J Vet Sci.  2011 Sep;12(3):203-207. 10.4142/jvs.2011.12.3.203.

Cytotoxicity of gamma-ray in rat immature hippocampal neurons

• Yang M
• Song MS
• Kim SH
• Kim JC
• Kim JS
• Shin T
• Moon C

• KMID: 1067390
• DOI: http://doi.org/10.4142/jvs.2011.12.3.203

Abstract

This in vitro study evaluated the detrimental effect of acute gamma (gamma)-irradiation on rat immature hippocampal neurons. Rat immature hippocampal neurons (0.5 day in vitro) were irradiated with 0~4 Gy gamma-rays. Cytotoxicity was analyzed using a lactate dehydrogenase release assay at 24 h after gamma-irradiation. Radiation-induced cytotoxicity in immature hippocampal neurons increased in a dose-dependent manner. Pre-treatments of pro-apoptotic caspase inhibitors and anti-oxidative substances significantly blocked gamma-irradiation-induced cytotoxicity in immature hippocampal neurons. The results suggest that the caspase-dependent cytotoxicity of gamma-rays in immature hippocampal cultured neurons may be caused by oxidative stress.

Keyword

caspase; cytotoxicity; gamma-ray; hippocampal neurons; oxidative stress

MeSH Terms

Amifostine/pharmacology
Animals
Antioxidants/pharmacology
Caspase 3/metabolism/radiation effects
Catechin/analogs & derivatives/pharmacology
Cell Survival/radiation effects
Cells, Cultured/cytology/enzymology/*radiation effects
Dose-Response Relationship, Radiation
Female
*Gamma Rays
Hippocampus/cytology/enzymology/*radiation effects
L-Lactate Dehydrogenase/radiation effects
Neurons/cytology/enzymology/*radiation effects
Poly(ADP-ribose) Polymerases/drug effects
Pregnancy
Rats
Rats, Sprague-Dawley

Figure

• 

  Fig. 1 Double-immunofluorescent images of Ki-67 and nestin (A~D) and doublecortin (DCX) and glial fibrillary acidic protein (GFAP) (E~H) in 0.5 day in vitro-cultured hippocampal neurons. (A) Immunolabeling of Ki-67 (red). (B) Immuno-labeling of nestin (green). (C) 4',6-diamidino-2-phenylindole (DAPI) staining (blue). (D) Merged photograph. (E) Immunolabeling of DCX (red). (F) Immuno-labeling of GFAP (green). (G) DAPI staining (blue). (H) Merged photograph. Scale bars = 30 µm.

• 

  Fig. 2 Dose-dependent pattern of γ-ray-induced cytotoxicity in immature hippocampal neurons. (A) Lactate dehydrogenase (LDH) assay as an indicator of cytotoxicity of γ-rays (0~4 Gy) at 24 h after irradiation in hippocampal neurons of 0.5 DIV. (B and C) Differential interference contrast images revealed that cellular shrinkage and loss in irradiated cells (2 Gy) was increased compared with sham-irradiated controls. The data in (A) represent the mean ± SE, n = 3 cultures per condition. Scale bars = 30 µm.

• 

  Fig. 3 Western blot analysis showing active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) expression in immature hippocampal neurons with γ-irradiation (2 Gy). (A) Representative immunoblots for detection of active caspase-3 (17 kDa), PARP (118 kDa) and cleaved PARP (89 kDa) at 24 h after γ-irradiation (IR, 2 Gy). (B) 2 Gy γ-irradiated cells displayed significant increase of LDH in the medium. However, pre-treatment of the caspase-family inhibitor Z-VAD-FMK or the caspase-3 specific inhibitor Z-DEVD-FMK significantly inhibited release of LDH in the medium of 2 Gy-irradiated cells. The data represents the mean ± SE, n = 3 cultures per condition.

• 

  Fig. 4 The inhibitory effects of anti-oxidative substances on γ-ray-induced cytotoxicity and increase of intracellular reactive oxygen species (ROS) in immature hippocampal neurons. (A and B) Pre-treatments of anti-oxidative substances, amifostine (Ami) and epigallocatechin gallate (EGCG) at 30 min before irradiation significantly inhibited LDH release in immature hippocampal neurons with all of treated doses compared with that in 2 Gy-irradiated controls. (C) Pre-treatments of amifostine (100 µM) and EGCG (10 µM) significantly blocked cellular levels of ROS by 2 Gy-irradiation. The data represent the mean ± SE, n = 3 cultures per condition. *p < 0.05, **p < 0.01 vs. 2 Gy-irradiated controls.

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