Abstract
It is important to examine the neurocytotoxic effects of radiation on the developing central nervous system (CNS) because of its high sensitiveness to radiation. Mammalian embryos develop in the uteri, hence, their developmental process cannot be examined directly. In contrast, medaka is an ideal model for studying the effects of developing CNS of vertebrates, because the transparency of their eggs and embryos makes it possible to detect morphological abnormalities in the CNS easily using a conventional stereomicroscope. Moreover, the smaller size of their embryos compared with that of mammalian embryos provides the advantage of easy examination of whole-mount specimens. Wild-type Hd-rR medaka embryos at developmental stage 28, when neural cells proliferate rapidly especially in the optic tectum (OT), were irradiated with 10Gy gamma-rays, which does not affect to the hatchability. Radiation-induced apoptosis were examined in whole-mount specimens using an acridine orange (AO) assay. At 8-10 h after the irradiation, we found distinct two types of AO-stained cells, namely, a single condensed nuclei and rosette-shaped clusters of them. This characteristic staining pattern was continually observed up to 35 h after the irradiation. Electron microscopic observation of irradiated OT at 24 h after the irradiation revealed that rosette-shaped clusters were aggregates of 10-15 apoptotic cells and these aggregated apoptotic cells were phagocytosed inside a single large cell.
In order to prevent the diffusion of damaging degradation products into the surrounding tissue, apoptotic neuronal cells are quickly phagocytosed by a resident immune cell, known as the microglia in the brain. The phagocytic motility of microglia for radiation-induced apoptosis in irradiated developing brain was not well studied. Here, we investigated how microglia act to phagocytose rosette-shaped clusters of apoptotic cells in Hd-rR irradiated brain at 24 h after the irradiation. The gene expressions of apolipoprotein-E (Apo-E) protein, which is specifically expressed in micriglia, determined by whole-mount in situ hybridization revealed higher levels of transcripts in the irradiated embryos especially in the eyes and OT than that of non-irradiated controls. Imaging in 3D of the microglial motility was based on the serial sections of irradiated in situ stained brain by utilizing 3D application tools for computational method.
