RESUMEN
The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T-cell depletion or positive selection of CD34(+) cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft-versus-host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN ) cells from donor G-CSF-mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720-treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory T cells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G-CSF-mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient.
Asunto(s)
Clorhidrato de Fingolimod/farmacología , Ingeniería Genética , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Animales , Ingeniería Genética/métodos , Supervivencia de Injerto/genética , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/patología , Factor Estimulante de Colonias de Granulocitos/farmacología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Depleción Linfocítica , Ratones , Ratones Noqueados , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Trasplante HomólogoRESUMEN
Painful peripheral neuropathy often occurs without apparent underlying cause. Gain-of-function variants of sodium channel Na(v)1.7 have recently been found in â¼30% of cases of idiopathic painful small-fiber neuropathy. Here, we describe mutations in Na(v)1.8, another sodium channel that is specifically expressed in dorsal root ganglion (DRG) neurons and peripheral nerve axons, in patients with painful neuropathy. Seven Na(v)1.8 mutations were identified in 9 subjects within a series of 104 patients with painful predominantly small-fiber neuropathy. Three mutations met criteria for potential pathogenicity based on predictive algorithms and were assessed by voltage and current clamp. Functional profiling showed that two of these three Na(v)1.8 mutations enhance the channel's response to depolarization and produce hyperexcitability in DRG neurons. These observations suggest that mutations of Na(v)1.8 contribute to painful peripheral neuropathy.
Asunto(s)
Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.8/genética , Neuralgia/genética , Adulto , Anciano , Sustitución de Aminoácidos/genética , Animales , Análisis Mutacional de ADN , Fenómenos Electrofisiológicos , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Humanos , Masculino , Ratones , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Neuralgia/fisiopatologíaRESUMEN
Axonal degeneration occurs in multiple neurodegenerative disorders of the central and peripheral nervous system. Although the underlying molecular pathways leading to axonal degeneration are incompletely understood, accumulating evidence suggests contributions of impaired mitochondrial function, disrupted axonal transport, and/or dysfunctional intracellular Ca(2+)-homeostasis in the injurious cascade associated with axonal degeneration. Utilizing an in vitro model of axonal degeneration, we studied a subset of mouse peripheral sensory neurons in which neurites were exposed selectively to conditions associated with the pathogenesis of axonal neuropathies in vivo. Rotenone-induced mitochondrial dysfunction resulted in neurite degeneration accompanied by reduced ATP levels and increased ROS levels in neurites. Blockade of voltage-gated sodium channels with TTX and reverse (Ca(2+)-importing) mode of the sodium-calcium exchanger (NCX) with KB-R7943 partially protected rotenone-treated neurites from degeneration, suggesting a contribution of sodium channels and reverse NCX activity to the degeneration of neurites resulting from impaired mitochondrial function. Pharmacological inhibition of the Na(+)/K(+)-ATPase with ouabain induced neurite degeneration, which was attenuated by TTX and KB-R7943, supporting a contribution of sodium channels in axonal degenerative pathways accompanying impaired Na(+)/K(+)-ATPase activity. Conversely, oxidant stress (H2O2)-induced neurite degeneration was not attenuated by TTX. Our results demonstrate that both energetic and oxidative stress targeted selectively to neurites induces neurite degeneration and that blockade of sodium channels and of reverse NCX activity blockade partially protects neurites from injury due to energetic stress, but not from oxidative stress induced by H2O2.
Asunto(s)
Axones/fisiología , Ganglios Espinales/fisiología , Enfermedades Mitocondriales/fisiopatología , Degeneración Nerviosa/fisiopatología , Neuritas/fisiología , Canales de Sodio/fisiología , Animales , Axotomía , Muerte Celular/fisiología , Células Cultivadas , Ganglios Espinales/citología , Humanos , Peróxido de Hidrógeno/toxicidad , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microtúbulos/fisiología , Neuritas/ultraestructura , Oxidantes/toxicidad , Rotenona/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Tetrodotoxina/toxicidad , Tiourea/análogos & derivados , Tiourea/farmacología , Desacopladores/farmacologíaRESUMEN
Microglia are motile resident immune cells of the central nervous system (CNS) that continuously explore their territories for threats to tissue homeostasis. Following CNS insult (e.g., cellular injury, infection, or ischemia), microglia respond to signals such as ATP, transform into an activated state, and migrate towards the threat. Directed migration is a complex and highly-coordinated process involving multiple intersecting cellular pathways, including signal transduction, membrane adhesion and retraction, cellular polarization, and rearrangement of cytoskeletal elements. We previously demonstrated that the activity of sodium channels contributes to ATP-induced migration of microglia. Here we show that TTX-sensitive sodium channels, specifically NaV 1.6, participate in an initial event in the migratory process, i.e., the formation in ATP-stimulated microglia of polymerized actin-rich membrane protrusions, lamellipodia, containing accumulations of Rac1 and phosphorylated ERK1/2. We also examined Ca(2+) transients in microglia and found that blockade of sodium channels with TTX produced a downward shift in the level of [Ca(2+) ]i during the delayed, slower recovery of [Ca(2+) ]i following ATP stimulation. These observations demonstrate a modulatory role of sodium channels on Ca(2+) transients in microglia that are likely to affect down-stream signaling cascades. Consistent with these observations, we demonstrate that ATP-induced microglial migration is mediated via Rac1 and ERK1/2, but not p38α/ß and JNK, dependent pathways, and that activation of both Rac1 and ERK1/2 is modulated by sodium channel activity. Our results provide evidence for a direct link between sodium channel activity and modulation of Rac1 and ERK1/2 activation in ATP-stimulated microglia, possibly by regulating Ca(2+) transients.
Asunto(s)
Adenosina Trifosfato/farmacología , Microglía , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Seudópodos/fisiología , Proteína de Unión al GTP rac1/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Transgénicos , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Seudópodos/efectos de los fármacos , Seudópodos/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
Small-fiber neuropathy (SFN) is characterized by injury to small-diameter peripheral nerve axons and intraepidermal nerve fibers (IENF). Although mechanisms underlying loss of IENF in SFN are poorly understood, available data suggest that it results from axonal degeneration and reduced regenerative capacity. Gain-of-function variants in sodium channel Na(V)1.7 that increase firing frequency and spontaneous firing of dorsal root ganglion (DRG) neurons have recently been identified in â¼30% of patients with idiopathic SFN. In the present study, to determine whether these channel variants can impair axonal integrity, we developed an in vitro assay of DRG neurite length, and examined the effect of 3 SFN-associated variant Na(V)1.7 channels, I228M, M932L/V991L (ML/VL), and I720K, on DRG neurites in vitro. At 3 days after culturing, DRG neurons transfected with I228M channels exhibited â¼20% reduced neurite length compared to wild-type channels; DRG neurons transfected with ML/VL and I720K variants displayed a trend toward reduced neurite length. I228M-induced reduction in neurite length was ameliorated by the use-dependent sodium channel blocker carbamazepine and by a blocker of reverse Na-Ca exchange. These in vitro observations provide evidence supporting a contribution of the I228M variant Na(V)1.7 channel to impaired regeneration and/or degeneration of sensory axons in idiopathic SFN, and suggest that enhanced sodium channel activity and reverse Na-Ca exchange can contribute to a decrease in length of peripheral sensory axons.
Asunto(s)
Axones/fisiología , Ganglios Espinales/fisiología , Variación Genética/genética , Canal de Sodio Activado por Voltaje NAV1.7/genética , Enfermedades del Sistema Nervioso Periférico/genética , Animales , Axones/patología , Muerte Celular/genética , Células Cultivadas , Ganglios Espinales/patología , Humanos , Enfermedades del Sistema Nervioso Periférico/patología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/patología , Células Receptoras Sensoriales/fisiologíaRESUMEN
Diabetic neuropathic pain imposes a huge burden on individuals and society, and represents a major public health problem. Despite aggressive efforts, diabetic neuropathic pain is generally refractory to available clinical treatments. A structure-function link between maladaptive dendritic spine plasticity and pain has been demonstrated previously in CNS and PNS injury models of neuropathic pain. Here, we reasoned that if dendritic spine remodeling contributes to diabetic neuropathic pain, then (1) the presence of malformed spines should coincide with the development of pain, and (2) disrupting maladaptive spine structure should reduce chronic pain. To determine whether dendritic spine remodeling contributes to neuropathic pain in streptozotocin (STZ)-induced diabetic rats, we analyzed dendritic spine morphology and electrophysiological and behavioral signs of neuropathic pain. Our results show changes in dendritic spine shape, distribution, and shape on wide-dynamic-range (WDR) neurons within lamina IV-V of the dorsal horn in diabetes. These diabetes-induced changes were accompanied by WDR neuron hyperexcitability and decreased pain thresholds at 4 weeks. Treatment with NSC23766 (N(6)-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride), a Rac1-specific inhibitor known to interfere with spine plasticity, decreased the presence of malformed spines in diabetes, attenuated neuronal hyperresponsiveness to peripheral stimuli, reduced spontaneous firing activity from WDR neurons, and improved nociceptive mechanical pain thresholds. At 1 week after STZ injection, animals with hyperglycemia with no evidence of pain had few or no changes in spine morphology. These results demonstrate that diabetes-induced maladaptive dendritic spine remodeling has a mechanistic role in neuropathic pain. Molecular pathways that control spine morphogenesis and plasticity may be promising future targets for treatment.
Asunto(s)
Aminoquinolinas/uso terapéutico , Espinas Dendríticas/patología , Neuropatías Diabéticas/patología , Plasticidad Neuronal/fisiología , Umbral del Dolor/fisiología , Pirimidinas/uso terapéutico , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Aminoquinolinas/administración & dosificación , Aminoquinolinas/farmacología , Animales , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/fisiología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/fisiopatología , Neuropatías Diabéticas/complicaciones , Neuropatías Diabéticas/tratamiento farmacológico , Neuropatías Diabéticas/fisiopatología , Inyecciones Espinales , Masculino , Plasticidad Neuronal/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/patología , Células del Asta Posterior/fisiopatología , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
Chronic neuropathic pain is affected by specifics of the precipitating neural pathology, psychosocial factors, and by genetic predisposition. Little is known about the identity of predisposing genes. Using an integrative approach, we discovered that CACNG2 significantly affects susceptibility to chronic pain following nerve injury. CACNG2 encodes for stargazin, a protein intimately involved in the trafficking of glutamatergic AMPA receptors. The protein might also be a Ca(2+) channel subunit. CACNG2 has previously been implicated in epilepsy. Initially, using two fine-mapping strategies in a mouse model (recombinant progeny testing [RPT] and recombinant inbred segregation test [RIST]), we mapped a pain-related quantitative trait locus (QTL) (Pain1) into a 4.2-Mb interval on chromosome 15. This interval includes 155 genes. Subsequently, bioinformatics and whole-genome microarray expression analysis were used to narrow the list of candidates and ultimately to pinpoint Cacng2 as a likely candidate. Analysis of stargazer mice, a Cacng2 hypomorphic mutant, provided electrophysiological and behavioral evidence for the gene's functional role in pain processing. Finally, we showed that human CACNG2 polymorphisms are associated with chronic pain in a cohort of cancer patients who underwent breast surgery. Our findings provide novel information on the genetic basis of neuropathic pain and new insights into pain physiology that may ultimately enable better treatments.
Asunto(s)
Canales de Calcio/genética , Neuralgia/genética , Animales , Canales de Calcio/metabolismo , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Ratones , Ratones Endogámicos , Fenotipo , Polimorfismo Genético , Sitios de Carácter Cuantitativo , Nervios Espinales/lesionesRESUMEN
Clostridium difficile-associated disease (CDAD) with frequent watery stools, sometimes with painful bowel movements, fever and sickness, is probably the major known cause of antibiotic-associated diarrhea and colitis, most probably depending on a disruption of the normal intestinal balance in the microbiome. In this study, we have inoculated a mixture of fecal microbes--as an enema--originating from a healthy Scandinavian middle-aged donor, regularly re-cultivated under strict anaerobic conditions for more than 10 years, to 32 patients. Twenty-two patients (69%) were durably cured. In those patients receiving the transplant by colonoscopy, four out of five were cured. To the best of our knowledge, this is the first time a fecal culture of microbes has retained the possibility for years to cure a substantial number of patients with CDAD.
Asunto(s)
Clostridioides difficile , Diarrea/microbiología , Diarrea/terapia , Heces/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Bacterias Anaerobias , Terapia Biológica , Infecciones por Clostridium/complicaciones , Infecciones por Clostridium/tratamiento farmacológico , Colon/microbiología , Colonoscopía , Enema , Femenino , Humanos , Masculino , Metagenoma/efectos de los fármacos , Persona de Mediana Edad , Recto/microbiología , RecurrenciaRESUMEN
BACKGROUND: Nociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX) and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals. RESULTS: Small diameter DRG neurons exhibited robust NCX2, but not NCX1 or NCX3 immunolabeling, and virtually all PGP 9.5-positive intra-epidermal free nerve terminals displayed NCX2 immunoreactivity. Sodium channel NaV1.1 was not detectable in free nerve endings. In contrast, the majority of nerve terminals displayed detectable levels of expression of NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Sodium channel immunoreactivity in the free nerve endings extended from the dermal boundary to the terminal tip. A similar pattern of NCX and sodium channel immunolabeling was observed in DRG neurons in vitro. CONCLUSIONS: NCX2, as well as NaV1.6, NaV1.7, NaV1.8 and NaV1.9, are present in most intra-epidermal free nerve endings. The presence of NCX2, together with multiple sodium channel isoforms, in free nerve endings may have important functional implications.
Asunto(s)
Epidermis/inervación , Terminaciones Nerviosas/química , Canales de Sodio/análisis , Intercambiador de Sodio-Calcio/análisis , Animales , Inmunohistoquímica , Masculino , Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Neuropéptidos , Nociceptores , Isoformas de Proteínas/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Neuropathic pain that develops after trauma to a nerve may be caused by altered transcription of genes in the damaged neurons. We have previously investigated the effect of nerve injury on the expression of six dorsal root ganglion (DRG) pain candidate molecules in five inbred mouse strains with different pain phenotypes after nerve injury. In this study, we present a detailed morphological examination of mRNA expression in the DRG in the same mouse strains. For Na(v) 1.9, TRPA1, and TRPM8, the size spectra of labeled neurons remained mostly unchanged after injury in all strains. However, in CBA, AKR, and C58 mice, injury caused a preferential downregulation of Na(v) 1.8 in large diameter neurons. In CBA mice there was a shift toward larger neuronal profiles expressing TRPV1 after injury, indicating de novo (or upregulated) expression of TRPV1 in a subpopulation of neurons that normally does not express this gene. Finally, in C58 mice there was a shift toward smaller P2X3-expressing neuronal profiles after injury, suggesting that a loss of P2X3 mRNA transcript occurred preferentially in medium-sized cells. We used a multivariate statistical model to compare the regulation patterns of the six DRG genes. Clustering patterns suggested that genes of similar phylogenetic origin and function are regulated similarly.
Asunto(s)
Ganglios Espinales/metabolismo , Neuralgia/metabolismo , Neuronas/metabolismo , Nervios Espinales/lesiones , Animales , Análisis Factorial , Ganglios Espinales/citología , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos CBA , Ratones Endogámicos , Análisis Multivariante , Neuralgia/genética , Neuronas/citología , Fenotipo , ARN Mensajero/metabolismo , Especificidad de la EspecieRESUMEN
BACKGROUND: Nerve injury-triggered hyperexcitability in primary sensory neurons is considered a major source of chronic neuropathic pain. The hyperexcitability, in turn, is thought to be related to transcriptional switching in afferent cell somata. Analysis using expression microarrays has revealed that many genes are regulated in the dorsal root ganglion (DRG) following axotomy. But which contribute to pain phenotype versus other nerve injury-evoked processes such as nerve regeneration? Using the L5 spinal nerve ligation model of neuropathy we examined differential changes in gene expression in the L5 (and L4) DRGs in five mouse strains with contrasting susceptibility to neuropathic pain. We sought genes for which the degree of regulation correlates with strain-specific pain phenotype. RESULTS: In an initial experiment six candidate genes previously identified as important in pain physiology were selected for in situ hybridization to DRG sections. Among these, regulation of the Na+ channel alpha subunit Scn11a correlated with levels of spontaneous pain behavior, and regulation of the cool receptor Trpm8 correlated with heat hypersensibility. In a larger scale experiment, mRNA extracted from individual mouse DRGs was processed on Affymetrix whole-genome expression microarrays. Overall, 2552 +/- 477 transcripts were significantly regulated in the axotomized L5DRG 3 days postoperatively. However, in only a small fraction of these was the degree of regulation correlated with pain behavior across strains. Very few genes in the "uninjured" L4DRG showed altered expression (24 +/- 28). CONCLUSION: Correlational analysis based on in situ hybridization provided evidence that differential regulation of Scn11a and Trpm8 contributes to across-strain variability in pain phenotype. This does not, of course, constitute evidence that the others are unrelated to pain. Correlational analysis based on microarray data yielded a larger "look-up table" of genes whose regulation likely contributes to pain variability. While this list is enriched in genes of potential importance for pain physiology, and is relatively free of the bias inherent in the candidate gene approach, additional steps are required to clarify which transcripts on the list are in fact of functional importance.
Asunto(s)
Neuralgia/genética , Neuropéptidos/genética , Canales de Sodio/genética , Canales Catiónicos TRPM/genética , Animales , Ganglios Espinales/fisiopatología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Canal de Sodio Activado por Voltaje NAV1.9 , ARN Mensajero/análisis , Especificidad de la EspecieRESUMEN
Voltage-gated sodium channels NaV1.7, NaV1.8 and NaV1.9 have been the focus for pain studies because their mutations are associated with human pain disorders, but the role of NaV1.6 in pain is less understood. In this study, we selectively knocked out NaV1.6 in dorsal root ganglion (DRG) neurons, using NaV1.8-Cre directed or adeno-associated virus (AAV)-Cre mediated approaches, and examined the specific contribution of NaV1.6 to the tetrodotoxin-sensitive (TTX-S) current in these neurons and its role in neuropathic pain. We report here that NaV1.6 contributes up to 60% of the TTX-S current in large, and 34% in small DRG neurons. We also show NaV1.6 accumulates at nodes of Ranvier within the neuroma following spared nerve injury (SNI). Although NaV1.8-Cre driven NaV1.6 knockout does not alter acute, inflammatory or neuropathic pain behaviors, AAV-Cre mediated NaV1.6 knockout in adult mice partially attenuates SNI-induced mechanical allodynia. Additionally, AAV-Cre mediated NaV1.6 knockout, mostly in large DRG neurons, significantly attenuates excitability of these neurons after SNI and reduces NaV1.6 accumulation at nodes of Ranvier at the neuroma. Together, NaV1.6 in NaV1.8-positive neurons does not influence pain thresholds under normal or pathological conditions, but NaV1.6 in large NaV1.8-negative DRG neurons plays an important role in neuropathic pain.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Neuralgia/genética , Animales , Femenino , Ganglios Espinales/metabolismo , Hiperalgesia/genética , Hiperalgesia/metabolismo , Masculino , Ratones , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.3/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/fisiología , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Tejido Nervioso/metabolismo , Neuralgia/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Canales de Sodio Activados por Voltaje/genética , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Peripheral neuropathy results from damage to peripheral nerves and is often accompanied by pain in affected limbs. Treatment represents an unmet medical need and a thorough understanding of the mechanisms underlying axonal injury is needed. Longer nerve fibers tend to degenerate first (length-dependence), and patients carrying pathogenic mutations throughout life usually become symptomatic in mid- or late-life (time-dependence). The activity of voltage-gated sodium channels can contribute to axonal injury and sodium channel gain-of-function mutations have been linked to peripheral neuropathy. Recent studies have implicated sodium channel activity, mitochondrial compromise, and reverse-mode Na(+)/Ca(2+) exchange in time- and length-dependent axonal injury. Elucidation of molecular mechanisms underlying axonal injury in peripheral neuropathy may provide new therapeutic strategies for this painful and debilitating condition.
Asunto(s)
Axones/patología , Mitocondrias/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Canales de Sodio/metabolismo , Animales , Axones/metabolismo , Humanos , Mitocondrias/patología , Mutación , Degeneración Nerviosa , Dolor/etiología , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/patología , Canales de Sodio/genéticaRESUMEN
We used a photochemical method to generate a partial ischemic injury to the infraorbital branch of the trigeminal nerve in rats. Following injury, rats developed a bilateral persistent hypersensitivity to mechanical stimulation in the territory innervated by the infraorbital nerve. In addition, spread of mechanical hypersensitivity beyond the facial region was noted. Heat hypersensitivity was also present, although to a lesser extent and of a shorter duration. In some rats, excessive facial grooming/scratching were observed. Morphological examination revealed a graded damage to the irradiated portion of the infraorbital nerve that was related to the duration of laser irradiation. Investigations of gene expression changes in injured trigeminal ganglion neurons of animals with behavioral signs of neuropathic pain demonstrated that the sodium channel alpha-subunit Na(v)1.3-absent in sham-operated animals-was expressed to a limited extent. mRNAs for Na(v)1.8 and Na(v)1.9 were reduced both with respect to proportions of expressing neurons and to intensities, whereas the beta 3 subunit was markedly upregulated. mRNA levels of p11, a regulatory factor that facilitates the surface expression of Na(v)1.8, were unchanged. Previous findings have shown that injury to the trigeminal nerve branches may elicit responses that differ from those of segmental spinal nerves. Despite this we conclude that the key sodium channel regulations that are reported as consequences of nerve damage in the dorsal root ganglia seem to appear also in the trigeminal ganglion. Thus, novel analgesic drugs designed to target the sodium channel subtypes involved could be of use for the treatment of orofacial pain.
Asunto(s)
Modelos Animales de Enfermedad , Dolor Facial/fisiopatología , Hiperestesia/fisiopatología , Trastornos Mentales/fisiopatología , Canales de Sodio/metabolismo , Ganglio del Trigémino/metabolismo , Adaptación Fisiológica , Animales , Dolor Facial/complicaciones , Hiperestesia/complicaciones , Masculino , Trastornos Mentales/etiología , Ratas , Ratas Sprague-DawleyRESUMEN
The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology. The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator. The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.
Asunto(s)
Animales Recién Nacidos , Transferencia de Embrión/veterinaria , Vida Libre de Gérmenes , Animales , Cruzamientos Genéticos , Transferencia de Embrión/instrumentación , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , EmbarazoAsunto(s)
Neuralgia/genética , Animales , Enfermedad Crónica , Costo de Enfermedad , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Variación Genética , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Humanos , Hiperalgesia/genética , Ratones , Ratones Endogámicos , Nociceptores , Insensibilidad Congénita al Dolor/genética , Polimorfismo GenéticoAsunto(s)
Diarrea/terapia , Enema/métodos , Enterocolitis Seudomembranosa/terapia , Heces/microbiología , Anciano , Anciano de 80 o más Años , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Terapia Biológica/métodos , Clostridioides difficile , Diarrea/microbiología , Enterocolitis Seudomembranosa/tratamiento farmacológico , Enterocolitis Seudomembranosa/microbiología , Femenino , Humanos , Metronidazol/efectos adversos , Metronidazol/uso terapéutico , Recurrencia , Resultado del Tratamiento , Vancomicina/efectos adversos , Vancomicina/uso terapéuticoRESUMEN
Peripheral nerve injury can result in formation of a neuroma, which is often associated with heightened sensitivity to normally innocuous stimuli as well as spontaneous dysesthesia and pain. The onset and persistence of neuropathic pain have been linked to spontaneous ectopic electrogenesis in axons within neuromas, suggesting an involvement of voltage-gated sodium channels. Sodium channel isoforms Na(V)1.3, Na(V)1.7 and Na(V)1.8 have been shown to accumulate in chronic painful human neuromas, while, to date, only Na(V)1.3 has been reported to accumulate within experimental neuromas. Although recent evidence strongly support a major contribution for Na(V)1.7 in nociception, the expression of Na(V)1.7 in injured axons within acute neuromas has not been studied. The current study examined whether Na(V)1.7 accumulates in experimental rat neuromas. We further investigated whether activated (phosphorylated) mitogen-activated protein (MAP) kinase ERK1/2, which is known to modulate Na(V)1.7 properties, is co-localized with Na(V)1.7 within axons in neuromas. We demonstrate increased levels of Na(V)1.7 in experimental rat sciatic nerve neuromas, 2weeks after nerve ligation and transaction. We further show elevated levels of phosphorylated ERK1/2 within individual neuroma axons that exhibit Na(V)1.7 accumulation. These results extend previous descriptions of sodium channel and MAP kinase accumulation within experimental and human neuromas, and suggest that targeted blockade of Na(V)1.7 or ERK1/2 may provide a strategy for amelioration of chronic pain that often follows nerve injury and formation of neuromas.
Asunto(s)
Axones/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuroma/metabolismo , Neoplasias del Sistema Nervioso Periférico/metabolismo , Neuropatía Ciática/metabolismo , Canales de Sodio/metabolismo , Animales , Western Blotting , Inmunohistoquímica , Canal de Sodio Activado por Voltaje NAV1.7 , Fosforilación/fisiología , Ratas , Ratas Sprague-Dawley , Nervio Ciático/metabolismoRESUMEN
The developmental pattern of sodium channel expression in neurons of primary sensory ganglia is likely reflected in the electrical behavior of these cells. Little information is available on how voltage-gated sodium channels in sensory neurons are expressed during development in the trigeminal-innervated craniofacial region, where sensitivity is fundamental during early stages of life. Using in situ hybridization, we here demonstrate a differential both regional and transcript-dependent distribution of sodium channel alpha- and beta-subunits between Embryonic day (E)15 and Postnatal day (P)5/6 in the rat trigeminal ganglion. Na(v)1.3 mRNA was strongly expressed at E15, but declined to low levels at P5/P6. Na(v)1.8 was expressed at E15, increased to reach maximum levels at P1 and then decreased. Na(v)1.9 mRNA was detected at E19, reached a maximum at P1, and was then reduced. beta1 mRNA showed a steady rise in expression from E17, while beta2 mRNA was widely expressed from P1. beta 3 mRNA was detected at E15, reached a maximum at E19 followed by a decrease in expression. In the ophthalmic TG portion, there was a higher expression level of Na(v)1.8 and Na(v)1.9 between E19 and P5/P6 as compared to the maxillary/mandibular part, indicating an unexpected positional difference in channel distribution. mRNA levels of p11, which facilitates the expression of Na(v)1.8, were high at all stages. These findings show that trigeminal ganglion sodium channel transcripts mature in steps that are specific for each transcript. They also raise the possibility that different facial regions could differ in the ability to transmit sensory signals during early life.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Neuropéptidos/genética , Canales de Sodio/genética , Ganglio del Trigémino/embriología , Ganglio del Trigémino/fisiología , Animales , Anexina A2/genética , Femenino , Hibridación in Situ , Canal de Sodio Activado por Voltaje NAV1.3 , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Neuralgia/genética , Neuralgia/fisiopatología , Embarazo , Subunidades de Proteína/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/fisiología , Ganglio del Trigémino/citologíaRESUMEN
Patients who have suffered nerve injury show profound inter-individual variability in neuropathic pain even when the precipitating injury is nearly identical. Variability in pain behavior is also observed across inbred strains of mice where it has been attributed to genetic polymorphisms. Identification of cellular correlates of pain variability across strains can advance the understanding of underlying pain mechanisms. Voltage-gated sodium channels (VGSCs) play a major role in the generation and propagation of action potentials in the primary afferents and are therefore of obvious importance for pain phenotype. Here, we examined the mRNA expression levels of the VGSC alpha-subunits Na(v)1.3, Na(v)1.5, Na(v)1.6, and Na(v)1.7, as well as the auxiliary VGSC-related molecule, Contactin. Dorsal root ganglia (DRG) and spinal cords from 5 inbred mouse strains with contrasting pain phenotype (AKR/J, C3H/HeJ, C57BL/6J, C58/J and CBA/J) were analyzed 7 days following sciatic and saphenous nerve transection. In the DRG, Na(v)1.6, Na(v)1.7 and Contactin were abundantly expressed in control animals. Following nerve injury, the residual mRNA levels of Na(v)1.6 (downregulated in two of the strains) correlated tightly to the extent of autotomy behavior. A suggestive correlation was also seen for the post-injury mRNA levels of Contactin (downregulated in all strains) with autotomy. Thus, our results suggest a contribution by DRG Na(v)1.6, and possibly Contactin to neuropathic pain in the neuroma model in mice.