**2. Role of omega-3 FAs in resolution of acute inflammation**

Remarkable progress in our understanding of the pathophysiology of acute inflammation has been achieved through basic science over the last 20 years. Resolution of acute inflammation is now considered to be an active biochemical process that is required to enable tissues to restore normal structure and function following an injury [14]. Interference with the resolution phase of acute inflammation may result in necrosis, chronic inflammation, fibrosis, and organ dysfunction. The resolution process is triggered at the beginning of inflammation by a temporal switch in lipid mediator classes, which is induced by cross talk between cells of the innate immune system and other cells in the inflammatory microenvironment [11].

A diverse range of biologically active pro-resolving and anti-inflammatory mediators are synthetized by complex pathways that involve several enzymes, including cyclooxygenase 2 (COX-2), cytochrome P450s and several lipoxygenases (LOX). The majority of these endogenous mediators are derived from the longchain omega-3 fatty acids (FAs) EPA and DHA and are members of the specialized pro-resolving mediator (SPM) superfamily [15]. SPMs represent an essential biochemical interface between inflammation and tissue repair and regeneration. The resolution of inflammation is a highly orchestrated adaptive process that depends on both the availability of SPMs precursors and the efficiency of the related biosynthetic pathways.

#### **2.1 Specialized pro-resolving mediators**

Each endogenous lipid mediator is structurally distinct and possesses specific biological functions which have been extensively studied in diverse experimental

**239**

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models. EPA is the precursor of the E-series resolvins (RvEs), which contains four main mediators (RvE1-RvE3 and 18-HEPE). DHA is the precursor of the D-series resolvins (RvDs), which contains six mediators (RvD1-RvD6), as well as the protectins (PD1-PDX) and the maresins (MaR1-MaR2); DHA derivatives are also known

SPMs are active at pico-nanogram ranges and exert pleiotropic actions at the inflammatory microenvironment. SPMs stimulate the clearance of bacteria, dead cells, and debris that is required during tissue repair. SPMs also reduce leukocyte transendothelial migration, platelet activation, and production of inflammatory cytokines, thus providing multi-organ protection. The maresins (macrophage mediators in resolving inflammation) exert potent phagocyte-directed actions that include phenotypic conversion of proinflammatory macrophages into macrophages that suppress inflammation and promote tissue regeneration [16]. Collectively, SPMs actively promote resolution of inflammation and recovery of tissue homeostasis [15]. Interestingly, resolution of systemic inflammation appears to have its counterpart in the CNS repre-

sented by different DHA-derived endogenous mediators (see Section 5.1).

**3. Incorporation and transport of omega-3 FAs**

trations of EPA and DHA in plasma and tissues.

functional pool due to the crucial roles of PBMCs in inflammation.

The biosynthetic pathways that generate SPMs are clinically relevant and have been comprehensively studied [15, 16]. Drugs that inactivate the enzymes involved in SPM biosynthesis, such as selective COX-2 inhibitors and certain LOX inhibitors, delay the return to homeostasis and are considered resolution antagonists. Importantly, selective COX-2 inhibitors were synthesized before the identification of inflammation resolution pathways and are currently widely used as anti-inflammatory agents. Aspirin and statins also modify COX-2 by acetylation and S-nitrosylation, respectively, which results in generation of longer-acting SPM R-epimers. Thus, aspirin and statins are resolution

Incorporation and transport of omega-3 FAs have been comprehensively described; here, we focus on the clinically relevant aspects [10, 17, 18]. Several studies have consistently shown that in vivo conversion of alpha linolenic acid (ALA), the short-chain omega-3 FAs from vegetable origin, to its bioactive long-chain derivatives (EPA and DHA) is very low in humans. In addition, the metabolism of omega-6 and omega-3 FAs is tightly linked, and thus a high dietary intake of omega-6 FAs further reduce the conversion of ALA to EPA and DHA and their biological effects. The body has also a limited capacity to store longchain omega-3 FAs; only very small amounts of EPA and DHA are present in adipose tissue, and the brain retains DHA for its own function. Thus, providing preformed EPA and DHA is the most efficient method of increasing the concen-

EPA and DHA are incorporated into different blood lipid fractions after absorption by the gastrointestinal tract or after release from intravenously infused fish oil-based lipid emulsions (FOLE). These lipid fractions reflect the diverse means by which FAs are transported in the circulation and execute their physiological functions. The fractions incorporated in the phospholipid coat of plasma lipoproteins and plasma nonesterified FAs (NEFAs) are considered transport pools. Notably, the NEFA pool seems to be the main DHA plasma fraction that supplies the brain. The NEFA pool also represents a direct source of FAs to cells for generation of SPMs, as this pool readily transfers to inflammatory tissue via edema formation [19]. The FAs fraction incorporated in peripheral blood mononuclear cells (PBMCs) represents a

*DOI: http://dx.doi.org/10.5772/intechopen.88297*

as docosanoids [15].

agonists [15, 16].

*Aneurysmal Subarachnoid Hemorrhage and Resolution of Inflammation DOI: http://dx.doi.org/10.5772/intechopen.88297*

*New Insight into Cerebrovascular Diseases - An Updated Comprehensive Review*

to identify novel therapeutic strategies [7, 8].

patients' functional outcome [13].

brain homeostasis.

ongoing pathophysiological process has been overlooked in SAH. The failure of the recent major NEWTON 2 clinical trial, of sustained intraventricular release of nimodipine, is the latest in a series of unsuccessful phase 3 randomized controlled trials (RCTs) to improve clinical outcome after SAH and further reinforces the need

Nutrition is essential to human health, and appropriate nutritional support is currently considered a standard of care for critically ill patients. Malnutrition including depletion of essential micronutrients—frequently occurs among critically ill patients and is associated with an increased risk of morbimortality [9]. However, the clinical relevance of key nutrient deficiencies in acute neurological illnesses has not been thoroughly investigated. EPA and DHA are essential constituents of endothelial and neuronal membranes, respectively, and also the precursors of key mediators involved in resolution of inflammation and endogenous neuroprotection [10, 11]. Although massive loss of brain DHA in SAH patients was first reported over 15 years ago, the pathological significance of this process and the role of inflammation resolution following SAH have largely been ignored [11, 12]. Therapeutic interventions aimed at stimulating inflammation resolution and recovering the homeostasis of the brain and other vital organs after SAH could improve

This chapter provides an overview of the potentially harmful consequences of selective deficiency of omega-3 FAs on brain structure and function in SAH patients. Moreover, given the possible clinical relevance to SAH and the growth and rupture of intracranial aneurysms (IAs), we provide a detailed discussion of recent findings on the role of omega-3 FAs in resolution of inflammation, with a focus on

Remarkable progress in our understanding of the pathophysiology of acute inflammation has been achieved through basic science over the last 20 years. Resolution of acute inflammation is now considered to be an active biochemical process that is required to enable tissues to restore normal structure and function following an injury [14]. Interference with the resolution phase of acute inflammation may result in necrosis, chronic inflammation, fibrosis, and organ dysfunction. The resolution process is triggered at the beginning of inflammation by a temporal switch in lipid mediator classes, which is induced by cross talk between cells of the innate immune system and other cells in the inflammatory microenvironment [11]. A diverse range of biologically active pro-resolving and anti-inflammatory mediators are synthetized by complex pathways that involve several enzymes, including cyclooxygenase 2 (COX-2), cytochrome P450s and several lipoxygenases (LOX). The majority of these endogenous mediators are derived from the longchain omega-3 fatty acids (FAs) EPA and DHA and are members of the specialized pro-resolving mediator (SPM) superfamily [15]. SPMs represent an essential biochemical interface between inflammation and tissue repair and regeneration. The resolution of inflammation is a highly orchestrated adaptive process that depends on both the availability of SPMs precursors and the efficiency of the related biosyn-

Each endogenous lipid mediator is structurally distinct and possesses specific biological functions which have been extensively studied in diverse experimental

**2. Role of omega-3 FAs in resolution of acute inflammation**

**238**

thetic pathways.

**2.1 Specialized pro-resolving mediators**

models. EPA is the precursor of the E-series resolvins (RvEs), which contains four main mediators (RvE1-RvE3 and 18-HEPE). DHA is the precursor of the D-series resolvins (RvDs), which contains six mediators (RvD1-RvD6), as well as the protectins (PD1-PDX) and the maresins (MaR1-MaR2); DHA derivatives are also known as docosanoids [15].

SPMs are active at pico-nanogram ranges and exert pleiotropic actions at the inflammatory microenvironment. SPMs stimulate the clearance of bacteria, dead cells, and debris that is required during tissue repair. SPMs also reduce leukocyte transendothelial migration, platelet activation, and production of inflammatory cytokines, thus providing multi-organ protection. The maresins (macrophage mediators in resolving inflammation) exert potent phagocyte-directed actions that include phenotypic conversion of proinflammatory macrophages into macrophages that suppress inflammation and promote tissue regeneration [16]. Collectively, SPMs actively promote resolution of inflammation and recovery of tissue homeostasis [15]. Interestingly, resolution of systemic inflammation appears to have its counterpart in the CNS represented by different DHA-derived endogenous mediators (see Section 5.1).

The biosynthetic pathways that generate SPMs are clinically relevant and have been comprehensively studied [15, 16]. Drugs that inactivate the enzymes involved in SPM biosynthesis, such as selective COX-2 inhibitors and certain LOX inhibitors, delay the return to homeostasis and are considered resolution antagonists. Importantly, selective COX-2 inhibitors were synthesized before the identification of inflammation resolution pathways and are currently widely used as anti-inflammatory agents. Aspirin and statins also modify COX-2 by acetylation and S-nitrosylation, respectively, which results in generation of longer-acting SPM R-epimers. Thus, aspirin and statins are resolution agonists [15, 16].

### **3. Incorporation and transport of omega-3 FAs**

Incorporation and transport of omega-3 FAs have been comprehensively described; here, we focus on the clinically relevant aspects [10, 17, 18]. Several studies have consistently shown that in vivo conversion of alpha linolenic acid (ALA), the short-chain omega-3 FAs from vegetable origin, to its bioactive long-chain derivatives (EPA and DHA) is very low in humans. In addition, the metabolism of omega-6 and omega-3 FAs is tightly linked, and thus a high dietary intake of omega-6 FAs further reduce the conversion of ALA to EPA and DHA and their biological effects. The body has also a limited capacity to store longchain omega-3 FAs; only very small amounts of EPA and DHA are present in adipose tissue, and the brain retains DHA for its own function. Thus, providing preformed EPA and DHA is the most efficient method of increasing the concentrations of EPA and DHA in plasma and tissues.

EPA and DHA are incorporated into different blood lipid fractions after absorption by the gastrointestinal tract or after release from intravenously infused fish oil-based lipid emulsions (FOLE). These lipid fractions reflect the diverse means by which FAs are transported in the circulation and execute their physiological functions. The fractions incorporated in the phospholipid coat of plasma lipoproteins and plasma nonesterified FAs (NEFAs) are considered transport pools. Notably, the NEFA pool seems to be the main DHA plasma fraction that supplies the brain. The NEFA pool also represents a direct source of FAs to cells for generation of SPMs, as this pool readily transfers to inflammatory tissue via edema formation [19]. The FAs fraction incorporated in peripheral blood mononuclear cells (PBMCs) represents a functional pool due to the crucial roles of PBMCs in inflammation.

#### **3.1 Omega-3 index**

The EPA and DHA content of red blood cells (RBCs) membrane which can be quantified by a specific and standardized analytical procedure—the HS-Omega-3 Index® methodology—reliably reflects the omega-3 FAs content of several tissues [20]. This omega-3 index has been increasingly utilized as a surrogate marker of omega-3 status. Long-term intake of EPA and DHA is the main predictor of the omega-3 index, but other factors influence its variability. Acute supplementation of omega-3 FAs does not modify the omega-3 index, as expected given the long lifetime of RBCs (100–120 days). In terms of clinical relevance, strong inverse correlations have been observed between the omega-3 index and cardiovascular morbimortality, particularly sudden cardiac death, as well as depression [21–23].

## **4. Role of EPA in microvascular thromboinflammation**

Microvascular inflammation is an early event in the pathogenesis of atherosclerosis and other inflammatory conditions and is inextricably linked to microthrombosis [24]. Eicosanoid metabolism and leukocyte-endothelial interactions are interrelated processes and in turn are major drivers of thromboinflammation [25, 26]. Inflammation stimulates eicosanoid synthesis and expression of cell-surface adhesion molecules through upregulation of the nuclear factor kappa B (NF-kB), a master transcription factor necessarily involved in the inflammatory response.

Most eicosanoids derived from the long-chain omega-6 FAs arachidonic acid (ARA), including prostaglandins, leukotrienes, and thromboxanes, are potent vasoconstrictors, platelet activators, and leukocyte chemotactic factors. Moreover, the expression of cell-surface adhesion molecules on endothelial and inflammatory cells is essential for leukocyte-endothelial interactions; rolling and adhesion on vascular surfaces are the first step in the recruitment of circulating leukocytes or platelets to sites of thromboinflammation [27].

EPA incorporated in the membrane phospholipids of inflammatory cells can modulate eicosanoid metabolism by replacing ARA as an eicosanoid precursor [25]. EPA-derived eicosanoids are significantly less potent than those derived from ARA, and nonesterified EPA can also directly inhibit the production of eicosanoids from ARA. In addition, EPA has been shown to decrease the expression of several cell-surface adhesion molecules on inflammatory cells. EPA appears to elicit these pleiotropic effects by modulating the activity of the NF-kB.

Additionally, recent mechanistic studies have shown that minor changes in the EPA content of endothelial membranes may markedly alter the biophysical properties of the membrane [28]. Furthermore, changes in membrane fluidity, thickness, and deformability induced by modifications to lipid dynamics and/or structural organization can profoundly impact endothelial function [29]. Given the intimate association between brain capillary pericytes and endothelial cells, it would not be surprising if EPA also incorporates into pericyte cell membranes and potentiates the function of these cells as regulators of brain homeostasis [30, 31].

#### **5. Unique function of DHA in the brain**

DHA is widely distributed throughout the human body in membrane phospholipids and is particularly abundant in neural tissues such as the brain and retina [10]. DHA represents 30–40% of the fatty acid content of the gray matter in the cortex and is absorbed into the brain by a specific transporter that is found in

**241**

neurogenesis.

*Aneurysmal Subarachnoid Hemorrhage and Resolution of Inflammation*

the endothelium of the blood-brain barrier (BBB) microvasculature [32, 33]. The functional significance of selective enrichment of DHA in neural tissues has been actively researched over the last few decades. DHA possesses unique biophysical and biochemical characteristics that make it particularly suitable for brain and retinal membranes. DHA has been highly conserved during evolution and is present throughout the entire spectrum of living organisms [34]. DHA has even been suggested to be a major determinant for evolution of the modern hominid brain

DHA is an essential component of neuronal membrane architecture and composition and promotes selective accumulation of phosphatidylserine (PS), a crucial phospholipid involved in intracellular signaling [36]. PS participates in the signaling events of several key enzymes, including protein kinase C, Raf-1 kinase, and Akt, which play essential roles in cell proliferation, differentiation, and survival. Thus, DHA significantly modulates the activity of essential cellular kinases in

In addition to its function as a unique building block of cell membranes, DHA is also a precursor for docosanoids and other bioactive endogenous derivatives in the neural tissue [37]. The number of recently identified DHA derivatives in neural tissue is increasingly growing and includes neuroprotectin D1 (NPD1), synaptamide, endocannabinoid epoxides, and elovanoids [38–40]. Collectively, the potent bioactive properties of these DHA derivatives contribute to preservation of normal neuronal function, tissue homeostasis, and neuronal survival [37–41]. In addition, the DHA derivatives exert a range of potent neuroprotective properties that include inhibition of proinflammatory gene expression and leukocyte infiltration. A striking hallmark of the DHA derivatives is their ability to potentiate microglial polarization from a proinflammatory phenotype to a surveillance-repair state and reduce NF-kB-mediated expression of inflammatory cytokines in the brain. Moreover, DHA derivatives contribute to BBB integrity and are neurogenic and synaptogenic [38, 42]. Thus, the DHA derivatives seem to be key mediators of the resolution of inflammation and recovery of homeostasis in the CNS microenvironment.

The concept of neurovascular unit emphasizes the intimate relationship between the brain and its vessels, particularly the coupling between neural activity and cerebral blood flow [43]. Although the role of DHA neurolipidomics in neurovascular coupling appears to be underestimated, substantial experimental evidence suggests that the morphologic and functional integrity of the neurovascular unit largely depends on high DHA enrichment [37, 40, 43]. Moreover, the potential role of EPA in microvascular function further supports the evolutionary importance of these essential nutrients to maintain efficient functional couplings between neural and vascular networks [28, 29]. A functional neurovascular unit may be crucial not only for neurovascular coupling but also for BBB integrity and

A regular dietary supply of DHA is required to preserve normal brain and retinal

function. Under physiologic conditions, the net DHA incorporation rate for the entire human brain is very low and equivalent to the net rate of DHA consumption by the brain (3.8 ± 1.7 mg/day) [44]. However, loss of DHA in pathological states or due to nutritional deficiency of omega-3 FAs may severely impair neurovascular

integrity and have far-reaching implications on normal brain function [36].

*DOI: http://dx.doi.org/10.5772/intechopen.88297*

due to its unique encephalization potential [35].

**5.1 DHA and endogenous neuroprotective signaling**

**5.2 DHA and neurovascular unit**

neuronal cells.

#### *Aneurysmal Subarachnoid Hemorrhage and Resolution of Inflammation DOI: http://dx.doi.org/10.5772/intechopen.88297*

*New Insight into Cerebrovascular Diseases - An Updated Comprehensive Review*

**4. Role of EPA in microvascular thromboinflammation**

factor necessarily involved in the inflammatory response.

pleiotropic effects by modulating the activity of the NF-kB.

function of these cells as regulators of brain homeostasis [30, 31].

**5. Unique function of DHA in the brain**

sites of thromboinflammation [27].

The EPA and DHA content of red blood cells (RBCs) membrane which can be quantified by a specific and standardized analytical procedure—the HS-Omega-3 Index® methodology—reliably reflects the omega-3 FAs content of several tissues [20]. This omega-3 index has been increasingly utilized as a surrogate marker of omega-3 status. Long-term intake of EPA and DHA is the main predictor of the omega-3 index, but other factors influence its variability. Acute supplementation of omega-3 FAs does not modify the omega-3 index, as expected given the long lifetime of RBCs (100–120 days). In terms of clinical relevance, strong inverse correlations have been observed between the omega-3 index and cardiovascular morbimortality, particularly sudden cardiac death, as well as depression [21–23].

Microvascular inflammation is an early event in the pathogenesis of atherosclerosis and other inflammatory conditions and is inextricably linked to microthrombosis [24]. Eicosanoid metabolism and leukocyte-endothelial interactions are interrelated processes and in turn are major drivers of thromboinflammation [25, 26]. Inflammation stimulates eicosanoid synthesis and expression of cell-surface adhesion molecules through upregulation of the nuclear factor kappa B (NF-kB), a master transcription

Most eicosanoids derived from the long-chain omega-6 FAs arachidonic acid (ARA), including prostaglandins, leukotrienes, and thromboxanes, are potent vasoconstrictors, platelet activators, and leukocyte chemotactic factors. Moreover, the expression of cell-surface adhesion molecules on endothelial and inflammatory cells is essential for leukocyte-endothelial interactions; rolling and adhesion on vascular surfaces are the first step in the recruitment of circulating leukocytes or platelets to

EPA incorporated in the membrane phospholipids of inflammatory cells can modulate eicosanoid metabolism by replacing ARA as an eicosanoid precursor [25]. EPA-derived eicosanoids are significantly less potent than those derived from ARA, and nonesterified EPA can also directly inhibit the production of eicosanoids from ARA. In addition, EPA has been shown to decrease the expression of several cell-surface adhesion molecules on inflammatory cells. EPA appears to elicit these

Additionally, recent mechanistic studies have shown that minor changes in the EPA content of endothelial membranes may markedly alter the biophysical properties of the membrane [28]. Furthermore, changes in membrane fluidity, thickness, and deformability induced by modifications to lipid dynamics and/or structural organization can profoundly impact endothelial function [29]. Given the intimate association between brain capillary pericytes and endothelial cells, it would not be surprising if EPA also incorporates into pericyte cell membranes and potentiates the

DHA is widely distributed throughout the human body in membrane phospholipids and is particularly abundant in neural tissues such as the brain and retina [10]. DHA represents 30–40% of the fatty acid content of the gray matter in the cortex and is absorbed into the brain by a specific transporter that is found in

**3.1 Omega-3 index**

**240**

the endothelium of the blood-brain barrier (BBB) microvasculature [32, 33]. The functional significance of selective enrichment of DHA in neural tissues has been actively researched over the last few decades. DHA possesses unique biophysical and biochemical characteristics that make it particularly suitable for brain and retinal membranes. DHA has been highly conserved during evolution and is present throughout the entire spectrum of living organisms [34]. DHA has even been suggested to be a major determinant for evolution of the modern hominid brain due to its unique encephalization potential [35].

DHA is an essential component of neuronal membrane architecture and composition and promotes selective accumulation of phosphatidylserine (PS), a crucial phospholipid involved in intracellular signaling [36]. PS participates in the signaling events of several key enzymes, including protein kinase C, Raf-1 kinase, and Akt, which play essential roles in cell proliferation, differentiation, and survival. Thus, DHA significantly modulates the activity of essential cellular kinases in neuronal cells.
