title: Apoptosis is not an invariable component of in vitro models of cortical cerebral ischaemia.
abstract: Characterising the mechanisms of cell death following focal cerebral ischaemia has been hampered by a lack of an in vitro assay emulating both the apoptotic and necrotic features observed in vivo. The present study systematically characterised oxygen-glucose-deprivation (OGD) in primary rat cortical neurones to establish a reproducible model with components of both cell-death endpoints. OGD induced a time-dependent reduction in cell viability, with 80% cell death occurring 24 h after 3 h exposure to 0% O2 and 0.5 mM glucose. Indicative of a necrotic component to OGD-induced cell death, N-methyl-D-aspartate (NMDA) receptor inhibition with MK-801 attenuated neuronal loss by 60%. The lack of protection by the caspase inhibitors DEVD-CHO and z-VAD-fmk suggested that under these conditions neurones did not die by an apoptotic mechanism. Moderating the severity of the insult by decreasing OGD exposure to 60 min did not reduce the amount of necrosis, but did induce a small degree of apoptosis (a slight reduction in cell death was observed in the presence of 10 uM DEVD-CHO). In separate experiments purported to enhance the apoptotic component, cells were gradually deprived of O2, exposed to 4% O2 (as opposed to 0%) during the OGD period, or maintained in serum-containing media throughout. While NMDA receptor antagonism significantly reduced cortical cell death under all conditions, a caspase-inhibitor sensitive component of cell death was not uncovered. These studies suggest that OGD of cultured cortical cells models the excitotoxic, but not the apoptotic component of cell death observed in vivo.
authors.affiliation: Fujisawa Institute of Neuroscience, Division of Neuroscience, University of Edinburgh, 1 George Square, Edinburgh, UK. P.Jones@ed.ac.uk
authors.forename: Paul Alexander
authors.initials: PA
authors.lastname: Jones
authors.forename: Gillian Ruth
authors.initials: GR
authors.lastname: May
authors.forename: Joyee Ann
authors.initials: JA
authors.lastname: McLuckie
authors.forename: Akinori
authors.initials: A
authors.lastname: Iwashita
authors.forename: John
authors.initials: J
authors.lastname: Sharkey
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002545:Brain Ischemia / Q000378:metabolism / Q000473:pathology*; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D016291:Dizocilpine Maleate / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D008954:Models, Biological; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D009842:Oligopeptides / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D018691:Excitatory Amino Acid Antagonists; D009842:Oligopeptides; C108637:aspartyl-glutamyl-valyl-aspartal; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D020169:Caspases; D019311:Staurosporine; D005947:Glucose; D010100:Oxygen