Ischemia and reperfusion in skeletal muscle injury mechanisms and treatment perspectives*
(Portuguese PDF version)

Marcos da Silveira,1 Winston Bonetti Yoshida2

1. M.Sc. Department of Surgery, School of Medicine of Botucatu, Universidade Estadual de São Paulo (UNESP), Botucatu, São Paulo, Brazil.
2. Ph.D. Adjunct and associate professor. Department of Surgery and Orthopedics, School of Medicine of Botucatu, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil.

* This research was financially supported by FAPESP, SP.

Correspondence:
Marcos da Silveira
Rua Arthur Verri, 411, Nova Jaboticabal
CEP 14887-018 - Jaboticabal, SP, Brazil
Phone: +55 (16) 3202.0683
E-mail: mda@unimedjaboticabal.com.br

ABSTRACT

This disease causes serious problems including many limb amputations and death. Ischemia leads to muscle cell energy failure, inflammatory reaction, and biochemical alterations. These are worsened by reperfusion, which triggers high free radical production and neutrophil activation, making local and systemic lesions more severe. There have been many studies trying to better understand alterations caused by ischemia and reperfusion, and to search for more efficient therapeutic alternatives. The objective of this study is to review current knowledge on the physiopathology and the different therapies used to reduce injuries caused by skeletal muscle ischemia and reperfusion in acute limb arterial occlusion as well as to review the experimental models used to study these alterations.

Key-words: ischemia, reperfusion, free radicals, skeletal muscle.

J Vasc Br 2004;3(4):367-78

The acute arterial occlusion (AAO) is the most common of all vascular diseases, accounting for 10 to 16% of cases. It is estimated that the incidence of non-traumatic AAO is around 14 cases per 100,000 inhabitants, and this number tends to get higher due to an increase in the population's life expectation and the increasing incidence of the atherosclerotic disease.1-3

The AAO is a severe condition that affects especially the elder population, which already suffers with other problems that increase morbidity, like diabetes, hypertension, and coronary or pulmonary diseases. In this population, the risks of limb amputation are around 10 to 30%, and of death is 15%, approximately.2

The AAO is a significant cause of prolonged length of hospital stay, increasing expenditures of the already scarce resources of the public healthcare system. The social cost is also high, because the amputee becomes extremely dependent on the family and social assistance.

The pathophysiological phenomena involved in AAO are closely associated with ischemia and reperfusion of the skeletal muscle. In the present study, we intend to make an extensive review on the literature about the topic, as well as to investigate the development of new therapeutic options for this disease.

PATHOPHYSIOLOGY

Ischemia

The occlusion of an artery immediately triggers an intense distal vasospasm, followed by proximal and distal secondary thrombosis. Due to stasis and loss of endothelial function, the thrombus occludes the collateral circulation, worsening the ischemia event. Deep venous thrombosis may occur in parallel, as a result of the slow venous flow and hypoxia, affecting the tissues patency.1-3

The intensity of the ischemia event will depend on the level of the arterial occlusion, of the collateral circulation and of the amount of oxygen required by the tissues involved.1-3 This is the reason why the ischemia time is crucial to determine the injuries intensity and the limb patency.1-3 The muscular tissue responds for almost 80% of the inferior limb mass and it has medium resistance to ischemia; nerves are less resistant; tendons, bones and skin are the most resistant tissues.1-3 Muscles are more resistant to ischemia than nerves because of their low metabolic activity at rest, the large storage of glycogen and high-energy phosphates, and because of their capacity to maintain basic cellular functions by means of the anaerobic glycolysis.

At the onset of ischemia, the biochemical alterations in the basic cellular functions can be reversed, however, when the ischemia time is longer, another sequence of reactions is triggered as a consequence of cellular energetic failure.4,5 The anaerobic metabolism prevails up to two hours of ischemia, increasing the amount of lactate and inorganic phosphate, and reduction of pH, adenosine triphosphate (ATP) and creatine (40% of the normal amount).After 3 hours of ischemia, the ATP levels fall drastically and acidosis gets worse. Some authors believe that when reperfusion is made within three hours of ischemia, the ATP levels can be restored with no tissue injuries.6-8 The time needed to deplete all ATP reserves is not precise, but it has been already demonstrated that ischemia time over 5 hours causes irreversible ischemia.9-11

The ATP synthesized by the anaerobic metabolism maintains the ion pumps, the membrane potential and the contractile function, although the production of lactic acid infuse into the interstitial space, causing edema and acidemia.12-14 When this energetic source is over, the pumps fail and the ionic gradient of the cells is altered, with potassium and magnesium release and sodium and calcium input into the intracellular medium, causing edema in the cell, matrix or mitochondrial crests.6,13,15 The increased offer of intracellular calcium activates the cytoplasmatic proteases (calpain). In combination with the high concentration of hypoxanthine resultant from the uninterrupted degradation of ATP, the proteases convert the enzyme xanthine dehydrogenase into the oxidase enzyme. This enzyme has an important role in reperfusion injuries. Calcium also activates the lysosomal enzymes that impair organelles and the phospholipase enzyme A2, which degradates the arachidonic acid, originating inflammation mediators like: leukotriens, prostaglandins, prostacyclins and thromboxanes.6,8,13,14 (Figure 1a). The leukotriene B4, produced by lipoxygenase enzymes, binds to specific receptors present in the neutrophils surface. It is then adhered and activated by means of the CD11/CD18 complex (clusters of differentiation). On their turn, the leukotriens C4 and D4 have been implicated as possible vasoconstrictors that impair perfusion.6,12-14 The cyclooxygenase pathway produces prostacyclin (PGI-2) in the endothelium; it is a vasodilator and also inhibits the platelet aggregation. Through this pathway, a great amount of thromboxane A2 is produced in the platelets. Thromboxane A2 is a potent platelet aggregator and vasoconstrictor that impairs the microcirculation.6,12-14 Of all the antagonistic affects of some endothelial and tissue mediators, in ischemia vasoconstriction, endothelial injury, platelet aggregation, chemotaxis and neutrophil activation (Figure 1b) are prevalent.6,12-14

click hereFigure 1a - Biochemical events of ischemia and reperfusion, neutrophils activation and mechanisms of injury .

click hereFigure 1b - Biochemical event, interaction between the activated neutrophils and the endothelium and mechanisms of ischemia and reperfusion injuries.

Reperfusion

Paradoxically, although reperfusion is critical for reverting ischemia, it worsens injuries that occurred during the ischemia period. The production of exceeding free-radicals (FR), especially by the xanthine oxidase system, and the intense participation of neutrophils increases the inflammatory reaction thereby promoting muscular edema formation, tissue necrosis, impairment of systemic clinical conditions and, sometimes, leads to limb loss and even death (Figure 2).13,16,17

click hereFigure 2 - Summarized schema of how free radicals act to produce tissue injuries.

In the respiratory chain, the nicotinamide adenine dinucleotide phosphate (NADPH) is the final acceptor of electrons, producing water. This reaction is catalyzed by the xanthine dehydrogenase enzyme. After ischemia, tissues were shown to contain an accumulation of xanthine oxidase. This enzyme uses the molecular oxygen, which is available as the final electron acceptor. This reaction produces singlet oxygen molecules, which are extremely unstable. Using these molecules, secondary chemical reactions produce superoxide, hydrogen peroxide and hydroxyl. These FR start the lipid peroxidation (Figure 3), which ends in the disintegration of membranes and consequent rupture and death of the cell. Other forms of FR production are the catecholamine autoxidation and the neutrophil NADPH-oxidase enzyme (Figure 1a).6,12-14,16-20

click hereFigure 3 - Schematic representation of lipid peroxidation of fatty acids in the cellular membrane. The free radical OH- takes an oxygen molecule from the fatty acid and produces the lipid radical. As it is unstable, it undergoes a molecular rearrangement and produces a conjugated diene, which can react with an oxygen molecule and produce a peroxyl radical. This radical takes a hydrogen molecule from the fatty acid originating a chain reaction ( )

The FR and the products of the inflammatory reaction attract neutrophils, which adhere to the endothelium. Adhesion occurs through an interaction between proteins like the selectins ELAM-1 and GMP-140) and immunoglobulin (ICAM-1 and VCAM-1), present in the endothelium, and proteins present in the neutrophil surface, known as leukocyte integrin (CD11 e CD18). Endothelium may become activated by cytokines, specially the interleukin, and the tumor necrosis factor (TNF). The leukocyte integrins are modulated by leukotriens LTB4, by the complement system C5a and the platelet activation factor (PAF) (Figure 1b).12,13,19

When neutrophils adhere to the endothelium, they are activated and release different proteolytic enzymes, such as myeloperoxidases, proteases, collagenases and elastases, which damage tissues and modify the function of the endothelium. Of these enzymes, we call the attention to the myeloperoxidase, which can catalyze the reaction between the hydrogen peroxide and the chloride ion, producing hypochlorite, a potent oxidation agent (Figures 1a and 1b).12,13,16,21-24

Cytokines of systemic action, like the TNF and interleukin-1, and metabolites of the arachidonic acid are released by activated neutrophils, increasing the inflammatory response by recruiting new cells. This way, mast cells in the interstitial space are activated and release granules containing histamine, proteases, proteoglycans, prostacyclins, leukotriens and kinins. These substances change the capillary patency, worsening the muscular and interstitial edemas. In the lungs, the mast cells are responsible for the formation of edema and promotion of hematogenesis, configuring as a contributor factor of respiratory insufficiency in severe cases of post-ischemia and reperfusion syndrome (IR).25,26 The mast cells are activated by the systemic circulation of inflammatory mediators. They synthesize and release nitric oxide (NO), a free radical gas with vasodilating and anti-platelet aggregation actions; it is also toxic for bacteria. Paradoxically, the NO can damage tissues, as in contact with the superoxide it generates a reaction that produces peroxynitrite and nitrogen dioxide. It can start the lipid peroxidation and potentialize inflammatory injuries in endothelial cells.25-27

As the inflammatory response propagates, releasing large amounts of chemotactic mediators, it causes an intense migration of leukocytes to the blood vessels of ischemic tissues and consequent endothelial adhesion and activation. This can obstruct the microcirculation vessels and make ischemia even worse.

Summing up, injuries resultant from ischemia followed by reperfusion occur due to an energetic failure of the cell, inflammatory reactions, and production of FR. Tissue mediators, neutrophils and mast cells contribute to potentialize injuries (Figure 2).

TREATMENT - EXPERIMENTAL MODELS

Some experimental models of IR in skeletal muscle are described in the literature: dog gracilis muscle,28 rat cremaster muscle,29 dog hind limb,30 rat hind limb,31,32 and experimental tourniquet ischemia.33 These models assessed muscles with different metabolic characteristics (slow and rapid contraction) submitted to IR. The outcomes were conflicting in the evaluation of ischemia resistance. It is worth mentioning that injuries were always worsened by reperfusion, regardless of the metabolic characteristics of the muscle.8,18,34-41 Thus, several aspects should be taken into account in the study of IR in the skeletal muscle, such as: animal studied, type of muscle, evaluation method, and ischemia and reperfusion time.18,41

Different drugs and methods used to attenuate the mechanisms of IR lesions were studied. In a way, they allowed a better understanding of the IR pathophisiology and of possible treatments.

Substances capable of neutralizing FR were also studied. Moreno,12 in a model of rat limb with 4-hours ischemia and 2-hours reperfusion, proved that alphatocopherol prevented the edema formation but did not avoid histological injuries in muscles. However, in another study, tocopherol used before ischemia increased the concentration of high-energy phosphates and the resistance to ischemia, also attenuating tissue injuries.42 In the same line of neutralizers, the polivitaminic solution (tocopherol, ascorbic acid, retinol and B complex) was tested in rabbit hind limbs undergoing IR. It presented positive results only when used before ischemia, reducing edema and malondialdehyde (MDA) and improving microcirculation. These effects were not observed when the solution was administered before reperfusion.43

The superoxide dismutase enzyme has been the endogenous scavenger which has been submitted to the majority of experimental studies. It acts over the superoxide radicals, converting them into water and oxygen. Lu et al. performed an experimental study with transgenic rats that produced superoxide dismutase. The hind limb was submitted to IR and injuries were attenuated. This was probably due to scavenging of the superoxide radical, which inhibits the adhesion molecules CD11b/CD18, and ICAM-1.44 Bowler & MacLaughlin tested the use of recombinant superoxide dismutase in a model of IR in the cremaster muscle of rats. They tested the muscle function with electric stimulation and observed it was maintained after the first 48 hours. Nevertheless, after 7 days of reperfusion, results got similar to the control group.45

Other drugs, routinely used in the clinical treatment of several diseases, were also tested.21,42,43,46-56 We highlight the use of pentoxifylline, a xanthine that inhibits phosphodiesterase. It also increases the availability of c-AMP (cyclic adenosine monophosphate), thereby reducing the release of cytokines, increasing the release of prostacyclins and decreasing the formation of FR, attenuating injuries resultant from IR. In a model of IR of the skeletal muscle of rats and dogs, c-AMP improved the transmembrane potential and the microcirculation, and reduced edema and leukocytes adhesion.46,47 The necrosis area and the production of PAF also decreased.48 Carvedilol is a nonselective beta-adrenergic blocking agent with antioxidant properties used in the management of heart failure. The use of carvedilol in rat hind limbs undergoing IR reduced the necrosis areas after 3 hours and 15 minutes of ischemia and 72 hours of reperfusion.49 Verapamil is routinely used to treat arrhythmias. It was compared to deferoxamine, an iron chelating, and was shown to improve the muscular function (strength and contraction time) in a model of IR in rat limbs.50 It was not shown to be better than deferoxamine.51,52 Another branch of experiments in the treatment of muscular IR analyzed the essential amino acids. Glycine is a neutral amino acid with cell-protective characteristics in the kidney, small intestine and liver. Ascher et al. used an isolated dog gracilis muscle undergoing IR. They demonstrated that the group managed with glycine presented edema reduction (weight before and after 48-hour of reperfusion). The necrosis areas were also attenuated; contraction and muscle strength were maintained and measured with tension transducers connected to tendons and by analyzing the response to electric stimuli.53 Positive results are claimed to be due to an increased offer of ATP resultant from higher storage of energy in the form of creatine-phosphate, preserving the mitochondrial integrity and maintaining the mechanism of cellular defense more effective.

Brigitte & Brisson54 studied the effects of adenosine pretreatment in IR canine gracilis muscle flaps, assessing the FR production through electron paramagnetic resonance.

Adenosine is an inhibitor of the transformation of xanthine dehydrogenase into xanthine oxidase, consequently reducing the production of FR. After 4 hours of ischemia and 15 minutes of reperfusion, the authors verified that pretreatment with adenosine could slightly reduce the production of FR, but it did not attenuate histological changes.

Inosine was also tested. It is a metabolite resultant of adenosine break by the adenosine-deaminase enzyme which has more active and stable and has longer half-life.55 Inosine accumulates in skeletal muscles after endovenous injection.

In normal conditions, it is almost undetectable and increases with inflammation. It has antiflammatory action and inhibits the cytokines production, acting in the adenosine-receptors. In the hind limb of rats submitted to 2 hours of ischemia and 3 hours of reperfusion, inosine reduced some cytokines (TNF-alpha; MIP-2), the myeloperoxidase and the intensity of tissue edema, as compared to the control group.55

There is a number of reports that highlight the importance of NO in the endothelial function, protection of microcirculation and angiogenesis, thereby an increase in the production of NO would reduce tissue lesions after IR periods. Quercetin, a bioflavonoid that scavenges superoxide and increases NO concentration, was used in an IR model of rabbit skeletal muscle. It was shown to improve microcirculation, reduced the FR production (superoxide dosage) and maintained the levels of NO elevated , as compared to the control.56

Transgenic rats with a high expression of the endothelial NO-synthetase gene were submitted to limb IR. Due to the superproduction of NO, there was not an increase of the superoxide radical. Moreover, we reported a reduction in the expression of leukocyte adhesion proteins (ICAM1 e VCAM1), besides it improved cohesion among the endothelial cells, decrease of tissue invasion by neutrophils and liquid enters interstitial space, as a consequence of reduction of vascular patency. Tissues viability was considerably better in transgenic rats than in the control group.57 Viability was identified with tetrazolium salts, which color viable and necrosis tissues differently. They can then be assessed quantitatively through microscopy or morphometry, or through the colorimetric method in a spectrophotometer. This technique has been extensively used in the protocols of IR studies.

The use of prostaglandin E1 decreased the superoxide and peroxinitrite concentrations in a study performed with rabbits submitted to 2 hours and 30 minutes of ischemia followed by 2 hours of reperfusion, as compared to the control groups. This potent vasodilator also maintained the concentrations of NO close to the pre-ischemic values. Prostaglandin E1 acts in the endothelium and preserves the function of the NO-synthetase enzyme. It maintains the adequate levels of NO to protect the cell with no additional damage.58 L-Arginine was tested in a rat model undergoing 4 hours of ischemia and 24 hours of reperfusion, as compared to a well known inhibitor of the NO-synthetase enzyme, L-NAME. In tests of tissues viability, the results confirmed that NO protects the cell.59

Another approach toward the reduction of IR injuries is about ischemic preconditioning.

Some studies evidenced that when the muscular tissue is submitted to repeated cycles of IR, it presents less intense injuries as compared to the control group.60,61 Saita et al.60, in a study to assess the muscle viability with the tetrazolium salts technique, reported that three to five cycles of 10-minutes IR protected the cell more than one or two cycles before a long period of 4 hours of ischemia and 24 hours of reperfusion of the hind limb of Wistar rats. Ischemia preconditioning increases the availability of adenosine, because as it is a product of ATP degradation and a mediator of the A1 receptor activation, it stimulates the opening of ATP-sensible potassium channels, favoring the input of potassium in the cell and hyperpolarizing the membrane. Other probable benefits would be: increase in the collateral blood flow, reduction of neutrophils free radicals, increase in the endothelial production of NO62 and prostacyclin, intensification of production and release of stress proteins, which increase the resistance to infarct, and probably involving the protein Kinase C (PKC).60 Downey & Cohen63 proposed another schema to explain the protection mechanism triggered by the ischemia preconditioning. They say that the receptors of the cells surface would be activated and would add a protein G to the phospholipase C, producing diacylglycerol. Diacylglycerol would activate PKC, which would produce a local and systemic anti-inflammatory effect that would also protect the lung.

Neutrophils are highly important in the mechanisms of IR injuries. Neutralizing antibodies of the adhesion protein CD18 were tested in short periods of muscle ischemia and presented positive results. However, in longer periods of terminal aorta grasping (over 45 minutes) in rats, the neutrophils activation happened with no mediation of the cellular adhesion complex CD18.64,65

One of the mechanisms that cause cellular death resultant from IR is the activation of the caspase cascade (cysteine-derived proteases), determinig cellular injuries that are not reversible with the DNA break. In this context, an inhibitor of protease z-VAD (Z-Val-Ala-DL-Asp-fluoromethylketone) was tested in a model of IR in rats. The IR event was produced as a result of infrarenal aortic cross-clamping. In the comparison of groups treated with CD-18 blockade and z-VAD, the inhibitor of proteases was more cell protective, regardless of the ischemia time, which varied from 15 to 120 minutes. This measurement was taken by analyzing the intensity of DNA injury and the dosages of creatinine phosphokinase. The study reported should open new research possibilities regarding the use of this proteases.64

Another alternative for IR injuries treatment is the immunoglobulin G-anti-Pecam 1. It decreases the transendothelial migration of the neutrophil, reducing injuries and the production of neutrophilic peroxynitrite. This drug was tested in an experimental model of IR of hind limb of rabbits submitted to 3 hours of ischemia by iliac artery grasping and 2 hours of reperfusion. The results of this methodology were based on the amount of nitrotyrosine in the tissues. This substance is a precursor of peroxynitrate, so, indirectly, it is possible to quantify the reduction of peroxynitrate and its injuries, once this anion is significantly produced with neutrophil superoxide and nitric oxide. When the muscular tissue is treated with monoclonal antityrosine antibodies and analyzed through computerized morphometry, it allows the comparison between treated groups and the control. The clinical use of this drug will depend, however, on further studies.66

The gradual and controlled reperfusion is a cost-effective and easily performed technique developed with the aim of slowly taking oxygen to ischemic tissues, decreasing reperfusion injuries. The authors compared groups of rapid reperfusion and gradual reperfusion; at each 30 seconds 25% of the initial arterial blood flow was released. MDA, myeloperoxidadse and histology were evaluated. The study concluded that this technique attenuated the effects of IR by reducing leukocytes activities and the production of superoxide radicals.67

Starting from a similar principle, controlled reperfusion has been used to prevent limbs amputation and reduce systemic alterations. It consists of starting reperfusion with reduced blood flow and using reperfusion solutions that comprise the multifactorial IR aggression. The composition of these solutions aim at limiting the calcium inflow, controlling hyperosmolarity, decreasing the FR production, increasing the concentration of glucose, restoring the intermediate amino acids in the citric acid cycle (aspartic and glutamic acids) and reversing the tissue acidose with a buffer solution [tris(hydroxymethyl)methylamine].71 esides the drugs presented above, others were tested in the reperfusion solution, as prostaglandin E1 (vasodilator) or the oxypurinol (inhibitor of xanthine oxidase) in an experimental work with rats. It presented hystologic alterations and decreased tissue lipoperoxidation, which was assessed by analyzing the dosage of MDA. An endothelium receptor antagonist, Bosentan, presented positive results in the controlled reperfusion, it reduced the tissues necrosis by increasing the number of patent capillaries, that is, reducing the spasm the endothelium generates.73 Before restoring the normal blood flow in the operated extremity, some other factors should be controlled, besides the type of solution used. They are: the pressure of reperfusion (smaller than 50 mmHg), and temperature, which should be kept at 37° C and time (30 minutes).

Other articles report the experimental use of a number of other substances that are aimed to attenuate IR injuries, like reperfusion with low leukocyte accumulation blood;74 acidic fibroblast growth factor (aFGF) as calcium inflow regulator;75 cyclosporin A as an immunodepressive;21 allopurinol as inhibitor of xanthine oxidase;76,77 inhibitor of TNF-alpha;78 inhibitor of thromboxane synthetase, which reduces the leukocytes activities;79 S-nitrose human serum albumin and S-nitroso-N-acetylcysteine, which increase the availability of NO and have an antioxidant action.80,82 Hyperbaric oxygen treatment has also been tested and presents positive outcomes, as it reduces injuries and improves microcirculation.83,84

TREATMENT - CLINICAL TRIALS

Although there is a wide number of experimental studies showing the enormous potential of different drugs and/or techniques in reperfusion injuries attenuation following ischemia, there are not many clinical trials.

Mannitol seems to be the most tested drug, as it has a benefic action in the microcirculation and neutralizes the hydroxyl radical. However, it was not shown to reduce tissue injuries or neutrophil adhesion and infiltration, despite the tissue patency occurring only after 60 minutes reperfusion and so has not been used with this purpose.85

Propofol is an endovenous anesthetic with antioxidant action that was used to attenuate lesions resultant from 60 minutes of ischemia followed by 20 minutes reperfusion in orthopedic surgeries. A 5 mg per kg weight was injected during ischemia.86 This study compared the concentrations of MDA, uric acid and catalase in the muscle tissue; the first two were reduced as compared to the control. Catalase dosages were not altered in the groups with ischemia, evidencing that propofol is effective in the reduction of lipid peroxidation products.86

Ihnken et al. reported a clinical case where they used the technique and solution of controlled reperfusion in 16-year-old patients. They have undergone complications in the surgical procedure, with 6-hour ischemia in the right lower limb. After controlled reperfusion, they had total recovery of the limb function with no systemic alterations.87

Vogt et al.88 also reported a clinical case in which an elder patient, with terminal aorta occlusion for 24 hours was submitted to aortofemoral revascularization concomitant with reperfusion with low-potassium solution, similar to the solution used in cardioplegia. A positive result with total recovery of the limb function was achieved, with no need of fasciotomy or presence of systemic complications.

Bayersdorf et al.68,70 reported a case in which 19 patients with serious and prolonged ischemia in the lower limbs after cardiac surgery were managed with controlled reperfusion. A successful outcome of 84% of cases with normal function restored and without systemic complications or compartmental syndrome was reported. This may be a path to be followed in the search for a better evolution of serious cases.

CONCLUSION

The pathophysiology of IR is multifactorial and interdependent. The ideal treatment should comprise as much as possible injuries mechanisms, thereby they should: improve the tissues resistance to ischemia; be able of reducing the FR production and scavenge them; inhibit the neutrophil and cytokines activation, as well as the activation of inflammation mediators; scavenge toxins produced in the anaerobic metabolism and, last, perform all these actions at the onset of reperfusion, when the patient is administered the treatment.

In the comprehensive and extensive review we made on the topic, it is noteworthy the non-standardization of studies as for the animals being studied, techniques for inducing ischemia, time of IR and methods for results evaluation. This makes difficult to compare results and choose drugs or methods to be used in bigger multicentric studies or in the clinical practice.

We conclude that a standard methodology should be adopted in the study of IR in order to make comparison studies more objective and useful for the development of more effective approaches to this serious and impairing disease.

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