteric nervous system. Studies of AAV-mediated gene transfer to enteric neurons and dorsal root ganglion neurons (DRG) have provided proofs-of-concept for the utility of AAV vectors for genetic manipulations of the intrinsic and extrinsic components of the enteric nervous system. Here we describe a method for AAV-mediated gene transfer to enteric neurons of the descending colon as well as colon-innervating DRG neurons by injection within the intestinal wall (intracolonic injection).
Asunto(s)
Dependovirus/genética , Sistema Nervioso Entérico/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Animales , Femenino , Expresión Génica , Genes Reporteros , Vectores Genéticos/administración & dosificación , Masculino , Ratones , Transducción Genética , TransgenesRESUMEN
Neuroplasticity in the dorsal horn after peripheral nerve damage contributes critically to the establishment of chronic pain. The neurosecretory protein VGF (nonacronymic) is rapidly and robustly upregulated after nerve injury, and therefore, peptides generated from it are positioned to serve as signals for peripheral damage. The goal of this project was to understand the spinal modulatory effects of the C-terminal VGF-derived peptide TLQP-62 at the cellular level and gain insight into the function of the peptide in the development of neuropathic pain. In a rodent model of neuropathic pain, we demonstrate that endogenous levels of TLQP-62 increased in the spinal cord, and its immunoneutralization led to prolonged attenuation of the development of nerve injury-induced hypersensitivity. Using multiphoton imaging of submaximal glutamate-induced Ca responses in spinal cord slices, we demonstrate the ability of TLQP-62 to potentiate glutamatergic responses in the dorsal horn. We further demonstrate that the peptide selectively potentiates responses of high-threshold spinal neurons to mechanical stimuli in singe-unit in vivo recordings. These findings are consistent with a function of TLQP-62 in spinal plasticity that may contribute to central sensitization after nerve damage.
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Hiperalgesia/metabolismo , Plasticidad Neuronal/fisiología , Péptidos/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Médula Espinal/metabolismo , Animales , Calcio/metabolismo , Hiperalgesia/etiología , Hiperalgesia/fisiopatología , Masculino , Ratones , Neuronas/metabolismo , Neuropéptidos/metabolismo , Dimensión del Dolor , Traumatismos de los Nervios Periféricos/complicaciones , Traumatismos de los Nervios Periféricos/fisiopatología , Ratas , Ratas Sprague-Dawley , Médula Espinal/fisiopatologíaRESUMEN
The mu opioid receptor (MOR) and metabotropic glutamate receptor 5 (mGluR5) are well-established pharmacological targets in the management of chronic pain. Both receptors are expressed in the spinal cord. MMG22, a bivalent ligand containing 2 pharmacophores separated by 22 atoms, which simultaneously activates MOR and antagonizes mGluR5, has been shown to produce potent reversal of tactile hypersensitivity in rodent models of lipopolysaccharide (LPS)-and bone cancer-induced chronic pain. This study assessed whether intrathecal MMG22 also is effective in reducing pain of neuropathic origin. Furthermore, we theorized that MMG22 should reduce hyperalgesia in nerve-injured mice in a manner consistent with a synergistic interaction between MOR and mGluR5. Several weeks after spared nerve injury, tactile hypersensitivity was reversed in mice by the intrathecal injection of MMG22 (0.01-10 nmol) but also by its shorter spacer analog, MMG10, with similar potency. The potencies of the bivalent ligands were 10- to 14-fold higher than those of the compounds upon which the bivalent structure was based, the MOR agonist oxymorphone and the mGluR5 antagonist MPEP. Coadministration of oxymorphone and MPEP demonstrated analgesic synergism, an interaction confirmed by isobolographic analysis. This study indicates that in the spared nerve injury-induced model of neuropathic pain, the 2 pharmacophores of the bivalent ligands MMG22 and MMG10 target MOR and mGluR5 as separate receptor monomers. The observed increase in the potency of MMG22 and MMG10, compared with oxymorphone and MPEP, may reflect the synergistic interaction of the 2 pharmacophores of the bivalent ligand acting at their respective separate receptor monomers.
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Analgésicos/uso terapéutico , Antagonistas de Narcóticos/farmacología , Neuralgia/tratamiento farmacológico , Receptor del Glutamato Metabotropico 5/efectos de los fármacos , Receptores Opioides mu/agonistas , Animales , Hiperalgesia/tratamiento farmacológico , Inyecciones Espinales/métodos , Ligandos , Masculino , Ratones , Antagonistas de Narcóticos/administración & dosificaciónRESUMEN
Innate resistance to Candida albicans in mucosal tissues requires the production of interleukin-17A (IL-17A) by tissue-resident cells early during infection, but the mechanism of cytokine production has not been precisely defined. In the skin, we found that dermal γδ T cells were the dominant source of IL-17A during C. albicans infection and were required for pathogen resistance. Induction of IL-17A from dermal γδ T cells and resistance to C. albicans required IL-23 production from CD301b(+) dermal dendritic cells (dDCs). In addition, we found that sensory neurons were directly activated by C. albicans. Ablation of sensory neurons increased susceptibility to C. albicans infection, which could be rescued by exogenous addition of the neuropeptide CGRP. These data define a model in which nociceptive pathways in the skin drive production of IL-23 by CD301b(+) dDCs resulting in IL-17A production from γδ T cells and resistance to cutaneous candidiasis.
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Células Dendríticas/inmunología , Inmunidad/inmunología , Interleucina-23/inmunología , Células Receptoras Sensoriales/inmunología , Piel/inmunología , Animales , Candida albicans/inmunología , Candida albicans/fisiología , Candidiasis/genética , Candidiasis/inmunología , Candidiasis/microbiología , Células Cultivadas , Células Dendríticas/metabolismo , Dermis/citología , Citometría de Flujo , Interacciones Huésped-Patógeno/inmunología , Inmunidad/genética , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/genética , Receptores de Péptido Relacionado con el Gen de Calcitonina/inmunología , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/metabolismo , Piel/metabolismo , Piel/microbiología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5). Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir) in thalamus was most prominent in medial geniculate nucleus, and otherwise limited to posterior nuclei of the dorsal and lateral margins. Labeling was also observed in neurons and astrocytes of the hippocampal formation and amygdaloid complex. In the hippocampal formation, GFP-ir was found in neuronal cell bodies of the rostral ventral portion, but was largely restricted to fiber-like staining in the molecular layer of dentate gyrus and stratum lacunosum-moleculare of the rostral dorsal region. GFP-ir was seen in neurons and astroglia throughout caudal cortex, whereas in rostral regions of neocortex it was limited to isolated neurons and non-neuronal cells. Labeling was also present in olfactory bulb. These results demonstrate that intrathecal delivery of AAV5 vector leads to transgene expression in discrete CNS regions throughout the rostro-caudal extent of the neuraxis. A caudal-to-rostral gradient of decreasing GFP-ir was present in choroid plexus and Purkinje cells, suggesting that spread of virus through cerebrospinal fluid plays a role in the resulting transduction pattern. Other factors contributing to the observed expression pattern likely include variations in cell-surface receptors and inter-parenchymal space.
RESUMEN
Adeno-associated virus serotype 9 (AAV9)-mediated gene transfer has been reported in central nervous system (CNS) and peripheral tissues. The current study compared the pattern of expression of Green Fluorescent Protein (GFP) across the mouse CNS and selected peripheral tissues after intrathecal (i.t.) or intravenous (i.v.) delivery of equivalent doses of single-stranded AAV9 vector. After i.t. delivery, GFP immunoreactivity (-ir) was observed in spinal neurons, primary afferent fibers and corresponding primary sensory neurons at all spinal levels. Robust transduction was seen in small and large dorsal root ganglion (DRG) neurons as well as trigeminal and vagal primary afferent neurons. Transduction efficiency in sensory ganglia was substantially lower in i.v. treated mice. In brain, i.v. delivery yielded GFP-immunoreactivity (-ir) primarily in spinal trigeminal tract, pituitary, and scattered isolated neurons and astrocytes. In contrast, after i.t. delivery, GFP-ir was widespread throughout CNS, with greater intensity and more abundant neuropil-like staining at 6 weeks compared to 3 weeks. Brain regions with prominent GFP-ir included cranial nerve nuclei, ventral pons, cerebellar cortex, hippocampus, pituitary, choroid plexus, and selected nuclei of midbrain, thalamus and hypothalamus. In cortex, GFP-ir was associated with blood vessels, and was seen in both neurons and astrocytes. In the periphery, GFP-ir in colon and ileum was present in the enteric nervous system in both i.v. and i.t. treated mice. Liver and adrenal cortex, but not adrenal medulla, also showed abundant GFP-ir after both routes of delivery. In summary, i.t. delivery yielded higher transduction efficiency in sensory neurons and the CNS. The observation of comparable gene transfer to peripheral tissues using the two routes indicates that a component of i.t. delivered vector is redistributed from the subarachnoid space to the systemic circulation.
RESUMEN
VGF (nonacronymic) is a granin-like protein that is packaged and proteolytically processed within the regulated secretory pathway. VGF and peptides derived from its processing have been implicated in neuroplasticity associated with learning, memory, depression, and chronic pain. In sensory neurons, VGF is rapidly increased following peripheral nerve injury and inflammation. Several bioactive peptides generated from the C-terminus of VGF have pronociceptive spinal effects. The goal of the present study was to examine the spinal effects of the peptide TLQP-21 and determine whether it participates in spinal mechanisms of persistent pain. Application of exogenous TLQP-21 induced dose-dependent thermal hyperalgesia in the warm-water immersion tail-withdrawal test. This hyperalgesia was inhibited by a p38 mitogen-activated protein kinase inhibitor, as well as inhibitors of cyclooxygenase and lipoxygenase. We used immunoneutralization of TLQP-21 to determine the function of the endogenous peptide in mechanisms underlying persistent pain. In mice injected intradermally with complete Freund adjuvant, intrathecal treatment with anti-TLQP-21 immediately prior to or 5hours after induction of inflammation dose-dependently inhibited tactile hypersensitivity and thermal hyperalgesia. Intrathecal anti-TL21 administration also attenuated the development and maintenance of tactile hypersensitivity in the spared nerve injury model of neuropathic pain. These results provide evidence that endogenous TLQP-21 peptide contributes to the mechanisms of spinal neuroplasticity after inflammation and nerve injury.
Asunto(s)
Hiperalgesia/metabolismo , Inflamación/metabolismo , Neuralgia/metabolismo , Neuropéptidos/metabolismo , Nocicepción/fisiología , Fragmentos de Péptidos/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Animales , Inhibidores de la Ciclooxigenasa/farmacología , Modelos Animales de Enfermedad , Adyuvante de Freund/envenenamiento , Calor , Hiperalgesia/inducido químicamente , Inflamación/inducido químicamente , Inyecciones Espinales , Inhibidores de la Lipooxigenasa/farmacología , Ratones , Factores de Crecimiento Nervioso , Nocicepción/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Nervio Peroneo/lesiones , Piel/efectos de los fármacos , Nervio Tibial/lesiones , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/ß-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.
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Neuropéptidos/metabolismo , Fragmentos de Péptidos/farmacología , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Composición Corporal , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/metabolismo , Masculino , Ratones , Células 3T3 NIH , Factores de Crecimiento Nervioso , Obesidad/inducido químicamente , Obesidad/metabolismo , Unión Proteica , Transporte de Proteínas , Receptores de Superficie CelularRESUMEN
BACKGROUND: Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. RESULTS: Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP. CONCLUSIONS: The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.
Asunto(s)
Dependovirus , Ganglios Espinales/fisiología , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Manejo del Dolor , Células Receptoras Sensoriales/fisiología , Animales , Proteínas Fluorescentes Verdes/genética , Inyecciones Espinales , Masculino , Ratones , Ratones Endogámicos C57BL , Punción EspinalRESUMEN
Peripheral tissue injury is associated with changes in protein expression in sensory neurons that may contribute to abnormal nociceptive processing. We used cultured dorsal root ganglion (DRG) neurons as a model of axotomized neurons to investigate early changes in protein expression after nerve injury. Comparing protein levels immediately after DRG dissociation and 24 h later by proteomic differential expression analysis, we found a substantial increase in the levels of the neurotrophin-inducible protein VGF (nonacronymic), a putative neuropeptide precursor. In a rodent model of nerve injury, VGF levels were increased within 24 h in both injured and uninjured DRG neurons, and the increase persisted for at least 7 d. VGF was also upregulated 24 h after hindpaw inflammation. To determine whether peptides derived from proteolytic processing of VGF participate in nociceptive signaling, we examined the spinal effects of AQEE-30 and LQEQ-19, potential proteolytic products shown previously to be bioactive. Each peptide evoked dose-dependent thermal hyperalgesia that required activation of the mitogen-activated protein kinase p38. In addition, LQEQ-19 induced p38 phosphorylation in spinal microglia when injected intrathecally and in the BV-2 microglial cell line when applied in vitro. In summary, our results demonstrate rapid upregulation of VGF in sensory neurons after nerve injury and inflammation and activation of microglial p38 by VGF peptides. Therefore, VGF peptides released from sensory neurons may participate in activation of spinal microglia after peripheral tissue injury.
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Neuropéptidos/metabolismo , Nociceptores/fisiología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Proteómica , Animales , Benzoxazoles/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Adyuvante de Freund , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Imidazoles/farmacología , Inflamación/inducido químicamente , Inflamación/metabolismo , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas Aferentes/metabolismo , Neuropéptidos/química , Neuropéptidos/farmacología , Técnicas de Cultivo de Órganos , Dimensión del Dolor/efectos de los fármacos , Péptidos/farmacología , Enfermedades del Sistema Nervioso Periférico/etiología , Enfermedades del Sistema Nervioso Periférico/patología , Piridinas/farmacología , Compuestos de Quinolinio/metabolismo , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Receptor trkA/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X3 , Transducción de Señal/efectos de los fármacos , Traumatismos de la Médula Espinal/metabolismo , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Agonists acting at alpha(2)-adrenergic and opioid receptors (alpha(2)ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although the existence of alpha(2)AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of heterooligomers has been proposed as a molecular basis for interactions between neuronal G-protein-coupled receptors. The relevance of heterooligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between alpha(2)ARs and ORs in the rat spinal cord to determine whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between alpha(2A)-adrenergic and delta-opioid receptors (DOP) on substance P (SP)-immunoreactive (-ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. alpha(2A)AR- and DOP-ir elements were colocalized in subcellular structures of 0.5 mum or less in diameter in isolated nerve terminals. Furthermore, coincubation of isolated synaptosomes with alpha(2)AR and DOP agonists resulted in a greater-than-additive increase in the inhibition of K(+)-stimulated neuropeptide release. These findings suggest that coexpression of the synergistic receptor pair alpha(2A)AR-DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein-protein interactions such as heterooligomerization.
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Células del Asta Posterior/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Opioides delta/metabolismo , Sustancia P/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Animales , Inmunohistoquímica , Masculino , Microscopía Confocal , Neuropéptidos/metabolismo , Nociceptores/metabolismo , Nociceptores/ultraestructura , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Opioides delta/agonistas , Piel/inervación , Sinaptosomas/metabolismoRESUMEN
Agmatine (decarboxylated arginine) was originally identified in the CNS as an imidazoline receptor ligand. Further studies demonstrated that agmatine antagonizes NMDA receptors and inhibits nitric oxide synthase. Intrathecally administered agmatine inhibits opioid tolerance and hyperalgesia evoked by inflammation, nerve injury, and intrathecally administered NMDA. These actions suggest an anti-glutamatergic role for agmatine in the spinal cord. We have previously reported that radiolabeled agmatine is transported into spinal synaptosomes in an energy- and temperature-dependent manner. In the present study, we demonstrate that agmatine is releasable from purified spinal nerve terminals upon depolarization. When exposed to either elevated potassium or capsaicin, tritiated agmatine (but not its precursor L-arginine or its metabolite putrescine) is released in a calcium-dependent manner. Control experiments confirmed that the observed release was specific to depolarization and not due to permeabilization of or degradation of synaptosomes. That capsaicin-evoked stimulation results in agmatine release implicates the participation of primary afferent nerve terminals. Radiolabeled agmatine also accumulates in purified spinal synaptosomal vesicles in a temperature-dependent manner, suggesting that the source of releasable agmatine may be vesicular in origin. These results support the proposal that agmatine may serve as a spinal neuromodulator involved in pain processing.
Asunto(s)
Vías Aferentes/metabolismo , Agmatina/metabolismo , Nociceptores/metabolismo , Terminales Presinápticos/metabolismo , Médula Espinal/metabolismo , Raíces Nerviosas Espinales/metabolismo , Vías Aferentes/efectos de los fármacos , Vías Aferentes/ultraestructura , Animales , Capsaicina/farmacología , Masculino , Fibras Nerviosas Amielínicas/efectos de los fármacos , Fibras Nerviosas Amielínicas/metabolismo , Fibras Nerviosas Amielínicas/ultraestructura , Nociceptores/efectos de los fármacos , Nociceptores/ultraestructura , Dolor/metabolismo , Dolor/fisiopatología , Cloruro de Potasio/metabolismo , Cloruro de Potasio/farmacología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/ultraestructura , Raíces Nerviosas Espinales/efectos de los fármacos , Raíces Nerviosas Espinales/ultraestructura , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Sinaptosomas/ultraestructuraRESUMEN
Lectins are proteins that bind to glycoproteins and glycolipids. The isolectin Griffonia simplicifolia I-B4 (IB4) recognizes terminal alpha-galactose and binds to a subset of small and medium-sized neurons in the dorsal root ganglia (DRG). Using one and two-dimensional gel electrophoresis, we have identified several proteins that bind IB4 in sciatic nerve, dorsal horn, and DRG. Treatment with the enzyme alpha-galactosidase reduces IB4 binding, strongly suggesting the binding is specific for the IB4 epitope. Mass spectrometric analysis of tryptic digests of alpha-galactosidase sensitive bands identified three proteins that bind IB4: the laminin beta 2 chain and the light and medium subunits of neurofilaments.
Asunto(s)
Ganglios Espinales/metabolismo , Lectinas de Plantas/metabolismo , Animales , Ganglios Espinales/química , Glicoproteínas/metabolismo , Griffonia , Masculino , Lectinas de Plantas/aislamiento & purificación , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Coloración y EtiquetadoRESUMEN
Morphine and other opioids have direct analgesic actions in the spinal cord and chronic spinal administration of opioid agonists is used clinically in patients suffering from severe, chronic pain. Neuropathic pain resulting from peripheral nerve injury is often less sensitive to opioid therapy than other forms of chronic pain in both humans and animal models. Changes in spinal mu-opioid receptor (MOR) expression have been demonstrated in animal models of neuropathic pain. However, these changes alone fail to account for the attenuation of opioid activity. Reduced expression of delta-opioid receptors (DOR) following peripheral nerve injury has been reported but most of these reports are limited to subjective observation. The magnitude and consistency of these changes is therefore unclear. In addition, previous studies did not evaluate the effects of nerve injury on behavioral measures to confirm induction of aberrant pain symptoms. We therefore performed quantitative image analysis to evaluate the effect of peripheral nerve injury on DOR-immunoreactivity in spinal cord sections from rats previously characterized for sensory responsiveness. We observed statistically significant decreases ipsilateral to nerve injury in all three models tested: sciatic nerve transection, chronic constriction injury of the sciatic nerve and L5/L6 spinal nerve ligation. These results suggest that decreases in the expression of DOR are a common feature of peripheral nerve injury.