Abstract
Background/Aim: Age-related cerebral microvascular loss is associated with reduced angiogenesis and leads to inadequate tissue perfusion in the brain. Vascular endothelial growth factor (VEGF), crucial for angiogenesis, declines with age. Exercise enhances microvascular density and blood flow, promoting brain health. The specific mechanisms underlying exercise-induced angiogenesis in aging brains remain unclear. This study investigated the impact of exercise on microvessel density, tissue perfusion, and VEGF-angiogenic signaling in aging brains.
Materials and Methods: Male rats were divided into three groups: an age-matched control, a sedentary control, and an exercise-trained group, which underwent swimming exercise. Regional cerebral tissue perfusion (CTP) was assessed in an in vivo study. Brain immunohistochemical staining of glucose transporter-1 (GLUT1) was used to determine microvessel density and endothelial metabolic state. Immunoassays of VEGF angiogenic proteins in isolated brain microvessels were conducted to reveal microvascular signaling mechanisms.
Results: Microvessel density and regional CTP in the sedentary control group were significantly reduced compared to the age-matched control group. The exercise-trained group demonstrated substantial increases in microvessel density and regional CTP compared to the sedentary control group. An increase in GLUT1 in the exercise group indicates microvascular metabolic restoration. VEGF angiogenic protein levels were significantly decreased in the sedentary control group compared to the age-matched control group; however, exercise training significantly increased these protein levels relative to the sedentary control group. Furthermore, we discovered a strong and significant positive correlation among the microvessel density, regional CTP, and VEGF angiogenic proteins across the three groups.
Conclusion: Swimming exercise training may protect against age-induced cerebral microvascular loss and insufficient brain tissue perfusion through the VEGF-mediated angiogenic signaling cascade. The increase in GLUT1 indicates restored structural and metabolic endothelial function in the aging brain.
Introduction
Growing evidence suggests that brain tissue perfusion and its functional capacity deteriorate significantly with age (1), leading to insufficient delivery of nutrients and oxygen, thereby increasing the risk of cerebrovascular alterations (2). Regular blood flow to the brain depends on maintaining microvascular density (3). However, angiogenesis – a crucial process in the revascularization of hypoperfused and ischemic tissues and in the repair of damaged tissues – is progressively disrupted by aging (4-6). The mechanisms underlying this microvascular deterioration are multifactorial. Among these, oxidative stress plays a central role in driving brain aging (7). This oxidative damage impairs angiogenic capacity and reduces cerebral blood flow (7, 8), probably accelerating neurovascular function decline. Collectively, the decreased microvascular density in aged brains is a significant factor that increases the susceptibility of vulnerable regions to ischemic brain damage (9).
Vascular endothelial growth factor (VEGF) is a central mediator promoting angiogenesis in both physiological and pathological conditions (10, 11). The angiogenic actions of VEGF occur through its tyrosine kinase receptors: VEGF receptor (VEGFR) 1 (Flt1), VEGFR2 (Flk-1; KDR as a human counterpart), and VEGFR3 (Flt4) (12). Flk-1 is the key VEGF receptor in endothelial cells, responsible for transducing VEGF angiogenic signals (13). In advanced age, the expression of VEGF and Flk-1 is decreased in brain tissues (14), as well as in the brain vasculature (15). VEGF initiates an angiogenic signaling cascade that includes phosphatidylinositol-3 kinase (PI3K)/Akt (10, 13, 16); however, the specific intracellular signaling pathways involved in VEGF/Flk-1 angiogenic signaling in aging brains remain poorly understood.
Brain endothelial cells are highly glycolytic, obtaining most of their energy from glucose breakdown via the glucose transporter 1 (GLUT1)-mediated uptake, which is essential for endothelial proliferation during vessel formation (17). During active cerebral angiogenesis, tip cells rely on glycolysis for proliferation in the glucose-limited, avascular microenvironment, making GLUT1 expression a powerful indicator of active vascular growth (18). VEGF has been shown to increase GLUT-1 gene expression and facilitate glucose transport in brain vascular endothelial cells, establishing a link between GLUT-1 expression and pro-angiogenic signals (19). GLUT1 expression decreases significantly with age in brain microvasculature, aligning with a decline in cerebral microvascular density (20). The link between brain glucose transport and angiogenesis in aging reflects metabolic-vascular disruption that significantly contributes to age-related cognitive decline and could be exacerbated in neurodegenerative diseases.
Exercise has been shown to benefit brain health in both human and animal studies, including increased cerebral tissue perfusion and angiogenesis, as well as improved memory and cognitive abilities (21-24). Moreover, physical exercise, a non-pharmaceutical intervention, improves cerebrovascular deterioration and age-related cognitive impairment in both humans and animals (25, 26). Several studies have shown that exercise training increases tissue perfusion and angiogenesis in aged brains (24, 27-29), potentially improving brain function. Increased angiogenesis with exercise training has been reported to be associated with the up-regulation of VEGF mRNA and protein (30). Our previous study demonstrated that exercise improves age-related hypoperfusion and microvascularization changes, accompanied by increased VEGF and reduced oxidative stress in the aged rat brain (8). However, our previous work focused on cortical vascularization, which may not fully capture the complexity of cerebral microvascular changes associated with aging. This study expands the investigation into microvessels within the brain parenchyma, providing a comprehensive assessment of age-related microvascular alterations and the impact of exercise. GLUT1, highly expressed in brain microvascular endothelial cells, serves as a reliable marker for measuring capillary density in the brain (31). GLUT1 reflects both glucose uptake and endothelial cell abundance and transporter expression, offering an integrated index of vascular integrity and metabolic support. Using GLUT1 staining enables the sensitive detection of age-related loss of cerebral microvessels and the evaluation of exercise-induced microvascular changes.
The aim of this study was to determine whether exercise training ameliorates age-related brain microvascular rarefaction and hypoperfusion by quantifying microvessel density using GLUT1 staining, examining VEGF/Flk-1/PI3K/Akt signaling in isolated brain microvessels, and evaluating cerebral perfusion.
Materials and Methods
Animals. Eight-week-old male Wistar rats were obtained from the National Laboratory Animal Center, Mahidol University (Nakhon Pathom, Thailand). They were housed in the Laboratory Animal Facility, Faculty of Medicine, Chulalongkorn University (Bangkok, Thailand), and maintained until they reached 4 months or 20 months of age. Rats were kept under a 12:12 h light-dark cycle with a controlled temperature of 22-24°C and humidity of 50-60%. Animals were fed standard laboratory chow, and tap water was available ad libitum. Rats were randomly divided into three experimental groups (n=5 per group): an age-matched control group (AC; 4 months old), a sedentary control group (SC; 20 months old), and an exercise-trained group (ET; 20 months old). Each animal served as a biological replicate, yielding five biological replicates per group. This study received approval from the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chulalongkorn University (No.14/2559), and adhered to the guidelines of the National Research Council of Thailand.
Exercise training program. The exercise training was conducted in accordance with the Resource Book for the Design of Animal Exercise Protocols (American Physiological Society, 2006) and a previously published protocol (32). Animals in the exercise-trained (ET) group were individually subjected to swimming exercise (once a day, 5 days a week, for 8 weeks) in a cylindrical, semi-transparent plastic container (50 cm diameter and 65 cm height) filled with water at a depth of 55 cm, maintained at a temperature of 35±2°C. For adaptation to exercise, the duration of swimming was 15 min in the first 2 days and progressively increased by 15 min until it reached 60 min over the first week. The exercise program was continued for ET rats at 60 min/day for the remainder of the 7-week training period. After swimming, the rats were dried before being returned to their cages. The sedentary control (SC) group was exposed to the same stress as the ET group, including daily transfer to the training room, being individually placed in the container with water at a depth of 5 cm maintained at a temperature of 35±2°C for 30 min at the same time points as that of the ET group, and drying after 30 min of immersion.
In vivo measurement of cerebral perfusion. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg), tracheostomized, and mechanically ventilated with room air supplemented with oxygen via a cannula using a rodent ventilator. To maintain physiological conditions, body temperature was kept at 37°C using a feedback-controlled rectal probe and a homeothermic blanket. The femoral vein was cannulated for additional anesthetic, while the femoral artery was cannulated to draw blood samples for physiological level monitoring of pH, PO2, and PCO2.
After securing the animal’s head in a custom-made stereotaxic frame, a high-speed micromotor drill (OmniDrill35, World Precision Instruments, Sarasota, FL, USA) was used to perform a 3-mm circular craniotomy at the center of the left parietal bone. Following the craniotomy, the dura mater flaps were carefully retracted to the edges of the cranial window, exposing the cortical surface. A custom metal ring was attached to the cranial window with dental cement, and the windows were filled with artificial cerebrospinal fluid.
After surgically exposing the cortical brain, real-time noninvasive assessment of regional cerebral tissue perfusion (CTP) was performed using a laser Doppler perfusion monitoring system (Periflux 5000, Perimed, Järfälla, Sweden). The optical needle probe was positioned over the cortical surface, avoiding large vessels. The laser Doppler signals were transferred to the PeriSoft software (Perimed) for the analysis of regional CTP. For each rat, CTP was measured at three different probe sites on the cortical surface. At each site, the probe remained in place for 90 sec, and CTP values were determined by averaging three consecutive 30-sec intervals. Then, the mean of these three site readings was calculated to give one CTP value per animal.
Immunohistochemical staining. After transcardial perfusion with 200 ml of ice-cold phosphate-buffered saline, a 2-mm-thick coronal section of brain tissue from the parietal lobe was subjected to immunohistochemical (IHC) staining of GLUT1, a marker of endothelial glucose uptake and microvessel density. IHC staining was performed using Leica Bond automated immunostaining system with Bond polymer refine detection kit (Leica Biosystems, Deer Park, IL,USA, DS9800) and Bond epitope retrieval solution 2 (ER2; Leica Biosystems, AR9640), following the manufacturer’s instructions. Briefly, after deparaffinization of 3-μm-thick formalin-fixed, paraffin-embedded brain sections at 72°C and washing with graded alcohols, the sections were pretreated with heat-induced antigen retrieval with ER2 at 100°C for 25 min. Endogenous peroxidase activities were blocked with peroxide block reagent provided in the detection kit for 5 min. Sections were then incubated with primary antibody, rabbit polyclonal anti-GLUT1 antibody (Cell Marque, Rocklin, CA, USA, 335A-15, 1:200) for 40 min at room temperature. After washing, the biotin-free polymeric horseradish peroxidase reagent was applied for 10 min, followed by 3,3-diaminobenzidine (DAB) chromogen for 5 min and counterstained with hematoxylin for 5 min. The slides were then dehydrated, cleared, and finally cover-slipped with permanent mounting medium.
Quantitative image analysis for microvessel density. IHC staining images were captured using an Eclipse Ni-U microscope (Nikon Corporation, Tokyo, Japan) at 100× magnification. A circular region of interest (ROI) covering the entire stained area was used, and this ROI was divided into eight equal parts. From each part, three non-overlapping images were captured, resulting in a total of 24 images per stained section. All images were captured under the same settings and conditions to ensure consistency across samples. Quantitative image analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) under blinded conditions, as previously described (33). Briefly, each IHC image was processed to separate brown DAB staining for GLUT1 protein expression from the blue hematoxylin counterstains. For thresholding the brown DAB-stained image, the minimum threshold value was set to zero, and the maximum threshold value was set to remove the background signal without affecting the actual signal. The software’s mean gray value option was used to calculate the percentage of the DAB signal area. Microvessel density was expressed as a percentage of the GLUT1-positive area. The microvessel density for each animal was calculated by averaging measurements from the 24 non-overlapping images. Two blinded observers independently analyzed all images, and the mean value from their assessments was used for analysis.
Isolation of brain microvessel fractions. The remaining perfused brain tissues, after removing part of the parietal cortex for immunohistochemical analysis, were processed to isolate microvessel fractions using a previous method (34). The microvessel isolation protocol was performed at a temperature of 4°C. The brain tissues were cut into small pieces and homogenized in 0.32 M sucrose solution containing 3 mM HEPES (pH 7.4) using a tissue grinder homogenizer (Glas-Col, Terre Haute, IN, USA). The brain homogenate was centrifuged at 1,000 × g for 10 min twice. The suspended pellet in the sucrose buffer was centrifuged at 100 × g for 10 min twice. Then the pooled supernatants from two consecutive centrifugations were centrifuged at 200 × g for 2 min. The pellet underwent two washes with the sucrose buffer and one wash with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), performed at 200 × g for 2 min. The pellet was centrifuged at 14,000 × g for 2 min after being resuspended in 1 ml of 0.1% BSA. The final pellet, which contained brain microvessel fractions, was stored at −80°C. To assess the purity of the isolated microvessel preparation, air-dried smears of the microvessel fractions were stained with hematoxylin and examined under a light microscope.
Immunoassays for VEGF angiogenic proteins. Post-nuclear supernatants were prepared by homogenizing isolated brain microvessel fractions in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors, followed by centrifugation at 1,000 × g for 5 min at 4°C. The resulting supernatants were aliquoted and stored at −80°C for later use in immunoassays of VEGF, Flk-1, PI3K, and phospho-Akt (p-Akt). Total protein concentration in the post-nuclear supernatants was measured using a bicinchoninic acid protein assay kit (23252; Pierce Biotechnology, USA). Protein levels of VEGF, Flk-1, PI3K, and p-Akt were measured using commercially available ELISA kits according to the manufacturer’s instructions. VEGF (MMV00), Flk-1 (MVR200B), and p-Akt (DYC887B-2) kits were obtained from R&D Systems (Minneapolis, MN, USA), and PI3K kit (E-EL-R0739) was obtained from Elabscience Biotechnology Co., Ltd (Wuhan, China). ELISA assays were performed in duplicate for each sample, and the average of each pair of wells was used for analysis.
Statistical analysis. Data are presented as mean±standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to assess statistical significance, followed by Tukey’s post hoc test for multiple comparisons. Correlations among physiological and molecular parameters were examined using Pearson’s correlation coefficient. A p-value of less than 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).
Results
Effect of exercise training on microvessel density and tissue perfusion in the aging brain. Figure 1A displays representative images of GLUT1 IHC staining in the brain from the AC, SC, and ET groups. Compared to the AC group, the SC group showed reduced GLUT1-positive microvessel coverage, which was restored in the ET group. Quantitative analysis of microvessel density, shown as the percentage of GLUT1-positive area (Figure 1B), demonstrated significantly lower values in the SC group than in the AC group (p<0.001), but significantly higher in the ET group (p<0.001). The increased GLUT1-positive staining observed in the exercise-trained group not only indicates preserved microvessel structure but also suggests enhanced endothelial glucose transport. Regional CTP measurements (Figure 1C) revealed a significant decrease in the SC group compared to the AC group (p<0.0001). Conversely, the ET group had notably higher regional CTP than the SC group (p=0.0139). Regression analysis indicated a strong positive correlation between regional CTP and microvessel density (r2=0.8122, p<0.0001) across all groups (Figure 1D), emphasizing a close link between structural changes in the cerebral microvasculature and perfusion capacity.
Microvessel density and tissue perfusion in the brain of AC, SC, and ET groups. (A) Representative immunohistochemical staining images of brain sections with GLUT1 antibody, delineating the boundaries of microvessels. Magnification 100×; scale bar=50 μm. (B) Percentage of GLUT1-positive area, determining microvessel density. (C) Regional CTP at the cortical surface, measured using a laser Doppler probe through the cranial window. (D) Correlation analysis of regional CTP and microvessel density in the three groups (r2=0.8122, p<0.0001). Data are mean±SEM. One-way ANOVA with Tukey’s post hoc test was used. ***p<0.001, ****p<0.0001 vs. AC, respectively; #p<0.05, ###p<0.001 vs. SC, respectively. AC: Age-matched control group; SC: sedentary control group; ET: exercise-trained group; GLUT1: glucose transporter-1; CTP: cerebral tissue perfusion.
Effect of exercise training on the expression of angiogenic signaling proteins in isolated microvessels of the aged brain. Figure 2 shows the impact of swimming exercise training on the expression of key angiogenic proteins that regulate cerebral angiogenesis during aging. The levels of VEGF and Flk-1 proteins in isolated brain microvessels were significantly lower in the SC group compared to the AC group (p<0.0001). However, exercise training reduced the aging-related decreases in VEGF and Flk-1 expression in the isolated brain microvessels (p=0.0008 and p=0.0493, respectively; Figure 2A and B). Additionally, the levels of PI3K and p-Akt proteins in isolated brain microvessels were significantly lower in SC rats compared to AC rats (p=0.0181 and p<0.0001, respectively; Figure 2C and D). The expression of p-Akt protein in isolated brain microvessels was significantly increased in ET rats (p<0.0282, Figure 2D), while the level of PI3K in these microvessels did not differ significantly between SC and ET rats (p=0.1351); however, it tended to be lower in ET rats than in SC rats (Figure 2C).
Expression of angiogenic signaling proteins in isolated microvessels of the brain of AC, SC, and ET groups. Concentrations of VEGF (A), Flk-1 (B), PI3K (C), and p-Akt (D) proteins were measured by immunoassays. One-way ANOVA with Tukey’s post hoc test was used. *p<0.05, ****p<0.0001 vs. AC, respectively; #p<0.05, ###p<0.001, vs. SC, respectively. AC: Age-matched control group; SC: sedentary control group; ET: exercise-trained group; VEGF: vascular endothelial growth factor.
Relationship between cerebral microvessel density and angiogenic signaling proteins in isolated brain microvessels. Considering changes in the expression of key angiogenic proteins, VEGF and Flk-1 are recognized as potent regulators of microvessel density maintenance. Here, a significant positive correlation between microvessel density and VEGF (r2=0.7796; p<0.0001) and Flk-1 (r2=0.8669; p<0.0001) protein expression in isolated brain microvessels (Figure 3A-B) was observed. Flk-1, which is activated by VEGF, engages in a critical PI3K/Akt signaling pathway involved in angiogenesis. Our results demonstrated a significant positive correlation between the expression of VEGF protein and the expression of PI3K (r2=0.6845; p=0.0009) and p-Akt (r2=0.7226; p=0.0002) proteins (Figure 3C-D).
Correlation analysis of microvascular alteration and angiogenic signaling proteins in the brain microvessel. (A-B) Positive correlation between microvessel density and VEGF (r2=0.7796, p<0.0001) and Flk-1 (r2=0.8669, p<0.0001) in AC, SC, and ET groups. (C-D) Positive correlation between VEGF and PI3K (r2=0.6845, p=0.0009) and p-Akt (r2=0.7226, p=0.0002) among the three groups. AC: Age-matched control group; SC: sedentary control group; ET: exercise-trained group; VEGF: vascular endothelial growth factor; PI3K: phosphatidylinositol-3 kinase.
Discussion
We revealed the protective effects of swimming training on brain vascularization, as demonstrated by increased microvessel density and improved tissue perfusion in aged rats, with these factors exhibiting a marked positive correlation. Microvessel density was determined by IHC staining for GLUT1, ensuring a reliable assessment of microvascular changes and endothelial metabolic function in response to exercise training. Additionally, we demonstrated that the key angiogenic proteins VEGF, Flk-1, PI3K, and p-Akt were up-regulated in aged brain microvessels following exercise training. The positive correlation between microvessel density and microvascular angiogenic proteins in aged brains suggests that the enhancement of cerebral perfusion by exercise training may be related to the up-regulation of the VEGF/Flk-1/PI3K/Akt signaling pathway.
Microvascular rarefaction in the brain during aging may contribute to reduced cerebral perfusion, leading to cerebrovascular pathogenesis (9). It has been demonstrated that age-related microvascular loss occurs in several brain regions, including the cerebral cortex, hippocampus, and corpus callosum (28, 35). Age-related global and regional CTP reductions have also been reported to occur in both cortical and subcortical areas, which are located in the vicinity of areas of reduced neural activity (36). Our present study demonstrated a significant positive relationship between cerebral microvessel density and regional CTP in advanced age, suggesting that a reduction in cerebral microvessel density is a key determinant of cerebral tissue perfusion, probably linked to age-related decline in functional outcomes of the brain.
VEGF is a well-known and crucial pro-angiogenic factor that stimulates both physiological and pathological angiogenic processes. VEGF exerts its effects mostly through binding to its receptor, VEGFR2, also known as Flk-1 (37). VEGF and Flk-1 expression in various organs, including the brain, significantly decreases with age (14, 15). Reduced VEGF/Flk-1 signaling has been linked to age-related angiogenesis impairment (38). Hypoxia-induced angiogenesis and up-regulation of VEGF protein are reportedly attenuated in aged mouse brains (5). Decreased Flk-1 expression is associated with reduced vascular endothelial growth factor-mediated angiogenesis in aged brains (4). Aging has also been reported to decrease the angiogenic capacity of isolated cerebrovascular endothelial cells (1). The current study revealed a significant reduction in VEGF and Flk-1 levels in isolated microvessels of the brains of SC rats. This suggests that a decrease in VEGF/Flk-1 signaling during aging occurs at the microvascular level, affecting angiogenesis, and possibly resulting in insufficient brain perfusion.
GLUT1 is widely recognized as a reliable marker for cerebral microvessels due to its strong endothelial localization and its ability to quantify microvessel density in both physiological and pathological conditions consistently (31, 39). Beyond its methodological application, GLUT1 plays a functional role in endothelial glucose uptake, angiogenic sprouting, and blood–brain barrier integrity (17). In the aging brain, reduced GLUT1 expression together with microvascular rarefaction has been associated with impaired cerebral glucose transport and cognitive decline (40). Our study showed that exercise training significantly increased GLUT1-positive staining in the brains of aged rats, suggesting not only structural preservation but also the restoration of endothelial metabolic function. This restoration corresponds with recent evidence indicating that intrinsic cellular rejuvenation pathways could reduce age-related cellular senescence and functional decline (41). A compensatory mechanism probably involves VEGF, which can restore GLUT1 expression and promote angiogenesis to sustain vascular and metabolic homeostasis (42, 43). VEGF has been shown to increase GLUT1 expression via the PI3K/Akt pathway (43, 44), which was also up-regulated in our exercised animals, supporting the exercise-induced links between VEGF signaling and angiogenic responses, as well as metabolic support. Importantly, aerobic exercise has been shown to increase VEGF and GLUT1 expression, enhance cerebral microvessel density, and improve tissue perfusion (42, 43), which is consistent with our observed improvements in microvascularization and cerebral blood flow.
Under both normoxic and hypoxic conditions, VEGF stimulation of angiogenesis is partly regulated by PI3K/Akt signaling (45). PI3K inhibitors have been shown to prevent VEGF-induced neovascularization both in vivo and in vitro (46, 47). In the current study, we demonstrated that the protein levels of VEGF, Flk-1, PI3K, and p-Akt were lower in isolated brain microvessels of aged rats. A positive linear relationship between microvessel density and VEGF/Flk-1, VEGF, and PI3K/Akt was also observed, indicating that VEGF, Flk-1, PI3K, and p-Akt levels in the brain microvasculature decreased with age. Our findings suggest a link between VEGF/Flk-1 and PI3K/Akt signaling in the brain microvasculature, which may contribute to the age-related decline in brain microvascular density.
Numerous studies have documented the angiogenic effects of exercise training on the brain during adulthood (8, 21, 27, 28). Exercise-induced increases in microvessel density in aged brains may have beneficial effects that eventually improve cerebral tissue perfusion (24, 29, 48). The present study demonstrated that an 8-week swimming exercise training program ameliorated the aging-induced decrease in microvessel density and tissue perfusion in the brain. Furthermore, the observed positive correlation between microvessel density and regional CTP indicates an increase in microvessel density, promoting cerebral perfusion. The effect of exercise training on increasing the microvascular density in the brain has been linked to increased VEGF protein levels (49), particularly in middle-aged and older adults (49, 50). In a recent study on 2-month-old mice, 1 hour of daily treadmill exercise for 5 weeks increased the microvascular density in the hippocampus, which is linked to the activation of the PI3K/Akt signaling pathway (51). Our current microvascular findings show that exercise training can increase VEGF, Flk-1, PI3K, and p-Akt protein expression in isolated microvessels of aged brains, which is closely correlated with increased microvessel density. This suggests that exercise training can improve cerebral tissue blood flow perfusion, which is related to the stimulation of cerebral neovascularization through the VEGF/Flk1/PI3K/Akt signaling pathway (Figure 4).
Proposed mechanism of the effects of swimming exercise training on tissue perfusion in aged brains via angiogenic VEGF/Flk-1/PI3K/Akt signaling and VEGF-induced glucose uptake. VEGF: Vascular endothelial growth factor; PI3K: phosphatidylinositol-3 kinase.
It is widely recognized that angiogenesis is regulated by the dynamic balance between vessel proliferation, driven by pro-angiogenic mechanisms, and vessel regression, mediated by anti-angiogenic mechanisms. In aged rats, exercise training has been reported to induce an increase in brain vascularization, which is associated with an increase in microvascular number through the angiogenesis process. The primary cause of the increase in microvascular number, according to researchers, is the proliferation of growing blood vessels, which is stimulated by pro-angiogenic factors such as VEGF, angiopoietin, and platelet-derived growth factor (27, 28, 49, 52, 53). Researchers have reported that exercise training can prevent vessel regression during advanced age by down-regulating anti-angiogenic factors; however, these findings are limited to cardiac and skeletal muscle tissues and do not apply to brain tissues (54, 55). Consequently, the current state of the matter is that it is uncertain whether the impact of exercise training on the angiogenesis process is a result of the down-regulation of anti-angiogenic factors or the inhibition of vessel regression. This uncertainty is consistent with evidence suggesting that age-related vascular decline may result from impairments in essential cellular homeostatic mechanisms, including mitophagy, a decline observed in other aging tissues as well (56). This is a subject that requires further investigation in the future.
Conclusion
The present study suggests that an 8-week swimming exercise training program can enhance microvessel density and tissue perfusion in aged rat brains, which involves the activation of the VEGF/Flk-1/PI3K/Akt signaling pathway at the microvascular level. Moreover, the use of GLUT1 IHC staining highlights that exercise not only counteracts age-related microvascular rarefaction but may also restore endothelial metabolic function through VEGF/PI3K/Akt–dependent up-regulation of GLUT1. This dual structural and functional restoration underscores the integrative role of VEGF and GLUT1 in maintaining cerebral perfusion during aging. Therefore, the findings emphasize the potential of regular aerobic exercise, such as swimming, as a non-pharmacological approach to enhance vascularization in the aging brain, providing a mechanistic basis for understanding how exercise supports microvascular remodeling and enhances tissue perfusion and glucose uptake in the aging brain.
Acknowledgements
The Authors would like to thank Ms. Maethinee Sakhakorn and Ms. Channipa Chanpakdee for their assistance during the research at the laboratory animal facility, and Mr. Thongbai Janseecha for his support in preparing brain tissue for immunohistochemical staining.
Footnotes
Author’s Contributions
S.V. conceived and designed the study, performed the experiments, analyzed the data, and drafted the manuscript. D.B. assisted with data collection and contributed to manuscript revision. S.P. contributed to study design, provided resources, supervised the work, and critically revised the manuscript. All authors read and approved the final version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest.
Funding
This work was supported by the Thailand Research Fund (TRF), the Commission on Higher Education (CHE), and Rangsit University (RSU) under the TRF-CHE-RSU Research Grant (MRG5680057) and by the Rangsit University Research Grant (RSUG29/2563).
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (Grammarly) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.
- Received November 19, 2025.
- Revision received December 13, 2025.
- Accepted January 29, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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