Determination of the Time Course and Extent of Neurotoxicity at Defined Temperatures in Cultured Neurons Using a Modified Multiwell Plate Fluorescence Scanner

• Published in: Journal of cerebral blood flow and metabolism Vol. 17; no. 4; pp. 455 - 463
• Main Authors: Sattler, Rita, Charlton, Milton P., Hafner, Mathias, Tymianski, Michael
• Format: Journal Article
• Language: English
• Published: London, England SAGE Publications 01.04.1997; Lippincott Williams & Wilkins
• Subjects: Animals; Biological and medical sciences; Cell Survival; Cells, Cultured; Cerebral Cortex - cytology; Cerebral Cortex - drug effects; Cerebral Cortex - enzymology; Coloring Agents; Degeneration. Regeneration. Wound healing. Graft; Dose-Response Relationship, Drug; Fluorescence; Fundamental and applied biological sciences. Psychology; Glutamic Acid - pharmacology; L-Lactate Dehydrogenase - metabolism; Mice - embryology; Neurons - cytology; Neurons - drug effects; Neurons - enzymology; Neurotoxins - pharmacology; Osmolar Concentration; Propidium; Temperature; Time Factors; Vertebrates: anatomy and physiology, studies on body, several organs or systems; Cytotoxicity assay; Propidium iodide; Neurotoxicity; Cell death; Excitotoxicity; Hypothermia; Cell culture; Vertebrata; Temperature; Mammalia; Neuron; Mouse; Animal; Rodentia; Fluorescence; Exploration; In vitro
• ISSN: 0271-678X; 1559-7016
• DOI: 10.1097/00004647-199704000-00011

The cellular and molecular mechanisms of hypoxic/ischemic neurodegeneration 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 provides for a high-throughput, accurate, and reproducible determination of the time course and degree of neurotoxicity in cultured cortical neurons at precisely defined temperatures. We used a fluorescence multiwell plate scanner, modified by us to permit the control of temperature, to perform serial quantitative measurements of propidium iodide (PI) fluorescence in cortical neuronal cultures exposed to excitotoxic insults. In validating this approach, we show that these time course measurements correlate highly with manual counts of PI-stained cells in the same cultures (r = 0.958, p < 0.0001) and with lactate dehydrogenase release (r = 0.964, p < 0.0001). This method represents an efficient approach to mechanistic and quantitative studies of cell death as well as a high-throughput technique for screening new neuroprotective therapies in vitro.