title: Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity.
abstract: Excitotoxic glutamate CNS stimulation can result in neuronal cell death. Contributing mechanisms and markers of cell death are the activation of caspase-3 and DNA fragmentation. It remains to be resolved to which extent both cellular reactions overlap and/or indicate different processes of neurodegeneration. In this study, mixed neuronal cultures from newborn mice pubs (0-24 h) were stimulated with glutamate, and the co-localization of active caspase-3 and DNA fragmentation was investigated by immunocytochemistry and the TUNEL nick-end labelling. In untreated cultures, 8% scattered neurons (marked by MAP-2) displayed activated caspase-3 at different morphological stages of degeneration. TUNEL staining was detected in 5% of cell nuclei including GFAP-positive astrocytes. However, co-localization of active caspase-3 with TUNEL was less than 2%. After glutamate stimulation (125 microM), the majority of neurons was dying between 12 and 24 h. The absolute number of active caspase-3 neurons increased only moderately but in relation of surviving neurons after 24 h from 8 to 36% (125 microM), to 53% (250 microM) or to 32% (500 microM). TUNEL staining also increased after 24 h following glutamate treatment to 37% but the co-localization with active caspase-3 remained at the basal low level of 2%. In our system, glutamate-mediated excitotoxicity effects the DNA fragmentation and caspase-3 activation. Co-localization of both parameters, however, is very poor. Active caspase-3 in the absence of TUNEL indicates a dynamic degenerative process, whereas TUNEL marks the end stage of severe irreversible cell damage regardless to the origin of the cell.
authors.affiliation: Institut für Pharmakologie, Christian-Albrechts-Universität, Hospitalstrasse 4, 24105, Kiel, Germany. sbrecht@pharmakologie.uni-kiel.de
authors.forename: S
authors.initials: S
authors.lastname: Brecht
authors.forename: M
authors.initials: M
authors.lastname: Gelderblom
authors.forename: A
authors.initials: A
authors.lastname: Srinivasan
authors.forename: K
authors.initials: K
authors.lastname: Mielke
authors.forename: G
authors.initials: G
authors.lastname: Dityateva
authors.forename: T
authors.initials: T
authors.lastname: Herdegen
mesh_terms: D000818:Animals; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000378:metabolism*; D018920:Coculture Techniques; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005189:False Positive Reactions; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D018698:Glutamic Acid / Q000633:toxicity*; D006624:Hippocampus / Q000166:cytology; D020287:In Situ Nick-End Labeling / Q000592:standards; D051379:Mice; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins / Q000032:analysis; D009457:Neuroglia / Q000737:chemistry / Q000166:cytology; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology*; D009498:Neurotoxins / Q000633:toxicity
chemical_list: D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; D009498:Neurotoxins; D018698:Glutamic Acid; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases