The present study used male Mongolian gerbils (Meriones unguiculatus) at postnatal month 3 (PM 3; 40–50 g) as a young-group, PM 12 (65–75 g) as an adult-group and PM 24 (85–95 g) as an aged-group (total, n=42; n=14/group). This model was selected due to its suitability for research on aging (22). The gerbils were obtained from the Experimental Animal Center of Kangwon National University (Chuncheon, Republic of Korea). The animals were housed under conventional conditions at an ambient temperature (23±3°C) and relative humidity (55±5%) under a 12-h light/dark cycle and were allowed free access to food and water. The study was conducted to minimize the number of gerbils. The procedures for animal handling and care adhered to guidelines in compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, The National Academies Press, 8th ed., 2011) (23). All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Kangwon National University (approval no. KW-160802-2).

Changes in Nurr1 protein levels during the normal aging process were examined in the hippocampi of gerbils (n=7/group). Western blot analysis was performed according to the method described in our previous studies (24,25). In brief, following euthanasia of the animals, hippocampi were removed. The hippocampi were homogenized and centrifuged, and the supernatants were subjected to western blot analysis. The tissues were homogenized in 50 mM phosphate-buffered saline (PBS; pH 7.4) containing 0.1 mM ethylene glycol-bis(2-aminoethylether)-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; Merck KGaA, Darmstadt, Germany). Following centrifugation at 16,000 × g for 20 min at 4°C, the protein level of Nurr1 in the supernatants was determined using a micro bicinchoninic acid protein assay kit (Sigma-Aldrich; Merck KGaA), with bovine serum albumin as the standard (Pierce; 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% SDS, 0.3% bromophenol blue and 30% glycerol. The aliquots were then loaded onto a 10% polyacrylamide gel. Following electrophoresis, the gels were transferred onto nitrocellulose transfer membranes. To reduce background staining, the membranes were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween-20 for 45 min at room temperature. Following three washes with PBS with Tween-20 (PBST; each for 5 min), the membranes were incubated with rabbit anti-Nurr1 (PA5-13416; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) overnight at 4°C. Following another three washes with PBST (each for 10 min), the membranes were incubated with peroxidase-conjugated donkey anti-rabbit immunoglobulin G (IgG; sc-2305; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature, followed by ECL reagents (Pierce; Thermo Fisher Scientific, Inc.). The resulting protein bands were scanned, and densitometric analysis for quantification of the bands was performed using Image J 1.59 software (National Institutes of Health, Bethesda, MD, USA), which was used to calculate relative optical density (ROD). The protein level of Nurr1 was normalized to that of β-actin (A5316; 1:5,000; Sigma-Aldrich; Merck KGaA). A ratio of the ROD was calibrated as a percentage, with the young-group designated as 100%.

The gerbils (n=7/group) were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection; JW Pharmaceutical Corporation, Seoul, Republic of Korea) and perfused transcardially with 0.1 M PBS (pH 7.4) followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were removed and postfixed with the same fixative for 7 h at room temperature. The brain tissue including the hippocampi was sectioned at 30-µm thickness with a cryostat.

To examine age-related changes in Nurr1 immunoreactivity in young, adult and aged hippocampi, immunohistochemical staining was performed according to the method described in our previous studies (24,25). Immunohistochemical staining for Nurr1 was performed using the rabbit anti-Nurr1 antibody (1:100) as the primary antibody overnight at 4°C. Following three washes with PBS (each for 10 min), the brain tissues were incubated with biotinylated goat anti-rabbit IgG (BA-1000; 1:200; Vector Laboratories, Burlingame, CA, USA) for 2 h at room temperature, and then streptavidin peroxidase complex (SA-5004; 1:200; Vector Laboratories) for 45 min at room temperature. To establish the specificity of the immunostaining, a negative control test without primary antibody was performed, which resulted in the absence of immunoreactivity in all structures.

A total of six sections at 120-µm intervals per animal were selected to quantitatively analyze Nurr1 immunoreactivity. Digital images of Nurr1 immunoreactive structures of the hippocampal regions were observed and captured with an Axio Imager 2 light microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a digital camera (Axiocam; Carl Zeiss). According to the method of previous studies (24,26), semi-quantification of the immunostaining intensity of Nurr1 was evaluated and analyzed using Image J 1.59. The mean intensity of Nurr1 immunoreactivity in the immunoreactive structures was measured using a 0–255 gray scale system (white to dark signal corresponding to 255 to 0). Based on this approach, the background density was subtracted, and 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.

CV staining was performed to investigate cellular distribution and morphology. In brief, according to the method of our previous study (27), the sections of the hippocampal regions were mounted on gelatin-coated microscopy slides. Cresyl violet acetate (Sigma-Aldrich; Merck KGaA) was dissolved at 1.0% (w/v) in distilled water, and glacial acetic acid (0.25% v/v) was added to this solution. The sections were stained with CV and dehydrated by immersing in serial ethanol baths. Finally, the stained sections were mounted with Canada balsam (Kanto Chemical, Co., Inc., Tokyo, Japan). A total of six sections at 120-µm intervals per animal were selected to identify the distribution of Nurr1 immunoreactivity in the hippocampus. Digital images of CV stained structures were observed and captured with the Axio Imager 2 microscope and digital camera.

Data are expressed as the mean ± standard error of the mean. Differences in the mean ROD among the groups were statistically analyzed using one-way analysis of variance followed by post hoc Bonferroni's multiple comparison tests using GraphPad InStat (version 3.05; GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was considered at P<0.05.

Results from western blot analysis indicated an age-related change of Nurr1 protein level in the gerbil hippocampus (Fig. 1). The protein expression of Nurr1 in the adult hippocampus was significantly decreased compared with that in the young hippocampus (P<0.05). In addition, the expression of Nurr1 in the aged hippocampus was significantly decreased compared with that in the adult and young hippocampi (P<0.05). Notably, the protein level of Nurr1 in the aged hippocampus was reduced by 73.6% compared with that in the young hippocampus.

Age-related changes in Nurr1 immunoreactivity (Table I and Fig. 2) were identified in the hippocampus proper (CA1-3 regions). In the young group, very strong Nurr1 immunoreactivity was primarily observed in pyramidal neurons of the stratum pyramidale (SP; Fig. 2A and D). In the adult group, Nurr1 immunoreactivity in pyramidal neurons was decreased compared with that in the young group (Table I and Fig. 2B and E). Furthermore, a marked reduction of Nurr1 immunoreactivity in the SP was identified in the aged group, compared with that in the adult group (Table I and Fig. 2C and F).

In the dentate gyrus (DG), Nurr1 immunoreactivity was primarily observed in granule cells of the granule cell layer (GCL; Fig. 2G, H and I). Similar to the change of Nurr1 immunoreactivity in the hippocampus proper, Nurr1 immunoreactivity in the DG gradually and markedly decreased during normal aging (Table I and Fig. 2G, H and I). Therefore, Nurr1 immunoreactivity decreased in the hippocampal regions in an age-dependent manner.

In the young, adult and aged groups, CV positive cells were well distributed, mainly in the SP of the hippocampal CA1-3 regions, and the granular cell layer (GCL) and polymorphic layer (PL) of the DG in the hippocampus (Fig. 3A-I). The distribution of Nurr1 immunoreactivity was concentrated to pyramidal neurons of the SP in the hippocampus proper and to granule cells of the GCL in the DG when comparing with the results of CV staining (Fig. 3).