calpain and Carbon-Monoxide-Poisoning

Carbon monoxide (CO) poisoning causes neuronal and glial apoptosis that can result in delayed neurological symptoms. The damage of brain cells can be prevented by oxygen therapy. Based on the central role of astrocytes in maintaining neuronal function and viability we investigated the toxic effects of 3000ppm CO in air followed by 24h of normoxia and evaluated the possible protective influence of 100% normobaric oxygen or 100% oxygen at a pressure of 3bar (hyperbaric) against CO poisoning in these cells. CO/normoxia caused a progressive decline of viability, increase in reactive oxygen species and decline of mitochondrial membrane potential and intracellular ATP levels in cultured rat astrocytes. Increased caspase-9, caspase-8 and calpain activity converged in activation of caspase-3/7. 1h treatment with oxygen disclosed pressure- and time-dependent efficacy in restoring astrocytic mitochondrial function and the prevention of apoptosis. The protective effect was most evident when the astrocytes were exposed to hyperbaric oxygen, but not normobaric oxygen, 1-5h after exposure to CO.

Topics: Animals; Apoptosis; Astrocytes; Calpain; Carbon Monoxide Poisoning; Caspase 3; Caspase 8; Caspase 9; Cell Survival; Cells, Cultured; Hyperbaric Oxygenation; Membrane Potential, Mitochondrial; Mitochondria; Neuroglia; Rats; Reactive Oxygen Species

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.

Topics: Animals; Annexin A5; Apoptosis; Calpain; Carbon Monoxide Poisoning; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Culture Media; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Hippocampus; Humans; Hypoxia, Brain; Immunoblotting; Immunohistochemistry; Membrane Potentials; Mice; Microfilament Proteins; Mitochondria; Neurons; Phosphatidylserines; Propidium; Signal Transduction; Trypan Blue