Preparation of AA was performed according to a method published in our previous report [2]. Briefly, a total of 40 g of AA was heated for 30 min with 1.8 L of distilled water (DW) under 150,000 Pa at 80℃. The extract was allowed to fully cool, and then was filtered first with paper (185 mm; Advantec, Japan) and then with a Nalgene Rapid-Flow Bottle Top Filter (0.2 µm pore membrane; Thermo Scientific, USA). The final AA extract was stored at −80℃ until use.

Twenty-four adult C57BL/6J mice (mean weight, 23.0 g; 7-week-old) were maintained at room temperature (23 ± 1℃) and 60% humidity under a 12 h light-dark cycle (light cycle from 7:00–19:00). The mice were separated into four groups, two that were provided a normal chow diet (ND) (2018S; Harlan, USA) and two that were given a high-fat diet (HD) (2018S; Harlan). All animals had ad libitum access to water. The AA extract (0.2 g AA/10 mL DW/kg) was carefully administered with an oral sonde (0.9 × 50 mm) to half of each food group, while the same amount of DW was administered to the other half. Body weight and food intake were recorded daily.

Mice were treated with 5′-bromo-2′-deoxyuridine (BrdU, Sigma-Aldrich, USA) at 7 weeks of age in order to label endogenous proliferating cells. BrdU (50 mg/kg) was administered intraperitoneally every 12 h for 3 days. The mice were sacrificed 28 days following the last BrdU injection, as it takes approximately 4 weeks for newly developed neurons to differentiate in adult mice.

All procedures involving animals were done in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC approval No. SCH15-0001) at Soonchunhyang University.

Blood samples were collected from the heart and centrifuged at 15,000 × g for 10 min. Then, the IL-6 level in serum was measured with a Mouse IL-6 ELISA Kit (Koma Biotech, Korea). We performed the test according to the instructions provided with the kit.

For Immunohistochemistry, brain tissue sections between −1.46 and −2.47 mm were made using the Bregma anatomical reference in mouse [16] for each animal. The sections were incubated overnight with primary antibodies specific for DCX (1:100; Santa Cruz Biotechnology, USA), and Iba-1 (1:1,000; Chemicon, USA). Sequentially, the sections were incubated with biotinylated secondary antibodies and streptavidin peroxidase complex (Vector Labs, USA). Antibody bindings were detected with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich). The number of positive DCX or Iba-1 cells for all groups were noted. In particular, the number of DCX-positive cell bodies as neuroblasts or DCX_Blasts were counted in the subgranular zone (SGZ) of the hippocampal dentate gyrus, and DCX-positive dendrites from the neuroblasts were also counted in the granular cell layer (GCL) and the molecular layer (ML) in the hippocampal dentate gyrus.

Brains were removed from animals and the hippocampal tissues were separated. All tissues were frozen with liquid nitrogen and homogenized in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Korea), and protein concentrations were determined with a SMART BCA kit (iNtRON Biotechnology). Lysates were separated with 10% SDS-PAGE and then transferred to PVDF membranes (Bio-Rad Laboratories, USA). The membranes were probed with primary antibodies against COX-2 (Cayman, USA), Iba-1 (Wako, Japan), and GAPDH (Cell Signaling Technologies, USA), then incubated overnight. After further washing, membranes were incubated with HRP-conjugated secondary antibody (Vector Labs, USA) and ECL substrate solution (Progma, USA). CheBi (Neoscience, Korea) was used for imaging.

For immunohistochemical analysis, brain tissue sections between −1.46 and −2.47 mm were made using the Bregma anatomical reference [16] for each animal. The sections were incubated overnight with primary antibodies specific for DCX (1:500; Santa Cruz Biotechnology). Sequentially, the sections were incubated with biotinylated secondary antibodies and streptavidin-peroxidase complex (Vector Labs). Antibody binding was detected with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich). The number of DCX-positive cells in all groups were recorded. Of special importance, the number of DCX-positive cell bodies as neuroblasts or DCX-Blasts were counted in the SGZ of the hippocampal dentate gyrus, and DCX-positive dendrites from the neuroblasts were also counted in the GCL and ML in the hippocampal dentate gyrus.

Brain tissues were cryoprotected by infiltration with 30% sucrose in phosphate buffer (pH 7.2). The frozen tissues were then serially sectioned on a cryostat (Thermo Scientific, USA) in 30 µm coronal sections containing the dentate gyrus of the hippocampus according to anatomical landmarks given by Bregma (−1.46 mm to −2.46 mm) in the mouse brain atlas [17] and they were then transferred to 24-well plates containing phosphate buffered saline (PBS).

Double immunofluorescence staining of the tissue samples was performed to confirm the presence of differentiating neuronal cells stained with DCX (a neuroblast marker), as well as differentiation from newly-generated cells stained with BrdU at the beginning of AA administration into mature neurons with NeuN staining (a neuronal nucleus marker). Double staining for cells with both BrdU and DCX signals was also performed. For BrdU and antibody staining of the sections, we incubated for 2 h in 50% formamide/2× SCC (0.3M NaCl, 0.03M sodium citrate) at 65℃, followed by incubation for 30 min in 2N HCl at 37℃, and then by a 10 min rinse in 0.1 M boric acid (pH 8.5). Following this, the sections were incubated in a mixture of goat anti-DCX (diluted 1:200, Santa Cruz Biotechnology)/rabbit anti-NeuN (1:5,000, Millipore, USA), rat anti-BrdU (diluted 1:400, Santa Cruz Biotechnology)/rabbit anti-NeuN (1:5,000), and a mixture of rat anti-BrdU (diluted 1:400)/goat anti-DCX (diluted 1:200) overnight at 4℃. They were then incubated in a mixture of Cy3-conjugated AffiniPure donkey anti-rat IgG (1:300, Jackson ImmunoResearch, USA) for BrdU staining, Alexa Fluor 488 donkey anti-goat IgG (1:300, Invitrogen, USA), and Alexa Fluor 594 donkey anti-goat IgG for DCX staining, and Alexa Fluor 488 donkey anti-rabbit IgG (1:300, Invitrogen) for NeuN staining for 2 h at room temperature. Immunoreactivity was observed with a fluorescence microscope (Olympus, USA) after the slides were mounted and coverslipped with Vector Shield media (Vector Labs). The DCX/NeuN double-merged images, BrdU/NeuN double-merged images and BrdU/DCX double-merged images were captured with the image analysis program in Meta Imaging series 7.1 software (Molecular Devices, USA).

The C. elegans CL4176 strain expressing muscle-specific human Aβ_1–42 [dvls27 (myo-3/Aβ_1–42/let UTR, rol-6)] was purchased from CGC (USA). Worms were cultured at 20℃ on NGM (Nematode Growth Media) agar plates (1.7% agar, 2.5 mg/mL peptone, 25 mM NaCl, 50 mM KH2PO4 pH 6.0, 5 µg/mL cholesterol, 1 mM CaCl2, and 1 mM MgSO4) seeded with Escherichia coli OP50 as the food source. For the paralysis assay, five CL4176 L4/young adult worms were transferred to a fresh NGM plate and were allowed to lay eggs for 2 h at 15℃. After eliminating the five adult worms, eggs were maintained at 15℃ for 5 days. Age-synchronized young adult worms were allowed to lay eggs on NGM plates spread with 100 µL of AA extract for 2 h at 15℃. The gravid adults were removed from the plate and the progeny were upshifted to 25℃ to induce Aβ peptide production. The number of paralyzed worms was scored every hour for 24 h after the temperature upshift. The nematodes were scored every hour until they were all paralyzed. To identify paralysis, each nematode was touched gently with a platinum loop. The nematode was considered paralyzed if it moved its head slightly or did not move at all after being touched. The numbers of paralyzed worms were compared between untreated control and AA-treated groups. The log-rank test was used for statistical analysis asymptotically efficient rank invariant test procedures were used as previously reported [16].

To ensure objectivity, all measurements were performed under blinded conditions by two observers per experiment and under identical conditions. For quantitation of immunoreactivity, the extent of the staining was measured in five sections per animal. The images of DCX, BrdU, and NeuN immunoreactive structures were taken by using a BX53 light microscope (Olympus) equipped with a digital camera (DP71, Olympus) connected to a personal computer and a monitor.

The data shown represent the experimental mean ± SE for each experimental group. Differences between means were analyzed by using repeated two-way analysis of variance and one-way analysis of variance followed by Bonferroni post hoc test and Duncan's new multiple range tests in order to determine differences between experimental groups. The measures were analyzed statistically by ANOVAs (with repeated measure when appropriate). Statistical analyses were performed by using JMP9 (SAS Institute, USA). P values < 0.05 were considered statistically significant.

Body weight measurement results showed that the HD/AA group weighed significantly less than did the HD/DW group; in contrast, the ND/DW and the ND/AA groups did not show any significant weight differences. The weight difference between the HD/AA and HD/DW groups began on day 16 following AA treatment and was maintained as the experiment progressed (panel A in Fig. 1). There were no notable differences in the first two weeks for the HD-fed groups.

On the other hand, food intake in both ND-fed groups (ND/DW and ND/AA) and in both HD-fed groups (HD/DW and HD/AA) did not show any significant differences throughout the experimental period (panel B in Fig. 1). There was a lower IL-6 level in the AA-administered group than in the DW-treated group in the HD-fed mice (Fig. 2). Serum of HD-fed mice showed higher IL-6 levels than did that of ND-fed mice when administered DW. However, the IL-6 level in AA-administered mice was much lower than that in DW mice when fed HD. The IL-6 levels in the ND-fed groups were similar, regardless of substance administered (Fig. 2).

The Iba-1 level of AA-administered mice was considerably lower than that in DW-administered mice (panels A and B in Fig. 3). However, this tendency was not observed in the western blot results. The Iba-1 positive cells abundance was significantly lower in the AA administered-groups than in the DW-treated groups in both ND and HD food types (panel B in Fig. 3).

Quantitation of inflammatory factors relative to GAPDH was performed by using western blots (panel C in Fig. 3). COX-2 and Iba-1 expression in the hippocampus of HD-fed mice were higher than the levels in ND-fed mice (panels D and E in Fig. 3). Overall, the expression of Iba-1 was slightly higher than that of COX-2 (p<0.05).

DCX immunoreactivity represents neuronal differentiation and the presence of neuroblasts. DCX-positive signals by DAB chromogen were shown in the hippocampal dentate gyrus. Neuroblasts (“DCX_Blast”) (panel B in Fig. 4) and dendrites from the DCX-positive cell bodies (“DCX_Branch”) were analyzed (panel C in Fig. 4). As shown in Fig. 4, DCX_Blast and DCX_Branch levels were higher in the AA-treated groups than in the DW-treated groups for both food-type groups. There were no significant differences between ND/DW and ND/AA in the DCX_Blast and DCX_Branch groups in the hippocampal dentate gyrus. The number of secondary- and tertiary-branched dendrites showed significant differences only between the HD/DW and HD/AA groups (p<0.05).

To investigate the extent of neuronal differentiation, neurons were double-stained with NeuN and DCX and labeled with BrdU in the hippocampal dentate gyrus. As shown in panels A and B in Fig. 5, DCX-positive cells were observed in the SGZ and GCL in the dentate gyrus in the AA-treated groups on ND and HD diets (panel B in Fig. 5; arrows). However, the length of dendrites, as assessed by DCX immunofluorescence, in the HD/DW group was shorter than that in the ND/DW group. For these experiments, BrdU was injected systemically at the beginning of AA administration, and BrdU-labeled cells (in red) were seen in the SGZ and GCL areas, while cells positive for NeuN were observed in the AA-treated groups in both ND and HD groups, but particularly in the HD/AA group (panel D in Fig. 5; arrows). The BrdU-positive cells had migrated into the GCL from the SGZ. Panels G and H in Fig. 5, respectively, show the number of BrdU-positive neurons in the whole hippocampus and in the GCL. In the hippocampus, BrdU expression in HD-fed mice was lower than that of ND-mice; however, in the HD-fed group, it appeared that AA-administered mice had a higher number of BrdU-positive cells (panel G in Fig. 5). Interestingly, and particularly in the GCL region, we observed that BrdU expression of AA-treated mice was notably increased over that of DW-treated mice, regardless of food type (panel H in Fig. 5).

A reliable and convenient screening for potential anti-AD agents uses the C. elegans paralysis and Aβ induction model. Compared to the DW-treated group (control), the AA-treated C. elegans group (AA) had a significantly higher percentage of “not paralyzed” animals (Fig. 6). In addition, the time (days) to paralysis after Aβ induction was significantly longer in the AA-treated group than in the control group.