High-Intensity Interval Exercise Regulates Neurotrophic Factors and Astrocyte Activity in the Hippocampus and Cerebral Cortex
Article information
Trans Abstract
PURPOSE
Exercise training is widely recognized as a complementary intervention that provides both therapeutic and preventive benefits for neurodegenerative diseases, given its strong association with brain health. Regulating the expression of neurotrophic factors can significantly enhance cognitive function and memory processes, particularly in conditions such as Alzheimer's disease (AD). However, the effects of high-intensity interval aerobic exercise training (HIE) on brain-derived neurotrophic factors and astrocyte activation in the cortex (CTX) and the hippocampus (HIP) remain unclear. Therefore, this study aimed to investigate whether HIE regulates the expression of neurotrophic factors and neuroinflammatory cytokines in the CTX and HIP.
METHODS
Eighteen-week-old female C57BL/6 mice were assigned to control (Cnt) and HIE groups. After 12 weeks of HIE, the brains were harvested for immunoblotting, immunofluorescence, real-time PCR (qRT-PCR), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays.
RESULTS
HIE increased the expression of glial cell line-derived neurotrophic factor (GDNF) protein in the CTX but not in the HIP. Additionally, HIE exerted a protective effect against neuroinflammation by significantly upregulating A2-specific astrocytic transcripts. These findings suggest that HIE promotes neurotrophic and anti-inflammatory responses in both CTX and HIP.
CONCLUSIONS
Our study results suggest that HIE exerts protective regulatory effects on neurotrophic factors, neuroinflammation, and A1/A2 type astrocytic reactivity in CTX and HIP of animal models. These findings suggest that HIE has beneficial effects on brain health. However, as these results were derived from animal studies, further clinical research is required to determine whether similar neuroprotective effects can be observed in humans.
INTRODUCTION
In the brain, astrocytes and microglia play a crucial role in the immune response within the central nervous system (CNS) [1]. Under various physiological and pathological conditions, astrocytes and microglia undergo morphological and functional changes to regulate neurotrophic factors, neuroplasticity, and neuroinflammation, potentially influencing cognitive function and overall brain health [2,3]. Crosstalk between astrocytes and microglia is tightly involved in brain homeostasis by releasing cytokines and chemokines. Glia-cell line-derived neurotrophic factor (GDNF) is known to be an essential astrocyte molecule for supporting motor neuron survival and the modulation of microglial activation [4,5]. Also, glial fibrillary acidic protein (GFAP) is an astrocyte marker, and ionized calcium-binding adapter molecule 1 (Iba-1), which is an identification of microglia, are indicative of various neurological conditions, including brain injury and neurodegenerative diseases [6]. The activated microglia and astrocytes can be polarized into pro-inflammatory (M1/ A1) and anti-inflammatory (M2/A2) phenotypes, releasing pro-inflammatory and neurotoxic cytokines, and secreting anti-inflammatory and neuroprotective cytokines, respectively [6,7]. Therefore, it is imperative to investigate the neurotrophic factors influencing the phenotypic alterations of microglia and astrocytes in brain health regulation.
There are conventional therapeutic approaches for managing neurodegenerative disorders that typically involve a combination of medication, physical and cognitive therapies, and lifestyle modifications [8,9]. A growing body of clinical evidence has documented the beneficial effects of exercise training on brain health, characterized by the facilitation of neurotrophic factor production and the attenuation of pro-inflammatory cytokine secretion [10,11]. Given the robust association between exercise and brain health, the release of neurotrophic factors, neurogenesis, and astrogenesis can bring substantial benefits for maintaining cognitive function and memory processes in the progression of neurodegenerative diseases, notably dementia, Alzheimer's disease (AD), and Parkinson's disease (PD) [12,13]. Exercise-induced astrocyte activation increases astrocyte end-foot on cerebral blood vessels, glutamate uptake, and the release of neurotrophic factors [4]. However, there is existing a knowledge gap not only in the exploration of neurotrophic, such as GDNF and GFAP expression, but also in the phenotype switch on microglia and astrocytes in response to different exercise modalities, which could be useful and practical knowledge to prescribe appropriate exercise intensity and duration suited for patients with neurodegenerative diseases to reduce their disease progression and symptoms [14,15].
Based on the evidence mentioned above, an increasing number of studies have defined the essential role of exercise training on overall brain health by showing a morphological and neurochemical alteration in various brain regions, and it still remains challenging to exert the underlying mechanism for astrocyte and microglia functions in response to different types of exercise for brain homeostasis. High-intensity interval training aerobic exercise (HIE), which is comprised of repeating short bouts of maximal aerobic exercise leading to increases in cardiorespiratory fitness, could help to ameliorate neural plasticity and cognitive impairment in AD brain [16,17]. Nevertheless, only limited studies have investigated the effects of HIE on neurotrophic factors and astrocyte activity in the brain. Therefore, the current study aimed to investigate the impact of HIE on modulating neurotrophic factors and the activity of neuroprotective astrocytes, ultimately providing rehabilitation programs for patients with neurodegenerative diseases. We hypothesize that HIE will positively regulate the expression of neurotrophic factors and the activation of astrocytes in the hippocampus and cerebral cortex, which play distinct roles in learning, memory retrieval, and consolidation.
METHODS
1. Experimental animals
Female C57BL/6 mice (18 weeks) were supplied from Central Lab (Animal Inc., Seoul, Korea) and were randomly assigned into control (Cnt, n=9) and high-intensity interval exercise training (HIE, n=9) groups. The mice were housed in an animal facility with a controlled temperature (22-23°C), under 12-hour of light/dark cycle, and were provided a standard chew with ad libitum access to food and water. 24 hours after the last exercise session (31-week-old), all mice were sacrificed by intraperitoneal injection of sodium pentobarbital (100 mg/kg), and their brains were immediately excised. All experimental protocols were approved by the Institutional Animal Care and Use Committee at Inha University (INHA 220822-837-6). The described experiments on animals were performed in agreement with the National Institutes of Health guidelines for the Use of Animals in Research.
2. HIE protocol
The exercise training protocol was previously described in detail [18]. At 19 weeks of age, mice were subjected to HIE three times a week on every other day for 12 weeks. Preceding HIE, mice were acclimated to running on a treadmill for a week. HIE consisted of a 5 m/min warm-up (5 min), followed by 7-13 bouts of main running sessions. Each running session consisted of 14-28 m/min of running for 2 minutes followed by 5-12 m/min of active rest for a minute. After the main exercise session, all mice ran at 5 m/min for 2 minutes cool-down. The main running speed during HIE increased gradually from 14 to 28 m/min over 12 weeks (Fig. 1). Therefore, the total exercise session time was gradually increased up to 49 minutes. During the experiment, sedentary groups of mice remained in the same room and condition. If mice failed to run on the treadmill during the training session, the running speed was set to the previous speed.
Schematic diagram of the exercise training protocols. After a week of acclimation on the motorized treadmill, mice were subjected to HIE three times a week on every other day for 12 weeks. Each HIE session, including running speed and time, was gradually increased over 12 weeks.
3. Immunofluorescence (IF)
The histological evaluation was performed on 40 μm-thick, contiguous brain tissue from fixed, frozen, cut brain tissue. Sections were permeabilized in PBS containing 0.2% Triton X-100 (Biosesang, Korea) for 10 minutes at room temperature (RT) and then incubated with anti-body-containing diluted solution (5% goat serum and 2% Triton X-100 in PBS) overnight at 4°C in the dark. The tissue sections were washed in 2% Triton-X in PBS and then treated with secondary antibodies for an hour at RT. Images were taken using an Olympus Fluoview FV1000 fluorescence microscope (Olympus, Tokyo, Japan). The fluorescence intensity was measured with Image J software.
4. Western Blot (WB) analysis
Each brain tissue was lysed using a cell grinder in ice-cold RIPA buffer (Thermo Fisher Scientific, MA, USA) containing Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), then incubated on ice for 30 minutes and centrifuged at 16,000×g for 20 minutes at 4°C. Supernatants were collected after centrifugation, and protein concentrations were measured using the BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples were mixed with 4X sample buffer (Bio-Rad, CA, USA) and then electrophoresed on polyacrylamide gels, which were then transferred to nitrocellulose membranes sequentially. After blocking with 5% skim milk, membranes were reacted with primary antibodies overnight at 4°C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at RT. Membranes were incubated with Electrochemiluminescence (ECL) substrate (Bio-Rad), and blots were analyzed using the ChemiDoc XRS+ system (Bio-Rad).
5. Reverse Transcription and Quantitative PCR (qPCR) analysis
Total RNA was extracted using TRIzol (Invitrogen). Extracted RNA concentration was measured spectrophotometrically using Nanodrop (DeNvix Inc., Wilmington, USA). Then, 1 μg of total RNA was converted to cDNA through reverse transcription using the HiSecriptTM RH(-) RT Premix Kit (Intron Biotechnology, Gyeonggi, Korea). Quantitative PCR was conducted on a CFX Connect Real-time PCR System using iQ SYBR Green Supermix (Bio-Rad). Relative mRNA levels were normalized to Gapdh level for each gene.
6. Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labelling (TUNEL) assay
TUNEL staining was conducted to assess neuronal cell death using an In Situ Cell Death Fluorescein Detection Kit (Roche Molecular Bio-chemicals, Mannheim, Germany), according to the manufacturer's instructions. The images were captured using an Olympus Fluoview FV1000 confocal microscopy (Tokyo, Japan). The number of apoptotic cells observed via TUNEL staining was quantified using ImageJ.
7. Statistical analysis
All data were analyzed by using GraphPad Prism 7 software (Graph Pad Software, San Diego, CA, USA). The groups were compared using unpaired two-tailed t-test to assess the statistical significance. p-values for the test are included in the figures. Data are expressed as mean±S. E.M. p <.05 was considered significant.
RESULTS
1. HIE differently regulates GDNF expression in the cerebral cortex (CTX) and hippocampus (HIP)
The cortex and hippocampus are important brain parts that control memory, thinking, and learning [19,20]. GDNF promotes differentiation, neurogenesis, and survival of neurons in brain regions. To investigate whether HIE regulates the expression of neurotrophic factors in the CTX and HIP regions, we analyzed the protein expression of GDNF in both brain regions. Immunoblotting and immunofluorescence results demonstrated that HIE did not alter GDNF protein expression in the HIP region compared to the Cnt group (Fig. 2A-D; no statistical difference), whereas it induced a significant elevation of GDNF protein expression in the CTX region (Fig. 3A, B; p =.0069, Fig. 3C, D; p <.0001). Furthermore, HIE did not promote cortical neuronal cell death in both brain regions, confirmed by using the TUNEL assay (Figs. 2E, 2F, 3E, and 3F; no statistical difference). Our results indicated that HIE only increases the GDNF expression in the cortex, but did not induce neuronal cell death in both brain regions.
HIE activity is not involved in GDNF and cell death in the HIP. (A) The protein expression of GDNF was examined in the HIP. (B) Quantification of relative GDNF protein expression in the HIP. (C) Representative IF images of the expression of GDNF in the HIP. (D) Quantification of relative GDNF intensity in the HIP. (E) Apoptotic neurons in the HIP were determined by TUNEL assay. (F) Number of TUNEL-positive cells. All values are represented as the means ± S.E.M of six independent mice and compared with the Cnt group. Unpaired two-tailed t-test was used for statistical analysis. n.s, non-significant; N.D, non-detected.
HIE activity elevates GDNF expressions without neuronal apoptosis in CTX. (A) The protein expression of GDNF was examined in the CTX. (B) Quantification of relative GDNF protein expression in the CTX. (C) Representative IF images of the expression of GDNF in the CTX. (D) Quantification of relative GDNF intensity in the CTX. (E) Apoptotic neurons in the CTX were determined by TUNEL assay. (F) Number of TUNEL-positive cells. All values are represented as the means ± S.E.M of six independent mice and compared with the Cnt group. Unpaired two-tailed t-test was used for statistical analysis. N.D, non-detected.
2. HIE modulates the neuroprotective activity of astrocytes in the HIP
To investigate the effect of HIE on the glial cells in the HIP, the protein expression levels of GFAP (astrocyte marker) and Iba-1 (microglia marker) were measured. Both immunoblotting and immunofluorescence data showed that HIE reduced the GFAP expression in the HIP of the brain (Fig. 4A, B; p =.0020, Fig. 4D, E; p =.0110). However, Iba-1 protein expression did not show statistical difference (Fig. 4A, C, D, and F). To examine the effect of HIE on neuroinflammation in the HIP, we investigated the mRNA expression of the A1- and A2-specific transcripts of astrocytes and cytokines. HIE significantly downregulated the mRNA expression levels of A1-specific transcript, which are implicated in neuroinflammation and neuronal cell death (Fig. 4G). Notably, the HIE group significantly increased A2-specific transcripts, which are known to function as neuroprotective (Fig. 4H). However, both mRNA levels of pro- (Tnfα, Il1α, and Il6) and anti-inflammatory (Il10 and C3) cytokine expressions were significantly elevated in HIE groups compared to Cnt (Fig. 4I). Collectively, these data confirmed that HIE regulates neuroinflammation by decreasing the expression of GFAP and A1-specific transcripts and increasing A2-specific transcripts in the HIP.
HIE activity induces neuroprotective effects by increasing A2-reactive astrocyte genes in HIP. (A) Representative IF images of the expression of GFAP and Iba-1 in the HIP. (B) Quantification of relative GFAP intensity in the HIP. (C) Quantification of relative Iba-1 intensity in the HIP. (D) The protein expression of GFAP and Iba-1 were examined in the HIP. (E) Quantification of relative GFAP protein expression in the HIP. (F) Quantification of relative Iba-1 protein expression in the HIP. (G-H) The relative mRNA levels of A1 and A2-specific transcripts. (I) The relative mRNA levels of cytokines. All values are represented as the means ± S.E.M of six independent mice and compared with the Cnt group. Unpaired two-tailed t-test was used for statistical analysis. n.s, non-significant.
3. HIE activity regulates the mRNA expression of astrocyte-related genes in the CTX
Increased GFAP is widely recognized as a hallmark of astrocyte activation, typically in response to brain injury, inflammation, or neurodegeneration [1,6]. Also, Iba-1 expression is upregulated during microglial activation, which is characterized by morphological changes and production of inflammatory mediators [7,15]. Our study results showed that HIE did not induce statistical differences in the protein expressions of GFAP and Iba-1 in the CTX (Fig. 5A-F). However, A1-specific transcripts mRNA expression was unchanged or significantly decreased in the HIE group compared to the Cnt (Fig. 5G). Furthermore, the HIE significantly increased A2-specific transcripts gene expressions compared with the Cnt (Fig. 5H). HIE also induced a significant elevation of mRNA levels of pro- (Il1α, Il1β, and Il6) and anti-inflammatory (Il10 and C1q) cytokine expressions in the CTX (Fig. 5I). These results suggest that HIE increases A2-specific transcriptional mRNA expression without the alteration of glial cell marker expression in the CTX.
HIE activity induces neuroprotective effects by increasing A2-reactive astrocyte genes in CTX. (A) Representative IF images of the expression of GFAP and Iba-1 in the CTX. (B) Quantification of relative GFAP intensity in the CTX. (C) Quantification of relative Iba-1 intensity in the CTX. (D) The protein expression of GFAP and Iba-1 were examined in the CTX. (E) Quantification of relative GFAP protein expression in the CTX. (F) Quantification of relative Iba-1 protein expression in the CTX. (G-H) The relative mRNA levels of A1 and A2-specific transcripts. (I) The relative mRNA levels of cytokines. All values are represented as the means ± S.E.M of six independent mice and compared with the Cnt group. Unpaired two-tailed t-test was used for statistical analysis. n.s, non-significant.
DISCUSSION
The hippocampus and cerebral cortex are integral components of the brain, each fulfilling crucial roles in diverse cognitive functions, memory consolidation, and learning processes [21]. Notably, the hippocampus region is responsible for the formation of new memories and plays a vital role in short and long-term memory consolidation, while the cerebral cortex is primarily responsible for managing sensory, motor, and linguistic functions [22]. These two brain regions harmoniously interplay, enabling various cognitive processes such as memory retention, spatial awareness, and sensory information processes [21]. Various exercise training modalities have been investigated as non-pharmacological strategies for treating neurodegenerative diseases and maintaining overall brain health [3,23]. HIE, a highly efficient and time-saving exercise modality that involves short bursts of intense exercise with brief recovery time, has recently gained growing attention [24]. Therefore, our study investigated that HIE would exert beneficial effects on brain health by modulating neurotrophic factors and neuroinflammatory responses in the hippocampus and cerebral cortex brain regions.
GDNF is an essential neurotrophic factor that regulates cognition, neuroplasticity, and angiogenesis and plays a crucial role in the development of learning and memory [25]. Furthermore, GDNF is recognized as a potent neurotrophic factor in the maintenance and repair of dopaminergic neurons damaged by PD [23]. Several studies have reported that heterogeneous exercise regimens, including treadmill training, rotarod walking, and voluntary wheel activity, promote GDNF expression in spinal cords, skeletal muscles, and brain [23,26,27], although the molecular mechanism responsible for this effect is unclear. Treadmill exercise training ameliorated reduced serum GDNF level and improved postural balance after 4 weeks of intervention in aged participants and restored downregulated expression of GDNF in substantia nigra pars compacta and striatum in PD mouse model [28]. However, there is a lack of studies investigating the direct evidence of high-intensity interval exercise on GDNF expression in brain regions, especially in the hippocampus and cortex, which are key brain regions for memory consolidation. Our study showed that GDNF expression was significantly elevated in the cortex from the HIE group, but not in HIP, compared with the Cnt group (Fig. 2A-D and 3A-D). Eo and Leem [29]. reported that exercise intensity and modality differently induced GDNF expression, which was observed only in moderate-intensity exercise, but not in the high-intensity treadmill running group in the mouse prefrontal cortex brain region. One study demonstrated that the protein content of GDNF in skeletal muscles is not altered in groups of involuntary high-velocity wheel and voluntary wheel running with resistance, but showed an elevation of GDNF in groups undergoing involuntary low-velocity or voluntary wheel running without resistance [27]. Previous studies and our data implied that GDNF expression might be differently altered in response to exercise modalities and in a region-dependent manner in rodent models. Unlike previous studies, our HIE intervention involved a progressive increase in running speed throughout the training period, which consequently altered the total exercise session as well. This is in contrast to several earlier studies that employed either moderate-intensity continuous exercise or shorter intervention periods. The extended duration and intensity in our study may have contributed to the differential effects observed in neurotrophic factor expression and astrocyte phenotype modulation in CTX and HIP in the brain. Therefore, further investigation is required to gain an in-depth understanding of HIE effects on the neurotrophic factor. In conclusion, our data suggested that HIE might promote neurogenesis and neuronal survival signaling CTX, not in HIP region in normal healthy mouse brains.
Astrocytes represent a form of neuroglial cell crucial in the maintenance and regulation of neural functionality [19]. Thus, the activation or activity of microglia and astrocytes in response to neuronal inflammation leads to neuronal toxicity and has significant implications in the pathogenesis of neurodegenerative diseases. A1- and A2-specific transcripts refer to distinct gene expression profiles associated with two reactive astrocyte phenotypes identified under different pathological conditions. A1-reactive astrocytes, typically induced by neuroinflammatory signals, exhibit a neurotoxic and inflammatory phenotype, leading to detrimental cell death [30]. In contrast, A2-reactive astrocytes are generally induced under neuroprotective conditions and express transcripts that promote tissue repair, synapse formation, and neuroprotection, which are involved in anti-inflammatory responses, extracellular matrix remodeling, and the secretion of certain neurotrophic factors, thereby promoting the survival and growth of neurons [31]. It has been demonstrated that appropriate exercise can induce astrocyte activation, alter astrocyte phenotype, and modulate astrocyte-mediated neuroinflammatory response [4]. In healthy control rodent models, aerobic exercise training increases the GFAP expression with elevation of A2 astrocyte markers in medial prefrontal cortex (mPFC) [29] and pericortex [32]. In contrast, 3 weeks of resistance exercise did not exert GFAP and Iba-1 expressions in the dentate gyrus in healthy control female Wistar rats [33]. Our data demonstrated that HIP exercise did not exert the alteration of GFAP expression, which is a maker of astrocyte reactivity, but positively modulates astrocyte phenotype via reduced A1 astrocyte (pro-inflammatory function) and increased A2 astrocyte (anti-inflammatory function) mRNA markers, both in CTX and HIP, further supporting exercise-induced neuroplasticity. Feng et al. [34] reported that high-intensity interval exercise training on the treadmill did not regulate GFAP and A1 astrocyte (C3d)/A2 astrocyte (S100A10) expressions in both cortex and hippocampus regions in healthy control mice. Also, a study reported that aerobic exercise differently induced gene expression of Gfap, Lif (a differentiation factor), Thbs2 (an astrocyte-secreted semantogenic factor), and Il6 in the brain region-specific manner [35]. Based on previous and our findings provide evidence that exercise modality and intensity differently regulate the astrocyte reactivity and phenotypes in the brain region-specific manner.
Unlike exercise-induced A1 to A2 astrocyte phenotype transition, the mRNA expression of the pro- and anti-inflammatory markers was elevated following HIE in our study. Even though pro-inflammatory gene expressions were significantly increased in both CTX and HIP after 12 weeks of HIE, our TUNEL assay data demonstrated that HIE did not induce apoptotic neuronal cell death in both brain regions with astrocyte phenotype transition, which partially supports that HIE might play a neuronal protective role, especially preventing programmed cell death, which is associated with neurodegenerative process. In contrast, High-intensity interval training reduced expression of both pro-inflammatory (TNFα, IL-6, IL-1β) and anti-inflammatory cytokines (IL-10) in the hippocampus in rats [36] and in human blood [37]. Notably, IL-6 is recognized as a pleiotropic cytokine with both pro- and anti-inflammatory properties [38], and its elevated expression may indicate a global response to exercise aimed at maintaining homeostasis [39]. However, the specific role of IL-6 in the brain in response to exercise has not been fully elucidated. A previous study reported that the mRNA level of Il6 was elevated only in the hippocampus, not in the cortex or cerebellum, after acute aerobic exercise training in a mouse model [40]. Additionally, a study showed that mRNA Il6 expression increased in a brain region-specific manner at 1 week and 2 weeks after exercise [35]. However, these results were obtained from acute exercise, which contrasts with our chronic exercise modality. Aforementioned, our exercise protocol involved progressive increases in both running speed and exercise sessions across the intervention, which may have led to an overall increase in exercise intensity. This elevated intensity could have activated global pro-inflammatory signaling; however, as a compensatory mechanism, anti-inflammatory signaling may also have been upregulated, thereby contributing to the maintenance or enhancement of physiological homeostasis in both brain regions. Therefore, further studies are required to elucidate the specific effect of exercise with different modalities on cytokine expression in a brain region-specific manner. Collectively, these findings suggest that exercise training may promote a conducive micro-environment that facilitates the transformation of activated astrocytes from A1 to A2 astrocytes, ultimately leading to the manifestation of neuroprotective effects.
CONCLUSION
In conclusion, the current study underscores the significance of exercise training as a non-pharmacological approach to mitigating the decline in neurophysiological function in the hippocampus and cerebral cortex. Although this study was conducted in a mouse model, HIE in-terventions exhibit protective regulatory effects on neuroinflammation and A1 and A2 type astrocytes reactivities, which are directly linked to brain health, suggesting that possible translational relevance for human brain health. However, the efficacy of exercise intervention for neurotrophic factors regulation might vary on the exercise type, intensity, and volume in the brain region-specific manner. Therefore, future clinical research is required to provide appropriate different modalities of exercise to establish its efficacy, safety, and translational applicability, and to achieve biological and clinical neuroprotective outcomes for people, particularly at risk for neurodegenerative diseases.
Notes
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Conceptualization: J Hong, SH Kim, SP Yun; Formal analysis: J Hong, SH Kim, J Kim; Data curation: SP Yun; Visualization: J Hong, SH Kim; Writing - original draft: J Hong, SH Kim; Writing - review and editing: YH Jung, SP Yun; Methodology: CS Kim, DH Park, HJ Kim, SW Park, JW Kim, YH Jung, SP Yun; Project administration: J Hong, SH Kim; Funding acquisition: SP Yun. All authors have read and agreed to the published version of the manuscript.