Mechanical injury to neuronal/glial cultures in microplates: role of NMDA receptors and pH in secondary neuronal cell death.
In vitro models of traumatic injury are useful adjuncts to animal models for studying mechanisms of post-traumatic cell death. Here we describe a new in vitro model in which reproducible levels of injury are delivered by a punch device that produces 28 parallel cuts in individual wells of 96-well microplates. Cell loss is measured by LDH assay or quantitative fluorometric assay for ethidium homodimer staining. Glial cultures show cell death restricted to the initial injury site, whereas neuronal/glial cultures demonstrate substantial spread of cell loss over time. We used this model to examine the role of pH and NMDA receptors in delayed post-traumatic injury. NMDA receptor blockade by dizocilpine (MK-801) or treatment with antisense oligodeoxynucleotides directed against NMDAR1 was neuroprotective. Decreased cell death was observed under acidic conditions whereas increased extracellular pH was associated with increased, MK-801 sensitive cell loss. Advantages of our model include: reproducible trauma induction; rapid measurements of cell injury; and use of 96-well microplates which reduce time and cost. This model appears to be well-suited for the study of selected mechanisms of post-traumatic neuronal injury as well as for screening potential neuroprotective agents.
Mukhin
A G
AG
Institute for Cognitive and Computational Sciences, Georgetown University Medical Center, Washington, DC 20007, USA.
Ivanova
S A
SA
Allen
J W
JW
Faden
A I
AI
D000818:Animals; D000831:Animals, Newborn; D016923:Cell Death; D002470:Cell Survival; D002478:Cells, Cultured; D002540:Cerebral Cortex; D018920:Coculture Techniques; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004622:Embryo, Mammalian; D006863:Hydrogen-Ion Concentration; D009457:Neuroglia / Q000473:pathology*; D009474:Neurons / Q000473:pathology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D016291:Dizocilpine Maleate