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The role of free radicals in the pathophysiology of ischemia and reperfusion in skin flaps: experimental models and treatment strategies
(Portuguese PDF version)

Eloísa Bueno Pires de Campos,1 Winston Bonetti Yoshida2

1. Plastic Surgeon. Hospital das Clínicas de Botucatu, Botucatu, SP, Brazil.
2. Adjunct and associate professor. Department of Surgery and Orthopedics, School of Medicine of Botucatu, Universidade Estadual Paulista, Botucatu, SP, Brazil.

Correspondence:
Winston Bonetti Yoshida
Departamento de Cirurgia e Ortopedia
Faculdade de Medicina de Botucatu - UNESP
CEP 18607-030 - Botucatu, SP, Brazil
Phone: +55 (14) 3811.6269
E-mail: winston@fmb.unesp.br

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ABSTRACT

With the development of microsurgery in the reconstructive plastic surgery area, former impossible restorations have now come true, including the free-tissue transfer. Knowledge accumulated so far indicates a 90 to 95% patency rate of vascular anastomosis in skin flaps. Occlusions are generally due to technical errors or vascular thrombosis. However, the blood flow restoration indicated in these occlusive situations may paradoxically induce to even more important lesions than the ischemia itself. Several studies show that oxygen-derived free radicals are involved in biochemical, inflammatory and cellular alterations that follow blood flow restoration. In this review, we focus on the main mechanisms responsible for these alterations, the experimental models used to study them and the treatment alternatives employed to attenuate lesions of ischemia and reperfusion.

Key-words: ischemia, reperfusion, surgical flaps, free radicals, antioxidants.

J Vasc Br 2004;3(4):357-66

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The development of microsurgery in the reconstructive plastic area, started by Buncke & Shuntz,¹ have opened up new possibilities for reconstruction surgeries. The free tissue transfer technique evolved and revolutionized the area. McLean & Buncke reported the first successful cranium coverage using omental transplantation in 1972.² In 1973, Daniel & Taylor³ described the transfer of a vascularized groin flap for the pre-tibial area, also using microsurgery.

In the meantime, surgical techniques were improved with the development of advanced microscopes, delicate surgical materials and very fine suture. Besides, deeper knowledge on the microvascular and nervous anatomy and the study of their physiology enabled ossibilities of new graft flaps.

Today, it is known that the frequency of patent vascular anastomosis in microsurgery is from 90 to 95%.^4-6 However, occlusions may occur as a result of technical mistakes or vascular thrombosis⁷ and, in these cases, they should be managed with thrombectomy, assessment of the anastomosis and blood flow restoration. The time elapsed since the occlusion onset andclinical diagnose up to the review is the time the flap remains ischemic, with no blood flow enough to fulfill the basic needs of the tissue..

Tissues injury may be reversed or not, depending on ischemia time and hypoxia. Some tissues, as peripheral nerves and muscles, are less resistant to hypoxia than the skin.⁸ Kerrigan & Daniel⁹ reported that the critical ischemia time would be the maximum time the free tissue could tolerate to still remain patent after reconstruction. The blood flow should be restored so that the ischemia event could be reversed, however, paradoxically, it could be responsible for even more serious lesions than those caused by the ischemia per se. The studies by Granger et al.¹⁰ evidenced that oxygen in the ischemic tissue lead to a series of biochemical, inflammatory and cellular alterations resultant especially from the formation of oxygen free-radicals.

The goal of the present review is to approach the main pathophysiological aspects of ischemia and reperfusion injuries, as well as to describe the balance between free-radicals and endogenous antioxidants, respectively in the genesis and prevention of alterations. We also discuss treatment strategies based especially on experimental models.

OXYGEN FREE RADICALS

A free radical is any atom or molecule with at least one unpaired electron in its outermost ring.¹¹ Unpaired electrons make atoms and molecules highly reactive because of their tendency to donate or receive electrons. In living organisms of aerobic metabolism, oxygen undergoes a tetravalent reduction by the mitochondrial electron-transport chain, producing water. This reaction is catalyzed by the cytochrome oxidase enzyme. In the process described below, a small fraction (1-2%) is reduced, producing the following free radicals: superoxide (O₂), hydroxyl (OH), hydrogen peroxide (H₂O₂) and hydroperoxyl (HO₂")12 (Figure 1).

Figure 1 - Tetravalent reduction of oxygen (O₂) in the mitochondrion and the formation of water (H₂O). Adapted from Cohen.13

The superoxide radical (O₂^-") results from a monovalent reduction of O2.^13,14 In isolation, the superoxide radical is not very reactive, therefore, not highly cytotoxic. However, as ^{it has a huge potential to generate secondary radicals, which are highly toxic and damaging, as the OH" radical.15-18}

The hydroperoxyl radical (HO₂") is the protonated form of the superoxide radical, that is, it contains the hydrogen proton. According to Halliwell¹¹ it is not more powerful than the superoxide in the destruction of biological membranes. The hydrogen peroxide (H₂O₂) is not a free radical, because it does not have unpaired electrons in its outermost ring, but it takes part in reactions that produce the OH" radical, either by Fenton or Haber-Weiss reaction. H₂O₂ has a long life time, it is able to go through lipid layers and it can react with the erythrocyte membrane and with iron-containing proteins.¹⁶ That is the reason why there is a recent tendency to name the whole of substances involved in these reactions as "reactive oxygen species" instead of oxygen free radicals.

The hydroxyl radical (HO") is extremely reactive and it is capable of extracting hydrogen atoms of the methylene group of fatty polyunsaturated acids, triggering the lipid peroxidation that causes the lysis of the cellular membrane. This radical can be produced in vivo by means of reactions of transition metal ions (Fe⁺⁺) with the hydrogen peroxide, through the Fenton and Haber-Weiss reaction.^17,18

Fenton reaction

Fe⁺⁺ + O₂ <---------> Fe⁺⁺⁺ + O₂-"

2 O₂^-" + 2H+ ----------> O₂ + H₂O₂

Fe⁺⁺ + H₂O₂ ----------> Fe^+++´+ OH- + OH "

Haber-Weiss reaction

Fe⁺⁺⁺ + O₂^-" ------------> Fe⁺⁺ + O₂

Fe⁺⁺ + H₂O₂ ------------> Fe⁺⁺⁺ + OH^- + OH"

O₂^-" + H₂O₂ ------------> O₂ + OH^- + OH"

According to Del Maestro,¹⁹ the most damaging effects of free radicals is the lipid peroxidation of the membrane. It is a complex phenomenon that starts with the abstraction of a hydrogen atom from the methylene group placed between the two unsaturated bands of the lipid molecule, producing a new lipid free radical with carbon in the centre, which in the presence of oxygen produces lipid peroxides or lipoperoxides. The metabolization of these products generates malonic dialdehyde, which can indirectly indicates the level of peroxidation (Figure 2).

Figure 2 - Representation of the lipid peroxidation of fatty acids of the cellular membrane (adapted from Del Maestro19).

The role of free radicals in the formation of ischemia and reperfusion injuries in the bowel, stomach, liver, pancreas, kidney, heart, skin and brain has been widely studied.^20-22

In a study about ischemia and reperfusion in the small intestine, Granger et al.¹⁰ proposed a mechanism that explains the origin of free radicals: " "Hypoxanthine is the first substrate for the xanthine oxidase oxidation, which occurs when the second substrate (oxygen) is supplied in the reperfusion, producing the reactive oxygen species". (Figura 3).

Figure 3 - Production of O₂ the reperfusion of ischemic tissues by the conversion of xanthine dehydrogenase into xanthine oxidase (adapted from Granger et al.10).

Free radicals can also originate from the production of superoxide radicals after the breaking of electrons in the mitochondrion or through the cyclooxigenase metabolism of arachidonic acid.²³ This mechanism would activate unspecific proteases and phospholipases induced by the accumulation of calcium within cells during reperfusion, leading to a synthesis of pro-inflammatory mediators: the platelet factor (PF) and the eicosanoid substances (leukotriens, thromboxanes and prostaglandins). Kerrigan & Stotland²⁴ reported that the most important mediators in the ischemia and reperfusion events are the PF, the leukotrien B₄ (LTB4) and the thromboxane A₂ (TXA₂), because they can increase the inflammatory response due to an increase in the peptide mediators, like cytokines, the tumor necrosis factor (TNF) and interleukin-1 (IL-1). The TXA₂ and the LTB₄ are, respectively, byproducts of the cyclooxygenase and lipoxygenase action over the arachidonic acid. According to Knight et al.,²⁵ the LTB4 is a potent chemotactic leukocyte factor that favors the interaction between neutrophils and the endothelial cell with a release of free radicals and proteolytic enzymes. The TXA₂ is a potent vasoconstrictor and platelet aggregator that decreases the capillary flow after reperfusion (Figure 4)

Figure 4 - Formation of sub-products of the arachidonic acid in the reperfusion period. (Adapted from Welbourn et al.28)

In vitro studies demonstrate that the interaction between the neutrophils and the endothelial cell are responsible for the release of pro-inflammatory mediators that trigger the inflammatory reperfusion injury.²⁶ An experimental study on myocardial ischemia and reperfusion by Ashraf & Zhai²⁷ showed that the relation between the neutrophil and the endothelial cell activates the production of the superoxide anion derived from the neutrophil in the NADPH oxidation to NADP. Besides, the neutrophil myeloperoxidases could convert H₂O₂ into hypochlorous acid (HOCL), which can damage tissues.

Figure 5 - Interaction between the neutrophil and the endothelial cell with oxygen free radicals release (adapted from Ashraf Zhai27).

According to Welbourn et al.,²⁸ the metabolites of the arachidonic acid, besides being chemotactic elements, favor the interaction between the neutrophil and the endothelial cell, making neutrophils produce more oxygen free radicals and proteolytic enzymes, affecting also the blood flow, as they act directly in the microcirculation. Augustin et al.²⁹ proposed that neutrophils, besides adhering to the endothelial cell causing injuries, could also plug in the microcirculation, obstructing the blood flow - the no reflow phenomenon, described by Ames et al.³⁰ with relation to the brain circulation and by May et al.³¹ regarding free experimental flaps. Another free radical closely associated with ischemia and reperfusion injuries is the nitric oxide (NO) gas.³² Initially, it was thought to be a relaxing factor derived from the endothelium,³³ but later it was recognized as being NO.³⁴ According to Moncada et al.,³⁵ it is produced by the endothelial cells, the macrophages and specific central neurons, and synthesized from the L-arginine, the O₂ and the NADPH by means of the NOS enzyme, which is activated by the inflow of calcium in the cell. It maintains the vascular tonus, inhibits the platelet aggregation and the adhesion of neutrophils and platelets on the vascular endothelium. Besides, NO is cytotoxic for certain microorganisms and tumor cells. In parallel, NO reacts with the superoxide radical, resultant from the ischemia and reperfusion, producing the nitrogen dioxide and the hydroxil radical via peroxynitrite anion (ONOO-).³⁶

	+	O2"-		ONOO-
ONOO-	+	H+	n	ONOOH
ONOOH	n	OH"	+	NO2
OH.	+	N2O	n	NO3-+ H

ANTIOXIDANTS

In normal conditions, free radicals are continuously produced in small quantities as metabolic byproducts or escapes from the oxidative phosphorylation. These radicals are naturally scavenged by an endogenous antioxidant defense system, enzymatic or non-enzymatic, which will prevent or repair the oxidative injury.³⁷

The preventive antioxidants include the enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), reduced glutathione (GSH) and vitamin E. The glutathione reductase (GSH-Rd), glutathione peroxidase (GSH-Px) and ascorbic acid are examples of repairing antioxidants.³⁸

The reduced glutathione (GSH, L-gamma-glutamyl-L-cysteinyl-glycine) is present in most cells and can be considered one of the most important agents of the antioxidation system of the cell. The glutathione reductase (GSH-Rd) is a flavoprotein that recovers the GSH when there is oxidation. The glutathione peroxidase (GSH-Px) catalyses the reduction of hydrogen peroxide (H₂O₂) and other organic peroxides. It can be found in the cytosol, in the mitochondrion and in the membrane, and contains selenium in its active site.³⁹

Catalase is a hemoprotein that catalyses the reduction of H₂O₂ to H2O and O₂. It is present in most tissues, but concentrates mainly in the liver, kidney, spleen and erythrocyte.⁴⁰

The SOD catalyses the dismutation of the superoxide radical into H₂O₂ and O₂ in the presence of the H+ proton . There are two forms of SOD: the zinc copper superoxide dismutase, present in the cytosol, and the manganese, present mainly in the mitochondrion.⁴¹

Besides the enzymatic antioxidant system, all substances that give or receive an electron from a free radical and deactivate it are considered to be antioxidants. Examples are the ascorbic acid (vitamin C), the beta-carotene, the uric acid, the vitamin E (alpha tocopherol), albumin, transferrin and mannitol. There are also those elements that indirectly have an antioxidant effect, as the allopurinol (inhibitor of xanthine oxidase), selenium (present in the glutathione peroxidase), deferoxamine (iron chelating agent), among others.⁴²

STRATEGIES FOR MANAGEMENT OF ISCHEMIA AND REPERFUSION

Based on the scientific experience accumulated over the years, which evidenced the role of oxygen free radicals in ischemia and reperfusion injuries, many different works were developed with the goal of lessening the hemodynamic and pathophysiological consequences of the vascular restoration after tissues ischemia. The main strategies adopted were:

• Supplementation of endogenous antioxidants
  
• Supplementation of exogenous antioxidants
  
• Supplementation of enzymatic components (selenium)
  
• Blockage of transition metals (Fe⁺⁺)
  
• Blockage of leukocytes activities
  
• Neutralization of the hypoxanthine action
  
• Anti-inflammatory medication, vasodilators and anti-platelets aggregators
  
• Monoclonal antibodies against adhesion glycoprotein of the neutrophil
  
• Supplementation of NO providers

Even though most of these strategies have been successful in lab tests, a consensus has not been reached in the clinical practice, due to a series of variables. A multifatorial approach, using a combination of antioxidant agents, may be the most adequate strategy for the majority of situations.⁴³ For Chen et al.,⁴⁴ the combination of antioxidant agents would reduce injuries in animal tissues; however it must be confirmed if the improvement in this protective effect is due to the interaction (synergism) among antioxidants or to an additional effect of each antioxidant in separate.

TREATMENT OF ISCHEMIA AND REPERFUSION IN TISSUE FLAPS

The majority of studies carried out in this area had an experimental character. The most used model is a rectangular myocutaneous flap that depends on epigastric vessels taken from the anterior abdomen of a rat. They are clamped in different times of ischemia and the reperfusion injuries are reported after 7 days, in general, by measuring the healthy and necrosed areas.

A number of pharmacological agents was tested in myocutaneous flaps submitted to experimental ischemia and reperfusion. Manson et al.⁴⁵ studied epigastric flaps, in rats, submitted to an 8-hour ischemia. After 7 days of reperfusion, they found a complete necrosis in the control group (without treatment), with a significant increase of survival of flaps treated with the SOD enzyme. In 1984, Im et al.⁴⁶ also used the model of epigastric flaps, in rats, using allopurinol, a xanthine oxidase inhibitor. Results show that the survival rate of the group approached was significantly higher than in the control group (saline solution). Besides, this work showed that the allopurinol blocked the activity of the xanthine oxidase enzyme, which is one of the origins of the superoxide radical, as we have already described. In the following year, these authors published a similar work, comparing the allopurinol against the superoxide dismutase. Both drugs showed to be effective.

Douglas et al.⁴⁷ performed a washout with Ringer lactate in the entire vascular region of epigastric flaps in rats, soon after ischemia. They observed that the survival rate and the obtainment of fluoresceine in the group submitted to the washout were higher than in the control group (without perfusion). In 1993, Israeli et al.⁴⁸ assessed the washout of the perfusion after ischemia with Ringer lactate, University of Wisconsin solution, verapamil, urokinase and iloprost, comparing it with the control group (with no perfusion). All solutions increased the flap blood flow.

Nishikawa et al.²² compared the deferoxamine (iron chelating agent) and hypertonic citrate in an experimental adipomusculocutaneous flap in rats. They did not find positive results as for survival rates of flaps in relation to the control group (without treatment). Besides, histology evidenced that necrosis, swelling and edema in all groups were not statistically different.

Zaccaria et al.⁴⁹ reported that vitamin C was effective in the enhancement of survival of epigastric flaps in rats, as compared to the control (saline solution) in the clinical assessment.

Fu & Jiao⁵⁰ opted for using mannitol and anisodamine (analogous to the atropine) in rats and observed that both were effective in the reduction of the necrosis. On the other hand, Atabey et al.⁵¹ showed that mannitol combined with cobalt was not effective to hinder the damaging action of cobalt in the lipid peroxidation measured through the levels of malondialdehyde.

The most recent studies have focused on drugs that act in neutrophils, as the immunosuppressive, which prevent the production of free radicals by the neutrophils. Cetinkale et al.⁵² used the FK 506 agent (an immunosuppressive drug) in rats and observed that it increased the survival of flaps compared with the control group (without treatment). Willemart et al.⁵³ had a positive answer using another immunosuppressive agent, the dexamethasone, which had a smaller necrosed area when compared to the group which received only physiological solution.

Another approach⁵⁴ compared two antihistamines: the diphenhydramine (H₁ blocker) and the cimetidine (H2 blocker). Both were effective and reduced the damaging actions of ischemia and reperfusion.

Akamatsu et al.⁵⁵ and Ueda et al.⁵⁶ observed that flaps were protected with sulphatide, a substance that connects to the D and P-selectine adhesion molecules. Cetin et al.⁵⁷ were successful using fucoidin, a potent inhibitor of neutrophil rolling.

A number of recent experimental works have demonstrated that the ischemia and reperfusion injuries were less severe with the use of Vitamin E and Vitamin A,⁵⁸ prostaglandin E1,⁵⁹ trimetazidine,⁶⁰ nitric oxide donors,^61,62 inhibitors of protein GIIb/IIIa,⁶³ and nitric oxide synthase expression.⁶⁴

We conclude that there is a great interest in the topic, especially by the plastic surgery area, which has to deal with ischemia and reperfusion of cutaneous flaps in microsurgery. Although a number of drugs have already presented satisfactory results in the experiments, in the clinical practice they were not carefully assessed and some may have important side effects. We have no doubt that these studies must be continued, especially those which aim at decreasing the damaging effects of tissue loss.

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