RESUMEN
In the past decade, signature clinical neuropathology of blast-induced traumatic brain injury has been under intense debate, but interface astroglial scarring (IAS) seems to be convincing. In this study, we examined whether IAS could be replicated in the rat brain exposed to a laser-induced shock wave(s) (LISW[s]), a tool that can produce a pure shock wave (primary mechanism) without dynamic pressure (tertiary mechanism). Under certain conditions, we observed astroglial scarring in the subpial glial plate (SGP), gray-white matter junctions (GM-WM), ventricular wall (VW), and regions surrounding cortical blood vessels, accurately reproducing clinical IAS. We also observed shock wave impulse-dependent meningeal damage (dural microhemorrhage) in vivo by transcranial near-infrared (NIR) reflectance imaging. Importantly, there were significant correlations between the degree of dural microhemorrhage and the extent of astroglial scarring more than 7 days post-exposure, suggesting an association of meningeal damage with astroglial scarring. The results demonstrated that the primary mechanism alone caused the IAS and meningeal damage, both of which are attributable to acoustic impedance mismatching at multi-layered tissue boundaries. The time course of glial fibrillary acidic protein (GFAP) immunoreactivity depended not only on the LISW conditions but also on the regions. In the SGP, significant increases in GFAP immunoreactivity were observed at 3 days post-exposure, whereas in the GM-WM and VW, GFAP immunoreactivity was not significantly increased before 28 days post-exposure, suggesting different pathological mechanisms. With the high-impulse single exposure or the multiple exposure (low impulse), fibrotic reaction or fibrotic scar formation was observed, in addition to astroglial scarring, in the cortical surface region. Although there are some limitations, this seems to be the first report on the shock-wave-induced IAS rodent model. The model may be useful to explore potential therapeutic approaches for IAS.
Asunto(s)
Astrocitos , Cicatriz , Meninges , Ratas Sprague-Dawley , Animales , Ratas , Astrocitos/patología , Masculino , Cicatriz/patología , Cicatriz/etiología , Meninges/patología , Rayos Láser/efectos adversos , Lesiones Traumáticas del Encéfalo/patología , Ondas de Choque de Alta Energía/efectos adversos , Traumatismos por Explosión/patología , Traumatismos por Explosión/complicaciones , Encéfalo/patologíaRESUMEN
BACKGROUND: The most common gene responsible for autosomal recessive retinitis pigmentosa (RP) is EYS. The manner of decay of genetically defective EYS gene transcripts varies depending on the type of mutation using our cellular model, which consists of induced photoreceptor-directed fibroblasts from EYS-RP patients (EYS-RP cells). However, disease-specific profiles have not been clarified in EYS-RP cells. Herein we investigated comprehensive gene expression patterns and restoration of altered expression by low molecular weight molecules in EYS-RP cells. METHODS: Using induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2, we employed qRT-PCR and DNA microarray analysis to compare expression levels of disease-related genes in EYS-RP cells. We investigated the effect of antiapoptotic or anti-endoplasmic reticulum (ER) stress/antioxidant reagents on the restoration of altered gene expression. RESULTS: Expression levels of phototransduction-related genes (blue opsin, rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD was extracted by global gene expression analysis, as a downregulated, retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or aging-related gene. Pathway enrichment analysis suggested that "complement and coagulation cascades," "ECM-receptor interaction" and "PI3K-Akt signaling pathway" could be involved in EYS-RP-associated pathogenesis. Among the matching/overlapping genes involved in those pathways, F2R was suggested as an EYS-RP-associated gene. The downregulation of CRYGD and F2R was completely restored by additional 4-PBA, an inhibitor of ER stress, and partially restored by metformin or NAC. In addition, 4-PBA normalized the expression level of cleaved caspase-3. CONCLUSIONS: Our cellular model may reflect the ER stress-mediated degenerative retina and serve as a pathogenesis-oriented cost-effective rescue strategy for RP patients.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Retinitis Pigmentosa , Análisis Costo-Beneficio , Análisis Mutacional de ADN , Proteínas del Ojo/metabolismo , Fibroblastos/metabolismo , Humanos , Mutación , Linaje , Fosfatidilinositol 3-Quinasas/genética , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Rodopsina/genéticaRESUMEN
We herein report a case of recurrent multifocal, distal-dominant-sensorimotor neuropathy with ophthalmoplegia, IgM anti-GM1 antibody, and pyrexia-associated relapse. The patient developed sensory disturbance in her limbs after febrile disease at 50 years old. She had experienced several similar episodes and was admitted to the hospital at 56 years old. Based on a pathological study and electrophysiological findings consistent with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), maintenance IVIg therapy was administered and produced partial improvement with no relapse at one-year follow-up. Immunohistochemical studies suggested the presence of IgG (not IgM) anti-myelin antibodies. Chronic neuropathy with ophthalmoplegia and pyrexia-associated relapse may be a unique variant of CIDP.
Asunto(s)
Oftalmoplejía , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante , Polirradiculoneuropatía , Enfermedad Crónica , Femenino , Fiebre , Gangliósidos , Humanos , Inmunoglobulina M , Fenotipo , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/complicaciones , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/diagnóstico , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/tratamiento farmacológico , RecurrenciaRESUMEN
We analysed the effect of adding cholesterol to glycolipid antigens on antibody activity with enzyme-linked immunosorbent assay in 123 subjects consisting of 96 patients with Guillain-Barré syndrome, 25 Miller Fisher syndrome, and two Bickerstaff brainstem encephalitis. The use of cholesterol-added GM1 antigens increased anti-GM1 activity in 11 out of 23 anti-GM1-positive patients and resulted in six out of 100 anti-GM1-negative patients becoming anti-GM1-positive. Enhancement of anti-GM1 activity by cholesterol addition was significantly associated with antecedent gastrointestinal infection. The use of cholesterol-added glycolipid antigens can increase the detection rate of anti-glycolipid antibodies and accurately evaluate the anti-glycolipid antibody activity in vivo.
Asunto(s)
Autoanticuerpos/sangre , Colesterol/administración & dosificación , Gangliósido G(M1)/sangre , Glucolípidos/sangre , Síndrome de Guillain-Barré/sangre , Síndrome de Miller Fisher/sangre , Encefalitis/sangre , Encefalitis/tratamiento farmacológico , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Síndrome de Guillain-Barré/tratamiento farmacológico , Humanos , Masculino , Síndrome de Miller Fisher/tratamiento farmacológico , Estudios RetrospectivosRESUMEN
Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 µM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.
Asunto(s)
Cerebelo/efectos de los fármacos , Imidazoles/toxicidad , Insecticidas/toxicidad , Neuronas/efectos de los fármacos , Nicotina/toxicidad , Nitrocompuestos/toxicidad , Piridinas/toxicidad , Transcriptoma/efectos de los fármacos , Animales , Cerebelo/embriología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Neonicotinoides , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/genéticaRESUMEN
Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.
RESUMEN
Redirecting differentiation of somatic cells by over-expression of transcription factors is a promising approach for regenerative medicine, elucidation of pathogenesis and development of new therapies. We have previously defined a transcription factor combination, that is, CRX, RAX and NEUROD, that can generate photosensitive photoreceptor cells from human iris cells. Here, we show that human dermal fibroblasts are differentiated to photoreceptor cells by the same transcription factor combination as human iris cells. Transduction of a combination of the CRX, RAX and NEUROD genes up-regulated expression of the photoreceptor-specific genes, recoverin, blue opsin and PDE6C, in all three strains of human dermal fibroblasts that were tested. Additional OTX2 gene transduction increased up-regulation of the photoreceptor-specific genes blue opsin, recoverin, S-antigen, CNGB3 and PDE6C. Global gene expression data by microarray analysis further showed that photoreceptor-related functional genes were significantly increased in induced photoreceptor cells. Functional analysis, that is, patch-clamp recordings, clearly revealed that induced photoreceptor cells from fibroblasts responded to light. Both the NRL gene and the NR2E3 gene were endogenously up-regulated in induced photoreceptor cells, implying that exogenous CRX, RAX, OTX2 and NEUROD, but not NRL, are sufficient to generate rod photoreceptor cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas del Ojo/genética , Fibroblastos/citología , Proteínas de Homeodominio/genética , Factores de Transcripción Otx/genética , Células Fotorreceptoras de Vertebrados/citología , Transactivadores/genética , Factores de Transcripción/genética , Diferenciación Celular , Dermis/citología , Dermis/metabolismo , Fibroblastos/metabolismo , Humanos , Iris/citología , Iris/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/citología , Retina/metabolismo , Transducción GenéticaRESUMEN
Dermatan sulfate (DS) is synthesized from chondroitin sulfate (CS) by epimerization of glucuronic acid of CS to yield iduronic acid. In the present study, the role of CS and DS was examined in mice that received transection of nigrostriatal dopaminergic pathway followed by injection of glycosaminoglycan degrading enzymes into the lesion site. Two weeks after injury, fibrotic and glial scars were formed around the lesion, and transected axons did not regenerate beyond the fibrotic scar. Injection of chondroitinase ABC (ChABC), which degrades both CS and DS, completely suppressed the fibrotic scar formation, reduced the glial scar, and promoted the regeneration of dopaminergic axons. Injection of the DS-degrading enzyme chondroitinase B (ChB) also yielded similar results. By contrast, injection of chondroitinase AC (ChAC), a CS-degrading enzyme, did not suppress the fibrotic and glial scar formation, but reduced CS immunoreactivity and promoted the axonal regeneration. Addition of transforming growth factor-ß1 (TGF-ß1) to a co-culture of meningeal fibroblasts and cerebral astrocytes induces a fibrotic scar-like cell cluster. The effect of TGF-ß1 on cluster formation was suppressed by treatment with ChABC or ChB, but not by ChAC. TGF-ß1-induced cell cluster repelled neurites of neonatal cerebellar neurons, but addition of ChABC or ChAC suppressed the inhibitory property of clusters on neurite outgrowth. The present study is the first to demonstrate that DS and CS play different functions after brain injury: DS is involved in the lesion scar formation, and CS inhibits axonal regeneration.
Asunto(s)
Axones/fisiología , Lesiones Encefálicas/metabolismo , Sulfatos de Condroitina/metabolismo , Cicatriz/metabolismo , Dermatán Sulfato/metabolismo , Regeneración Nerviosa/fisiología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Axones/patología , Lesiones Encefálicas/patología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos ICR , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Acetamiprid (ACE) and imidacloprid (IMI) belong to a new, widely used class of pesticide, the neonicotinoids. With similar chemical structures to nicotine, neonicotinoids also share agonist activity at nicotinic acetylcholine receptors (nAChRs). Although their toxicities against insects are well established, their precise effects on mammalian nAChRs remain to be elucidated. Because of the importance of nAChRs for mammalian brain function, especially brain development, detailed investigation of the neonicotinoids is needed to protect the health of human children. We aimed to determine the effects of neonicotinoids on the nAChRs of developing mammalian neurons and compare their effects with nicotine, a neurotoxin of brain development. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of cerebellar neurons from neonatal rats allow for examinations of the developmental neurotoxicity of chemicals because the various stages of neurodevelopment-including proliferation, migration, differentiation, and morphological and functional maturation-can be observed in vitro. Using these cultures, an excitatory Ca(2+)-influx assay was employed as an indicator of neural physiological activity. Significant excitatory Ca(2+) influxes were evoked by ACE, IMI, and nicotine at concentrations greater than 1 µM in small neurons in cerebellar cultures that expressed the mRNA of the α3, α4, and α7 nAChR subunits. The firing patterns, proportion of excited neurons, and peak excitatory Ca(2+) influxes induced by ACE and IMI showed differences from those induced by nicotine. However, ACE and IMI had greater effects on mammalian neurons than those previously reported in binding assay studies. Furthermore, the effects of the neonicotinoids were significantly inhibited by the nAChR antagonists mecamylamine, α-bungarotoxin, and dihydro-ß-erythroidine. CONCLUSIONS/SIGNIFICANCE: This study is the first to show that ACE, IMI, and nicotine exert similar excitatory effects on mammalian nAChRs at concentrations greater than 1 µM. Therefore, the neonicotinoids may adversely affect human health, especially the developing brain.
Asunto(s)
Cerebelo/citología , Imidazoles/farmacología , Insecticidas/farmacología , Neuronas/metabolismo , Nicotina/farmacología , Nitrocompuestos/farmacología , Piridinas/farmacología , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Bungarotoxinas/farmacología , Calcio/metabolismo , Cerebelo/metabolismo , Dihidro-beta-Eritroidina/farmacología , Humanos , Mecamilamina/farmacología , Neonicotinoides , Neurotoxinas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Traumatic damage to the central nervous system (CNS) destroys the blood-brain barrier (BBB) and provokes the invasion of hematogenous cells into the neural tissue. Invading leukocytes, macrophages and lymphocytes secrete various cytokines that induce an inflammatory reaction in the injured CNS and result in local neural degeneration, formation of a cystic cavity and activation of glial cells around the lesion site. As a consequence of these processes, two types of scarring tissue are formed in the lesion site. One is a glial scar that consists in reactive astrocytes, reactive microglia and glial precursor cells. The other is a fibrotic scar formed by fibroblasts, which have invaded the lesion site from adjacent meningeal and perivascular cells. At the interface, the reactive astrocytes and the fibroblasts interact to form an organized tissue, the glia limitans. The astrocytic reaction has a protective role by reconstituting the BBB, preventing neuronal degeneration and limiting the spread of damage. While much attention has been paid to the inhibitory effects of the astrocytic component of the scars on axon regeneration, this review will cover a number of recent studies in which manipulations of the fibroblastic component of the scar by reagents, such as blockers of collagen synthesis have been found to be beneficial for axon regeneration. To what extent these changes in the fibroblasts act via subsequent downstream actions on the astrocytes remains for future investigation.
Asunto(s)
Sistema Nervioso Central/patología , Cicatriz/patología , Cicatrización de Heridas , Animales , Axones/patología , Humanos , Regeneración Nerviosa , Neuroglía/patologíaRESUMEN
The expression of purinergic receptors (P2X) on rat vestibular ganglion neurons (VGNs) was examined using whole-cell patch-clamp recordings. An application of adenosine 5'-triphosphate (ATP; 100microM) evoked inward currents in VGNs at a holding potential of -60mV. The decay time constant of the ATP-evoked currents was 2-4s, which is in between the values for rapidly desensitizing subgroups (P2X1 and P2X3) and slowly desensitizing subgroups (P2X2, P2X4, etc.), suggesting the heterogeneous expression of P2X receptors. A dose-response experiment showed an EC(50) of 11.0microM and a Hill's coefficient of 0.82. Suramin (100microM) reversibly inhibited the ATP-evoked inward currents. Alpha, beta-methylene ATP (100microM), a P2X-specific agonist, also evoked inward currents but less extensively than ATP. An application of adenosine 5'-dihosphate (ADP; 100microM) evoked similar, but much smaller, currents. The current-voltage relationship of the ATP-evoked conductance showed pronounced inward rectification with a reversal potential more positive than 0mV, suggesting non-selective cation conductance. However, the channel was not permeable to a large cation (N-methyl-d-glucamine) and acidification (pH 6.3) had little effect on the ATP-evoked conductance. RT-PCR confirmed the expression of five subtypes (P2X2-P2X6) in VGNs. The physiological role of P2X receptors includes the modulation of excitability at the synapses between hair cells and dendrites and/or trophic support (or also neuromodulation) from supporting cells surrounding the VGNs.
Asunto(s)
Ganglios Sensoriales/fisiología , Neuronas/fisiología , Receptores Purinérgicos P2/fisiología , Nervio Vestibular/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Relación Dosis-Respuesta a Droga , Potenciales Evocados Auditivos/efectos de los fármacos , Potenciales Evocados Auditivos/fisiología , Ligandos , Modelos Animales , Conducción Nerviosa/fisiología , Técnicas de Placa-Clamp , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X , Suramina/farmacologíaRESUMEN
After central nervous system (CNS) injury, meningeal fibroblasts migrate in the lesion center to form a fibrotic scar which is surrounded by end feet of reactive astrocytes. The fibrotic scar expresses various axonal growth-inhibitory molecules and creates a major impediment for axonal regeneration. We developed an in vitro model of the scar using coculture of cerebral astrocytes and meningeal fibroblasts by adding transforming growth factor-beta1 (TGF-beta1), a potent fibrogenic factor. Addition of TGF-beta1 to this coculture resulted in enhanced proliferation of fibroblasts and the formation of cell clusters which consisted of fibroblasts inside and surrounded by astrocytes. The cell cluster in culture densely accumulated the extracellular matrix molecules and axonal growth-inhibitory molecules similar to the fibrotic scar, and remarkably inhibited the neurite outgrowth of cerebellar neurons. Therefore, this culture system can be available to analyze the inhibitory property in the lesion site of CNS.
Asunto(s)
Axones/fisiología , Fibroblastos/fisiología , Inhibición Neural/fisiología , Neuronas/citología , Análisis de Varianza , Animales , Animales Recién Nacidos , Antígenos/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Bromodesoxiuridina/metabolismo , Recuento de Células , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Corteza Cerebral/citología , Sulfatos de Condroitina/metabolismo , Técnicas de Cocultivo , Fibroblastos/efectos de los fármacos , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Meninges/citología , Proteínas del Tejido Nervioso/metabolismo , Inhibición Neural/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteoglicanos/metabolismo , Pirazoles/farmacología , Pirroles/farmacología , Ratas , Ratas Sprague-Dawley , Receptor EphB2/metabolismo , Semaforina-3A/metabolismo , Tenascina/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
The fibrotic scar which is formed after traumatic damage of the central nervous system (CNS) is considered as a major impediment for axonal regeneration. In the process of the fibrotic scar formation, meningeal fibroblasts invade and proliferate in the lesion site to secrete extracellular matrix proteins, such as collagen and laminin. Thereafter, end feet of reactive astrocytes elaborate a glia limitans surrounding the fibrotic scar. Transforming growth factor-beta1 (TGF-beta1), a potent scar-inducing factor, which is upregulated after CNS injury, has been implicated in the formation of the fibrotic scar and glia limitans. In the present study, expression of receptors to TGF-beta1 was examined by in situ hybridization histochemistry in transcortical knife lesions of the striatum in the mouse brain in combination with immunofluorescent staining for fibroblasts and astrocytes. Type I and type II TGF-beta receptor mRNAs were barely detected in the intact brain and first found in meningeal cells near the lesion 1 day postinjury. Many cells expressing TGF-beta receptors were found around the lesion site 3 days postinjury, and some of them were immunoreactive for fibronectin. After 5 days postinjury, many fibroblasts migrated from the meninges to the lesion site formed the fibrotic scar, and most of them expressed TGF-beta receptors. In contrast, few of reactive astrocytes expressed the receptors throughout the postinjury period examined. These results indicate that meningeal fibroblasts not reactive astrocytes are a major target of TGF-beta1 that is upregulated after CNS injury.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Meninges/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Astrocitos/patología , Cicatriz/patología , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratones , Proteínas Serina-Treonina Quinasas/genética , Transporte de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genéticaRESUMEN
Pleiotrophin (PTN) is highly expressed in the nervous system during embryogenesis; however, little is known about its functional role in neural development. By using whole mount in situ hybridization, we observed that the expression pattern of PTN was similar to that of Wnt3a; PTN mRNA was abundant in the nervous tissue along the dorsal midline and in the forelimb and hindlimb buds of embryonic mice (E8.5-E12.5). Treatment with recombinant PTN (100ng/ml) induced phosphorylation of glycogen synthase kinase 3beta (GSK3beta), nuclear localization of beta-catenin and up-regulation of growth-associated protein (GAP)-43 mRNA in cultured embryonic mouse (E14.5) neurons. Furthermore, recombinant PTN enhanced neurite outgrowth from cortical explants embedded in Matrigel. These PTN-induced biochemical changes and neurite outgrowth were attenuated by the co-treatment with anti-anaplastic lymphoma kinase (ALK) antibodies, but not with anti-protein tyrosine phosphatase (PTP)zeta antibodies. These findings imply that ALK is involved in the PTN signaling on neural development.
Asunto(s)
Encéfalo/citología , Encéfalo/embriología , Proteínas Portadoras/farmacología , Citocinas/farmacología , Proteína GAP-43/genética , Neuritas/efectos de los fármacos , Neuronas/citología , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Quinasa de Linfoma Anaplásico , Animales , Cateninas/metabolismo , Células Cultivadas , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Femenino , Proteína GAP-43/metabolismo , Ganglios Espinales/citología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas ReceptorasRESUMEN
There are a number of kinase inhibitors that regulate components of the neovasculature. We previously reported the use of transforming growth factor (TGF)-beta inhibitor on neovasculature in stroma-rich tumor models to increase the intratumoral distribution of nanoparticles. Here, we compared the effects of two other kinase inhibitors, imatinib and sorafenib, with TGF-beta inhibitor (LY364947) on extravasation of a modeled nanoparticle, 2 MDa dextran. We first used a mouse model of neoangiogenesis, the Matrigel plug assay, to compare neovasculature formed inside of and around Matrigel plugs (intraplug and periplug regions, respectively). Intraplug vasculature was more strongly pericyte covered, whereas periplug vasculature was less covered. In this model, TGF-beta inhibitor exhibited the most potent effect on intraplug vasculature in increasing the extravasation of dextran, whereas sorafenib had the strongest effect on periplug vasculature. Although imatinib and TGF-beta inhibitor each reduced pericyte coverage, imatinib also reduced the density of endothelium, resulting in a decrease in overall delivery of nanoparticles. These findings were confirmed in two tumor models, the CT26 colon cancer model and the BxPC3 pancreatic cancer model. The vasculature phenotype in the CT26 model resembled that in the periplug region, whereas the latter resembled that in the intraplug region. Consistent with this, sorafenib most potently enhanced the accumulation of nanoparticles in the CT26 model, whereas TGF-beta inhibitor did in the BxPC3 model. In conclusion, the appropriate strategy for optimization of tumor vasculature for nanoparticles may differ depending on tumor type, and in particular on the degree of pericyte coverage around the vasculature.
Asunto(s)
Bencenosulfonatos/farmacología , Nanopartículas , Neoplasias/irrigación sanguínea , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Animales , Benzamidas , Línea Celular Tumoral , Extravasación de Materiales Terapéuticos y Diagnósticos , Humanos , Mesilato de Imatinib , Ratones , Ratones Endogámicos BALB C , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Transducción de Señal , Sorafenib , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
We observed that recombinant ciliary neurotrophic factor (CNTF) enhanced survival and neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. Among other neurotrophic factors (NGF and GDNF) and interleukin (IL)-6 cytokine members [IL-6, LIF, cardiotrophin-1, and oncostatin M (OSM)] at the same concentration (50 ng/ml), CNTF, as well as LIF and OSM, displayed high efficacy for the promotion of the number of viable neurons and neurite-bearing cells. CNTF enhanced the number of neurite-bearing cells in both small neurons (soma diameter <30 microm) and large neurons (soma diameter > or =30 microm), whereas NGF and GDNF promoted that in only small neurons. Western blot analysis revealed that CNTF induced phosphorylation of STAT3, Akt, and ERK1/2 in the neurons. Furthermore, the neurite outgrowth-promoting activity of CNTF was diminished by co-treatment with Janus kinase (JAK) 2 inhibitor, AG490; STAT3 inhibitor, STA-21; phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor, LY294002; and mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, in a concentration-dependent manner. Its survival-promoting activity was also affected by AG490, STA-21, and LY294002 at higher concentrations, but not by PD98059. These findings suggest the involvement of JAK2/STAT3, PI3K/Akt, and MEK/ERK signaling pathways in CNTF-induced neurite outgrowth, where the former two pathways are thought to play major roles in mediating the survival response of neurons to CNTF.
Asunto(s)
Factor Neurotrófico Ciliar/farmacología , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Factor Neurotrófico Ciliar/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Femenino , Flavonoides/farmacología , Ganglios Espinales/crecimiento & desarrollo , Inmunohistoquímica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfolinas/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Compuestos Policíclicos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Tirfostinos/farmacologíaRESUMEN
Individual primary vestibular afferents exhibit spontaneous activity the regularity of which can vary from regular to irregular. Different aspects of vestibular responsiveness have been associated with this dimension of regularity of resting discharge. Isolated rat vestibular ganglion cells (VGCs) showed heterogeneous intrinsic firing properties during sustained membrane depolarization: some neurons exhibited a strong adaptation generating just a single or a few spikes (phasic type), whereas other neurons showed moderate adaptation or tonic firing (tonic type). Tonic discharging VGCs were rare at postnatal days 5-7 and increased up to approximately 60% of neurons during postnatal 2-3 wk. To explore the major factors responsible for the discharge regularity of primary vestibular afferents, we investigated the contribution of K+ channels to the firing properties of isolated rat VGCs. Phasic firing became tonic firing in the presence of 4-aminopyridine or alpha-dendrotoxin, indicating that Kv1 potassium channels control the firing pattern of the phasic VGCs. Tetraethylammonium decreased the number of spikes during step current stimuli in all types. Blockade of Ca2+-activated K+ channels decreased the number of spikes in tonic VGCs. Our results suggest that Kv1 channels are critical both in determining the pattern of spike discharge in rat vestibular ganglion neurons and in their proportional change during maturation.
Asunto(s)
Ganglios/fisiología , Neuronas/fisiología , Canales de Potasio de la Superfamilia Shaker/fisiología , Nervio Vestibular/fisiología , 4-Aminopiridina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Interpretación Estadística de Datos , Venenos Elapídicos/farmacología , Electrofisiología , Ganglios/citología , Potenciales de la Membrana/efectos de los fármacos , Neuronas Aferentes/fisiología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Compuestos de Tetraetilamonio/farmacología , Nervio Vestibular/citologíaRESUMEN
Fez-like (Fezl), the forebrain embryonic zinc finger-like protein, is a transcriptional repressor selectively expressed in the deep layers of the developing cortex. We examined the thalamocortical and corticofugal pathways in Fezl-deficient fetal mice by using immunohistochemistry and by axonal labeling with the lipophilic dyes DiI and DiA, with special attention to the spatiotemporal relation between thalamocortical and corticofugal axons. In normal mice, thalamic and cortical axons meet in the internal capsule between embryonic day (E) 13.5 and E14.5 and fasciculate with each other as they extend to their targets, the cortex and thalamus, respectively. In Fezl-deficient mice, most of the thalamic and cortical axons stop in the internal capsule and at the pallial-subpallial boundary at E14.5, respectively. This abnormality is transient, and the thalamic and cortical axons reach their targets at E15.5, although the number of thalamic axons is remarkably reduced in the cortical anlage. Double labeling with DiI and DiA demonstrated close apposition of the thalamic and cortical axons in the subpallium and pallium as well as in the external capsule of this mutant after E15.5. Because the expression of genes that define the pallial-subpallial boundary and guidance molecules of thalamocortical axons did not show remarkable changes in Fezl-deficient mice, abnormal formation of thalamocortical pathway in this mutant may be caused by the defect of axons of cortical efferent neurons that express Fezl.