Mechanisms and therapeutic potential of hyperbaric oxygen inducing autophagy in cerebral ischemia-reperfusion injury

Abstract

Cerebral ischemia-reperfusion injury (CIRI) is a complex pathological process involving oxidative stress, inflammation, and dysregulated autophagy. Hyperbaric oxygen therapy (HBO) has emerged as a promising intervention that mitigates neuronal injury by modulating autophagy. This review summarizes current evidence on the mechanisms of HBO through the AMPK-mTOR, HIF-1α-mTOR, and PI3K-AKT pathways, emphasizing its dual role in either promoting cell survival or exacerbating injury depending on autophagic flux. Preclinical studies demonstrate that HBO upregulates LC3-II and Beclin-1 while downregulating p62, indicating enhanced autophagy, whereas clinical trials (e.g., OPENS-2) suggest synergistic effects when combined with endovascular therapy. However, challenges remain regarding the lack of standardized biomarkers and optimal treatment parameters. Future research should focus on integrating HBO with specific autophagy modulators and conducting large-scale randomized controlled trials to validate its therapeutic potential in CIRI.

Introduction

Ischemia-reperfusion injury refers to the metabolic dysfunction and structural damage of brain cells that occur following a period of cerebral ischemia and subsequent restoration of blood perfusion, leading to further aggravation of tissue and organ injury [1]. Cerebral ischemia-reperfusion injury (CIRI) is commonly observed in ischemic stroke, hypoxic-ischemic encephalopathy, and post-cardiopulmonary resuscitation brain injury. In recent years, various therapeutic strategies have been developed to alleviate the secondary injury caused by reperfusion; however, most have shown limited efficacy. Therefore, it is imperative to explore more effective interventions for preventing and treating CIRI. Autophagy plays a critical role in the pathophysiological process of CIRI and represents a promising therapeutic target. Hyperbaric oxygen therapy (HBO) has demonstrated neuroprotective effects against CIRI, partly through the modulation of autophagy. This review summarizes the mechanisms by which HBO regulates autophagy in CIRI and discusses its clinical translational challenges.

Overview of CIRI

The pathogenesis of CIRI is multifactorial, involving calcium overload, mitochondrial dysfunction, inflammatory activation, apoptosis, accumulation of oxygen-free radicals, and excessive release of excitatory amino acids [1]. During the ischemic phase, insufficient cerebral oxygen and blood supply force neurons to rely on anaerobic glycolysis for energy, resulting in intracellular accumulation of sodium and calcium ions. Calcium influx into mitochondria through calcium transporters inhibits adenosine triphosphate (ATP) synthesis, leading to mitochondrial impairment. Meanwhile, elevated intracellular calcium triggers massive release of the excitatory neurotransmitter glutamate. The binding of glutamate to N-methyl-D-aspartate receptors promotes the generation of reactive oxygen species (ROS) and reactive nitrogen species, causing oxidative and nitrosative stress.

When reperfusion occurs, microglia, astrocytes, and macrophages become activated, releasing large amounts of inflammatory cytokines such as TNF-α, IL-6, and IL-1β [2]. These inflammatory mediators disrupt mitochondrial integrity, damage vascular endothelial cells, and increase the permeability of the blood-brain barrier, ultimately resulting in neuronal and tissue injury. The main pathophysiological mechanisms are illustrated in Figure 1.

Figure 1.

Major pathophysiologic processes in cerebral ischemia-reperfusion injury. Illustration created by Figdraw. IL: interleukin; TNF: tumor necrosis factor; ATP: adenosine triphosphate; ROS: reactive oxygen species; RNS: Reactive nitrogen species; Glu: glutamate; NMDA: N-methyl-d-aspartate.

Role of autophagy in CIRI

During CIRI, multiple cell death pathways - including inflammation [3], apoptosis, and autophagy - interact and influence neuronal survival [4]. Autophagy is a lysosome-dependent process in which autophagosomes engulf damaged organelles and misfolded proteins, forming autolysosomes that degrade and recycle cellular components. Numerous studies have confirmed [5] that autophagy is a key mechanism in the development and repair processes of CIRI. Regulating autophagy can thus promote neuronal recovery during reperfusion.

Under physiological conditions [6], autophagy maintains cellular homeostasis by eliminating damaged organelles and proteins. However, under stress, the activation of autophagy may exert either protective or detrimental effects depending on its intensity and duration.

Mitochondrial autophagy (mitophagy) selectively removes dysfunctional mitochondria [7], which are central to energy production, calcium homeostasis, and ROS regulation [8]. By clearing damaged mitochondria, mitophagy maintains mitochondrial quality and prevents the release of pro-apoptotic factors such as cytochrome c under ischemic conditions [9,10]. Notably, mitophagy may exert different effects in distinct temporal phases of injury: it appears protective during the reperfusion phase, when clearance of damaged mitochondria limits oxidative stress and neuronal apoptosis.

Mechanisms of HBO therapy in CIRI

HBO therapy is an adjunctive intervention in which patients breathe 100% oxygen at 2-3 atmospheres absolute (ATA) to elevate arterial oxygen tension and oxygen dissolution in plasma. This enhances cellular respiration and maintains ATP synthesis in ischemic and hypoxic tissues [11]. HBO also reduces oxidative stress, suppresses inflammation, promotes phagocytic clearance of damaged cells, and recruits reparative cells. Clinically, HBO has been widely used in the management of craniocerebral trauma, limb replantation, sepsis, and wound healing [12].

Experimental and clinical studies have demonstrated that inhaling pure oxygen at 2-3 ATA increases its solubility in plasma by approximately 12.6-21.7 fold compared with normal atmospheric pressure [13]. The neuroprotective efficacy of HBO depends on the therapeutic window, frequency, and duration of treatment [13]. Optimal outcomes are achieved when HBO at 2.0 ATA is administered immediately after middle cerebral artery occlusion (MCAO) and continued for more than six hours, significantly improving survival in rodent models [14]. Moreover, repeated HBO sessions initiated 48 hours after brain injury can still confer therapeutic benefits [15], and delayed HBO administration may attenuate inflammation and reduce neuronal injury [16]. Recent studies further reveal that HBO alleviates oxidative stress, inflammation, apoptosis, amyloid-β deposition, and cholinergic dysfunction, thereby providing multifaceted neuroprotection [17].

Mechanisms of interaction between HBO and autophagy

Autophagy is a highly conserved cellular process essential for maintaining intracellular homeostasis. It functions by degrading and recycling damaged proteins and organelles, thereby serving as a primary pathway for the clearance of senescent or dysfunctional cellular components. Adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are two key signaling molecules that regulate cellular metabolism, energy balance, and autophagy [18].

mTOR acts as a central inhibitor of autophagy [19], modulating the expression and activity of microtubule-associated protein 1 light chain 3 (LC3), p62, and Beclin-1. LC3 exists in two forms, LC3-I and LC3-II, and an increased LC3-II level or LC3-II/LC3-I ratio reflects enhanced autophagic activity [15]. The mechanistic target of rapamycin (mTOR) forms two distinct complexes - mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [20]. mTORC1 acts as the primary regulator of energy-consuming cellular processes: under nutrient-rich conditions, it promotes cell growth and biosynthesis, while under nutrient deprivation, its inhibition triggers autophagy to recycle intracellular components [21,22].

mTOR suppresses autophagy both by inhibiting its early stages and by regulating lysosomal degradation via transcriptional control of structural and catalytic factors [23]. When mTOR is inhibited, autophagic recycling restores intracellular energy levels, which can subsequently reactivate mTORC1 - indicating that autophagy operates both downstream and upstream of mTOR. The intricate feedback regulation between mTOR and autophagy has been well characterized at the cellular level [24]. Importantly, HBO therapy has been shown to influence autophagy. For example, HBO alleviated neuropathic pain in rats by activating autophagy through the AMPK signaling pathway [25]. However, the precise mechanisms underlying HBO-autophagy crosstalk remain incompletely understood (Figure 2).

Figure 2.

Interaction between hyperbaric oxygen (HBO) therapy and autophagy.

AMPK/mTOR signaling pathway

AMPK and mTOR are major regulators of the autophagy process [26]. AMPK is a trimeric enzyme complex consisting of a catalytic α-subunit and regulatory β- and γ-subunits. The α-subunit is predominantly expressed in brain tissue, and its activation depends on the intracellular AMP/ATP ratio. Once activated, AMPK stimulates multiple metabolic pathways that enhance energy production and inhibit energy consumption [27].

mTOR, a serine/threonine kinase, modulates cell growth, metabolism, and proliferation. Its activation promotes the synthesis of macromolecules such as proteins, nucleotides, and lipids, while suppressing autophagy [28]. AMPK can directly activate the ULK1 complex to initiate autophagy and can also inhibit mTOR to promote autophagy under energy-deficient conditions, thus maintaining a dynamic equilibrium between anabolic and catabolic processes [29-31].

Wang et al. conducted a systematic transcriptomic analysis and found that HBO significantly activated the AMPK/mTOR signaling pathway, promoting autophagosome formation as evidenced by increased expression of Beclin-1 and LC3 proteins [32].

mTOR/HIF-1α signaling pathway

HIF-1α is a transcription factor that responds to ischemic and hypoxic stress and plays a pivotal role in angiogenesis, tumor progression, and hypoxia-induced autophagy [33]. HIF-1α is regulated downstream of mTOR, which enhances its transcriptional activity, stimulates protein synthesis, accelerates metabolism, and modulates autophagic responses [34].

Experimental results from Wang et al. showed that HBO effectively reduced cerebral infarction and edema by modulating the expression of mTOR, LC3-II, and HIF-1α in the hippocampus, thereby influencing autophagic activity and improving ischemia-reperfusion outcomes [35].

PI3K/AKT/mTOR signaling pathway

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR signaling pathway plays a critical role in autophagy regulation following acute central nervous system injury. Akt, a serine-threonine kinase, is activated downstream of PI3K [29]. PI3K links Akt and mTOR through inactivation of the tuberous sclerosis complex subunit 2 (Tsc2) [30].

Evidence suggests that HBO induces autophagy via the Akt/Tsc2/mTOR pathway, thereby alleviating neuropathic pain [31]. The PI3K/AKT/mTOR axis is one of the most crucial signaling pathways involved in autophagy regulation [36]. Upon activation, PI3K generates the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), which recruits phosphoinositide-dependent kinase 1 (PDK1) and Akt through their PH domains. This interaction results in phosphorylation of Akt by PDK1, leading to Akt activation [37,38]. Activated Akt subsequently phosphorylates and activates mTOR to form mTORC1, which governs protein synthesis and autophagy [39].

Previous studies demonstrated that the PI3K/AKT/mTOR pathway contributes to cerebral ischemic injury in Sprague-Dawley rats; pharmacological inhibition of PI3K or mTOR altered autophagy levels and neuronal survival [40]. Moreover, HBO has been shown to mediate the Akt/Tsc2/mTOR pathway to induce autophagy and relieve neuropathic pain [41,42]. Therefore, evaluating the expression of autophagy-related proteins such as PI3K, Akt, and mTOR can reflect autophagic activity in CIRI and its modulation by HBO.

Preclinical and clinical evidence associated with autophagy in CIRI treatment

Animal studies

Autophagy exhibits a dual nature: appropriately activated autophagy supports neuronal survival after focal cerebral ischemia [43], whereas excessive activation can trigger cell death [6]. Preclinical studies have shown that HBO enhances autophagic activity, thereby exerting neuroprotective effects against cerebral ischemic injury. In one study, intracerebroventricular administration of the autophagy inhibitor 3-methyladenine (3-MA) prior to HBO preconditioning suppressed the HBO-induced upregulation of LC3-II and Beclin-1, and consequently attenuated its neuroprotective effects. In contrast, rapamycin pretreatment increased LC3-II and Beclin-1 expression after reperfusion, mimicking the neuroprotective effects of HBO preconditioning [44].

Lu et al. reported that autophagy blockade via activation of extracellular signal-regulated kinase 1/2 (ERK1/2) expanded the cerebral infarct area and aggravated CIRI in MCAO rats [45]. Similarly, Chen et al. found that HBO attenuated myocardial ischemia-reperfusion injury by inhibiting excessive autophagy through activation of the mTOR pathway [46].

Clinical trials

Clinically, HBO serves as a versatile adjunctive therapy for CIRI. Its combination with traditional Chinese medicine, targeted temperature management, endovascular therapy, and transcranial magnetic stimulation (TMS) has shown enhanced therapeutic outcomes.

In the OPENS-2 trial, the combination of normobaric high-oxygen therapy and endovascular treatment significantly improved 90-day modified Rankin Scale scores, with a favorable safety profile. Combined HBO also delayed the transition of the ischemic penumbra to the infarct core compared with thrombolysis alone [47].

Furthermore, both repetitive TMS (rTMS) and HBO were shown to facilitate recovery in comatose patients. HBO not only repaired neuronal injury but also provided metabolic energy that augmented neurotransmitter synthesis induced by rTMS. The combination of rTMS and HBO was markedly more effective in promoting awakening than HBO monotherapy, highlighting its potential for broader clinical adoption [15].

Clinical efficacy testing of biomarkers

The expression of autophagy-related proteins serves as an important biomarker for evaluating both CIRI pathogenesis and HBO efficacy [48]. Among these, p62 - a substrate and regulator of autophagy, is negatively correlated with autophagic activity. Clinical studies have reported that elevated peak serum p62 levels in patients with traumatic brain injury were significantly associated with poor neurological outcomes (P<0.05), suggesting that excessive accumulation of autophagosomes may aggravate secondary brain injury. This finding identifies p62 as a potential molecular marker for monitoring autophagy imbalance [49].

In animal models exploring HBO’s therapeutic mechanisms, dynamic regulation of p62 and other autophagy markers has been confirmed [50]. HBO pretreatment markedly downregulated p62 expression while increasing the LC3-II/LC3-I ratio and Beclin-1 expression. Notably, inhibition of cystatin C (CysC) abolished HBO-induced p62 reduction and concomitantly suppressed LC3-II and Beclin-1 upregulation, indicating that CysC may be a key mediator in HBO-induced autophagy regulation. These results suggest that dynamic changes in p62 may reflect the reparative capacity of HBO on autophagic balance.

Future studies should explore the translational potential of these biomarkers using multimodal assessments - such as cerebrospinal fluid p62 quantification and PET/MRI autophagy imaging - to refine the optimal therapeutic window for HBO intervention.

Controversies and future directions

Despite extensive preclinical evidence, research on HBO-mediated modulation of autophagy in CIRI remains limited. Although numerous basic studies have demonstrated HBO’s neuroprotective effects through autophagy-related pathways, clinical translation is still insufficient. Most investigations focus on isolated autophagy markers rather than comprehensive monitoring of autophagic flux [51].

Clinically, current trials remain limited by small sample sizes, the absence of standardized efficacy evaluation systems, and a lack of studies examining the synergistic effects of HBO with neuroprotective agents or autophagy modulators. Moreover, the bidirectional role of autophagy - protective under moderate activation but detrimental when excessive - complicates interpretation of HBO’s dose-response relationship. Therefore, determining the optimal HBO dosage, treatment frequency, and timing for precise autophagy regulation remains an urgent priority.

In summary, HBO confers neuroprotection in CIRI by bidirectionally modulating autophagy through the AMPK/mTOR, HIF-1α, and related signaling pathways. However, its clinical efficacy is constrained by the therapeutic time window and the dynamic balance of autophagy. Future research should elucidate the interactions among autophagy, inflammation, and apoptosis regulated by HBO [52] - for instance, its influence on NLRP3 inflammasome activation via Beclin-1-dependent mechanisms [53].

Large, multicenter randomized controlled trials are warranted to evaluate combination regimens integrating HBO with autophagy modulators or neuroprotective agents. For patients without contraindications to pneumatic therapy, HBO should be administered alongside peripheral monitoring of autophagic activity to enable individualized treatment. As clinical evidence accumulates, a precise HBO-based therapeutic framework centered on autophagy modulation may significantly improve neurological outcomes in CIRI.

Although HBO shows considerable promise for managing cerebral ischemia-reperfusion injury, its mechanisms of action remain incompletely understood. Determining optimal treatment timing, dosing parameters, and safety profiles is essential before widespread clinical adoption. Nevertheless, accumulating experimental evidence continues to support HBO as a promising adjunctive strategy for stroke therapy.

Disclosure of conflict of interest

None.