RESUMEN
BACKGROUND: Labor subjects the fetus to an hypoxic episode and concomitant adrenomodullary catecholamine surge that may provide protection against the hypoxic insult. The beta1-adrenergic agonist dobutamine protects against hypoxia/aglycemia induced neuronal damage. We aimed to identify the associated protective biological processes involved. RESULTS: Hippocampal slices from 6 days old mice showed significant changes of gene expression comparing slices with or without dobutamine (50 mM) in the following two experimental paradigms: (1) control conditions versus lipopolysacharide (LPS) stimulation and (2) oxygen-glucose deprivation (OGD), versus combined LPS/OGD. Dobutamine depressed the inflammatory response by modifying the toll-like receptor-4 signalling pathways, including interferon regulatory factors and nuclear factor κ B activation in experimental paradigm 1. The anti-oxidant defense genes superoxide dismutase 3 showed an upregulation in the OGD paradigm while thioredoxin reductase was upregulated in LPS paradigm. The survival genes Bag-3, Tinf2, and TMBIM-1, were up-regulated in paradigm 1. Moreover, increased levels of SOD3 were verified on the protein level 24 h after OGD and control stimulation in cultures with or without preconditioning with LPS and dobutamine, respectively. CONCLUSIONS: Neuroprotective treatment with dobutamine depresses expression of inflammatory mediators and promotes the defense against oxidative stress and depresses apoptotic genes in a model of neonatal brain hypoxia/ischemia interpreted as pharmacological preconditioning. We conclude that beta1-adrenoceptor activation might be an efficient strategy for identifying novel pharmacological targets for protection of the neonatal brain.
Asunto(s)
Antioxidantes/farmacología , Dobutamina/farmacología , Expresión Génica/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/genética , Animales , Hipocampo/efectos de los fármacos , Ratones Endogámicos BALB C , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuroprotección/efectos de los fármacos , Neuroprotección/fisiología , Fármacos Neuroprotectores/farmacología , Oxidantes/metabolismo , Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Inflammation acting in synergy with brain ischemia aggravates perinatal ischemic brain damage. The sensitizing effect of pro-inflammatory exposure prior to hypoxia is dependent on signaling by TNF-α through TNF receptor (TNFR) 1. Adrenoceptor (AR) activation is known to modulate the immune response and synaptic transmission. The possible protective effect of α and ß AR activation against neuronal damage caused by tissue ischemia and inflammation, acting in concert, was evaluated in murine hippocampal organotypic slices treated with lipopolysaccharide (LPS) and subsequently subjected to oxygen-glucose deprivation (OGD). METHOD: Hippocampal slices from mice were obtained at P6, and were grown in vitro for 9 days on nitrocellulose membranes. Slices were treated with ß1(dobutamine)-, ß2(terbutaline)-, α1(phenylephrine)- and α2(clonidine)-AR agonists (5 and 50 µM, respectively) during LPS (1 µg/mL, 24 h) -exposure followed by exposure to OGD (15 min) in a hypoxic chamber. Cell death in the slice CA1 region was assessed by propidium iodide staining of dead cells. RESULTS: Exposure to LPS + OGD caused extensive cell death from 4 up to 48 h after reoxygenation. Co-incubation with ß1-agonist (50 µM) during LPS exposure before OGD conferred complete protection from cell death (P < 0.001) whereas the ß2-agonist (50 µM) was partially protective (p < 0.01). Phenylephrine was weakly protective while no protection was attained by clonidine. Exposure to both ß1- and ß2-agonist during LPS exposure decreased the levels of secreted TNF-α, IL-6 and monocyte chemoattractant protein-1 and prevented microglia activation in the slices. Dobutamine remained neuroprotective in slices exposed to pure OGD as well as in TNFR1-/- and TNFR2-/- slices exposed to LPS followed by OGD. CONCLUSIONS: Our data demonstrate that activation of both ß1- and ß2-receptors is neuroprotective and may offer mechanistic insights valuable for development of neuro-protective strategies in neonates.
Asunto(s)
Isquemia Encefálica/metabolismo , Encefalitis/metabolismo , Glucosa/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Lipopolisacáridos/farmacología , Oxígeno/metabolismo , Receptores Adrenérgicos beta/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Isquemia Encefálica/patología , Muerte Celular , Citocinas/metabolismo , Dobutamina/farmacología , Encefalitis/patología , Hipocampo/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Fenilefrina/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Terbutalina/farmacología , Técnicas de Cultivo de TejidosRESUMEN
Inflammation and ischemia have a synergistic damaging effect in the immature brain. The role of tumor necrosis factor (TNF) receptors 1 and 2 in lipopolysaccharide (LPS)-induced sensitization and tolerance to oxygen-glucose deprivation (OGD) was evaluated in neonatal murine hippocampal organotypic slices. Hippocampal slices from balb/c, C57BL/6 TNFR1(-/-), TNFR2(-/-), and wild-type (WT) mice obtained at P6 were grown in vitro for 9 days. Preexposure to LPS immediately before OGD increased propidium iodide-determined cell death in regions CA1, CA3, and dentate gyrus from 4 up to 48 h after OGD (P<0.001). Extending the time interval between LPS exposure and OGD to 72 h resulted in tolerance, that is reduced neuronal cell death after OGD (P<0.05). Slices from TNFR1(-/-) mice showed neither LPS-induced sensitization nor LPS-induced tolerance to OGD, whereas both effects were present in slices from TNFR2(-/-) and WT mice. Cytokine secretion (TNFalpha and interleukin-6) during LPS exposure was decreased in TNFR1(-/-) slices and increased in TNFR2(-/-) as compared with WT slices. We conclude that LPS induces sensitization or tolerance to OGD depending on the time interval between exposure to LPS and OGD in murine hippocampal slice cultures. Both paradigms are dependent on signaling through TNFR1.
Asunto(s)
Glucosa/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Lipopolisacáridos/farmacología , Oxígeno/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Animales , Animales Recién Nacidos , Hipocampo/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/inmunología , Ratones , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/deficiencia , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Resuscitation with pure oxygen at birth after fetal asphyxia may aggravate brain damage by inducing pro-inflammation. The toll-like receptors (TLRs) may serve a pro-inflammatory role in hyperoxemia during ischemia-reperfusion. Sixteen near-term fetal sheep (132-136 d) were subjected to 10 min of cord occlusion, delivery and mechanical ventilation with 100% O2 (n = 8), or 21% O2 (n = 8) for 30 min followed by normoxemia for 90 min. Eight sheep fetuses were delivered immediately with inspired O2 targeted at normoxemia for 120 min (controls). Levels and distributions of mRNAs for IL-1beta, TNF-alpha, IL-12p40, IL-18, IL-6, IL-10, IFN-gamma, TLR-2, -3 and -4 in cerebral tissue at 2 h after birth were evaluated with real-time polymerase chain reaction (PCR) and in situ hybridization. Expressions of IL-1beta, IL-12p40, TLR-2, and TLR-4 were increased in cortex/subcortex after resuscitation with 100% O2 compared with 21% O2 (all p < 0.05) and to controls (all p < 0.05). Increased cellular expression of IL-1beta was localized to sub-meningeal cortical layers and to sub-cortical white matter. Hyperoxic resuscitation at birth following fetal asphyxia induces a cerebral pro-inflammatory response with an up-regulation of TLR-2 and -4. These may be early events leading to increased tissue damage after exposure to hyperoxemia at birth.