RESUMEN
Severe hypoxia was shown to induce apoptotic death in developing brain neurons, whereas mild hypoxia was demonstrated to stimulate neurogenesis. Since the apoptotic process may share common pathways with mitosis, expression profiles of proteins involved in apoptosis or the cell cycle were analyzed by immunohistochemistry and/or western blotting, in relation with cell outcome of cultured neurons from fetal rat forebrain subjected to either lethal (6 h) or non-lethal (3 h) hypoxia (95% N(2)/5% CO(2)). Hypoxia for 6 h led to apoptosis that was inhibited by the cell cycle blocker olomoucine. Transient overexpression of proliferating cell nuclear antigen was followed by increasing expression of p53, p21, Bax and caspases, whereas Bcl-2 and heat shock proteins were progressively repressed. Conversely, a 3-h hypoxic insult initiated neuronal mitosis, with increased thymidine incorporation. In these conditions, levels of proliferating cell nuclear antigen, Rb, Bcl-2 and heat shock proteins were persistently elevated, while expression of p53, p21, Bax and caspases gradually decreased. These data confirm that hypoxia promotes cell cycle activation, whatever the stress intensity. This process is then aborted following apoptosis-inducing hypoxia, whereas sublethal insult would trigger neurogenesis, at least in developing brain neurons in vitro, by stimulating timed expression of neurogenic and survival-associated proteins.
Asunto(s)
Fragmentación del ADN/fisiología , Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/fisiopatología , Neuronas/citología , Neuronas/metabolismo , Animales , Caspasa 1/metabolismo , Caspasa 3 , Caspasas/metabolismo , Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Supervivencia Celular/fisiología , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Femenino , Feto , Proteínas HSP70 de Choque Térmico/metabolismo , Hipoxia Encefálica/patología , Embarazo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Prosencéfalo/citología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2RESUMEN
To address the influence of oxidative stress and defense capacities in the effects of transient hypoxia in the immature brain, the time course of reactive oxygen species generation was monitored by flow cytometry using dihydrorhodamine 123 and 2',7'-dichlorofluorescein-diacetate in cultured neurons issued from the fetal rat forebrain and subjected to hypoxia/reoxygenation (6 h/96 h). Parallel transcriptional and activity changes of superoxide dismutases, glutathione peroxidase and catalase were analyzed, in line with cell outcome. The study confirmed hypoxia-induced delayed apoptotic death, and depicted increased mitochondrial and cytosolic productions of free radicals (+30%) occurring over the 48-h period after the restoration of oxygen supply, with sequential stimulations of superoxide dismutases. Whereas catalase mRNA levels and activity were augmented by cell reoxygenation, glutathione peroxidase activity was transiently repressed (-24%), along with reduced glutathione reductase activity (-27%) and intracellular glutathione depletion (-19%). Coupled with the neuroprotective effects of the glutathione precursor N-acetyl-cysteine (50 microM), these data suggest that hypoxia/reoxygenation-induced production of reactive oxygen species can overwhelm glutathione-dependent antioxidant capacity, and thus may contribute to the resulting neuronal apoptosis.
Asunto(s)
Apoptosis/fisiología , Depuradores de Radicales Libres/metabolismo , Hipoxia Encefálica/enzimología , Líquido Intracelular/enzimología , Neuronas/enzimología , Prosencéfalo/enzimología , Daño por Reperfusión/enzimología , Animales , Apoptosis/efectos de los fármacos , Asfixia Neonatal/enzimología , Asfixia Neonatal/patología , Asfixia Neonatal/fisiopatología , Catalasa/genética , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Células Cultivadas/patología , Feto , Fluoresceínas/farmacocinética , Colorantes Fluorescentes/farmacocinética , Radicales Libres/metabolismo , Glutatión Peroxidasa/genética , Humanos , Hipoxia Encefálica/embriología , Hipoxia Encefálica/fisiopatología , Recién Nacido , Líquido Intracelular/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Prosencéfalo/patología , Prosencéfalo/fisiopatología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Rodaminas/farmacocinética , Superóxido Dismutasa/genética , Factores de TiempoRESUMEN
Both hypoxia and bilirubin are common risk factors in newborns, which may act synergistically to produce anatomical and functional disturbances of the CNS. Using primary cultures of neurons from the fetal rat brain, it was recently reported that neuronal apoptosis accounts for the deleterious consequences of these two insults. To investigate the influence of hypoxia, bilirubin, or their combination on the outcome of neuronal cells of the immature brain, and delineate cellular mechanisms involved, 6-d-old cultured neurons were submitted to either hypoxia (6 h), unconjugated bilirubin (0.5 microM), or to combined conditions. Within 96 h, cell viability was reduced by 22.7% and 24.5% by hypoxia and bilirubin, respectively, whereas combined treatments decreased vital score by 34%. Nuclear morphology revealed 13.4% of apoptotic cells after hypoxia, 16.2% after bilirubin, and 22.6% after both treatments. Bilirubin action was specifically blocked by the glutamate receptor antagonist MK-801, which was without effect on the consequences of hypoxia. Temporal changes in [(3)H]leucine incorporation rates as well as beneficial effects of cycloheximide reflected a programmed phenomenon dependent upon synthesis of selective proteins. The presence of bilirubin reduced hypoxia-induced alterations of cell energy metabolism, as reflected by 2-D-[(3)H]deoxyglucose incorporation, raising the question of free radical scavenging. Measurements of intracellular radical generation, however, failed to confirm the antioxidant role of bilirubin. Taken together, our data suggest that low levels of bilirubin may enhance hypoxia effects in immature neurons by facilitating glutamate-mediated apoptosis through the activation of N:-methyl-D-aspartate receptors.
Asunto(s)
Bilirrubina/toxicidad , Encéfalo/efectos de los fármacos , Hipoxia de la Célula , Ácido Glutámico/toxicidad , Animales , Encéfalo/citología , Células Cultivadas , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/efectos de los fármacosRESUMEN
Many catechol derivatives are currently used as drugs, even if they produce reactive oxygen species that may cause tissue damage. Among them, apomorphine, a potent dopamine agonist, displays efficient anti-parkinsonian properties, but the consequences of its oxidant and toxic properties have been poorly investigated on in vitro models. In the present work, we investigated apomorphine cytotoxicity by incubating cultures of rat glioma C6 cells and primary cultures of neurons with different concentrations of the drug. Apomorphine-promoted cell death was proportional to its concentration and was time-dependent. The ED(50) of apomorphine on C6 cell death after 48 hr was about 200 microM. The cytotoxic effects induced by apomorphine were correlated to its autoxidation, which leads to the formation of reactive oxygen species, semiquinones, quinones, and a melanin-like pigment. C6 cells that underwent treatment with 400 microM apomorphine for 6 hr displayed features of necrosis, including loss of membrane integrity, degeneration of mitochondria, and DNA fragmentation. Thiols, such as cysteine, N-acetyl-L-cysteine, and glutathione, significantly protected cultured neurons and C6 cells against apomorphine-induced cytotoxicity. Thiols also inhibited apomorphine autoxidation. These data strongly suggest that apomorphine cytotoxicity towards neurons and C6 cells results from an intracellular oxidative stress.
Asunto(s)
Antioxidantes/farmacología , Apomorfina/toxicidad , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Sustancias Protectoras/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN/efectos de los fármacos , ADN/metabolismo , Daño del ADN , Agonistas de Dopamina/toxicidad , Interacciones Farmacológicas , Electroforesis en Gel de Agar , Neuroglía/ultraestructura , Neuronas/ultraestructura , Oxidación-Reducción/efectos de los fármacos , Proteínas/efectos de los fármacos , Proteínas/metabolismo , RatasRESUMEN
Following hypoxia/reoxygenation (6h/96h), cultured neurons from the embryonic rat forebrain undergo delayed apoptosis. To evaluate the participation of oxidative stress and defense mechanisms, temporal evolution of intraneuronal free radical generation was monitored by flow cytometry using dihydrorhodamine 123, in parallel with the study of transcriptional, translational, and activity changes of the detoxifying enzymes Cu/Zn-SOD and Mn-SOD. Two distinct peaks of radical generation were depicted, at the time of reoxygenation (+ 27%) and 48 h later (+ 25%), respectively. Radical production was unaffected by caspase inhibitors YVAD-CHO or DEVD-CHO, which prevented neuronal damage, suggesting that caspase activation is not an upstream initiator of radicals in this model. Cell treatment by vitamin E (100 microM) displayed significant neuroprotection, whereas the superoxide generating system xanthine/xanthine oxidase induced apoptosis. Transcript and protein levels of both SODs were reduced 1 h after the onset of hypoxia, but activities were transiently stimulated. Reoxygenation was associated with an increased expression (139%), but a decreased activity (21%) of the inducible Mn-SOD, whereas Cu/Zn-SOD protein and activity were low and progressively increased until 48 h post-hypoxia, when the second rise in radicals occurred. In spite of a temporal regulation of SODs, which parallels radical formation, oxidative stress might account for neurotoxicity induced by hypoxia.
Asunto(s)
Apoptosis/fisiología , Hipoxia de la Célula/fisiología , Neuronas/citología , Neuronas/fisiología , Prosencéfalo/fisiología , Aerobiosis , Animales , Inhibidores de Caspasas , Células Cultivadas , Embrión de Mamíferos , Radicales Libres/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Prosencéfalo/citología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Vitamina E/farmacología , Xantina/metabolismo , Xantina Oxidasa/metabolismoRESUMEN
Increased amounts of bilirubin, the end product of heme degradation, are known to be detrimental to the central nervous system, especially in preterm newborns. In an attempt to delineate the cellular mechanisms by which unconjugated bilirubin exerts its toxic effects on neuronal cells in the developing brain, bilirubin (0.25-5 microM) was added to the extracellular medium of 6-day-old primary cultured neurons from the embryonic rat forebrain, and cell alterations were studied over the ensuing 96 h. Bilirubin decreased cell viability dose dependently with an ED(50) around 1 microM. At the dose of 0.5 microM, it triggered delayed cell death that affected 24% of the neurons. Nuclear incorporation of the fluorescent dye DAPI (4,6-diamidino-2-phenylindole) depicted the presence of apoptosis (16%). Apoptosis features were confirmed by DNA fragmentation reflected by a progressive loss of [(3)H]thymidine and sequential changes in macromolecular synthesis, as shown by the time course of [(3)H]leucine incorporation, as well as by the beneficial effects of cycloheximide and caspase inhibitors. In parallel, treatments with glutamate receptor antagonists showed that MK-801, but not NBQX, protected neurons against bilirubin neurotoxicity, suggesting a role for NMDA receptors in bilirubin effects. Coupled with previous work about glutamate toxicity in the same culture model, these data support the hypothesis that low levels of free bilirubin may promote programmed neuronal death corresponding to an apoptotic process which involves caspase activation and requires the participation of NMDA receptors, along with bilirubin-induced inhibition of protein kinase C activity.
Asunto(s)
Apoptosis/efectos de los fármacos , Bilirrubina/toxicidad , Neuronas/citología , Neuronas/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Apoptosis/fisiología , Caspasas/metabolismo , Células Cultivadas , Cicloheximida/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Colorantes Fluorescentes/farmacocinética , Indoles/farmacocinética , Masculino , Neuronas/enzimología , Oligopéptidos/farmacología , Embarazo , Prosencéfalo/citología , Prosencéfalo/embriología , Proteína Quinasa C/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Timidina/metabolismo , Timidina/farmacología , TritioRESUMEN
In neuronal cultures from the forebrain of 14-d-old rat embryos, transient hypoxia (95% N2/5% CO2, 37 degrees C) for 6 h has been shown to trigger delayed apoptotic death through sequential changes in protein synthesis, whereas preconditioning by a brief episode of hypoxia can rescue neurons. Because hypothermia has been reported to be neuroprotective, the present study was designed to test the influence of reduced temperature on the consequences of lethal hypoxia in our culture model, and cellular mechanisms involved were compared with those underlying preconditioning effects. After 6 d in vitro, cultures were subjected to hypoxia for 6 h. They were either placed at 32 degrees C concomitantly with hypoxia for 6 h or preconditioned the day before by a 1-h episode of hypoxia. The hypoxic insult decreased cell viability by 38% at 96 h after reoxygenation, and 23% of the neurons showed morphologic features of apoptosis. Both hypothermia and preconditioning prevented neuronal death and reduced apoptosis. Preconditioning led to time-dependent changes in leucine incorporation, with persistent overexpression of the survival proteins Bcl-2 and heat-shock protein 70. It also increased thymidine incorporation, in line with induction of the cofactor for DNA polymerase, proliferating cell nuclear antigen. Hypothermia reduced basal apoptosis and necrosis, but did not affect thymidine incorporation, and abolished hypoxia-associated protein synthesis. Therefore, both treatments were protective against neuronal injury consecutive to hypoxia in developing brain neurons in vitro. Whereas preconditioning activated a program that stimulated the expression of anti-apoptotic gene products and regulatory components of the cell cycle, hypothermia did not trigger active processes, but depressed cell activity, which in turn may impair the apoptotic phenomenon.
Asunto(s)
Apoptosis , Hipotermia , Hipoxia/patología , Precondicionamiento Isquémico , Prosencéfalo/patología , Animales , Células Cultivadas , ADN/biosíntesis , Femenino , Inmunohistoquímica , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Biosíntesis de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
To further explore the contribution of caspase-1/interleukin-1beta-convening enzyme in the consequences of hypoxia in developing brain neurons, its temporal expression profile was analysed by immunohistochemistry and western blotting in cultured neurons from the embryonic rat forebrain subjected to a hypoxic stress (95% N2/5% CO2 for 6 h), and proteolytic activity of caspase-1 was monitored as a function of time by measuring the degradation of a selective colorimetric substrate (N-acetyl-Tyr-Val-Ala-Asp-p-nitroanilide). In addition, the influence of pre- and posthypoxic treatments by caspase-1 inhibitors (N-acetyl-Tyr-Val-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone) was tested on cell outcome. Hypoxia led to delayed apoptotic neuronal death, with an elevation of the expression of both pro-caspase-1 and caspase-1 active cleavage product (ICE p20) for up to 96 h after cell reoxygenation. As reflected by cleavage of the specific substrate, caspase-1 activity progressively increased between 24 h and 96 h posthypoxia, and was blocked by inhibitors in a dose-dependent fashion. The inhibitory compounds, including when given 24 h after hypoxia, prevented neuronal death, reduced apoptosis hallmarks and also increased the number of mitotic neurons, suggesting they might promote neurogenesis. Similar observations were made when neurons were exposed to a sublethal hypoxia (i.e. 3 h). These data emphasize the participation of caspase-1 in neuronal injury consecutive to oxygen deprivation, and provide new insight into the possible cellular mechanisms by which caspase inhibitors may protect developing brain neurons.
Asunto(s)
Encéfalo/enzimología , Encéfalo/patología , Caspasa 1/fisiología , Hipoxia/enzimología , Hipoxia/patología , Animales , Western Blotting , Encéfalo/embriología , Caspasa 1/metabolismo , Núcleo Celular/ultraestructura , Supervivencia Celular , Células Cultivadas , Senescencia Celular/fisiología , Inhibidores de Cisteína Proteinasa/farmacología , Inmunohistoquímica , Neuronas/enzimología , Neuronas/fisiología , Neuronas/ultraestructura , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Since caspase members have been identified as effectors of apoptosis, the role of CPP32/caspase-3 was further explored in cultured neurons from the embryonic rat forebrain submitted to a 6-h hypoxia which has previously been shown to induce apoptotic death within four days after reoxygenation, whereas a shorter aggression (i.e., for 3 h) leads by the same time to an increased number of living neurons, suggesting that sublethal hypoxia may promote neurogenesis. Neuronal expression of the active cleavage product of CPP32 (CPP32 p20) increased specifically after hypoxia for 6 h to finally reach 985% over control normoxic values at 96 h post-insult, while a 3-h hypoxia triggered the inducible stress protein HSP70 that has been shown to inhibit caspase-3. Proteolytic activity of caspase-3 was progressively stimulated by lethal hypoxia, as reflected by the degradation of two selective substrates, including poly (ADP-ribose) polymerase (PARP). Caspase-3 activity was blocked specifically and dose-dependently by the peptide inhibitor, DEVD-CHO, that reduced the number of apoptotic cells and prevented the hypoxia-induced decrease in cell viability, including when given 24 h post-insult. Interestingly, in these conditions, the inhibitory compounds enhanced the number of mitotic neurons. These data emphasize the critical role of caspase-3 in neuronal injury consecutive to hypoxia. Whereas caspase inhibitors may provide benefit over a broad therapeutic window, they might allow developing neurons to complete their cell cycle initiated in response to stress, as it is the case for sublethal hypoxia.
Asunto(s)
Apoptosis , Encéfalo/enzimología , Caspasas/metabolismo , Precursores Enzimáticos/metabolismo , Neuronas/enzimología , Animales , Western Blotting , Caspasa 3 , Hipoxia de la Célula , Supervivencia Celular , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Reparación del ADN , Femenino , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Glutamate NMDA receptor has been implicated in brain developmental processes as well as in excitotoxicity and seizure mediation. A previous study has shown that an acute episode of seizures for 30 min in rats altered NMDA receptor characteristics, mainly in the very immature animal. In order to assess whether receptor modifications may also account for long-lasting cerebral disabilities, medium- and long-term consequences of repeated seizures in developing rats on brain NMDA receptor properties were investigated. Seizures were induced once a day for 3 consecutive days, either from post-natal day 5 (P5) to P7 or from P15 to P17. NMDA receptors were then analysed at P15, P25 and P60 (adulthood) by measuring specific binding of [3H]MK-801 on brain membrane preparations. In addition, allosteric modulation of NMDA receptors by exogenous glutamate and glycine was investigated. Seizures from P5 to P7 led to a 22% increase in the density of [3H]MK-801 binding sites measured at P15, but did not affect NMDA receptor density or affinity at P25 or P60. P15-P17 seizures led to a 21% decrease in the density of binding sites and to a 33% decrease in receptor dissociation constant at P25, while they were without effect at P60. Moreover, P5-P7 and P15-P17 seizures were both associated with a suppression of the glutamate/glycine-induced receptor activation at P60. These modifications might account for long-term alterations in cerebral excitability or plasticity after early convulsive disorders, with regards to altered cognitive capacities, epileptogenesis and brain susceptibility to recurrent seizures.
Asunto(s)
Envejecimiento/metabolismo , Animales Recién Nacidos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsiones/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Sitios de Unión , Unión Competitiva , Maleato de Dizocilpina/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/farmacología , Glicina/farmacología , Ratas , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Recurrencia , Factores de TiempoRESUMEN
In order to assess long-lasting consequences of recurrent seizures during development, the effects of repeated seizures in developing rats were investigated on brain adenosine A1 and A2A receptors. The characteristics of A1 and A2A receptors were analyzed by measuring the binding of the selective agonists [3H]CHA (N6-cyclohexyladenosine) and [3H]CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine), respectively, on cerebral membrane preparations, whereas receptor coupling to G-proteins was examined by using a GTP analogue (Gpp(NH)p; guanylyl-5'-imidodiphosphate). Seizures were induced by bicuculline once a day at two different developmental stages: either from postnatal day 5 to postnatal day 7 (P5-P7) or from P15 to P17. Adenosine receptors were then studied at P15, P25 and P60. P5-P7 seizures led to an increase in A1 receptor density at P60 and to a decrease in their coupling to G-proteins at P15, but they did not affect A2A receptors. P15-P17 seizures decreased the coupling of A1 receptors to G-proteins at P25 and P60, reduced the density of A2A receptors at P25 and increased their affinity at P60. These results depict a persistent sensitivity of both A1 and A2A brain adenosine receptors to repeated seizures, with selective receptor alterations according to the cerebral maturational stage when seizures occur. In respect to the neuromodulatory and anticonvulsant properties of adenosine, such changes might be implicated in long-term functional brain reorganization after early seizures and future susceptibility to convulsive disorders.
Asunto(s)
Receptores Purinérgicos P1/fisiología , Convulsiones/fisiopatología , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Animales Recién Nacidos/fisiología , Bicuculina/farmacología , Química Encefálica/fisiología , Convulsivantes/farmacología , Femenino , Guanilil Imidodifosfato/farmacología , Técnicas In Vitro , Cinética , Membranas/metabolismo , Fenetilaminas/metabolismo , Embarazo , Agonistas del Receptor Purinérgico P1 , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A2A , Receptores Purinérgicos P1/metabolismo , Convulsiones/inducido químicamenteRESUMEN
Previous studies have demonstrated that transient hypoxia (6 h) induces apoptotic death in cultured neurons isolated from the fetal rat forebrain. Since activation of c-Jun N-terminal kinases (JNKs) and subsequent phosphorylation of c-Jun are suspected to be involved in the apoptotic pathway in several cell types, the time course of activator protein-1 (AP-1) DNA-binding, in line with induction of the AP-1 components and JNK activation, was examined during hypoxia/reoxygenation in the same model. Gel shift analysis depicted the presence of functional AP-1 transcription factors in both control and hypoxic neurons. One hour after the onset of hypoxia, all AP-1 components were markedly overexpressed. They include c-Jun, Jun B, Jun D, c-Fos and Fos-related antigens. Whereas, only c-Jun remained elevated for up to 96 h post-reoxygenation, time at which neurons were injured, other gene products showed patterned induction/repression as hypoxia progressed and then during the post-reoxygenation period, with Fos-related antigens being finally induced at 96 h. Only JNK1 was constitutively detected in cultured neurons, and its expression was inhibited during hypoxia. Nonetheless, both JNK1 and JNK3 were markedly, but transiently, induced at 48 h post-reoxygenation, when apoptosis-related morphological features became apparent. These data support the hypothesis that transient hypoxia, independently of ischemia, may trigger apoptosis through JNK signaling pathway in developing brain neurons.
Asunto(s)
Apoptosis/fisiología , Hipoxia Encefálica/fisiopatología , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Neuronas/citología , Proteínas Quinasas/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Anticuerpos , Encéfalo/irrigación sanguínea , Encéfalo/embriología , Encéfalo/enzimología , Química Encefálica/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Femenino , Ataque Isquémico Transitorio/fisiopatología , MAP Quinasa Quinasa 4 , Masculino , Neuronas/química , Neuronas/enzimología , Embarazo , Proteínas Quinasas/análisis , Proteínas Quinasas/inmunología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/fisiopatología , Factor de Transcripción AP-1/análisis , Factor de Transcripción AP-1/inmunologíaRESUMEN
To assess long-term metabolic consequences of recurrent ictal events arising during development, seizures were repeatedly generated in rats at different stages of cerebral maturation. Seizures were induced by i.p. injections of bicuculline for three consecutive days, starting from postnatal day 5 (P5), when the brain is very immature, or from P15, a period at which the brain is more structurally organized. Local cerebral metabolic rates for glucose were measured in 74 structures at P15, P25 and in adults (P60), by the autoradiographic method using 2-D-[14C]deoxyglucose. Repeated seizures in P5 to P7 pups led to a reduction (16-34%) of glucose consumption at P15, mainly significant in sensory, motor and functionally non-specific areas as well as in cerebellar nuclei. Selective decreases in metabolic activity were still recorded in adults, mostly in auditory system (20%) and cerebellar nuclei (27%). Seizures generated from P15 to P17 led to an overall mortality rate of 62% (versus 22% at P5 to P7). Surviving animals exhibited reduced metabolic rates for glucose (by 7-27%) at P25, significant in 23 structures, and depicting pronounced changes in limbic, hypothalamic, sensory and white matter areas, whereas brain functional activity finally returned to basal values at P60. Therefore, while younger rats seemed to better tolerate repeated bicuculline-induced seizures than older animals, the reverse was true for long-term metabolic effects, and the more immature the brain when seizures arise, the more persistent the functional consequences.
Asunto(s)
Envejecimiento/metabolismo , Bicuculina/toxicidad , Encéfalo/metabolismo , Metabolismo Energético , Convulsiones/metabolismo , Animales , Vías Auditivas/crecimiento & desarrollo , Vías Auditivas/metabolismo , Autorradiografía , Peso Corporal , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Dióxido de Carbono/sangre , Radioisótopos de Carbono , Desoxiglucosa/metabolismo , Metabolismo Energético/efectos de los fármacos , Vías Olfatorias/crecimiento & desarrollo , Vías Olfatorias/metabolismo , Tamaño de los Órganos , Especificidad de Órganos , Oxígeno/sangre , Presión Parcial , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/fisiopatología , Vías Visuales/crecimiento & desarrollo , Vías Visuales/metabolismoRESUMEN
Brain reperfusion and/or reoxygenation may be of particular importance in the etiology of neuronal damage after hypoxic-ischemic insult in neonates, especially with reference to the generation of free radicals. To investigate this issue, the influence of either standard reoxygenation or transient hyperoxia was studied on the consequences of severe hypoxia in a model of cultured neurons isolated from the fetal rat brain. Culture dishes were exposed for 6 h to hypoxia (95% N2/5% CO2). They were then placed under normoxia (95% air/5% CO2) or hyperoxia (95% O2/5% CO2) for 3 h, and finally returned to normoxia. Control cultures were kept under normoxic conditions. Cell morphology, protein concentrations, lactate dehydrogenase leakage, energy metabolism, as reflected by specific transport and incorporation of 2-D-[3H]deoxyglucose, as well as superoxide radical formation were analyzed as a function of time. Po2 values in the cell incubating medium were decreased by 78% by hypoxia and increased by 221% by hyperoxia. No morphologic alteration could be noticed before 72 h posthypoxia, when cell degeneration became apparent, with a concomitant reduction in protein contents. Hypoxia-reoxygenation induced a transient cellular hypermetabolism, as shown by a 36% increase in 2-D-[3H]deoxyglucose uptake 24 h after hypoxia, and then a 23% decrease below control values at 72 h. It also led to a sharp increase in the formation of superoxide radicals (+108%). Transient hyperoxia during reoxygenation did not exacerbate these events, and thus would not enhance their deterimental effects on cell integrity.
Asunto(s)
Neuronas/metabolismo , Oxígeno/metabolismo , Superóxidos/metabolismo , Animales , Hipoxia de la Célula , Células Cultivadas , Desoxiglucosa/farmacocinética , Metabolismo Energético , Espacio Extracelular/metabolismo , Femenino , Humanos , L-Lactato Deshidrogenasa/metabolismo , Masculino , Neuronas/citología , Embarazo , Prosencéfalo/citología , Prosencéfalo/embriología , Ratas , Ratas Sprague-Dawley , ReperfusiónRESUMEN
Kernicterus is characterized by the accumulation of bilirubin mainly into subcortical brainstem nuclei. Inasmuch as premature infants are more susceptible to kernicterus, we hypothesized that the cerebral permeability to bilirubin could vary by cerebral region and with age. Therefore, in the present study, we measured the blood-to-brain transfer constant (Ki) of [3H]bilirubin in 6-8 rats at postnatal age 10 (P10) or 21 d (P21) in basal conditions and after a bilirubin perfusion to explore age-related and bilirubin-induced changes in the cerebral permeability to the dye. Blood-to-brain transfer of [3H]bilirubin was measured in 39 brain regions by quantitative autoradiography in 15-min experiments. Rats exposed to unlabeled bilirubin received a loading dose of 160 mg/kg over 15 min followed by a 90-min bilirubin perfusion at a speed of 64 mg/kg/h. At P10, cerebral permeability to bilirubin ranged from 0.07 to 0.12 microL/g/min, except in the auditory nerve, dentate nucleus, hypothalamus, and thalamus where it reached 0.41-0.47 microL/g/min. At P21, Ki of bilirubin was significantly lower than at P10 and ranged from 0.03-0.06 microL/g/min in most brain areas. In P10 bilirubin-exposed rats, permeability to bilirubin significantly increased over control levels in all brain regions but three. The largest increases (> 350%) were recorded in the sensory regions, most limbic areas, hypothalamus, and thalamus. At P21, hyperbilirubinemia induced increases in blood-to-brain transfer of bilirubin of 50-200% in 16 brain areas, except in the hippocampus, sensory-motor cortex, and thalamic nuclei where they reached 200-433%. Thus, it appears that the immature rat brain (P10) is very permeable to bilirubin. The increased permeability with preexposure to the dye, especially in brain regions which are affected in infants with kernicterus, could be related either to the large decrease in the value of the albumin:bilirubin ratio between control (15-16) and hyperbilirubinemic conditions (1.7-1.8) and/or to an increased permeability to bilirubin.
Asunto(s)
Bilirrubina/farmacocinética , Encéfalo/metabolismo , Hiperbilirrubinemia/metabolismo , Factores de Edad , Animales , Volumen Sanguíneo , Femenino , Hiperbilirrubinemia/fisiopatología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The regional cerebral metabolic consequences of bilirubin intoxication are not well known. With the quantitative autoradiographic [14C]2-deoxyglucose (2DG), we studied the effect of moderate or severe bilirubin infusion on local cerebral metabolic rates for glucose utilization (LCMRglcs) in 10 (P10) and 21 day-old (P21) rats. After an 80 or 160 mg/kg loading dose of bilirubin administered over 15 min, the speed of bilirubin infusion was reduced to 32 or 64 mg/kg/h for the following 105 min, for moderate or severe intoxication, respectively. This infusion protocol led to plasma bilirubin concentrations of 100-200 nmol/ml (moderate intoxication) or 200-300 nmol/ml (severe intoxication). Cerebral bilirubin concentration was 10 nmol/g at P10 and undetectable at P21 in moderate hyperbilirubinemia while it reached 22-34 nmol/g at both ages during severe hyperbilirubinemia. At P10, bilirubin infusion, moderate or severe, induced significant decreases in LCMRglcs in 17 and 15 brain regions of the 24 studied, respectively. At P21, moderate hyperbilirubinemia induced a decrease in LCMRglcs in only 2 regions, auditory cortex and auditory nerve. Conversely, at that age, severe bilirubin intoxication led to significant decreases in LCMRglcs in all regions studied. These results demonstrate that metabolic changes induced by bilirubin are directly correlated to its entry into the brain which occurs without any alteration in the blood-brain barrier. Indeed, the effects of the dye are quite discrete during moderate hyperbilirubinemia at P21 when no bilirubin is detectable in the brain while they are massive during severe hyperbilirubinemia at P21 and at both levels of intoxication at P10 when bilirubin has entered the brain in measurable amounts.
Asunto(s)
Bilirrubina/toxicidad , Encéfalo/metabolismo , Desoxiglucosa/metabolismo , Ictericia Neonatal/metabolismo , Animales , Corteza Auditiva/metabolismo , Bilirrubina/metabolismo , Humanos , Recién Nacido , Cinética , Ratas , Albúmina Sérica/metabolismo , Nervio Vestibulococlear/metabolismoRESUMEN
While the accumulation of bilirubin in specific brain regions has been well characterized at autopsy in kernicteric infants, data on the regional effects of early cerebral bilirubin intoxication are still missing. Therefore, the quantitative autoradiographic [14C]2-deoxyglucose technique was applied to the measurement of the effects of a bilirubin infusion on local cerebral metabolic rates for glucose (LCMRglc) in immature rats. A loading dose of 80 mg/kg bilirubin was first administered to the animals over 15 min. Thereafter, the velocity of the infusion was reduced to 32 mg/kg/h and the infusion was continued for 105 min. The animals were studied at two ages, postnatal day 10 (P10) and P21. The [14C]2-deoxyglucose was injected to the animals 45 min before the end of the infusion. Bilirubin infusion led to plasma concentrations ranging from 100 to 200 mumol/l at both ages and to brain amounts of 10-16 nmol/g at P10 while bilirubin was not detectable in brain at P21. Hyperbilirubinemia induced widespread decreases in LCMRglcs at P10 and had rather limited consequences on cerebral glucose utilization at P21. At P10, decreases in LCMRglcs were mostly prominent in regions that have been shown to preferentially accumulate bilirubin in kernicteric infants. In conclusion, there appears to be a good correlation between these metabolic data and regional brain permeability to bilirubin.
Asunto(s)
Envejecimiento/metabolismo , Animales Recién Nacidos/metabolismo , Bilirrubina/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Hiperbilirrubinemia/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Glucosa/metabolismo , Ratas , Ratas Sprague-Dawley , Albúmina Sérica/metabolismo , Distribución TisularRESUMEN
In order to further analyze the development of glutamatergic pathways in neuronal cells, the expression of excitatory amino acid receptors was studied in a model of neurons in primary culture by measuring the specific binding of L-[3H]glutamate under various incubation conditions in 8-day-old intact living neurons isolated from the embryonic rat forebrain, as well as in membrane preparations from these cultures and from newborn rat forebrain. In addition, the receptor responsiveness to glutamate was assessed by studying the uptake of tetraphenylphosphonium (TPP+) which reflects membrane polarization. In the presence of a potent inhibitor of glutamate uptake, the radioligand bound to a total number of sites of 36.7 pmol/mg protein in intact cells incubated in a Tris buffer containing Na+, Ca2+, and Cl-, with a Kd around 2 microM. In the absence of the above ions, [3H]glutamate specific binding diminished to 14.2 pmol/mg protein with a Kd-value of 550 nM. Under both of the above conditions, similar Kd were obtained in membranes isolated from cultures and from the newborn brain. However, Bmax-values were significantly lower in culture membranes than in intact cells or newborn membranes. Displacement studies showed that NMDA was the most potent compound to inhibit [3H]glutamate binding in membranes obtained from cultured neurons as well as from the newborn brain, whereas quisqualate, AMPA, kainate and trans-ACPD were equally effective.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Neuronas/metabolismo , Prosencéfalo/metabolismo , Receptores de Glutamato/metabolismo , Animales , Animales Recién Nacidos , Unión Competitiva , Membrana Celular/metabolismo , Células Cultivadas , Embrión de Mamíferos , Femenino , Feto , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Cinética , N-Metilaspartato/farmacología , Neuronas/citología , Compuestos Onio/metabolismo , Compuestos Onio/farmacología , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/farmacología , Potasio/farmacología , Embarazo , Prosencéfalo/citología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/análisis , TritioRESUMEN
The quantitative autoradiographic [14C]iodoantipyrine technique was applied to the measurement of rates of local cerebral blood flow (LCBF) during audiogenic seizures in Wistar AS rats belonging to a genetic strain selected at the Centre de Neurochimie (Strasbourg, France) for their sensitivity to sound. Seizures were elicited in native rats never exposed to sound (single audiogenic seizures) or in rats previously exposed to 10-40 seizure-inducing sound stimulations until generalization of the seizure to forebrain areas (referred to as "kindled animals"). During single audiogenic seizures, rates of LCBF increased over control values in all areas but the genu of the corpus callosum. The highest increases in LCBF (180-388%) were recorded in the inferior and superior colliculus, reticular formation, monoaminergic cell groupings, especially the substantia nigra, posterior vegetative nuclei, and many thalamic and hypothalamic regions. The lowest increases were seen in forebrain limbic regions and cortical areas. In kindled animals, LCBF rates increased over control levels in 67 areas of the 75 studied. LCBF increases were generally of a lower amplitude in kindled than in naive rats. Differences between the two groups of seizing rats were located mostly in brain-stem regions, mainly the inferior colliculus, reticular formation, substantia nigra, and posterior vegetative nuclei. Conversely, rates of LCBF were similar in forebrain areas of naive and kindled animals. In conclusion, the present data show that there is a good correlation between the structures known to be involved in the expression of audiogenic seizures (inferior colliculus, reticular formation, substantia nigra mainly) and the large increase in LCBF during single audiogenic seizures, while rates of LCBF increase to a lesser extent in forebrain areas not involved in this type of seizures. The circulatory adaptation to kindled seizures is rather a decreased response in brain-stem regions and no change in the forebrain, although the kindling process induces a generalization of the seizure from brain-stem to anterior regions.
Asunto(s)
Circulación Cerebrovascular , Excitación Neurológica , Convulsiones/fisiopatología , Estimulación Acústica , Animales , Tronco Encefálico/fisiopatología , Ratas , Ratas Wistar , Convulsiones/etiologíaRESUMEN
The quantitative autoradiographic [14C]-iodoantipyrine technique was applied to measure the effects of a 30-min period of pentylenetetrazol (PTZ)-induced status epilepticus (SE) on local cerebral blood flow (LCBF) in rats 10 (P10), 14 (P14), 17 (P17), and 21 (P21) days after birth. The animals received repetitive, timed injections of subconvulsive doses of PTZ until SE was reached. At P10, SE induced a 32 to 184% increase in the rates of LCBF affecting all structures studied. In P14- and P17 PTZ-treated rats, LCBF values significantly increased in two-thirds of the structures belonging to all systems studied and were not changed by SE in the parietal cortex, dorsal hippocampus, and dentate gyrus. At P21, rates of LCBF were still increased in 48 of the 73 structures studied; however, LCBF values were decreased by SE in most cortical areas, the hippocampus, and the dentate gyrus. CBF and cerebral metabolic rate for glucose (CMRglc) remained coupled in both controls and PTZ-exposed rats. Our results show that changes in LCBF with seizures are age dependent. At the most immature ages, P10 and P14, both LCBF and local CMRglc (LCMRglc) values are largely increased by long-lasting seizures. At P17 and P21, the blood flow response to SE becomes more heterogeneous, with specific decreases in the hippocampus and cortex at P21. The absence of mismatch between LCBF and LCMRglc in PTZ-exposed rats at all ages may explain at least partly why the immature brain is more resistant to seizure-induced brain damage than the adult brain.