RESUMEN
Massive production of free radicals (FR) has been associated with a variety of pathological conditions in the central nervous system (CNS). We have used the FR generating compound hydrogen peroxide (H2O2) in organotypic hippocampal slice cultures to model oxidative injury in the brain. Necrotic cell death was monitored for up to 48 h using propidium iodide (PI) and confocal microscopy. A 1 h exposure to H2O2 (0.5-2.5 mM) caused a dose-dependent, and region specific cell death in hippocampal slice cultures. Glial cells demonstrated a high degree of vulnerability to H2O2. During the initial 3 h post-injury period, regions of the slice where glial cell bodies predominated showed massive cell death. The majority of neurons in the pyramidal layers were spared, though at later time points they appeared damaged as well. Carboxy-dichlorofluorescein imaging revealed a corresponding early increase in ROS generation in glial cells compared to pyramidal neurons. Immunohistochemistry of PI labeled slices identified astrocytes as the cells most sensitive to H2O2 toxicity. In dissociated cell cultures of hippocampal astrocytes and neurons, astrocytes also exhibited a significantly higher sensitivity to H2O2 than neurons. Hydrogen peroxide-induced cytotoxicity in all regions of the hippocampal slice culture was significantly attenuated by pre-treatment with antioxidants (alpha-tocopherol and glutathione), and was not prevented by blockade of Ca2+ influx, or NMDA channel activation. Cyclosporin A, an inhibitor of mitochondrial permeability transition, reduced cytotoxicity in glial areas by more than 50%, while in the CA2-CA3 pyramidal layers a much smaller, but still significant, attenuation of cytotoxicity was observed. Our results suggest that mitochondria are primary targets of H2O2 toxicity, particularly in astrocytes.
Asunto(s)
Isquemia Encefálica/metabolismo , Radicales Libres/metabolismo , Hipocampo/metabolismo , Degeneración Nerviosa/metabolismo , Neuroglía/metabolismo , Estrés Oxidativo , Animales , Animales Recién Nacidos , Antioxidantes/farmacología , Muerte Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Colorantes Fluorescentes , Hipocampo/efectos de los fármacos , Hipocampo/patología , Peróxido de Hidrógeno/toxicidad , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Necrosis/metabolismo , Degeneración Nerviosa/inducido químicamente , Neuroglía/efectos de los fármacos , Neuroglía/patología , Técnicas de Cultivo de Órganos , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Células Piramidales/patología , Ratas , Factores de TiempoRESUMEN
In monolayers of cultured rat astrocytes a number of agents that induce oxidative stress act synergistically with exposure to copper leading to rapid depolarization of the mitochondrial membrane potential (Psi m) and increased reactive oxygen species (ROS) production. Copper sensitized astrocytes to the action of menadione, an intracellular generator of superoxide anion radical, exogenous hydrogen peroxide (H2O2) and rotenone, an inhibitor of mitochondrial electron transport chain complex I. However, significant differences were observed in the ability to modulate the copper-enhanced oxidative stress depending on which stressor was used. The inhibitor of mitochondrial permeability transition cyclosporin A attenuated the effect of copper and rotenone, but had no protective action in the case of H2O2/copper and menadione/copper combinations. The H2O2 scavenger pyruvate was effective at protecting mitochondria against damage associated with the combined exposure to H2O2/copper and menadione/copper but not to the rotenone/copper combination. The antioxidant Trolox was ineffective at protecting against any of these actions and indeed had a damaging effect when combined with copper. The membrane-permeable copper chelator neocuproine combined with sensitizing concentrations of menadione caused a decrease in Psi m, mimicking the action of copper. Penicillamine, a membrane-impermeable copper chelator, was effective at reducing copper sensitization. Endogenous copper, mobilized during periods of oxidative stress, may play a role in the pathophysiology of brain injury. Our results suggest that this might be particularly dangerous in dysfunctional conditions in which the mitochondrial electron transport chain is compromised.
Asunto(s)
Astrocitos/metabolismo , Cobre/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/farmacología , Astrocitos/citología , Astrocitos/efectos de los fármacos , Células Cultivadas , Quelantes/farmacología , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Potenciales de la Membrana/efectos de los fármacos , Oxidantes/metabolismo , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Fenantrolinas/farmacología , Ratas , Ratas Wistar , Desacopladores/farmacologíaRESUMEN
Mitochondrial membrane potential (Deltapsi(M)) plays a key role in coordinating mitochondrial function and cell biology in general. In astrocytes, Deltapsi(M) is an important indicator of the health of these brain cells and their response to traumatic and hypoxic injury. We have shown previously how fluorescent signals can be measured from cells attached to a coverslip in a standard cuvette with a fluorometer and modulated using a cuvette perfusion system (Pflugers Arch-Eur. J. Physiol. 421 (1992) 400). Here we report on how this method can be employed to characterize the actions of a number of potentiometric fluorescent cationic dyes, including JC-1, Rh123 and TMRM, for their ability to monitor Deltapsi(M) in primary cultures of intact astrocytes. All dyes detected the reversible depolarization produced by brief exposure to the mitochondrial uncoupler protonophore FCCP, which short circuits and dissipates Deltapsi(M). Qualitatively similar responses were measured after treatment with either azide, an inhibitor of complex IV in the mitochondrial respiratory chain, or the oxidant H(2)O(2). Cell depolarization with high potassium modified the responses to FCCP. The time courses of these responses differed in a manner dependent on the particular dye used and in a way that correlated with expected permeation rate. The merits and pitfalls of these different potentiometric dyes for monitoring Deltapsi(M) are discussed.