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

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].

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

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

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

understanding of the clinically observed positive effects of HBO [90].

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 microangiopathy of retina, kidney, and peripheral neuropathy and next with macroangiopathy causing myocardial infarction, stroke, hypertension, and peripheral artery lesion. Patients with DM have progressive cerebrovascular atherosclerosis and increased cerebral vascular reaction to vascular constrictors, a deregulated reaction to vascular dilators and damaged automatic regulation of brain-blood stream. Damaged endothelium and vascular motor function of small arteries can lead to hypoperfusion of certain areas of the brain in diabetic patients.

acute ischemic stroke. The question of later and repetitive administration of HBO shows some promising results; however, they are still based on a few clinical cases and lack scientific proof and larger number of cases. Multiple repetitive HBO has positive effect on cognitive recovery after stroke and metabolism of temporal lobe. In one clinical trial, HBO combined with antidepressants showed better results than any of these therapies alone. HBO reduces cerebrovascular vasospasm and secondary brain infarctions after aneurismal subarachnoid hemorrhage (SAH). In intracerebral hemorrhage patients, HBO also provided improvement

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

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

97

When one thinks about treating acute stroke in diabetic patients with HBO, a few still unanswered questions arise, mostly due to the paucity of experiments in these settings. There are a few experiments conducted in animal models, but they vary in criteria for its use. In humans, we can rely only on a small number of cases with very diverse inclusion criteria and different results. Therefore, we can only draw some direct and more indirect conclusions about it from

There is a question of optimal model of animal stroke in diabetic animals. The most commonly used experimental model of stroke in rats is a model of middle cerebral artery occlusion (MCAO) by intra-luminal suture. There are variations of this model in terms of use of permanent or transitory MCA occlusion-induced ischemia. The duration of occlusion varies in models from permanent MCAO to transitory MCAO (t-MCAO) of 180, 120, 105, or 60 minutes [111]. Taking into account the observed differences in clinical presentation of diabetic vs. nondiabetic patients with stroke, there are few issues that variations in experimental approach to stroke study are brought to light. For example, in diabetic rat stroke models, the

The usual duration of t-MCAO used in non-diabetic rats was 60-120 minute [112]. In diabetic rats the same duration of t-MCAO produced massive stroke with malignant brain edema, devastating neurological deficits (such as inability to move, eat and drink) that become worse over time, leading to unconsciousness and death of animals within the first 24 hours (mostly due to massive edema and a rise in intracranial pressure). If ischemia lasts too long, laser Doppler flowmetry (LDF) finds lesser than expected reperfusional values. This brain vascular sign could be a marker of point of no return in stroke treatment [111]. Therefore (to develop the adequate diabetic female rat model, using transitory middle cerebral artery occlusion (t-MCAO) that would produce treatable stroke conditions in rats with diabetes), one has to significantly shorten the duration of t-MCAO to avoid already-irreversible brain infarct with brain vascular derangement. One study suggests that 30-minute t-MCAO could be a more appropriate stroke model than the usual 60-120 minute t-MCAO models, consistently producing medium-sized stroke, which affects 30–50% of ischemic hemisphere [111] (865443). Similarly, patients with the most severe strokes of the whole MCA territory and high National Institute of Health Stroke Score (NIHSS) not only are poor candidates for treatment with thrombolysis and mostly die due to brain edema and complications of dysphagia and immo-

In conclusion, it is questionable to compare results of artery occlusion for rats with and with-

if started early, and the patient is stable [110].

experiments on nondiabetic stroke experiments.

same duration of MCAO as in nondiabetic rat models is used.

bility, but also have higher risk of secondary hemorrhage.

out diabetes, even if the duration of t-MCAO is equal.

The principles of HBO are based on physical laws and mechanisms of oxygen transport in human body. At sea level (1 ATA), almost all hemoglobin is saturated with oxygen, and HBO can increase its saturation only slightly. However, HBO increases the amount of oxygen dissolved in plasma from 0.3 to 5.6% at 2.5 ATA, and due to this mechanism, it increases tissue oxygenation even in areas where erythrocytes cannot pass [104]. Due to oxygen pressure gradient, HBO promotes diffusion of oxygen to longer distances in ischemic region. HBO<sup>2</sup> raises oxygenation of ischemic penumbra by 20% and improves mitochondrial function [105, 106]. Single or multiple exposures to HBO create environment of intermittent relative hypoxia that can not only prepare tissue for longer hypoxia but also save tissue until other salvation strategies (such as thrombolysis, mechanical thrombectomy, stenting, and endarterectomy) take effect [47, 107]. Not only oxygen in ischemic core and penumbra itself plays a vital role in surviving tissues; HBO also influences on many different pathophysiological mechanisms. HBO improves oxygen delivery to ischemic brain tissue due to the higher arterial blood-brain oxygen gradient.

In animal models, it stabilizes blood-brain barrier (BBB) and therefore reduces brain edema formation. It improves brain microcirculation and brain metabolism, creating sufficient energy and ion homeostasis needed for survival of cells until reperfusion or collateral circulation creation. Some concern was about vasoconstriction of arteries under HBO. This can be applied to normal, but not ischemic vessels, where secondary vasodilatation is salvation mechanism and vasoconstriction does not appear. HBO actually improves microcirculation in ischemic areas [108, 109]. HBO reduces poststroke inflammation by various mechanisms, reduces the number of brain cells undergoing apoptotic pathways and necrotic death, and if applied early, it can reduce ischemia-reperfusion injury and reduce oxidative stress. These combined effects reduce brain edema and modulate cerebral vascular flow resulting in reduced intracranial pressure. Longer effects of HBO include promotion of angiogenesis and neurogenesis in ischemic tissues with positive effect on neurorehabilitation. In numerous animal experimental models, HBO was effective in reducing brain infarction after stroke. However, few human studies were so successful.

HBO has been used in humans in many different stroke types (hemorrhagic, ischemic, large and small artery stroke, global ischemia, etc.) using different pressures, protocols of application (single or multiple) and in different poststroke time windows. Due to these inconsistent standards, some studies showed lack of effect and other benefits. Another point of concern is that only the small number of these studies were well-designed randomized controlled trials and that their limitations include the small number of patients, which means that precise conclusions cannot be drawn. Some cautious conclusions could be suggested. HBO is so far the only effective early treatment of air embolism (mostly after surgery). HBO early after stroke improves recovery after stroke, but this effect progressively decreases if treatment is applied later. The most significant results are achieved in first 3 hours after stroke (similar to thrombolysis and other revascularization trials). Time window for HBO is 3–6 hours in acute ischemic stroke. The question of later and repetitive administration of HBO shows some promising results; however, they are still based on a few clinical cases and lack scientific proof and larger number of cases. Multiple repetitive HBO has positive effect on cognitive recovery after stroke and metabolism of temporal lobe. In one clinical trial, HBO combined with antidepressants showed better results than any of these therapies alone. HBO reduces cerebrovascular vasospasm and secondary brain infarctions after aneurismal subarachnoid hemorrhage (SAH). In intracerebral hemorrhage patients, HBO also provided improvement if started early, and the patient is stable [110].

microangiopathy of retina, kidney, and peripheral neuropathy and next with macroangiopathy causing myocardial infarction, stroke, hypertension, and peripheral artery lesion. Patients with DM have progressive cerebrovascular atherosclerosis and increased cerebral vascular reaction to vascular constrictors, a deregulated reaction to vascular dilators and damaged automatic regulation of brain-blood stream. Damaged endothelium and vascular motor function of small

The principles of HBO are based on physical laws and mechanisms of oxygen transport in human body. At sea level (1 ATA), almost all hemoglobin is saturated with oxygen, and HBO can increase its saturation only slightly. However, HBO increases the amount of oxygen dissolved in plasma from 0.3 to 5.6% at 2.5 ATA, and due to this mechanism, it increases tissue oxygenation even in areas where erythrocytes cannot pass [104]. Due to oxygen pressure gradi-

genation of ischemic penumbra by 20% and improves mitochondrial function [105, 106]. Single or multiple exposures to HBO create environment of intermittent relative hypoxia that can not only prepare tissue for longer hypoxia but also save tissue until other salvation strategies (such as thrombolysis, mechanical thrombectomy, stenting, and endarterectomy) take effect [47, 107]. Not only oxygen in ischemic core and penumbra itself plays a vital role in surviving tissues; HBO also influences on many different pathophysiological mechanisms. HBO improves oxygen delivery to ischemic brain tissue due to the higher arterial blood-brain oxygen gradient. In animal models, it stabilizes blood-brain barrier (BBB) and therefore reduces brain edema formation. It improves brain microcirculation and brain metabolism, creating sufficient energy and ion homeostasis needed for survival of cells until reperfusion or collateral circulation creation. Some concern was about vasoconstriction of arteries under HBO. This can be applied to normal, but not ischemic vessels, where secondary vasodilatation is salvation mechanism and vasoconstriction does not appear. HBO actually improves microcirculation in ischemic areas [108, 109]. HBO reduces poststroke inflammation by various mechanisms, reduces the number of brain cells undergoing apoptotic pathways and necrotic death, and if applied early, it can reduce ischemia-reperfusion injury and reduce oxidative stress. These combined effects reduce brain edema and modulate cerebral vascular flow resulting in reduced intracranial pressure. Longer effects of HBO include promotion of angiogenesis and neurogenesis in ischemic tissues with positive effect on neurorehabilitation. In numerous animal experimental models, HBO was effective in

raises oxy-

arteries can lead to hypoperfusion of certain areas of the brain in diabetic patients.

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

ent, HBO promotes diffusion of oxygen to longer distances in ischemic region. HBO<sup>2</sup>

reducing brain infarction after stroke. However, few human studies were so successful.

HBO has been used in humans in many different stroke types (hemorrhagic, ischemic, large and small artery stroke, global ischemia, etc.) using different pressures, protocols of application (single or multiple) and in different poststroke time windows. Due to these inconsistent standards, some studies showed lack of effect and other benefits. Another point of concern is that only the small number of these studies were well-designed randomized controlled trials and that their limitations include the small number of patients, which means that precise conclusions cannot be drawn. Some cautious conclusions could be suggested. HBO is so far the only effective early treatment of air embolism (mostly after surgery). HBO early after stroke improves recovery after stroke, but this effect progressively decreases if treatment is applied later. The most significant results are achieved in first 3 hours after stroke (similar to thrombolysis and other revascularization trials). Time window for HBO is 3–6 hours in When one thinks about treating acute stroke in diabetic patients with HBO, a few still unanswered questions arise, mostly due to the paucity of experiments in these settings. There are a few experiments conducted in animal models, but they vary in criteria for its use. In humans, we can rely only on a small number of cases with very diverse inclusion criteria and different results. Therefore, we can only draw some direct and more indirect conclusions about it from experiments on nondiabetic stroke experiments.

There is a question of optimal model of animal stroke in diabetic animals. The most commonly used experimental model of stroke in rats is a model of middle cerebral artery occlusion (MCAO) by intra-luminal suture. There are variations of this model in terms of use of permanent or transitory MCA occlusion-induced ischemia. The duration of occlusion varies in models from permanent MCAO to transitory MCAO (t-MCAO) of 180, 120, 105, or 60 minutes [111]. Taking into account the observed differences in clinical presentation of diabetic vs. nondiabetic patients with stroke, there are few issues that variations in experimental approach to stroke study are brought to light. For example, in diabetic rat stroke models, the same duration of MCAO as in nondiabetic rat models is used.

The usual duration of t-MCAO used in non-diabetic rats was 60-120 minute [112]. In diabetic rats the same duration of t-MCAO produced massive stroke with malignant brain edema, devastating neurological deficits (such as inability to move, eat and drink) that become worse over time, leading to unconsciousness and death of animals within the first 24 hours (mostly due to massive edema and a rise in intracranial pressure). If ischemia lasts too long, laser Doppler flowmetry (LDF) finds lesser than expected reperfusional values. This brain vascular sign could be a marker of point of no return in stroke treatment [111]. Therefore (to develop the adequate diabetic female rat model, using transitory middle cerebral artery occlusion (t-MCAO) that would produce treatable stroke conditions in rats with diabetes), one has to significantly shorten the duration of t-MCAO to avoid already-irreversible brain infarct with brain vascular derangement. One study suggests that 30-minute t-MCAO could be a more appropriate stroke model than the usual 60-120 minute t-MCAO models, consistently producing medium-sized stroke, which affects 30–50% of ischemic hemisphere [111] (865443). Similarly, patients with the most severe strokes of the whole MCA territory and high National Institute of Health Stroke Score (NIHSS) not only are poor candidates for treatment with thrombolysis and mostly die due to brain edema and complications of dysphagia and immobility, but also have higher risk of secondary hemorrhage.

In conclusion, it is questionable to compare results of artery occlusion for rats with and without diabetes, even if the duration of t-MCAO is equal.

The only effective pharmacological therapy of acute ischemic stroke in humans is thrombolysis with recombinant tissue plasminogen activator, but DM is sometimes an exclusion criterion in recurrent stroke treatment. The time window for the therapy is narrow, and no other pharmacological agents have demonstrated efficacy in improving outcomes after ischemic stroke [1–4, 100, 102]. Thus, the searches for alternative approaches are welcomed. HBO [113] improves oxygen delivery and postischemic metabolism, restores ion pump function, and allows time for collateral circulation to develop [107]. In normal tissue, it causes vasoconstriction, but in ischemic brain tissue, it increases microvascular flow and improves oxygen dissolution and transport [109]. Time window for HBO application may be up to 6 hours [108], which is longer than the time window for thrombolytic therapy. HBO raises oxygenation of ischemic penumbra by 20% and improves mitochondrial function [107, 108]. It has anti-inflammatory effect by reducing expression of cyclooxygenase-2 and reduces the number of intercellular adhesion molecules and therefore reduces adhesion and infiltration of leukocytes [24]. However, guidelines do not recommend HBO treatment for acute ischemic stroke due to somewhat inconclusive data [102]. Some data imply that the intervention may be harmful causing middle ear trauma, epileptic seizures, and claustrophobia, while others found no firm evidence that HBO improves clinical outcomes for acute stroke. However, the main disadvantage of these trials used in meta-analysis was delay from stroke onset to initiation of HBO and the need for care delivery in a specialized chamber [114].

To conclude, HBO is currently not recommended for patients with acute ischemic stroke outside of clinical trials (except caused by air embolism).

animal experimental models of diabetes. However, this represents only a part of the complete picture, and further studies are necessary to completely elucidate all the mechanisms

Endothelial dysfunction ↑ NO bioavailability [20–23]

↑ Antioxidant defense systems (?)

Renin-angiotensin system ↑ Vascular reactivity to ANG-(1–7) [2, 3, 11] Physical effects ↑ Dissolved oxygen in plasma and tissues [104–106]

**Table 1.** Major potential mechanisms of HBO-induced vascular functional changes in diabetic animal models.

↑Angiogenic mediators

Arachidonic acid metabolites ↑ EETs synthesis, CYP epoxygenase expression, vascular sensitivity to EETs (?)

↓ 20-HETE

**Effect References**

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

, Zrinka Mihaljevic1

1 Department of Physiology and Immunology, Faculty of Medicine, University of Osijek,

4 Department of Diagnostic and Interventional Radiology, Osijek University Hospital,

5 Department for Heart and Vascular Diseases, Osijek University Hospital, Osijek, Croatia

, Sanela Unfirer1,3,

[2, 3, 11, 13, 47]

99

[2, 70–74]

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

[1, 2, 90–94]

involved in the effects of HBO on blood vessels.

Inflammation ↓ Proinflammatory mediators

The authors have no conflict of interest to declare.

, Mihael Mišir1,2, Martina Mihalj1

\*Address all correspondence to: aleksandar\_mf@yahoo.com

2 Neurological Clinic, Osijek University Hospital, Osijek, Croatia

3 Emergency Department, Osijek University Hospital, Osijek, Croatia

Dijana Kibel1,4 and Aleksandar Kibel1,5\*

**Conflict of interest**

**Target group of mechanisms or** 

Oxidative stress ↑ ROS

**single mechanism**

**Author details**

Ivana Jukic1

Osijek, Croatia

Osijek, Croatia

On the other hand, some preclinical experiments suggest that if administered shortly after the stroke, HBO is highly effective treatment of stroke in diabetic female rats, even in the presence of long-term untreated DM [109]. Experiments that did not show effectiveness of HBO were possibly unsuccessful due to the unrecognizing the vulnerability of neurons. They used prolonged ischemia and applied HBO treatment too late after stroke.
