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

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.

2.1 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.

3.1 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

3.3 Histological results