Controlled reperfusion decreased reperfusion induced oxidative stress and evoked inflammatory response in experimental aortic-clamping animal model

Abstract

Revascularization after long term aortic ischaemia in vascular surgery induces reperfusion injury accompanied with oxidative stress and inflammatory responses. The hypothesis of this study was that the aortic occlusion followed by controlled reperfusion (CR) can reduce the ischaemia-reperfusion injury, the systemic and local inflammatory response induced by oxidative stress.

Animal model was used. Control group: animals underwent a 4-hour infrarenal aortic occlusion followed by continuous reperfusion. Treated group: animals were treated with CR: after a 4-hour infrarenal aortic occlusion we made CR for 30 minutes with the crystalloid reperfusion solution (blood: crystalloid solution ratio 1:1) on pressure 60 Hgmm. Blood samples were collected different times. The developing oxidative stress was detected by the plasma levels of malondialdehyde, reduced glutathion, thiol groups and superoxide dismutase. The inflammatory response was measured by phorbol myristate acetate-induced leukocyte reactive oxygen species production and detection of change in myeloperoxidase levels. The animals were anaesthetized one week after terminating ligation and biopsy was taken from quadriceps muscle and large parenchymal organs.

CR significantly reduced the postischaemic oxydative stress and inflammatory responses in early reperfusion period. Pathophysiological results: The rate of affected muscle fibers by degeneration was significantly higher in the untreated animal group. The infiltration of leukocytes in muscle and parenchymal tissues was significantly lower in the treatedgroup.

CR can improve outcome after acute lower-limb ischaemia. The results confirm that CR might be also a potential therapeutic approach in vascular surgery against reperfusion injury in acute limb ischaemia. Supported by OTKA K108596.

Keywords

Controlled reperfusion reperfusion injury aortic ischaemia

1 Introduction

1.1 Acute limb ischemia

Acute limb ischemia (ALI) is a medical emergency, that needs immediate medical attention and its outcome ranges from simple revascularization to limb amputation. It is defined as a decreased tissue perfusion that can endanger the patient’s life and extremities, but lasting less than 14 days [27]. Although there is little information on the incidence of acute limb ischemia in the general population, it is estimated to be 14 out of a 100,000 and to create 10% to 16% of the vascular workload [15].

Despite the past medical evolvement in the treatment of ALI, here especially to mention the Fogarthy catheter and the Thrombolysis, the course of the disease has remained largely unchanged. Patients presenting, often have a particularly severe short-term outlook, both in terms of loss of the leg as well as mortality, including high amputation rates of 10% to 30% and a mortality rate of around 15%.

1.2 Pathomechanism of reperfusion injury

Reperfusion is the restoration of blood flow to the ischemic tissue. Despite the unquestioned benefit of reperfusion to an ischemic tissue, reperfusion itself can elicit a cascade of adverse reactions that paradoxically injures tissue [10]. The inflammatory responses following reperfusion varies greatly. If muscle tissue death is uniform, little inflammatory response will result. In most instances of reperfusion, which follows thrombotic or embolic occlusion, there will be a variable degree of ischemic damage in the zone where collateral blood flow is possible [2]. The extent of this region will determine the magnitude of the inflammatory response, whether it is local or systemic.

The pathophysiology of the ischemic reperfusion injury (IRI) is complex [35]. With start of reperfusion, washout of accumulated lactate and protons from the ischemic tissue causes an increase in intracellular calcium followed by an elevated Nitrous oxide system (NOS) activity. Sudden availability of oxygen leads to an oxidative burst with the generation of free radicals. The inflammatory aspect of IRI includes both, the cellular and humoral components of the immune system [18]. Moreover, mechanisms of IRI may be organ-dependent, with similar but distinct pathways involved in different organs. During the last decade, there has been an explosion of research investigating the role of leukocytes and leukocyte adhesion molecules in IRI [3]. Multiple mechanisms have been postulated since then for the leukocyte-mediated tissue injury that occurs after ischemic-reperfusion. Microvascular occlusion [13], release of oxygen free radical [23], cytotoxic enzyme release [32], increased vascular permeability [8] and increased cytokine release [1] have all been demonstrated to contribute to leukocyte-induced tissue injury [17].

In addition to the before mentioned processes, large amounts of muscular waste products may enter the systemic circulation. This has been described as a part of the reperfusion syndrome and is associated with multi-organ failureand death [11].

1.3 Principle of controlled reperfusion

As experimental studies on isolated rat hind limbs have shown, cellular integrity and biochemical function is preserved after 4 hours of warm ischemia and that the severe metabolic changes just occurred after the onset of uncontrolled (full) reperfusion [7].

This opened the possibility to counter reperfusion injuries by adjusting the timing and the technique of reperfusion itself. Controlled reperfusion has two major advantages:

First, to lower the perfusion pressure, as animal models examining myocardial ischemia have shown that reduction of initial reperfusion pressure alone leads to improved functional and metabolic recovery [30]. In another animal model dealing with skeletal muscle ischemia, a reduction of the reperfusion blood flow has shown to reduce edema formation and muscle injury [34].

Secondly, to change the compound the reperfusion fluid, instead of highly oxygenated pure blood, a blood-crystalloid mixed solution is used. This crystalloid reperfusion solution is aimed to decrease reperfusion induced pathological changes. Lower oxygen content leads to weaker oxidative stress. Glucose is added, to provide a substrate for anaerobic metabolism and as a hyperosmolar substance to reduce edema generation. With glutamate, aspartate and amino acid precursors, substances of the Krebs-cycle are substituted to ensure a more effective oxidative metabolism with the onset of reperfusion. Also Allopurinol should be supplemented, to reduce the generation of oxygen-derived free radicals. Finally, Sodium citrate is complemented to reduce intracellular calcium.

Controlled reperfusion should be performed during the first half hour after revascularization, as there is evidence that most additional tissue injuries are caused by uncontrolled reperfusion during the first 20 to 30 minutes [16].

1.4 Aims

We hypothesized that controlled reperfusion using a simple arterial blood bag perfusion system should reduce reperfusion injury and thus facilitates the return of normal function [33].

We used an experimental (pig) animal model with an infrarenal aortic occlusion and performed controlled reperfusion, observing the effects on oxidative stress and inflammatory pathways compared to uncontrolled reperfusion.

Furthermore, we examined with histological examinations the protective effect of controlled reperfusion on organs sensitive to reperfusion injury like skeletal muscle, kidneys, lungs, the liver and the heart.

2 Materials and methods

2.1 Study protocol

We used ten Yorkshire pigs for this animal model. Five of these animals underwent a four-hour infrarenal aortic occlusion followed by continuous reperfusion without any further therapy. This population served as our control group (CG).

The other five animals underwent controlled reperfusion. After the four-hour infrarenal occlusion we performed the controlled reperfusion during the first 30 minutes and then started the continuous reperfusion with normal blood flow.

2.2 Surgical preparation

These protocols were approved by the Institutional Animal Care and Use Committee of the Uniformed Services, the University of the Health Sciences and they conform to the standards in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publications No. 85-23, Revised 1996).

Yorkshire pigs of either sex (male-female 1:1 ratio), age of 10 weeks, weighing between 18 and 22 kg that were free of clinically evident diseases, entered this study. The pigs were sedated with an intramuscular injection of 15 mg/kg ketamine hydrochloride (Vetalar, Fort Dodge) and anesthetized with pentobarbital sodium (30 mg/kg, Sigma; St. Louis, MO). The pigs were placed on a homoeothermic blanket control unit (Harvard Apparatus; Holliston, MA) designed to maintain a core body temperature of at least 37°C, constantly measured by a thermistor probe placed in the animals rectum.

Variation in temperature between animals was minimized with careful monitoring.

An intratracheal intubation was performed, and the pigs were mechanically ventilated (Harvard) with room air supplemented with oxygen (1 liter/min). A saline filled catheter was placed in the right external jugular vein for drug administration and fluid infusions. A 9-Fr catheter introducer (Catheter Sheath Introducer System, Cordis; Miami, FL) was placed in the right carotid artery. Through this introducer catheter, a Mikro-Tip Dualpressure Transducer catheter (model SPC-780C, Millar) was inserted to measure aortic and ventricular pressure and to permit simultaneous electronic differentiation to record the alteration in pressure change over time (dP/dt). End-tidal CO₂ was monitored continuously (Hewlett- Packard, model 78356A; Palo Alto, CA), arterial blood gases were measured periodically, and ventilator parameters were adjusted as needed to maintain blood gases within physiological ranges (pO2:83-108Hgmm, pH: 7,35-7,45). Slow intravenous infusion (1,5 ml/min) of normal saline maintained hydration throughout the surgery and additional anesthetics were administered as needed.

After those preparations, a median laparatomy was prepared, and with gentle retraction of the bowels we isolated the infrarenal aorta. We administered heparin to prevent any formation of thrombi. We clamped the aorta with DeBakey clamps. Lack of pulsation showed the effectiveness of clamping. After the four hours long ischemic period, we removed the clamps and checked the restoration of continuous blood flow by observing the pulsation of the iliac arteries.

2.3 Management of controlled reperfusion

The equipment used to apply the controlled limb reperfusion, consisted of two blood bags (each capable of holding 1 liter), crystalloid solution, a blood line, and a reperfusion line (both made of 0.25-inch polyester tubes).

Controlled limb reperfusion was performed as follows:

After the aortic occlusion and before restoration of blood flow, a 10-Charrier (CH) (1 CH is equivalent to.33 mm) cannula was inserted proximally of the occlusion into the aorta, another 10-CH cannula was inserted distally of the obstruction. The proximal cannula was connected to the blood line (aorta), and oxygenated blood was drawn into the first blood bag where it was mixed with the crystalloid reperfusion solution (blood: reperfusion solution ratio, 1:1. The composition of the crystalloid solution is given in Table 1.

According to the hemodynamic status (blood pressure: 85–115 Hgmm, heart rate: 110–140/min) of the animals, either 200 ml or 300 ml of blood were taken each cycle. After the blood-reperfusion solution had been done in the blood bag, the reperfusion line was connected to this bag. After all air had been expelled from the reperfusion line, controlled reperfusion was initiated via the distal cannula. A 12-gauge cannula was inserted into the aorta distally to the reperfusion cannula intended for continuous pressure control. Perfusion pressure was kept strictly at 60 mmHg. In most cases, the blood reperfusion solution was returned to the legs by gravity alone. If necessary, a pressure-cuffed bag was used to achieve a perfusion pressure close to 60 mmHg. Also the perfusion pressure was adjusted by changing the height of the blood bag. The procedure (cycle) was repeated for 30 minutes. The number of cycles performed depended on the flow that could be achieved (usually 2-3 cycles). After removal of the cannulas, the arteriotomy was closed with a direct suture, and normal blood flow was established and checked with the aortic and femoral pulse detection. During the experiment, blood samples were taken as indicated in Table 1.

Avoiding the crystalloid infusion generated diluting effect in the controlled reperfusion group we added 600 ml physiological salina into peripherial vein to the animals of the full reperfusion group in the first 30 min of reperfusion.

2.4 Measuring ischemic and reperfusion injury

The main parameters that are involved in reperfusion injury are widely known. One can divide them into oxidative stress parameters and inflammatory parameters (Table 2). One oxidative stress parameter is a lipid peroxidase marker like Malondialdehyde (MDA) that increases in ischemic situations. The other contributors are anti-oxidant enzymes, that naturally decrease in ischemic environments, namely Glutathione (GSH), Superoxide dismutase (SOD) and plasmaThiol groups (–SH groups). Leucocyte free radical production and Myeloperoxid (MPO) activity are summarized as inflammatory parameters.

Malondialdehyde (MDA) is one of the most frequently used indicators of lipid peroxidation [24]. It was measured in anticoagulated whole blood by a photometric method [25].

Reduction of Glutathione (GSH) and plasma Thiol (-SH) groups were measured after reaction with Ellmann’s reagent and according to the method of Sedlak and Linsday,from whole, by EDTAanticoagulated, blood [29]. The results were collected via photometry.

Measurement of Superoxide dismutase (SOD) activity in red blood cells is based on the spontaneous conversion of adrenalin to adrenochrom, which itself is a colorful complex. SOD is able to block this reaction and thereby was used to provide us with information’s about the quantity of SOD activity in different tissues [21].

Leucocyte free radical production was determined with aChrono- log lumino- aggregometer. This chemiluminescense method is based upon the reaction of luminol with free radicals. Phorbol-12-myristate-13-acetate (PMA) was used to induce free radical production and the resulting light output was recorded on a chart recorder. The peak value of free radical production was calculated from the recorded curve, and the results were related to the white blood cell counts(WBC). The slope of the steep part of elevation in radical production could also be determined [14].

Plasma Myeloperoxidase (MPO) level is a sign of neutrophil granulocyte activation and it wasmeasured with a spectrophotometer. Myeloperoxidase was determined from 200 μl anticoagulated blood (EDTA plasma) that wascentrifuged at 2000 g followed byaddition of 10.9 ml 0,1M sodium- citrate, 5 μl 0. 05% Triton-X-100, 1 ml 1 mM H2O2 and 100 μl 0,1% o-dianisidine.After incubation for 1 min. at 37 Celsius with1 ml of 35% perchloric acid, the amount of MPO could be measured photometrical at 560nM [5].

2.5 Hematology test

Red blood cell counts (RBC), White blood cellcounts (WBC), Platelet numbers, Hemoglobin concentrations and Hematocrit levels were measured by Minitron automatic analysator (Diatron Ltd, Budapest, Hungary).

2.6 Statistical analysis

Data were expressed as mean±SE, or percentages. For the analysis of data, paired and unpaired Student’s t-test, and one-way analysis of variance (ANOVA) were used. Statistical significance was established at p < 0.05.

2.7 Histological examinations

Animals from both groups were anesthetized 7 days after terminating ligation and biopsies were taken from the quadriceps muscle and large parenchymal organs, namely the liver, kidneys, lungs and the heart. Large and small intestines were also biopsied.

The definite aim of the biopsies was to register any quantitative and qualitative differences between the two animal groups. We concentrated first and over all on any transformations happening in the striated muscular tissue. There was no intention in this study, to make statements concerning pathogenesis or pathologies not caused by ischemia. We took transversal and longitudinal cut biopsies from the striated muscular tissue. The fragments of muscle did not contain any well-identifiable fascia, as it was prepared into 5-6 paraffin-embedded blocks. From each block, several sample slices were prepared and stained immunohistochemically and with Hematoxylin & Eosin (H.E.). We examined all available organs from each animal.

The specimens of the biopsied tissue were created with the following method:

The fresh tissue was fixed in 10% neutral buffered formalin. Sample preparation was performed with a tissue processor equipment (Thermo Shandon Path centre, Thermo Fisher Scientific Inc., Waltham, MA, USA). Sectioning was performed with a sledge microtome (5 μm, Reichert Optische Werke AG, Vienne, Austria) from the paraffin-embedded blocks, and staining was carried out with a carousel-type slide stainer (Thermo Varistain 24-4, Thermo Fisher Scientific Inc., Waltham, MA, USA) with H.E., at the County Hospital of Baranya, Department of Pathology, Pécs, Hungary. We used immune-histochemical staining for actin, desmin and calponin with antibodies available in trade, which were produced against human antigens. We expected results mostly from actin and desmin antibodies. To determine the quantitative data of normal and degenerative fibers, we performed our applications in blocks of cross sections of areas showing definitive fiber changes.

3 Results

3.1 Hemodynamic data

Hemodynamic data was summarized in Table 3. Baseline heart rate, systolic and diastolic pressures were not significantly different among the various groups. In the reperfusion period controlled reperfusion group showed significant lower data of systolic and diastolic blood pressure.

3.2 Oxidative stress results

3.2.1 Malondialdehyde

The plasma MDA concentration was elevated in the full (ischemic-) reperfusion group right after the beginning of reperfusion and decreased until the 24th hour of reperfusion. This elevation was much milder in controlled reperfusion group. We measured a significant difference between the two groups. On the end of the investigationtime we detected a recurrent elevation in the reperfusion group, but lower than in the early reperfusion period. (Fig. 1)

3.2.2 Plasma thiol groups

We observed changes in the – SH group in the first hour of reperfusion. There was a significant decrease in the full (ischemic-) reperfusion group, but these changes could not be seen in the controlled reperfusion group. The measured values in the 24th hour were similar to the values before reperfusion. A second decrease was detected on the 7th day in the full (ischemic-) reperfusion group (Fig. 2).

3.2.3 Glutathione

GSH plasmalevels decreased significantly in the early reperfusion period until the 24th hour. We did not detected differences between the two groups. On the 7th day of reperfusion, the measured values in both groups reached the start values (Fig. 3).

3.2.4 Superoxide dismutase

We did not measure changes in SOD activity during the ischemic period and in the early reperfusion phase. A significant decrease in SOD activity was detected in the 24th hour of reperfusion in the full (ischemic-) reperfusion group. This decrease could not be detected in the controlled reperfusion group. On the last measurement, both groups showed the same values as prior the ischemia, and there was no difference between them (Fig. 4).

3.2.5 Free radical production of leucocytes

We detected a significant increasedvelocity of leukocyte radical production in the early period (15th and in the 60th minutes) in the full (ischemic-) reperfusion group. These changes could not be observed in the controlled reperfusion group. In the 24th hour we measured physiological values in both groups (Fig. 5).

3.2.6 Maximum free radical production

The maximum free radical production first elevated in the full (ischemic-) reperfusion group in the 15th minute of reperfusion and this elevation could be detected during the complete examination period. Significant differences between the groups could be measured in the 1st hour of reperfusion. On the seventh day we measured increased values in both groups (Fig. 6).

3.2.7 Myeloperoxidase

We detected a significant increase of plasma MDA concentrations in the full (ischemic-) reperfusion group in the 24th hour. At this time, there was no elevation in the controlled reperfusion group, and a significant difference could be seen between the 2 groups. We could even measure elevated values at the end of the experimental period (Fig. 7).

3.3 Histological results

3.3.1 Muscle tissue

During the histological evaluation, we could observe the same tissue changes characterizing degeneration in both animal groups, but in the full (ischemic-) reperfusion group immediately after reperfusion. In the group treated with controlled reperfusion, we also recognized degeneration, but changes appeared in bigger areas in the full (ischemic-) reperfused group, and only moderately in the controlled reperfused group.

In the full (ischemic-) reperfused group of animals, the basic striated muscular structure was mainly kept within the tissue, there was no fibrosis and necrosis that could be defined with absolute certainty. Also significant inflammation could not be observed. At the same time, the muscle tissues showed well-observable size and shape differences. Regular morphology was seen within the normal stained muscle fibers, showing also a strong desmin immune-histochemical reaction that covered the full area of fibers. The fibers were swollen, irregularly-shaped and the interstitial space between the fibers was pressed and decreased. The fibers stained appeared paler, less even and slightly basophile. In some places, the sarcoplasm seemed tattered and the nucleuses were decreased.

The rate of affected muscle fibers with degeneration was 61.7% in the full (ischemic-) reperfusion group (617:383), and it exceeded 50% in all areas. The rate of damaged fibers was 42.4% (424:576) in the controlled reperfused group, and it stayed below 50% in all examined areas (Figs. 8 and 9).

3.3.2 Kidneys

Transformations could only consistently be observed in the kidneys. Here, the smaller veins were definitely diluted in numerous places, sometimes in groups, and in other places only single veins were affected. Transformations in the cortex were shown in each individual of both animal groups. Only one animal of the full (ischemic-) reperfused group had signs that could be detected in the marrow and around the collective tubes. We could not demonstrate any other consistent changes in the kidneys (Fig. 10).

3.3.3 Lungs, liver and heart

It was remarkable, that the smaller veins were dilated in the liver, lungs, and heart (Figs. 11–16). We observed dilated veins in both groups, but not in all individuals and its occurrence were random. Smaller necrotic patches could be observed sporadically in the liver, around the portal areas and partly in the centro-lobular regions accompanied by reactive inflammatory processes (Fig. 10). Those changes appeared in small numbers and also randomized. We also discovered in a heart specimen of one individual of the controlled perfused animal group some fibrinoid necrosis in a central vessel.

In summary, all described histological transformations do not give a strong enough evidence to draw any long term results, like the save exclusion of any distal vessel obliteration described in otherstudies [4]. At best, they indicate that it is worth to do further experiments with higher numbers of animals, to ensure if our findings were accidental or regular events.

4 Discussion

Acute lower-limb ischaemia is probably the most common reason for emergency admission to a vascular surgery unit. The predominant reasons for acute limb ischaemia are embolism of cardiac or arterial (eg, aortic aneurysm with adherent thrombus) origin and arterial in situ thrombosis of arteriosclerotic vessels. Re-establishment of arterial blood flow to the compromised leg is essential for limb salvage. With the introduction of Fogarty catheter in the early 1960 s, a simple surgical technique for the revascularization of occluded vessels was introduced into clinical practice. In combination with other surgical techniques for revascularization, such as local thrombo-endarterectomy and bypass-procedures, vascular surgeons today have a variety of therapeutic options for revascularization of acute ischemic limbs. Through the last decades, interventional treatment options such as thrombolytic therapy have become another therapeutic option in treating acute limb ischaemia.

Despite improvements in revascularization techniques, the results of surgical and interventional treatments have remained unsatisfactory, with high amputation rates and high mortality [26]. The poor results of revascularization therapy alone may be mainly due to additional reperfusion injury [9]. Reperfusion injury is descriptive of the fact that the reperfusion of ischemic tissue, which is absolutely necessary for tissue salvage, causes further tissue damage that in turn can result in tissue apoptosis and necrosis. However, a prerequisite for evaluating different reperfusion protocols is the achievement of a complete revascularization. Even controlled reperfusion will result in amputation if revascularization cannot be achieved because of obliteration of distal vessels. The therapeutic principle of controlled reperfusion has been used successfully in treating myocardial ischaemia and has been shown to improve clinical outcome [4].Experimental studies on isolated rat hindlimbs have shown that cellular integrity and biochemical function is preserved after 4 hours of warm ischaemia. The severe changes occur after the onset of uncontrolled reperfusion [7]. Reduction of initial reperfusion pressure alone resulted in improved functional and metabolic recovery in an animal model of myocardial ischaemia [30]. In an animal model of skeletal muscle ischaemia, a reduction of reperfusion blood flow was shown to reduce edema generation and muscle injury [34].

Controlled reperfusion, with reduced reperfusion pressure and modification of the initial reperfusate, can reduce the local consequences of reperfusion injury such as depletion of high-energy phosphates and local swelling. This is accompanied by improvement in the return of contractile function [6]. The systemic complications of reperfusion, such as release of muscle proteins and potassium into the systemic circulation, could be reduced in an animal model of acute lower-limb ischaemia [22].

By using a simple blood bag reperfusion system, as we did in this study, two main principles of controlled limb reperfusion - a reduction of initial reperfusion pressure and modification of the composition of the initial perfusate - can be achieved in clinical practice with minimal technical effort. As there is evidence that most additional tissue injury caused by uncontrolled reperfusion occurs during the first 20 to 30 minutes, a 30-minute interval for controlled reperfusion was chosen [16]. Several well-known biochemical changes occur during ischaemia and reperfusion. With the onset of ischaemia, aerobic metabolism is suspended and anaerobic metabolism is activated. This leads to a breakdown in high-energy phosphates, an increase in intracellular lactate, and intracelluar acidosis [19]. The Krebs cycle loses intermediates [28]. With the start of reperfusion, the washout of lactate and protons from the ischemic tissue leads to an increase in intracellular calcium. An oxidative burst with the generation of oxygen-derived free radicals occurs with the return of oxidative metabolism [20].

The crystalloid reperfusion solution is aimed to counter these changes. Glucose is added to provide a substrate for anaerobic metabolism and as a hyperosmolar substance to reduce edema generation. With glutamate and aspartate, amino acid precursors of the Krebs-cycle are added to ensure more effective oxidative metabolism with the onset of reperfusion. Allopurinol is added to reduce the generation of oxygen-derived free radicals. Sodium citrate is added to reduce intracellular calcium. The results of our study shows that controlled reperfusion using this simplified reperfusion system can be safely performed in any operating room. Controlled reperfusion has been used before with good results. All the techniques described so far required the use of a heart-lung machine or roller pumps [31]. This required an enormous technical effort and limited the use of controlled reperfusion to large vascular surgerycenters.

Most patients with acute lower-limb ischaemia will not present at centers capable of providing these techniques.

After a period of severe ischaemia, reperfusion of the involved leg results in the washout of large amounts of muscular waste products into the systemic circulation. This has been described as a part of the reperfusion syndrome and is associated with multiorgan failure and death [9]. Experimental studies on isolated rat hindlimbs showed that controlled reperfusion significantly reduces the amount of metabolic waste products [6]. The duration of ischaemia in the animals included in this study was quite long. Clinical studies on controlled reperfusion for severe lower-limb ischaemia have showed good results [12].

5 Conclusion

Our results strongly support the hypothesis that controlled reperfusion can improve outcome after acute severe lower-limb ischaemia even though this study was limited by the small number of animals. We have seen that controlled reperfusion significantly reduced the postischaemic oxidative stress and inflammatory responses in the early reperfusion period.

Our pathohistological results confirm, that controlled reperfusion has real beneficial effect not only on the ischaemic skeletal muscle, but also protects against reperfusion syndrome in the lung, kidney and liver.

Our results confirm, that controlled reperfusion might be also a potential therapeutic approach in vascular surgery against reperfusion injury in acute limb ischaemia.

Footnotes

Acknowledgments

The study was supported by OTKA- K67731; AOKKA-34039-28/2009; OTKA: K78434.

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