DETERMINATION OF THE TIME-COURSE AND EXTENT OF NEUROTOXICITY AT DEFINED TEMPERATURES IN CULTURED NEURONS USING A MODIFIED MULTIWELL PLATE FLUORESCENCE SCANNER

The cellular and molecular mechanisms of hypoxic/ischemic neurodegener ation are sensitive to numerous factors that modulate the time course and degree of neuronal death. Among such factors is hypothermia, which can dramatically protect neurons from injury. To examine and control for temperature-dependent effects, we developed a technique that provi des for a high-throughput, accurate, and reproducible determination of the time course and degree of neurotoxicity in cultured cortical neur ons at precisely defined temperatures. We used a fluorescence multiwel l plate scanner, modified by us to permit the control of temperature, to perform serial quantitative measurements of propidium iodide (PI) f luorescence in cortical neuronal cultures exposed to excitotoxic insul ts. In validating this approach, we show that these time course measur ements correlate highly with manual counts of PI-stained cells in the same cultures (r = 0.958, p < 0.0001) and with lactate dehydrogenase r elease (r = 0.964, p < 0.0001). This method represents an efficient ap proach to mechanistic and quantitative studies of cell death as well a s a high-throughput technique for screening new neuroprotective therap ies in vitro.