コメットアッセイにより検出された興奮性神経細胞死における急性DNA断片化

抄録

Excitotoxic neuronal death induced by glutamate is classified into two categories; one is acute cell death which is known as necrosis and the other is delayed cell death which is thought to be apoptosis. In hippocampal neurons, we previously observed within 20 min, small granular structures inside the nuclei, with video-enhanced constract-differential interference contrast microscope, following continuous exposure to 1 mM glutamate. To investigate whether this granulation is the concequence of nuclear DNA fragmentation, we analysed it using Comet assay. This assay is also called single cell gel electrophoresis which is a rapid and sensitive method for detection of DNA damage on the individual cell level. We employed visual scoring to assess DNA damage; grade 1(no damage) to grade 5(severe damge) in this assay. Necrosis induced by high concentration of glutamate for 1 h significantly increased the DNA damage to grade 4, while staurosporine, an apoptosis inducer, increased the incidence of grade 5. The results suggested that: 1) the nuclear granule structure in necrotic process was the result of DNA fragmentation; 2) the sizes of DNA fragments in necrotic process were larger than those in apoptosis. [Jpn J Physiol 55 Suppl:S232 (2005)]