Optimization of single-cell gel electrophoresis (SCGE) for quantitative analysis of neuronal DNA damage

Neuronal death can be induced by DNA-damaging agents and occurs by apoptosi s involving a specific signal-transduction pathway. However, to our knowled ge, methods for the quantitative determination of DNA damage in individual neurons have trot yet been described. Here we optimize the single-cell gel electrophoresis (SCGE) or "comet"-assay to measure DNA damage,within indivi dual neurons growing in dissociated cell culture. In addition, Mle have,wri tten a macro for the NIH Image program to determine the tail moment of indi vidual comets. We have calibrated this method using gamma-irradiated (0-16 Gy) cerebral cortical neurons from the rat central nervous system. Neuronal DNA damage (in the form of DNA strand breaks) occurs in a linear; dose-dep endent manner, which can be quantitatively determined in. vitro using the S CGE assay. These data demonstrate that the SCGE assay is an effective metho d to measure DNA damage in individual neurons and may be highly useful for the study of neuronal DNA damage formation, repair and apoptosis.