**2. Pathophysiology of I/R injury**

Tissue perfusion is the most important parameter of flaps. When it is interrupted for a period of time and abruptly restored, I/R injury occurs. As long as there is timely reperfusion, ischemia results in reversible cellular damage. However, restoration of blood flow after a period of time results in an incident whereby reperfusion ends up with greater tissue injury than that which is produced by ischemia itself. I/R injury is a complex interplay between biochemical, cellular, and vascular endothelial factors. Although the clinical sequelae are organ specific, it may also involve systemic inflammatory responses [6].

The tissue damage in I/R injury is like a double-edged sword and is divided into two parts: ischemia injury and reperfusion injury. Ischemic injury may initially cause hypoxia and hyponutrition. After prolonged ischemia, the metabolic products from cells are retained and cause metabolic acidosis. When the blood supply is reestablished, local inflammation and reactive oxygen species production increase. Those lead to an activation of neutrophils and a consecutive adhesion between granulocytes and endothelial cells causing segmental vessel occlusion in postcapillary venules, transendothelial leukocyte migration, nitric oxide (NO) depletion, and the release of tissue-damaging enzymes leading to secondary injury [7] Reactive oxygen species are potent oxidizing and reducing agents that directly damage cellular membranes by lipid peroxidation [8]. The cell response is dependent on the severity of total tissue injury [9]. Cell damage induced by prolonged ischemia-reperfusion injury may lead to apoptosis, autophagy, and necrosis (**Figure 1**). Cell survival systems (control ROS generation and cell damage) are activated in short duration of I/R injury [10]. Moderate I/R injury may cause cell dysfunction by autophagy and also activate recovery systems for survival [11]. But if damage is severe, cell death may be induced via apoptotic or necrotic pathways [12].

I/R injury has effect on the microcirculation of the entire flap because of the inflammatory process and the rise in ROS in the early stages of reperfusion [13]. For this reason, insufficient microcirculation usually occurs mainly in the distal parts of the flap, which is a common cause of partial flap necrosis.

As mentioned above, total flap necrosis is most often caused by thrombosis of the pedicle causing vascular insufficiency. Immediate revision of the anastomosis is crucial to reestablish blood flow. Nevertheless, such complications can increase

**389**

experimental.

from happening.

**3.1 Free radical formation**

*The Effect of Antioxidants on Ischemia-Reperfusion Injury in Flap Surgery*

I/R injury, which can lead to intravascular hemoconcentration, swelling of endothelium, increase in interstitial edema as well as inflammatory reactions because of the reperfusion injury. After a critical period, I/R injury can lead to a no-reflow

Several methods and techniques are described to protect flap from the dangerous effects of the I/R injury or minimize the stress during and after ischemia. Tissue conditioning, which is the most acceptable one, consists of preoperative, perioperative, and postoperative techniques to adapt the tissue to the ischemic stress.

Nitric oxide donation is another technique that NO administered through inhalation. NO plays a protective role via its antioxidative and anti-inflammatory functions [17], but it is not in common use in flap surgery and still remains

There are also studies aiming to prevent anti-inflammatory mediators released by leukocytes. Anti-leukocyte therapy limits leukocyte-mediated I/R injury and has focused on inhibition of inflammatory mediator release or receptor engagement, leukocyte adhesion molecule synthesis, or leukocyte-endothelial adhesion [18]. Many drugs act in this manner and have been shown to be very effective.

Antioxidants that have been extensively studied in I/R injury are also found to be effective in various studies [19]. Because high amount of ROS is the primary supplement of the injury, it can neutralize the effect or prevent the mechanism

Free radicals were discovered less than 50 years ago [20]. At first, they were assumed as completely harmful. Later on, advantageous biological effects of free radicals were reported. In recent studies, the role of free radicals is being researched commonly both in physiological conditions and in diseases. In molecular biology, a molecule that has an unpaired electron in its outer valence orbital that needs an extra electron to restore stability is called a free radical. They are short lived and highly reactive. The situation of instability because of the unpaired electron creates energy that has to be released instantly. In cell physiology, normal low levels of free radicals are used in autophagy, cell signaling, and antimicrobial oxidative bursts [21]. But, at higher free radical levels, the interaction with neighbor molecules

phenomenon, which also leads to complete flap loss [14–16].

**3. Free radical and antioxidant connection**

Multiple methods were described for tissue conditioning in **Table 1**.

*DOI: http://dx.doi.org/10.5772/intechopen.85500*

• Ischemic preconditioning • Remote ischemic preconditioning • Pharmaceutical preconditioning • Thermic preconditioning

• Extracorporeal shock waves

• Surgical delay • Growth factors

• Stem cells

*Types of tissue conditioning.*

**Table 1.**

*The Effect of Antioxidants on Ischemia-Reperfusion Injury in Flap Surgery DOI: http://dx.doi.org/10.5772/intechopen.85500*


#### **Table 1.**

*Antioxidants*

**2. Pathophysiology of I/R injury**

involve systemic inflammatory responses [6].

may be induced via apoptotic or necrotic pathways [12].

the flap, which is a common cause of partial flap necrosis.

Tissue perfusion is the most important parameter of flaps. When it is interrupted for a period of time and abruptly restored, I/R injury occurs. As long as there is timely reperfusion, ischemia results in reversible cellular damage. However, restoration of blood flow after a period of time results in an incident whereby reperfusion ends up with greater tissue injury than that which is produced by ischemia itself. I/R injury is a complex interplay between biochemical, cellular, and vascular endothelial factors. Although the clinical sequelae are organ specific, it may also

The tissue damage in I/R injury is like a double-edged sword and is divided into two parts: ischemia injury and reperfusion injury. Ischemic injury may initially cause hypoxia and hyponutrition. After prolonged ischemia, the metabolic products from cells are retained and cause metabolic acidosis. When the blood supply is reestablished, local inflammation and reactive oxygen species production increase. Those lead to an activation of neutrophils and a consecutive adhesion between granulocytes and endothelial cells causing segmental vessel occlusion in postcapillary venules, transendothelial leukocyte migration, nitric oxide (NO) depletion, and the release of tissue-damaging enzymes leading to secondary injury [7] Reactive oxygen species are potent oxidizing and reducing agents that directly damage cellular membranes by lipid peroxidation [8]. The cell response is dependent on the severity of total tissue injury [9]. Cell damage induced by prolonged ischemia-reperfusion injury may lead to apoptosis, autophagy, and necrosis (**Figure 1**). Cell survival systems (control ROS generation and cell damage) are activated in short duration of I/R injury [10]. Moderate I/R injury may cause cell dysfunction by autophagy and also activate recovery systems for survival [11]. But if damage is severe, cell death

I/R injury has effect on the microcirculation of the entire flap because of the inflammatory process and the rise in ROS in the early stages of reperfusion [13]. For this reason, insufficient microcirculation usually occurs mainly in the distal parts of

As mentioned above, total flap necrosis is most often caused by thrombosis of the pedicle causing vascular insufficiency. Immediate revision of the anastomosis is crucial to reestablish blood flow. Nevertheless, such complications can increase

**388**

**Figure 1.**

*Ischemia-reperfusion mechanism.*

*Types of tissue conditioning.*

I/R injury, which can lead to intravascular hemoconcentration, swelling of endothelium, increase in interstitial edema as well as inflammatory reactions because of the reperfusion injury. After a critical period, I/R injury can lead to a no-reflow phenomenon, which also leads to complete flap loss [14–16].

Several methods and techniques are described to protect flap from the dangerous effects of the I/R injury or minimize the stress during and after ischemia. Tissue conditioning, which is the most acceptable one, consists of preoperative, perioperative, and postoperative techniques to adapt the tissue to the ischemic stress. Multiple methods were described for tissue conditioning in **Table 1**.

Nitric oxide donation is another technique that NO administered through inhalation. NO plays a protective role via its antioxidative and anti-inflammatory functions [17], but it is not in common use in flap surgery and still remains experimental.

There are also studies aiming to prevent anti-inflammatory mediators released by leukocytes. Anti-leukocyte therapy limits leukocyte-mediated I/R injury and has focused on inhibition of inflammatory mediator release or receptor engagement, leukocyte adhesion molecule synthesis, or leukocyte-endothelial adhesion [18]. Many drugs act in this manner and have been shown to be very effective.

#### **3. Free radical and antioxidant connection**

Antioxidants that have been extensively studied in I/R injury are also found to be effective in various studies [19]. Because high amount of ROS is the primary supplement of the injury, it can neutralize the effect or prevent the mechanism from happening.

#### **3.1 Free radical formation**

Free radicals were discovered less than 50 years ago [20]. At first, they were assumed as completely harmful. Later on, advantageous biological effects of free radicals were reported. In recent studies, the role of free radicals is being researched commonly both in physiological conditions and in diseases. In molecular biology, a molecule that has an unpaired electron in its outer valence orbital that needs an extra electron to restore stability is called a free radical. They are short lived and highly reactive. The situation of instability because of the unpaired electron creates energy that has to be released instantly. In cell physiology, normal low levels of free radicals are used in autophagy, cell signaling, and antimicrobial oxidative bursts [21]. But, at higher free radical levels, the interaction with neighbor molecules

such as lipids, proteins, and DNA for releasing the energy causes damage. So, free radicals are the products of normal cellular metabolism. In order to reach stability, an electron has to be stolen. The attacked molecule engages itself in a chain reaction to steal an electron from another molecule.

In aerobic organisms, oxygen free radicals launch autocatalytic reactions that finally damage the living cell. The unsaturated carbon-carbon double bonds in the exposed end groups are particularly sensitive to free radicals forming a covalent single bond at a carbon atom to form a free radical at the opposite carbon atom [22]. Free radicals interact with molecular cross-linking for increased structural organization by reducing the transport of oxygen. ROS can be produced from endogenous or exogenous sources. Endogenous ROS is produced in different cellular organs where oxygen consumption is high such as mitochondria, peroxisomes, and endoplasmic reticulum. Most of the intracellular ROS are derived from mitochondria. The amount of free radicals is determined by many factors. In periods of irregular hypoxia in mitochondrial energy synthesis, excess electron production can develop free radicals that can damage lipids, proteins, and greatly increase molecular size in increasing vicious cycles to further reduce oxygen availability for mitochondria during energy synthesis. Another major type of free radical in a living cell is reactive nitrogen species (RNS). Nitric oxide (NO) radical is formed by the enzyme nitric oxide synthase and involves in smooth muscle relaxation and various other cGMP-dependent functions [23].

Free radicals are prominent in many pathological conditions such as cancer, diabetes, cardiovascular diseases, neurodegenerative diseases, cataracts, asthma, rheumatoid arthritis, inflammation, burns, intestinal tract diseases, progerias, and ischemic and postischemic pathologies. In particular, ROS is substantial for the pathogenesis of atherosclerosis. Low density lipoprotein (LDL) accumulates within plaques and contributes to the inflammatory state when ROS concentration is high and ROS oxidizes neighbor LDLs [24]. Also, it is believed that aging is a process mediated by free radicals [25]. At the present time, each chemical step has been investigated meticulously in order to prevent cell damage and clarify free radicals.

Different reactive oxygen species are formed in biological tissue (**Table 2**). Superoxide radical (O2 − ) can be formed by adding an extra electron to the oxygen molecule. Hydroxyl radical (⋅OH) can be formed in two different ways. First, it can be formed from O2 − and H2O2 with a reaction catalyzed by a metal such as iron (Fe). Second, it can be formed from singlet oxygen (<sup>1</sup> O2) reaction. Moreover, oxygen free radicals may also be formed by polymorphonuclear leukocytes in ischemic tissue.

#### **3.2 Human body defense mechanisms**

Although accumulation of this substances are harmful to cell viability, it is important to know that human body is equipped with a defense system consisting of several antioxidative enzymes, to fight with these ROS. Superoxide dismutase


**391**

**Table 3.**

*The Effect of Antioxidants on Ischemia-Reperfusion Injury in Flap Surgery*

these enzymes. SOD catalyzes dismutation reaction where O2

(SOD), glutathione peroxidase, glutathione reductase and catalase are some of

O2 + H2O2 molecules. Catalase (heme-containing enzyme) also catalyzes the H2O2 reaction. H2O2 can also be reduced by glutathione peroxidase (GSH-P) which is selenium dependent enzyme, transforms reduced glutathione to oxidized glutathione. After that reaction oxidized glutathione is transformed into reduced for with help of nicotinamide adenine dinucleotide phosphate (NADPH). Additionally, NADPH regenerated from glucose 6-phosphate catalyzed by the enzyme glucose

The biochemical reaction of glutathione (GSH) is crucial. An intermolecular disulfide non-radical end product, glutathione disulfide (GSSG), is formed, which can either be exported from the cells or transformed back to glutathione by the combined action of glutathione reductase and the NADPH cofactor. Glutathione can react directly with ROS and RNS by its thiol group; also, it can aim the disulfide bridges formed inside and between proteins by the action of free radicals [26].

Antioxidants are molecules against free radicals and are capable of securing or deactivating free radicals before damaging the cells. There are many antioxidant systems that work synergistically with each other to protect the body's organs and organ systems against free radical damage. There are highly complex enzymatic and non-enzymatic antioxidants: the enzymes such as SOD, glutathione peroxidase, and catalase, as well as non-enzymatic compounds such as α-tocopherol (vitamin E), β-carotene, ascorbic acid (vitamin C), and glutathione. Referred enzymes aim free radicals to delocalize their proteins into side chains and peptide bonds. Also, antioxidants may be endogenous or exogenous, such as part of a diet or dietary supplement. As we know aging is related to free radicals, nutrients rich with antioxidants contend with aging. Under oxidative stress, endogenous antioxidants may not be sufficient and dietary antioxidants may be required to maintain optimal cellular functions. According to literature, exogenous antioxidants comprise the secondary defense system against oxygen free radicals. Moreover, it is believed that ischemia-reperfusion is associated with generation of excess amounts of reactive oxygen species, the removal of which is beyond the capacity of the existing antioxidant defense system [19]. So, contribution of secondary defense system is crucial for the injury associated with

Some dietary compounds that do not neutralize free radicals but increase endogenous activity can also be classified as antioxidants. An antioxidant should eliminate free radicals and be absorbed easily, and chelate redox metals at physiologically

+ 2H ➔ O2 + H2O2

2GSH + H2O2 ➔ GSSG + H2O

Glucose 6-P + NADP+ ➔ gluconate 6-P + NADPH + H+

2H2O2 ➔ 2H2O + O2

−

Glutathione reductase GSSH + NADPH + H+ ➔ 2GSH + NADP+

*Antioxidative enzymes and catalyzed reactions in human body.*

−

transforms into

*DOI: http://dx.doi.org/10.5772/intechopen.85500*

6-phosphate dehydrogenase (**Table 3**).

**3.3 Antioxidants**

ischemia-reperfusion.

**Human body antioxidative enzymes** Superoxide dismutase (SOD) 2O2

Catalase (heme-dependent

GSH-P (selenium-dependent

Glucose 6-phosphate dehydrogenase (G6PD)

enzyme)

enzyme)

**Table 2.** *Reactive oxygen species.*

#### *The Effect of Antioxidants on Ischemia-Reperfusion Injury in Flap Surgery DOI: http://dx.doi.org/10.5772/intechopen.85500*

(SOD), glutathione peroxidase, glutathione reductase and catalase are some of these enzymes. SOD catalyzes dismutation reaction where O2 − transforms into O2 + H2O2 molecules. Catalase (heme-containing enzyme) also catalyzes the H2O2 reaction. H2O2 can also be reduced by glutathione peroxidase (GSH-P) which is selenium dependent enzyme, transforms reduced glutathione to oxidized glutathione. After that reaction oxidized glutathione is transformed into reduced for with help of nicotinamide adenine dinucleotide phosphate (NADPH). Additionally, NADPH regenerated from glucose 6-phosphate catalyzed by the enzyme glucose 6-phosphate dehydrogenase (**Table 3**).

The biochemical reaction of glutathione (GSH) is crucial. An intermolecular disulfide non-radical end product, glutathione disulfide (GSSG), is formed, which can either be exported from the cells or transformed back to glutathione by the combined action of glutathione reductase and the NADPH cofactor. Glutathione can react directly with ROS and RNS by its thiol group; also, it can aim the disulfide bridges formed inside and between proteins by the action of free radicals [26].
