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

We describe a novel method for 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 x-ray. 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. From 7 to 10 h after irradiation with 5 Gy x-ray, we found two morphologically different types of AO-stained structures, namely, small single nuclei and rosette-shaped nuclear clusters. Electron microscopy revealed that these two distinct types of structures were single apoptotic cells with condensed nuclei and aggregates of apoptotic cells, respectively. From 10 to 30 h after irradiation, a similar AO-staining pattern was observed. The numbers of AO-stained rosette-shaped nuclear clusters and AO-stained single nuclei increased in a dose-dependent manner in the optic tectum. We used the number of AO-stained rosette-shaped nuclear clusters/optic tectum as an index of the degree of radiation-induced brain cell death at 20-24 h after irradiation. The results showed that the number of rosette-shaped nuclear clusters/optic tectum in irradiated embryos exposed to 2 Gy or higher doses was highly significant compared to the number in nonirradiated control embryos, whereas no difference was detected at 1 Gy. Thus, the threshold dose for brain cell death in medaka embryos was taken as being between 1-2 Gy, which may not be so extraordinarily large compared to those for rodents and humans. The results show that medaka embryos are useful for quantitative evaluation of developmental neurocytotoxic effects of radiation.