Evaluation of preconditioning treatments to protect near-pure cortical neuronal cultures from in vitro ischemia induced acute and delayed neuronal death.
We evaluated the efficacy of cycloheximide, heat stress, NMDA receptor blockade (MK801/AP-5), oxygen--glucose deprivation, hypoxia, hypothermia and TNFalpha preconditioning to protect cortical neurons from in vitro ischemic insults that result in acute necrotic and delayed apoptotic neuronal death. Preconditioning treatments were performed 22--24 h before in vitro ischemia. In vitro ischemia was carried out in 96-well microtitre strip-plates by washing neuronal cultures with a balanced salt solution containing 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Glutamate receptor blockers were present during in vitro ischemia to induce delayed neuronal death. Cycloheximide, heat stress, MK801 and oxygen--glucose deprivation preconditioning were neuroprotective in both acute and delayed in vitro ischemic neuronal death models. AP-5 preconditioning and a 12 h post-MK801 preconditioning interval protected neurons from acute ischemic neuronal death only. Hypoxia, TNFalpha and hypothermic preconditioning provided no neuronal protection in the in vitro ischemia models. This study has confirmed for the first time that several preconditioning treatments can protect neurons from in vitro ischemia induced acute necrotic and delayed apoptotic neuronal death. In addition, a unique feature of this study is the finding that preconditioning could be induced in near-pure primary cortical neuronal cultures, thus confirming that ischemic tolerance is an intrinsic property of neurons and provides a simplified culture system for identifying neuroprotective proteins.
Meloni
Bruno P
BP
Department of Neurosurgery/Sir Charles Gairdner Hospital, Centre for Neuromuscular and Neurological Disorders/The University of Western Australia, QEII Medical Centre, Nedlands 6009, Western Australia, Australia. meloni@cyllene.uwa.edu.au
Majda
Bernadette T
BT
Knuckey
Neville W
NW
D000818:Animals; D002545:Brain Ischemia / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy*; D016923:Cell Death / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects* / Q000378:metabolism / Q000503:physiopathology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005333:Fetus; D005947:Glucose / Q000172:deficiency; D018882:Heat Stress Disorders / Q000378:metabolism / Q000503:physiopathology; D007036:Hypothermia, Induced; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology; D019194:Ischemic Preconditioning / Q000379:methods*; D009410:Nerve Degeneration / Q000188:drug therapy* / Q000503:physiopathology / Q000517:prevention & control; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018691:Excitatory Amino Acid Antagonists; D018696:Neuroprotective Agents; D011500:Protein Synthesis Inhibitors; D016194:Receptors, N-Methyl-D-Aspartate; D014409:Tumor Necrosis Factor-alpha; D005947:Glucose