**2. Potential cellular and molecular mechanisms underlying HBOT**

Increasing number of animal studies on HBOT in experimental TBI revealed a myriad of diverse mechanisms that may underlie neuroprotective effects of HBOT. Researchers suggested that many of these cellular and molecular mechanisms and signaling pathways work in parallel, or together, contributing to repair of the injured brain [6, 7, 23, 25, 28]. These mechanisms involve: (1) alleviation of secondary injury; (2) increasing of tissue oxygenation; (3) reducing of neurodegeneration; (4) decreasing of apoptosis; (5) regulation of oxidant/antioxidant status; (6) reduction of oxidative stress; (7) attenuation of reactive gliosis (microgliosis and astrogliosis) and glial scarring; (8) reducing of inflammation; (9) enhancement of neuronal plasticity; (10) promoting of synaptogenesis, neurogenesis and angiogenesis.

### **2.1. HBOT suppresses development of secondary brain damage**

**1. Introduction**

treatment [20–23].

Traumatic brain injuries (TBI) are one of the leading causes of death and chronic disability especially among the working population, and represent an important public health problem worldwide. Globally, about 10 million people are affected by TBI every year with projections that TBI will be one of the major causes of death and disability by the year 2020 [1]. Since TBI is a complex injury that encompasses a broad spectrum of symptoms and disabilities, the manifestations of head injury may be clinically very variable ranging from mild, to moderate or severe, depending on the extent and duration of damage to the brain. Many cognitive, physical and psychological skills can be affected, exerting a devastating impact on the patients and their family [2]. Over the years more than 30 phase III clinical trials failed emphasizing the urgent need for efficient treatment modalities and new directions in the future research to improve posttraumatic morbidity and mortality. Considering the complexity of TBI it is reasonable to assume that only combination of different treatment protocols could provide better prognosis for recovery to all forms of TBI [3]. In this view, hyperbaric oxygenation (HBO) or hyperbaric oxygen therapy (HBOT) appeared as an adjunctive therapy that may have the synergistic effect with other treatment protocols, suggesting that combining therapies with HBOT could provide better results than either alone [4]. According to definition given by the Undersea and Hyperbaric Medical Society (UHMS), hyperbaric medicine is a therapeutic approach in which a patient breathes 100% oxygen intermittently, while the pressure of the treatment chamber is higher than ambient (1 atmosphere absolute, 1 ATA = 101.3 kPa) [5–7]. In comparison to the normobaric conditions increased oxygen supply under hyperbaric conditions enables easier diffusion of oxygen into the injured tissue [8]. Accordingly, the HBOT can be used to obtain 100% saturation of hemoglobin and to significantly elevate the volume of physically dissolved oxygen fraction in blood plasma. This increased blood oxygen level then can penetrate to ischemic areas and perilesioned tissue more deeply than under normobaric conditions [9–11]. Thus, the HBOT has found its place, as the primary or adjuvant therapy in

26 Hyperbaric Oxygen Treatment in Research and Clinical Practice - Mechanisms of Action in Focus

the treatment protocols for different clinical conditions [12, 13].

neuroprotective effects of hyperbaric oxygenation after the brain injury.

On the other hand, opinions about usage of HBOT as adjunctive therapy for the treatment of patients with brain injuries are still controversial [14–16]. In this way HBO is a very motivating therapeutic modality, which is known to produce oxidative stress by itself [17], but reduces oxidative stress when used in pathological conditions [18, 19]. The main concern in HBOT is oxidative stress and/or oxygen toxicity that can affect multiple organs. However, these side-effects are dependent on treatment parameters – pressure and duration of the

Substantial amount of evidences has been published indicating that HBOT can interfere with the processes that are following brain injury and moderate its consequences [14, 24–27]. Recent results of experimental and clinical studies and potential mechanisms of HBOT in TBI are reviewed by Wang et al. [28] and Hu et al. [7]. However, knowledge about the exact mechanisms by which HBOT exerts its beneficial effects is still deficient. Therefore, data presented in this chapter are meant to put more light on cellular and molecular mechanisms underlying TBI involves primary and secondary injury. Primary injury occurs at the time of the impact and is the result of immediate mechanical damage of neural pathways followed by a permanent neuronal lost. The site of mechanical impact is called the "core". Surrounding regions consist of neuronal tissue that have not been directly affected by trauma and are often addressed to as "penumbra area". Neurons inside this zone are at risk due to a cascade of events, known as secondary injury that involves: impaired blood flow (limited or not at all), inflammation, development of edema, acidosis and hemorrhage, and the loss of most of their connections with the other neurons [11, 21, 22]. Secondary degeneration can also progress into the surrounding intact regions of the brain. Compromised blood flow and insufficient oxygen supply leads to tissue hypoxia and the resulting energy failure, which initiates a cascade of cellular events that culminate with neuronal cell apoptosis [23]. Thus, the consequence of secondary injury is degeneration of neurons that previously have not been exposed to trauma [29–31]. Most of the neurotherapeutic strategies are directed toward the containment of the secondary processes and the preservation and reactivation of the penumbra area and perilesioned region [30]. Cumulative evidence have proved that HBOT may reduce development of secondary brain damage and prevent neuronal apoptosis in animal models of TBI [32, 25], ischemic stroke [33–37], and hypoxia-ischemia [38–40], which was manifested by diminishing of brain infarction area and improvement of neurological deficits. Recently, Baratz-Goldstein et al. [41] demonstrated that both immediate (initiated 3 h post-injury) and delayed treatments with HBO (initiated 7 days post-injury) have a potential to prevent a neuronal loss in mouse model of moderate TBI.

### **2.2. HBOT reduces neuronal degeneration and prevents apoptosis after brain injury by regulation of oxidant/antioxidant status and reduction of oxidative stress**

One of the main processes in this pathological cascade is oxidative stress that develops in the cells which have been exposed to trauma and in the cells at "penumbra area". Reactive oxygen species (ROS) are one of the products of oxidative stress [2] that are responsible for cellular damaging and apoptosis. The first line of the defense against ROS are enzymes located in mitochondria, such as manganese superoxide dismutase (SOD2) [42]. In our previous study, we have shown that repetitive HBOT influenced the pattern of SOD2 expression both on gene and protein level in cortical stab injury model (CSI) of TBI [43]. We applied HBO protocol of 60 min exposure to 100% oxygen at 2.5 ATA, once a day for 3 or 10 consecutive days. HBOT significantly increased mRNA levels of SOD2 at both time points compared to the corresponding lesioned group. Exposure to HBOT for 3 days down-regulated SOD2 protein levels in the injured cortex, while after 10 days of HBOT an up-regulation of SOD2 was observed. Using double-immunofluorescence staining we have demonstrated that HBOT attenuated SOD2 expression both in neuronal and astroglial cells surrounding the lesion site. Staining of the injured cortex with Fluoro-Jade®B (as a marker of degenerating neurons) revealed that HBOT significantly decreased the number of degenerating neurons in the injured cortex, and this effect was more pronounced after 10 consecutive HBOT. In according to this, we concluded that antioxidative and neuroprotective effect of HBOT is in part due to its influence on expression pattern of SOD2 [43].

In this chapter, using the cortical suction ablation (CSA) model of brain injury, described in our previous publications [44, 45], and the same HBOT protocol, we demonstrated that 10 repetitive HBOT altered activities of antioxidant enzymes and reduced lipid peroxidation, thereby preventing neuronal degeneration and apoptosis. Oxidant/antioxidant status in the injured cortex after HBOT is presented in **Figure 1**. HBOT significantly increased glutathioneperoxidase (GPX) activity in the injured cortex compared to all other groups. Injury markedly lowered the level of superoxide dismutase (SOD) activity, while HBOT returned SOD activity to almost control levels. The content of Malondialdehyde (MDA), which was used as an indicator of lipid peroxidation and reflects the membrane damage caused by ROS, was the highest in the tissue samples from injured cortex. HBOT initiated statistically significant reduction of MDA levels, pointing to preservation of membrane integrity. The similar trend of changes of MDA levels was determined in the serum indicating that serum concentrations of MDA may be used as a marker of degree of brain damage.

To evaluate the effect of HBOT on neurodegeneration/apoptosis we performed doubleimmunofluorescence staining: neurons undergoing degeneration were visualized with Fluoro-Jade®B, while NeuN (neuronal cell nuclei) was used as a marker of neuronal cell bodies. As it is shown in **Figure 2**, in the perilesioned cortex a huge number of neurons undergoing degeneration (**Figure 2A**, **E**) was significantly reduced after the HBOT (**Figure 2B**, **F**). Moreover, when the cortical sections from the injured (L) group were observed at higher magnification the formation of apoptotic bodies (**Figure 2G**, **I**, asterisks) in the neuronal nuclei was observed, indicating that they have entered into the process of apoptosis. On the contrary, in the sections from LHBO group the majority of neurons have healthy nuclei in which the nucleolus was clearly visible (**Figure 2H**, **J**, arrow heads). These results indicate that increased activities of antioxidant enzymes and reduction of lipid peroxidation underlies observed neuroprotective and anti-apoptotic effects of HBOT. Similarly, Li et al. [51], in the rat model of brain ischemia-reperfusion injury (IRI), have shown that HBO preconditioning lessened neuronal injury, reduced the level of MDA and increased the antioxidant activity of catalase (CAT) and SOD. They suggested that an up-regulation of the antioxidant enzyme activities after HBO preconditioning may play an important role in the generation of tolerance against IRI.

Maintaining proper mitochondrial function is essential for cellular function, since ROS are formed in mitochondria when energy metabolism is compromised. Niizuma and co-workers [52] demonstrated that mitochondrial dysfunction and oxidative stress may determine neuronal

death/survival after stroke and neurodegeneration. Therefore, a lot of studies have been conducting with purpose of finding out whether HBOT have a role in the preservation of mitochondrial function and integrity [25, 32, 53–55]. Palzur, Vlodavsky and their colleagues,

vs. L, # P < 0.05 vs. C, † P < 0.05 vs. CHBO, ‡ P < 0.05 vs. S, § P < 0.05 vs. SHBO.

**Figure 1.** Effects of HBOT and cortical injury on the activities of antioxidant enzymes and lipid peroxidation in the injured cortex. Glutathione-peroxidase (GPX) activity is measured using coupled enzyme method by measuring the decrease of NADPH at 340 nm [46] and is expressed as unites per milligram of protein (U/mg). One unit (U) catalyzes the oxidation by

 of 1.0 μmol of reduced glutathione to oxidized glutathione per minute. Total superoxide dismutase (SOD) activity is determined at room temperature according to the method of Misra and Fridovich [47], and is measured at 480 nm. One unit of SOD is defined as the amount of enzyme that inhibits the speed of oxidation of epinephrine for 50%. The results are expressed as U/mg of protein. Malondialdehyde (MDA) content is determined both in the injured cortex and serum. Thiobarbituric acid (TBA) reactive product MDA is used as an indicator of lipid peroxidation. MDA content is measured according to the method of Aruoma et al. [48] as described previously in Mladenovic et al. [49]. The absorbance of the organic phase is read at 532 nm. The values are expressed as nmol of MDA/mg of protein, using a standard curve of 1,1,3,3-tetramethoxypropane. Total protein was quantified according to Lowry's method [50] using bovine serum albumin as standard. Values are mean ± SD from n ≥ 4 independent determinations performed in duplicate. Acronyms for the groups (n = 8 per group) are as follows: C – intact control, CHBO intact control subjected to the HBO protocol, S – sham surgery, SHBO sham control HBO group, L - injured group and LHBO - injured group subjected to the HBO protocol. Significant difference from corresponding group \* P < 0.05

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cortical stab injury model (CSI) of TBI [43]. We applied HBO protocol of 60 min exposure to 100% oxygen at 2.5 ATA, once a day for 3 or 10 consecutive days. HBOT significantly increased mRNA levels of SOD2 at both time points compared to the corresponding lesioned group. Exposure to HBOT for 3 days down-regulated SOD2 protein levels in the injured cortex, while after 10 days of HBOT an up-regulation of SOD2 was observed. Using double-immunofluorescence staining we have demonstrated that HBOT attenuated SOD2 expression both in neuronal and astroglial cells surrounding the lesion site. Staining of the injured cortex with Fluoro-Jade®B (as a marker of degenerating neurons) revealed that HBOT significantly decreased the number of degenerating neurons in the injured cortex, and this effect was more pronounced after 10 consecutive HBOT. In according to this, we concluded that antioxidative and neuroprotective effect of HBOT

In this chapter, using the cortical suction ablation (CSA) model of brain injury, described in our previous publications [44, 45], and the same HBOT protocol, we demonstrated that 10 repetitive HBOT altered activities of antioxidant enzymes and reduced lipid peroxidation, thereby preventing neuronal degeneration and apoptosis. Oxidant/antioxidant status in the injured cortex after HBOT is presented in **Figure 1**. HBOT significantly increased glutathioneperoxidase (GPX) activity in the injured cortex compared to all other groups. Injury markedly lowered the level of superoxide dismutase (SOD) activity, while HBOT returned SOD activity to almost control levels. The content of Malondialdehyde (MDA), which was used as an indicator of lipid peroxidation and reflects the membrane damage caused by ROS, was the highest in the tissue samples from injured cortex. HBOT initiated statistically significant reduction of MDA levels, pointing to preservation of membrane integrity. The similar trend of changes of MDA levels was determined in the serum indicating that serum concentrations of MDA may

To evaluate the effect of HBOT on neurodegeneration/apoptosis we performed doubleimmunofluorescence staining: neurons undergoing degeneration were visualized with Fluoro-Jade®B, while NeuN (neuronal cell nuclei) was used as a marker of neuronal cell bodies. As it is shown in **Figure 2**, in the perilesioned cortex a huge number of neurons undergoing degeneration (**Figure 2A**, **E**) was significantly reduced after the HBOT (**Figure 2B**, **F**). Moreover, when the cortical sections from the injured (L) group were observed at higher magnification the formation of apoptotic bodies (**Figure 2G**, **I**, asterisks) in the neuronal nuclei was observed, indicating that they have entered into the process of apoptosis. On the contrary, in the sections from LHBO group the majority of neurons have healthy nuclei in which the nucleolus was clearly visible (**Figure 2H**, **J**, arrow heads). These results indicate that increased activities of antioxidant enzymes and reduction of lipid peroxidation underlies observed neuroprotective and anti-apoptotic effects of HBOT. Similarly, Li et al. [51], in the rat model of brain ischemia-reperfusion injury (IRI), have shown that HBO preconditioning lessened neuronal injury, reduced the level of MDA and increased the antioxidant activity of catalase (CAT) and SOD. They suggested that an up-regulation of the antioxidant enzyme activities after HBO preconditioning may play an important role in the generation of toler-

Maintaining proper mitochondrial function is essential for cellular function, since ROS are formed in mitochondria when energy metabolism is compromised. Niizuma and co-workers [52] demonstrated that mitochondrial dysfunction and oxidative stress may determine neuronal

is in part due to its influence on expression pattern of SOD2 [43].

28 Hyperbaric Oxygen Treatment in Research and Clinical Practice - Mechanisms of Action in Focus

be used as a marker of degree of brain damage.

ance against IRI.

**Figure 1.** Effects of HBOT and cortical injury on the activities of antioxidant enzymes and lipid peroxidation in the injured cortex. Glutathione-peroxidase (GPX) activity is measured using coupled enzyme method by measuring the decrease of NADPH at 340 nm [46] and is expressed as unites per milligram of protein (U/mg). One unit (U) catalyzes the oxidation by H2 O2 of 1.0 μmol of reduced glutathione to oxidized glutathione per minute. Total superoxide dismutase (SOD) activity is determined at room temperature according to the method of Misra and Fridovich [47], and is measured at 480 nm. One unit of SOD is defined as the amount of enzyme that inhibits the speed of oxidation of epinephrine for 50%. The results are expressed as U/mg of protein. Malondialdehyde (MDA) content is determined both in the injured cortex and serum. Thiobarbituric acid (TBA) reactive product MDA is used as an indicator of lipid peroxidation. MDA content is measured according to the method of Aruoma et al. [48] as described previously in Mladenovic et al. [49]. The absorbance of the organic phase is read at 532 nm. The values are expressed as nmol of MDA/mg of protein, using a standard curve of 1,1,3,3-tetramethoxypropane. Total protein was quantified according to Lowry's method [50] using bovine serum albumin as standard. Values are mean ± SD from n ≥ 4 independent determinations performed in duplicate. Acronyms for the groups (n = 8 per group) are as follows: C – intact control, CHBO intact control subjected to the HBO protocol, S – sham surgery, SHBO sham control HBO group, L - injured group and LHBO - injured group subjected to the HBO protocol. Significant difference from corresponding group \* P < 0.05 vs. L, # P < 0.05 vs. C, † P < 0.05 vs. CHBO, ‡ P < 0.05 vs. S, § P < 0.05 vs. SHBO.

death/survival after stroke and neurodegeneration. Therefore, a lot of studies have been conducting with purpose of finding out whether HBOT have a role in the preservation of mitochondrial function and integrity [25, 32, 53–55]. Palzur, Vlodavsky and their colleagues,

using cortical deformation model of TBI and the HBO protocol which consisted of two successive 45 min sessions at 2.8 ATA, have shown that increased concentrations of oxygen in the cells lead to preservation of mitochondrial integrity due to significant decrease of the loss of mitochondrial trans-membrane potential. Additionally, HBOT reduced the release of pro-apoptotic mediators Cytochrome C (Cyt C) and the Bcl-2-associated X protein (Bax) from mitochondria, and up-regulated the expression of anti-apoptotic protein Bcl-2 (B-cell lymphoma 2), consequently alleviating neuronal apoptosis in the injured brain tissue [56]. Zhou et al. [57] have emphasized that maintenance of mitochondrial function is one of the

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TBI leads to the impairment of cerebral oxygen delivery and consumption [58]. So, the main problem is how to make cerebral hyperoxia, which is possible either under normobaric (NBH) or hyperbaric conditions. Clinical trials have shown that hyperbaric O2 has better effect than NBH on oxidative cerebral metabolism due to its ability to produce a brain tissue

Oxidative stress, and/or oxygen toxicity as unwanted side-effects of HBOT, as well as the fact that inhalation of pure oxygen at high pressures may lead to generation of ROS led researches to investigate which anti-oxidants can be used during HBO therapy. Studies have shown that hydrogen gas (H2) could be useful for this purpose. It alleviates oxygen toxicity due to reduction of hydroxyl radical levels [59]. Even, adding of inert gases, such as argon or xenon during

HBO treatments can potentially make further improvement of cerebral lesions [60].

**2.3. HBOT attenuates reactive microgliosis, astrogliosis and glial scarring after brain** 

microglia to more ramified, resting form (**Figure 3B** upper rectangle, **D** inset, **F** inset).

overcoming detrimental effects of gliosis and providing its beneficial effects.

HBO-induced suppression of microgliosis and astrogliosis was reported to give an account to beneficial effects of HBO treatment in different rat models of TBI [41, 67, 68], cerebral ischemia [69], neuropathic and inflammatory pain [70, 71]. In contrast, Lee et al. [72] reported that prolonged HBOT may increase degree of gliosis indicating that longer oxygen cycling might help

After the injury astrocytes become rapidly activated during the process of "reactive astrogliosis" and accumulate around the lesion site, acting as a barrier that impedes neuroregeneration and neurite outgrowth, and isolates intact CNS tissue from secondary lesions [61, 62]. Proportionally to the severity of injury, they undergo cell proliferation, hypertrophy, increased expression of glial fibrillary acidic protein (GFAP) and vimentin, and exhibit an enhancement of immune-modulating capacities [2, 44, 63–66]. In our recently published paper [66] we have shown that repetitive HBOT attenuates reactive astrogliosis, prevent glial scar formation, and down-regulates GFAP and vimentin gene, protein and tissue expression in the perilesioned cortex. Similarly, Baratz-Goldstein et al. [41] demonstrated that both immediate and delayed HBOT have a potential to reduce reactive astrogliosis in mice model of TBI. Here, we reported that HBOT reduces reactive microgliosis around the lesion site as well (**Figure 3B**). Besides decreasing the number of activated microglia, HBOT also alters the morphology of activated

PO2 > or = 200 mm Hg, which represents a graduated effect [14].

most important effects of HBOT.

**injury**

**Figure 2.** HBOT reduces neurodegeneration and prevents apoptosis in the injured cortex. Fluoro-jade®B staining (green) is performed in order to visualize neuronal cells undergoing degeneration and cell death, while NeuN (red) is used as a marker of neuronal cell bodies. Procedure for double-immunofluorescence staining was as described in Parabucki et al. [43]. Cortical sections were incubated with mouse anti-NeuN (1:200, Milipore, USA) and then with 0.0004% solution of Fluoro-Jade®B (FJB, Chemicon International, Temecula, CA, USA) dissolved in 0.1% acetic acid. The slides were examined with Carl Zeiss AxioVert microscope with AxioCam monochromatic camera (Zeiss, Goettingen, Germany), equipped with ApoTome software for optical sectioning. (**A, C, E**) A huge number of NeuN+ neurons in the perilesioned cortex were co-stained with FJB (**A**, yellow fluorescence) indicating that they are undergoing neurodegeneration. Strikingly, when the cortical sections from the injured (L) group were observed at higher magnification, the formation of apoptotic bodies (**G, I,** asterisks) in the neuronal nuclei was observed, suggesting that they have entered into the process of apoptosis. (**B, D, F**) after 10 repetitive HBOT the number of NeuN+ /FJB+ (**B)** and FJB+ (**F**) neurons was negligible. Correspondingly, in the LHBO cortical sections the majority of neurons have healthy nuclei in which the nucleolus was clearly visible (**H, J,** arrow heads). Rectangles indicate where the high magnification images are taken from. Scale bars: (A-J) 50 μm.

using cortical deformation model of TBI and the HBO protocol which consisted of two successive 45 min sessions at 2.8 ATA, have shown that increased concentrations of oxygen in the cells lead to preservation of mitochondrial integrity due to significant decrease of the loss of mitochondrial trans-membrane potential. Additionally, HBOT reduced the release of pro-apoptotic mediators Cytochrome C (Cyt C) and the Bcl-2-associated X protein (Bax) from mitochondria, and up-regulated the expression of anti-apoptotic protein Bcl-2 (B-cell lymphoma 2), consequently alleviating neuronal apoptosis in the injured brain tissue [56]. Zhou et al. [57] have emphasized that maintenance of mitochondrial function is one of the most important effects of HBOT.

TBI leads to the impairment of cerebral oxygen delivery and consumption [58]. So, the main problem is how to make cerebral hyperoxia, which is possible either under normobaric (NBH) or hyperbaric conditions. Clinical trials have shown that hyperbaric O2 has better effect than NBH on oxidative cerebral metabolism due to its ability to produce a brain tissue PO2 > or = 200 mm Hg, which represents a graduated effect [14].

Oxidative stress, and/or oxygen toxicity as unwanted side-effects of HBOT, as well as the fact that inhalation of pure oxygen at high pressures may lead to generation of ROS led researches to investigate which anti-oxidants can be used during HBO therapy. Studies have shown that hydrogen gas (H2) could be useful for this purpose. It alleviates oxygen toxicity due to reduction of hydroxyl radical levels [59]. Even, adding of inert gases, such as argon or xenon during HBO treatments can potentially make further improvement of cerebral lesions [60].
