Rapid and simple method for quantitative evaluation of neurocyototoxic effects of radiation on developing medaka brain

We describe a novel method of rapid and quantitative evaluation of the degree of radiation-induced apoptosis in the developing brain of medaka (Oryzias latipes). Embryos at stage 28 were irradiated with 1, 2, 3.5, and 5 Gy of X-rays. Twenty to twenty-four hours after the irradiation, living embryos were stained with a vital dye, acridine orange (AO), for 1-2 h, and whole-mount brains were examined under an epifluorescence microscope. The numbers of AO-stained rosette-shaped nuclear clusters and AO-stained single nuclei increased in a dose-dependent manner in the optic tectum. Electron microscopy revealed that the AO-stained rosette-shaped nuclear clusters were the nuclei of aggregated apoptotic cells. We used the number of AO-stained rosette-shaped nuclear clusters/tectum as an index of the degree of radiation-induced brain cell death. The results showed that the number of rosette-shaped nuclear clusters/optic tectum in irradiated embryos exposed to higher than 2 Gy was highly significantly different from that in nonirradiated control embryos, whereas no difference was detected at 1 Gy. Thus, the threshold dose for brain cell death in medaka embryos is about 1 Gy and this dose is probably comparable to those for rodents and humans. The results show that medaka embryos are useful for quantitative evaluation of developmental neurocytotoxic effects of radiation