RESUMEN
The central histaminergic neuron system is an important regulator of activity stages such as arousal and sleep. In several epilepsy models, histamine has been shown to modulate epileptic activity and histamine 1 (H1) receptors seem to play a key role in this process. However, little is known about the H1 receptor-mediated seizure regulation during the early postnatal development, and therefore we examined differences in severity of kainic acid (KA)-induced status epilepticus (SE) and consequent neuronal damage in H1 receptor knock out (KO) and wild type (WT) mice at postnatal days 14, 21, and 60 (P14, P21, and P60). Our results show that in P14 H1 receptor KO mice, SE severity and neuronal damage were comparable to those of WT mice, whereas P21 KO mice had significantly decreased survival, more severe seizures, and enhanced neuronal damage in various brain regions, which were observed only in males. In P60 mice, SE severity did not differ between the genotypes, but in KO group, neuronal damage was significantly increased. Our results suggest that H1 receptors could contribute to regulation of seizures and neuronal damage age-dependently thus making the histaminergic system as a challenging target for novel drug design in epilepsy.
Asunto(s)
Neuronas/patología , Receptores Histamínicos H1/deficiencia , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Factores de Edad , Animales , Susceptibilidad a Enfermedades/metabolismo , Susceptibilidad a Enfermedades/patología , Femenino , Ácido Kaínico/toxicidad , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/fisiología , Estado Epiléptico/inducido químicamenteRESUMEN
Birth asphyxia and hypoxia-ischemia (HI) are important factors affecting the normal development and maturation of the central nervous system (CNS). Depending on the maturity of the brain, HI-induced damage at different ages is region-selective, the white matter (WM) peripheral to the lateral ventricles being selectively vulnerable to damage in premature infants. As a squeal of primary or secondary HI in the preterm infant, the brain injury comprises periventricular leukomalasia (PVL), accompanied by neuronal and axonal damage, which affects several brain regions. Premature delivery and improved neonatal intensive care have led to a survival rate of about 75% to 90% of infants weighting under 1500g both in Europe and in the United States. However, about 5-10% of these survivors exhibit cerebral palsy (CP), and many have cognitive, behavioral, attentional or socialization deficits. In this review, we first shortly discuss developmental changes in the expression of the excitatory glutamate receptors (GluRs), and then in more detail elucidate the contribution of GluRs to oligodendrocyte (OL) damage both in experimental models and in preterm human infants. Finally, therapeutic interventions targeted at GluRs at the young age are discussed in the light of results obtained from recent experimental HI animal models and from humans.
Asunto(s)
Asfixia/fisiopatología , Ácido Glutámico/metabolismo , Hipoxia-Isquemia Encefálica/fisiopatología , Transducción de Señal , Asfixia/metabolismo , Asfixia/terapia , Humanos , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/terapia , Receptores de Glutamato/metabolismo , Tasa de SupervivenciaRESUMEN
BACKGROUND: Status epilepticus (SE) is proposed to lead to an age-dependent acute activation of a repertoire of inflammatory processes, which may contribute to neuronal damage in the hippocampus. The extent and temporal profiles of activation of these processes are well known in the adult brain, but less so in the developing brain. We have now further elucidated to what extent inflammation is activated by SE by investigating the acute expression of several cytokines and subacute glial reactivity in the postnatal rat hippocampus. METHODS: SE was induced by an intraperitoneal (i.p.) injection of kainic acid (KA) in 9- and 21-day-old (P9 and P21) rats. The mRNA expression of interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), matrix metalloproteinase-9 (MMP-9), glial-derived neurotrophic factor (GDNF), interferon gamma (IFN-γ), and transforming growth factor-beta 1 (TGF-ß1) were measured from 4 h up to 3 days after KA injection with real-time quantitative PCR (qPCR). IL-1ß protein expression was studied with ELISA, GFAP expression with western blotting, and microglial and astrocyte morphology with immunohistochemistry 3 days after SE. RESULTS: SE increased mRNA expression of IL-1ß, TNF-α and IL-10 mRNA in hippocampus of both P9 and P21 rats, their induction being more rapid and pronounced in P21 than in P9 rats. MMP-9 expression was augmented similarly in both age groups and GDNF expression augmented only in P21 rats, whereas neither IFN-γ nor TGF-ß1 expression was induced in either age group. Microglia and astrocytes exhibited activated morphology in the hippocampus of P21 rats, but not in P9 rats 3 d after SE. Microglial activation was most pronounced in the CA1 region and also detected in the basomedial amygdala. CONCLUSION: Our results suggest that SE provokes an age-specific cytokine expression in the acute phase, and age-specific glial cell activation in the subacute phase as verified now in the postnatal rat hippocampus. In the juvenile hippocampus, transient increases in cytokine mRNA expression after SE, in contrast to prolonged glial reactivity and region-specific microglial activity after SE, suggest that the inflammatory response is changed from a fulminant and general initial phase to a more moderate and specific subacute response.
Asunto(s)
Envejecimiento/fisiología , Citocinas/genética , Hipocampo/fisiopatología , Neuroglía/metabolismo , ARN Mensajero/metabolismo , Estado Epiléptico/fisiopatología , Animales , Citocinas/metabolismo , Hipocampo/fisiología , Neuroglía/citología , Ratas , Ratas Sprague-Dawley , Estado Epiléptico/metabolismoRESUMEN
In the postnatal rodent hippocampus status epilepticus (SE) leads to age- and region-specific excitotoxic neuronal damage, the precise mechanisms of which are still incompletely known. Recent studies suggest that the activation of inflammatory responses together with glial cell reactivity highly contribute to excitotoxic neuronal damage. However, pharmacological tools to attenuate their activation in the postnatal brain are still poorly elucidated. In this study, we investigated the role of inflammatory mediators in kainic acid (KA)-induced neuronal damage in organotypic hippocampal slice cultures (OHCs). A specific cyclooxygenase-2 (COX-2) inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398) was used to study whether or not it could ameliorate neuronal death. Our results show that KA treatment (24 h) resulted in a dose-dependent degeneration of CA3a/b pyramidal neurons. Furthermore, COX-2 immunoreactivity was pronouncedly enhanced particularly in CA3c pyramidal neurons, microglial and astrocyte morphology changed from a resting to active appearance, the expression of the microglial specific protein, Iba1, increased, and prostaglandin E2 (PGE2) production increased. These indicated the activation of inflammatory processes. However, the expression of neither proinflammatory cytokines, i.e. tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), nor the anti-inflammatory cytokine IL-10 mRNA was significantly altered by KA treatment as studied by real-time PCR. Despite activation of an array of inflammatory processes, neuronal damage could not be rescued either with the combined pre- and co-treatment with a specific COX-2 inhibitor, NS-398. Our results suggest that KA induces activation of a repertoire of inflammatory processes in immature OHCs, and that the timing of anti-inflammatory treatment to achieve neuroprotection is a challenge due to developmental properties and the complexity of inflammatory processes activated by noxious stimuli. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/farmacología , Agonistas de Aminoácidos Excitadores/efectos adversos , Hipocampo/efectos de los fármacos , Inflamación/inducido químicamente , Ácido Kaínico/efectos adversos , Degeneración Nerviosa/inducido químicamente , Animales , Técnicas de Cultivo de Célula , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Dinoprostona/análisis , Dinoprostona/fisiología , Agonistas de Aminoácidos Excitadores/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , Hipocampo/patología , Inflamación/metabolismo , Inflamación/fisiopatología , Ácido Kaínico/metabolismo , Ácido Kaínico/farmacología , Masculino , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Nitrobencenos/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacologíaRESUMEN
OBJECTIVE: The purpose of this study was to analyze event related potentials mismatch negativity (MMN) and P3a in childhood cancer patients at the time of diagnosis (Study 1) and after treatment (Study 2) to evaluate their clinical usefulness in screening potential treatment-related neurotoxicity. METHODS: The MMN and P3a to phonetic stimuli were examined in 27 childhood cancer patients with age- and sex-matched controls. Neuropsychological tests were also studied. RESULTS: The MMN peak amplitude was attenuated in the patient group at Study 1. Between the studies, poorer enhancement of the MMN peak amplitude correlated with deterioration in the Verbal intelligence quotient (IQ) in leukaemia patients. In addition, prolongation of the MMN peak latency correlated significantly with deterioration in the Full Scale and Performance IQ in the patient group. Deterioration in the Arithmetic subtest and Performance IQ correlated negatively with the age at diagnosis. CONCLUSIONS: The MMN changes between the studies associated with deterioration in the neuropsychological tests indicating that the method could be clinically useful. The performance of the younger patients was more likely to deteriorate during the treatment. SIGNIFICANCE: Changes in the MMN response during cancer treatment seem to be of clinical importance as indicates of the cognitive outcome of childhood cancer patients.
Asunto(s)
Antineoplásicos/efectos adversos , Trastornos del Conocimiento/diagnóstico , Trastornos del Conocimiento/fisiopatología , Electroencefalografía/métodos , Potenciales Evocados Auditivos/fisiología , Estimulación Acústica/métodos , Adolescente , Factores de Edad , Niño , Preescolar , Trastornos del Conocimiento/inducido químicamente , Progresión de la Enfermedad , Electroencefalografía/efectos de los fármacos , Potenciales Evocados Auditivos/efectos de los fármacos , Femenino , Humanos , Masculino , Neoplasias/tratamiento farmacológico , Pruebas Neuropsicológicas/normas , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Valor Predictivo de las Pruebas , Procesamiento de Señales Asistido por ComputadorRESUMEN
Molecular mechanisms involved in epileptogenesis in the developing brain remain poorly understood. The gene array approach could reveal some of the factors involved by allowing the identification of a broad scale of genes altered by seizures. In this study we used microarray analysis to reveal the gene expression profile of the laser microdissected hippocampal CA1 subregion one week after kainic acid (KA)-induced status epilepticus (SE) in 21-day-old rats, which are developmentally roughly comparable to juvenile children. The gene expression analysis with the Chipster software generated a total of 1592 differently expressed genes in the CA1 subregion of KA-treated rats compared to control rats. The KEGG database revealed that the identified genes were involved in pathways such as oxidative phosporylation (26 genes changed), and long-term potentiation (LTP; 18 genes changed). Also genes involved in Ca(2+) homeostasis, gliosis, inflammation, and GABAergic transmission were altered. To validate the microarray results we further examined the protein expression for a subset of selected genes, glial fibrillary protein (GFAP), apolipoprotein E (apo E), cannabinoid type 1 receptor (CB1), Purkinje cell protein 4 (PEP-19), and interleukin 8 receptor (CXCR1), with immunohistochemistry, which confirmed the transcriptome results. Our results showed that SE resulted in no obvious CA1 neuronal loss, and alterations in the expression pattern of several genes during the early epileptogenic phase were comparable to previous gene expression studies of the adult hippocampus of both experimental epileptic animals and patients with temporal lobe epilepsy (TLE). However, some changes seem to occur after SE specifically in the juvenile rat hippocampus. Insight of the SE-induced alterations in gene expression and their related pathways could give us hints for the development of new target-specific antiepileptic drugs that interfere with the progression of the disease in the juvenile age group.
Asunto(s)
Perfilación de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Células Piramidales/metabolismo , Células Piramidales/patología , Estado Epiléptico/genética , Estado Epiléptico/patología , Envejecimiento/genética , Envejecimiento/patología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Análisis por Conglomerados , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Ácido Kaínico , Potenciación a Largo Plazo/genética , Masculino , Microdisección , Neuronas/metabolismo , Neuronas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Transducción de Señal/genética , Estado Epiléptico/inducido químicamente , Transmisión Sináptica/genéticaRESUMEN
The central histaminergic neuronal system is a powerful modulator of brain activity, and its functional disturbance is related to e.g. epilepsy. We have recently shown in the slice culture system that histaminergic neurons attenuate kainic acid (KA)-induced epileptiform activity and neuronal damage in the hippocampus through histamine 1 (H1) receptors. We now further examined the role of H1 receptors in the regulation of KA-induced seizures and neuronal damage in immature 9-day-old H1 receptor knock out (KO) mice. In the H1 receptor KO mice, behavioral seizures were significantly more severe and duration of seizures was significantly longer when compared to the wild type (WT) mice at the KA dose of 2mg/kg. Moreover, neuronal damage correlated with seizure severity, and it was significantly increased in the thalamus and retrosplenial granular cortex (RGC) of the KO mice. The H1 receptor antagonist triprolidine treatment supported these findings by showing significantly increased seizures severity and neuronal damage in the septum, thalamus, CA3 region of the hippocampus, and RGC in the KA-treated WT mice. Our present novel findings suggest that H1 receptors play a pivotal role in the regulation of seizure intensity and duration as well as seizure-induced neuronal damage in the immature P9 mice.
Asunto(s)
Susceptibilidad a Enfermedades/fisiopatología , Antagonistas de los Receptores Histamínicos H1/uso terapéutico , Receptores Histamínicos H1/metabolismo , Convulsiones/tratamiento farmacológico , Triprolidina/uso terapéutico , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Encéfalo/patología , Recuento de Células/métodos , Distribución de Chi-Cuadrado , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fluoresceínas , Ácido Kaínico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Compuestos Orgánicos , Receptores Histamínicos H1/deficiencia , Convulsiones/inducido químicamente , Convulsiones/genética , Convulsiones/patologíaRESUMEN
Epilepsy is a common neurological disorder that occurs more frequently in children than in adults. The extent that prolonged seizure activity, i.e. status epilepticus (SE), and repeated, brief seizures affect neuronal structure and function in both the immature and mature brain has been the subject of increasing clinical and experimental research. Earlier studies suggest that seizure-induced effects in the immature brain compared with the adult brain are different. This is manifested as differences in neuronal vulnerability, cellular and synaptic reorganization and regenerative processes. The focus of this review is first to give a short overview of currently used experimental models of epilepsy in immature rats, and then discuss more thoroughly seizure-induced acute and sub-acute cellular and molecular alterations, highlight the contribution of inflammatory-like reactions and intracellular cytoskeleton to the insult, and reveal changes in the structure and function of inhibitory GABA(A) and excitatory glutamate receptors. The role of seizure-activated reparative, plastic processes, synaptic remodelling, neurogenesis as well as the long-term consequences of seizures are briefly outlined. The main emphasis is put on studies carried out in experimental animals, and the focus of interest is the hippocampus, the brain area of great vulnerability in epilepsy. In vitro studies are discussed only to limited extent. Collectively, recent studies suggest that the deleterious effects of seizures may not solely be a consequence of neuronal damage and loss per se, but could be due to the fact that seizures interfere with the highly regulated developmental processes in the immature brain.
Asunto(s)
Daño Encefálico Crónico/fisiopatología , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiopatología , Epilepsia/fisiopatología , Degeneración Nerviosa/fisiopatología , Animales , Encéfalo/metabolismo , Daño Encefálico Crónico/etiología , Daño Encefálico Crónico/metabolismo , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Encefalitis/etiología , Encefalitis/metabolismo , Encefalitis/fisiopatología , Epilepsia/metabolismo , Humanos , Degeneración Nerviosa/etiología , Degeneración Nerviosa/metabolismo , Plasticidad Neuronal/fisiología , Ratas , Receptores de Neurotransmisores/metabolismo , Sinapsis/metabolismoRESUMEN
PURPOSE: Epileptic seizures lead to age-dependent neuronal damage in the developing brain, particularly in the hippocampus, but the mechanisms involved have remained poorly elucidated. In this study, we investigated the contribution of apoptosis and inflammatory processes to neuronal damage after status epilepticus (SE) in postnatal rats. METHODS: SE was induced by an intraperitoneal injection of kainic acid (KA) in 21- and 9-day-old (P21 and P9) rats. The expression of Bax, Bcl-2 and caspase-3, markers for apoptosis, and cyclooxygenase-2 (COX-2), an indicator for activation of inflammatory processes, were studied from 6 h up to 1 week after SE by Western blotting and immunocytochemistry. Neuronal damage was verified by Fluoro-Jade B staining. RESULTS: In P21 rats, SE resulted in neuronal damage in the CA1 neurons of the hippocampus. COX-2 expression was extensively, but transiently, increased and its immunoreactivity pronouncedly enhanced in several hippocampal subregions, amygdala, and piriform cortex by 24 h after SE. The expression of Bax and caspase-3 remained unchanged, whereas the antiapoptotic factor Bcl-2 transiently decreased by 24 h. Single caspase-3 positive neurons appeared in the CA1 region of both control and KA-treated rats. In P9 rats, no neuronal death was detected, and COX-2 expression and immunoreactivity remained at the control level. DISCUSSION: Our results suggest that SE provokes age-specific effects on COX-2 expression. This together with the activation of putative inflammatory processes may contribute to neuronal cell death in the hippocampus of postnatal rats, whereas caspase-dependent apoptosis seems not to be involved in the death process.
Asunto(s)
Corteza Cerebral/enzimología , Ciclooxigenasa 2/metabolismo , Hipocampo/enzimología , Hipocampo/patología , Neuronas/patología , Estado Epiléptico/patología , Factores de Edad , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Apoptosis/fisiología , Caspasa 3/metabolismo , Caspasa 3/fisiología , Muerte Celular/fisiología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Ciclooxigenasa 2/fisiología , Encefalitis/enzimología , Encefalitis/metabolismo , Encefalitis/patología , Inducción Enzimática/fisiología , Hipocampo/metabolismo , Ácido Kaínico , Neuronas/enzimología , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/enzimología , Estado Epiléptico/inducido químicamente , Estado Epiléptico/enzimología , Proteína Letal Asociada a bcl/farmacologíaRESUMEN
PURPOSE: Children with epilepsy are in risk for cognitive impairment, but reliable methods, other than neuropsychological testing, to verify such a decline are few. The purpose of this study was to assess the effect of infrequent seizures on cognitive skills in children with non-symptomatic focal epilepsy taking antiepileptic medication but still having infrequent seizures. METHODS: EEG (electroencephalogram) brain electric oscillatory responses of the 4-6Hz, 6-8Hz, 8-10Hz and 10-12Hz EEG frequency bands were studied. These responses, assessed by means of the event-related desynchronization (ERD) and synchronization (ERS) method, were recorded in 6 children with epilepsy (mean age 11.3 years) and in 11 control children (mean age 12 years) while they performed an auditory memory task. All subjects also underwent a comprehensive neuropsychological test battery. RESULTS: The differences in the 4-6Hz ERD/ERS responses between encoding and recognition were smaller in the children with epilepsy as compared to those of the control children. In the 6-8Hz frequency band, the responses of the two groups dissociated most notably in the frontal electrodes. No statistically significant differences in the alpha frequency range (8-12Hz) were observed between the groups. CONCLUSIONS: Significant alterations in the lower EEG frequency (4-8Hz) ERD/ERS responses in children with epilepsy during auditory memory processing, as compared to age-matched, healthy children may suggest that seizures affect memory and underlying brain processes, indexed also by poorer performance particularly in neuropsychological subtests related to language functions.
Asunto(s)
Sincronización Cortical , Electroencefalografía , Epilepsia/fisiopatología , Epilepsia/psicología , Memoria/fisiología , Desempeño Psicomotor/fisiología , Estimulación Acústica , Anticonvulsivantes/uso terapéutico , Percepción Auditiva/fisiología , Encéfalo/fisiopatología , Niño , Femenino , Humanos , Masculino , Pruebas NeuropsicológicasRESUMEN
In this study, we analyzed the spatiotemporal expression patterns of the high-molecular weight (MAP2a and b) and low-molecular weight (MAP2c and d) cytoskeletal microtubule-associated protein-2 (MAP2) isoforms with Western blotting, and the cellular localization of the high-molecular weight MAP2 isoforms with immunocytochemistry in the hippocampi of 1- to 21-day-old rats. Moreover, the temporal profile (from 30 min to 1 week) of MAP2 isoform reactivity to kainic acid-induced status epilepticus was studied in P9 rats. During development, the expression of the high-molecular weight MAP2 isoforms significantly increased, while the low-molecular weight isoforms decreased, the most prominent changes occurring during the second postnatal week. This developmental increase in the high-molecular weight MAP2 expression was also confirmed with immunocytochemistry, which showed increased immunoreactivity, particularly in the molecular layers of the dentate gyrus, and in CA1 and CA3 stratum radiatum. In 9-day-old rats, status epilepticus resulted in a rapid transient increase (about 210%) in the high-molecular weight MAP2 expression, without any effect on the low-molecular weight MAP2. Moreover, disturbed dendritic structure in the CA1 and CA3 stratum radiatum was manifested as formation of varicosities 3h after the kainic acid treatment. The strictly developmentally regulated MAP2 isoform expression suggests different functional roles for these proteins during the postnatal development in the rat hippocampus. Moreover, high-molecular weight MAP2s may play a role in nerve cell survival during cell stress.
Asunto(s)
Envejecimiento/fisiología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Estado Epiléptico/metabolismo , Animales , Animales Recién Nacidos , Axones/metabolismo , Convulsivantes , Giro Dentado/metabolismo , Hipocampo/fisiopatología , Inmunohistoquímica , Ácido Kaínico , Peso Molecular , Isoformas de Proteínas/metabolismo , Células Piramidales/metabolismo , Ratas , Ratas Sprague-Dawley , Estado Epiléptico/inducido químicamente , Estado Epiléptico/fisiopatologíaRESUMEN
BACKGROUND: Meconium aspiration-induced hypertensive lung injury is frequently associated with neuronal damage. Inhaled nitric oxide (iNO) is widely used in the treatment of pulmonary hypertension, but its effects on the brain are poorly known. AIMS: The aim of this study was to determine the effects of iNO treatment on the neuronal tissue after meconium aspiration. STUDY DESIGN: 71 anesthetized, catheterized and ventilated newborn piglets were studied for 6 h. Thirty-five piglets were instilled with a bolus of human meconium intratracheally and 36 piglets with saline instillation served as controls. Nineteen meconium piglets and 17 control piglets were continuously treated with 20 ppm of iNO, started at 30 min after the insult. The extent of neuronal injury was analysed histologically, and the levels of brain tissue lipid peroxidation products, reduced glutathione (GSH), myeloperoxidase activity and oxidized DNA were analysed as indicators of oxidative stress. RESULTS: iNO treatment diminished the pulmonary hypertensive response caused by meconium aspiration, but did not change systemic or carotid hemodynamics. NO administration was associated with reduced neuronal injury and diminished amount of oxidized DNA in the hippocampus of the meconium piglets. Further, iNO treatment was associated with decreased level of GSH in the cortex, but no change in lipid peroxidation production or myeloperoxidase activity was detected in any of the studied brain areas. CONCLUSIONS: Our results suggest that iNO treatment may inhibit DNA oxidation and neuronal injury in the hippocampus, associated with newborn meconium aspiration.
Asunto(s)
Asfixia Neonatal/tratamiento farmacológico , Interneuronas/patología , Síndrome de Aspiración de Meconio/fisiopatología , Óxido Nítrico/uso terapéutico , Estrés Oxidativo/fisiología , 8-Hidroxi-2'-Desoxicoguanosina , Administración por Inhalación , Análisis de Varianza , Animales , Asfixia Neonatal/etiología , Presión Sanguínea , Gasto Cardíaco , Estudios de Casos y Controles , Cromatografía Líquida de Alta Presión , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Glutatión/metabolismo , Frecuencia Cardíaca , Humanos , Recién Nacido , Peroxidación de Lípido/fisiología , Óxido Nítrico/administración & dosificación , Peroxidasa/metabolismo , Espectrofotometría Ultravioleta , Sus scrofa , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The cytoskeleton controls the architecture and survival of the central nervous system neurons by maintaining the stability of axons, dendrites and cellular architecture, and any disturbance in this genuine structure could compromise cell survival. The developmentally regulated intracellular intermediate filament protein neurofilament (NF), composed of the light (NF-L), medium (NF-M) and high (NF-H) molecular weight isoforms, is expressed abundantly in nerve cells but its significance in nerve cell survival in stress situations in the brain is unknown. We have used Western blotting, immunocytochemistry, and Fluoro-Jade B and thionine stainings to clarify the effect of kainic acid (KA) treatment on NF protein stability, and its importance for neuronal survival in hippocampal slice cultures. The contribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA glutamate receptor subtypes, calpain proteases and L-type Ca2+-channels to these processes were also assessed. Our results indicated that KA-induced degradation of NF was a fast process, similarly affecting all three NF proteins. It was effectively inhibited by the AMPA/KA receptor antagonist CNQX and the calpain inhibitor MDL-28170, whereas the Ca2+-channel blocker nifedipine and the NMDA receptor antagonist MK-801 had no significant effect. Moreover, KA-induced neuronal damage was effectively decreased in cultures treated with CNQX and MDL-28170. Our results suggest that the stability of NF proteins is an important factor contributing to neuronal survival after excitotoxic injury, and that both AMPA/KA receptor antagonists and calpain inhibitors might serve as neuroprotectants against this type of insult in the immature hippocampus.
Asunto(s)
6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas de Neurofilamentos/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Western Blotting , Calpaína/antagonistas & inhibidores , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Electroforesis en Gel de Poliacrilamida , Hipocampo/efectos de los fármacos , Inmunohistoquímica , Ácido Kaínico/farmacología , Microscopía Confocal , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidores , Receptores de Ácido Kaínico/antagonistas & inhibidoresRESUMEN
We have studied the effects of AMPA/kainate receptor agonists on GABA(A) receptor subunit mRNA expression in vitro in cultured rat cerebellar granule cells (CGCs). Kainate (KA) (100 microM) and high K(+) (25 mM) dramatically up-regulated delta subunit mRNA expression to 500-700% of that in control cells grown in low K(+) (5 mM). KA or high K(+) had no effect on the expression of the other major GABA(A) receptor subunits alpha1, alpha6, beta2, beta3 or gamma2. Up-regulation of delta mRNA was also detected with the AMPA receptor-selective agonist CPW-399 and to a lesser extent with the KA receptor-selective agonist ATPA. AMPA/kainate receptor-selective antagonist DNQX completely inhibited KA-, CPW-399- and ATPA-induced delta mRNA up-regulation indicating that the effects were mediated via AMPA and KA receptor activation. NMDA receptor-selective antagonist MK-801 inhibited 76% of the KA- and 57% of the CPW-399-induced delta up-regulation suggesting that KA and CPW-399 treatments may induce glutamate release resulting in NMDA receptor activation, and subsequently to delta mRNA up-regulation. In CGCs, delta subunit is a component of extrasynaptic alpha6betadelta receptors that mediate tonic inhibition. Up-regulation of delta during prolonged glutamate receptor activation or cell membrane depolarization may be a mechanism to increase tonic inhibition to counteract excessive excitation.
Asunto(s)
Cerebelo/citología , Neuronas/fisiología , Receptores de GABA-A/metabolismo , Receptores de Glutamato/fisiología , Regulación hacia Arriba/fisiología , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Neuronas/efectos de los fármacos , Potasio/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Receptores de Glutamato/clasificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sales de Tetrazolio , Tiazoles , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The central histaminergic neuron system inhibits epileptic seizures, which is suggested to occur mainly through histamine 1 (H1) and histamine 3 (H3) receptors. However, the importance of histaminergic neurons in seizure-induced cell damage is poorly known. In this study, we used an organotypic coculture system and confocal microscopy to examine whether histaminergic neurons, which were verified by immunohistochemistry, have any protective effect on kainic acid (KA)-induced neuronal damage in the developing hippocampus. Fluoro-Jade B, a specific marker for degenerating neurons, indicated that, after the 12 h KA (5 microM) treatment, neuronal damage was significantly attenuated in the hippocampus cultured together with the posterior hypothalamic slice containing histaminergic neurons [HI plus HY (POST)] when compared with the hippocampus cultured alone (HI) or with the anterior hypothalamus devoid of histaminergic neurons. Moreover, alpha-fluoromethylhistidine, an inhibitor of histamine synthesis, eliminated the neuroprotective effect in KA-treated HI plus HY (POST), and extracellularly applied histamine (1 nM to 100 microM) significantly attenuated neuronal damage only at 1 nM concentration in HI. After the 6 h KA treatment, spontaneous electrical activity registered in the CA1 subregion contained significantly less burst activity in HI plus HY (POST) than in HI. Finally, in KA-treated slices, the H3 receptor antagonist thioperamide enhanced the neuroprotective effect of histaminergic neurons, whereas the H1 receptor antagonists triprolidine and mepyramine dose-dependently decreased the neuroprotection in HI plus HY (POST). Our results suggest that histaminergic neurons protect the developing hippocampus from KA-induced neuronal damage, with regulation of neuronal survival being at least partly mediated through H1 and H3 receptors.
Asunto(s)
Convulsivantes/toxicidad , Hipocampo/efectos de los fármacos , Histamina/farmacología , Ácido Kaínico/toxicidad , Neuronas/fisiología , Fármacos Neuroprotectores/farmacología , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/fisiología , Técnicas de Cocultivo , Hipocampo/citología , Histamina/biosíntesis , Histamina/fisiología , Antagonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacología , Hipotálamo Anterior/citología , Hipotálamo Posterior/citología , Imidazoles/farmacología , Metilhistidinas/farmacología , Microscopía Confocal , Técnicas de Cultivo de Órganos , Piperidinas/farmacología , Pirilamina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Histamínicos H1/efectos de los fármacos , Receptores Histamínicos H1/fisiología , Receptores Histamínicos H3/efectos de los fármacos , Receptores Histamínicos H3/fisiología , Tiourea/análogos & derivados , Tiourea/farmacología , Triprolidina/farmacologíaRESUMEN
The hippocampus has become one of the most extensively studied areas of the mammalian brain, and its proper function is of utmost importance, particularly for learning and memory. The hippocampus is the most susceptible brain region for damage, and its impaired function has been documented in many human brain diseases, e.g. hypoxia, ischemia, and epilepsy regardless of the age of the affected patients. In addition to experimental in vivo models of these disorders, the investigation of basic anatomical, physiological, and molecular aspects requires an adequate experimental in vitro model, which should meet the requirements for well-preserved representation of various cell types, and functional information processing properties in the hippocampus. In this review, the characteristics of organotypic hippocampal slice cultures (OHCs) together with the main differences between the in vivo and in vitro preparations are first briefly outlined. Thereafter, the use of OHCs in studies focusing on neuron cell death and synaptic plasticity is discussed.
Asunto(s)
Citoprotección , Hipocampo/citología , Modelos Biológicos , Plasticidad Neuronal , Neuronas/citología , Animales , Muerte Celular , Técnicas de Cultivo de ÓrganosRESUMEN
AIM: Meconium aspiration-induced hypertensive lung injury, especially when connected with perinatal asphyxia, has been associated with brain damage. We aimed to determine the neuronal injury induced by pulmonary meconium contamination alone and with concurrent asphyxia. METHODS: 36 anaesthetized and ventilated newborn piglets were haemodynamically monitored for 6 h. Seven piglets without concurrent asphyxia and seven piglets with asphyxia were instilled with a bolus of human meconium intratracheally. Seven piglets had only asphyxia and 15 piglets served as controls. The brains were studied histologically. RESULTS: Meconium aspiration did not change systemic haemodynamics acutely, while its combination with asphyxia diminished the abrupt postasphyxic systemic hypertensive peak and resulted in a transient increase in carotid artery flow, not seen after isolated asphyxia. Systemic pressure declined after 4 h in all insulted groups, but only isolated asphyxia was associated with a sustained decrease in carotid artery flow. Arterial oxygenation remained normal, except during the acute insults. Brain examination after meconium instillation indicated neuronal injury, especially in the CA3 region of the hippocampus. Asphyxia resulted in neuronal injury in the cortical, cerebellar and hippocampal hilus regions. CONCLUSION: Severe meconium aspiration itself may result in hippocampal neuronal injury.
Asunto(s)
Asfixia Neonatal/patología , Asfixia Neonatal/fisiopatología , Encefalopatías/patología , Encefalopatías/fisiopatología , Hipocampo/patología , Síndrome de Aspiración de Meconio/patología , Síndrome de Aspiración de Meconio/fisiopatología , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Asfixia Neonatal/etiología , Biopsia con Aguja , Modelos Animales de Enfermedad , Hipocampo/ultraestructura , Humanos , Inmunohistoquímica , Recién Nacido , Microscopía Electrónica , Fotomicrografía , Probabilidad , Valores de Referencia , Sensibilidad y Especificidad , PorcinosRESUMEN
BACKGROUND: Meconium aspiration-induced hypertensive lung injury has been associated with neuronal damage in the newborn, but the mechanisms of the injury are poorly known. AIMS: The aim of the study was to determine the contribution of oxidative stress to the brain damage after pulmonary meconium contamination. STUDY DESIGN: Sixteen anesthetized and ventilated newborn piglets were studied for 6 h. Eight piglets were instilled with a bolus of human meconium intratracheally and eight piglets with saline instillation served as controls. Brain tissue lipid peroxidation products (TBARS), reduced glutathione (GSH), myeloperoxidase activity and oxidized DNA were analyzed as indicators of oxidative stress. RESULTS: Meconium aspiration did not change the systemic or carotid hemodynamics, but caused a well-established pulmonary hypertensive response. Sustained increase in additional oxygen demand was also observed after meconium insult, but no actual hypoxemia or hypercarbia was evident during the whole study period. Myeloperoxidase activity was elevated in the cerebellum after pulmonary meconium instillation, whereas concentrations of peroxidation products and glutathione were similar in the cortical, cerebellar and hippocampal regions of the two groups. Still, the amount of oxidized DNA was increased in the hippocampus of the meconium-aspirated piglets when compared to controls. CONCLUSIONS: Our data thus suggest that oxidative injury associated with pulmonary, but not systemic, hemodynamic disturbances may contribute to hippocampal damage after meconium aspiration in newborns.
Asunto(s)
Hipocampo/fisiopatología , Síndrome de Aspiración de Meconio/fisiopatología , Estrés Oxidativo/fisiología , Análisis de Varianza , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Glutatión/metabolismo , Hipocampo/metabolismo , Humanos , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/fisiopatología , Recién Nacido , Síndrome de Aspiración de Meconio/metabolismo , Peroxidasa/metabolismo , Sus scrofa , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
PURPOSE: Seizures induce progressive morphologic and functional changes in particular in the hippocampus, but whether and at what stage the hippocampus is affected in children with focal, temporal, nonintractable epilepsy is poorly known. We have now studied eventual metabolic and volume changes in the hippocampus of children with nonsymptomatic focal epilepsy taking antiepileptic medication (AEDs) but still having infrequent seizures. METHODS: Quantitative proton magnetic resonance spectroscopy ((1)HMRS) and volumetric MRI were used to study the hippocampal region of 11 pediatric outpatients (age 10 to 17 years) with cryptogenic localization-related epilepsy, and eight healthy volunteers (age 9 to 16 years) served as controls. The spectra were obtained bilaterally from the hippocampi by using the 1.5-T MR imager. The spectral resonance lines of N-acetyl group (NA), creatine and phosphocreatine group (Cr), choline-containing compounds (Cho), and myoinositol (mI) were analyzed quantitatively. The volume of the hippocampus was semiautomatically calculated. RESULTS: The mean concentration of NA was significantly decreased both in the focus side (9.02 +/- 2.00 mM) and in the nonfocus side (8.88 +/- 2.09 mM) of the patients compared with the controls (10.76 +/- 1.86 mM), in particular if the children had a history of generalized tonic-clonic seizures. The mean concentrations of Cho, Cr, and mI did not differ significantly between the patients and controls. Moreover, the mean hippocampal volume of the focus side of patients was significantly reduced compared with that of the controls. CONCLUSIONS: Metabolic changes in hippocampi were detected in children with nonsymptomatic localization-related epilepsy and infrequent seizures. Reduced NA could reflect neuronal metabolic dysfunction and/or neuronal damage, as indicated by our volumetric findings.
Asunto(s)
Ácido Aspártico/análogos & derivados , Epilepsias Parciales/diagnóstico , Epilepsias Parciales/patología , Hipocampo/metabolismo , Hipocampo/patología , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Adolescente , Ácido Aspártico/metabolismo , Niño , Colina/metabolismo , Creatina/metabolismo , Epilepsias Parciales/metabolismo , Femenino , Lateralidad Funcional , Humanos , Imagen por Resonancia Magnética/estadística & datos numéricos , Masculino , Neuronas/metabolismo , Neuronas/patología , Fosfocreatina/metabolismoRESUMEN
The preparation of hippocampal slices results in loss of input neurons to dentate granule cells, which leads to the reorganization of their axons, the mossy fibers, and alters their functional properties in long-term cultures, but its temporal aspects in the immature hippocampus are not known. In this study, we have focused on the early phase of this plastic reorganization process by analyzing granule cell function with field potential and whole cell recordings during the in vitro maturation of hippocampal slices (from 1 to 17 days in vitro, prepared from 6 to 7-day-old rats), and their morphology using extracellular biocytin labelling technique. Acute slices from postnatal 14-22-day-old rats were analyzed to detect any differences in the functional properties of granule cells in these two preparations. In field potential recordings, small synaptically-evoked responses were detected at 2 days in vitro, and their amplitude increased during the culture time. Whole cell voltage clamp recordings revealed intensive spontaneous excitatory postsynaptic currents, and the susceptibility to stimulus-evoked bursting increased with culture time. In acutely prepared slices, neither synaptically-evoked responses in field potential recordings nor any bursting in whole cell recordings were detected. The excitatory activity was under the inhibitory control of gamma-aminobutyric acid type A receptor. Extracellularily applied biocytin labelled dentate granule cells, and revealed sprouting and aberrant targeting of mossy fibers in cultured slices. Our results suggest that reorganization of granule cell axons takes place during the early in vitro maturation of hippocampal slices, and contributes to their increased excitatory activity resembling that in the epileptic hippocampus. Cultured immature hippocampal slices could thus serve as an additional in vitro model to elucidate mechanisms of synaptic plasticity and cellular reactivity in response to external damage in the developing hippocampus.