Male ICR mice, 6 months of age were housed in a temperature-controlled animal room with a 12-h light-dark cycle and allowed access to food and water ad libitum. All experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (USA), and were approved and monitored by the Ethics Committee of Animal Experiments at Chungnam National University, Daejeon, Korea. Prior to the experiments, the mice were left undisturbed for 7 days and were randomly assigned to 1 of 4 experimental groups: i) the saline (0.9% NaCl)-treated group (n=16); ii) the group treated with 5 mg/kg Rh₁ (n=16); iii) the group treated with 10 mg/kg Rh₁ (n=16); and iv) the behavioral test group (n=30, passive avoidance test; n=30, water maze test). The behavioral test group was subdivided into 3 groups (control, 5 mg/kg Rh₁ and 10 mg/kg Rh₁; 10 mice per group) and the other 3 groups were subdivided into 2 groups (n=8 per group) for the evaluation of neurogenesis and cell survival in the hippocampus.

Mice in the saline-treated group (0.9% NaCl) and the ginsenoside (FuSong County Natural Biotechnology, Co., Ltd., Fusong, China)-treated groups (Rh₁, 5 and 10 mg/kg body weight) were orally administrated saline and ginsenoside, respectively for a period of 3 months. Rh₁ doses were converted between adult human (60 kg) and mouse (20 g) body weights, using the body surface area normalization method, as previously described (20). The selected doses corresponded to 2–3 and 5–6 g of ginseng per day in an adult human (60 kg body weight). Considering that the long-term intravenous administration of ginsenosides would cause inflammation or anxiety, we selected oral administration, even though this would be associated with less bioactivity.

The thymidine analogue, BrdU, is incorporated into the DNA of dividing cells and can be detected immunohistochemically in their progeny. The behavioral tests were performed at the end of the drug administration. To determine the effects of ginsenoside Rh₁ on neurogenesis, BrdU (100 mg/kg body weight) was administered to the mice twice per day for 3 consecutive days prior to sacrifice; to determine the effects of ginsenoside Rh₁ on cell survival, BrdU was administered to the mice twice per day for 3 consecutive days (20 days prior to sacrifice) during the the 3rd month of the treatment period. Following the evaluation of neurogenesis and cell survival, the animals (8 per group) were sacrificed, the brains were excised and the brain tissue was then subjected to immunohistochemical and protein expression analysis. The overall experimental protocol is presented in Fig. 1.

Mice from the different groups were subjected to a Morris water maze test for 5 consecutive days in the terminal phase of the administration process. The escape platform (diameter, 10 cm; height, 24 cm) was hidden 1 cm below the surface of the water, which had been made opaque by the addition of non-toxic black paint. Each animal was subjected to 4 experimental trails per day, each lasting 60 sec and each time commencing from 4 different starting points that randomly varied each day. If an animal was not able to find the platform it was manually place on it at the end of the trail. The animals were allowed to rest on the platform for 15 sec. A probe test was performed on day 6.

The passive avoidance test was performed in identical compartments. The illuminated compartment (20×20×20 cm) contained a 100 W bulb, and the floor of the non-illuminated compartment (20×20×20 cm) was composed of 2 mm stainless steel rods at 1 cm intervals. These 2 compartments were separated by a guillotine door (5×5 cm). For the acquisition trials, the mice were initially placed in the illuminated compartment and the door was opened 15 sec later. When the mouse entered the non-illuminated compartment, the door was closed and an electrical foot shock (0.5 mA) of 3 sec in duration was delivered through the stainless steel rods. Twenty-four hours after the acquisition trial, the mice were again placed in the illuminated compartment for the retention trials. The time taken for a mouse to enter the non-illuminated compartment after the opening of the door was termed as the step-through latency time in the retention trials. If a mouse did not enter the non-illuminated compartment within 180 sec, it was assumed that the mouse had remembered the single training trial.

The mice were sacrificed and the brains were removed after the final BrdU injection. The brains were fixed in 4% phosphate-buffered paraformaldehyde for 12 h. The brain tissues were then embedded in paraffin and cut into sections. The sections were mounted on glass slides and stored overnight at 42°C. Following deparaffinization with xylene and rehydration in a graded series of ethanol, the sections were rinsed in 0.01 M phosphate-buffered saline (PBS). BrdU is a widely used S-phase marker of neurogenesis.

For BrdU-immunostaining, the sections were hydrolyzed with 2 N hydrochloride (HCl) in PBS (pH 7.4) at 37°C for 15 min, and then stained using the Invitrogen BrdU staining kit (Invitrogen, Carlsbad, CA, USA). The sections were incubated in serum blocking solution, in a 1:50 dilution of a mouse monoclonal antibody against BrdU (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and incubated overnight at 4°C in biotinylated secondary antibody at room temperature for 30 min, and finally in streptavidin-peroxidase conjugate at room temperature for 20 min. After each step, the sections were rinsed with PBS. The sections were then incubated in 3,3′-diaminobenzidine (DAB) solution. Subsequently, the sections were incubated in 1% ferric chloride solution at room temperature for 5 min. BrdU-positive nuclei exhibited deposits of dark brown or black-colored precipitates. The sections were counterstained with hematoxylin and cover-slipped with histomount.

Every 10th section throughout the hippocampus was processed for BrdU immunohistochemistry. Using this spacing ensures that the same neuron will not be counted in two sections. All BrdU-labeled cells in the dentate gyrus (granule cell layer) and hilus were counted in each section. To distinguish single cells within clusters, all counts were performed at ×400 magnification under a light microscope (Olympus BX-41; Olympus, Tokyo, Japan), omitting cells in the outermost focal plane. A cell was counted as being in the SGZ of the dentate gyrus if it was touching or in the SGZ. Cells that were located more than two cells away from the SGZ were classified as hilar. The total number of BrdU-labeled cells per section was determined and multiplied by 6 to obtain the total number of cells per dentate gyrus.

The mice were anesthetized and decapitated after 3 months of treatment. The hippocampus dissected from each animal was homogenized ultrasonically in protein extraction buffer (PRO-PREP™ 17081; iNtRON Biotechnology, Seongnam, Korea). The supernatant was collected after centrifugation at 15,000 rpm for 30 min at 4°C. Following quantification, the samples (20 μg protein per lane) were subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 15% gel and electrophoretically transferred onto PVDF membranes (Millipore, Billerica, MA, USA) using a trans-blot device (Bio-Rad, Hercules, CA, USA) at a 15 V constant current overnight at 4°C. The PVDF membranes were soaked in 5% skim milk in PBS solution for 2 h at room temperature to block non-specific binding, rinsed in PBST, and incubated with a rabbit polyclonal anti-SNAP-25 antibody (diluted 1:300 in 5% skim milk in TBST; Santa Cruz Biotechnology, Inc.) overnight at 4°C. The membranes were then washed 3 times for 10 min each in PBST and incubated for 2 h with a secondary antibody, goat anti-rabbit IgG (1:10,000; Santa Cruz Biotechnology, Inc.). After washing twice for 15 min in PBST, the signal was detected using an ECL system. Western blot analysis for β-actin was performed using the same procedure using a goat polyclonal anti-actin antibody (1:1,000; Santa Cruz Biotechnology, Inc.) as the primary antibody. The blots were quantified using image analysis software (ImageJ). Bond intensity values were expressed as a percentage of the control average.

Data are expressed as the means ± SEM. Statistical differences were assessed by one-way analysis of variance (ANOVA) using repeated measures where appropriate. The post-hoc Duncan’s test was carried out where appropriate. The level for a statistically significant difference was set at P<0.05.

For the Morris water maze, repeated ANOVA (time × group) revealed a significant effect decrease in escape latency (in days) following treatment with Rh₁ [5 mg/kg, F(2,24)=3.72, P=0.067; 10 mg/kg, F(3,42)=11.65, P<0.0001].

In fact, all groups, including the control group showed a general decrease in overall latency throughout the acquisition phase (Fig. 2A).

In the probe tests, the group treated with 10 mg/kg ginsenoside Rh₁ showed a significant increase in the number of crosses (number of times mouse crosses the platform location) in the target quadrant (P<0.05), whereas the group treated with 5 mg/kg ginsenoside Rh₁, did not show statistically significant results (P>0.48) compared with the control group (Fig. 2B).

ANOVA for the time spent in the platform quadrant yielded significant results for the groups treated with Rh₁ [5 mg/kg, F(1,14)=13.62, P=0.071; 10 mg/kg, F(1,14)=17.85, P<0.002]. Post-hoc comparisons revealed that a general increase in the time spent in the target quadrant throughout the acquisition phase (Fig. 2C).

For the passive avoidance tests, no differences were observed among all the groups in the step-through latency during the acquisition trials (Fig. 3). ANOVA for the step-through latency during the retention trials revealed significant differences among the groups treated with Rh₁ [5 mg/kg, F(1,16)=0.59, P<0.031; 10 mg/kg, F(1,16)=0.40, P<0.01].

The animals were administered ginsenoside Rh₁ for 3 months and sacrificed after the final BrdU injection. Analysis of the number of BrdU-labeled cells demonstrated that the long-term administration of ginsenoside Rh₁ had no statistically significant effect on the number of BrdU-positive cells in the dentate gyrus (5 mg/kg, P>0.30; 10 mg/kg, P>0.47) (Fig. 4A).

To specially determine the effects of long-term ginsenoside Rh₁ administration on cell survival, a BrdU injection was administered on the 1st day of the 3rd month. ANOVA revealed an overall significant effect in the number of BrdU-positive cells [F(2,18)=51.87, P<0.0003], and post-hoc tests revealed that treatment with 10 mg/kg Rh₁ yielded significant results (P<0.05) compared with the control group (Fig. 4B). The results of BrdU immunohistochemistry are presented in Fig. 5. These results suggest that the long-term administration of Rh₁ increased cell survival in the hippocampus.

The mice were sacrificed and BDNF protein expression was quantified by western blot analysis. BDNF density was measured in the hippocampus (Fig. 6). BDNF density in the control group was 100±2.8% and in the groups treated with 5 mg/kg and 10 mg/kg Rh₁ was 103.2±2.4 and 112±3.7% of the control, respectively. Treatment with 10 mg/kg Rh₁ yielded statistically significant results compared with the control group (P<0.05).