RESUMEN
BACKGROUND: Circular RNA (circRNA) is highly expressed in the brain tissue, but its molecular mechanism in cerebral ischemia-reperfusion remains unclear. Here, we explored the role and underlying mechanisms of circRNA antisense non-coding RNA in the INK4 locus (circ_ANRIL) in oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell injury. RESULTS: The expression of circ_ANRIL in OGD/R-induced human brain microvascular endothelial cells (HBMECs) was significantly up-regulated, while that of miR-622 was significantly down-regulated. Overexpression of circ_ANRIL significantly inhibited the proliferation of OGD/R-induced HBMECs and aggravated OGD/R-induced cell apoptosis. Moreover, circ_ANRIL overexpression further increased the secretion of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 in OGD/R-treated HBMECs. The results of bioinformatics analysis and luciferase reporter assay indicated that circ_ANRIL served as an miR-622 sponge to negatively regulate the expression of miR-622 in OGD/R-treated HBMECs. Additionally, circ_ANRIL silencing exerted anti-apoptotic and anti-inflammatory effects by positively regulating the expression of miR-622. Furthermore, inhibition of OGD/R-induced activation of the nuclear factor (NF)-κB pathway by circ_ANRIL silencing was significantly reversed by treatment with miR-622 inhibitor. CONCLUSIONS: Knockdown of circ_ANRIL improved OGD/R-induced cell damage, apoptosis, and inflammatory responses by inhibiting the NF-κB pathway through sponging miR-622.
Asunto(s)
Hipoxia Encefálica , MicroARNs , ARN Circular , Daño por Reperfusión , Apoptosis , Encéfalo , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Células Endoteliales , Glucosa/metabolismo , Humanos , Hipoxia Encefálica/metabolismo , Inflamación , MicroARNs/genética , MicroARNs/fisiología , Oxígeno , ARN Largo no Codificante , Daño por Reperfusión/metabolismoRESUMEN
BACKGROUND: Circular RNA (circRNA) is highly expressed in the brain tissue, but its molecular mechanism in cerebral ischemia-reperfusion remains unclear. Here, we explored the role and underlying mechanisms of circRNA antisense non-coding RNA in the INK4 locus (circ_ANRIL) in oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell injury. RESULTS: The expression of circ_ANRIL in OGD/R-induced human brain microvascular endothelial cells (HBMECs) was significantly up-regulated, while that of miR-622 was significantly down-regulated. Overexpression of circ_ANRIL significantly inhibited the proliferation of OGD/R-induced HBMECs and aggravated OGD/R-induced cell apoptosis. Moreover, circ_ANRIL overexpression further increased the secretion of interleukin (IL)-1ß, IL-6, tumor necrosis factor-a, and monocyte chemoattractant protein-1 in OGD/R-treated HBMECs. The results of bioinformatics analysis and luciferase reporter assay indicated that circ_ANRIL served as an miR-622 sponge to negatively regulate the expression of miR-622 in OGD/R-treated HBMECs. Additionally, circ_ANRIL silencing exerted anti-apoptotic and anti-inflammatory effects by positively regulating the expression of miR-622. Furthermore, inhibition of OGD/R-induced activation of the nuclear factor (NF)-kB pathway by circ_ANRIL silencing was significantly reversed by treatment with miR-622 inhibitor. CONCLUSIONS: Knockdown of circ_ANRIL improved OGD/R-induced cell damage, apoptosis, and inflammatory responses by inhibiting the NF-κB pathway through sponging miR-622.
Asunto(s)
Humanos , Daño por Reperfusión/metabolismo , Hipoxia Encefálica/metabolismo , MicroARNs/fisiología , MicroARNs/genética , ARN Circular , Oxígeno , Encéfalo , Apoptosis , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Células Endoteliales , ARN Largo no Codificante , Glucosa/metabolismo , InflamaciónRESUMEN
Brain damage caused by perinatal asphyxia is dangerous for neonatal infants, but the mechanism by which it occurs remains elusive. In this study, microRNA-152 (miR-152) expression was induced by low oxygen levels in rat models of hypoxia brain damage, as well as in human brain microvascular endothelial cells (HBMECs) cultured in vitro. Analysis of the sequence of miR-152 revealed that the phosphatase and tensin homolog gene (PTEN) is probably the target of miR-152 both in humans and rats. When HBMECs were transfected with miR-152 mimics, PTEN expression was inhibited at both the mRNA and protein levels. Moreover, transfection with the miR-152 mimic also inhibited apoptosis induced by hypoxia. Furthermore, expression of the pro-apoptotic gene Bax was downregulated while the anti-apoptotic gene Bcl2 was upregulated after miR-152 mimic transfection. Taken together, these results indicate that miR-152 induced by hypoxia suppresses cell apoptosis and acts as a protective factor during hypoxia by repressing PTEN.
Asunto(s)
Células Endoteliales/enzimología , Hipoxia Encefálica/metabolismo , MicroARNs/biosíntesis , Oxígeno/metabolismo , Fosfohidrolasa PTEN/genética , Animales , Apoptosis/genética , Encéfalo/irrigación sanguínea , Hipoxia de la Célula/fisiología , Células Endoteliales/metabolismo , Células HEK293 , Humanos , Hipoxia Encefálica/patología , Masculino , Microvasos/enzimología , Microvasos/metabolismo , Modelos Animales , Fosfohidrolasa PTEN/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 µM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti-inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A1 receptor, phosphatidylinositol-3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin-sensitive G-protein-coupled signaling, blockade of adenosine A2A receptors (A2A R), but not via A1 receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.
Asunto(s)
Encefalitis , Guanosina/metabolismo , Hipocampo/inmunología , Hipocampo/metabolismo , Hipoxia Encefálica , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Encefalitis/tratamiento farmacológico , Encefalitis/inmunología , Encefalitis/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Glucosa/farmacología , Ácido Glutámico/farmacocinética , Guanosina/farmacología , Hipocampo/citología , Hipoxia Encefálica/tratamiento farmacológico , Hipoxia Encefálica/inmunología , Hipoxia Encefálica/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Masculino , Potencial de la Membrana Mitocondrial/fisiología , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Técnicas de Cultivo de Órganos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Oxígeno/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Receptor de Adenosina A1/metabolismo , Sinaptotagminas , TritioRESUMEN
Hypoxia at birth is a major source of brain damage and it is associated with serious neurological sequelae in survivors. Alterations in the extracellular turnover of glutamate (Glu) and acetylcholine (ACh), two neurotransmitters that are essential for normal hippocampal function and learning and memory processes, may contribute to some of the neurological effects of perinatal hypoxia. We set out to determine the immediate and long-lasting effects of hypoxia on the turnover of these neurotransmitters by using microdialysis to measure the extracellular concentration of Glu and ACh in hippocampus, when hypoxia was induced in rats at postnatal day (PD) 7, and again at PD30. In PD7 rats, hypoxia induced an increase in extracellular Glu concentrations that lasted for up to 2.5 h and a decrease in extracellular ACh concentrations over this period. By contrast, perinatal hypoxia attenuated Glu release in asphyxiated rats, inducing a decrease in basal Glu levels when these animals reached PD30. Unlike Glu, the basal ACh levels in these animals were greater than in controls at PD30, although ACh release was stimulated less strongly than in control animals. These results provide the first evidence of the initial and long term consequences of the hypoxia on Glu and ACh turnover in the brain, demonstrating that hypoxia produces significant alterations in hippocampal neurochemistry and physiology.
Asunto(s)
Acetilcolina/metabolismo , Espacio Extracelular/metabolismo , Glutamatos/metabolismo , Hipoxia Encefálica/metabolismo , 4-Aminopiridina/farmacología , Animales , Animales Recién Nacidos , Química Encefálica/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Femenino , Hipocampo/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Embarazo , Ratas , Convulsiones/metabolismoRESUMEN
The enhanced carotid body (CB) chemosensory response to hypoxia induced by chronic intermittent hypoxia (CIH) has been attributed to oxidative stress, which is expected to increase the expression of chemosensory modulators including chemoexcitatory pro-inflammatory cytokines in the CB. Accordingly, we studied the time-course of the changes in the immunohistological expression of TNF-α, IL-1ß, IL-6, ET-1, iNOS, eNOS and 3-nitrotyrosine in the CB, along with the progression of enhanced CB chemosensory responses to acute hypoxia in male Sprague-Dawley rats exposed to CIH (5%O2, 12 times/h per 8h) for 7, 14 and 21 days. Exposure to CIH for 7 days resulted in a sustained potentiation of CB chemosensory responses to acute hypoxia, which persisted until 21 days of CIH. The chemosensory potentiation was paralleled by an increased 3-nitrotyrosine expression in the CB. On the contrary, CIH produced a transient 2-fold increase of ET-1 immunoreactivity at 7 days, a decrease in eNOS immunoreactivity, and a delayed but progressive increase of TNF-α, IL-1ß and iNOS immunoreactivity, which was not associated with changes in systemic plasma levels or immune cell invasion within the CB. Thus, present results suggest that the local expression of chemosensory modulators and pro-inflammatory cytokines in the CB may have different temporal contribution to the CB chemosensory potentiation induced by CIH.
Asunto(s)
Cuerpo Carotídeo/metabolismo , Citocinas/biosíntesis , Endotelina-1/biosíntesis , Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/patología , Mediadores de Inflamación/metabolismo , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Animales , Cuerpo Carotídeo/enzimología , Cuerpo Carotídeo/patología , Citocinas/sangre , Modelos Animales de Enfermedad , Endotelina-1/sangre , Hipoxia Encefálica/enzimología , Inflamación/sangre , Inflamación/enzimología , Inflamación/metabolismo , Mediadores de Inflamación/sangre , Masculino , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Regulación hacia Arriba/fisiologíaRESUMEN
Cerebral palsy (CP) is a disorder of locomotion, posture and movement that can be caused by prenatal, perinatal or postnatal insults during brain development. An increased incidence of CP has been correlated to perinatal asphyxia and maternal infections during gestation. The effects of maternal exposure to low doses of bacterial endotoxin (lipopolysaccharide, LPS) on motor behavior and hind leg muscle morphology were examined in young adult rats. Prenatal exposure to LPS was also studied in association with perinatal anoxia (PA) and/or combined with subsequent sensorimotor restriction (SR) and all possible combinations of the three conditions. Rats exposed to LPS, PA and SR alone or combined (LPS + PA, LPS + SR, PA + SR, and LPS + PA + SR) showed deficits in balance and coordination when tested on the Rotarod. The SR groups, with or without other insults, (SR, LPS + SR, PA + SR, and LPS + PA + SR) exhibited the greatest motor deficits, characterized by the reduced ability to perform the horizontal ladder and suspended bar tests on postnatal day 29 (P29) and P45. Histological assessment revealed substantial morphological alterations in the slow ankle extensor soleus muscle of all SR rats. Soleus myofibers presented a reduction in cross-sectional area (CSA), an increase in sarcomere length and a decrease in sarcomere density. The CSA of the fast flexor tibialis anterior muscle was only decreased by the association of all treatments (LPS, PA, SR), but no differences were found in sarcomere length and density when compared to control. A slow-to-fast fiber type transition was only observed in the soleus and tibialis anterior muscles in the SR groups. These results suggest that exposure to LPS during the prenatal period, PA, SR alone or in combination has various degrees of consequences on motor behavior and muscle morphology. These data corroborate the concept that early experience-dependent movements play the most important role in shaping motor behavior and that reduced or anomalous sensorimotor experience can contribute to the development of aberrant motor behavior and muscle morphology.
Asunto(s)
Parálisis Cerebral/embriología , Hipoxia Encefálica/embriología , Hipoxia Encefálica/etiología , Lipopolisacáridos/toxicidad , Actividad Motora , Músculo Esquelético/embriología , Efectos Tardíos de la Exposición Prenatal/etiología , Animales , Animales Recién Nacidos , Parálisis Cerebral/etiología , Parálisis Cerebral/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Hipoxia Encefálica/metabolismo , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Efectos Tardíos de la Exposición Prenatal/patología , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1-NR2A and NR1-NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis.
Asunto(s)
Hipoxia Encefálica/metabolismo , ARN Mensajero/biosíntesis , Receptores de N-Metil-D-Aspartato/biosíntesis , Colículos Superiores/metabolismo , Animales , Embrión de Pollo , Técnicas In Vitro , Receptores de N-Metil-D-Aspartato/genética , Colículos Superiores/embriologíaRESUMEN
Brain ischemia causes more extensive injury in hyperglycemic than normoglycemic subjects, and the increased damage is to astroglia as well as neurons. In the present work, we found that in cortical astrocytes from rat or mouse, reoxygenation after hypoxia in a medium mimicking interstitial fluid during ischemia increases hemichannel activity and decreases cell-cell communication via gap junctions as indicated by dye uptake and dye coupling, respectively. These effects were potentiated by high glucose during the hypoxia in a concentration-dependent manner (and by zero glucose) and were not observed in connexin 43(-/-) astrocytes. The responses were transient and persistent after short and long periods of hypoxia, respectively. The persistent responses were associated with a progressive reduction in cell viability that was prevented by La(3+) or peptides that block connexin 43 (Cx43) hemichannels or by inhibition of p38 MAP kinase prior to hypoxia-reoxygenation but not by treatments that block pannexin hemichannels. Block of Cx43 hemichannels did not affect the reduction in gap junction mediated dye coupling observed during reoxygenation. Cx43 hemichannels may be a novel therapeutic target to reduce cell death following stroke, particularly in hyperglycemic conditions.
Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Conexina 43/metabolismo , Glucosa/metabolismo , Hiperglucemia/metabolismo , Hipoxia Encefálica/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Comunicación Celular/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/fisiopatología , Conexina 43/efectos de los fármacos , Colorantes Fluorescentes/farmacocinética , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Glucosa/toxicidad , Células HeLa , Humanos , Hiperglucemia/fisiopatología , Hipoxia Encefálica/fisiopatología , Lantano/farmacología , Ratones , Oxígeno/metabolismo , Oxígeno/farmacología , Péptidos/metabolismo , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatologíaRESUMEN
UNLABELLED: Perinatal asphyxia occurs in approximately 0.3% full-term newborn babies, and this percentage has not decreased despite medical advances. There are now evidences indicating that neurosteroids are important in neurodevelopment showing neuroprotective effects. We studied the potential protective effect of allopregnanolone (Allo) in vitro using organotypic cultures from neocortex, striatum, and hippocampus. Immunocytochemistry and confocal microscopy showed an increase of the glial fibrillary acidic protein (GFAP) signal in the studied brain areas after hypoxia. Western blot studies supported these results (hippocampus, 193%; neocortex, 306%; and striatum, 231%). Twenty-four-hour pretreatment with Allo showed different effects at the brain areas studied. In the hippocampus and the neocortex, 24-h pretreatment with Allo 5x10(-6) M showed to be neuroprotective as there was a significant decrease of the GFAP signal compared to control cultures exposed to hypoxia. Pretreatment with 5x10(-8) M Allo attenuated the astrogliosis response in the hippocampus and the neocortex in a nonsignificant way. Allo pretreatment at all doses did not show to affect the astrogliosis triggered by hypoxia in the striatum. Cell survival was analyzed by measuring LDH. After 1 h of hypoxia, all cultures showed a nonsignificant increase of LDH, which was greater after 24 h of hypoxia (hippocampus, 180%; striatum-cortex co-cultures, 140%). LDH levels have no changes by Allo pretreatment before hypoxia. CONCLUSION: 24 h pretreatment with 5x10(-6) M of Allo does not change neuronal viability but it prevents astrogliosis induced by hypoxia in the hippocampus and the neocortex.
Asunto(s)
Astrocitos/efectos de los fármacos , Gliosis/tratamiento farmacológico , Hipoxia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Pregnanolona/farmacología , Prosencéfalo/efectos de los fármacos , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiopatología , Citoprotección/efectos de los fármacos , Citoprotección/fisiología , Relación Dosis-Respuesta a Droga , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/metabolismo , Gliosis/fisiopatología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/fisiopatología , L-Lactato Deshidrogenasa/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Neocórtex/fisiopatología , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Fármacos Neuroprotectores/uso terapéutico , Técnicas de Cultivo de Órganos , Pregnanolona/uso terapéutico , Prosencéfalo/metabolismo , Prosencéfalo/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
PRIMARY OBJECTIVE: To report Magnetic Resonance Imaging (MRI) and/or Magnetic Resonance Spectroscopy (MRS) findings in subjects with hypoxic encephalopathy caused by drowning in recent literature and to compare them with non-specific hypoxic encephalopathy. METHOD: Systematic review of the Medline Database for bibliographic citations from 1996 to 2008. RESULTS: The studies included in this review described a total of 68 victims of drowning. From those, 58 performed MRS with a decrease of N-Acetyl-Aspartate/Creatine ratio in 75.86% (n = 44), and presence of lactate in 65.52% (n = 38) of the cases. MRI data was available in 46 cases. The main finding was brain edema in 78.26% (n = 36) and abnormalities of MRI signal in basal ganglia in 75% (n = 27) of the cases. Worse clinical outcomes were reported in conjunction with degree of MRI and MRS alterations. The findings were more consistent in the latter. Comparing these results with literature from non-specific hypoxic brain injury, the drowning process is apparently more variable. CONCLUSIONS: We found a trend to a more variable pattern of brain injury as seen by MRI/MRS in victims of drowning, which may reflect the nature of the aggression. Possibly there are different mechanisms involved in aquatic submersion, such as temperature, not present in pure hypoxic injury.
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Lesiones Encefálicas/diagnóstico , Ahogamiento , Hipoxia Encefálica/diagnóstico , Espectroscopía de Resonancia Magnética/métodos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Lesiones Encefálicas/metabolismo , Niño , Preescolar , Ahogamiento/metabolismo , Femenino , Humanos , Hipoxia Encefálica/metabolismo , Lactante , Ácido Láctico/metabolismo , Masculino , Estados UnidosRESUMEN
Synaptic dysfunction has been associated with neuronal cell death following hypoxia. The lack of knowledge on the mechanisms underlying this dysfunction prompted us to investigate the morphological changes in the postsynaptic densities (PSDs) induced by hypoxia. The results presented here demonstrate that PSDs of the rat neostriatum are highly modified and ubiquitinated 6 months after induction of hypoxia in a model of perinatal asphyxia. Using both two dimensional (2D) and three dimensional (3D) electron microscopic analyses of synapses stained with ethanolic phosphotungstic acid (E-PTA), we observed an increment of PSD thickness dependent on the duration and severity of the hypoxic insult. The PSDs showed clear signs of damage and intense staining for ubiquitin. These morphological and molecular changes were effectively blocked by hypothermia treatment, one of the most effective strategies for hypoxia-induced brain injury available today. Our data suggest that synaptic dysfunction following hypoxia may be caused by long-term misfolding and aggregation of proteins in the PSD.
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Hipotermia Inducida/métodos , Hipoxia Encefálica , Neostriado/metabolismo , Sinapsis/metabolismo , Ubiquitinas/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Calbindinas , Modelos Animales de Enfermedad , Tomografía con Microscopio Electrónico/métodos , Femenino , Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/patología , Hipoxia Encefálica/terapia , Masculino , Microscopía Inmunoelectrónica/métodos , Neostriado/patología , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Embarazo , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100/metabolismo , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Sinapsis/ultraestructura , Factores de Tiempo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We assessed immunoreactivity (IR) in the cerebral cortex (CC), hippocampus (Hipp), and striatum (ST) of a growth-associated protein, GAP-43, and of proteins of the synaptic vesicle fusion complex: VAMP-2, Syntaxin-1, and SNAP-25 (SNARE proteins) throughout postnatal development of rats after submitting the animals to acute global postnatal hypoxia (6.5% O(2), 70 min) at postnatal day 4 (PND4). In the CC only the IR of the SNARE protein SNAP-25 increased significantly with age. The hypoxic animals showed the same pattern of IR for SNAP-25, although with lower levels at PND11, and also a significant increase of VAMP-2. SNAP-25 (control): PND11 P < 0.001 vs. PND18, 25, and 40, SNAP-25 (hypoxic): P < 0.001 vs. PND18, 25, and 40; VAMP-2 (hypoxic): P < 0.05 PND11 vs. PND18, and P < 0.01 vs. PND25 and PND40; one-way ANOVA and Bonferroni post-test. In the Hipp, SNAP-25 and syntaxin-1 increased significantly with age, reaching a plateau at PND25 through PND40 in control animals (one-way ANOVA: syntaxin-1: P = 0.043; Bonferroni: NS; SNAP-25: P = 0.013; Bonferroni: P < 0.01 PND11 vs. PND40). Hypoxic rats showed higher levels of significance in the one-way ANOVA than controls (syntaxin-1: P = 0.009; Bonferroni: P < 0.05 PND11 vs. PND25 and P < 0.001 PND11 vs. PND40). In the ST, GAP-43 differed significantly among hypoxic and control animals and the two-way ANOVA revealed significant differences with age (F = 3.23; P = 0.037) and treatment (F = 4.84; P = 0.036). VAMP-2 expression also reached statistical significance when comparing control and treated animals (F = 6.25, P = 0.018) without changes regarding to age. Elevated plus maze test performed at PND40 indicated a lower level of anxiety in the hypoxic animals. At adulthood (12 weeks) learning, memory and locomotor abilities were identical in both groups of animals. With these results, we demonstrate that proteins of the presynaptic structures of the ST are sensitive to acute disruption of homeostatic conditions, such as a temporary decrease of the O(2) concentration. Modifications in the activity of these proteins could contribute to the long term altered responses to stress due to acute hypoxic insult in the neonatal period.
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Cuerpo Estriado/metabolismo , Proteína GAP-43/metabolismo , Hipoxia Encefálica/metabolismo , Regulación hacia Arriba/fisiología , Enfermedad Aguda , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Trastornos de Ansiedad/etiología , Trastornos de Ansiedad/metabolismo , Trastornos de Ansiedad/fisiopatología , Cámaras de Exposición Atmosférica , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/fisiopatología , Hipoxia Encefálica/fisiopatología , Inmunohistoquímica , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Ratas , Ratas Sprague-Dawley , Estrés Fisiológico/metabolismo , Estrés Fisiológico/fisiopatología , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Here we investigated the effects of estradiol replacement in ovariectomized female rats using hippocampal slices exposed to oxygen-glucose deprivation (OGD). OGD induced lactate dehydrogenase (LDH) release to the incubation medium, what was assumed as a parameter of cellular death. In the estradiol-treated group the LDH release was markedly decreased by 23% as compared to the vehicle-treated group. In attempt to study a possible mechanism by which estradiol acts, we investigated some parameters of oxidative stress. In both vehicle-treated and estradiol-treated groups, OGD significantly increased the free radical production by 34% and 16%, respectively, although no significant differences on total antioxidant capacity were observed. Interestingly, estradiol replacement prevented the significant reduction in tryptophan and tyrosine contents caused by OGD observed in vehicle-treated animals. Our results show that estradiol replacement in ovariectomized female rats decreases cellular susceptibility to an ischemic-like injury and suggest a role for the hormone on protein damage prevention.
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Estradiol/farmacología , Terapia de Reemplazo de Estrógeno , Glucosa/deficiencia , Hipocampo/metabolismo , Hipoxia Encefálica/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores , Animales , Antioxidantes/metabolismo , Estradiol/metabolismo , Femenino , Radicales Libres/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/patología , Hipoxia Encefálica/patología , L-Lactato Deshidrogenasa/metabolismo , Proteínas del Tejido Nervioso/efectos de los fármacos , Ovariectomía , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Triptófano/efectos de los fármacos , Triptófano/metabolismo , Tirosina/efectos de los fármacos , Tirosina/metabolismoRESUMEN
CNS exposure to hypoxia impairs excitatory and inhibitory neurotransmission. Our aim was to determine variations induced by normobaric acute hypoxic hypoxia (8% O(2) for 60 min) on the NMDA receptor complex, as well as their potential reversibility after normoxic recovery. To this end, [3H]MK-801 binding assays to a synaptic membrane fraction isolated from chick optic lobes were performed. Previous studies throughout development had disclosed a characteristic age-dependent pattern. Results at embryonic day (ED) 12 and 18 indicated two distinct MK-801 binding sites. Hypoxic treatment failed to alter either the high affinity site dissociation constant (K(d)) or its maximal binding capacity (B(max)), whereas the low affinity site B(max) was significantly decreased (50% and 30% at ED12 and 18, respectively), without alteration in its K(d) values. Hypoxic embryos restored for 48 h at ED12 to normoxic conditions displayed unchanged MK-801 binding reduction, unlike those treated likewise at ED18 whose values fully recovered control levels. To conclude, hypoxic treatment reduces low affinity MK-801 B(max) in the NMDA receptor which proves irreversible up to ED12. Such early neuronal vulnerability may be due to post-transcriptional changes, to endocytosis followed by receptor degradation, or alternatively to cell death.
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Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipoxia Encefálica/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Enfermedad Aguda , Animales , Embrión de Pollo , Pollos , Maleato de Dizocilpina/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Sinapsis/metabolismo , TritioRESUMEN
1. In the present work we describe the short term effects of mild neonatal hypoxia on the synapse as assessed by the immunoreactivity (IR) of two synaptic proteins: rab 3A and synaptobrevin (VAMP). 2. Using the sensitive methodology of immunoblotting, we measured rab 3A and VAMP-IR in homogenates from the cerebral cortex, hippocampus, and corpus striatum of control (breathing room air) and hypoxiated (breathing 95.5% N2-6.5% O2 for 70 min) 4-day-old rats at 1, 2, and 6 h after the end of the hypoxia. Immunostaining with examination by light microscopy was performed using the synaptic protein-specific antibodies on fixed brain sections from animals belonging to the same litter and submitted to hypoxia. 3. A transient increase of VAMP-IR was observed in the hippocampus and corpus striatum, and for rab 3A in the striatum, 1 h after initiating reoxygenation. At the following time points the values returned to control levels. This effect was less clearly observed in the immunostained sections. 4. Mild hypoxia has an effect on sensitive brain regions, eliciting an increase in the IR of at least two proteins involved in the synaptic vesicle cycle. The transient nature of this effect possibly indicates the activation of endogenous neuroprotective mechanisms.
Asunto(s)
Hipoxia Encefálica/metabolismo , Proteínas de la Membrana/metabolismo , Proteína de Unión al GTP rab3A/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Femenino , Proteína GAP-43/análisis , Proteína GAP-43/inmunología , Immunoblotting , Inmunohistoquímica , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Embarazo , Proteínas R-SNARE , Ratas , Ratas Sprague-Dawley , Proteína de Unión al GTP rab3A/análisis , Proteína de Unión al GTP rab3A/inmunologíaRESUMEN
The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day x 21 days) applied to adult male mice on GABA(A) receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [3H]GABA specifically bound to GABA(A) receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (Bmax control, 4.70+/-0.19, hypoxic, 3.33+/-0.10 pmol/mg protein), with no effect on GABA binding sites affinity (Kd control: 159.3+/-13.3 nM, hypoxic: 164.2+/-15.1 nM). Decreased B(max) values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA(A) receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5alpha-pregnan-3alpha-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA(A) receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.
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Corteza Cerebral/metabolismo , Desoxicorticosterona/análogos & derivados , Hipoxia Encefálica/metabolismo , Receptores de GABA-A/metabolismo , Animales , Ansiolíticos/metabolismo , Bicuculina/metabolismo , Desoxicorticosterona/metabolismo , Antagonistas del GABA/metabolismo , Moduladores del GABA/metabolismo , Masculino , Ratones , Pentobarbital/metabolismoRESUMEN
We measured the activities of Na(+)K(+) ATPase and of enzymes of the glycolytic pathway, Krebs cycle, and the respiratory chain in cerebral cortex of mice exposed to chronic hypoxia for three weeks and compared their values with those of sea level controls. There were no differences in Na(+)K(+) ATPase activity or in the activity of glycolytic enzymes. In the Krebs cycle, a 66% increase of succinate dehydrogenase activity was found due to a lower Km. In contrast, respiratory chain cytochrome oxidase activity was reduced by 12% in mice exposed to hypoxia. This suggested that the metabolic demand would be satisfied despite the respiratory chain depression (cytochrome oxidase), probably due to anaerobic energy production within the mitochondria (succinate dehydrogenase).
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Corteza Cerebral/metabolismo , Metabolismo Energético/fisiología , Hipoxia Encefálica/metabolismo , Animales , Enfermedad Crónica , Ciclo del Ácido Cítrico , Transporte de Electrón , Complejo IV de Transporte de Electrones/metabolismo , Masculino , Ratones , Mitocondrias/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Succinato Deshidrogenasa/metabolismoRESUMEN
Synapsins are phosphoproteins related to the anchorage of synaptic vesicles to the actin skeleton. Hypoxia-ischemia causes an increased calcium influx into neurons through ionic channels gated by activation of glutamate receptors. In this work seven-day-old Wistar rats were submitted to hypoxia-ischemia and sacrificed after 21 hours, 7, 30, or 90 days. Synaptosomal fractions were obtained by Percoll gradients and incubated with 32P (10 microCi/g). Proteins were analysed by SDS-PAGE and radioactivity incorporated into synapsin 1 was counted by liquid scintillation. Twenty-one hours after hypoxia-ischemia we observed a reduction on the in vitro phosphorylation of synapsin 1, mainly due to hypoxia, rather than to ischemia; this effect was reversed at day 7 after the insult. There was another decrease in phosphorylation 30 days after the event interpreted as a late effect of hypoxia-ischemia. No changes were observed at day 90. Our results suggest that decreased phosphorylation of synapsin 1 could be related to neuronal death that follows hypoxia-ischemia.
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Isquemia Encefálica/metabolismo , Hipoxia Encefálica/metabolismo , Sinapsinas/metabolismo , Sinaptosomas/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Isquemia Encefálica/patología , Muerte Celular , Femenino , Hipoxia Encefálica/patología , Masculino , Fosforilación , Ratas , Ratas WistarRESUMEN
The NMDA receptor subtype is the major excitatory mediator for glutamate neurotoxicity. To assess its participation in the noxious effects of postnatal hypoxia, we have characterized the binding of the ionophoric marker of NMDA receptor, dizocilpine (MK-801). Binding of (+)-3-[125I]MK-801 to NMDA brain receptors under nonequilibrium conditions was quantified by in vitro autoradiography in rats exposed to hypoxia induced by 93% N2/6.5% O2 exposure for 70 min on Postnatal Day 4. Acute and long-lasting effects were investigated at 4 h after injury and on Postnatal Day 40. At the acute stage, a transient decrease in binding was found in several specific brain areas, hypothalamus, amygdaloid nuclei, entorhinal cortex, perirhinal cortex, and hippocampus, and no differences were found in temporal cortex, thalamus, and geniculate nucleus, when compared to sham-treated animals. At this early age, there was no increase of binding when slices from both groups were incubated in the presence of glutamate and glycine (Glu/Gly), positive allosteric modulators of MK-801 binding. In the 40-day-old brains, the binding to the NMDA receptors of hypoxiatreated animals was not different with respect to controls in most of the areas studied, but the Glu/Gly stimulation of binding in hypoxic rats showed a reduced, or absent, response to the allosteric modulators. In contrast, control rats showed a remarkable increase of the specific binding induced by the presence of the modulators in the incubation buffer. Binding of (+)-3-[125I]MK-801 was also performed at a higher concentration to clarify whether the altered response to Glu/Gly may be due to differences in the number of channels; however, the density of NMDA receptors at this concentration was similar in both control and hypoxia-treated rats. We conclude that the effect of exposure of newborn rats to hypoxia can generate acute and long-lasting effects on the NMDA receptor. The deleterious action of this kind of noxa on the CNS could be exerted by interference with normal glutamatergic transmission and hence over normal growth and development.