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, (C) 1998 Wiley-Liss, Inc.