Effectiveness of carbon-ion beams for apoptosis induction in rat primary immature hippocampal neurons

Kaminuma, Takuya*; Suzuki, Yoshiyuki*; Shirai, Katsuyuki*; Mizui, Toshiyuki*; Noda, Shinei*; Yoshida, Yukari*; Funayama, Tomoo; Takahashi, Takeo*; Kobayashi, Yasuhiko; Shirao, Tomoaki*; Nakano, Takashi*

The direct biological effects of radiation, particularly accelerated heavy particle ions, on neurons are not fully known. Hence, the direct effect of carbon-ion beams on immature neurons was investigated by comparing to the effect of X-rays using primary hippocampal neurons. Primary neurons were prepared from hippocampi of fetal rats at embryonic day 18 from timed pregnant Wistar rats and cultured with Banker's methods. At 7 Days (DIV), the cells were irradiated with 140 kV X-ray and 18.3 MeV/amu carbon-ion beams (LET = 108 keV/m). The cells were fixed with 4% paraformaldehyde at 12 hours after irradiation. Then, the cells were treated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and DAPI staining for measuring the percentage of apoptosis (apoptotic index: AI). AI in sham-irradiated hippocampal neurons was 18%. The value of AI (AIs) of the cells irradiated with X-rays at 10 or 30 Gy were 15% or 23%, respectively. AI in cells irradiated with carbon-ion beams at 1 Gy, 3 Gy, 5 Gy and 10 Gy were 22%, 23%, 24% and 33%, respectively. AI was significantly increased by carbon-ion beams at 10 Gy (p 0.001). The apoptosis of hippocampal neurons increased in a dose-dependent manner following both X-ray and carbon-ion beams irradiation. Carbon-ion beams were about 10-fold more effective than X-rays for apoptosis induction in immature hippocampal neurons.