Male Mongolian gerbils (Meriones unguiculatus) were obtained from the Experimental Animal Center, Kangwon National University (Chuncheon, South Korea) at postnatal month (PM) 3, representing young animals, PM 6 and PM 12, representing adult animals, and PM 24, representing aged animals. The animals (n=14 in each group) were housed in a conventional state under adequate temperature (23°C) and humidity (60%) control, with a 12-h light/12-h dark cycle, and were provided with free access to food and water. The procedures for handling and caring for animals adhered to the guidelines in compliance with the Guide for the Care and Use of Laboratory Animals (30), and they were approved by the Institutional Animal Care and Use Committee at Kangwon National University (Chuncheon, South Korea; approval no. KW-130424-3). All experiments were performed in a manner to minimize the number of animals used and to minimize the suffering caused by the procedures used in the present study.

To examine changes in the protein levels of Redd1, mTOR and phosphorylated (p-)mTOR in the hippocampus between the adult and aged groups, animals in each age group (n=7) were used, and western blot analysis was performed, according to the method used in our previous studies (6,23,31). In brief, following sacrifice of the animals by cervical dislocation, the hippocampus was removed. The hippocampal tissues were homogenized in 50 mM phosphate-buffered saline PBS (pH 7.4) containing 0.1 mM ethylene glycol bis (2 aminoethyl ether)-N,N,N′,N′ tetraacetic acid (pH 8.0), 0.2% Nonidet P-40, 10 mM ethylendiamine tetraacetic acid (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl floride and 1 mM dithiothreitol (DTT; all from Sigma-Aldrich). Following centrifugation at 16,000 × g for 20 min at 4°C, the protein levels were determined in the supernatants using a Micro BCA protein assay kit (Sigma-Aldrich), with bovine serum albumin as the standard (Pierce Biotechnology, Inc.; Thermo Fisher Scientific Inc., Waltham, MA, USA). Aliquots containing 20 µg total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 3 mM DTT, 6% sodium dodecyl sulfate, 0.3% bromophenol blue and 30% glycerol (all from Sigma-Aldrich). The aliquots were then loaded onto a 10% polyacrylamide gel. Following electrophoresis, the gels were transferred onto nitrocellulose transfer membranes (Pall Life Sciences, East Hills, NY, USA). To reduce background staining, the membranes were incubated with 5% non-fat dry milk (Sigma-Aldrich) in PBS containing 0.1% Tween 20 (PBST; Sigma-Aldrich) for 45 min at room temperature. Following washing with PBS (×3), the membranes were incubated with polyclonal rabbit anti-human mTOR (cat. no. 2972; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), polyclonal rabbit anti-human phospho-mTOR (Ser2448; cat. no. 2971; dilution, 1:1,000; Cell Signaling Technology, Inc.), polyclonal rabbit anti-human Redd1 (cat. no. 10638-1-AP; dilution, 1:500; Proteintech, Chicago, IL, USA) or monoclonal mouse anti-human β-actin (cat. no. A5316; dilution, 1:5,000; Sigma-Aldrich) overnight at 4°C. Following washing with PBST (x3), the membranes were incubated with peroxidase-conjugated donkey anti-rabbit IgG (cat. no. sc-2305) or goat anti-mouse IgG (cat. no. sc-2031; dilution, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 hr at room temperature. An enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.) was used to detect the protein expression. The results of the western blot analyses were scanned, and densitometric analysis for the quantification of the bands was performed using Image J 1.46 software (ImageJ, National Institutes of Health, Bethesda, MD, USA), which was used to determine the relative optical density (ROD). The levels of mTOR, p-mTOR and Redd1 were normalized with the level of β-actin. A ratio of the ROD was calibrated as the percentage, with the PM 3 group designated as 100%.

For the immunohistochemical analyses, the animals (n=7 per group) were anesthetized with zoletil 50 (30 mg/kg; Virbac, Carros, France) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.4; Sigma-Aldrich), followed by 4% paraformaldehyde (Sigma-Aldrich) in 0.1 M phosphate-buffer (pH 7.4). The brain tissues were removed, cryoprotected and serially sectioned on a cryostat (CM1520; Leica Microsystems; Wetzlar, Germany) into 30 µm coronal sections, and these were collected into six-well plates containing PBS.

According to the method used in our previous study (6,23,31), immunohistochemical staining for Redd1 was performed using the polyclonal rabbit anti-human Redd1 primary antibody (1:200; cat. no. 10638-1-AP; Proteintech) a overnight at 4°C. Following washing with PBS (×3), the brain tissues were incubated with biotinylated goat anti-rabbit IgG (BA-1000; 1:200; Vector Laboratories, Burlingame, CA, USA) for 2 hr at room temperature and streptavidin peroxidase complex (SA-5004; 1:200; Vector Laboratories) for 45 min at room temperature. In order to establish the specificity of the immunostaining, a negative control assessment was performed using pre-immune serum (S-2012; 5%; Vector Laboratories, Inc., Burlingame, CA, USA), which was used in place of the primary antibody. The negative control resulted in the absence of immunoreactivity in any structures (data not shown).

A total of seven sections, with 90-µm intervals, per animal were selected to quantitatively analyze Redd1 immunoreactivity. Digital images of the hippocampal subregions were captured under an AxioM2 light microscope (Carl Zeiss AG, Jena, Germany) equipped with a digital camera (Axiocam; Carl Zeiss AG) connected to a PC monitor. Semi-quantification of the immunostaining intensities in the pyramidal cells of the hippocampus proper and in the granular cells of the dentate gyrus were evaluated using ImageJ 1.46 software (National Institutes of Health), according to the method of our previous study (6,31). The mean intensity of the immunostaining in each of the immunoreactive structures was measured using a 0–255 gray scale system (32,33), in which white to dark signal corresponded to values between 255 and 0, respectively). Based on this approach, the level of immunoreactivity was scaled as −, ±, +, ++ or +++, representing no staining (gray scale value: ≥200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), strong (gray scale value: 50–99) or very strong (gray scale value: ≤49), respectively.

Data are expressed as the mean ± standard error of the mean. Differences between the mean ROD values among the groups were statistically analyzed using one-way analysis of variance, followed by Bonferroni's post-hoc multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

From the results of the western blot analysis, no significant differences were observed in the levels of mTOR in the hippo-campus among the experimental groups (Fig. 1A). However, the levels of p-mTOR in the hippocampus decreased in an age-dependent manner (Fig. 1A).

By contrast, the protein level of Redd1 in the hippocampus increased gradually during normal aging. The protein levels of Redd1 were significantly increased in the PM 6 and PM 12 groups (133.2 and 142.5%, respectively), compared with the PM3 group. In addition, the protein level of Redd1 was markedly higher at PM 24, compared with the PM 6 and PM 12 group (132.7% of the PM 12 group; Fig. 1B).

In the PM 3 group, weak Redd1 immunoreactivity was observed in the pyramidal neurons of the stratum pyramidale (SP) of the hippocampus proper (CA1-3 regions; Table I; Fig. 2A). In particular, Redd1 immunoreactivity in the pyramidal neurons was detected in the cytoplasm, but not in the nucleus. In the PM 6 group, Redd1 immunoreactivity was increased; and moderate Redd1 immunoreactivity was observed in the SP of the hippocampus proper, compared with that in the PM 3 group (Table I; Fig. 2B). The Redd1 immunoreactivity in the hippocampus proper of the PM 12 group was similar to that observed in the PM 6 group (Table I; Fig. 2C). In the PM 24 group, Redd1 immunoreactivity in the SP of the hippocampus proper was markedly higher, compared with the PM 6 and PM 12 groups (Table I, Fig. 2D).

In the dentate gyrus of the PM 3 group, weak Redd1 immunoreactivity was observed in the granule cells of the granule cell layer (GCL) and in the polymorphic cells of the polymorphic layer (PL; Table I; Fig. 3A). The age-related change in Redd1 immunoreactivity in the GCL was similar to that observed in the hippocampus proper. In the GCL, the levels of Redd1 immunoreactivity were moderate in the PM 6 and PM 12 groups, and strong in the PM 24 group (Table I; Fig. 3B-D). However, no marked changes in Redd1 immunoreactivity were observed in the polymorphic cells of the PL among the experimental groups (Fig. 3).