RESUMEN
Melatonin has been shown to down-regulate inflammatory responses and provide neuroprotection. However, the mechanisms underlying the anti-inflammatory properties of melatonin are poorly understood. In the present work, we studied the modulatory effect of melatonin against pro-inflammatory cytokines in glial cell cultures. Treatment with pro-inflammatory cytokines mainly tumor necrosis factor-alpha, interleukin 1-beta, and interferon-gamma induces an increase in inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. Pre-treatment with melatonin produced an inhibitory effect on iNOS expression and NO production. The biochemical studies revealed that cytokine treatment favors the activation of several pathways, such as mitogen-activated protein kinases (MAPKs), STAT1, and STAT3; however, the anti-inflammatory effect of melatonin was accompanied only by a decrease in p38 MAPK activity. Likewise, SB203580 a p38 kinase inhibitor inhibits NO production. These data indicate that the anti-inflammatory action of melatonin in glial cells after stimulation with pro-inflammatory cytokines may be in part, attributable to p38 inhibition which down-regulates iNOS expression and NO production.
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Antiinflamatorios/farmacología , Citocinas/fisiología , Sistema de Señalización de MAP Quinasas , Melatonina/farmacología , Neuroglía/metabolismo , Óxido Nítrico/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Guanilato Ciclasa , Mediadores de Inflamación/farmacología , Mediadores de Inflamación/fisiología , Ratones , Ratones Endogámicos C57BL , Neuroglía/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIMS: 3-Nitropropionic acid (3-NP) is a toxin that replicates most of the clinical and pathophysiological symptoms of Huntington's disease, inducing neurodegeneration in the striatum due to the inhibition of mitochondrial succinate dehydrogenase. Different pathways have been implicated in the cell death induced by 3-NP in rodents. One of them is the Jun-N-terminal kinase (JNK) pathway, which may play a role in the neurodegenerative process in different diseases. Moreover, the lack of one isoform of JNK (JNK3) has been associated with neuroprotection in different experimental models of neurodegeneration. Therefore, in the present study the role of JNK3 in the experimental Huntington's model induced by 3-NP administration was evaluated. METHODS: 3-NP was intraperitoneally administered once a day for 3 days to wild-type and Jnk3-null mice. Coronal brain sections were used to determine cell death and astrogliosis in striatum. Western blots were performed to determine the involvement of different pathways in both wild-type and Jnk3-null mice. RESULTS: Although JNK activation was observed following 3-NP administration, the results indicate that the lack of JNK3 does not confer neuroprotection against 3-NP toxicity. Thus, other pathways must be involved in the neurodegeneration induced in this model. One of the possible pathways towards 3-NP-induced apoptosis could involve the calpains, as their activity was increased in wild-type and Jnk3-null mice. CONCLUSION: Although JNK3 is a key protein involved in cell death in different neurodegenerative diseases, the present study demonstrates that the lack of JNK3 does not confer neuroprotection against 3-NP-induced neuronal death.
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Cuerpo Estriado/enzimología , Enfermedad de Huntington/enzimología , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Degeneración Nerviosa/enzimología , Animales , Western Blotting , Convulsivantes/toxicidad , Modelos Animales de Enfermedad , Activación Enzimática , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Degeneración Nerviosa/inducido químicamente , Nitrocompuestos/toxicidad , Propionatos/toxicidadRESUMEN
Taurine, 2-aminoethanesulfonic acid, is present at high concentrations in many invertebrate and vertebrate systems, and it has several biological functions. In addition, it has been related to a neuroprotective role against several diseases, such as epilepsy. It has been reported that taurine induces a decrease of calbindin-D28k, calretinin, and parvalbumin protein levels in the hippocampus 3 days after administration. In the present work we hypothesized that the decrease of these proteins could alter the action of kainic acid (KA) and make mice more susceptible to excitotoxicity. Therefore, we treated mice with taurine and after 3 days treated them with KA. The results showed that taurine pretreatment did not induce a major susceptibility to KA. Moreover, neurodegeneration was reduced in pretreated mice. However, astrogliosis was similar to that observed in mice treated only with KA. The immunohistochemistries for calbindin-D28k, calretinin, and parvalbumin showed that these proteins were reduced as a consequence of KA treatment and of taurine treatment. However, mice pretreated with taurine prior to KA administration presented the same reduction in these proteins as mice treated with only taurine or only KA.
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Proteínas de Unión al Calcio/antagonistas & inhibidores , Resistencia a Medicamentos/efectos de los fármacos , Ácido Kaínico/agonistas , Neurotoxinas/agonistas , Parvalbúminas/antagonistas & inhibidores , Proteína G de Unión al Calcio S100/antagonistas & inhibidores , Taurina/toxicidad , Animales , Calbindina 1 , Calbindina 2 , Calbindinas , Proteínas de Unión al Calcio/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Resistencia a Medicamentos/fisiología , Epilepsia/inducido químicamente , Epilepsia/tratamiento farmacológico , Epilepsia/metabolismo , Masculino , Ratones , Ratones Endogámicos , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Parvalbúminas/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Taurina/metabolismoRESUMEN
Taurine, 2-aminoethanesulfonic acid, is present at high concentrations in many invertebrate and vertebrate systems and has several biological functions. In addition, it has been related to a neuroprotective role against several diseases such as epilepsy. In the present work, we treated mice with taurine and examined its effects on the expression of proteins in the hippocampus associated with calcium regulation. Taurine treatment alters the presence of calbindin-D28k, calretinin, and parvalbumin in the brain, mainly in the hippocampus. It also reduced CaMKII activity, indicating that taurine could alter calcium signaling pathways. However, the activity of calpain, a protease related to apoptosis induced by calcium signalling, did not change. The concentration of taurine in the hippocampus was also unaffected by the treatment. These results provide new insight into the role of taurine in calcium homeostasis.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Hipocampo/efectos de los fármacos , Parvalbúminas/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Taurina/farmacología , Animales , Calbindina 1 , Calbindina 2 , Calbindinas , Calpaína/metabolismo , Cromatografía Líquida de Alta Presión , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Ratones , Neuronas/metabolismo , Espectrometría de Fluorescencia , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
An experimental model based on kainic acid (KA) injections replicates many phenomenological features of human temporal lobe epilepsy, the most common type of epilepsy in adults. Taurine, 2-aminoethanesulfonic acid, present in high concentrations in many invertebrate and vertebrate systems, is believed to serve several important biological functions. In addition, it is believed to have a neuroprotective role against several diseases. In the present study, an experimental mouse model based on taurine pretreatment prior to KA administration has been improved to study whether taurine has a neuroprotective effect against KA-induced behavior and cell damage. Under different treatments tested, taurine's most neuroprotective effects were observed with intraperitoneal taurine injection (150 mg/kg dosage) 12 hr before KA administration. Thus, a reduction in or total absence of seizures, together with a reduction in or even disappearance of cellular and molecular KA-derived effects, was detected in mice pretreated with taurine compared with those treated only with KA. Moreover, the use of tritiated taurine revealed taurine entry into the brain, suggesting possible changes in intracellular:extracellular taurine ratios and the triggering of pathways related to neuroprotective effects.
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Anticonvulsivantes/farmacología , Epilepsia/prevención & control , Fármacos Neuroprotectores/farmacología , Taurina/farmacología , Análisis de Varianza , Animales , Anticonvulsivantes/administración & dosificación , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Calpaína/metabolismo , Muerte Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Epilepsia/inducido químicamente , Immunoblotting , Inmunohistoquímica , Inyecciones Intraperitoneales , Ácido Kaínico , Masculino , Ratones , Neuroglía/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Taurina/administración & dosificación , Taurina/análisis , Taurina/sangreRESUMEN
Zac1, a new zinc-finger protein that regulates both apoptosis and cell cycle arrest, is abundantly expressed in many proliferative/differentiation areas during brain development. In the present work, we studied Zac1 gene expression and protein in experimental seizure models following i.p. injection of pentylenetetrazole (PTZ) or kainic acid (KA). Following KA treatment, an early and intense up-regulation of Zac1 is detected in the limbic areas, such as the hippocampus, cortex and amygdaloid and hypothalamic nuclei. Pre-treatment with MK-801, an antagonist of the NMDA receptors, fully blocks the effect of KA in the hippocampus, whereas it only attenuates KA-induced Zac1 up-regulation in the other areas of the limbic system. A reduced induction is obtained with PTZ-treated animals, specifically in the entorhinal and piriform cortices as well as in amygdaloid and hypothalamic nuclei. Thus, Zac1 is highly induced in the seizure models that generate strong neuronal stimulation and/or extensive cell damage (cell death), reinforcing its putative role in the control of the cell cycle and/or apoptosis. Moreover, strong induction is observed in the granular cells of the dentate gyrus (which are resistant to neurodegeneration) and in some glial cells of the dentate gyrus and subventricular zone, suggesting that Zac1 may be implicated in the mechanisms of neural plasticity following injury.
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Proteínas de Ciclo Celular/metabolismo , Sistema Límbico/metabolismo , Neuronas/metabolismo , Convulsiones/fisiopatología , Factores de Transcripción/metabolismo , Animales , Apoptosis , Proteínas de Ciclo Celular/genética , Convulsivantes/farmacología , Maleato de Dizocilpina/farmacología , Sinergismo Farmacológico , Antagonistas de Aminoácidos Excitadores/farmacología , Expresión Génica/efectos de los fármacos , Genes Supresores de Tumor , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Ácido Kaínico/farmacología , Sistema Límbico/patología , Masculino , Ratones , Ratones Endogámicos ICR , Pentilenotetrazol/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/metabolismo , Convulsiones/patología , Distribución Tisular , Factores de Transcripción/genética , Dedos de ZincRESUMEN
Taurine and zinc possess neurotrophic and neuroprotective properties, and they have been demonstrated to interact in the central nervous system (CNS). The aim of this work was to determine taurine, hypotaurine, and zinc levels during postnatal development and any possible significant correlation between them in selective areas of the CNS with differential taurine level regulation and intrinsic capacity to proliferate. Taurine and hypotaurine content (nM/region) and concentration (nM/mg protein) and total zinc levels were determined in the retina, hippocampus, and dentate gyrus of the rat at postnatal days 5, 10, 15, 20, 30, and 50. Taurine and hypotaurine increased during development in the retina without significant correlation between them. In the hippocampus there was a progressive decrease, and in the dentate gyrus there was an initial increase and a posterior decrease of taurine and hypotaurine levels. Correlation between the two amino acids was observed at P10, P15, and P50 for the hippocampus and at P15, P30, and P50 for the dentate gyrus. The variations in total zinc levels followed a biphasic behavior, with an early decrease and later increase. Significant and positive correlation of zinc and taurine was only observed in the hippocampus at P30 and P50 and negative in the dentate gyrus at P30. No significant correlation was obtained for the retina. The maintenance of taurine levels in specific CNS areas does not seem to be related to the availability of the precursor, hypotaurine, which might have a role by itself. There are critical postnatal periods during which there is a preservation of taurine, hypotaurine, or zinc levels. It seems that these requirements could be related to zinc-taurine interactions.
Asunto(s)
Giro Dentado/metabolismo , Hipocampo/metabolismo , Retina/metabolismo , Taurina/análogos & derivados , Taurina/metabolismo , Zinc/metabolismo , Animales , Ratas , Ratas Sprague-DawleyRESUMEN
Using in situ hybridization, we analyzed the expression pattern of the Zac1 gene in mouse brain during the embryonic and postnatal development. Zac1 is a new gene that regulates extensive apoptosis and cell cycle arrest through unrelated pathways. At embryonic stages, strong expression was observed in brain areas with active proliferation (ventricular zone and numerous neuroepithelius) and in nervous system (neural retina and neural tube). In addition, some areas with differentiation activity were noticeably labeled such as arcuate nucleus and amygdaloid region of the brain together with other embryonic sites (hindlimb, forelimb and somites). From P0 onwards, the expression appeared in some proliferative areas, such as subventricular zone and cerebellum (external granular layer and Purkinje cells) and in some synaptic plasticity areas, such as the dorso and ventromedial hypothalamic nuclei, arcuate nucleus, ventral thalamic nucleus.
Asunto(s)
Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Genes Supresores de Tumor , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción , Animales , Apoptosis/genética , Apoptosis/fisiología , Encéfalo/citología , Ciclo Celular/genética , Ciclo Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Dedos de Zinc/genética , Dedos de Zinc/fisiologíaRESUMEN
The neuronal diversity of the subplate and developing white matter in the mouse was studied using a variety of neuronal markers. The subplate was first visible in lateral cortical areas at E13, coinciding with the emergence of the cortical plate. During prenatal development, this layer was formed by morphologically heterogeneous neurons, subsets of which were immunoreactive for GABA- and calcium-binding proteins. From E18 onwards, a few subplate cells also contained neuropeptides. Colocalization experiments demonstrated that the percentages of neurons immunoreactive for each antigen were similar to those described in adult neocortex. By E15, subplate cells had received synaptic contacts. Moreover, a second early-neuronal population was conspicuous from E13 in the lower intermediate zone: the intermediate-subventricular population. Unlike subplate cells, these neurons were morphologically uniform, smaller and horizontally oriented. Nevertheless, a few of these cells also appeared within the ventricular zone, with a perpendicular/ oblique orientation. Most of these cells were GABA-positive and showed calbindin immunoreactivity. At the electron microscopic level, no synaptic contacts were found in these neurons. Tracing studies using DiI showed that subplate neurons were the first to send axons outside the neocortex towards the ganglionic eminence at E13. At E14, subplate axons and ingrowing thalamic fibers met in the striate primordium. Subplate cells retained their projection to the thalamus during prenatal development. Thalamocortical axons reached the subplate at E15, and 1 day later began to invade the upper cortical layers. Early callosal axons, in contrast, did not run through the subplate to reach the contralateral hemisphere, nor did subplate cells send out callosal fibers. Callosal axons ran just above the subventricular zone, intermingled with the intermediate-subventricular neuronal population. We conclude that the subplate neuronal population has a chemical heterogeneity reminiscent of that of the adult cortex and is crucial to the establishment of thalamocortical relationships, whereas the intermediate-subventricular neurons constituted a particular GABAergic population, which includes resident cells and tangentially migrating postmitotic neurons spatially related to the development of callosal connections.
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Vías Aferentes/citología , Cuerpo Calloso/citología , Neocórtex/citología , Neocórtex/embriología , Neuronas/citología , Vías Aferentes/embriología , Vías Aferentes/metabolismo , Animales , Animales Recién Nacidos , Axones/ultraestructura , Calbindina 2 , Calbindinas , Cuerpo Calloso/embriología , Cuerpo Calloso/metabolismo , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Ratones , Ratones Endogámicos , Proteínas Asociadas a Microtúbulos/metabolismo , Neocórtex/metabolismo , Neuroglía/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Neuropéptido Y/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Tálamo/citología , Tálamo/embriología , Tálamo/metabolismo , Tubulina (Proteína)/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The initial ingrowth of corticothalamic and thalamocortical projections was examined in mice at embryonic and perinatal stages. Fibers, in fixed brains, were labeled with the carbocyanine dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocianine perchlorate (DiI). By E13, the corticofugal fibers had entered the lowest intermediate zone through which they ran, turned over the corpus striatum, and left the cortex. The fibers were arranged in scattered bundles throughout the corpus striatum. At E14 corticofugal axons reached the internal capsule and at E14.5-E15 they established contact within the thalamus. Meanwhile, the thalamocortical afferents reached the neocortex at E13. At this time fibers ran tangentially within the intermediate zone, immediately underneath the cortical plate. By E14, the fibers had started to invade the subplate and, by E15, thalamocortical fibers had begun their radial growth into the cortex. Such radial growth proceeded steadily, invading each cortical layer as it differentiated cytoarchitectonically from the dense cortical plate. The first retrogradely labeled cells were detected at the cortical plate at E15. By the day of birth (E20), thalamocortical fibers had formed a dense branching system within layers VI and V. Our observations indicate that, in mice, the thalamic axons reach the cortex before corticothalamic projections enter the thalamic nuclei. Moreover, the results suggest that the pathway followed by each fiber system is different. By DiI injections into the internal capsule we have also determined that subplate cells are the first to send axons to the thalamus.
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Neocórtex/embriología , Tálamo/embriología , Animales , Animales Recién Nacidos , Carbocianinas , Desarrollo Embrionario y Fetal , Colorantes Fluorescentes , Ratones , Microscopía Fluorescente , Neocórtex/citología , Neocórtex/crecimiento & desarrollo , Fibras Nerviosas/ultraestructura , Vías Nerviosas/citología , Vías Nerviosas/embriología , Neuronas/citología , Tálamo/citología , Tálamo/crecimiento & desarrolloRESUMEN
We have analyzed the developmental pattern of beta-galactosidase (beta-gal) expression in the cerebral cortex of the beta 2nZ3'1 transgenic mouse line, which was generated using regulatory elements of the beta 2-microglobulin gene and shows ectopic expression in nervous tissue. From embryonic day 10 onward, beta-gal was expressed in the medial and dorsal cortices, including the hippocampal region, whereas lateral cortical areas were devoid of labeling. During the period of cortical neurogenesis (embryonic days 11-17), beta-gal was expressed by selective precursors in the proliferative ventricular zone of the neocortex and hippocampus, as well as by a number of migrating and postmigratory neurons arranged into narrow radial stripes above the labeled progenitors. Thus, the transgene labels a subset of cortical progenitors and their progeny. Postnatally, radial clusters of beta-gal-positive neurons were discernible until postpartum day 10. At this age, the clusters were 250 to 500 microns wide, composed of neurons spanning all the cortical layers and exhibiting several neuronal phenotypes. These data suggest molecular heterogeneity of cortical progenitors and of the cohorts of postmitotic neurons originating from them, which implies intrinsic molecular mosaicism in both cortical progenitors and developing neurons. Furthermore, the data show that neurons committed to the expression of the transgene migrate along very narrow, radial stripes.
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Movimiento Celular , Corteza Cerebral/embriología , Heterogeneidad Genética , Células Madre , Microglobulina beta-2/biosíntesis , Animales , Genes Reporteros , Inmunohistoquímica , Operón Lac , Ratones , Ratones Transgénicos , Mitosis , Neuronas/fisiología , Fenotipo , Coloración y Etiquetado , Microglobulina beta-2/genética , beta-Galactosidasa/biosíntesisRESUMEN
The presence of migrating callosal neurons during the development of the murine cerebral cortex was studied using biocytin and the lipophilic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate as retrograde tracers. After injections of biocytin in the presumptive somatosensory cortex of newborn mice which were analysed one day later, many anterogradely labelled fibres coursed towards the contralateral hemisphere through the corpus callosum. Retrogradely labelled callosal cells were also observed. Most callosal neurons corresponded to immature pyramidal cells. In addition, a few biocytin-labelled callosal neurons displayed extremely fusiform shapes, vertical orientation and a short, single process emerging from the apical side of the perikaryon. At the electron microscopic level, these cells had features identical to those described for migrating callosal neurons. Twenty-four hours after birth, these migrating neurons were almost exclusively observed in the upper, dense aspect of the cortical plate (presumptive layers II-III) and only very exceptionally in the infragranular layers. No retrogradely labelled cell resembling migrating neurons were noticed after injections on postnatal days 2 or 5. To study migrating callosal neurons at embryonic stages, crystals of the lipophilic dye were injected in the corpus callosum or the contralateral white matter in embryos aged 17, 18 and 19 days, corresponding to the initial development of the corpus callosum in mice. Whereas callosal migrating neurons were not detected at embryonic days 17 and 18, injections of the lipophilic dye on embryonic day 19 revealed the presence of labelled migrating neurons in the infragranular layers. To corroborate further that these cells are migrating neurons, [3H]thymidine was administered on embryonic days 16 and 17, and labelled mice were injected with biocytin on embryonic day 19 or the first postnatal day. Retrogradely labelled callosal neurons resembling migrating neurons were autoradiographically labelled. These results indicate that the specification of certain neuronal types and the emergence of their cell type-specific characteristics occur shortly after postmitotic neurons leave the ventricular zone, before being positioned within the cortical plate.
Asunto(s)
Axones/fisiología , Corteza Cerebral/crecimiento & desarrollo , Neuronas/clasificación , Animales , Autorradiografía , Femenino , Humanos , Recién Nacido , Lisina/análogos & derivados , Lisina/farmacología , Masculino , Ratones , TimidinaRESUMEN
Cortical layers VI to II develop between two layers of older neurons, the marginal and subplate zones, which are believed to have unique roles in cortical development. While subplate cells have been found essential for the establishment of thalamocortical relationships, the function of the marginal zone and in particular of the neurons of Cajal-Retzius has not been elucidated. Here we show that an antibody against the calcium-binding protein calretinin labels the population of Cajal-Retzius cells throughout their life in the murine cerebral cortex. In prenatal and early postnatal stages, Cajal-Retzius cells were found evenly distributed throughout the murine cerebral cortex. Cajal-Retzius-like neurons were also found in the developing hippocampus and dentate gyrus, which indicates that they may have a general function in cortical development. From P8 onward Cajal-Retzius cells disappeared from the neocortex and hippocampus, at the same time as degenerating immunoreactive neurons were observed. Calretinin-positive Cajal-Retzius cells were glutamate immunoreactive and their presumed axon terminals formed asymmetric synapses. These observations indicate that Cajal-Retzius cells may provide a tonic excitatory input, essential for the maturation of cortical neurons. Furthermore, since neuronal migration has been shown to be dependent on glutamate receptors, we propose that Cajal-Retzius cells releasing glutamate may direct migrating neuroblasts toward the marginal lamina, therefore creating the "inside-out" sequence of cortical development.
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Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Animales , Bromodesoxiuridina , Calbindina 2 , Recuento de Células , Corteza Cerebral/crecimiento & desarrollo , Femenino , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Inmunohistoquímica , Ratones , Degeneración Nerviosa/fisiología , Neurotransmisores/metabolismo , Embarazo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Monospecific rabbit antisera obtained through experimental immunization with previously purified proteins were used in the ultrastructural localization of two hydatid fluid antigens, in brood capsules and protoscoleces of Echinococcus granulosus of human origin. The antigen-antibody reaction was revealed by a colloidal gold based method. Reaction was evident in the connective region of the germinal membrane and in the parenchyma of the protoscoleces. Both antigen 5 and antigen B were located in the interstitial material between the parenchymal cells and precisely associated with disorganized areas. The brood capsule wall and the brood capsule contents, the tegument of the protoscoleces, the parenchymal cells, the muscle cells, the calcareous corpuscles and the hooks did not contain antigen 5 or antigen B. Label was not observed in the lumen of the collecting ducts or in the flame cells, although antigen 5 was evident in the periluminal cytoplasm. The origin of the antigens and their release are discussed.
Asunto(s)
Antígenos Helmínticos/análisis , Equinococosis/inmunología , Echinococcus/inmunología , Animales , Echinococcus/ultraestructura , Humanos , Sueros Inmunes/inmunología , Inmunohistoquímica , Microscopía ElectrónicaRESUMEN
Cells displaying highly condensed pyknotic nuclei, the most characteristic feature of apoptosis, are considered as dead cells in neural tissue. The present study aimed to devise methods that could allow the neurogenetic and phenotypic characterization of dying pyknotic cells. In the first set of experiments, pregnant mice were labeled at embryonic days E10-E16 with pulses of 5'-bromodeoxyuridine visualization of BrdU after an immunoperoxidase reaction. In addition to normal, healthy immunopositive nuclei, these preparations displayed a number of pyknotic nuclei that were immunoreactive for BrdU. Both the regional and the temporal distribution of BrdU-positive pyknotic cells were coincidental with the peaks of dead cells in neural tissue. For example, pulses of BrdU at E10-E11 resulted in the visualization of immunoreactive pyknotic cells in the subplate and white matter of the cerebral cortex in early postnatal (P) animals. Thus, the times of generation (birthdates) of cells subjected to degenerative processes can be unequivocally identified. In the second set of experiments, brain sections from unlabeled littermates were immunostained for a variety of neural and glial markers and counterstained with bisbenzimide, to find antigens which, by being present in degenerate pyknotic cells, could indicate the phenotype of such cells. Although no pyknotic cells were positively immunostained for neurofilaments, neuropeptide Y, somatostatin, vasoactive intestinal polypeptide, or vimentin, a number of pyknotic cells were found to be immunoreactive for microtubule-associated protein 2, gamma-aminobutyric acid, calbindin 28KD, and glial fibrillary acidic protein. The percentage of pyknotic cells labeled with neural antigens accounted for more than 20% of the total number of pyknotic cells in a given brain region. In contrast, GFAP-positive pyknotic cells represented up to 50% of the total pyknotic cell population. The method shown here has enabled us to determine that both neurons and glial cells undergo degeneration during normal development.
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Antígenos/metabolismo , Apoptosis , Bromodesoxiuridina , Senescencia Celular , ADN/metabolismo , Sistema Nervioso/citología , Animales , Femenino , Inmunohistoquímica , Ratones , Sistema Nervioso/inmunología , Fenotipo , EmbarazoRESUMEN
Parvalbumin (PARV), a Ca(2+)-binding protein believed to play a role in neuronal excitability, is contained in certain GABAergic inhibitory neurons of the cerebral cortex. Here we report that expression of PARV in the developing neocortex of rats and mice occurs with a sequence which does not follow the usual 'inside-out' gradient of cortical development. Thus, PARV-immunoreactive neurons appear first in layer V and only thereafter in the remaining cortical layers. An adult-like pattern of immunoreactivity is reached simultaneously in layers II-III and VIb. These observations indicate that the mechanisms regulating the functional maturation of PARV-containing inhibitory neurons are different from those that generally govern developmental processes in the cortex.
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Corteza Cerebral/metabolismo , Neuronas/metabolismo , Parvalbúminas/biosíntesis , Animales , Hipocampo/metabolismo , Inmunohistoquímica , Ratones , Parvalbúminas/inmunología , Ratas , Ratas Wistar , Corteza Somatosensorial/inmunología , Corteza Somatosensorial/metabolismo , Ácido gamma-Aminobutírico/fisiologíaAsunto(s)
Fiebre Botonosa/complicaciones , Uveítis/etiología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The absorption of a preparation of the antiviral drug 5-iodo-2'-deoxyuridine (idoxuridine or IDU] has been assessed in rats. A 40% w/v solution of 3H-IDU in dimethyl sulfoxide (DMSO) was topically applied to rats; cutaneous absorption was determined by morphometric evaluation of autoradiographic preparations of the skin. Passage of the drug to the bloodstream was followed using radioisotopic techniques. In addition, the potential irritative action of the same preparation of IDU was investigated in a more suitable animal model. A predictive guinea pig sensitization study was performed according to the Magnusson and Kligman method. 3H-IDU was detected in the epidermis of the rat 60 min after local application. However, it was not possible to determine noticeable levels of the radiolabelled drug in the circulating blood. Experiments on guinea pigs showed slight transient erythematous responses in three animals of the test group following first challenge, and in one animal of the test group and one of the control group, following the second challenge. The overall results of the sensitization test were negative. These results give experimental support for the safety of the IDU preparation studied.