HBOT has been observed to cause molecular changes in the central nervous system (CNS) of subjects afflicted with neurological conditions, including changes in mitochondrial function, white matter, neuroinflammation, oxidative stress, and CBF. In this section, we discuss HBOT’s mechanism from a neurobiological point of view and present some recent studies in this field (Figure 1
2.1. The Impact of HBOT on Mitochondrial Properties
The mitochondrion is a vital cell organelle that is responsible for supplying energy to cells by generating ATP [38
]. It also mediates cellular processes such as apoptosis [39
] and proliferation [40
], and takes part in neuronal functions such as synaptic plasticity [41
The mitochondria produce ATP via an electron-transport chain, in which oxygen has an important role as the last electron acceptor in the chain. In this process, a proton gradient is created as protons are pumped from the mitochondrial matrix to the intermembrane space. The high concentration of protons in the intermembrane space creates an electrical potential and a chemical gradient of protons in the membrane, which is vital for maintaining membrane potential and for the ATP-production process. When the membrane’s integrity (i.e., its structure or function) is compromised, an apoptotic pathway can be initiated inside the cell.
In neurons, there is a great need for well-functioning mitochondria, as neuronal activity consumes a large amount of energy and neurons have only small energy reserves [42
]. While there are several conditions that may cause mitochondrial dysfunction, such as mutations in the mitochondrial DNA that amplify with age [44
], this organelle’s function relies heavily on oxygen consumption. Reduction in oxygen levels, such as a hypoxic state, can damage energy production [45
] and cause lactate aggregation in the tissue as well as other metabolic changes [8
]. Accordingly, several studies have considered the use of HBOT to treat neuronal conditions related to mitochondrial dysfunction, by increasing the amount of oxygen arriving to the mitochondria.
HBOT was found to facilitate the correction of mitochondrial abnormalities such as those in mitochondrial metabolism [46
], improve the integrity of compromised mitochondrial membranes [47
], and inhibit secondary cell death by causing the transfer of mitochondria from astrocytes to neurons [48
]. Some studies measured HBOT’s impact according to changes in ATP levels after treatment [34
]. For example, Hu et al. [46
] studied a rat model for strokes, induced by middle cerebral artery occlusion combined with hyperglycemia to induce ischemia and hemorrhagic transformation. Following HBOT, ATP-expression levels were measured by the enzyme-linked immunosorbent assay (ELISA) and the results showed significantly upregulated ATP expression, along with an increase in NAD+ expression, an important marker of energy metabolism. In addition, they observed an increase in nicotinamide phosphoribosyltransferase (NAMPT) activity, which is a production-limiting protein of NAD+, and upregulation of Sirt1 expression, which is an upstream protein of p53 and NF-κB, the latter related to cell apoptosis and inflammation, respectively (Figure 2
Concomitant reduction in the expression of p53 and NF-κB strengthened the fact that an ATP/NAD+/Sirt1 pathway had been activated by HBOT. Furthermore, administration of NAD+ exhibited an effect similar to that of HBOT, and administration of ATP synthase inhibitor, NAMPT inhibitor, or Sirt1 small interfering RNA (siRNA) prevented HBOT‘s positive effect. Activation of this specific pathway by HBOT reduced cell necrosis and improved neurological function [46
Additional studies considered other aspects of HBOT’s effects on mitochondria. Palzur et al. [47
] examined HBOT’s effects on mitochondrial integrity and the activity of the mitochondrial apoptotic pathway in a rat model of TBI. They created lesions on a single hemisphere and used the uninjured hemisphere for comparison. A statistical analysis was performed comparing three different groups: A control group that was not treated with HBOT and went through dynamic cortical deformation in one hemisphere, a HBOT-treated group that went through the same procedure as the first group, and a sham group that underwent surgery without dynamic cortical deformation. The results showed that mitochondrial integrity, measured by mitochondrial transmembrane potential, was significantly restored after HBOT, as seen by lower differences in transmembrane potential between both hemispheres in the treated group compared to the untreated control group. Furthermore, in the untreated control group, activity of both caspase-3 and caspase-9, proteins known to mediate apoptosis through the mitochondria’s apoptotic pathway, was increased as compared to the treated group in which their activity was significantly reduced. However, caspase-8, a protein known to initiate apoptosis, showed no significant difference in activity between the treated and untreated groups. These results were consistent with the previous work, in which an increase in the expression of proteins Bcl-2 and Bcl-xL, both of which inhibit apoptosis, was measured after HBOT in the penumbra of a TBI rat model [50
]. In addition, no significant change was measured in the amount of the proapoptotic protein Bax [50
]. Together, these studies strongly indicate that HBOT specifically influences the mitochondrion’s intrinsic apoptotic pathway, as Bcl-2, caspase-3, and caspase-9 are involved in this particular pathway, whereas caspase-8 is mostly involved in the extrinsic apoptotic pathway [51
] (Figure 2
Lastly, an in vitro study was performed to determine HBOT’s effect on mitochondrial transfer from astrocytes to neurons [48
]. The researchers placed primary rat neuronal cells, co-cultured with astrocytes, in a HBOT chamber, and then exposed the cells to either tumor necrosis factor-alpha (TNF-α) or lipopolysaccharide to create a brain injury or stroke-like environment during the secondary cell death stage. They observed that the preliminary use of HBOT increases cell viability and induces higher mitochondrial transfer from astrocytes to neurons, suggesting that mitochondrial transfer, activated by HBOT, protects neurons from secondary cell death [48
Today, there are many studies focusing on the effects of HBOT on mitochondrial properties in the context of TBI and stroke. Mitochondrial impairment is also a common phenomenon in NDDs such as ASD [53
], and thus a natural direction for future research would be to examine HBOT’s effectiveness on those disorders as well.
2.2. The Impact of HBOT on Alterations in White Matter
Myelin is the outer layer that envelopes neurons [55
]. It is comprised mostly of lipids and is produced by OLs in the CNS [56
]. Myelin plays an important role in isolating the electrical information passing through an axon, which is crucial for neuronal connections [55
]. This isolation layer is key to the proper transmission of information between neurons, as evidenced by neurological conditions that include myelination deficits [58
Evidence from numerous studies suggests a connection between hypoxia and myelination deficits [7
]. Back et al. discovered that late OL precursors are extra-susceptible to oxygen deficiency, as reflected by high cell death [65
]. Hence, hypoxia may result in a reduction in the number of cells differentiated to mature myelinating OLs. In Cree et al. [60
], administration of the drug clemastine to hypoxic injured mice resulted in an elevated number of myelinating OLs and improved myelin ultrastructure. Together, these findings suggest that HBOT can ameliorate the damage caused by hypoxia to white matter, and might ease some of the symptoms in neurological disorders related to white matter deficiency and hypoxia.
One work investigated the effect of HBOT on nerve fiber regeneration in human TBI subjects with post-concussion syndrome [66
], which is a set of symptoms that are common after TBI. The effects were observed using diffusion tensor imaging (DTI) [67
], a magnetic resonance imaging (MRI) technique that can assess changes in white matter in live human subjects. Along with major improvements in clinical symptoms, they observed an increase in the fractional anisotropy, a value that represents the degree of anisotropy in the diffusion of a voxel, in diverse brain regions such as the corpus callosum, internal capsule, and midbrain. They also observed decreased mean diffusivity, a value that represents the total diffusion in a voxel, mainly in the white matter of the frontal lobe [66
], both indicative of restored neuronal fibers.
In animal models, several studies have investigated HBOT’s influence on myelin basic protein (Mbp) and remyelination. Baratz-Goldstein et al. [68
] studied these effects in a mouse model of mild TBI, applying either immediate or delayed HBOT treatment. They found that both treatments ameliorated the reduced Mbp expression and demyelination compared to the untreated control group, and that both the treated groups and the sham group had the same measured amount of Mbp expression [68
]. Differences between the treated groups and the control group were already observed 10 days after the beginning of the treatment, meaning that HBOT led to a rather fast recovery, even when limiting the length of the treatment to four days. In Kraitsy et al.’s work [69
], HBOT resulted in upregulation of the expression levels of two specific isoforms of Mbp which are important to myelin structure, upregulation of proteolipid protein (Plp), and increased remyelination processes.
Although these and other studies show the important effects of HBOT on white matter in the nervous system, there is a lack of mechanistic research performed directly on OLs, which could be key to understanding the mechanism acting in HBOT and myelination processes.
2.3. The Impact of HBOT on Neuroinflammation
Neuroinflammation in the brain is a reaction of the CNS immune cells and the peripheral immune system when the brain experiences trauma, injury or other pathological conditions. It is mainly created by microglia and astrocytes [70
], or by infiltrating peripheral immune system cells when the blood–brain barrier is compromised [72
], and those cells secrete various proinflammatory cytokines [71
]. While neuroinflammation is a necessary process for tissue repair, a prolonged state of hyperactivity of the immune system, usually referred to as chronic inflammation, may cause extensive tissue damage, as can happen in AD [74
], autism [75
], CP [78
], and TBI [80
Hypoxia is a major factor in inducing neuroinflammation, whereby it mediates the activation of cells from the innate immune system, resulting in upregulated secretion of proinflammatory cytokines and increased immune cell aggregation [4
]. On the other hand, chronic neuroinflammation can also cause or increase a hypoxic state, as evidenced by the upregulated expression of hypoxia-inducible factor 1-alpha (Hif1-α), thereby creating a positive-feedback cycle between the two conditions [83
One of the neurodegenerative disorders that is highly associated with neuroinflammation is AD. In AD, large amounts of amyloid plaques accumulate in the brain. These plaques may incur microgliosis, in which activated microglia surround the plaques and secrete proinflammatory cytokines [85
]. Shapira et al. [28
], studied the influence of HBOT on an AD mouse model and found less microglia around the amyloid plaques after the treatment, along with less proinflammatory cytokines such as TNF-α, as shown by immunofluorescence staining in the hippocampus. Those researchers suggested that the reduction in the amount of cytokines might be due to the reduced amount of microglia. Moreover, a morphological examination showed that HBOT enhances microglial process extension and increases the number of sprouting microglia around the plaques, which could indicate a change in the microglia’s activation state and function. These findings emerged in parallel to a reduction in hypoxia levels and in the amount of amyloid plaques, and improvement in cognitive- and anxiety-like-related behaviors [28
Several studies have found that aside from the decrement in the secretion of proinflammatory cytokines, HBOT attenuates neuroinflammation via enhancement of immune cell secretion of anti-inflammatory cytokines [28
]. In animal models, HBOT increased the expression levels of mRNA encoding the anti-inflammatory cytokine interleukin (IL-4), as measured by real-time quantitative PCR [28
], and increased the concentration of the anti-inflammatory cytokine IL-10 in the cortices, as measured by ELISA [88
]. Changes in anti-inflammatory cytokines were found in human patients with various neurological conditions as well [89
The anti-inflammatory effect of HBOT can promote tissue repair and prevent secondary cell death by hindering the apoptotic pathway. This was shown in a TBI mouse model, in which increased expression of the anti-inflammatory cytokine IL-10 following HBOT resulted in reduced caspase-3 activity, along with a reduction in Bax-expression levels [88
]. Together, these findings suggest that HBOT has the ability to not only stop future damage, but also to initiate a healing process in the tissue.
2.4. The Impact of HBOT on Oxidative Stress
Oxidative stress is the result of an imbalance of reactive oxygen species (ROS) inside the cell. Oxidative stress can have several causes, a primary one being the release of ROS from the mitochondria in a hyperoxic state [90
]. Although ROS can be beneficial under normal conditions to several cell processes, such as regulation of synaptic plasticity [92
] and molecular signaling [93
], abnormal ROS levels can cause DNA fragmentation and crosslinking of proteins, which can lead to cell apoptosis [93
]. One of the greatest concerns in using HBOT is an increase in oxidative stress, because of the high administration of oxygen and the potential for hyperoxia.
There are conflicting results on HBOT’s effect on oxidative stress; some studies have shown that after HBOT, there is an increase in antioxidants which are highly important for balancing ROS concentration [95
]; others have indicated an increase in ROS and induction of oxidative stress, in accordance with the higher oxygen supply [97
]. However, some of the latter studies were performed with either repeated consecutive exposures [99
], which deviates from the standard protocol, or with the relatively high baric pressure of four bars [97
], performed on animals, which is higher than the clinically approved pressure levels. In addition, it was shown that 24 h after the end of the treatment, the elevated ROS returned to baseline [100
], and the DNA damage incurred by the elevation in ROS was reversible [102
Despite the rise in ROS, several studies on cerebral ischemia and other conditions have shown that after HBOT, there is an increase in a variety of antioxidants [95
]. Both Wada et al. [96
] and Nie et al. [103
] demonstrated an increase in antioxidants such as superoxide dismutase (SOD) and catalase—important antioxidant enzymes—mediated by HBOT preconditioning, in rodent models for cerebral ischemia and spinal ischemia, respectively.
As already noted, an increase in ROS can result in positive outcomes, and not only cell toxicity. ROS may assist in initiating signaling in cellular pathways involved in angiogenesis via stabilization of Hif1-α, which in turn increases the production of vascular endothelial growth factor (VEGF) [104
]. This further solidifies the relationship between HBOT and angiogenesis, which is discussed in detail in Section 2.5
Although HBOT can increase ROS concentrations, its use with the right protocol has been established as safe [101
]. Furthermore, the aforementioned elevation in antioxidants may counteract this increase in ROS, creating a balance in which the oxidative stress state remains at the same level as prior to treatment. Wada et al. [96
] suggested that ROS upregulation may enhance the expression of antioxidants such as SOD, and even create ischemic tolerance (Figure 2
2.5. The Impact of HBOT on Induction of Angiogenesis and Changes in CBF
When the blood supply to a certain brain area is reduced, the high oxygen demand in that region might not be met, which could lead to a hypoxic state. In autism, for example, reduction in CBF was found in specific brain regions related to language, such as the temporal lobes [6
], and to occur during specific activities, such as observing facial expressions [108
The therapeutic value of HBOT in increasing CBF and angiogenesis—the process by which new blood vessels are created—in wounds has been firmly established [105
]. HBOT’s effect on these aspects in neurological conditions has been studied mainly by using various imaging techniques, such as positron emission tomography (PET) [109
], single-photon emission computed tomography (SPECT), and functional MRI [110
]. Most of the studies performed on humans used SPECT imaging and found increased blood flow, along with symptom improvement, in various neurological conditions [27
Tal et al. [66
] observed increased CBF and cerebral blood volume after HBOT in human TBI subjects, as measured by dynamic susceptibility contrast enhancement MRI imaging, potentially indicating angiogenesis. In other works, evidence of induction of angiogenesis by HBOT was found through the investigation of molecular markers. Two studies that measured such markers in the CNS [111
] showed upregulation of VEGF mRNA, which is responsible for, among other things, vascularization. Angiogenesis could be a central mechanism by which HBOT increases CBF, as well as vascular repair.
Overall, the discussed studies have shown that HBOT can assist in vascular repair. In addition to achieve a direct supply of oxygen, there are strong indications that HBOT can indirectly increase the amount of oxygen reaching a tissue by encouraging angiogenesis in the CNS.