Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity

Excitotoxic glutamate CNS stimulation can result in neuronal cell death. Co ntributing mechanisms and markers of cell death are the activation of caspa se-3 and DNA fragmentation. It remains to be resolved to which extent both cellular reactions overlap and/or indicate different processes of neurodege neration. In this study, mixed neuronal cultures from newborn mice pubs (0- 24 h) were stimulated with glutamate, and the co-localization of active cas pase-3 and DNA fragmentation was investigated by immunocytochemistry and th e TUNEL nick-end labelling. In untreated cultures, 8% scattered neurons (ma rked by MAP-2) displayed activated caspase-3 at different morphological sta ges of degeneration. TUNEL staining was detected in 5% of cell nuclei inclu ding GFAP-positive astrocytes. However, co-localization of active caspase-3 with TUNEL was less than 2%. After glutamate stimulation (125 muM), the ma jority of neurons was dying between 12 and 24 h. The absolute number of act ive caspase-3 neurons increased only moderately but in relation of survivin g neurons after 24 h from 8 to 36% (125 muM), to 53% (250 muM) or to 32% (5 00 muM). TUNEL staining also increased after 24 h following glutamate treat ment to 37% but the co-localization with active caspase-3 remained at the b asal low level of 2%. In our system, glutamate-mediated excitotoxicity effe cts the DNA fragmentation and caspase-3 activation. Co-localization of both parameters, however, is very poor. Active caspase-3 in the absence of TUNE L indicates a dynamic degenerative process, whereas TUNEL marks the end sta ge of severe irreversible cell damage regardless to the origin of the cell. (C) 2001 Elsevier Science BY All rights reserved.