Effectiveness of Carbon-ion Beams for Apoptosis Induction in Rat Primary Immature Hippocampal Neurons

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  • KAMINUMA Takuya
    Department of Radiation Oncology, Gunma University Graduate School of Medicine
  • SUZUKI Yoshiyuki
    Department of Radiation Oncology, Gunma University Graduate School of Medicine
  • SHIRAI Katsuyuki
    Department of Radiation Oncology, Gunma University Graduate School of Medicine
  • MIZUI Toshiyuki
    Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine
  • NODA Shin-ei
    Department of Radiation Oncology, Gunma University Graduate School of Medicine
  • YOSHIDA Yukari
    Gunma University Heavy Ion Medical Center
  • FUNAYAMA Tomoo
    Microbeam Radiation Biology Group, Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency
  • TAKAHASHI Takeo
    Department of Radiation Oncology, Gunma University Graduate School of Medicine
  • KOBAYASHI Yasuhiko
    Microbeam Radiation Biology Group, Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency
  • SHIRAO Tomoaki
    Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine
  • NAKANO Takashi
    Department of Radiation Oncology, Gunma University Graduate School of Medicine

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Abstract

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 in vitro 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 In Vitro (DIV), the cells were irradiated with 140 kV X-ray and 18.3 MeV/amu carbon-ion beams (LET = 108 keV/μm). 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.

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