Mechanical injury to neuronal/glial cultures in microplates: role of NMDA receptors and pH in secondary neuronal cell death.

Abstract

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.