For the surgeon, the tourniquet is a common tool in surgical procedures of the limbs. It facilitates a less bloody surgical setting, but ischaemia causes an increase in tissue pressure and cell damage [1,2]. After deflating the tourniquet, reperfusion of ischaemic tissue takes place gradually and in an incomplete fashion as a result of microvascular dysfunction, which causes local and systemic organ damage by oxidative stress elements [1–3].
Reperfusion of an acutely ischaemic lower limb may lead to systemic complications such as adult respiratory distress syndrome, and renal and hepatic dysfunction . Activation of polymorphonuclear leukocytes and increases in circulatory proinflammatory cytokines are postulated to be important mediators in local and remote organ damage . Many theories have been suggested to explain ischaemia/reperfusion damage. Rapid free oxygen radicals derived from xanthine oxidase is the most validated mechanism . Ischaemia/reperfusion injury is accompanied by disturbances in vasomotility and microvascular permeability. Deficiency of vasodilator nitric oxide due to its consumption by vasoconstrictor oxygen-derived free radicals (OFRs) results in microvascular constriction and prominent reduction of blood flow in reperfused tissue. OFRs also promote the formation of inflammatory responses causing leukocyte ‘rolling and sticking’ . In addition, altered rheological conditions lead to thrombus formation in microvessels. These mechanisms contribute to the development of the no-reflow phenomenon . Concurrently, OFRs damage endothelial cells, enhance microvascular protein efflux, and potentiate endothelial permeability, causing interstitial reperfusion oedema .
Neutrophil or platelet aggregation, hypoxic vasoconstriction, increase in capillary permeability, and structural changes in the endothelium have also been shown . Catalase and glutathione systems play a compensatory role against oxidative stress. Many drugs have been tested to explain or to prevent ischaemia/reperfusion damage . Blood volume has a crucial role in the maintenance of haemodynamic balance and tissue oxygenation. New studies investigating ischaemia/reperfusion injury and antioxidant pharmacology, with the aim of improving our understanding, show promising results in terms of preventing regional or distant organ damage [1,2,9].
Hydroxyethyl starch (HES) 130/0.4, 6%, not only has a role in replacement of the liquid deficits due to trauma, bleeding or shock, but is also effective in enhancing the tissue oxygen tension and regulation of microcirculation [9,11]. The aim of the present study was to investigate the effect of HES 130/0.4 6% on ischaemia/reperfusion injury. For this purpose, myeloperoxidase (MPO), malondialdehyde (MDA), nitrite, nitrate, and reduced glutathione (GSH) levels were measured in muscle tissue samples during the ischaemia/reperfusion period.
Material and method
After obtaining the approval of the local animal ethics committee, the study was conducted at the Experimental and Surgical Animal Research Laboratory in a manner similar to the acute ischaemia/reperfusion model described by Hardy et al.. Fourteen New Zealand rabbits were used. Anaesthesia was performed by intramuscular ketamine HCl (Ketalar) injected in the left forefoot at a dose of 30 mg kg−1. Anaesthesia was maintained with intravenous ketamine HCl if required. Arterial and venous lines were inserted through the ear in all test subjects. All rabbits were given oxygen support at a rate of 3 l min−1 by mask. Haemodynamic parameters of blood pressure and heart rate were recorded via their left forefoot at 30 min intervals during the procedure.
Both hind legs of all rabbits were shaved starting from the abdominal midline, and the surgical area was scrubbed with povidone iodine preceded by wrapping under sterile conditions. The mean weight of the rabbits was 3.2 kg (2.8–4.1 kg).
Test participants were randomized into two groups of seven as study and control groups. Normal saline was infused in the control group (group S), whereas HES 130/0.4 solution was infused in the study group (group HES). In all periods, fluids were given continuously in both groups. According to the maximum safe total HES dose reported in the literature, 70 ml kg−1 total fluid was given to both groups . The amount of fluid was calculated as 70 ml kg−1 total dose; one-third was given at anaesthesia until the tourniquet was inflated (approximately 45 min), another third during ischaemia (3 h), and the remaining third during reperfusion (2 h). In both groups, after the first dose was infused and before the tourniquet was inflated, muscle biopsy sampling was performed at the triceps surae muscle of the lower extremity of the left hind leg (sampling A). Thereafter, using an Esmarch bandage, a tourniquet was applied to the right hind leg of all rabbits at the level of the hip joint in order to arrest arterial circulation and to empty the venous system of that side's extremity.
At the end of 3 hours' ischaemia and before the tourniquet was deflated, ischaemic muscle biopsy sampling was performed at the triceps surae muscle (sampling B). After the tourniquet was deflated, biopsy was performed in the same muscle group following 2 hours' reperfusion (sampling C) and sent to the biochemistry laboratory with the aim of determining nitrite, nitrate, GSH, MPO and MDA levels. In all test subjects, arterial blood gases and haematocrit levels were measured during A, B, and C muscle biopsy sampling (A–C arterial blood gases) and their results were recorded.
Myeloperoxidase, malondialdehyde, nitrite, nitrate, and reduced glutathione determination in muscle tissue
Tissue samples were immediately frozen and stored at −80°C until nitric oxide metabolites, MDA, MPO activity and GSH were measured. In the working day, samples were homogenized in buffer on ice and assayed for nitric oxide metabolites, MDA, MPO and GSH contents using the method as described. Nitrite and nitrate contents were measured by the modified Griess method . The method developed by Uchiyama and Mihara  was used for MDA determination. GSH was measured by a colorimetric method . MPO was assayed using a spectrophotometric technique . For MDA and GSH measurements, protein content was determined by the Lowry method .
Lipid peroxidation in the rabbit muscle was measured as thiobarbituric acid-reactive material. After isolation of muscle, tissue homogenates (10% w/v) were prepared by homogenizing tissue in cold 1.15% KCl. In a test tube, 0.5 ml of tissue homogenates was mixed with 3 ml of 1% orthophosphoric acid. After addition of 1 ml of 0.67% thiobarbituric acid, the mixture was heated in boiling water for 45 min. The colour formed was extracted into 4 ml of n-butanol and the absorption was measured at 532 nm, using 10, 5, 2.5, 1.25, 0.625 nmol ml−1 1, 1, 3, 3-tetraethoxypropane as the standard. MDA was calculated as an indicator of tissue lipid peroxide levels (nmol mg−1 protein).
Reduced GSH was determined by the method of Ellman. Metaphosphoric acid (0.5 mol l−1; W/V = 1/5) was added to the homogenate and the mixture was centrifuged at 3500 rpm. Three millilitres of supernatant was treated with 2.0 ml of phosphate buffer (0.1 mol l−1, pH 8.0), 5 ml distilled water, and 0.02 ml of DTNB reagent (39.6 ml of 5,5′-dithiobisnitro benzoic acid in 10 ml 0.1 mol l−1 pH 7 phosphate buffer). The absorbance was read at 412 nm, using 80, 90, 100, and 125 μmol l−1 reduced GSH as the standard. Tissue reduced GSH levels were calculated (μmol mg−1 protein).
Fifty microlitres of homogenate and 50 μl of distilled water were pipetted into each tube. Two millilitres of Lowry stock reagent were added to each tube and incubated for 15 min at room temperature; 0.2 ml of Folin's reagent (Folin–Ciocalteau reagent, diluted 1: 1 before use) was added to each tube and incubated for 60 min at room temperature. The resultant colour was read in a spectrophotometer at 750 nm; 1, 2.5, 5 mg ml−1 bovine serum albumin (BSA) was used as the standard.
Tissue nitrite and nitrate
Three hundred microlitres of KH2PO4/K2HPO4 buffer (pH 7.5), 50 μl of 2 mmol l−1 NADPH, 50 μl of 50 μmol l−1 FAD and 50 μl of 1 unit ml−1Aspergillus nitrate reductase were added to 300 μl homogenate. This was incubated at room temperature for 1 h followed by the addition of 500 μl of 0.2 mol l−1 ZnSO4 and 70 μl 2 mol l−1 NaOH to deproteinate the sample. After centrifugation, 0.75 ml of the supernatant was added to 1 ml of 1% sulphanilic acid (in 4 mol l−1 HCl). After 10 min at room temperature, 0.75 ml of freshly prepared 1% N-naphthylethylene diamine was also added. The resultant colour change was measured at 548 nm using a spectrophotometer. Nitrite concentration was calculated from 5, 12.5, 25, 50, and 100 μmol l−1 sodium nitrite standards.
MPO activity was assayed spectrophotometrically by determining the decomposition of hydrogen peroxide using o-dianisidine as the hydrogen donor. Tissue samples of approximately 50 mg were taken, weighed and homogenized three times for 30 s at 4°C in 1 ml of ice-cold 0.5% hexadecyltrimethylammonium bromide in 50 mmol l−1 phosphate buffer (pH 6). The homogenate was subjected to three freeze/thaw cycles and centrifuged for 15 min at 40 000 × g. MPO activity was determined by the addition of 0.1 ml of the supernatant to 2.9 ml of 50 mmol l−1 phosphate buffer containing 0.167 mg m l−1o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The change in absorbance at 460 nm over a 5 min period was measured at 25°C. The data are expressed as the change in absorbance min−1 g−1 wet weight.
Data analysis was performed by using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, Illinois, USA). The Shapiro Wilks test was used to determine whether or not the continuous variables were normally distributed. Data are shown as the means ± SD. pH, PaO2 and PCO2 means were compared with baseline by using the Student's t test. Haematocrit means were compared by using the Mann–Whitney U test and Freidman test. Repeated MDA, MPO, nitrite, nitrate, and GSH were evaluated by using repeated measures of ANOVA followed by Bonferroni adjustment for all multiple comparison tests. When P values of the interaction terms from the variance analysis were statistically significant, the mean percentage or actual differences according to baseline were compared by using the Student's t or Mann–Whitney U test, wherever appropriate. A P value less than 0.05 was considered statistically significant.
During the procedure, there were no statistically significant differences between groups in terms of heart rate (HR) and blood pressure levels (no data were given) (P > 0.05). The test participants in the S and HES groups showed a similar difference in time (A–B–C phases); the effect of these phases on PaO2, PCO2, and pH levels was not statistically significant (P > 0.05). PaO2 levels (mean ± SD, mmHg) at sample times A, B, C in group S were 184.4 ± 81.9, 153.6 ± 119.9 and 176.6 ± 102.4, and in group HES were 207.9 ± 107.2, 187.9 ± 46.1 and 197.9 ± 73.6, respectively. PCO2 levels at sample times A, B, C in group S were 48.5 ± 6.5, 38.1 ± 4.3 and 41.8 ± 16.2, and in group HES were 47.1 ± 4.9, 46.6 ± 12.9 and 47.9 ± 10.4, respectively. pH levels in group S in periods A, B and C were 7.37 ± 0.02, 7.34 ± 0.04 and 7.29 ± 0.08, respectively, and in group HES were 7.38 ± 0.1, 7.33 ± 0.1 and 7.30 ± 0.09, respectively. Differences in haematocrit values in the preischaemic, ischaemic and reperfusion sample periods were determined not to be statistically significant between the two groups and between periods in each group (P > 0.05) [group S, A-B-C period (28.1 ± 3.3, 30.1 ± 4.7, 26.4 ± 3.4); group HES, A-B-C (30.0 ± 4.6, 28.3 ± 2.1, 28.4 ± 3.9)].
Nitrite, nitrate and GSH levels did not show statistically significant differences between groups (P > 0.05). In both groups nitrite levels were significantly (P = 0.034) higher in phase C than in phase B (Table 1).
There were no statistically significant differences between groups in MPO values in periods A, B and C (P = 0.050, P = 0.399 and P = 0.083 respectively, Table 1). When the actual differences in MPO values according to baseline (between baseline – A and reperfusion – C samplings) were taken into account, the mean result was a decrease in the HES group (−0.03 ± 0.15) compared with an increase in the S group (0.31 ± 0.25), and this difference was statistically significant (P = 0.011, Fig. 1).
Evaluation of the MPO values while passing from period A to B (−0.14 ± 0.14, −0.03 ± 0.03) and from period B to C (0.10 ± 0.09, 0.34 ± 0.26) showed no statistically significant difference between groups (P = 0.073, P = 0.053, respectively, Fig. 1).
There was no statistically significant difference between the groups or within the groups through the phases in terms of MDA values (P > 0.05, Table 1).
The present study provides some evidence that 6% HES 130/0.4 treatment in a rabbit model of lower limb ischaemia/reperfusion seems to attenuate the leukocyte recruitment into muscle tissues, which is postulated to be important in local and remote organ damage .
In experimental ischaemia/reperfusion model studies, ischaemia duration has varied between 2.5 and 4 h with 2 h reperfusion [10,11,20]. In an experimental model, 2.5 h of ischaemia/2 h of reperfusion led to the development of ischaemia/reperfusion injury, expressed by interstitial oedema formation, microvascular constriction and microvessel plugging with neutrophils. These morphologic alterations led to development of the no-reflow phenomenon, expressed by a reduction in blood flow . In this study, to demonstrate the effects of HES 130/0.4 solution on microcirculation, we preferred an animal ischaemia/reperfusion model as 3 h ischaemia and 2 h reperfusion. HES solution has regulator effects on microcirculation and on tissue oxygen tension [21,22]. HES 130 reduced arteriolar and venular leukocyte adherence, whereas NaCl resuscitation had no effect in normotensive endotoxaemic animals . Improved tissue oxygen tension (pti O2) was found in patients undergoing abdominal surgery in whom 6% HES 130/0.4 solution was used . In another study, microperfusion was improved and pti O2 was significantly more increased in patients treated with HES 130/0.4 solution when compared with those treated with crystalloid solutions . The results of Rittoo et al.'s  study showed better perioperative pulmonary function in patients treated with HES after abdominal aortic aneurysm surgery when compared with Gelofusine. HES solution has a repair effect on the microcirculation when used for volume replacement, and may modify and reduce the degree of ischaemia/reperfusion injury. We administered the dose of 70 ml kg−1 HES 130/0.4 to test its effect on ischaemia/reperfusion injury. This is the first experimental study on this subject and we chose to use the maximal dose in the literature . We could not determine a statistically significant difference between groups in terms of haematocrit, blood pressure and heart rate during the procedures. Both solutions were given continuously.
When the tourniquet is inflated, acute ischaemia develops and as reperfusion is obtained by deflating the tourniquet, OFRs are formed, leading to reperfusion injury. As a result of reperfusion, cellular components are damaged with the stimulation of neutrophil adhesion and activation under the influence of reactive molecules. These neutrophils secrete free radicals and proteases, thus leading to further tissue damage. The resultant MPO is an indicator of damage. The tissue MPO activity is directly proportional to the neutrophil count [9,13]. We detected tissue MPO as a marker of neutrophil oxygen radical production. In the present study, the MPO level in group S increased after 2 h reperfusion, whereas it decreased in group HES. This may be attributed to the regulatory effect of the HES solution infused in the preischaemic and reperfusion periods. In vivo, in normotensive endotoxemia, HES 130 was effective in preventing leukocyte adherence, attenuating capillary perfusion failure, and reducing macromolecular leakage. HES 130 (16 ml kg−1) was used 3 h after endotoxaemia and showed an in-vivo protective effect on endotoxin-induced microcirculatory disorder . In the present study during the ischaemic period (tourniquet application) the ischaemic limb was excluded from the systemic vascular bed and was not reached by the HES solution. Perhaps only one-third of the total dose of HES 130 solution that is used in this study could be infused during the reperfusion period to obtain a protective effect.
Corticosteroids and allopurinol prevented an increase in MPO levels without affecting OFRs . The effect of HES solution on MPO levels and ischaemia/reperfusion was similar to results reported above.
Ischaemia of the extremities is associated with lipid peroxidation, an autocatalytic mechanism leading to oxidative destruction of cell membranes, which may lead to the production of toxic reactive metabolites and cell death. Lipid peroxidation, as a free radical-generating system, may be closely related to ischaemia/reperfusion-induced tissue damage; MDA is an indicator of the degree of lipid peroxidation [24,25]. The results of this study showed that the difference in MDA levels was not statistically significant at the end of the reperfusion period in either group with respect to baseline. The reason may be that the number of experimental animals was inadequate to indicate statistical significance of MDA. Samples C were taken at the end of the reperfusion period, which may be a contributing factor. Another possible factor may be the anaesthetic agent. There have been controversial results about ketamine [10,26]. In the present study, both groups were anaesthetized with ketamine at induction and during maintenance of anaesthesia, and we observed no increase in MDA in the reperfusion period, though an increase in nitrite levels in the reperfusion period was observed in both groups. Although ketamine was used for anaesthesia in both groups, the MPO level was decreased only in the HES group in the reperfusion period.
In ischaemia/reperfusion injury, the decrease in vasodilating nitric oxide leads to microvascular constriction, and blood supply of the perfused tissue is also decreased. In the ischaemic tissue, the decrease in nitric oxide along with the increase in its derivatives such as nitrite and nitrate suggest that oxidative stress is increased . Nitrite and nitrate were evaluated in the present study and the nitrite parameters were increased in the reperfusion period in both groups. Nanobashvili et al. also determined that antioxidative treatment did not change nitric oxide production or its effects. Similarly, HES solution did not change the nitrite level in the present study.
It has been reported that ischaemia for more than 2 h in man causes irreversible damage to the muscle because of a shortage of adenosine triphosphate (ATP) . In the present study, muscle reduced GSH levels were measured to investigate the impact of a longer ischaemia time followed by reperfusion. The GSH results of the reperfusion period were not statistically significantly different from the baseline in either group, but we observed a decline in GSH levels compared with baseline. These results were obtained after a 2 h reperfusion period. Westman et al. studied leg muscle GSH and its redox status after a longer ischaemia period followed by reperfusion in elective aorto-bifemoral bypass surgery. They also found a decline in GSH levels in the reperfusion period; however, there was a significant difference compared with baseline after 24 h. In this study, samples were taken after a 2 h reperfusion, so this may be the reason for the insignificant statistical GSH results.
In conclusion, the results of this study showed that 6% HES 130/0.4 solution has a positive effect on ischaemia/reperfusion injury. The regulatory effect of HES solution on the microcirculaton prevents neutrophil sequestration and generation of OFRs in the reperfused tissue. In the saline group, the MPO level was increased in the reperfusion period even though 70 ml kg−1 solution was infused.
To further clarify the beneficial effects of the HES molecule, future studies should be planned including a third group with another colloid, for example gelatin or dextran, or another HES group with lower administered volume to determine any support for the positive results obtained in this study.
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