RESUMEN
The neurotoxic effects of carbon monoxide (CO) are well known. Brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity, while the direct effect of CO on intracellular targets remains poorly understood. In the present study, we have investigated the pathways leading to neural cell death induced by in vitro exposure to CO using a gas exposure chamber that we have developed. Mouse hippocampal neurons (HT22) and human glial cells (D384) were exposed to concentrations of CO ranging from 300 to 1000 ppm in the presence of 20% oxygen. Cytotoxicity was observed after 48 h exposure to 1000 ppm, corresponding to approximately 1 microM CO in the cultured medium, as measured by gas chromatography. CO induced cell death with characteristic features of apoptosis. Exposed cells exhibited loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, nuclei with chromatin condensation, and exposure of phosphatidyl serine on the external leaflet of the plasma membrane. CO also triggered activation of caspase and calpain proteases. Pre-incubation with either the pancaspase inhibitor Z-VAD-fmk (20 microM) or the calpain inhibitor E64d (25 microM) reduced by 50% the occurrence of apoptosis. When pre-incubating the cells with the two inhibitors together there was an additional reduction in the number of cells with apoptotic nuclei. These data suggest that CO causes apoptosis via activation of parallel proteolytic pathways involving both caspases and calpains. Furthermore, pre-treatment with the antioxidant MnTBAP (100 microM) significantly reduced the number of apoptotic nuclei, pointing to a critical role of oxidative stress in CO toxicity.
Asunto(s)
Apoptosis/efectos de los fármacos , Intoxicación por Monóxido de Carbono/patología , Hipoxia Encefálica/patología , Neuronas/patología , Animales , Anexina A5/metabolismo , Calpaína/antagonistas & inhibidores , Calpaína/metabolismo , Proteínas Portadoras/metabolismo , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Medios de Cultivo , Citocromos c/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipocampo/patología , Humanos , Immunoblotting , Inmunohistoquímica , Potenciales de la Membrana/efectos de los fármacos , Ratones , Proteínas de Microfilamentos/metabolismo , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosfatidilserinas/farmacología , Propidio , Transducción de Señal/efectos de los fármacos , Azul de TripanoRESUMEN
In order to demonstrate separated flow in vivo, a method for the computerized analysis of cineangiographies has been developed, tested in vitro, and compared with LDV. A pulsatile flow was created in a glass model bifurcation, and velocity profiles were obtained with LDV at several phase angles. The flow was cinefilmed during contrast injection and the images were digitized. The computer then transformed the image sequence into parametric images representing arrival times of the contrast. The separation regions demonstrated with LDV were identified as areas with delayed contrast arrival. A preliminary analysis of a cineangiography in vivo is also included.