• HIGHLIGHTS
- Treadmill exercise mitigated cognitive impairment in aged mice, as shown by the passive avoidance test.
- Exercise increased microvessel length and reduced fragmentation in the brains of aged mice while decreasing microglia and astrocyte activation, thus reducing neuroinflammation.
- Exercise mitigated brain microvascular and blood-brain barrier damage by preventing declines in tight junction proteins and platelet-derived growth factor receptor beta in aged mice.
INTRODUCTION
Aging is an inevitable biological process associated with various physiological changes throughout the body [1]. Among these changes, cognitive impairment is particularly significant because it affects quality of life and daily functioning. A primary cause of age-related cognitive impairment is dysfunction of the cerebral vascular system [2]. Cognitive impairment is characterized primarily by damage to microvessels, which are crucial for maintaining brain health. Microvessels, the smallest blood vessels in the brain, deliver oxygen and essential nutrients to brain cells and remove metabolic waste products. This process is vital for maintaining a stable and healthy neural environment. However, age-related damage to microvessels can impair cerebral blood flow, adversely impacting brain function [3]. Additionally, inefficient waste removal can result in neuroinflammation and neuronal damage. These microvascular changes ultimately exacerbate cognitive impairment [4].
The neurovascular unit (NVU) comprises endothelial cells, pericytes, astrocytes, neurons, and microglia, which function collaboratively to maintain the integrity of the blood-brain barrier (BBB) and ensure proper central nervous system function [5]. This complex interaction is essential for protecting the brain from fluctuations in blood composition and potential toxins. The BBB plays a critical role in sustaining the brain’s microenvironment by tightly regulating the influx of substances and ensuring normal neuronal function [6]. The BBB consists of endothelial cells, basement membranes, pericytes, and astrocytic end-feet lining the brain’s microvessels. Damage to these microvessels can compromise BBB integrity. When the BBB is impaired, harmful inflammatory and toxic substances from the bloodstream can infiltrate brain tissue [7]. Damage to the BBB can activate microglia, the primary immune cells of the brain, and astrocytes, which provide structural and functional support to neurons [8]. Activated glial cells initiate a neuroinflammatory response, and excessive neuroinflammation can damage neurons, exacerbating cognitive impairment [9]. Furthermore, chronic inflammation resulting from overactivated microglia and astrocytes can exacerbate BBB permeability, allowing more harmful substances to infiltrate brain tissue and accelerating neurodegeneration [8].
Exercise is widely recognized for its numerous benefits to cognitive function and brain health, primarily through positive effects on cerebral blood flow and neurovascular health [10]. Regular physical activity supports cognitive abilities such as attention, memory, and problem-solving by promoting neurogenesis and strengthening neural connections [11]. Exercise increases microvascular restoration by improving endothelial function and reducing arterial stiffness, thus facilitating cerebral blood flow and nutrient delivery to the brain [12]. These effects are critical for maintaining BBB integrity and mitigating age-related microvascular dysfunction. Consequently, exercise provides significant neuroprotective effects crucial for the prevention and management of neurodegenerative diseases associated with aging. Despite the well-documented benefits of exercise on general health and cognitive function, the mechanisms by which physical activity influences neurovascular health and mitigates age-related cognitive decline remain underexplored. Aging is associated with microvascular dysfunction and BBB disruption, which contribute to cognitive impairment and increase the risk of neurodegenerative diseases.
The present study aims to evaluate the effects of treadmill exercise on age-related cognitive impairment. Specifically, we investigated whether an 8-week treadmill exercise regimen in aged mice could ameliorate cognitive dysfunction by restoring neurovascular integrity, including that of the BBB, and by suppressing the activation of microglia and astrocytes in the brain. This research seeks to elucidate the mechanisms by which physical exercise can serve as a non-pharmacological intervention to counteract the effects of aging on the NVU.
MATERIALS AND METHODS
Animals
Nine-week-old and 12-month-old C57BL/6J male mice were purchased from Koatech (Korea) and allowed to acclimate to the laboratory environment for 1 week prior to the start of the experiment. The 12-month-old mice were then raised until they reached 20 months of age. Prior to exercise training, the mice were randomly assigned to 3 groups: young control (young, 9 weeks old, n =6), aging control (aging, 20 months old, n =6), and aging with exercise training (aging+Ex, 20 months old, n=6). All animals were housed under controlled conditions at a temperature of 22°C ±2°C, 60% humidity, and a 12-hour light-dark cycle. Food was provided ad libitum.
Treadmill Exercise Protocol
The 20-month-old mice in the exercise group underwent treadmill training sessions, each lasting 60 minutes, conducted once daily over an 8-week period. Before training began, mice in the aging+Ex group were allowed 3 to 5 minutes to acclimate to the treadmill apparatus. Each exercise session consisted of an initial 10 minutes at 5 m/min with no incline, followed by 10 minutes at 8 m/min without incline, and concluded with 40 minutes at 10 m/min without incline. Random assignment to the treadmill exercise group occurred prior to the training program’s initiation. Mice not participating in the exercise regimen were housed in the facility equipped with the treadmill apparatus but did not engage in training [13].
Passive Avoidance Test
The passive avoidance test is used to evaluate learning and memory in rodent models of neurodegenerative disorders. In this study, the apparatus consisted of 2 chambers separated by a wall with a guillotine door. During the training phase (day 1), mice were acclimated by allowing them to explore the dark chamber for 60 seconds. Subsequently, when the light was turned on, the mice instinctively moved to the dark chamber. Upon entry into the dark chamber, the guillotine door closed, and a mild electric shock (0.5 mA for 2 seconds) was delivered through the stainless-steel grid floor. During the retention test (day 2), the procedure was repeated, and latency to enter the dark chamber was recorded up to a maximum of 300 seconds. The latency times served as a measure of memory retention.
Tissue Preparation
After completion of the experiment, the animals were deeply anesthetized by a trained researcher in accordance with approved protocols and guidelines. Once adequate anesthesia was confirmed, euthanasia was performed via exsanguination. Subsequently, the animals were perfused with 50mM phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA). The brains were then collected, post-fixed in 4% PFA, and dehydrated in a 30% sucrose solution. After 3 days, the brains were cryosectioned into 30-µm-thick coronal sections using a freezing microtome (CM3050S; Leica Biosystems GmbH, Germany) at -23°C.
Immunohistochemistry and Immunofluorescence
Brain sections were processed using the free-floating method to visualize target proteins via immunohistochemistry and immunofluorescence techniques. For immunohistochemistry, endogenous peroxidase activity was blocked using a 3% hydrogen peroxide solution in PBS for 20 minutes at room temperature. The sections were then blocked for 1 hour with 1% bovine serum albumin (BSA) and 10% normal rabbit and goat serum in PBS. Brain tissues were incubated overnight at 4°C with primary antibodies targeting ionized calcium-binding adapter molecule 1 (Iba-1) (1:1,000; Abcam, UK), glial fibrillary acidic protein (GFAP) (1:1,000; Santa Cruz Biotechnology, USA), and platelet endothelial cell adhesion molecule-1 (PECAM-1) (1:1,000; LSBio, LifeSpan BioSciences, USA). Following primary antibody incubation, sections were treated with anti-rabbit and goat secondary antibodies (1:200; Vector Laboratories, USA) for 1 hour at room temperature. An antibody-biotin-avidin-peroxidase complex was then administered for 1 hour for amplification. Antibody binding was visualized using a DAB kit, applied for 3 minutes.
Lycopersicon esculentum lectin labeled with DyLight 488 (Thermo Fisher Scientific, USA) was injected intracardially at a concentration of 100 µg/mL over 60 seconds. For immunofluorescence, brain sections were washed and blocked with 3% BSA for 2 hours at room temperature. Sections were then incubated overnight at 4°C with primary antibodies against Iba-1, GFAP, and platelet-derived growth factor receptor beta (PDGFRβ) (1:1,000, Abcam). Tissues were washed and incubated with Alexa Fluor 594-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated rabbit anti-goat IgG (1:2,000; Molecular Probes, USA) for 60 minutes at room temperature. Following incubation, the tissues were washed again and mounted onto gelatin-coated slides. Coverslips were applied using 4’,6-diamidino-2-phenylindole. Fluorescence staining was examined with a confocal microscope (LSM 700, Zeiss, Germany). Image-Pro Plus (Media Cybernetics, USA) and ImageJ (National Institutes of Health, USA) were used for quantitative analysis of the immunohistochemistry data.
Western Blotting
Proteins were extracted from the mouse brain tissues and motor cortex using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific). Protein concentration was determined using a colorimetric protein assay kit (Bio-Rad, USA). For protein separation, 25-µg samples were electrophoresed on 8%–12% sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST; 10 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 2 hours at room temperature. Following blocking, the membranes were incubated overnight at 4°C with primary antibodies, including anti-zonula occludens-1 (ZO-1) (1:200; Lifespan Biosciences, USA), anti-PDGFRβ (1:1,000; Abcam), anti-occludin (1:200; Novus Biologicals, USA), anti-claudin-9 (1:500; Proteintech, USA), and anti-β-actin (1:5,000; Santa Cruz Biotechnology). After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-mouse) for 2 hours at room temperature. Protein bands were detected by chemiluminescence (Clarity Western ECL Substrate; Bio-Rad) and visualized using an imaging system (ChemiDoc; Bio-Rad). Relative protein expression levels were analyzed using Image-Pro Plus and ImageJ.
Statistical Analysis
All data are expressed as mean ±standard error of the mean. Statistical analyses were performed using IBM SPSS Statistics ver. 26.0 (IBM Corp., USA). Differences among groups were evaluated using 1-way analysis of variance followed by the Tukey post hoc test for multiple comparisons. A P-value less than 0.05 was considered to indicate statistical significance.
RESULTS
Treadmill Exercise Improves Cognitive Impairment in Aged Mice
To evaluate the effects of exercise on age-related cognitive impairment, we used the passive avoidance test (Fig. 1). Treadmill exercise was found to alleviate cognitive impairment in aged mice. Compared to young mice, the aged mice exhibited decreased latency time, indicating impaired cognitive function. However, an 8-week treadmill exercise intervention significantly increased latency time in aged mice compared to the non-exercising aging group (F = 12.116, P < 0.05). These results suggest that treadmill exercise can mitigate cognitive impairment associated with aging.
Treadmill Exercise Reduces Aging-Induced Microvascular Damage in the Cortex
PECAM-1 is expressed in all endothelial cells, including those in the brain’s microvascular system. This protein is crucial for maintaining vascular structure, facilitating angiogenesis, regulating platelet function, and controlling leukocyte and vascular permeability. PECAM-1, specifically expressed on the surface of brain microvascular endothelial cells, serves as a valuable marker for assessing microvessel length and fragmentation. In the aged mice, microvessel length in the cortex was decreased, but treadmill exercise intervention significantly extended this length (F = 50.239, P < 0.001). Furthermore, while aging was associated with an increase in microvessel fragmentation of less than 20 μm, treadmill exercise significantly reduced this fragmentation (F =17.029, P <0.05). These results indicate that treadmill exercise effectively alleviates age-related microvascular damage (Fig. 2).
Treadmill Exercise Alleviates Activation of Microglia and Astrocytes in the Brain
To investigate aging-induced activation of microglia and astrocytes, we evaluated the expression of Iba-1 and GFAP in the brain (Fig. 3). Aged mice exhibited increased expression of Iba-1 in the cortex and GFAP in the hippocampal CA1 region compared to young mice. However, an 8-week treadmill exercise intervention significantly reduced the expression of both Iba-1 (F=50.814, P<0.001) and GFAP (F=36.989, P<0.001) in aged mice. These results indicate that exercise intervention can mitigate the increased activation of microglia and astrocytes associated with aging.
Treadmill Exercise Reduces Aging-Induced BBB Damage in the Cortex
BBB damage is observed in the brains of aged mice. ZO-1, occludin, and claudin-9 are major tight junction proteins that constitute the BBB and play key roles in regulating the paracellular permeability of epithelial and endothelial barriers. Their expression decreases with age, resulting in increased BBB permeability. PDGFRβ, a receptor expressed by pericytes, is essential for maintaining BBB integrity and brain homeostasis. It regulates blood flow, aids in forming tight junctions between endothelial cells, and contributes to the repair and stability of the vascular network. Decreased PDGFRβ expression with age weakens the BBB, increasing its permeability and allowing harmful substances to enter the brain; these changes promote neuroinflammation and neurodegenerative disease. In the cortex of aged mice, protein levels of ZO-1 (F=25.356, P<0.001), PDGFRβ (F =13.385, P < 0.01), occludin (F =15.006, P <0.05), and claudin-9 (F=20.312, P<0.05) were reduced compared to young mice. However, treadmill exercise intervention in aged mice attenuated the decreases in these proteins. These results suggest that treadmill exercise can mitigate BBB damage caused by aging (Fig. 4).
DISCUSSION
Regular exercise is a potent complementary intervention for age-related cognitive decline, alongside pharmacological treatments. A recent study demonstrated that a 12-week exercise regimen could reduce cognitive decline in naturally aging rats [14]. Regular physical activity increases cerebral blood flow, which is often diminished with aging, thereby promoting the delivery of essential oxygen and nutrients to brain tissue and facilitating the removal of metabolic byproducts associated with aging [15]. When aging compromises the BBB, its permeability increases, allowing potentially harmful substances such as toxins and inflammatory molecules to enter the brain from the bloodstream [16, 17]. In response to these intrusions and the resulting stress on the brain’s microenvironment, astrocytes and microglia become activated. This activation can lead to chronic neuroinflammation, exacerbating cognitive impairment and contributing to neurodegenerative processes [9].
Aerobic training helps maintain the BBB by supporting the expression of tight junction proteins during aging [18, 19]. In this study, 20-month-old mice exhibited improved microvascular integrity and reduced BBB damage following an 8-week treadmill exercise regimen. The exercise also decreased neuroinflammation by reducing the activation of astrocytes and microglia, as evidenced by lowered levels of Iba-1 and GFAP in specific brain regions. Overall, regular treadmill exercise appears to improve cognitive function in aging by promoting brain vascular health, preserving BBB integrity, and reducing neuroinflammation.
Cognitive impairment due to aging has been shown to result from damage to microvessels in the brain [20]. This microvascular damage adversely impacts the NVU, which consists of endothelial cells, pericytes, astrocytes, neurons, and microglia, resulting in damage to the BBB and increased permeability. This altered permeability is primarily caused by damage to tight junction proteins [21]. In the present study, we observed that in 20-month-old mice, brain microvessels were shortened and fragmented, and the levels of tight junction proteins such as ZO-1, occludin, and claudin-9 —components of the BBB — were reduced. These tight junction proteins are crucial in regulating BBB permeability [17]. Reduced expression of ZO-1, occludin, and claudin-9 disrupts tight junctions and increases BBB permeability, allowing potentially harmful substances to enter the brain. Together, ZO-1, occludin, and claudin-9 form a complex network that supports the structural integrity of the BBB [6]. A decline in the expression of proteins forming the BBB complex may exacerbate barrier leakage, thereby contributing to neuroinflammation and neural injury [22]. Maintaining ZO-1, occludin, and claudin-9 expression is therefore crucial for protecting the brain from age-related damage and preserving cognitive function. This study revealed that treadmill exercise reduced microvascular damage, improved BBB integrity, and decreased the activation of microglia and astrocytes involved in neuroinflammation. Our previous research demonstrated that regular treadmill exercise prior to cerebral ischemia can protect against BBB damage caused by chronic cerebral hypoperfusion and improve cognitive function [23]. Accordingly, sustained physical activity contributes to the integrity of brain microvasculature and the BBB, particularly throughout the aging process.
Damage to the BBB due to aging increases hyperactivity of astrocytes and microglia in the brain, contributing to cognitive impairment [24]. Astrocytes typically maintain homeostasis, support neurons, and regulate BBB integrity. However, when activated, they can produce inflammatory cytokines and other molecules that contribute to neuroinflammation. Microglia, which respond to injury or pathogens, release inflammatory cytokines and function as phagocytic cells to remove debris and damaged cells when activated. Excessive activation of astrocytes and microglia due to aging intensifies neuroinflammation, adversely affecting neuronal health and function [9]. Neuroinflammation exacerbates cognitive decline and microvascular dysfunction, compromising cerebral blood flow and the NVU while increasing BBB permeability. This study indicates that treadmill exercise effectively reduces astrocyte and microglial activation in aged mice, helping to mitigate these effects. Previous studies have shown that exercise can alleviate cognitive impairment by modulating neuroinflammation, reducing microglial activation, and decreasing the production of pro-inflammatory cytokines and reactive oxygen species, thus exhibiting neuroprotective effects in neurodegenerative diseases [25, 26]. This reduction in microglial activation suggests decreased neuroinflammation, which may protect neural integrity and improve cognitive function.
In conclusion, this study emphasizes the role of regular treadmill exercise in alleviating cognitive decline associated with aging. Exercise not only mitigates brain microvascular and BBB damage but also reduces neuroinflammation by decreasing excessive activation of astrocytes and microglia. These results suggest that regular physical activity may serve as a nonpharmacological strategy to combat cognitive decline by maintaining the integrity of microvascular and BBB structures and reducing neuroinflammation.
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