Pregnant Sprague-Dawley rats were obtained from a specific pathogen-free colony at Oriental Inc. (Korea). The method used to primary hippocampal neuron culture has been previously described in detail [12]. Briefly, hippocampi were dissected from postnatal day 0 rats. After dissection, tissues were chopped and digested with 10 units/mL papain (Worthington, USA) and 100 units/mL DNase I (Roche, Switzerland) in dissociation buffer at 37℃ for 30 min. The digestion was triturated with Neurobasal A medium (Invitrogen, USA). The cells were seeded at a density of 4 × 10⁶ cells/well on poly-D-lysine hydrobromide (150 µg/mL; Sigma-Aldrich, USA) coated 4-well plates. Two hours after plating, Neurobasal A was replaced with growth medium including Neurobasal A, 1 × B27 supplement (Invitrogen, USA), 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 0.5 mM glutamine.

Hippocampal cultured neurons [0.5 day in vitro (DIV)] on a cover slip were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) in phosphate-buffered saline (PBS, pH 7.4) for 20 min at room temperature. The cells were blocked with 2% normal goat serum and 1% bovine serum albumin, allowed to react with rabbit monoclonal anti-Ki-67 (DRM004, 1 : 200; Acris Antibodies GmbH, Germany) and rabbit polyclonal anti-doublecortin (DCX) antibody (1 : 400; Cell Signaling Technology, USA) overnight at 4℃, and incubated with tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit IgG (1 : 50; Sigma-Aldrich, USA) for 1 h at room temperature. The cells were incubated with either mouse monoclonal anti-nestin (1 : 200; Chemicon International, USA) or mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1 : 500; Sigma-Aldrich, USA) for 1 h at room temperature, and then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1 : 50; Sigma-Aldrich, USA) for 1 h at room temperature. For counterstaining, 1 mg/mL 4',6-diamidino-2-phenylindole 2HCl (DAPI; Thermo Fisher Scientific, USA) in PBS was added and incubated at room temperature for 5 min. The immunofluorescence-stained specimens were examined using a ProgRes CFscan fluorescence digital camera (Jenoptik, Germany).

Cultured neurons at 0.5 DIV were irradiated with 0, 0.5, 2, or 4 Gy of ¹³⁷Cs-generated γ-rays using a Gamma-cell Elan 3000 (Nordion International, Canada). The mean dose rate of the γ-rays was 3.1 Gy/min. Control samples were sham-irradiated with 0 Gy. The cells were assayed at 24 h post-irradiation. To evaluate the radioprotective effects of anti-oxidative substances, including amifostine (0~100 µM; a representative agent of radioprotactants; Sigma-Aldrich, USA) and epigallocatechin gallate (EGCG, 0~10 µM; the main ingredient of green tea polyphenols; Sigma-Aldrich, USA) on immature hippocampal neurons, both substances were pre-treated at 30 min before irradiation.

Cytotoxicity was evaluated using a lactate dehydrogenase (LDH) release assay. A commercially available LDH-cytotoxicity assay kit from Biovision (USA) was used as recommended by the manufacturer. The optical density values were quantified by an Emax microplate reader (Molecular Devices, USA) for absorbance at a wavelength of 490 nm. To monitor intracellular accumulation of ROS, the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA; Invitrogen, USA) was used. Intracellular ROS production was quantified by a fluorescent microplate reader with excitation at 485 nm and emission at 535 nm.

Medium was removed and sodium dodecyl sulfate (SDS) sample buffer (4×) was added to each culture. The cells were scraped and sonicated for 10 sec. The samples were heated to 100℃ for 10 min. The samples were then separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the resolved proteins were transferred to a nitrocellulose membrane and blocked with 1% normal goat serum and 0.5% bovine serum albumin in PBS-T (PBS, 0.1% Tween 20) for 30 min at room temperature. The membranes were then incubated with a 1 : 1,000 dilution of either rabbit anti-cleaved (active) caspase-3 antibody (Cell Signaling Technology, USA) or rabbit anti-poly (ADP-ribose) polymerase (PARP) antibody (Cell Signaling Technology, USA) in PBS-T overnight at 4℃. After extensive washing and incubation with a 1 : 10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories, USA) for 1.5 h at room temperature, the signals were visualized using chemiluminescence methods (SuperSignal West Pico; Thermo Fisher Scientific, USA). For normalization purposes, the membranes were reprobed with a 1 : 20,000 dilution of β-actin antibody (Sigma-Aldrich, USA). Several exposure times were used to obtain the signals in the linear range. The bands were quantified using Scion Image Beta 4.0.2 for Windows XP software (Scion, USA).

The data are reported as the mean ± SE. The data was analyzed using one-way ANOVA followed by a Student-Newman-Keuls post hoc test for multiple comparisons. In all cases, a p value < 0.05 was considered significant.

To confirm the cell phenotypes of 0.5 DIV hippocampal cultured cells, double-immunofluorescent staining was performed. We first detected Ki-67, nestin, DCX and GFAP, which are immunohistochemical markers for proliferating cells, neural stem cells, immature progenitor neurons and astrocytes, respectively, in 0.5 DIV hippocampal cells. The double-immunofluorescent results showed that Ki-67-positive cells were co-localized to nestin immunoreactivity (approximately 15% of total cells; Figs. 1A~D), but DCX-positive cells (approximately 80% of total cells) were rarely matched with GFAP immunoreactivity (< 5% of total cells; Figs. 1E~H) in 0.5 DIV hippocampal cells.

The cytotoxicity of γ-irradiation in immature hippocampal cultured neurons was estimated 24 h after irradiation by 0~4 Gy by the LDH release assay (Fig. 2). The cytotoxicity increased in a dose-dependent pattern within the range of irradiation applied.

To determine whether radiation-induced cell death in immature hippocampal neurons was associated with the caspase-dependent pathway, caspase-3 activation and caspase-specific PARP cleavage were assessed by Western blot and the effects of caspase inhibitors on radiation-induced cytotoxicity were examined by LDH release assay (Fig. 3). Active caspase-3 and cleaved PARP markedly increased in immature hippocampal neurons at 24 h after 2 Gy γ-irradiation (Fig. 3A). However, both the caspase-family inhibitor Z-VAD-FMK and the caspase-3 specific inhibitor Z-DEVD-FMK significantly blocked LDH release from γ-irradiated immature hippocampal neurons (Fig. 3B).

We further examined whether anti-oxidative substances, including amifostine (well-known as a representative radioprotective agent [8]) and EGCG could inhibit the cytotoxicity of γ-rays in immature hippocampal neurons. Pre-treatment of amifostine and EGCG significantly blocked increase of LDH release (Figs. 4A and B) and intracellular ROS levels (Fig. 4C) caused by 2 Gy-irradiation.