Document Type : Research Paper
Authors
Department of Exercise Physiology, Faculty of Sport Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
Abstract
Background and Purpose
Ischemic stroke, resulting from vascular occlusion, induces hypoxia and neuronal death, leading to widespread functional impairments. In response to oxygen deprivation, hypoxia-inducible factor-1 alpha (HIF-1α) is stabilized and translocated to the nucleus, where it upregulates target genes such as vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), and erythropoietin (EPO), thereby promoting angiogenesis and neuronal survival. The activity of HIF-1α is further modulated by intracellular signaling cascades including PI3K/Akt/mTOR, MAPK/ERK, JAK/STAT, and cAMP/PKA pathways. Aerobic exercise, a cost-effective nonpharmacological strategy, enhances cerebral blood flow, activates AMP-activated protein kinase (AMPK), and elevates neurotrophic factors like brain-derived neurotrophic factor (BDNF). These changes augment HIF-1α expression, stimulate angiogenesis and neurogenesis, and attenuate post-ischemic inflammation. Preclinical studies confirm that regular aerobic training improves cognitive function, promotes hippocampal neurogenesis, and offers substantial protection against cerebral ischemia. Adenosine, an endogenous nucleoside produced during ATP hydrolysis under stress, regulates immune responses and exerts neuroprotective effects via its four receptors (A1, A2A, A2B, A3). Activation of A2A receptors enhances HIF-1α expression and repair gene activation through PI3K/Akt and cAMP/PKA signaling, while A1 receptor engagement reduces infarct size and mitigates neurological deficits by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2)/NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling and pyroptosis. The ATP–adenosine axis thereby shifts the inflammatory milieu toward neuroprotection. Despite strong evidence supporting the individual benefits of aerobic exercise and adenosine, their combined effects on HIF-1α–mediated neuroprotection remain insufficiently studied. This investigation aims to evaluate the combined impact of aerobic exercise and adenosine administration on hippocampal HIF-1α gene expression in a rat model of ischemic stroke, assessing potential synergistic effects on neuronal survival and cognitive recovery.
Materials and Methods
This experimental laboratory study employed a multi-group post-test design involving fifty adult male Wistar rats (8–10 weeks old, 240–270 g). Animals were acclimatized for one week under controlled conditions (22 ± 3 °C; 55 ± 3% humidity; 12-hour light/dark cycle), with ad libitum access to water and standard chow (5 g per 100 g body weight). Ischemic stroke was induced under intraperitoneal anesthesia with ketamine (60 mg/kg) and xylazine (4–5 mg/kg) by occluding the right common carotid artery for 45 minutes using a vascular clamp. Neurological deficits were verified by stereomicroscopic assessment, followed by a 5-minute reperfusion period. Rats were randomized into five groups (n=10 each): Sham (surgical procedure without occlusion), Stroke + Control (ischemia + saline), Stroke + Aerobic Exercise (AE), Stroke + Adenosine (Ad), and Stroke + Adenosine + Aerobic Exercise (Ad + AE). Adenosine (0.3 mg/kg) or saline was administered intraperitoneally 3 hours prior to exercise sessions daily. After three treadmill familiarization sessions (15 m/min for 10–15 minutes), rats underwent an eight-week aerobic protocol: five sessions weekly, each comprising a 5-minute warm-up, progressively increasing main bouts (from 20 m/min for 18 minutes in week 1 to 30 m/min for 50 minutes by week 8), and a 3-minute cool-down. Forty-eight hours post-intervention and following a 12-hour fast, hippocampal tissue was harvested. Total RNA was isolated using Qiagen kits; cDNA synthesis was performed with Fermentas reagents and Oligo dT primers. HIF-1α expression levels were quantified via reverse transcription quantitative PCR (RT-qPCR). Statistical analysis employed one-way ANOVA followed by Tukey’s post hoc tests in SPSS v26, with significance at p ≤ 0.05.
Findings
Aerobic exercise, with or without adenosine, induced the most substantial body weight reductions (2.76% in the Stroke + Ad + AE group and 2.18% in the Stroke + AE group) and significantly elevated hippocampal HIF-1α expression compared to Sham, Stroke + Control, and Stroke + Ad groups (P < 0.01). No statistically significant difference was found between the Stroke + AE and Stroke + Ad + AE groups (P = 0.1), nor did the Stroke + Ad group differ from Sham or Stroke + Control groups. These results indicate that aerobic exercise is the principal stimulus for HIF-1α upregulation in this model.
Conclusion
Ischemic stroke, precipitated by sudden vascular occlusion, triggers hypoxic injury, neuronal death, and functional deficits. HIF-1α stabilization and nuclear translocation activate genes such as VEGF, GLUT1, and EPO, vital for angiogenesis and neuroprotection. This gene expression is intricately regulated by signaling pathways including PI3K/Akt/mTOR, MAPK/ERK, JAK/STAT, and cAMP/PKA. Aerobic exercise enhances cerebral perfusion, activates AMPK and SIRT1, and increases neurotrophic factors like BDNF, collectively promoting HIF-1α expression and suppressing inflammation. Exercise-induced shear stress stimulates endothelial nitric oxide synthase (eNOS), augmenting nitric oxide synthesis, which improves tissue perfusion and alleviates secondary ischemic damage. Adenosine, released during ATP catabolism, modulates immune response and cell survival through A1 and A2A receptor pathways. Activation of A2A receptors potentiates HIF-1α expression via PI3K/Akt and cAMP/PKA mechanisms, whereas A1 receptor activation diminishes infarct volume by repressing pyroptosis and NLRP3 inflammasome pathways. Although both interventions possess neuroprotective capabilities, this study demonstrates that aerobic training predominantly drives HIF-1α upregulation, with adenosine alone exerting limited effects and no additive benefit when combined with exercise. Future studies should optimize adenosine dosing regimens and administration timing to potentiate synergistic neuroprotection. These findings endorse aerobic exercise as a key nonpharmacological intervention to attenuate ischemic brain injury, promote cognitive recovery, and inform rehabilitation protocols for stroke survivors.
Ethical Considerations
Aerobic exercise constitutes an effective nonpharmacological modality offering neuroprotection in ischemic stroke by modulating molecular pathways centered on HIF-expression, its combined use with aerobic exercise may enhance cellular repair mechanisms. These data support the integration of structured physical training with or without adjunct pharmacological agents to advance innovative stroke rehabilitation strategies and improve neuronal outcomes.
Conflicts of Interest
This research was conducted as part of a doctoral dissertation without external funding. The authors report no conflicts of interest related to this work.
Acknowledgement
We express our sincere gratitude to all individuals who supported us throughout this research.
Keywords
Subjects
