Astronauts exploring space beyond a low Earth orbit are exposed to cosmic ray flux including high-LET radiation, which has a greater biological effect on tissues than low-LET radiation. The United States National Research Council's Space Studies Board has concluded that further experimentation is essential to determine if CNS damage is a significant risk in terms of estimating health risks for crew members (Setlow 1999
). We have examined the acute biological effects, especially those concerning apoptosis, after exposure of the developing OT to high-LET iron-ions, using intact vertebrate medaka embryos.
In our previous studies, we developed a simple and quick AO-staining assay to detect and quantify apoptotic cells in the irradiated OT (Yasuda et al. 2008
). The response of radiation-induced apoptosis that was observed after exposure to iron-ions using the AO-staining method was similar to that observed after exposure to X-rays in our previous study with respect to its characteristic morphology and position (Yasuda et al. 2008
). Thus, AO-stained rosette-shaped clusters were observed particularly in the marginal OT area (see Results). An in vivo study in rodents showed that the extent and area of acute cell death in the ventricular area of fetal rat brain observed after exposure to 1.5 Gy carbon-ions were similar to those after exposure to 2.0-2.5 Gy X-ray irradiation (Inoue et al. 2000
). Furthermore, an in vitro study of neuronal cells showed that changes in morphological characteristics such as apoptosis and cell viability after exposure to carbon-ions were not significantly different from those after exposure to X-rays (Al-Jahdari et al. 2009
). These in vivo and in vitro findings showed that the kinds of neurocytotoxic effects on developing neurons resulting from exposure to high-LET radiation do not differ from those resulting from exposure to low-LET X-ray irradiation. Neuronal apoptosis in the irradiated developing OT increased in a dose-dependent manner after exposure to both iron-ions and X-ray radiation, as seen in our previous study (). However, the quantitative difference between them was significant. The threshold dose for developmental neurocytotoxic effects after exposure to iron-ions was determined as being 0.2-0.5 Gy, which is a lower dose than that for X-ray-induced damage, as was shown in our previous study of irradiated embryos determined at 1-2 Gy (Yasuda et al. 2008
). This finding indicates that prenatal exposure to the iron-ion radiation induced higher developmental neurocyototoxic effects on the developing CNS than did X-ray irradiation.
Almost all the available data indicate that RBE values in vitro and in vivo for cell killing, mutations, and cancer induction in animals increase with LET to a value in the neighborhood of 2-4 at LET values around 100-200 keV/μm (National Council on Radiation Protection and Measurements [NCRP] 1989
, Setlow 1999
). However, high-LET radiation has a large uncertainty depending on the applied charged-particle species, the type of tissue used and its maturation, and the end-point used. One investigation of the effects of high-atomic number nuclei on tumor induction in the Harderian gland in mice indicated an RBE of 20-40 at high values of LET (Alpen et al. 1993
The RBE value of iron-ions with an LET of 200 keV/μm in the present study for the induction of apoptosis in the developing OT was estimated to be 3.7-4.2, which was within the range of values reported previously (NCRP 1989
, Setlow 1999
). An in vitro study of human neuronal progenitor cells (Netra 2) indicated that the RBE value of iron-ions with an LET of 148 keV/μm for the induction of apoptosis was 3.4 (Guida et al. 2005
). Both in vivo and in vitro findings showed that exposure to iron-ion radiation induces higher neurocytotoxic effects on the developing brain than low-LET X-rays.
Even though a large number of apoptotic cells were induced in the iron-ion irradiated OT at 24 h after irradiation, histological examination during the hatching period (stage 39, 6-7 days after irradiation) showed no abnormalities in the CNS ( & ). Our present study demonstrated that the iron-ion irradiated embryos could overcome the radiation-induced damage completely during their development, which is in agreement with our previous findings in X-ray irradiated embryos (Yasuda et al. 2009
). An in vivo experiment with adult rodents showed that neural precursor cells, immature neurons, in the hippocampal dentate gyrus undergo apoptosis shortly after iron-ion irradiation with 1-3 Gy in a dose-dependent manner (Rola et al. 2004
). Furthermore, no recovery from neuronal damage was observed 3 months after exposure and damage worsened with time up to 9 months later (Rola et al. 2008
). This report from rodent studies indicated that high-LET radiation has a significant and long-lasting effect on the neurogenesis. This is obviously in contrast to our present results, which demonstrate a recovery from neuronal damage during development up to hatching. This contradiction is believed to arise for the following two reasons. First, as the radiosensitivity of medaka is much lower than that of mammals (Abrahamson et al. 1973
, Ishikawa and Hyodo-Taguchi 1997
), the detrimental effects of 1.5 Gy iron-ions on medaka embryos would be smaller than the effects of 1-3 Gy iron-ions on mice. Second, because our present study demonstrated that radiation effects on the CNS of embryo but not on adult fish, the capacity for eliminating damaged neuronal cells and regenerating them with neuronal progenitor cells might be superior (Kriegstein and Alvarez-Buylla 2009
) than that seen in 2.5-month-old adult mice (Rola et al. 2008
). For eliminating the neuronal damaged cells, it is essential that the dying neurons are quickly phagocytosed by microglia which are resident immune cells of the CNS (Kettenmann 2007
, Peri and Nusslein-Volhard 2008
). It has been reported in zebrafish embryo that microglia in the embryonic brain at steady and healthy state showed a surprisingly swift wandering behavior (Herbomel et al. 2001
). This unexpected behavior of restlessly wandering suggest that microglia in embryonic brain may be constantly patrolling for immune and possibly also developmental and trophic surveillance. This unique behavior of microglia in embryonic brain would result in superior capacity of eliminating damaged neuronal cells in contrast to the adult organism, however, we need further studies about behavior of microglia in irradiated embryonic brain to warrant the capacity of eliminating radiation-induced neuronal damages.
Although histological examinations at the hatching period (stage 39, 6-7 days after irradiation) showed no abnormalities in the CNS in our present study, it is possible that subtle structural changes in the CNS that cannot be detected by histological examination were manifested as behavioral alterations later in life. Behavioral tests are non-invasive measures for the study of alterations after prenatal radiation exposure, and are a sensitive indicator of teratogenic activity (Pecaut et al. 2004
, Wang et al. 2007
). Experimental data showed that the central dopaminergic system and behaviors mediated by this system are disrupted in iron-ion irradiated rats, as such irradiation induced cognitive declines in spatial learning and memory (Rabin et al. 2000
, Shukitt-Hale et al. 2000
). These adverse behavioral and neuronal effects are similar to those seen in aged animals, which might be related to an increase in the release of reactive oxygen species (Shukitt-Hale et al. 2000
). Moreover, these cognitive declines are associated with specific areas of brain signaling deficits, such as synaptic vesicle proteins, which are important in cognition (Denisova et al. 2002
). If these decrements in behaviors also occur in humans, they may impair the ability of astronauts to perform critical tasks. Further investigation to elucidate the effects of embryonic iron-ion irradiation on behaviors found later in adult medaka is warranted in future studies.
To our knowledge, this present study is the first report regarding the effects of high-energy iron-ions on the embryonic brain in vivo using medaka, or any other intact vertebrate, that are relevant to the aerospace radiation environment. Our present results clearly indicate that the AO-staining method is a useful tool for quantifying apoptosis in the developing CNS after exposure to high- and low-LET radiation. Thus, medaka embryos are a useful model for investigating embryonic neuronal damage associated with high- and low-LET radiation.