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Ischemia-reperfusion injuries in skeletal muscles: pathophysiology and new therapeutic trends focused on controlled reperfusion
(Portuguese PDF version)

José Bitu-Moreno¹, Ieda Francischetti², Ludvig Hafner³

1. Assistant Professor, and Head Professor of Vascular Surgery and Angiology, School of Medicine of Marília, State of São Paulo (FAMEMA).
2. Assistant Professor, School of Medicine of Marília, State of São Paulo (FAMEMA).
3. Assistant Professor, Angiology and Vascular Surgery, School of Medicine of Marília, State of São Paulo (FAMEMA).

Correspondence:
José Bitu-Moreno
Pça. Athos Fragata, 25/502
CEP 17501-220 - Marília - SP
E-mail: jbmoreno@famema.br

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ABSTRACT

Between 1992 and 1995, 645 individuals who were submitted to necropsy had their abdominal aortas dissected. Variations of arterial diameter were analyzed in this segment. In order to avoid underestimation of the aortic diameter, a device that stretches the aortic wall by means of controlled intraluminal pressure was designed. With respect to the diameter, aortas were considered as normal, with aneurysm, ectasia, arteriomegaly or hypoplasia.

Key words: ischemia, reperfusion, skeletal muscles
Palavras-chave: isquemia, reperfusão, músculos esqueléticos.

J Vasc Br 2002;1(2):113-8.

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INTRODUCTION

In 1911, Labey (apud Beyersdorf)¹ successfully carried out the first direct embolectomy in a patient with acute arterial embolism (AAE). Paradoxically, with this surgery, complications that were nonexistent until then began to arise. Some of these complications seemed to depend on revascularization, that is, they occurred with sudden reperfusion of ischemic muscles and consisted of tense edema,^2-4 capillary stasis,⁴ hemorrhage,²,³ acute inflammatory exudation⁴ and myoglobinemia.⁵

In addition, this author described a phenomenon that is currently known as no-reflow phenomenon, in which areas of the muscle inexplicably remained without perfusion after having been reperfused. This phenomenon was believed to originate from the thrombotic occlusion of microcirculation. However, in 1948, Harman4 disproved this by conducting experiments with rabbits whose pelvic limbs were submitted to tourniquet application for two to eight hours. Harman showed the existence of stasis and absence of thrombus signs in microcirculation. In order to explain the existence of stasis, several researchers suggested that the edema could be the major component of the reperfusion syndrome.⁶,⁷. Given this concept, measures that could mitigate the formation of edema would result in better limb viability.⁸ As a matter of fact, the no-reflow phenomenon occurred immediately after reperfusion, whereas a hemodynamically important edema would take longer to develop. In 1951, Burton⁹ introduced the concept of "critical high opening pressure". This author suggested that the critical opening pressure became overly high after prolonged ischemia to such an extent that the skeletal muscle arterioles would not open to the blood flow. The explanation to this increase in peripheral vascular resistance could be based on muscle edema, arterial spasm, and on the effect of depletion of energy on the actin-myosin interaction. Arterial spasm had already been advocated by other authors³,¹⁰ as the cause of postischemic muscle necrosis; however, studies on the correlation between vessel diameter and the amount of blood flow, as well as clinical and angiographic demonstrations conducted by Harman,⁴ in 1948, ruled out spasm as a relevant component. Some evidence bolstering the hypothesis of "critical high opening pressure" was not convincing.

Other authors have also shown the presence of postischemic or reperfusion injuries. In 1946, Meneely et al. (apud Harman),⁴ as a result of transient occlusions and reperfusion of the coronary artery in experimental models, showed progressive myocardial injuries by means of electrocardiographic studies after blood flow was reestablished. Harman & Gwinn,¹¹ in 1949, described a great increase in the severity of muscle fiber injury on ischemic rabbit's foots after tourniquet removal. Dahlback & Rais¹² observed histological evidence of tissue injury after an ischemia. The intensity of the injury progressed the longer the reperfusion phase was.

Nonetheless, Haimovici,¹³ in 1960, systematized and characterized these complications as a syndrome, which he called "myonephrotic metabolic syndrome," also known today as "reperfusion syndrome," "tourniquet syndrome" and "postischemic syndrome." In 1962, Cormier & Legrain¹⁴ also described this syndrome and, because of that, it is also known as "Legrain-Cormier-Haimovici syndrome."

In 1963, indirect embolectomy with Fogarty catheter15 became the treatment of choice for AAE, and was more widely employed due to its ease of use and surgical success. On the other hand, this led to a higher frequency of reperfusion injuries and the mortality rates remained very high.^16-21

The muscular, morphological and functional disorders that result from ischemic episodes longer than three hours include weakened ability to develop tension; mitochondrial edema; ruptured organization of sarcomeres; and escape of cytosolic enzymes to the bloodstream (Figures 1, 2 and 3). These disorders deteriorate with reperfusion.²²,²³ In microcirculation, we observe margination; leukocyte adhesion and infiltration; edema and endothelial cell denudation; increase in microvascular permeability; as well as the no-reflow phenomenon.⁷,²¹,^24-26

Figure 1 - Electronic microscopy of normal muscle. Well-preserved muscle cells with normal sarcomeres (S), glycogen (G), tubular system (T) and mitochondria (M) (X31,900).

Figure 2 - Electronic microscopy of the soleus muscle after four hours of complete ischemia and two hours of reperfusion. Quite edematous mitochondria (M), with loss of dense matrix and dilation of the reticular system (R) (X21,000).

Figure 3 - Electronic microscopy of the soleus muscle after four hours of complete ischemia and two hours of reperfusion. Disorganized myofibrils, mitochondria (*) with irreversible injuries, edematous myofibrils, separation of myofibrils, dilated reticular system (R) (X6,500).

ETIOLOGY OF INJURIES

When reperfusion has been regarded as lesional, what are the factors that cause such injuries?

As suggested evidence, we have the reintroduction of molecular oxygen into ischemic muscles as the major harmful reperfusion agent. Roberts et al.²⁷ carried out experiments with some canine skeletal muscles and described the following findings: in total ischemia, the values of ATP and of transmembrane potential difference normalized after reperfusion; in partial ischemia, in spite of adequate intracellular ATP levels, the transmembrane potential difference was not normal, showing a direct association with reperfusion time. Perry & Fantini²⁸ used analogous experimental models and administered superoxide dismutase (SOD) immediately before reperfusion and observed that this enzyme prevented the progressive deterioration of the transmembrane potential difference.

In 1987, Walker et al.²⁹ demonstrated that the reperfusion of the gracilis muscle of dogs with artificial plasma, to which erythrocyte and oxygen were gradually added, resulted in significant reduction of postischemic necrosis associated with normoxic reperfusion.

Ratych et al.³⁰ submitted cultures of pulmonary arterial endothelial cells of rats to anoxia, followed by reoxygenation. Without anoxia, in the control group, the cells remained viable throughout the experiment, with minimal evidence of injury. Anoxia followed by reoxygenation produced injury in 71% ± 6% of the cells. The authors also added SOD and catalase to the cell cultures before anoxia or before reperfusion. The results showed that they were efficient in attenuating the injury caused by anoxia-reoxygenation. The fact that they were equally effective, both before anoxia and before reoxygenation, confirmed that the injuries primarily occurred due to reperfusion and that they were mediated by some process dependent on the formation of oxygen free radicals (OFR).

Korthuis et al.³¹ carried out studies with experimental reperfusion using oxygenated and nonoxygenated blood. Reperfusion with oxygenated blood caused more extensive and evident injuries to the microcirculation of the skeletal muscles than those provoked by reperfusion with nonoxygenated blood. In addition, when the nonoxygenated blood was replaced with oxygenated blood, microcirculation injuries became evident.

Therefore, the evidence points to the molecular oxygen in the reperfusion fluid as a potentially harmful agent. However, what is the injury mechanism like?

INJURY MECHANISM

Harris et al.²³ used a model of canine gracilis muscle submitted to ischemia followed by reperfusion and observed that the products of lipidic peroxidation were present in great amounts at reperfusion in the animals in which the muscles showed irreversible injuries (seven hours of ischemia); these products were not found and a reversible muscle ischemic injury occurred (two hours of ischemia).

Lindsay et al.³² found low levels of lipidic peroxidation products in canine skeletal muscles after ischemia. A small but significant increase was observed at the end of ischemia and a great and substantial increase was noted at reperfusion. Given that free radicals stimulate the formation of these products, when acting on lipids, and that cell membranes have lipids as fundamental components in their structure, the authors suggested that OFRs act primarily on membrane lipids, causing extensive cellular injuries. There has been some strong evidence of the participation of free radicals.

Korthuis et al.²⁴ submitted a model of canine gracilis muscle to four hours of ischemia followed by two hours of reperfusion, by using alopurinol, catalase, SOD, dimethyl sulfoxide (DMSO), diphenhydramine or cimetidine, separately, as pretreatment, with the aim of minimizing ischemia-reperfusion injuries. Alopurinol, SOD, DMSO and, at a lower proportion, catalase, proved effective in preventing the increase of microvascular permeability. The use of DMSO also prevented the increase of peripheral vascular resistance and the subsequent reduction of blood flow in the reperfused muscle.

Since alopurinol inhibits the xanthine oxidase enzyme and minimizes ischemia-reperfusion injuries to the skeletal muscles, as in the previous experiment,²⁴ the evidence points to the crucial role of xanthine oxidase as a source of OFR in this process, as its role in the pathogenesis of ischemia-reperfusion injuries is well-known, especially in intestines.³³ Smith et al.²⁶ conducted an experiment with skeletal muscles of rats submitted to two hours of ischemia and 30 minutes of reperfusion. In this study, both the inhibition of xanthine oxidase by oxipurinol and its depletion by a diet poor in molybdenum and supplemented with tungsten were able to minimize the increase of microvascular permeability in the ischemia-reperfusion process.

Still characterizing and trying to confirm the increase of microvascular permeability as a result of the action of harmful free radicals, more specifically the hydroxyl radical, Smith et al.²⁵ provoked a two-hour ischemia, followed by 30 minutes of reperfusion, in skeletal muscles of rats. By using deferoxamine and apotransferrin as pretreatment, the authors attempted to prevent the formation of the hydroxyl radical produced by Haber-Weiss (or Fenton) reaction, which uses iron for this purpose. They observed, therefore, a remarkable attenuation in the increase of microvascular permeability, and the hydroxyl radical was again confirmed as being quite harmful.

Blebea et al.³⁴ carried out an experiment with a canine model of gracilis muscle submitted to six hours of ischemia with the aim of removing the hydroxyl radical formed at reperfusion rather than preventing its formation. For this purpose, they used mannitol as a remover and found a significant muscle protection against postischemic injuries.

Walker et al.,²⁹ aside from the gradual reintroduction of oxygen into the reperfusion fluid, also added a combination of SOD, catalase and mannitol, and obtained a more significant protection against postischemic muscle necrosis.

In addition to the aforementioned experiments, which show the important role of reperfusion and the action of OFRs as the probable key elements that cause the injuries, other mechanisms with potential participation in the pathophysiology of reperfusion injuries were also investigated. Among these mechanisms we have the activation of granulocytes, phospholipase A, complement, and endothelin, etc.

Granulocytes also produce OFR and other toxic substances in postischemic tissues. Physiologically, these reactive oxidants produced during chemotaxis and phagocytosis are primarily antimicrobial and are essential for the intracellular destruction of microorganisms. However, the activity of these oxidants is not limited to the intracellular environment; they go into the extracellular environment during the activation of phagocytosis. The activated granulocytes secrete proteolytic enzymes (peroxidase, elastase, protease, etc.), synthesize prostaglandin, release free radicals and, in clusters, they occlude microcirculation.³⁴,³⁶

The action of free radicals or the effects of ischemia can activate phospholipase A in cell membranes, with consequent formation of leukotrienes, among which we have leukotriene B4, which is bound to specific receptors on the surface of granulocytes, producing a series of responses, with a greater formation of free radicals and proteases.

In reperfused tissues, leukotrienes and C5a activate granulocytes, with exposure of adhesion molecules on the cell surface, especially ß2 CD11 and CD18 integrins, which can bind to ICAM-1 and to E-selectin in the activated endothelium, and promote the transmigration of granulocytes into this tissue.³⁷ Models of extremity splanchnic ischemia-reperfusion in rats and of myocardial infarction induced in rats, in which specific antagonists of leukotriene B4 were used, showed reduced accumulation of granulocytes and an increase in animal survival rate.³⁸,³⁹

The interaction of free radicals with the vascular endothelium can also lead to the formation of other inflammatory process mediators, such as PAF (platelet-activating factor), in addition to complement activation products. In an experimental model of intestinal ischemia-reperfusion, specific antagonists of PAF receptors showed a positive effect on the reduction of endothelial adhesion and on the extravascular transmigration of granulocytes.⁴⁰

These antagonists also protected skeletal muscles of rabbits from reperfusion injuries,⁴¹ and protected the myocardium of rats⁴² and rabbits⁴³ submitted to acute infarction.

Complement activation products, accompanied by a large number of granulocytes, were observed during the reperfusion of ischemic skeletal muscles.⁴⁴ Homozygous animals with C3 and C4 deficiency showed less microvascular permeability in ischemia-reperfusion experiments than normal animals.⁴⁵

Nitric oxide (NO), a substance released by the vascular endothelium, seems to play a crucial role in the maintenance of vascular homeostasis as a vasodilator,⁴⁶ platelet aggregation inhibitor⁴⁷, and neutrophil aggregation and adhesion inhibitor⁴⁰ and also as a direct remover of O2- anions.⁴⁸ NO is synthesized from L-arginine, O2 and NADPH, by nitric oxide synthase. Protective effects of exogenous L-arginine were demonstrated in a series of myocardial ischemia-reperfusion models.⁴⁹ In skeletal muscles of rabbits, NO concentration, quantified by the NO2/NO3 ratio reached 89% of its initial value immediately after ischemia, with a later decrease to 77% after one hour of reperfusion. A significant correlation between low NO concentration, reduced regional blood flow and high mortality was observed at reperfusion.⁵⁰

There is some evidence that the narrow relationship between NO and endothelin has an important effect on ischemia and reperfusion. Endothelin is a powerful vasoconstrictor and stimulates granulocyte adhesion to the vascular endothelium, interfering with ß2-integrins. Among other factors, ischemia and the reduced levels of NO can stimulate the release of endothelin, with consequent vasoconstriction and deterioration of ischemia. Some reports point out that there is an increase in the number of endothelin receptors by means of a calcium-dependent mechanism in ischemia-reperfusion and that endothelin levels increase in patients with acute myocardial infarction, and are higher when there is low production of NO.⁵¹

NO can react with the O2- radical and originate a strong nitrogen dioxide oxidant and an OH radical by means of a peroxinitrite anion (ONOO(-)). These elements can trigger lipidic peroxidation.⁵²

Even with the exposure of injury mechanisms, after one century of investigation and experimental studies, the morbidity and mortality rates of AAE are still high.

PREVENTIVE SUBSTANCES

As a result of this investigation, several substances have already been used, clinically as well, in an attempt to minimize injuries. Among the substances that have already been used are the so-called water-soluble endogenous antioxidants (SOD, catalase, glutathione peroxidase), fat-soluble antioxidants (tocopherols, carotenoids, quinones), alopurinol (xanthine oxidase), mannitol, calcium channel blockers, specific antagonists of leukotrienes and PAF receptors, iron chelators, leukocyte filters and different forms of leukocyte depletion, polynitroxyl-albumin (PNA), pyruvate, L-arginine, combination of drugs, controlled reperfusion, ischemic preconditioning, etc.²⁴,²⁵,²⁸,²⁹,³⁴,^38-45,^49-50,^53-75

Alpha-tocopherol (AT) acts in vivo as an antioxidant and free radical blocker. In several studies, AT has been described as a protection against acute pathological disorders caused by reperfusion injuries in different organs. The addition of a water-soluble alpha-tocopherol analog to the UW (University of Wisconsin) solution reduced reperfusion injuries and improved the endothelial viability of the lungs.60

Recent studies show that polynitroxyl-albumin (PNA) reduced the infarction area in focal cerebral ischemia, inhibited ischemia-reperfusion injuries induced by leukocyte adhesion to endothelial cells,⁶¹,⁶² reduced lipidic peroxidation and mitigated the injuries caused by free radicals in transplanted pig's heart.⁶² PNA is able to regenerate inactive nitroxides. The nitroxides had properties similar to those of superoxide dismutase, oxidized the reduced metal ions and reacted with the central carbon of free radicals.63-66 However, nitroxides have an antioxidant capacity that is limited by their quick intracellular inactivation.

There is strong evidence that pyruvate acts as an antioxidant and improves myocardial function during reperfusion.⁶⁷,⁶⁸ Other studies have shown that the pretreatment with pyruvate reduces ischemia-reperfusion injuries in skeletal muscles,⁷⁰ which could potentially prevent postischemic injuries to these tissues.⁷¹

Adenosine given at the beginning of reperfusion reduced microvascular dysfunction in skeletal muscles by means of a mechanism that seems to originate from the ability of adenosine to inhibit leukocyte adhesion and migration.⁶⁹

Other experimental models have shown that there was a remarkable development of new blood vessels in ischemic muscles when they received pedicle grafts, to which angiogenic factors were added.⁷² One should not forget that this is a developing region and that the adequate development of these new vessels occurred within one week, a time period that is too long for limbs with severe ischemia.

Beyersdorf et al. have experimentally reported, in isolated models of rat limbs1 and in in vivo pig models⁵⁷ that, by modifying and carefully controlling the composition of the reperfusion solution and reperfusion conditions, they could have a significant improvement of the metabolism, structure and function of limbs after an important ischemia.

Another study⁷³ has described a controlled reperfusion technique in which the venous return was drained. The authors cannulated the femoral artery and vein; The initial venous return was disregarded. Systemic complications were less frequent than complications caused by Beyersdorf arterio-arterial controlled reperfusion.

Experimental results also showed the beneficial effects of ischemic preconditioning: short periods of ischemia, prior to a longer ischemia, make tissues more resistant, and prevent endothelial dysfunction and activation of inflammatory cells, associated with the ischemia-reperfusion process.⁷⁴,⁷⁵ This technique, however, does not seem suitable for our patients and lies outside the scope of this review, since our patients usually present with arterial occlusion and prolonged ischemia.

The truth is that neither an efficient treatment nor a large-scale multicenter prospective study on these drugs have been available so far. This has led to the combined use of antioxidants and to the tendency towards a multifactorial approach, in which ischemia-reperfusion is regarded as a complex phenomenon, a great inflammatory response. The use of controlled reperfusion proposed by Beyersdorf et al. can be an alternative in such situations.¹,^57-59

CONTROLLED REPERFUSION

The aim of controlled reperfusion is to prevent amputation and allow the limb to function normally again, in addition to reducing the systemic effects of the process. It is difficult to establish a dividing line between ischemia and reperfusion. There is a relationship of dependence and causality between them, since the intensity of the injuries attributed to reperfusion seems to be proportionally related to the length of ischemic episodes. At the same time, therapeutic interventions applied only during reperfusion reduce the intensity of tissue injury, thus implying better recovery of the extremity.⁵⁸,⁵⁹ In this sense, the success of some therapeutic schemes indicates that even a later treatment, at the end of ischemia and at reperfusion, could still determine the success of revascularization.

The composition of the reperfusion solution was modified in order to satisfy the following principles: limit of calcium inflow; control of hyperosmolarity; reduction in the production of free radicals; increase in glucose concentration; reestablishment of amino acids that are the precursors of citric acid cycle intermediate products (i.e. glutamate and aspartate); and reversion of tissue acidosis with a buffer solution, TRIS (tris(hydroxymethyl)methylamine). The controlled reperfusion conditions included reperfusion pressures and smaller flow pressures (< 50 mmHg), normal body temperature (37°C), and longer duration of reperfusion (30 min) so as to allow for cell recovery before the normal flow was reestablished. The composition of the solution to be mixed with the patient's blood was chosen with the aim of maximum protection of the skeletal muscle, thus preventing undesirable side effects.

The controlled reperfusion of extremities is therefore based on the hypothesis that if we control the composition and perfusion conditions of the solution that first comes in contact with the endothelium and with the postischemic tissues, there is a reduction in reperfusion injuries caused by free radicals, pro-inflammatory substances, toxic metabolites released in metabolic acidosis, etc. In this sense, when we consider the ischemia-reperfusion process as a large inflammatory response, there are a lot of possibilities in terms of improving these solutions. In addition to the experimental studies mentioned above, clinical assays have also reported the therapeutic success of this approach.⁵⁹,⁷³,^76-80

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