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

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

**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

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,** 

(**B)** and FJB+

LHBO cortical sections the majority of neurons have healthy nuclei in which the nucleolus was clearly visible (**H, J,** arrow

/FJB+

heads). Rectangles indicate where the high magnification images are taken from. Scale bars: (A-J) 50 μm.

neurons in the perilesioned cortex were

(**F**) neurons was negligible. Correspondingly, in the

with ApoTome software for optical sectioning. (**A, C, E**) A huge number of NeuN+

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

**F**) after 10 repetitive HBOT the number of NeuN+

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 overcoming detrimental effects of gliosis and providing its beneficial effects.

**2.4. HBOT prevents spreading of the neuroinflammation in the injured tissue**

Inflammation is an important part of the pathophysiology of TBI and has a pivotal role in the extent of neuronal injury and repair. It is postulated that the initiation, progression and resolution of inflammation in TBI is multifaceted. These processes involve migration, recruitment and infiltration of leukocytes following blood-brain barrier (BBB) disruption and activation of resident immune cells of the CNS (microglia, astrocytes). Microglia and astrocytes then acquire immunological function and secrete inflammatory mediators such as pro- and anti-inflammatory cytokines, chemokines, adhesion molecules, complement factors, ROS and other factors [58, 73, 74]. The accumulation of neutrophils around the site of injury and their infiltration into the injured brain area is crucial for the initiation and progression of inflammation and the extent of secondary brain damage since they may release free oxygen and nitrogen radicals and pro-inflammatory cytokines [54, 58]. Neutrophils initially attach to vascular endothelium via binding to the endothelial intercellular adhesion molecules (ICAMs). In our previously mentioned paper [66] we have demonstrated injury-induced increase of gene and tissue expression of ICAM-1 (Intercellular Adhesion Molecule-1, CD54), an adhesion molecule that is important for trans-endothelial migration of neutrophils and propagation of inflammation [75, 76]. Using double- immunofluorescence staining we demonstrated its localization on various type of cells (astrocytes, vascular endothelium, neurons, activated microglia/macrophages and neutrophils), around the blood vessels, and in the proximity and within the lesion site. Ten successive treatments with HBO significantly decreased ICAM-1 mRNA expression returning it to control levels, while increased ICAM-1 immunoreactivity around the lesion site was diminished [66]. Herein, using double-immunofluorescence staining we have shown that HBOT reduced expression of ICAM-1 on activated microglia within the lesion site (**Figure 3H**). Furthermore, HBOT increased number of ramified/resting microglia in the perilesioned cortex (**Figure 3B**, upper rectangle). Interestingly, they do not express ICAM-1(**Figure 3H**, inset). These data indicate that HBOT by reducing ICAM-1 expression and targeting the passage of immune cells through the BBB via inhibition of cell adhesion molecules may contribute to dampen the neuroinflammatory response to TBI. Several studies also have shown that HBOT reduces the expression of ICAM-1 and adhesion of neutrophils to the endothelium, which is correlated with improved neurological outcome [54, 77–80].

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CD40 ligand (CD40L, also termed CD154, or GP39) and its counter receptor CD40 (a membrane protein that belongs to the tumor necrosis factor (TNF) receptor family) are well-known regulators of pro-inflammatory and immune responses in the CNS [81], and are members of CD40/ CD40L/ICAM-1 deleterious cascade of events after TBI. Given that CD40/CD40L dyad fosters neuroinflammation, it is suggested that CD40/CD40L interaction may be involved in modulating the outcome from injuries of the brain [82–84]. Accordingly, strategies aimed at suppressing CD40/CD40L/ICAM-1 expression may attenuate inflammation and neuronal damage after TBI, which will ultimately be of benefit in recovery [85]. In our recent paper [66] we have for the first time shown that HBOT prevents injury-induced up-regulation of expression of CD40 and its ligand CD40L on microglia/macrophages, neutrophils, cortical neurons and reactive astrocytes. These results indicate that repetitive HBOT, by limiting expression of inflammatory mediators,

supports formation of more permissive environment for repair and regeneration.

Data of many studies has been shown that HBO suppress various mediators of inflammation [54, 67, 86, 87] indicating that the decreased brain edema, blood-brain barrier leakage, cell

**Figure 3.** HBOT reduces reactive microgliosis in the perilesioned cortex and down-regulates ICAM-1 expression on microglial cells. (**A**) and (**B**) Effect of HBOT on reactive microgliosis in the injured cortex is determined using mouse anti-CD68 antibody (ED1, 1:100, Abcam, Cambridge, MA, USA) as a marker of activated microglia/macrophages. After using appropriate peroxidase linked secondary antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), the products of immunoreactions were visualized with 3′3-diaminobenzidine (DAB, Dako, Glostrup, Denmark). Immunohistochemical and immunofluorescence staining was performed as described in Lavrnja et al. [66]. (**A**) after cortical injury a huge number of activated microglia/macrophages are seen around and within the lesion site. (**B**) HBOT (60 min exposure to 100% oxygen at 2.5 ATA) initiated daily for 10 consecutive days attenuated reactive microgliosis and alters morphology of activated microglia to more ramified, resting form (**B**, upper rectangle). (**C**–**H**) expression of ICAM-1 (green) on microglial cells stained with Iba1 (red) in cortices of injured (L) and injured group subjected to HBO protocol (LHBO) is visualized with immunofluorescence double-labeling. Cortical sections were incubated with goat anti-ICAM1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:500, Abcam, Cambridge, MA, USA) antibody, while nuclei were counterstained with DAPI (Invitrogen, Grand Island, NY, USA). All sections were photographed with Carl Zeiss Axiovert microscope with AxioCam monochromatic camera (Zeiss, Goettingen, Germany), equipped with ApoTome software for optical sectioning. (**C**, **E**, **G**) activated microglia characterized with round morphology showed up-regulation of ICAM-1 expression (**G**). (**D**, **F**, **H**) repetitive HBOT downregulated ICAM-1 expression on activated microglia within the lesion site. Microglial cells with ramified morphology (insets to **D** and **F**) do not express ICAM-1 (insets to **D** and **H**). Rectangles indicate where the high magnification images are taken from. Scale bars: (**A**, **B**) 50 μm (**C**–**H**) 5 μm.

#### **2.4. HBOT prevents spreading of the neuroinflammation in the injured tissue**

Inflammation is an important part of the pathophysiology of TBI and has a pivotal role in the extent of neuronal injury and repair. It is postulated that the initiation, progression and resolution of inflammation in TBI is multifaceted. These processes involve migration, recruitment and infiltration of leukocytes following blood-brain barrier (BBB) disruption and activation of resident immune cells of the CNS (microglia, astrocytes). Microglia and astrocytes then acquire immunological function and secrete inflammatory mediators such as pro- and anti-inflammatory cytokines, chemokines, adhesion molecules, complement factors, ROS and other factors [58, 73, 74]. The accumulation of neutrophils around the site of injury and their infiltration into the injured brain area is crucial for the initiation and progression of inflammation and the extent of secondary brain damage since they may release free oxygen and nitrogen radicals and pro-inflammatory cytokines [54, 58]. Neutrophils initially attach to vascular endothelium via binding to the endothelial intercellular adhesion molecules (ICAMs). In our previously mentioned paper [66] we have demonstrated injury-induced increase of gene and tissue expression of ICAM-1 (Intercellular Adhesion Molecule-1, CD54), an adhesion molecule that is important for trans-endothelial migration of neutrophils and propagation of inflammation [75, 76]. Using double- immunofluorescence staining we demonstrated its localization on various type of cells (astrocytes, vascular endothelium, neurons, activated microglia/macrophages and neutrophils), around the blood vessels, and in the proximity and within the lesion site. Ten successive treatments with HBO significantly decreased ICAM-1 mRNA expression returning it to control levels, while increased ICAM-1 immunoreactivity around the lesion site was diminished [66]. Herein, using double-immunofluorescence staining we have shown that HBOT reduced expression of ICAM-1 on activated microglia within the lesion site (**Figure 3H**). Furthermore, HBOT increased number of ramified/resting microglia in the perilesioned cortex (**Figure 3B**, upper rectangle). Interestingly, they do not express ICAM-1(**Figure 3H**, inset). These data indicate that HBOT by reducing ICAM-1 expression and targeting the passage of immune cells through the BBB via inhibition of cell adhesion molecules may contribute to dampen the neuroinflammatory response to TBI. Several studies also have shown that HBOT reduces the expression of ICAM-1 and adhesion of neutrophils to the endothelium, which is correlated with improved neurological outcome [54, 77–80].

CD40 ligand (CD40L, also termed CD154, or GP39) and its counter receptor CD40 (a membrane protein that belongs to the tumor necrosis factor (TNF) receptor family) are well-known regulators of pro-inflammatory and immune responses in the CNS [81], and are members of CD40/ CD40L/ICAM-1 deleterious cascade of events after TBI. Given that CD40/CD40L dyad fosters neuroinflammation, it is suggested that CD40/CD40L interaction may be involved in modulating the outcome from injuries of the brain [82–84]. Accordingly, strategies aimed at suppressing CD40/CD40L/ICAM-1 expression may attenuate inflammation and neuronal damage after TBI, which will ultimately be of benefit in recovery [85]. In our recent paper [66] we have for the first time shown that HBOT prevents injury-induced up-regulation of expression of CD40 and its ligand CD40L on microglia/macrophages, neutrophils, cortical neurons and reactive astrocytes. These results indicate that repetitive HBOT, by limiting expression of inflammatory mediators, supports formation of more permissive environment for repair and regeneration.

**Figure 3.** HBOT reduces reactive microgliosis in the perilesioned cortex and down-regulates ICAM-1 expression on microglial cells. (**A**) and (**B**) Effect of HBOT on reactive microgliosis in the injured cortex is determined using mouse anti-CD68 antibody (ED1, 1:100, Abcam, Cambridge, MA, USA) as a marker of activated microglia/macrophages. After using appropriate peroxidase linked secondary antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), the products of immunoreactions were visualized with 3′3-diaminobenzidine (DAB, Dako, Glostrup, Denmark). Immunohistochemical and immunofluorescence staining was performed as described in Lavrnja et al. [66]. (**A**) after cortical injury a huge number of activated microglia/macrophages are seen around and within the lesion site. (**B**) HBOT (60 min exposure to 100% oxygen at 2.5 ATA) initiated daily for 10 consecutive days attenuated reactive microgliosis and alters morphology of activated microglia to more ramified, resting form (**B**, upper rectangle). (**C**–**H**) expression of ICAM-1 (green) on microglial cells stained with Iba1 (red) in cortices of injured (L) and injured group subjected to HBO protocol (LHBO) is visualized with immunofluorescence double-labeling. Cortical sections were incubated with goat anti-ICAM1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:500, Abcam, Cambridge, MA, USA) antibody, while nuclei were counterstained with DAPI (Invitrogen, Grand Island, NY, USA). All sections were photographed with Carl Zeiss Axiovert microscope with AxioCam monochromatic camera (Zeiss, Goettingen, Germany), equipped with ApoTome software for optical sectioning. (**C**, **E**, **G**) activated microglia characterized with round morphology showed up-regulation of ICAM-1 expression (**G**). (**D**, **F**, **H**) repetitive HBOT downregulated ICAM-1 expression on activated microglia within the lesion site. Microglial cells with ramified morphology (insets to **D** and **F**) do not express ICAM-1 (insets to **D** and **H**). Rectangles indicate where the high

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

magnification images are taken from. Scale bars: (**A**, **B**) 50 μm (**C**–**H**) 5 μm.

Data of many studies has been shown that HBO suppress various mediators of inflammation [54, 67, 86, 87] indicating that the decreased brain edema, blood-brain barrier leakage, cell apoptosis and improved neurological outcome are closely related to the inhibitory effect of HBOT on inflammation after TBI [7]. During the early stage of TBI, effect of HBOT in reducing inflammation was achieved by increasing anti-inflammatory cytokine interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) expression, decreasing of the RNA and protein levels of caspase-3, interleukin-8 (IL-8), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1) and transforming growth-interacting factor (TGIF), as well as via reduction of the expression of matrix metalloproteinase-9 (MMP9) [28, 54, 67, 68, 88–90]. Recently, Geng et al. [91] demonstrated that HBOT suppressed protein expression of inflammasome components and reduced the levels of interleukin-1β (IL-1β), interleukin-18 (IL-18) and high-mobility group box 1 (HMGB1) protein in the injured brain tissues and serum. Based on these results authors assumed that HBOT may diminish the inflammatory response after TBI by inhibiting the activation of inflammasome signaling. Latest results of Meng et al. [86] showed that HBOT significantly increased the expression of nuclear factor (erythroid-derived 2)- related factor 2 (Nrf2) and heme oxygenase-1, and inhibited the expression of Toll-like receptor 4 and nuclear factor-kappa B in a rat TBI model [87]. Furthermore, HBOT decreases expression of nNOS, eNOS and iNOS (neuronal, endothelial and inducible nitric oxide synthases) mRNA in the cortex after acute traumatic cerebral injury [92].

HBOT-induced enhancement of SYP expression after hypoxia-ischemia and proposed that the induction of synaptic plasticity and reducing of the ultrastructural damage may underlie

Hyperbaric Oxygen Therapy in Traumatic Brain Injury: Cellular and Molecular Mechanisms

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Nowadays, stem cells are in the center of attention. Stimulation of neurogenesis after HBOT and influence of stem cells mobilization on motor and cognitive performances is demonstrated in numerous studies [67, 95–97]. Thus, Shandley et al. [97] showed that cognitive improvement observed after treatment with HBO in patients with mild to moderate TBI is correlated with stem cell mobilization. Based on these findings they hypothesized that stem cells, mobilized by HBOT treatment, are recruited to repair damaged neuronal tissue. Yang et al. [98] and Wang et al. [99] reported that HBOT promotes the migration and differentiation of endogenous neural stem cells (NSCs) in neonatal rats with hypoxic-ischemic (HI) brain damage. Authors have shown that after HBOT, an increase in newly generated neurons, oligodendrocytes and remyelination was observed in the HI group treated with HBO compared to the untreated HI rats. Further, it was suggested that HBOT-stimulated proliferation of NSCs protects the learning and memory ability of the HI rats [100]. In our recent preliminary reports [101, 102] we also noticed that HBOT when applied after brain injury promoted endogenous NSCs to migrate to the site of injury and differentiate into mature neurons, contributing to improved neurofunctional recovery of the injured brain. Moreover, we demonstrated that HBOT alters morphology

Mu et al. [103] suggested that activation of several signaling pathways and transcription factors (Wnt, hypoxia-inducible factors - HIFs, and cAMP response element-binding - CREB) play an important role in HBOT-induced neurogenesis. Furthermore, it was assumed that endogenous neurogenesis, enhanced by application of delayed HBO in the late-chronic phase

Interestingly, combining of HBOT with bone marrow stem cells (BMSCs) transplantation showed synergistic effect and had favorable influence in improving rehabilitation after rat spinal cord injury [104]. The same combination of HBOT and BMSCs transplantation proved to be more effective for repair of cognitive and neurological functions after TBI than monotherapy [105]. Similarly, long course of HBO treatments (for 3 weeks) promote the mobilization and migration of BMSCs to ischemic brain, stimulate expression of trophic factors and

Besides neurogenesis HBOT may improve the outcome of TBI by stimulating angiogenesis [67, 95, 106, 107]. Using brain perfusion imaging, Tal et al. [106] demonstrated that 60 daily HBOT sessions stimulate cerebral angiogenesis in post-TBI patients, which induced significant improvement in the global cognitive scores. These data strongly suggest that one of the ways in which HBOT can induce neuroplasticity is angiogenesis. Given that HBOT was initiated 6 months to 27 years after the injury, obtained results imply that HBOT may improve perfusion to the chronic damaged brain tissue even months to years after the injury. Recent results of the same group [107] showed that in addition to the increased cerebral blood flow

rehabilitation mechanisms of HBOT.

**2.6. HBOT promotes neurogenesis and angiogenesis after TBI**

of neuronal precursors to more matured morphology [102].

of stroke, is possibly mediated by ROS/HIF-1α/β-catenin pathway [96].

neurogenesis, and help in neuronal repair after ischemic stroke [72].

## **2.5. HBOT improves neurofunctional recovery of the injured brain by enhancing neuronal plasticity and synaptogenesis**

Growing number of studies have reported that, irrespective to diversity of protocols, HBO therapy applied after TBI improved neurological status including motor and cognitive function, as well as learning and memory abilities, indicating that the best prognosis is achieved by earlier and continuous HBO treatment [11, 41, 45, 57, 67, 88, 93]. On the other hand, Baratz-Goldstein et al. [41] demonstrated that delayed treatment with HBO (initiated 7 days post-injury) also lead to improvement in learning abilities in mice model of moderate TBI. Additionally, in their recent publication Lim et al. [90] suggested that HBO treatment may ameliorate TBI-induced depression-like behavior in rats.

In our previously published paper [45] we have demonstrated that HBOT improves recovery of locomotor performances and sensorimotor integration after cortical injury in rats by enhancing neuroplastic responses and promoting synaptogenesis. Using growth-associated protein 43 (GAP43) and synaptophysin (SYP) as markers of axonal sprouting and synaptogenesis, respectively, we were the first to demonstrate that HBOT induces over-expression of GAP43 and SYP in the neurons surrounding the injury site. Given that an increase in GAP43 and SYP expression occurs concomitantly with improvement of locomotor abilities, we suggested that mechanisms underlying HBOT action involve promoting of axonal sprouting and the formation of new functional synaptic circuits. This implies that axonal reorganization and synapse remodeling contribute to observed functional recovery. Recent results of Zhang et al. [93] that HBOT-induced increase of GAP43 and synaptophysin expression underlies observed enhancement of learning abilities in the controlled cortical impact (CCI) model of rat brain injury confirmed our assumptions. Furthermore, Chen and Chen [94] detected HBOT-induced enhancement of SYP expression after hypoxia-ischemia and proposed that the induction of synaptic plasticity and reducing of the ultrastructural damage may underlie rehabilitation mechanisms of HBOT.

## **2.6. HBOT promotes neurogenesis and angiogenesis after TBI**

apoptosis and improved neurological outcome are closely related to the inhibitory effect of HBOT on inflammation after TBI [7]. During the early stage of TBI, effect of HBOT in reducing inflammation was achieved by increasing anti-inflammatory cytokine interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) expression, decreasing of the RNA and protein levels of caspase-3, interleukin-8 (IL-8), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1) and transforming growth-interacting factor (TGIF), as well as via reduction of the expression of matrix metalloproteinase-9 (MMP9) [28, 54, 67, 68, 88–90]. Recently, Geng et al. [91] demonstrated that HBOT suppressed protein expression of inflammasome components and reduced the levels of interleukin-1β (IL-1β), interleukin-18 (IL-18) and high-mobility group box 1 (HMGB1) protein in the injured brain tissues and serum. Based on these results authors assumed that HBOT may diminish the inflammatory response after TBI by inhibiting the activation of inflammasome signaling. Latest results of Meng et al. [86] showed that HBOT significantly increased the expression of nuclear factor (erythroid-derived 2)- related factor 2 (Nrf2) and heme oxygenase-1, and inhibited the expression of Toll-like receptor 4 and nuclear factor-kappa B in a rat TBI model [87]. Furthermore, HBOT decreases expression of nNOS, eNOS and iNOS (neuronal, endothelial and inducible nitric oxide synthases) mRNA in the

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

**2.5. HBOT improves neurofunctional recovery of the injured brain by enhancing** 

Growing number of studies have reported that, irrespective to diversity of protocols, HBO therapy applied after TBI improved neurological status including motor and cognitive function, as well as learning and memory abilities, indicating that the best prognosis is achieved by earlier and continuous HBO treatment [11, 41, 45, 57, 67, 88, 93]. On the other hand, Baratz-Goldstein et al. [41] demonstrated that delayed treatment with HBO (initiated 7 days post-injury) also lead to improvement in learning abilities in mice model of moderate TBI. Additionally, in their recent publication Lim et al. [90] suggested that HBO treatment may ameliorate TBI-induced

In our previously published paper [45] we have demonstrated that HBOT improves recovery of locomotor performances and sensorimotor integration after cortical injury in rats by enhancing neuroplastic responses and promoting synaptogenesis. Using growth-associated protein 43 (GAP43) and synaptophysin (SYP) as markers of axonal sprouting and synaptogenesis, respectively, we were the first to demonstrate that HBOT induces over-expression of GAP43 and SYP in the neurons surrounding the injury site. Given that an increase in GAP43 and SYP expression occurs concomitantly with improvement of locomotor abilities, we suggested that mechanisms underlying HBOT action involve promoting of axonal sprouting and the formation of new functional synaptic circuits. This implies that axonal reorganization and synapse remodeling contribute to observed functional recovery. Recent results of Zhang et al. [93] that HBOT-induced increase of GAP43 and synaptophysin expression underlies observed enhancement of learning abilities in the controlled cortical impact (CCI) model of rat brain injury confirmed our assumptions. Furthermore, Chen and Chen [94] detected

cortex after acute traumatic cerebral injury [92].

**neuronal plasticity and synaptogenesis**

depression-like behavior in rats.

Nowadays, stem cells are in the center of attention. Stimulation of neurogenesis after HBOT and influence of stem cells mobilization on motor and cognitive performances is demonstrated in numerous studies [67, 95–97]. Thus, Shandley et al. [97] showed that cognitive improvement observed after treatment with HBO in patients with mild to moderate TBI is correlated with stem cell mobilization. Based on these findings they hypothesized that stem cells, mobilized by HBOT treatment, are recruited to repair damaged neuronal tissue. Yang et al. [98] and Wang et al. [99] reported that HBOT promotes the migration and differentiation of endogenous neural stem cells (NSCs) in neonatal rats with hypoxic-ischemic (HI) brain damage. Authors have shown that after HBOT, an increase in newly generated neurons, oligodendrocytes and remyelination was observed in the HI group treated with HBO compared to the untreated HI rats. Further, it was suggested that HBOT-stimulated proliferation of NSCs protects the learning and memory ability of the HI rats [100]. In our recent preliminary reports [101, 102] we also noticed that HBOT when applied after brain injury promoted endogenous NSCs to migrate to the site of injury and differentiate into mature neurons, contributing to improved neurofunctional recovery of the injured brain. Moreover, we demonstrated that HBOT alters morphology of neuronal precursors to more matured morphology [102].

Mu et al. [103] suggested that activation of several signaling pathways and transcription factors (Wnt, hypoxia-inducible factors - HIFs, and cAMP response element-binding - CREB) play an important role in HBOT-induced neurogenesis. Furthermore, it was assumed that endogenous neurogenesis, enhanced by application of delayed HBO in the late-chronic phase of stroke, is possibly mediated by ROS/HIF-1α/β-catenin pathway [96].

Interestingly, combining of HBOT with bone marrow stem cells (BMSCs) transplantation showed synergistic effect and had favorable influence in improving rehabilitation after rat spinal cord injury [104]. The same combination of HBOT and BMSCs transplantation proved to be more effective for repair of cognitive and neurological functions after TBI than monotherapy [105]. Similarly, long course of HBO treatments (for 3 weeks) promote the mobilization and migration of BMSCs to ischemic brain, stimulate expression of trophic factors and neurogenesis, and help in neuronal repair after ischemic stroke [72].

Besides neurogenesis HBOT may improve the outcome of TBI by stimulating angiogenesis [67, 95, 106, 107]. Using brain perfusion imaging, Tal et al. [106] demonstrated that 60 daily HBOT sessions stimulate cerebral angiogenesis in post-TBI patients, which induced significant improvement in the global cognitive scores. These data strongly suggest that one of the ways in which HBOT can induce neuroplasticity is angiogenesis. Given that HBOT was initiated 6 months to 27 years after the injury, obtained results imply that HBOT may improve perfusion to the chronic damaged brain tissue even months to years after the injury. Recent results of the same group [107] showed that in addition to the increased cerebral blood flow and volume, HBOT improved both white and gray microstructures pointing to regeneration of nerve fibers. These micro structural changes correlate with the significant improvement in the memory, executive functions, information processing speed and global cognitive scores.

assays; RJ contributed substantially to the study by literature search and writing of the manuscript; PB carried out the HBOT and with MDj provided critical revision of the manuscript. All

, Rada Jeremic4

Hyperbaric Oxygen Therapy in Traumatic Brain Injury: Cellular and Molecular Mechanisms

, Marina Djelic4

and

http://dx.doi.org/10.5772/intechopen.75025

37

, Danijela Krstic<sup>3</sup>

1 Department of Neurobiology, Institute for Biological Research "Sinisa Stankovic",

2 Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade,

3 Institute of Medical Chemistry, Faculty of Medicine, University of Belgrade, Belgrade,

4 Institute of Medical Physiology "Richard Burian", Faculty of Medicine, University of

[1] Hyder AA, Wunderlich CA, Puvanachandra P, Gururaj G, Kobusingye OC. The impact of traumatic brain injuries: A global perspective. NeuroRehabilitation. 2007;**22**(5):341-353

[2] Pekovic S, Subasic S, Nedeljkovic N, Bjelobaba I, Filipovic R, Milenkovic I, et al. Molecular basis of brain injury and repair. In: Ruzdijic S, Rakic LJ, editors. Neurobiological Studies

[3] Margulies S, Hicks R. Combination therapies for traumatic brain injury: Prospective considerations. Journal of Neurotrauma. 2009;**26**(6):925-939. DOI: 10.1089/neu.2008.0794

[4] Michalski D, Küppers-Tiedt L, Weise C, Laignel F, Härtig W, Raviolo M, et al. Long-term functional and neurological outcome after simultaneous treatment with tissue-plasminogen activator and hyperbaric oxygen in early phase of embolic stroke in rats. Brain

[5] Feldmeier JJ, editor. Hyperbaric Oxygen Therapy: 2003 Committee Report, Rev. Ed. Kensington (MD): Undersea and Hyperbaric Medical Society, Inc.; 2003. p. 137

[6] Brkic DP, Pekovic MS, Krstic ZD, Jovanovic ST. Hyperbaric oxygenation as an adjuvant therapy for traumatic brain injury: A review of literature. Periodicum Biologorum.

Research. 2009;**15, 1303**:161-168. DOI: 10.1016/j.brainres.2009.09.038

– From Genes to Behaviour. Kerala, India: Research Signpost; 2006. pp. 143-165

authors read and approved the final manuscript.

\*, Sanja Dacic<sup>2</sup>

University of Belgrade, Belgrade, Serbia

\*Address all correspondence to: spekovic@ibiss.bg.ac.rs

**Author details**

Sanja Pekovic1

Predrag Brkic4

Belgrade, Serbia

**References**

Belgrade, Belgrade, Serbia

2014;**116**(1):29-36

Serbia
