The first pre-clinical study evaluating the effects of HBOT preconditioning on a gerbil ischemic stroke model showed that the therapy conferred tolerance to ischemia and prevented neuronal death [
90]. The following studies using other animal models showed that HBOT was protective against transient, not permanent, stroke, and protection was conferred in a dose-dependent manner [
91]. A treatment of five sessions (2.5 atmosphere absolute [ATA], 1 h) over consecutive days was more effective than three sessions at rescuing functional deficits in rats after middle cerebral artery occlusion (MCAO) 24 h after the last session [
91]. These results have been replicated by other studies, including one that showed that four sessions of HBOT (2.5 ATA, 1 h, twice a day) offered neurological and histopathological protection from MCAO 24 h after the last session [
92]. More aggressive treatments (3.5 ATA, 1 h, five consecutive days) have also provided significant histopathological signs of neuroprotection [
93]. Other pre-clinical studies have explored the therapeutic window for HBOT, which suggests neuroprotection can be achieved by treatment 24 h before ischemia, but not 72 h [
94]. However, it is important to note that intensity and number of sessions may play a bigger role in treatment effects, and the therapeutic window must be further investigated. In the following sections, we will discuss the potential mechanisms underlying neuroprotection provided by HBOT preconditioning.
5.1. Preparation for Oxidative Stress
HBOT primarily generates neuroprotection through its interactions with an oxidative preconditioning factor [
95]. Long-term subjection to hypoxic environments engenders drastic oxidative strain, the disarray of the antioxidant network, and ultimate cell damage. Initial exposure of brain tissue to non-lethal hypoxia via HBOT preconditioning can shield neurons from future ischemic injury by fortifying tissue against oxidative stress. As evidence suggests, oxidative toxicity may be spurred by ROS and reactive nitrogen species (RNS) (e.g., peroxynitrite and NO
2) upregulation in the CNS. Fortunately, cells can combat ROS escalation through defense mechanisms induced by antioxidant enzymes. Superoxide dismutase (SOD) sequesters superoxide, catalase/peroxidases neutralize hydrogen peroxide, and glutathione S-transferase off set lipid peroxides. Auxiliary enzymes, such as glutathione reductase (GRX) and glucose-6-phosphate dehydrogenase (G6PD), also contribute to brain tissue defense. Although sparse numbers of ROS can be beneficial, as they promote antioxidant enzyme pathways that make up the adaptive cellular response, lethal amounts of ROS, engendered by hyperoxia, surpass the cellular antioxidative potential and generate oxidative damage. Ischemic injury in the brain induces oxidation of proteins, lipid peroxidation and augmented DNA mutation, leading to cell membrane impairment, disturbances in metabolism, and tissue death [
96]. ROS levels are higher with HBOT, due to increased oxygen partial pressure and upregulated H
2O
2 produced by mitochondria [
42]. Moreover, non-lethal oxidative stress spurred by HBOT serves as a protective procedure, stimulating antioxidative activity [
95].
An in vivo investigation utilizing a focal cerebral ischemia model found that HBOT preconditioning elevated SOD and CAT mechanisms in cerebral tissue, leading to increased survival rates, as well as ameliorated neurological function and cell damage [
92]. Notably, the stroke-afflicted penumbra and hippocampus demonstrated diminished levels of lipid peroxidation and oxidative stress biomarker, malondialdehyde (MDA) [
92]. In the same way, HBOT preconditioning with a spinal cord ischemia experimental model upregulated SOD and CAT processes. However, activation of the CAT inhibitor, 3-amino-1, 2, 4- triazole, prior to ischemic stroke, abolished the beneficial effects of HBOT, like spinal cord resilience to oxidative stress decreased significantly [
97]. When dimethylthiourea, a free radical scavenger, was delivered to the spinal cord ahead of HBOT, the elevated SOD and CAT activity was eliminated [
97]. Moreover, HBOT preconditioning spurs preliminary oxidative stress that prompts antioxidative mechanisms from enzymes, leading to increased resistance of tissue to future ischemic damage.
By suppressing GRX and G6PD and elevating glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST) pathways, frequent non-lethal HBOT preconditioning imparts neuroprotection against oxidative damage in the central nervous system [
98]. Therefore, in an indirect manner, HBOT attenuates oxygen toxicity through the repression of G6PD mechanisms. Importantly, HBOT preconditioning bolsters antioxidative processes and dilutes enzymatic activity of pro-oxidants, as HBOT-induced diminishment of G6PD can be correlated with the truncation of GRX and the augmentation of GSH-Px activity.
Under normal conditions, HBOT promotes neuronal rehabilitation and neuroprotection by upregulating heat shock proteins (HSPs), particularly HSP70 [
99]. HSP70 inhibits protein build-up, restores slightly denatured proteins, attenuates inflammation, and hinders apoptosis, all of which contribute to neuroprotection [
100]. In addition, as displayed in vitro, HBOT fortifies the expression of HSP32, shielding neuronal tissue from oxidative damage, and oxygen-glucose deprivation (OGD) [
92,
101]. HSP32 or heme oxygenase-1 breaks down heme into carbon monoxide (CO), biliverdin, and ferrous iron. Hemoprotein oxidation, such as hemoglobin, myoglobin, and neuroglobin oxidation, engender the formulation of free heme. An iron atom lies in the middle of the heme molecule and can interact with H
2O
2 to form deleterious hydroxyl radicals. HSP32 catalyzes heme molecules, leading to the generation and build-up of ferritin release, which in turn, engenders the removal of iron, thereby safeguarding tissue from oxidative injury [
92,
101]. Importantly, HSP32 is known to be incited by ROS and nitric oxide (NO), as the ROS/p38/MAPK/Nrf2 pathway and MEK1/2Bach1- regulated negative feedback modulate HSP32 activity [
102]. Moreover, free radical production spurred by oxidative stress should promote HSP mechanisms. At very low concentrations, free radicals produced by mitochondria and NOXs serve a regulatory ole in cellular activity—their physiological role in cellular activity ties in with the use of HBOT. Nonetheless, evidence points to the idea that response to HBOT may be cell-specific. For instance, one HBOT subjection in healthy males demonstrated no elevation in HSP72 activity in peripheral blood mononuclear cells (PBMC) [
103].
Additionally, HBOT may impart neuroprotection against oxygen toxicity via an increase in NrF2-mediated antioxidant gene expression. Notably, more than 200 antioxidants and cytoprotective genes can be turned on by the Nrf2 pathway [
104]. HBOT not only promotes Nrf2 activity, but also upregulates essential proteins involved with intracellular GSH production and transport (e.g., GST, MRPI, GCL, cGT), the assembly/disassembly of macromolecules (e.g., HSPA1A, HSP32), and antioxidant enzymes (e.g., SOD1, GST), which are all target genes of Nrf2 [
31,
102,
105,
106,
107]. In addition, by bolstering the expression of SirT1 in more than three various ways, HBOT preconditioning imparts neuroprotection. Firstly, Sirt1 expression can be increased by mediating the fasting-induced initiation of Nrf2 signaling upstream through the regulation of the PPAR-ƴ/PGC1-1α complex that attaches to the Nrf2 promoter, stimulating expression. Secondly, SirT1 expression is mediated via repression of apoptosis, spurred by the upregulation of protein anti-apoptotic Bcl-2 expression, depletion of cleaved caspase-3, which is pro-apoptotic, and the removal of acetyl groups from p53. Thirdly, expression of SirT1 can be modulated through the augmentation of FoxO, which in turn, elevates SOD and CAT activity under oxidative stress [
107,
108,
109,
110].
Indeed, HBOT preconditioning can be linked to the inflation of nitric oxide, [
111,
112] as shown in
Figure 1. Serving as a key neurotransmitter, NO is generated by NO synthase (NOS) and is a critical agent of neuroprotection and neurotoxicity [
113]. Following cerebral ischemia, endothelial NOS (eNOS) secretion of NO is beneficial, as it stimulates vasodilation. Conversely, once ischemia evolves further, NO generated by hyperactivity of neuronal NOS (nNOS) and iNOS expression lead to cerebral injury. NO released by eNOS and iNOS promote synaptic plasticity and neuronal development, whereas NO secreted by nNOS has the opposite effect, attenuating neurogenesis [
114]. Since NO improves the vasodilation of the cerebrovasculature, it may fortify the oxygenation of tissues. Furthermore, NO possesses the capacity to favor or impair apoptosis. NO may also regulate cellular metabolism in the presence of dysfunctional mitochondria. On the other hand, it may escalate the transit of ROS to tissues as well. Additionally, NO may react with free radicals to generate toxic oxidant peroxynitrite and engender nitrosative injury. Importantly, preconditioning with HBOT elevates antioxidant enzymatic activity and represses peroxynitrite primarily in the hippocampus, demonstrating HBOT’s protective capabilities [
98].
As low amounts of NO displays beneficial effects after stroke, high concentrations of NO produced via iNOS or eNOS may augment neuroinflammation and neurotoxicity. NO provides these negative effects through various mechanisms, including cGMP, cAMP, G-protein, JAK/STAT, and MAPK dependent pathways. Moreover, NO is also believed to modulate specific gene expression, further exacerbating inflammation, and toxicity [
115].
Aside from NO’s capacity to cause inflammation, HBOT-induced upregulation of eNOS and nNOS mRNA and protein, along with increased NO in the hypothalamus and hippocampus, may amplify convulsion susceptibility following consecutive HBOT subjections, which may exacerbate the risk of seizures in successive HBOT exposures [
112]. Notably, the nonspecific NOS inhibitor, L-NAME, eliminated HBOT-induced neuroprotection, indicating that elevations in Mn-SOD, CAT, and Bcl-2, as well as apoptosis inhibition, may be regulated by NO [
69]. Furthermore, following HBOT preconditioning, NO bears both neuroprotective and neurotoxic effects, and thus, further investigation into the mechanisms of NO after HBOT pre-treatment is warranted.
5.2. Reduction of Apoptosis, Activation of Autophagy, and Promotion of Cell Survival
ROS molecules possess the ability to react with molecular components to initiate apoptosis or necrosis. Inhibiting major redox systems, such as thioredoxin reductases (TrxR), results in the production of ROS and increased cell apoptosis [
116]. PTSD models in rats revealed the upregulation of TrxR-1 and TrxR-2 mRNA in the hippocampus in addition to decrease levels of apoptosis of neurons after HBOT [
117]. Additionally, HBOT preconditioning reduced cellular necrosis by modulating mitochondrial pathways. Specifically, cytoplasm cytochrome c levels, as well as capase-3 and capase-9 activity were reduced, upregulating Bcl-2 and Bax proteins linked with improved brain recovery [
93,
118,
119,
120,
121]. Inducing BDNF and inhibiting p38/MAPK phosphorylation also reduced the early onset of apoptosis and apoptosis progression [
93,
122]. Therefore, HBOT preconditioning in stroke evidently limits apoptosis progression by promoting anti-apoptotic activity and protein expression.
In addition to initiating apoptosis, ROS also moderates starvation-induced autophagy via class III phosphoinositide 3-kinase pathway, which sabotages the survival mechanism. ROS-induced autophagy was demonstrated when HBOT preconditioning upregulated protein expression levels of LC3-II and Beclin 1, causing autophagosomes to form in the ischemic penumbra post-ischemia in rat brain models [
123]. Additionally, HBOT preconditioning enhanced cell survival by downregulating MMP-9 expression, inhibited CA1 cell damage, and promoted healthy functional performance [
122]. Furthermore, preconditioning HBOT can activate Wnt signaling pathway, upregulate HIF-1, and secrete vascular endothelial growth factor (VEGF) to mitigate cell loss. HBOT increased levels of VEGF, VEGFR2, MEK1/2, Raf-1, and phosphor-extracellular signal-regulated kinase (ERK) ½ protein that further improved neurological functions [
123].
5.4. Preservation of Blood-Brain Barrier, Edema Minimization, and Angiogenesis
HBOT demonstrated preservation of the blood-brain barrier (BBB) and minimizes edema after the onset of surgical brain injuries (SBI), stroke (either ischemic or hemorrhagic), and TBI [
42,
125,
126,
127,
128,
129]. These protective mechanisms exist due to the suppression of inflammatory responses by lowering hemorrhage volumes and reducing NLRP3 inflammasome expression to recover cognitive functions. Furthermore, HBOT preconditioning also relieved neurological dysfunctions and reduced blood volumes by reducing HIF-1α, MMP-2, and MMP-9 [
42,
129]. However, HBOT preconditioning may overexpress HSP-70 in the hippocampus, which could lead to cognitive deficits and oxidative stress [
130]. Preconditioning of HBOT could bring protective effects of microvascular endothelial cell protection by increasing Nrf2 and HSP32 activity. Recent studies, however, reveal the therapeutic effects of HBOT on infarction volume, BBB, and transformed hemorrhage in the absence of the mentioned proteins in the focal cerebral ischemia model [
129].
HBOT preconditioning may reduce edema via downregulation of aquaporin 4 (AQP-4) expression, which possess the mechanism to hinder hemorrhage and preserve neural tissue [
131]. Cultured astrocytes post-HBOT revealed an increase in AQP-4 and VEGF, demonstrating the ability to modulate BBB openings. This mechanism may introduce a possible treatment option for drug transportation into the CNS [
132]. Additionally, HBOT promotes the p44/42 pathway to help prevents the development of brain edema post-intracerebral hemorrhage; the activation of the pathway correlates to the cerebral ischemic tolerance that was observed [
125]. In vitro models highlight the protective abilities of HBOT for BBB integrity when occluding and ZO-1 activities were regulated in hypoxic settings [
133]. Alongside protecting BBB integrity and minimizing edema, HBOT may also protect energy metabolism and tissue perfusion by stabilizing glucose levels, preventing glutamate levels from increasing, lowering lactate/pyruvate ratios, and increasing Ang-2 activity. Protecting energy metabolism gave therapeutic effects, including increased microvessel density, reduced brain injury, and alleviated post-ischemic neurological deficits [
44,
134].