Male Sprague-Dawley rats weighting 250~280 g each were purchased from Samtaco Animal Corp. (Kyungki-do, Korea). The animals were allowed to acclimatize themselves for at least 7 days prior to the experimentation. The animals were housed in individual cages under light-controlled conditions (12/12-hr light/dark cycle) and at a 23℃ room temperature. Food and water were available ad libitum. All the experiments were approved by the Kyung Hee University Institutional animal care and use committee. The rats were allowed at least 1week to adapt to their environment before the experiments.

All the 6-year Korean white and red ginseng roots (P. ginseng C.A. Meyer) were purchased from the largest Korean ginseng market of Geumsan country (Chungcheong Province, South Korea). BG was manufactured by repeatedly steaming WG at 98℃ for 3 h in a pottery apparatus and drying it at 60℃ for 15 h. The dried BG samples (40 g) were immersed in a 200 ml of dH₂O and added 2 L of 80% ethanol and then mixed 500 ml of dH₂O. For the extraction, diethyl ether was used three times.

Focal cerebral ischemia was induced using the intraluminal filament technique. Anesthesia was administered with 3% isoflurane in 30% O₂/70% N₂O, and it was maintained throughout the operation with 0.5~0.6% isoflurane delivered via a nose mask. The right common carotid artery was exposed through a midline cervical incision. A heparinized intraluminal filament (φ0.28 mm, rounded tip) was introduced via the external carotid artery. The rectal temperature was monitored and maintained at 37℃ using a heating pad (Harvard Homeothermic Blanket Control Unit, 50-7061). After 120 min of occlusion, the filament was gently pulled out and the external carotid artery was permanently closed by cauterization. In the sham-operated rats, the right common carotid artery was exposed and the external carotid artery was opened without introducing the filament into the internal carotid artery. After the operation, the animals were allowed to wake up in the incubator (30℃) and they were then moved to their home cages. The rats were divided into four groups. The experimental group was treated with BG100 [100 mgkg^-1, p.o., BG100+ISCH group (n=6)] and BG400 [400 mgkg^-1, p.o., BG400+ISCH group (n=6)] for 2 weeks after the induction of ischemia. The control group was treated with saline [0.1 mg kg^-1, p.o., SAL+ISCH group (n=6)] for 2 weeks after the induction of ischemia. The black ginseng and saline were administered orally everyday in the morning. The naïve normal group (Normal, n=6) was not treated with any drug and operation. The water maze tests were performed the last (second) week.

The swimming pool of the Morris water maze was a circular water tank 200 cm in diameter and 35 cm deep. It was filled to a depth of 21 cm with water at 23±2℃. A platform 15 cm in diameter and 20 cm in height was placed inside the tank with its top surface being 1.5 cm below the surface of the water. The pool was surrounded by many cues that were external to the maze [18]. A CCD camera was equipped with a personal computer for the behavioral analysis. Each rat was received four daily trials. For 6 consecutive days, the rats were tested with three acquisition tests. For the acquisition test, the rat was allowed to search for the hidden platform for 180 s and the latency to escape onto the platform was recorded. The animals were trained to find the platform that was in a fixed position during 6 days for the acquisition test. The inter-trial interval time was 1 min. The performance of the test animals in each water maze trial was assessed by a personal computer for the behavioral analysis (S-mart program, Spain).

Briefly, the rats were anesthetized (sodium pentobarbital, 100 mg/kg, ip), they were perfused transcardially with heparinized phosphate-buffered saline (PBS; pH 7.4) for 30 s and this was followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10~15 min. The brains were postfixed in the same fixative overnight, cryoprotected in 30% sucrose solution in PBS, embedded and serially sectioned on a cryostat (Leica, Germany) at 30µm thickness in the coronal plane and then they were collected in PBS. One set of sections that represented different region of the cortex was dehydrated, rehydrated and stained with cresyl violet (ICN Biomedicals, Aurora, USA) to assess the cell loss and the morphology of the hippocampal area.

The sections were stained for NADPH-d activity as previously described [19,20]. In brief, free-floating sections were incubated at 37℃ for 30 min in 0.05 M PBS containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium (Sigma, MO, USA) and 0.1 mg/ml β-NADPH (Sigma). The sections were washed three times with 0.05 M PBS and then they were mounted on gelatin-coated slides. The slides were air-dried overnight at room temperature, rinsed twice with distilled water and dried again. Coverslips were mounted using permount solution. The brain sections were analyzed using the atlas by Paxinos and Watson. The staining intensities of the sections that stained specifically for NADPH-d were quantitatively assessed according to a microdensitometrical method based on optical density and using an image analyzer (Multiscan, Fullerton, CA, USA) [21].

The primary antibodies against the following specific antigen were used: cholineacetyl transferase (sheep polyclonal ChAT, concentration 1:2,000; Cambridge Research Biochemicals, Wilmington, D.E.). The primary antibody was prepared at a dilution in 0.3% PBST, 2% blocking serum and 0.001% kehole limpit hemocyanin (Sigma, USA). The sections were incubated in the primary antiserum for 72 h at 4℃. After three more rinses in PBST, the sections were placed in Vectastain Elite ABC reagent (Vector laboratories, Burlingame, CA) for 2 h at room temperature. Following a further rinsing in PBS, the tissue was developed using diaminobenzadine (Sigma, USA) as the chromogen. Images were captured using a DP2-BSW imaging system (Olympus, CA, USA) and they were processed using Adobe Photoshop. For measuring cells that were immunoreactive for ChAT, a grid was placed on the CA1 and CA3 areas of the hippocampus according to the method of Paxinos et al. [21]. The number of cells was counted at 100× magnification using a microscope rectangle grid that measured 200×200 mm². The cells were counted in three sections per rat within the hippocampal CA1 and CA3 areas.

Statistical comparisons were done for the behavioral and histochemical studies using one-way ANOVA and repeated measures of ANOVA, respectively, and Tukey post hoc was done. All of the results are presented as means±S.E.M., and we used SPSS 15.0 for Windows for analysis of the statistics. The level of significance was set at p<0.05.

The results of the evaluations of the cresyl violet positive neurons per section, from different hippocampal formations, are shown in Fig. 2. Post-hoc comparisons indicated that the cresyl violet activity in the hippocampus of the SAL+ISCH group was significantly lower than that of the normal group (p<0.05). In particular, there were significant differences in both the CA1 (F_3,37=17.2, p=0.001) and CA3 (F_3,37=10.6, p=0.001) areas. However, the number of cresyl violet positive neurons in the BG100+ISCH group was higher than that of the SAL+ISCH group, and particularly in the CA1 (p=0.001) and CA3 (p=0.001) areas. However, the number of cresyl violet positive neurons in the hippocampus of the BG100+ISCH group was not significantly different compared to that of the SAL+ISCH group, but there was a slight trend for a significant interaction effect on the expression of the cresyl violet positive neurons in the hippocampus. We also found that the tissue damage of the cortex was significantly decreased in the BG treated groups compared to that of the SAL+ISCH group.

The results of the evaluations of the NADPH-d-positive neurons per section, from different hippocampal formations, are shown in Fig. 3. Post-hoc comparisons indicated that the number of NADPH-d-positive neurons in the hippocampus of the SAL+ISCH group were noticeably less than that of the normal group (p<0.001). In particular, there were significant differences in both the CA3 area (F_3,19=13.6, p=0.001) and the dentate gyrus (F_3,19=7.1, p=0.003). However, the number of NADPH-d-positive neurons of the BG100+ISCH group was higher than that of the SAL+ISCH group, both in the CA3 area (p=0.001) and the DG (p=0.001).

The results of the evaluations of the ChAT immunoreactive cells per section, from different hippocampal formations, are shown in Fig. 4. Post-hoc comparisons indicated that the ChAT activity in the hippocampus of the SAL+ISCH group was significantly lower than that of the normal group (p<0.05). In particular, there were significant differences in both the CA1 (F_3,16=4.9, p=0.02) and CA3 (F_3,16=9.3, p=0.001) areas. However, the ChAT reactivity in the BG treated groups was higher than that of the SAL+ISCH group, and particularly in the CA3 area (BG 100, p=0.05; BG 400, p=0.014). However, the ChAT reactivity in the hippocampal CA1 area showed no significant differences among the groups.