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Introduction

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by progressive cognitive impairment and impaired learning and memory. AD is the most common form of dementia. As the world's population continues to age and the number of elderly people increases, the number of patients with AD is expected to reach 140 million worldwide by 2050(1). AD will become one of the most important diseases that affect the health of the elderly population. However, due to the complexity of the pathogenesis of AD and the side effects and tolerability of drugs for its treatment, no effective method or reasonable drug treatment has been developed yet to prevent and treat this disease. Therefore, developing methods to prevent or delay the onset of AD is particularly important.

Mild cognitive impairment (MCI) refers to a condition in which an individual has obvious memory and/or other cognitive defects but is still able to care for him/herself and has not yet reached the diagnostic criteria for dementia, adjusted for age and education level. MCI is currently described as a transitional state between normal aging and AD (2-5). The data showed that the probability of progression to AD in normal elderly individuals was 1-2%, whereas in patients with MCI was as high as 10-15% (6). Therefore, early diagnosis of MCI can aid the identification of individuals at high risk of dementia, and active, effective interventions in patients with MCI can prevent or delay the development of AD.

The senescence-accelerated mouse prone 8 (SAMP8) mouse model is considered a suitable, naturally derived animal model of late-onset AD (7-9). A previous study on age-related changes in SAMP8 mice using a grading score, the Morris water maze (MWM) test, Golgi staining, Nissl staining and immunohistochemical staining was conducted (10). The results demonstrated that SAMP8 mice exhibited obvious signs of aging, MCI, and slight neuropathological changes starting at 5 months of age and showed signs similar to those associated with AD at 7 months old compared with senescence-accelerated mouse resistant 1 (SAMR1) mice. The present study confirmed that SAMP8 mice began to enter the MCI phase at 5 months of age, which suggested that middle-aged SAMP8 mice could be used as a reasonable animal model to examine the transition from MCI to AD (10).

Aging is the result of interactions between genetic and environmental factors. Enriched environment (EE) has been widely used to study brain structural remodeling and functional recovery after an acute brain injury, such as cerebral ischemia, since it was proposed by Hebb in 1947(11). With advances in research, the effects of EE on neurodegenerative disease rehabilitation have attracted widespread attention. Animal experiments have demonstrated that EE can improve the neurological functions and cognitive performances associated with various neurodegenerative diseases, including AD and Parkinson's disease (12). In addition, it has been reported that lifestyle modifications, including diet, exercise, and cognitive stimulation, may be effective in slowing the progression of MCI to dementia (13). In the present study, the effects of EE exposure on the cognitive functions and neuropathological changes in the hippocampus of SAMP8 mice were evaluated during the MCI phase (5-months old) to determine whether EE exposure can delay the progression from MCI to AD.

Materials and methods

Animals and groups

A total of 30 male SAMP8 mice in the MCI phase (5-months old) were randomly categorized into either the P8 + EE group (n=15) or the P8 + standard environment (SE) group (n=15). Mice were housed under EE or SE conditions for 60 days. As a homologous control, 15 male senescence-accelerated mouse resistant 1 (SAMR1) mice aged 5 months (R1 + SE group) were housed under SE conditions for 60 days. All mice were provided by the experimental animal center of The First Hospital Affiliated to Tianjin Medical University. They received a standard rodent diet and water ad libitum and were housed at room temperature (18-22˚C) at a relative humidity of 65-70%. No significant differences in animal body mass were observed among the groups (P>0.05). All experimental procedures followed the guiding principles of mammalian neuroscience research and were approved by the Ethics Committee for Animal Experiments at the Changzhi Medical College.

Environments

SE: Mice were housed in standard polyethylene squirrel cages (30x20x15 cm; five mice per cage), with the top covered by stainless-steel wire. The cages only included nesting material.

EE: Mice were housed in large polyethylene squirrel cages (40x25x30 cm; five mice per cage) with the top covered by stainless-steel wire. The cages contained a running wheel, a swing, a slide, a tunnel, and several toys with different colors and shapes, in addition to nesting material. Cages were cleaned and the contents were rearranged once each day at a fixed time, and a 12-h light/dark cycle was implemented (lights on at 7:00 AM).

MWM test

The MWM comprises three components: A circular pool, a platform, and a video capture system (SMART 3.0 Behavioral Video Analysis Software, Panlab). The pool was 120 cm in diameter and 60 cm in height and was filled with water (20±1˚C) made opaque by the addition of carbon ink. The pool was divided into four quadrants. The platform (10 cm in diameter) was placed in the center of quadrant IV (the target quadrant) and was hidden 1 cm below the water surface. After the mice were housed in the maze laboratory for 2 days, each mouse was placed in the pool near the wall of one quadrant and was allowed to swim freely for 120 sec to adapt to the water temperature and gain familiarity with the task; using this test, mice with existing non-cognitive impairments were identified. The MWM test was conducted daily from 8:00 am to 12:00 pm, and the objects around the pool, including the experimenter, remained in the same location throughout the test.

Positioning navigation test

The mice were gently placed in the water at the midpoint of each quadrant, by turns, facing the pool wall. The video capture system tracked mouse movements and recorded the time required for each mouse to find the platform (escape latency). Each mouse was allowed to swim until it found the platform, for a maximum duration of 120 sec, and was allowed to rest on the platform for 20 sec. If the mice did not find the platform within 120 sec, they were guided to the platform and were maintained on the platform for 20 sec. The test was conducted once a day for 4 consecutive days.

Space exploration test

On the fifth day, the platform was removed from the pool to evaluate the memories of the mice regarding the platform location. Keeping all other factors unchanged, the mice were gently placed in the water at the midpoint of quadrant III, facing the pool wall. The video capture system recorded the swimming paths, time spent in the target quadrant, and number of times the platform location was crossed during each 120-sec test session.

Transmission electron microscopy

From each group, five mice were randomly selected and anesthetized with pentobarbital sodium (80 mg/kg) by intraperitoneal injection, before opening the thorax to expose the heart. An infusion needle was inserted into the left ventricle while the right atrial appendage was cut. At room temperature, the mice were perfused with 0.9% normal saline until clear liquid flowed out, and fixed with a 4% glutaraldehyde solution at the same temperature until the liver became white and the body became stiff. The brain was removed and postfixed for 24 h in the same fixative. The hippocampal CA1 region was examined and removed under a stereoscopic microscope (magnification, x240), referring to the Paxinos mouse brain stereotactic atlas. The CA1 region was fixed with 1% osmium acid at 4˚C for 1 h, dehydrated in a graded acetone (50%, 15 min; 60%, 15 min; 70%, 15 min; 80%, 15 min; 90%, 15 min; and 100%, 15 min; x2) at room temperature, embedded in araldite, and sliced into semi-thin sections (100-nm thick). Superfluous tissues surrounding the hippocampal CA1 region were removed under the microscope based on toluidine blue staining. Consecutive ultrathin (70-nm thick) sections were prepared and stained with uranium acetate and lead citrate, for 30 min each at 37˚C. Ultrastructures were observed under an H-7500 transmission electron microscope (Hitachi, Ltd.), and three photographs were randomly taken from clear sections (x25,000 magnification). A square-shaped area, defined as a counting frame, was randomly drawn onto each photograph. Synapses were identified according to the synaptic confirmation standard (14) and counted according to the stereological ‘forbidden line’ method (15). The synaptic numerical density (Nv, number/µm3) and surface density (Sv, µm2/µm3) were calculated using a stereological method and an image analysis system (16). Nv was calculated according to the formula Nv = Na/(L/Ko + T); where, Na is the total number of synapses per square micrometer, L is the average length of synaptic junction, T is the thickness of ultrathin section, and Ko is the correction coefficient to correct the overestimation of synaptic junction band length caused by Holmes effect (calculated as follows: Ko = 1+3T/2L). Sv was calculated according to the formula Sv = 2Ni/LtKo; where, Ni is the number of crossing points between synaptic junction and test line, Lt is the total length of test line, and Ko is the correction coefficient to correct the overestimation of synaptic junction band length caused by Holmes effect (calculated as aforementioned).

Preparation and staining of tissue sections

The remaining 10 mice in each group were anesthetized with pentobarbital sodium (80 mg/kg) by intraperitoneal injection, before opening the thorax to expose the heart. An infusion needle was inserted into the left ventricle, and the right atrial appendage was simultaneously cut. At room temperature, the mice were perfused with 0.9% normal saline until the clear liquid flowed out, followed by a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer at a pH of 7.4 until the liver became white and the body became stiff. The brain was carefully removed and fixed with the same fixative as aforementioned for 24 h. The tissue block between the optic chiasm and the superior colliculus was cut and divided into two halves along the median sagittal plane. The left tissue block was used for Nissl staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and immunohistochemical staining, whereas the right tissue block was used for Golgi staining. The left tissue block was dehydrated in a graded ethanol series (70%, 2 h; 80%, 2 h; 90%, overnight; 95%, 90 min x2; and 100%, 40 min x2), cleared in xylene (20 min x2), and embedded in paraffin; it was then cut serially from the cranial to the caudal side into 5-µm sections. Five successive sections were taken at intervals of 50 µm and mounted on glass slides coated with polylysine to prepare five sets of sections. One set of sections was used for Nissl staining, another set was used for TUNEL staining, and the remaining three sets were used for immunohistochemical staining of anti-synaptophysin (anti-SYN), anti-Aβ, and anti-APP.

Golgi staining

After immersion in mordant solution for 3 days, followed by immersion in 1.5% AgNO3 solution at room temperature for 5 days in a dark environment, the right tissue block was dehydrated in a graded ethanol series (70%, 2 h; 80%, 2 h; 90%, overnight; 95%, 90 min x2; and 100%, 40 min x2), cleared in xylene (20 min x2), embedded in paraffin, and cut serially into 60-µm sections. For each mouse, 12 randomly chosen neurons fully impregnated, unobscured by neighboring neurons, from 4 sections were analyzed, and the average value was used as the result for this mouse. The lengths (in µm) of the secondary and tertiary apical dendrites in the hippocampal CA1 region were measured using digitized images under x1,000 magnification, the number of dendritic spines was independently counted by two different investigators, and the results were cross-checked to preclude systematic analytical errors. In addition, the dendritic spine density was calculated (number/µm) (17).

Nissl staining

A set of sections was conventionally deparaffinized in xylene (10 min x2), dehydrated in a graded ethanol series (100%, 3 min x2; 95%, 3 min; 90%, 3 min; 80%, 3 min; and 70%, 3 min), soaked in 1% cresyl violet at 60˚C for 30 min, and then washed with distilled water. The sections were differentiated in 95% ethanol until the Nissl body appeared to be clear, dehydrated rapidly twice in 100% ethanol, cleared twice in xylene, and coverslipped using a neutral balsam. The numbers of pyramidal cells per field under x400 magnification using light microscope (Olympus Corporation) in the hippocampal CA1 region were counted.

TUNEL staining

Another set of sections was conventionally deparaffinized in xylene (10 min x2), dehydrated in a graded ethanol series (100%, 3 min x2; 95%, 3 min; 90%, 3 min; 80%, 3 min and 70%, 3 min), and incubated with protease K for 30 min at room temperature. After washing with PBS, the sections were incubated with 50 µl TUNEL reaction mixture in a wet box at 37˚C for 60 min. After further washing with PBS, the sections were incubated with 50 µl of transformant-peroxidase in a wet box at 37˚C for 30 min. Finally, the sections were stained using 3,3'-diaminobenzidine, counterstained using hematoxylin, and coverslipped using a neutral balsam [in situ cell apoptosis test kit; Roche Diagnostics (Shanghai) Co., Ltd.]. The number of TUNEL-positive cells and the number of pyramidal cells were counted separately in each field under x400 magnification using a light microscope (Olympus Corporation) in the hippocampal CA1 region. The apoptotic index (AI) was calculated by dividing the number of TUNEL-positive cells by the number of pyramidal cells (18).

Immunohistochemical staining

The remaining three sets of sections were conventionally deparaffinized in xylene (10 min x2), dehydrated in a graded ethanol series (100%, 3 min x2; 95%, 3 min; 90%, 3 min; 80%, 3 min; 70%, 3 min), and immersed in 3% H2O2 at room temperature for 20 min to block the endogenous peroxidase activity. After being subjected to antigen retrieval with citrate buffer solution (OriGene Technologies, Inc.) for 15 min in a microwave, the sections were incubated with 10% goat serum (OriGene Technologies, Inc.) in a wet box at room temperature for 10 min to block nonspecific binding. This was followed by overnight incubation with rabbit anti-SYN polyclonal antibody (1:200; cat. no. ab14692; Abcam), rabbit anti-Aβ polyclonal antibody (1:200; cat. no. bs-0107R; BIOSS), and rabbit anti-APP monoclonal antibody (1:200; cat. no. ab32136; Abcam) at 4˚C. After washing with PBS, the sections were incubated with biotinylated goat anti-rabbit immunoglobulin G (OriGene Technologies, Inc.) at room temperature for 1 h. The hippocampal CA1 region was imaged under a light microscope (Olympus Coporation; magnification, x400). Image-Pro Plus 6.0 (Media Cybernetics, Inc.) was used to determine the average optical density (OD) for SYN, Aβ and APP in the hippocampal CA1 region.

Data analysis

Repeated measures analysis of variance (ANOVA) was used to analyze the main effects of environment and day (1, 2, 3 and 4 days) on escape latency in the positioning navigation test. One-way ANOVA was used to analyze the effects of environment on the time spent in the target quadrant and the number of platform crossings in the space exploration test. Dendritic spine density, Nv, Sv, number of neurons, and the OD of SYN, Aβ, and APP were tested for normality (Kolmogorov-Smirnov test) and equal variance (Levene's test). If both normal distribution and homogeneity of variance were found, comparisons were analyzed using one-way ANOVA, and Cohen's d test was used for pair-wise comparisons. All data are presented as the mean ± standard deviation. The AI was generated using a Cartesian inspection test. All statistical analyses were performed using SPSS version 25.0 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of EE exposure on the cognitive functions of SAMP8 mice during the MCI phase, assessed using the MWM test

During the positioning navigation test, the average escape latencies on training days 1, 2, 3 and 4 in the P8 + EE group were significantly shorter compared with those in the P8 + SE group (all P<0.01) but significantly longer compared with the R1 + SE group (all P<0.01) (Fig. 1A).

Figure 1 Effects of different environments on the spatial learning and memory abilities of mice assessed using the Morris water maze test. Data are presented as latencies to identify the hidden platform over 4 consecutive training days (A) in the positioning navigation test, whereas the time spent in the target quadrant (C) and the number of platform crossings (D) were assessed to evaluate memory in the space exploration test. Photographs indicate the swimming paths (B) in the space exploration test. Each point or column represents the mean ± SD. **P<0.01 vs. the P8 + SE and R1 + SE groups. SE, standard environment; EE, enhanced environment.

During the space exploration test, the mice in the R1 + SE and P8 + EE groups primarily swam in or near the target quadrant, whereas the mice in the P8 + SE group primarily swam along the pool wall and rarely swam near the target quadrant (Fig. 1B). The time spent in the target quadrant was longer in the P8 + EE group compared with the P8 + SE group (P<0.01) but shorter compared with the R1 + SE group (P<0.01) (Fig. 1C). The number of platform crossings significantly increased in the P8 + EE group compared with that in the P8 + SE group (P<0.01) but was fewer than that in the R1 + SE group (P<0.01) (Fig. 1D).

Effects of EE exposure on neuronal loss in the hippocampal CA1 region in SAMP8 mice during the MCI phase

TUNEL staining resulted in the nuclei of apoptotic cells being stained brown (Fig. 2C). As shown in Fig. 2A, the AI of the P8 + EE group was significantly decreased compared with that of the P8 + SE group (P<0.01), but was significantly higher than that of the R1 + SE group (P<0.01).

Figure 2 Effects of different environments on neuronal loss in the hippocampal CA1 region of mice. EE exposure resulted in (A) a significant decrease in the AI value and (B) a significant increase in the number of neurons. Photomicrographs show TUNEL staining (C) and Nissl staining (D) Scale bar, 100 µm (C and D) **P<0.01 vs. P8 + SE and R1 + SE groups. EE, enriched environment; AI, apoptotic index; SE, standard environment; OD, optical density.

The Nissl staining results (Fig. 2B and D) showed that the number of neurons in the hippocampal CA1 region was significantly higher in the P8 + EE group (P<0.01) compared with that in the P8 + SE group but remained significantly lower than that in the R1 + SE group (P<0.01).

Effects of EE exposure on synaptic plasticity in the hippocampal CA1 region of SAMP8 mice during the MCI phase

Golgi staining was used to observe secondary and tertiary apical dendrites in the hippocampal CA1 region (Fig. 3E). The lengths of secondary and tertiary apical dendrites were measured, the numbers of dendritic spines were counted, and dendritic spine densities were determined. As shown in Fig. 3A, the dendritic spine density of the P8 + EE group was significantly increased compared with that of the P8 + SE group (P<0.01) but was significantly lower than that of the R1 + SE group (P<0.01).

Figure 3 Effects of different environments on synaptic plasticity in the hippocampal CA1 region of mice. EE exposure increased the dendritic spine density (A) Nv (B) Sv (C) and expression of SYN (D) in the hippocampal CA1 region. Photomicrographs show representative images of Golgi staining (E) transmission electron microscopy (F) and immunohistochemical staining (G) Scale bars, 50 µm (E) and 100 µm (G) Each column represents the mean ± SD. **P<0.01 compared with the P8 + SE and R1 + SE groups. EE, enriched environment; SE, standard environment; Nv, synaptic numerical density; SV, surface density; SYN, synaptophysin.

Transmission electron microscopy was used to observe the ultrastructure of excitatory synapses in the hippocampal CA1 region (Fig. 3F). The Nv and Sv were calculated using a stereological method and an image analysis system. The results showed significant differences in the Nv and Sv of synapses in the hippocampal CA1 region between the P8 + EE and P8 + SE groups (all P<0.01), and the Nv and Sv of synapses in the hippocampal CA1 region in the P8 + EE group were significantly lower than those in the R1 + SE group (all P<0.01) (Fig. 3B and C).

The immunohistochemical staining of SYN showed that immune reaction products, observed as a brownish-yellow color, were distributed throughout the neuropil of pyramidal cells but not in the nucleus and cytoplasm (Fig. 3G). The OD of the immune reaction products in each group was measured and found that the expression of immune reaction products in the P8 + EE group was significantly increased compared with that in the P8 + SE group (P<0.01), but was significantly lower than that in the R1 + SE group (P<0.01) (Fig. 3D).

Effects of EE exposure on the deposition of Aβ and the expression of APP in the hippocampal CA1 region of SAMP8 mice during the MCI phase

Immunohistochemical staining for Aβ showed that Aβ-immunoreactive granules, which appeared as a dark yellow color, were primarily distributed in the cytoplasm and protuberances (Fig. 4C). Aβ was deposited in large amounts in the P8 + SE group, resembling senile plaques (Fig. 4C). The Aβ-immunoreactive granules in the hippocampal CA1 region in the P8 + EE group were observed at significantly reduced numbers and densities compared with those in the P8 + SE group (Fig. 4C). In contrast, the deposition of Aβ was rarely observed in the R1 + SE group (Fig. 4C). The OD of Aβ in the P8 + EE group was significantly decreased compared with that in the P8 + SE group (P<0.01) but remained significantly higher than that in the R1 + SE group (P<0.01) (Fig. 4A).

Figure 4 Effects of different environments on the deposition of Aβ and the expression of APP in the hippocampal CA1 region of mice. EE exposure resulted in a significant decline in the deposition of Aβ (A) and the expression of APP (B) Photomicrographs show the immunohistochemical staining of Aβ (C) and APP (D) Scale bar, 100 µm (C and D) Each column represents the mean ± SD. **P<0.01 vs. the P8 + SE and R1 + SE groups. EE, enriched environment; SE, standard environment; APP, amyloid-precursor protein; Aβ, amyloid-β; OD, optical density.

The immunohistochemical staining of APP revealed that immune reaction products, observed in brown color, were distributed in the cytoplasm and the axons of neurons (Fig. 4D). The measurement of the OD of the immune reaction products in each group showed that the expression of immune reaction products in the P8 + EE group was significantly decreased compared with the P8 + SE group (P<0.01) but was significantly higher than that in the R1 + SE group (P<0.01) (Fig. 4B).

Discussion

In humans, the aging process is accompanied by a decline in cognitive function, which is often associated with progressive declines in the functions of multiple systems, including the visual, auditory, olfactory, masticatory and motor systems. An active lifestyle that includes mental and social aspects could improve the functions of these systems; this has been shown to have positive effects on the brain regions that mediate cognitive function, thereby mitigating declines in cognitive ability (19,20). The concept of EE was first introduced by the American scholar Hebb in 1947(11). For more than 70 years, EE has been demonstrated to be beneficial for the brain, particularly the hippocampus, at the molecular, cellular and behavioral levels. At the behavioral level, EE not only improves spatial, non-spatial and long-term memory in normal animals (21-23) but also ameliorate human-like neurological symptoms in animal models of human neurological pathology, including the cognitive decline associated with AD (24-28). Currently, accumulated evidence has demonstrated that EE exposure can mitigate cognitive decline in young SAMP8 mice (29-31). Griñán-Ferré et al (29,30) raised SAMP8 mice under EE conditions from 21 days after birth until 3 months of age and found a better generalized performance in SAMP8 mice raised under EE conditions compared with those raised under SE conditions, assessed by behavioral and learning tests. Yuan et al (31) housed 3-month-old female SAMP8 mice under EE conditions for 3 months, and the results demonstrated that EE exposure improved cognitive performance, assessed by the MWM test and a step-down avoidance experiment. In the present study, compared with mice under SE conditions, mice under EE conditions were housed in large, polyethylene squirrel cages, that provided opportunities to perform more activities, including a running wheel, a swing and a slide, increasing the opportunities for voluntary exercise and likely increased aerobic exercise. In addition, mice housed under the EE conditions had access to a tunnel and several toys with different colors and shapes. Additionally, the cages were cleaned and positions of the objects rearranged once a day at a fixed time to enhance visual stimulation. After 2 months, the learning and memory abilities of middle-aged SAMP8 and SAMR1 mice housed under different environments were evaluated using the MWM test. The results showed that mice in the P8 + EE group exhibited shorter escape latencies compared with mice in the P8 + SE group during the positioning navigation test. P8 + EE mice also spent more time in the target quadrant compared with mice in the P8 + SE group during the space exploration test. Moreover, the number of platform crossings increased significantly for the P8 + EE group compared with that for the P8 + SE group. However, a significant difference was observed between mice in the P8 + EE and R1 + SE groups for escape latencies, time spent in the target quadrant, and the number of platform crossings. These behavioral results indicated that EE exposure could be used as a rehabilitation strategy to mitigate the cognitive impairments of SAMP8 mice during the MCI phase and to delay the progression from MCI to AD.

The hippocampus is an important structure in limbic system, which is associated with learning, memory and cognition. The hippocampal CA1 region is an important part of learning and memory, information processing and signal transmission. It is responsible for spatial learning and memory (32,33). The selective vulnerability of distinct regions of the brain is a critical factor in neurodegenerative disorders. In Alzheimer's disease (AD), neurons in hippocampus situated in medial temporal lobe are immensely damaged (34). Neurons in the hippocampal CA1 region are the most sensitive of the entire forebrain, and are considered as the most vulnerable neurons of the hippocampus (35,36). It is the most sensitive area to hypoxia and other damage, also known as vulnerable area (35). In the present study, neuropathological changes in the hippocampal CA1 region were examined after the mice were exposed to different environments for 60 days.

Compared with the number of neurons in the hippocampus of age-matched normal elderly people, the hippocampus of patients with AD is significantly reduced (37). Autopsies of patients with AD and experiments performed in animal models of AD have revealed higher numbers of apoptotic cells in the hippocampus (38), which suggests that abnormal apoptosis is responsible for neuronal losses in the hippocampus of patients with AD. Studies have shown that EE exposure may reduce abnormal cell apoptosis (39,40) and cause changes in the morphology and structure of the brain at both macro and micro levels, such as increasing the thickness of the cerebral cortex, area of the cerebral sulcus, and number of neurons (41,42). Griñan-Ferré et al demonstrated that EE exposure decreased the levels of Bax and antiapoptotic Bcl-2 proteins and reduced caspase-3 activity, thereby preventing neuronal apoptosis and loss in SAMP8 mice (29). In agreement with these results, we found that in the hippocampal CA1 region, apoptotic neurons were reduced significantly, as assessed by TUNEL staining, and existing neurons were significantly increased, as assessed by Nissl staining, in the P8 + EE group compared with the P8 + SE group. However, these indicators, including AI and the number of existing neurons, also differed significantly between the P8 + EE and R1 + SE groups. Mitochondria play a pivotal role in the process of integrating all types of death signal stimulation and determining cell fate. By releasing cytochrome c and other related apoptotic factors, mitochondria can initiate caspase protein cascade reaction and induce cell apoptosis; it can also produce a large number of free radicals and destroy the electron transmission of respiratory chain to cause ATP synthesis disorder and induce cell necrotic death (43). Combined with these findings, it is speculated that EE condition may decrease apoptosis by affecting mitochondrial function, including caspase activity and ATP production; this hypothesis was be verified through relevant experiments in the present study.

The synaptic plasticity is the neurobiological basis for learning and memory activities (44,45). Synapses, which are the basic unit of transmission of information in the central nervous system, form connections and transmit electrical and chemical signals between neurons (46). Structural and functional damage to synapses has been identified as one of the pathological bases of learning and memory impairment in patients with AD (47,48). Dendritic spines, which are protrusions that extend outward from the neuronal dendrite, form the structural bases of neuronal connections in the brain and are the primary sites of synapse formation (49). Dendritic spines accept and transfer information, and their upregulation can increase communication and transmission (50,51). During learning and memory processes, changes in synaptic structural plasticity are frequently accompanied by changes in dendritic spines, including formation, expansion, atrophy and abscission. Accumulating studies have identified changes in the morphology and quantity of dendritic spines associated with neurodegenerative diseases such as AD (52,53). Several studies have demonstrated that the dendritic spine density of the secondary and tertiary dendrites in hippocampal neurons is significantly lower in SAMP8 mice compared with SAMR1 mice (10,17). EE exposure can modulate the shape, number and function of dendritic spines (54,55) and considerably increase dendritic spine densities of neocortical, hippocampal and cerebellar neurons (56-58). In the present study, it was found that the dendritic spine density in SAMP8 mice raised under EE conditions was significantly increased compared with that in SAMP8 mice raised under SE conditions, but was significantly lower compared with that in SAMR1 mice raised under SE conditions. To further elucidate the effects of EE exposure on the synapses in SAMP8 mice, the ultrastructure of the hippocampal CA1 region was examined using transmission electron microscopy and calculated synaptic count indices, including Nv and Sv, using a stereological method and an image analysis system. The results showed that EE exposure increased the density of synaptic contacts on the dendritic spines of hippocampal CA1 neurons. However, significant differences in Nv and Sv were observed between the P8 + EE and R1 + SE groups.

SYN is a glycoprotein that is located on the membranes of presynaptic vesicles, and SYN expression can accurately and objectively reflect the distribution and density of synapses (59), since SYN is closely associated with synapse formation and reconstruction (60). The degree of cognitive decline in animal models of AD has been correlated with changes in SYN expression in the hippocampus (17,61-64). The present study demonstrated that EE exposure increased the expression of SYN in the hippocampal CA1 region, which further indicates that EE exposure was able to increase neuronal synapses and improve synaptic plasticity in SAMP8 mice. Collectively, these results suggest that the observed declines in learning and memory abilities in SAMP8 mice are closely associated with synaptic plasticity in the hippocampal CA1 region and that EE exposure effectively regulates synaptic plasticity and reduces synaptic loss.

The deposition of Aβ is a common pathological change identified in brain regions associated with neurodegenerative diseases such as AD and MCI (65), and the present results showed that the average Aβ absorbance in the P8 + SE group significantly increased compared with that in the R1 + SE group. Accumulating research has demonstrated that EE exposure may decrease the Aβ content in the hippocampus. Male APPswe X PS1E9 mice were exposed to an EE for 5 months, starting at the age of 1 month, and the results of biochemical and histological analyses showed decreases in both the steady-state levels of Aβ peptides and Aβ deposition in the brains of mice exposed to EE compared with mice exposed to SE (66). Four months of continuous EE stimulation in 30-day-old female TgCRND8 mice resulted in significant reductions in Aβ plaques in the neocortex and hippocampus (67). Berardi et al (68) found that AD11 mice exposed to EE from 2 to 7 months of age exhibited marked reductions in the presence of Aβ clusters in the hippocampus. Ziegler-Waldkirch et al (69) exposed pregnant 5xFAD mice to EE for 18 days and found that hippocampal and cortical Aβ plaque pathology was significantly decreased compared with that in pregnant mice housed under SE conditions, reaching an Aβ-plaque load similar to that in the non-pregnant control group, which was housed under SE conditions. A review examining the neuroprotective effects of physical activity on the brain in patients with AD concluded that physical activity may improve cognitive dysfunction by reducing Aβ deposition in the brain (70). In the present study, it was also found that EE exposure significantly decreased the deposition of Aβ in the hippocampal CA1 region in SAMP8 mice. Aβ is a product of APP that is formed under the action of β-secretase or γ-secretase. Previous studies have shown that EE exposure significantly decreases APP levels in the hippocampus (29,71). In addition, the present study demonstrated that EE exposure significantly decreased the expression of APP in the hippocampal CA1 region in SAMP8 mice, indicating that EE exposure can decrease the production and deposition of Aβ, which is likely mediated by decreasing APP expression. Aβ is a neurotoxic peptide that can promote neuronal loss and mediate synaptic loss and dysfunction in specific regions by disrupting the mitochondrial membrane potential, decreasing ATP generation, enhancing intracellular reactive oxygen species production and activating apoptosis factors and mitophagy (46,72-79).

Based on the aforementioned findings, it is speculated that EE exposure may effectively decrease neuronal loss and regulate neuronal synaptic plasticity by reducing the expression of APP and the deposition of Aβ in the hippocampal CA1 region, so as to mitigate cognitive decline in SAMP8 mice during the MCI phase and delay the progression from MCI to AD. EE alone can play a role in ameliorating human-like neurological symptoms observed in animal models of human neurological pathology. Remarkably, the combination of drugs and EE effectively reduces drug-associated side effects and can promote the therapeutic effects of the drugs (71,76,80). Therefore, as a low-cost, low-risk and very effective method, EE exposure should attract widespread attention for the rehabilitation of neurodegenerative diseases.

Acknowledgements

Not applicable.

Funding

Funding: This study was supported by the Scientific Research Program of Health Commission in Shanxi of China (grant no. 2015158; to JZL) and the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi of China (grant no. 2019L0676 to JZL). Funders had no involvement in the study design; data collection, analysis and interpretation, manuscript writing or decision to submit the paper for publication.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

Study design: JZL and XHH. Experiment implementation: JZL, HPW and ML. Data analysis: XML and ZBW. Manuscript writing: JZL and XHH. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All experimental procedures followed the guiding principles of mammalian neuroscience research and were approved by the Ethics Committee for Animal Experiments at Changzhi Medical College (approval no. DW2015018).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References