The animal experiments were approved by the Institutional Animal Care and Use Committee of Anhui Medical University (Hefei, China). Sprague Dawley rats were obtained from the Shanghai B&K Universal Group Limited (Shanghai, China). Female adolescent rats (3 months; 210–260 g) and female aged rats (16-months; 490–540 g) rats were used in the present study (17 animals in each group). All animals were housed in standard cages with a 12 h light/dark cycle and had ad libitum access to food and water throughout the investigation. The animals were allowed to acclimate to their new surroundings for 1 week prior to the commencement of the behavioral experiments.

The water maze consisted of a plastic black circular tank (160 cm in diameter; 60 cm high). An overhead video camera connected to a computer and ANY-maze video tracking system software (Stoelting Company, Wood Dale, IL, USA) was used to track the path of movement of the rat. The water was maintained at a constant temperature of 22–25°C. A circular escape platform (10 cm diameter) was placed 2 cm below the surface of the water, in the middle of one of the four quadrants. The general assessment process has been described in detail previously (21,22). In the training trials, the animals were placed into the tank from the four quadrants each day, with a 5 min trial interval, for 4 days. Each trial lasted until the rats climbed onto the hidden platform within 90 sec, the rats were guided to the platform if they had not climbed onto the platform within 90 sec. The escape latency onto the platform was recorded to indicate the learning performance. Following the training trials, the platform was removed from the pool and each rat received one swim probe trial of 60 sec. The swimming distance (m) and the mean speed (m/sec) were recorded as an indication of motor behavior. The swimming duration in the quadrant of the platform (sec), swimming distance in the quadrant of the platform (m), number of crossings of the platform and time of first entry onto the platform (sec) were recorded as an indicator of memory aptitude.

The levels of serum estradiol and LH were measured using an automated microparticle enzyme immunoassay kit and analysis instrument (AxSYM; Abbott Diagnostics, Inc., Abbott Park, IL, USA). The AxSYM method is a heterogeneous immunoassay, in which estradiol and LH from the specimens bind to rabbit polyclonal anti-estradiol antibodies and anti-LH antibodies, which are linked to microparticles. Following removal of the unbound materials, estradiol and LH alkaline phosphatase conjugate were added and bound to available sites. Following washing with PBS, 4-methylum-belliferyl phosphate was added and the fluorescent product was measured. In this method, the intensity of the signal is inversely proportional to the concentrations of estradiol and LH in the specimens (23,24).

The animals were sacrificed via cervical dislocation (eight rats in each group). The brain was immediately dissected in half along the coronal line, one half of which was frozen (−8°C) for immunoblot analysis and the other was used to form a 10% physiological saline homogenate. The homogenate was centrifuged at 4,000 x g for 10 min, and the supernatant was collected. The activity of SOD and the level of MDA were detected using a spectrophotometer (SpectraMax 190; Molecular Devices Corp., Sunnyvale, CA, USA), according to the SOD and MDA assay kits (Niajing Jiancheng Bioengineering Institute, Nanjing, China).

Dihydroethidium (DHE) microfluorography (Beyotime Institute of Biotechnology, Haimen, China) was used to determine the production of ROS in the brain tissue (25). DHE is a cell permeable dye, which is oxidized to ethidium bromide and associated products by superoxide. Ethidium bromide is trapped intracellularly by intercalation into DNA and can then be identified using red fluorescence (26,27). In the present study, DHE was injected via the vena caudalis (100 µM; 1 ml/100 g) for 30 min prior to sacrifice of the rat (n=8; four rats/group). The brains were removed from the skull and transferred to a freezer at −20°C. Subsequently, the brains were sectioned at a thickness of 20 µm using a cryostat section cutter (Leica, Wetzlar, Germany) at −20°C and mounted onto glass slides. The sections were washed with phosphate buffered saline three times and examined using a fluorescence microscope (Olympus IX71; Olympus, Tokyo, Japan) equipped with a custom filter set for detection of DHE oxidation products (red). The production of ROS in the cortex and the hippocampal CA1 and CA3 regions were assessed using previously described methods (25). The exposure time was 900 ms for all sections. A total of four sections per group were assessed for ROS quantitative analysis. The mean fluorescence intensities of three consecutive fields (magnification, ×400) in the cortex, and hippocampal CA1 and CA3 regions, were performed in a blinded-manner using the Image-Pro Plus 6.0 (Media Cybernetics, Inc., Silver Spring, MD, USA) analysis system to indicate the production of ROS, with the data expressed in relative fluorescence units.

Following the morris water maze assessment, the animals (n=10; five rats/group) were sacrificed and the brains were removed. The brains were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin sections were cut at 5 µm and mounted onto glass slides. Prior to immunostaining, the sections were deparaffinized and rehydrated in water. The endogenous peroxidase activity was inhibited by incubation with 0.3% hydrogen peroxide for 30 min. Subsequently, the sections were incubated with normal goat serum at room temperature for 30 min, following which the sections were incubated with primary antibodies overnight at 4°C. The primary rabbit polyclonal antibodies were protein kinase C α (PKCα; BS1577; 1:200; Bioworld Technology, St. Louis Park, MN, USA), p47phox (BS4600; 1:200; Bioworld Technology), NOX2 (BS5674; 1:100; Bioworld Technology) and RAC1 (24072-1-AP; 1:100; ProteinTech, Chicago, IL, USA). Immunostaining was visualized using a peroxidase method with an ABC kit (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). The sections were resin-mounted and observed under a microscope (Olympus IX71; Olympus). The positive cells were stained brown. A total of five sections in each group were assessed for quantitative analysis and a total of three consecutive fields (magnification, ×400) of the cortex, and hippocampal CA1 and CA3 regions, in each section were observed. The mean optical density of the positive neurons of the hippocampal CA1 and CA3 regions and the cortex in each section were measured using the Image-Pro Plus 6.0 analysis system to determine the expression levels of PKCα, p47phox, NOX2 and RAC1 (22).

Tissue from the cortex and the hippocampus (100 mg) were homogenized in 1 ml radioimmunoprecipitation assay lysis buffer; Beyotime Institute of Biotechnology). Equal quantities of protein (50 µg) were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were washed in Tris-buffered saline with Tween 20 (TBS-T) and further incubated with the antibodies against NOX2 (1:500), p47phox (1:500), RAC1 (1:500) and β-actin (1:1,000) overnight at 4°C. The membranes were thoroughly washed with TBS-T and then incubated with anti-rabbit immunoglobulin G antibody conjugated to horseradish peroxidase (1:10,000) for 1 h. Following washing with TBS-T, the immunoreactive bands were visualized using an enhanced chemiluminesense kit (Amersham Biosciences, Little Chalfont, UK). The Tanon4500 imaging system (Shanghai Tanon Science & Technology Co., Ltd., Shanghai, China) was used to visualize protein bands, and densitometry was performed using ImageJ software, version 1.43 (National Institutes of Health, Bethesda, MD, USA). The ratio of the target band to that of β-actin was determined to calculate the relative intensity of the expression of p47phox, NOX2 and RAC1.

Data are expressed as the mean ± standard deviation. The SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) statistical software package was used to analyze the results. Statistical comparisons between groups were performed using Student's t-test with Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

A Morris water maze assessment was performed to assess the learning and memory skills in the rats, as described above. In the learning and memory training experiments, the mean escape latencies in the 3-and 16-month-old groups on the first day were significantly different (40.02±13.74, vs. 58.89±8.75, respectively; P=0.005). No significant differences were observed in the training experiments between day 2 and day 4 in the 3-month and 16-month-old groups (Fig. 1A). In the probe trial experiments (Fig. 1B), the swimming distance (14.16±1.56, vs. 11.30±1.90; P=0.007; Fig. 1C), mean swimming speed (0.24±0.03, vs. 0.19±0.03; P=0.007; Fig. 1D), and the swimming duration and distance to the platform (Fig. 1E and 1F; P=0.006 and P=0.003) were significantly different in the 3-month and 16-month-old groups, respectively. In addition, the number of crossings (3.88±1.55, vs. 2.13±1.3; P=0.035; Fig. 1G) and the time of first entry onto the platform (13.71±12.76, vs. 35.01±17.74; P=0.02; Fig. 1H) in the 3-month and 16-month-old groups, respectively were significantly different.

Levels of estradiol, which decline during the menopause have been associated with learning and memory impairments (28,29). To investigate the possible links between memory impairments in the menopause and the levels of serum estradiol and LH, the levels of serum estradiol and LH in the 3-month and 16-month-old rats were examined. The results revealed that, compared with the 3-month-old rats, the level of serum estradiol was significantly decreased in the 16-month-old rats (137.86±26.10, vs. 91.15±25.99, respectively; P=0.0008; Fig. 2A). No significant differences were identified in the level of serum LH between the 3-month and 16-month-old rats (Fig. 2B).

Compared with the 3-month-old group, the SOD activity was significantly decreased (P=0.03) and the MDA content was significantly increased (P=0.009) in the 16-month-old rats (Fig. 3).

DHE was used to detect the production of ROS in the cortex and hippocampal CA1 and CA3 regions in the 3-month and 16-month-old rats (27). A low level of fluorescence was detected in the cortex and hippocampal CA1 and CA3 regions in the 3-month rats (Fig. 4A), indicating mild ROS production. However, in the 16-month-old group, the production of ROS was significantly increased in the cortex and hippocampal CA1 and CA3 regions (Fig. 4A and 4B; P=0.001, P=0.024 and P=0.009, respectively).

NOX family members are important sources for the generation of ROS in neurodegenerative diseases. Therefore, the expression levels of PKCα, which activates NOX2, the NOX2 membrane subunit, the p47phox cytosolic subunit and RAC1 were investigated in the frontal cortex and hippocampal CA1 and CA3 regions using immunohistochemistry. The PKCα immunostaining results revealed that the expression of PKCα was significantly increased in the cortex and the hippocampal CA1 and CA3 regions in the 16-month-old group (Fig. 5A and 5B; P<0.05). The NOX2 immunostaining results revealed lower expression levels of NOX2 in the cortex and the hippocampal CA1 and CA3 regions in the 3-month-old rats, as the immunoreactive staining was light. Compared with the 3-month old group, the numbers of NOX2 immunoreactive cells were significantly increased in the cortex and the hippocampal CA1 and CA3 regions in the 16-month-old rats, in which staining was marked (Fig. 5C and 5D; P<0.05). The p47phox and RAC1 immunostaining results also revealed that the p47phox cytosolic subunit and RAC1, particularly p47phox, were expressed in the cortex and hippocampus in the two groups. However, compared with the 3-month group, the expression levels of p47phox and RAC1, particularly p47phox, were significantly increased in the cortex and the hippocampal CA1 and CA3 regions in the 16-month old female rats (Fig. 5E and 5F; P<0.05 and P<0.01 for p47phox; Fig. 5G and 5H; P<0.05 for RAC1).

The expression levels of NOX2, p47phox and RAC1 were also assessed in the frontal cortex and the hippocampus using immunoblotting (Fig. 6A). The results revealed that the expression levels of NOX2, p47phox and RAC1 were also significantly increased in the 16-month-old rats (Fig. 6B; P<0.05).