**6. Inflammation**

group and higher protein expression of CYP450-4A1/A2/A3 in both HBO groups when compared with their respective controls. Also in this study, there was not oxidative stress caused by HBO because thiobarbituric acid-reactive substances (TBARSs) were elevated in DM group but were normal in the healthy HBO group. This difference between studies is probably a result of different experimental protocols (intermittent hyperbaric oxygenation—2 hours, 4

Same authors investigate HBO effect on microcirculation (middle cerebral arteries) in diabetic animal model, 6-week duration of DM. Preliminary results shown impaired vasodilation response in diabetic rats and restored vasodilation after HBO exposure. Using inhibitors such as indomethacin (COX), NG-monomethyl-L-arginine (L-NMMA) (NOS), and clotrimazole (nonselective CYP 450 inhibitor), they notice shift in vasodilation mechanisms from mainly NO pathway toward two other pathways COX/CYP 450 because in both HBO groups, L-NMMA

In normal condition, vasodilation response to hypoxia is made by activating cyclooxygenase (COX) and production of prostacyclin (PGI2) [61]. There is evidence that CYP 450-epoxigenase enzyme in minor part causes vasodilation in healthy vessels [62]. Experiments on middle cerebral arteries (MCAs) of 6 weeks diabetic rats that underwent HBO exposure were used to evaluate the effect of HBO in acute hypoxia. They used COX inhibitor indomethacin and selective CYP 450 epoxygenase inhibitor MS-PPOH. COX inhibition partially preserved vasodilation in HBO groups, and eliminated vasodilation in response to hypoxia in the presence of MS-PPOH in both HBO groups suggests that HBO activates CYP450-epoxigenase in MCAs of healthy and DM rats and shifts vasodilation mechanisms in response to acute hypoxia [63].

Life on Earth is impossible without oxygen that is in our atmosphere, which consists of 21% oxygen. Paradoxically, oxygen can also potentially be very toxic for organisms that use it. Free radical formation occurs continuously in cells as a consequence of both enzymatic and nonenzymatic reactions [64]. The main compartments of these kinds of reactions in cells are mitochondria. Mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mitochondria are the site of significant reactive oxygen species (ROS) production [65]. The term "ROS" is generally used to describe reactive molecules containing oxygen. Such molecules have many common and similar characteristics; they also exhibit very different features, resulting in potentially beneficial or even toxic effects [66]. On the other hand, the term reactive oxygen species (ROS) can be defined as highly reactive oxygen-centered chemical species containing one or two unpaired electrons, where an unpaired electron is one that exists in an atomic or molecular orbital alone. The unpaired electron containing chemical species can also be called "free radicals." Furthermore, the term "ROS" can also be used as a "collective term" to include both radicals and nonradicals, the latter being devoid of unpaired electrons. So, ROS is classified into two categories: (1) oxygen-centered radicals and (2) oxygen-cen-

O2−), hydroxyl radical

). Oxygen-centered nonradicals are

days at 2.0 atm abs vs. 90 minutes, 7 days at 2.4 atm abs in Matsunami study [59]).

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

did not blocked vasodilation to acetylcholine. Further investigation is necessary [60].

**5. Effects on oxidative stress [reactive oxygen species (ROS)]**

tered nonradicals. Oxygen-centered radicals include superoxide anion (<sup>∙</sup>

), and peroxyl radical (ROO<sup>∙</sup>

(∙

OH), alkoxyl radical (RO<sup>∙</sup>

Pathological effects of DM on the vascular wall include enhanced ROS production and endothelial activation leading to inflammation, atherogenesis, and vascular dysfunction, which further results in clinical impairment of the micro- and macrocirculation. Interestingly, positive therapeutic effects of HBO<sup>2</sup> , such as antioxidative and anti-inflammatory effects, have been attributed to the enhanced ROS production induced by the HBO<sup>2</sup> treatment [1].

Numerous studies on experimental DM animal models revealed ongoing vascular inflammation under diabetic/hyperglycemic conditions, characterized by (a) increased proinflammatory cytokine levels, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α); (b) endothelial activation followed by increased expression of vascular cellular adhesion molecule-1 (VCAM-1); and (c) increased leukocyte homing to the vessels and tissues induced by excessive secretion of chemokines like monocyte chemoattractant protein (MCP-1) [75–77]. In addition to that, same noxa that lead to inflammation also precipitate development of vascular dysfunction, marked by substantial decrease in NO bioavailability, which is discussed in more detail elsewhere in this chapter [78]. Studies on diabetic (db/db) and control (db/+) mice have shown that DM prolongs the inflammatory response to a bacterial stimulus through cytokine dysregulation, particularly the TNF-α [79]. Similar results were also obtained from experiments using type 1 DM animal model (mice receiving multiple low-dose streptozotocin treatments), suggesting that the observed proinflammatory status of diabetic mice is predominately linked to hyperglycemia rather than pathomechanism involved in the development of a specific type of DM [80]. Additionally, impaired function of macrophages, including reduced efferocytosis and anti-inflammatory cytokine expression, has been attributed to the prolonged and ineffective resolution of inflammation in the wounds of diabetic mice, which is a leading complication in diabetic humans [81]. This was further confirmed by intravital microscopy that allowed researchers to real-time follow-up leukocytes in live diabetic and healthy control mice, which was followed by leukocyte isolation and functional tests that all together revealed enhanced recruitment but defective function of leukocytes during the inflammation in mouse models of type 1 and type 2 DM resulting in defective bacterial clearance [82]. Studies have also shown that hyperglycemia changes the intrinsic TCR-induced naïve T activation to increased T cell responsiveness in diabetes [83]. In the kidneys, the observed proinflammatory condition in DM animals has been linked to oxidative stress-induced JNK activation [84]. It has also been shown that diabetic condition facilitates binding of monocytes to vascular smooth muscle cells and their subsequent differentiation through induction of key chemokines in the vasculature, which can lead to enhanced atherogenesis [85]. In addition, endothelial cells (EC) express pattern-recognition receptors including Toll-like receptors (TLR) that have a central role in recognizing pathogens and damage signals and initiating immune responses [86]. It seems that in the vessels of diabetic animals/individuals, increased oxidative stress, free fatty acids, and hyperglycemia are directly involved in the pathogenesis of vascular inflammation via several cellular mechanisms, including TLR-mediated activation of protein kinase C (PKC) and NF-κB pathways resulting in increased expression of the proinflammatory molecules such as IL-6 and TNF-α. In turn, secretion of cytokines IL-1 and TNF-α increases NF-κB activity and production of cellular adhesion molecules by endothelial cells, further aggravating the inflammation [87].

and collagen deposition [91]. A study on an experimental wound model revealed increased synthesis of vascular endothelial growth factor (VEGF) in damaged tissue during HBO<sup>2</sup>

which is the most specific growth factor for neovascularization [92]. It is controversial that

It has been shown that HBO inhibits ischemia reperfusion induced β2-integrin-dependent adhesion of neutrophils to the endothelium by blocking CD18 surface polarization and through S-nitrosation of β2-integrin, with no effect on the cell-surface expression of β2-integrins [93]. Studies on monocyte-macrophages retrieved from healthy humans and animals exposed to HBO in vivo or cells exposed to HBO under in vitro condition revealed lower stimulus-

Studies on ApoE KO mice that exhibit accelerated atherosclerosis and related complica-

ens delayed hypersensitivity response to oxLDL challenge. The same studies demonstrated significant reduction in the production of proinflammatory cytokines, along with marked increase in the constitutive production of the anti-inflammatory cytokine IL-10 in splenocytes stimulated by LPS [95]. This effect was independent of antigen specificity, as indicated by

Approximately 25% of all stroke patients have DM and 40% have hyperglycemia, which is associated with worse neurologic outcome as well as higher risk of recurrence of stroke [96, 97]. Diabetic patients, compared to nondiabetics, are known to be more sensitive to cerebral ischemia. Thus, the same duration of ischemia results in more severe neurologic deficits and larger brain infarcts in diabetic patients. Female patients with DM have 4.8-fold higher risk for developing ischemic stroke than the general population (compared to 3.7-fold for men) and more often suffer fatal strokes (standardized mortality ratios of 3.1 for males and 4.4 for females) [98–100]. The outcome is frequently lethal, regardless of any therapy undertaken, including recombinant tissue plasminogen activator (rtPA) and mechanical thrombectomy. Possible underlying causes are chronic hyperglycemia, which leads to free oxygen radicals and cytokines production and increases ischemic brain cells predisposition to apoptosis [101]. In addition, the intimal artery thickening and arteriolar occlusion occur in diabetes, contributing by impaired vascular function to inadequate tissue perfusion. Moreover, DM is, in some cases, such as treatment of recurrent stroke with thrombolysis, one of the exclusion criteria [102].

A total of 90–95% diabetic patients are type 2 DM of noninsulin dependence and 5–10% are type 1 DM of insulin dependence. Type 2 DM patients have asymptomatic period of hyperglycemia for about 4–7 years that leads to most important problems—chronic complications of diabetes, leading to disability and premature death [103]. First diabetic complications are associated with

reduces the circulating levels of antibodies to MDALDL and damp-

Mechanisms of HBO-Induced Vascular Functional Changes in Diabetic Animal Models

induced proinflammatory cytokine production upon exposure to HBO<sup>2</sup>


HBO2

impaired NOS activity in the bone marrow [1].

tions showed that HBO<sup>2</sup>

polyclonal activation of T cells.

**7. The role of HBO in stroke**

,

95

[1, 94].

http://dx.doi.org/10.5772/intechopen.76569

Some of the beneficial anti-inflammatory effects of HBO include reduced proinflammatory cytokine expression, suppressed development of T helper cells, shrinking of spleen and lymph nodes, decreased responses to antigens, recruitment and differentiation of circulating stem cells, and reduced frequencies of circulating leukocytes [88, 89]. However, these effects were mainly observed in studies exploring experimental animal models of colitis, while in the particular case of DM, data on the effects of HBO on the vascular inflammation are scarce. This is in contrast to our knowledge about the effects of the HBO on the wound-healing mechanisms that have been subjects of intensive investigations for many years, which lead to profound understanding of the clinically observed positive effects of HBO [90].

Beneficial effects of HBO on the wound-healing processes include facilitation of the neovascularization through enhanced regional angiogenic stimuli and increased recruitment and differentiation of circulating stem cells from the bone marrow [1]. Under ischemic and hyperglycemic conditions, HBO further promotes wound repair by increasing tissue perfusion and collagen deposition [91]. A study on an experimental wound model revealed increased synthesis of vascular endothelial growth factor (VEGF) in damaged tissue during HBO<sup>2</sup> , which is the most specific growth factor for neovascularization [92]. It is controversial that HBO2 -induced oxidative stress leads to hypoxia-inducible factor (HIF)-1 and 2 mediated transcriptions of many genes involved with neovascularization, including stromal-derived factor-1 (SDF-1) and its counterpart ligand, CXCR4, as well as VEGF [1]. These effects could be especially beneficial for DM individuals whose stem cell mobilization is compromised by impaired NOS activity in the bone marrow [1].

It has been shown that HBO inhibits ischemia reperfusion induced β2-integrin-dependent adhesion of neutrophils to the endothelium by blocking CD18 surface polarization and through S-nitrosation of β2-integrin, with no effect on the cell-surface expression of β2-integrins [93]. Studies on monocyte-macrophages retrieved from healthy humans and animals exposed to HBO in vivo or cells exposed to HBO under in vitro condition revealed lower stimulusinduced proinflammatory cytokine production upon exposure to HBO<sup>2</sup> [1, 94].

Studies on ApoE KO mice that exhibit accelerated atherosclerosis and related complications showed that HBO<sup>2</sup> reduces the circulating levels of antibodies to MDALDL and dampens delayed hypersensitivity response to oxLDL challenge. The same studies demonstrated significant reduction in the production of proinflammatory cytokines, along with marked increase in the constitutive production of the anti-inflammatory cytokine IL-10 in splenocytes stimulated by LPS [95]. This effect was independent of antigen specificity, as indicated by polyclonal activation of T cells.
