RESUMEN
Accumulating evidence points to roles for oxidative stress, amyloid beta (Aß), and mitochondrial dysfunction in the pathogenesis of Alzheimer's disease (AD). In neurons, the base excision repair pathway is the predominant DNA repair (BER) pathway for repairing oxidized base lesions. Apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional enzyme with DNA repair and reduction-oxidation activities, has been shown to enhance neuronal survival after oxidative stress. This study seeks to determine 1) the effect of Aß25-35 on reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels, 2) the activities of respiratory complexes (I, III, and IV), 3) the role of APE1 by ectopic expression, and 4) the neuromodulatory role of ginkgolide B (GB; from the leaves of Ginkgo biloba). The pro-oxidant Aß25-35 peptide treatment increased the levels of ROS/RNS in human neuroblastoma IMR-32 and SH-SY5Y cells, which were decreased after pretreatment with GB. Furthermore, the mitochondrial APE1 level was found to be decreased after treatment with Aß25-35 up to 48 hr, and the level was increased significantly in cells pretreated with GB. The oxidative phosphorylation (OXPHOS; activities of complexes I, III, and IV) indicated that Aß25-35 treatment decreased activities of complexes I and IV, and pretreatment with GB and ectopic APE1 expression enhanced these activities significantly compared with Aß25-35 treatment. Our results indicate that ectopic expression of APE1 potentiates neuronal cells to overcome the oxidative damage caused by Aß25-35 . In addition, GB has been shown to modulate the mitochondrial OXPHOS against Aß25-35 -induced oxidative stress and also to regulate the levels of ROS/RNS in the presence of ectopic APE1. This study presents findings from a new point of view to improve therapeutic potential for AD via the synergistic neuroprotective role played by APE1 in combination with the phytochemical GB.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ginkgólidos/farmacología , Lactonas/farmacología , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Humanos , Complejos Multienzimáticos/metabolismo , Neuroblastoma/patología , Fosforilación Oxidativa/efectos de los fármacos , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , TransfecciónRESUMEN
Animal and human studies show that in-utero exposure to preeclampsia alters fetal programming and results in long-term adverse cardiovascular outcomes in the offspring. Human epidemiologic data also suggest that offspring born to preeclamptic mothers are also at risk of adverse long term neurodevelopmental outcomes. Pravastatin, a hydrophilic lipid-lowering drug with pleiotropic properties, was found to prevent the altered cardiovascular phenotype of preeclampsia and restore fetal growth in animal models, providing biological plausibility for its use as a preventive agent for preeclampsia. In this study, we used a murine model of preeclampsia based on adenovirus over-expression of the anti-angiogenic factor soluble Fms-like tyrosine kinase 1, and demonstrated that adult offspring born to preeclamptic dams perform poorly on assays testing vestibular function, balance, and coordination, and that prenatal pravastatin treatment prevents impairment of fetal programming.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Trastornos Neurológicos de la Marcha/prevención & control , Pravastatina/uso terapéutico , Preeclampsia/fisiopatología , Efectos Tardíos de la Exposición Prenatal/prevención & control , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Femenino , Desarrollo Fetal/efectos de los fármacos , Trastornos Neurológicos de la Marcha/etiología , Humanos , Masculino , Ratones , Equilibrio Postural/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/etiología , Trastornos Psicomotores/etiología , Trastornos Psicomotores/prevención & control , Reflejo/efectos de los fármacos , Factores Sexuales , Transducción Genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Neonatal hypoxia-ischemia (HI) is a common occurrence in preterm and low-birth-weight infants, and the incidence of low-birth-weight and preterm births is increasing. Characterization of brain injury after HI is of critical importance in developing new treatments that more accurately target the injury. After severe HI, neuronal cells undergo necrosis and secondary apoptosis of the surrounding cells as a result of neuroinflammation. We sought to characterize the biochemical pathways associated with cell death after HI. Bax, a cell death signaling protein, is activated after HI and translocates to the nucleus, endoplasmic reticulum, and mitochondria. The translocation patterns of Bax affect the resultant cell death phenotype (necrotic or apoptotic) observed. Although Bax is known to oligomerize once it is activated, less is known about the factors that control its translocation and oligomerization. We hypothesize that Bax kinase-specific phosphorylation determines its oligomerization and intracellular localization. Using well-established in vivo and in vitro models of neonatal HI, we characterized Bax oligomerization and multiorganelle translocation. We found that HI-dependent phosphorylation of Bax determines its oligomerization status and multiorganelle localization, and, ultimately, the cell death phenotype observed. Understanding the mechanisms of Bax translocation will aid in the rational design of therapeutic strategies that decrease the trauma resulting from HI-associated inflammation.
Asunto(s)
Núcleo Celular/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/patología , Neuronas/citología , Proteína X Asociada a bcl-2/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Recuento de Células , Modelos Animales de Enfermedad , Embrión de Mamíferos , Retículo Endoplásmico/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Masculino , Mitocondrias/metabolismo , Neuroblastoma/patología , Neuronas/ultraestructura , Fosforilación , Embarazo , Ratas , Ratas Wistar , Fracciones Subcelulares , Treonina/metabolismoRESUMEN
Mild traumatic brain injury (mTBI), particularly mild "blast type" injuries resulting from improvised exploding devices and many sport-caused injuries to the brain, result in long-term impairment of cognition and behavior. Our central hypothesis is that there are inflammatory consequences to mTBI that persist over time and, in part, are responsible for resultant pathogenesis and clinical outcomes. We used an adaptation (1 atmosphere pressure) of a well-characterized moderate-to-severe brain lateral fluid percussion (LFP) brain injury rat model. Our mild LFP injury resulted in acute increases in interleukin-1α/ß and tumor necrosis factor alpha levels, macrophage/microglial and astrocytic activation, evidence of heightened cellular stress, and blood-brain barrier (BBB) dysfunction that were evident as early as 3-6 h postinjury. Both glial activation and BBB dysfunction persisted for 18 days postinjury.
Asunto(s)
Conmoción Encefálica/patología , Inflamación/patología , Animales , Barrera Hematoencefálica/patología , Encéfalo/patología , Conmoción Encefálica/complicaciones , Citocinas/análisis , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Inmunoensayo , Inflamación/etiología , Masculino , Microscopía Confocal , Actividad Motora/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.
Asunto(s)
Cisteína/química , Óxido Nítrico/química , Proteómica/métodos , Animales , Compuestos de Boro/química , Calorimetría , Cisteína/metabolismo , Femenino , Fluorescencia , Hipoxia/metabolismo , Hipoxia/patología , Isquemia/metabolismo , Isquemia/patología , Luminiscencia , Maleimidas/química , Óxido Nítrico/metabolismo , Perfusión , Fosforilación , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
Exposure to hypoxia and hyperoxia in a rodent model of perinatal ischemia results in delayed cell death and inflammation. Hyperoxia increases oxidative stress that can trigger inflammatory cascades, neutrophil activation, and brain microvascular injury. Here we show that 100% oxygen resuscitation in our rodent model of perinatal ischemia increases cortical COX-2 protein levels, S-nitrosylated COX-2cys526, PGE2, iNOS and 5-LOX, all components of the prostaglandin and leukotriene inflammatory pathway.
Asunto(s)
Hipoxia-Isquemia Encefálica/fisiopatología , Oxígeno/metabolismo , Prostaglandinas/metabolismo , Animales , Muerte Celular , Ciclooxigenasa 2/metabolismo , Femenino , Humanos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/fisiología , Embarazo , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
Neonatal hypoxia/ischemia (HI) is a common cause of cognitive and behavioral deficits in children with hyperoxia treatment (HHI) being the current therapy for newborn resuscitation. HI induces cerebral edema that is associated with poor neurological outcomes. Our objective was to characterize cerebral edema after HI and determine the consequences of HHI (40% or 100% O(2)). Dry weight analyses showed cerebral edema 1 to 21 days after HI in the ipsilateral cortex; and 3 to 21 days after HI in the contralateral cortex. Furthermore, HI increased blood-brain barrier (BBB) permeability 1 to 7 days after HI, leading to bilateral cortical vasogenic edema. HHI failed to prevent HI-induced increase in BBB permeability and edema development. At the molecular level, HI increased ipsilateral, but not contralateral, AQP4 cortical levels at 3 and up to 21 days after HI. HHI treatment did not further affect HI-induced changes in AQP4. In addition, we observed developmental increases of AQP4 accompanied by significant reduction in water content and increase permeability of the BBB. Our results suggest that the ipsilateral HI-induced increase in AQP4 may be beneficial and that its absence in the contralateral cortex may account for edema formation after HI. Finally, we showed that HI induced impaired motor coordination 21 days after the insult and HHI did not ameliorate this behavioral outcome. We conclude that HHI treatment is effective as a resuscitating therapy, but does not ameliorate HI-induced cerebral edema and impaired motor coordination.
Asunto(s)
Edema Encefálico/etiología , Edema Encefálico/terapia , Hipoxia-Isquemia Encefálica/complicaciones , Hipoxia-Isquemia Encefálica/terapia , Terapia por Inhalación de Oxígeno/métodos , Resucitación/métodos , Animales , Animales Recién Nacidos , Acuaporina 4/metabolismo , Barrera Hematoencefálica/metabolismo , Agua Corporal , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Permeabilidad Capilar , Modelos Animales de Enfermedad , Discinesias/complicaciones , Discinesias/metabolismo , Discinesias/terapia , Lateralidad Funcional , Hipoxia-Isquemia Encefálica/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Prenatal perturbation of brain circulation and oxygenation is a leading cause of perinatal brain damage affecting about 0.3-0.9% of births. Hypoxia-ischemia (HI) in preterm human infants at gestational week 23-32 results in neurodevelopmental abnormalities in childhood, presenting as learning disability, seizure activity, motor impairment and in the most severe cases, death. Here, we examined the potential of MgSO4 treatment, prior to foetal hypoxia, to attenuate hypoxia induced damage in a murine model of maternal hypoxia. We studied the time course of maternal hypoxia and MgSO4 pre-treatment effects on cerebellar tissue by means of DNA microarray analyses. Mild hypoxia induced minor expression changes in most genes. However, there were 5 gene sets which were down-regulated by maternal hypoxia. MgSO4 pre-treatment abrogated these decreases in gene. A cell cycle gene set which responded immediately (2 h) to hypoxia, showed a delayed response (24 h) when MgSO4 pre-treatment was given. Similar proportions of cell death were observed in all groups before P7, where combined hypoxia and MgSO4 treatment increased cell death in the internal granule layer. There were a higher number of BrdU positive cells at the end of hypoxic episodes and a down-regulation of Reelin signaling, compared to control. MgSO4 pre-treatment prevented the enhancement of cell proliferation due to hypoxia and increased Reelin levels. Altogether, MgSO4 pre-treatment both reduced the number of genes differentially affected by hypoxia and delayed the responses to hypoxia. In addition, MgSO4 pre-treatment modified the nature of the transcriptional response; while hypoxia induced down-regulation of gene sets, MgSO4 pre-treatment mostly up-regulated them. The dual reaction to the MgSO4 treatment may be the source of the ambiguity in observations reported for affected newborns.
Asunto(s)
Cerebelo/metabolismo , Hipoxia Fetal/tratamiento farmacológico , Hipoxia Fetal/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Sulfato de Magnesio/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos C57BL , Proteína ReelinaRESUMEN
Prenatal hypoxia ischemia is a major cause of neurodevelopmental impairment in the newborn, associated with risk for motor, behavioral and cognitive impaired outcomes. We used an established mouse model of maternal hypoxia to examine the immediate molecular responses of signaling pathways associated with both cell death and neurogenesis. We also characterized responses to maternal pre-treatment with MgSO(4). Maternal hypoxia at embryonic day 17 (E17) failed to trigger inflammation or cell death in fetal brain at 24 h after hypoxia. However, maternal hypoxia decreased levels of neuronal migration signaling: Reelin (53% of control), Disabled 1 (Dab1, 77% of control), and amyloid precursor protein (APP, 64% of control) 2 h after the insult. These changes persisted for 24 h. At later times, Reelin levels in hippocampi of newborns in the maternal hypoxia-treated group increased compared to controls. Full protection from maternal hypoxia effects on hippocampal Reelin levels resulted from maternal pre-treatment with MgSO(4). Hypoxia and MgSO(4) increased radial and lateral migration distance in the CA1 four days after the insult, while in the DG the hypoxia treatment alone increased migration. Maternal hypoxia and MgSO(4) pre-treatment also stimulated hippocampal expression of genes related to neurogenesis, such as BDNF and NeuroD4. Taken together, the long-term neurodevelopmental outcome of prenatal and perinatal hypoxia may depend on perturbation of developmental signals that affect neuronal migration.
Asunto(s)
Movimiento Celular/fisiología , Hipoxia Fetal/fisiopatología , Hipocampo/embriología , Hipocampo/fisiopatología , Hipoxia Encefálica/embriología , Hipoxia Encefálica/fisiopatología , Neuronas/fisiología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Recién Nacidos , Moléculas de Adhesión Celular Neuronal/metabolismo , Muerte Celular/fisiología , Movimiento Celular/efectos de los fármacos , Fármacos del Sistema Nervioso Central/farmacología , Giro Dentado/efectos de los fármacos , Giro Dentado/embriología , Giro Dentado/fisiopatología , Modelos Animales de Enfermedad , Encefalitis/embriología , Encefalitis/fisiopatología , Proteínas de la Matriz Extracelular/metabolismo , Hipoxia Fetal/tratamiento farmacológico , Hipocampo/efectos de los fármacos , Hipoxia Encefálica/tratamiento farmacológico , Sulfato de Magnesio/farmacología , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Neurogénesis/fisiología , Neuronas/efectos de los fármacos , Distribución Aleatoria , Proteína Reelina , Serina Endopeptidasas/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
During embryonic and early postnatal development the combination of cell proliferation, migration, survival and cell death is intimately regulated. In the mouse embryo, significant numbers of primordial germ cells, the founder cells of the gametes, fail to migrate correctly to the genital ridges early in histogenesis. Studies in Bcl-2 associated X protein null mice (Bax(-/-)) have shown that the pro-apoptotic Bax gene is required for the programmed cell death of germ cells left in ectopic locations during and after germ cell migration. Independent studies carried out in the central nervous system of Bax(-/-) mice have shown impaired and ectopic neuronal migration in the cerebellum and olfactory bulb during development and in the adult hippocampus. Taken together, these evidences identify Bax as a major mechanism in ectopic cell death and are the subject of this review.
Asunto(s)
Muerte Celular/fisiología , Embrión de Mamíferos/fisiología , Proteína X Asociada a bcl-2/metabolismo , Animales , Encéfalo/anatomía & histología , Encéfalo/embriología , Movimiento Celular/fisiología , Embrión de Mamíferos/citología , Células Germinativas/citología , Células Germinativas/fisiología , Ratones , Ratones Noqueados , Proteína X Asociada a bcl-2/genéticaRESUMEN
Neonatal (P7) brain hypoxia-ischemia (HI) induces intracellular Bax protein shifts to the nucleus, mitochondria, and endoplasmic reticulum (ER), where it triggers the activation of the respective cell death signaling cascades. When compared with HI-treated rat pups, 100% O(2) resuscitation of HI-treated rat pups increases HI-induced ER Bax levels, ER-mediated cell death signaling, and resultant lesion volume and inflammation due to increased necrotic-like cell death. To better characterize the role of Bax intracellular shuttling ER cell death signaling and necrotic-like cell death, we used rotenone-treated P5 neuronal cortical cultures to increase ER Bax levels and subsequent cell death signaling. We treated P5 primary cortical neurons with 25 microM and 100 microM rotenone as an apoptotic or necrotic-like stimulus, respectively, and measured intracellular organelle Bax levels and the subsequent activation of ER/mitochondrial cell death signaling. The 25 microM rotenone treatment promptly increased nuclear Bax levels followed by a later increase in mitochondrial Bax levels and caspase-mediated cleavage of alpha-fodrin. The 100 microM rotenone treatment also resulted in an early increase in nuclear Bax levels followed by a subsequent increase in ER Bax levels and calpain-mediated cleavage of alpha-fodrin. After pretreatment with the immunosuppressive and neuroprotective FK506, there was a delay in Bax intracellular shifts and cell death signaling for both the 25 and 100 microM rotenone treatments. These results suggest that the different outcomes of apoptotic-like vs. necrotic-like cell death resulting from the treatment of neuronal cultures with rotenone at 25 and 100 microM rotenone reflect changes in the intracellular trafficking of Bax among different organelles.
Asunto(s)
Apoptosis/fisiología , Necrosis/metabolismo , Neuronas/metabolismo , Rotenona/farmacología , Proteína X Asociada a bcl-2/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/fisiopatología , Proteínas de Microfilamentos/metabolismo , Necrosis/inducido químicamente , Necrosis/fisiopatología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Orgánulos/efectos de los fármacos , Orgánulos/metabolismo , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Desacopladores/farmacologíaRESUMEN
Endothelial nitric oxide synthase (eNOS) activation with subsequent inducible NOS (iNOS), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX2) activation is essential to statin inhibition of myocardial infarct size (IS). In the rat, the peroxisome proliferator-activated receptor-gamma agonist pioglitazone (Pio) limits IS, upregulates and activates cPLA2 and COX2, and increases myocardial 6-keto-PGF1alpha levels without activating eNOS and iNOS. We asked whether Pio also limits IS in eNOS-/- and iNOS-/- mice. Male C57BL/6 wild-type (WT), eNOS-/-, and iNOS-/- mice received 10 mg.kg(-1).day(-1) Pio (Pio+) or water alone (Pio-) for 3 days. Mice underwent 30 min coronary artery occlusion and 4 h reperfusion, or hearts were harvested and subjected to ELISA and immunoblotting. As a result, Pio reduced IS in the WT (15.4+/-1.4% vs. 39.0+/-1.1%; P<0.001), as well as in the eNOS-/- (32.0+/-1.6% vs. 44.2+/-1.9%; P<0.001) and iNOS-/- (18.0+/-1.2% vs. 45.5+/-2.3%; P<0.001) mice. The protective effect of Pio in eNOS-/- mice was smaller than in the WT (P<0.001) and iNOS-/- (P<0.001) mice. Pio increased myocardial Ser633 and Ser1177 phosphorylated eNOS levels in the WT and iNOS-/- mice. iNOS was undetectable in all six groups. Pio increased cPLA2, COX2, and PGI2 synthase levels in the WT, as well as in the eNOS-/- and iNOS-/-, mice. Pio increased the myocardial 6-keto-PGF1alpha levels and cPLA2 and COX2 activity in the WT, eNOS-/-, and iNOS-/- mice. In conclusion, the myocardial protective effect of Pio is iNOS independent and may be only partially dependent on eNOS. Because eNOS activity decreases with age, diabetes, and advanced atherosclerosis, this effect may be relevant in a clinical setting and should be further characterized.
Asunto(s)
Fármacos Cardiovasculares/farmacología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/enzimología , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico Sintasa de Tipo II/deficiencia , Tiazolidinedionas/farmacología , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Ciclooxigenasa 2/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Immunoblotting , Oxidorreductasas Intramoleculares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III/genética , Fosfolipasas A2 Citosólicas/metabolismo , Fosforilación , Pioglitazona , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismoRESUMEN
Decreased cerebral blood flow (CBF) has been observed following the resuscitation from neonatal hypoxic-ischemic injury, but its mechanism is not known. We address the hypothesis that reduced CBF is due to a change in nitric oxide (NO) and superoxide anion O(2)(-) balance secondary to endothelial NO synthase (eNOS) uncoupling with vascular injury. Wistar rats (7 day old) were subjected to cerebral hypoxia-ischemia by unilateral carotid occlusion under isoflurane anesthesia followed by hypoxia with hyperoxic or normoxic resuscitation. Expired CO(2) was determined during the period of hyperoxic or normoxic resuscitation. Laser-Doppler flowmetry was used with isoflurane anesthesia to monitor CBF, and cerebral perivascular NO and O(2)(-) were determined using fluorescent dyes with fluorescence microscopy. The effect of tetrahydrobiopterin supplementation on each of these measurements and the effect of apocynin and N(omega)-nitro-L-arginine methyl ester (L-NAME) administration on NO and O(2)(-) were determined. As a result, CBF in the ischemic cortex declined following the onset of resuscitation with 100% O(2) (hyperoxic resuscitation) but not room air (normoxic resuscitation). Expired CO(2) was decreased at the onset of resuscitation, but recovery was the same in normoxic and hyperoxic resuscitated groups. Perivascular NO-induced fluorescence intensity declined, and O(2)(-)-induced fluorescence increased in the ischemic cortex after hyperoxic resuscitation up to 24 h postischemia. L-NAME treatment reduced O(2)(-) relative to the nonischemic cortex. Apocynin treatment increased NO and reduced O(2)(-) relative to the nonischemic cortex. The administration of tetrahydrobiopterin following the injury increased perivascular NO, reduced perivascular O(2)(-), and increased CBF during hyperoxic resuscitation. These results demonstrate that reduced CBF follows hyperoxic resuscitation but not normoxic resuscitation after neonatal hypoxic-ischemic injury, accompanied by a reduction in perivascular production of NO and an increase in O(2)(-). The finding that tetrahydrobiopterin, apocynin, and L-NAME normalized radical production suggests that the uncoupling of perivascular NOS, probably eNOS, due to acquired relative tetrahydrobiopterin deficiency occurs after neonatal hypoxic-ischemic brain injury. It appears that both NOS uncoupling and the activation of NADPH oxidase participate in the changes of reactive oxygen concentrations seen in cerebral hypoxic-ischemic injury.
Asunto(s)
Corteza Cerebral/irrigación sanguínea , Circulación Cerebrovascular , Endotelio Vascular/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Óxido Nítrico/metabolismo , Terapia por Inhalación de Oxígeno , Resucitación , Superóxidos/metabolismo , Acetofenonas/farmacología , Animales , Animales Recién Nacidos , Biopterinas/análogos & derivados , Biopterinas/farmacología , Dióxido de Carbono/metabolismo , Circulación Cerebrovascular/efectos de los fármacos , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Inhibidores Enzimáticos/farmacología , Espiración , Hipoxia-Isquemia Encefálica/patología , Hipoxia-Isquemia Encefálica/fisiopatología , Hipoxia-Isquemia Encefálica/terapia , Flujometría por Láser-Doppler , Microscopía Fluorescente , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Terapia por Inhalación de Oxígeno/efectos adversos , Ratas , Ratas Wistar , Resucitación/efectos adversos , Factores de TiempoRESUMEN
Perinatal hypoxia-ischemia (HI) occurs in 0.2%-0.4% of all live births, with 100% O(2) resuscitation (HHI) remaining a standard clinical treatment. HI produces a broad spectrum of neuronal death phenotypes ranging from a more noninflammatory apoptotic death to a more inflammatory necrotic cell death that may be responsible for the broad spectrum of reported dysfunctional outcomes. However, the mechanisms that would account for this phenotypic spectrum of cell death are not fully understood. Here, we provide evidence that Bcl-2-associated X protein (Bax) can shuttle to different subcellular compartments in response to HI, thus triggering the different organelle-associated cell death signaling cascades resulting in cell death phenotype diversity. There was an early increase in intranuclear and total nuclear Bax protein levels followed by a later Bax redistribution to the mitochondria and endoplasmic reticulum (ER). Associated with the organelle-specific Bax shuttling time course, there was an increase in nuclear phosphorylated p53, cytosolic cleaved caspase-3, and caspase-12. When HI-treated P7 rats were resuscitated with 100% O(2) (HHI), there were increased lesion volumes as determined by T2-weighted magnetic resonance imaging with no change in cortical apoptotic signaling compared with HI treatment alone. There was, however, increased inflammatory (cytosolic-cleaved interleukin-1beta) and necrotic (increased nuclear 55-kDa-cleaved PARP-1 [poly-ADP-ribose 1] and decreased nuclear HMGB1 [nuclear high-mobility group box 1]) after HHI. Furthermore, HHI increased ER calpain activation and ER Bax protein levels compared with HI alone. These data suggest that 100% O(2) resuscitation increases Bax-mediated activation of ER cell death signaling, inflammation, and lesion volume by increasing necrotic-like cell death. In light of these findings, the use of 100% O(2) treatment for neonatal HI should be reevaluated.
Asunto(s)
Infarto Encefálico/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/terapia , Degeneración Nerviosa/metabolismo , Terapia por Inhalación de Oxígeno/efectos adversos , Proteína X Asociada a bcl-2/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Infarto Encefálico/etiología , Infarto Encefálico/fisiopatología , Compartimento Celular/fisiología , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Hiperoxia/etiología , Hiperoxia/metabolismo , Hiperoxia/fisiopatología , Hipoxia-Isquemia Encefálica/fisiopatología , Mediadores de Inflamación/metabolismo , Necrosis/etiología , Necrosis/metabolismo , Necrosis/fisiopatología , Degeneración Nerviosa/etiología , Degeneración Nerviosa/fisiopatología , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Transporte de Proteínas/fisiología , Ratas , Ratas Wistar , Transducción de Señal/fisiologíaRESUMEN
The examination of Bcl-2-associated X protein (Bax) protein's role in the activation of cognate nuclear, mitochondrial and ER cell death signaling cascades and the resulting effects on cell death phenotype in the brain after neonatal hypoxia-ischemia (HI) requires an understanding of neonatal HI insult and progression, as well as, its dysfunctional outcomes. In addition, knowledge of key concepts of oxidative stress, a major injurious component of HI, and the different cell death phenotypes (i.e. apoptosis and necrosis) will aid the design of appropriate useful experimental paradigms. Here we discuss organelle cell death signaling cascades in the context of the different cell death phenotypes associated with animal models of neonatal hypoxia ischemia and tissue culture models used in the study of hypoxia ischemia, focusing on the intracellular shifts of the Bcl-2 associated X protein (Bax) in the hypoxic brain.
Asunto(s)
Isquemia Encefálica/patología , Encéfalo/patología , Muerte Celular , Hipoxia Encefálica/patología , Animales , Animales Recién Nacidos , Estrés Oxidativo , Ratas , Transducción de Señal , Proteína X Asociada a bcl-2/fisiologíaRESUMEN
Long-term functional impairments due to spinal cord injury (SCI) in the rat result from secondary apoptotic death regulated, in part, by SCI-induced decreases in protein levels of the antiapoptotic protein Bcl-xL. We have shown that exogenous administration of Bcl-xL spares neurons 24 h after SCI. However, long-term effects of chronic application of Bcl-xL have not been characterized. To counteract SCI-induced decreases in Bcl-xL and resulting apoptosis, we used the TAT protein transduction domain fused to the Bcl-xL protein (Tat-Bcl-xL), or its antiapoptotic domain BH4 (Tat-BH4). We used intrathecal delivery of Tat-Bcl-xL, or Tat-BH4, into injured spinal cords for 24 h or 7 days, and apoptosis, neuronal death and locomotor recovery were assessed up to 2 months after injury. Both, Tat-Bcl-xL and Tat-BH4, significantly decreased SCI-induced apoptosis in thoracic segments containing the site of injury (T10) at 24 h or 7 days after SCI. However, the 7-day delivery of Tat-Bcl-xL, or Tat-BH4, also induced a significant impairment of locomotor recovery that lasted beyond the drug delivery time. We found that the 7-day administration of Tat-Bcl-xL, or Tat-BH4, significantly increased non-apoptotic neuronal loss and robustly augmented microglia/macrophage activation. These results indicate that the antiapoptotic treatment targeting Bcl-xL shifts neuronal apoptosis to necrosis, increases the inflammatory response and impairs locomotor recovery. Our results suggest that a combinatorial treatment consisting of antiapoptotic and anti-inflammatory agents may be necessary to achieve tissue preservation and significant improvement in functional recovery after SCI.
Asunto(s)
Apoptosis/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Proteína bcl-X/administración & dosificación , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Conducta Animal , Antígeno CD11b/metabolismo , Modelos Animales de Enfermedad , Esquema de Medicación , Ensayo de Inmunoadsorción Enzimática/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Productos del Gen tat/administración & dosificación , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/patología , Factores de TiempoRESUMEN
The potential negative impact of early blood oxygenation on development of specific cognitive and motor outcomes in children born at very low birth weight (VLBW; 1000-1500g) has not been examined even though these infants are exposed to varying durations and amounts of oxygen as part of their neonatal care. While this is the largest group of preterm infants, they receive much less research attention than extremely low birth weight infants (ELBW<1000g). Although neonatologists are questioning the routine use of oxygen therapy for all neonates, research has focused primarily on the more medically fragile ELBW infants. To date there are no systematic studies available to guide decision making for oxygen supplementation for a large segment of the preterm infant population. The aim of the present study was to determine if there is an association between blood oxygenation in the first 4h of life and specific cognitive and motor skills in preterm infants with acute respiratory disorders but no severe intracranial insult using a selected cohort from a longitudinal study children recruited in 1991 and 1992 designed to examine the role of biological immaturity as defined by gestational age and parenting in development. From this cohort, 55 children had acute respiratory disorders without severe intracranial insult. Of these, 35 children had at least one partial pressure of oxygen obtained from arterial blood (PaO2) during the first 4h of life as part of their clinical care. Higher early PaO2 values were associated with lower impulse control and attention skills in the elementary school age period. Models that were examined for relations between PaO2 values that also included birth weight and parenting quality across the first year of life revealed that higher PaO2 remained associated with impulse control but not attention skills. Birth weight was not associated with any outcomes. These results suggest that hyperoxia may be a risk factor for developmental problems that are not expressed until school age.
Asunto(s)
Oxígeno/sangre , Nacimiento Prematuro/fisiopatología , Trastornos Respiratorios/sangre , Trastornos Respiratorios/etiología , Peso al Nacer , Presión Sanguínea , Niño , Desarrollo Infantil/fisiología , Preescolar , Trastornos del Conocimiento/etiología , Estudios de Cohortes , Femenino , Humanos , Masculino , Examen Neurológico , Estudios RetrospectivosRESUMEN
Hypoxic newborns have traditionally been given supplemental oxygen, and until recently, guidelines for neonatal resuscitation recommended that 100% oxygen be used. Exposure to 100% oxygen after hypoxic injury, however, may exacerbate oxidative stress. The current study evaluated the effect of exposure to 100, 40 or 21% oxygen after neonatal hypoxic-ischemic injury on the severity of brain injury after long-term survival. The severity of histological brain injury was not different in animals exposed to 100% oxygen versus room air. Male animals treated with 40% oxygen post-hypoxia had the lowest mean total histology scores, but this was not statistically significant due to the large variation in injury within each treatment group. These results support the growing number of studies in human infants and experimental animals that show no benefit of 100% oxygen over room air for neonatal resuscitation. Our results suggest that post-hypoxia treatment with 40% oxygen may be beneficial, particularly in males. Further studies of the effects of different concentrations of oxygen on brain injury are warranted and should have sufficient power to examine sex differences.