Yasuda Takako, Yoshimoto Masami, Maeda Keiko, Matsumoto Atsuko, Maruyama Kouichi, Ishikawa Yuji
Environmental Radiation Effects Research Group, Research Center for Radiation Protection, National Institute of Radiological Sciences, Chiba, Japan.
J Radiat Res. 2008 Sep;49(5):533-40. doi: 10.1269/jrr.08030. Epub 2008 Jul 24.
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.
我们描述了一种用于快速定量评估青鳉（Oryzias latipes）发育中大脑辐射诱导凋亡程度的新方法。对处于28期的胚胎进行1、2、3.5和5 Gy的X射线照射。将存活的胚胎用活性染料吖啶橙（AO）染色1 - 2小时，然后在落射荧光显微镜下检查整个大脑。在用5 Gy X射线照射后7至10小时，我们发现了两种形态不同的AO染色结构，即小的单核和玫瑰花结状核簇。电子显微镜显示，这两种不同类型的结构分别是核浓缩的单个凋亡细胞和凋亡细胞聚集体。照射后10至30小时，观察到类似的AO染色模式。在视顶盖中，AO染色的玫瑰花结状核簇和AO染色的单核数量呈剂量依赖性增加。我们将照射后20 - 24小时AO染色的玫瑰花结状核簇数量/视顶盖作为辐射诱导脑细胞死亡程度的指标。结果表明，与未照射的对照胚胎相比，接受2 Gy或更高剂量照射的胚胎中玫瑰花结状核簇/视顶盖的数量差异极显著，而在1 Gy时未检测到差异。因此，青鳉胚胎脑细胞死亡的阈值剂量被认为在1 - 2 Gy之间，与啮齿动物和人类相比，这个剂量可能不算特别大。结果表明，青鳉胚胎可用于定量评估辐射对发育中的神经细胞毒性作用。
