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Protective Effect of Antithrombin III Against Lung and Myocardial Injury in Lower-Limb Ischemia–Reperfusion Syndrome
Protective Effect of Antithrombin III Against Lung and Myocardial Injury in Lower-Limb IschemiaeReperfusion Syndrome Neophytos A. Zambas, Christos D. Karkos, Apostolos G. Kambaroudis, Dimitrios G. Karamanos, Charalampos T. Spyridis, and Thomas S. Gerassimidis, Thessaloniki, Greece
Background: Restoration of blood flow to an acutely ischemic limb can trigger systemic inflammation. We investigated whether antithrombin III (AT-III) exerts a protective action against remote lung and myocardial injury in an experimental animal model of lower-limb ischemiae reperfusion. Methods: Ischemia was induced by lower-limb arterial occlusion for 6 hours in 60 male Wistar rats. Animals were divided into those receiving AT-III (dose, 250 mg/kg) 30 minutes before the reperfusion (group A, n ¼ 30) and those receiving placebo (group B, n ¼ 30). Animals were then sacrificed, and lung and myocardial tissue samples were taken at baseline, 30 minutes, and 4 hours after reperfusion. Levels of malondialdehyde (MDA), a compound used as indirect index of oxygen free radicals, were estimated in lung and myocardium, and the two groups were compared at different time points using the independent sample t test. Results: Animals administered AT-III had significantly lower levels of lung MDA compared with the placebo group at baseline and at 30 minutes, but not at 4 hours (P ¼ 0.001, P ¼ 0.01, and P ¼ 0.9, respectively), indicating a protective action of AT-III against remote lung injury early in the reperfusion phase. With regard to myocardial MDA levels, no statistically significant differences existed between the AT-III and placebo groups at baseline, at 30 minutes, and at 4 hours (P ¼ 0.07, P ¼ 0.07, and P ¼ 0.2, respectively) after reperfusion. Conclusions: In this experimental animal model, AT-III appears to exert a protective effect against remote ischemiaereperfusion injury in the lung tissue, but not in the myocardium.
INTRODUCTION Acute lower-limb ischemia followed by reperfusion may be encountered in several clinical circumstances, such as abdominal aortic aneurysm repair, acute thromboembolism of the lower-limb arteries, trauma surgery, and in crushed victims being brought to the
Presented as an abstract at CHEST 2009, Annual Meeting of the American College of Chest Physicians, San Diego, CA, October 31 to November 5, 2009. 5th Department of Surgery, Medical School, Aristotle University of Thessaloniki, Hippocratio Hospital, Thessaloniki, Greece. Correspondence to: Christos D. Karkos, MD, FRCS, PhD, EBIR, 5th Department of Surgery, Medical School, Aristotle University of Thessaloniki, Hippocratio Hospital, Konstantinoupoleos 49, Thessaloniki 546 42, Greece; E-mail: [email protected] Ann Vasc Surg 2012; 26: 566–570 DOI: 10.1016/j.avsg.2012.01.004 Ó Annals of Vascular Surgery Inc.
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emergency room. Such clinical scenarios are associated with high mortality and morbidity rates owing to a systemic inflammatory response and multiple organ dysfunction occurring during the reperfusion phase. In particular, remote injury to the lungs and the myocardium after lower-limb ischemiaereperfusion is believed to represent one of the major causes for this high mortality.1,2 The pathogenesis of remote ischemiaereperfusion injury is complex; to date, no treatment has been proven to attenuate these deleterious effects. Systemic inflammatory response to ischemiae reperfusion involves polymorphonuclear leukocyte sequestration in the lungs and increased pulmonary microvascular permeability.3 Ischemiaereperfusion also triggers a chain of events that may result in cellular death caused by oxygen free radicals released in response to the exposure of oxygen.4,5
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In specific, oxygen free radicals are believed to cause lipid peroxidation, resulting in damage to the biological membranes. End products of lipid peroxidation include aldehydes, hydrocarbon gases, and malondialdehyde (MDA).4 Because quantifying oxygen free radicals is difficult because of their reactive nature and short lives, it has been suggested that MDA can be used instead to estimate the extent of oxidant damage to tissues.4,5 Previous investigators have used MDA levels to estimate the protective effects of pharmacological agents, such as clopidogrel, in ischemiaereperfusion of the muscle and remote ischemiaereperfusion of the lungs and liver.5 Antithrombin III (AT-III) is a serine protease inhibitor, which, in addition to its anticoagulant function, is believed to exhibit an anti-inflammatory activity in sepsis and in ischemiaereperfusion.6e8 However, the mechanism of its action is unclear. It has been suggested that pretreatment with AT-III may attenuate acute lung injury in intra-abdominal sepsis, probably by inhibiting leukocyte recruitment into the lung.6 Similarly, a study of a feline mesentery ischemiaereperfusion model demonstrated that AT-III can be used therapeutically after reperfusion to attenuate the leukocyte recruitment response in inflammation.7 The potential protective effect of AT-III as an antioxidant by decreasing the free radicals has not previously been investigated in remote ischemiaereperfusion injury. In this study, we investigated the possible anti-inflammatory action of AT-III on remote lung and myocardial injury by assessing MDA levels in an experimental animal model of hind limb ischemiaereperfusion.
METHODS Sixty male Wistar rats weighing between 250 and 300 g were included in this study. The rats were cared for in accordance with the Guide for the Care and Use of Laboratory Animals,9 kept under a 12-hour light/dark cycle, and permitted ad libitum access to standard laboratory rodent chow and tap water for 2 weeks before the beginning of the experimental procedure. The rats were anesthetized with ether (diethyl ether, Panreac Quimica SAU, Barcelona, Spain) and then weighted. Ischemia was induced by the application of a polypropylene suture (Prolene 0, Ethicon, Inc, a Johnson & Johnson company, Gargrave, England) on the right hind limb, and the absence of flow was checked by Doppler ultrasound (Huntleigh Diagnostics Ltd, South Glamorgan, UK). A previous in-house study, using both Doppler ultrasound and arteriography,
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verified that the tourniquet-like application of a nylon suture produces almost complete limb ischemia. Similarly, previous studies by other investigators had shown that this method reduces blood flow by 98% and leads to acute ischemia.5,10,11 AT-III (or placebo) was administered intravenously through the penile vein 5.5 hours after the induction of ischemia and 30 minutes before the reperfusion. The animals were divided in two groups of 30. Group A received AT-III (dose, 250 mg/kg) and group B received placebo (NaCl, 0.9%). Reperfusion was begun after 6 hours of ischemia by removing the nylon suture. After the rats had been anesthetized, a midline sternotomy was performed. The lungs and heart were removed, and MDA levels were determined. The two groups were further divided into three subgroups of 10, depending on the time point when lung and myocardium tissue samples had been taken for determination of MDA levels after blood flow restoration: subgroups A1 and B1 immediately after reperfusion (‘‘zero’’ time), subgroups A2 and B2 at 30 minutes, and subgroups A3 and B3 at 4 hours after restoration of blood flow. Determination of MDA levels was performed using a selective third-order derivative method.12 In brief, 1-g samples were thoroughly homogenized (Polytron homogenizer, PCU, Kinematica, Lucerne, Switzerland) with 5 mL of 5% aqueous trichloroacetic acid, and 2 mL of 0.8% butylated hydroxytoluene in hexane was added and the mixture was centrifuged. The top layer was discarded, and a 2.5-mL aliquot from the bottom layer was mixed with 1.5 mL of 0.8% aqueous two-thiobarbituric acid to be further incubated at 70 C for 30 minutes. After incubation, the mixture was cooled down to room temperature, and conventional spectrophotometric analysis (Shimadzu, Model UV-160A, Tokyo, Japan) was performed in the range of 400 to 650 nm, with a scanning speed of 480 nm/min. Thirdorder derivative spectra were obtained by electronic differentiation (derivative difference setting, 21 nm) of the conventional absorption spectra of samples from both groups. MDA concentration (nmol/g wet tissue) was calculated on the basis of the third-order derivative peak height at 532 nm by referring to the slope and intercept data of the computed least-squares fit of standard calibration curve. Repeated measures analysis of variance with Tukey post hoc tests was used to study the change in MDA levels during the study in the two groups. Two-sided P values less than 0.05 were considered statistically significant. Statistical analysis was performed using Statistica 8 (StatSoft Inc., Tulsa, OK).
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RESULTS MDA levels in lung tissue (nmol/g) at the different study points are depicted in Figure 1. Comparison of the AT-III versus the collapsed placebo groups (group A and B) produced statistically significant differences (P ¼ 0.005). Measurements changed significantly over time (P ¼ 0.007). There was a significant time group interaction (P ¼ 0.04). Tukey post hoc tests for the comparison of the two groups at different time points revealed that lung MDA levels in the AT-III group were significantly reduced compared with the placebo group at 0 and at 30 minutes, but not at 4 hours (Table I). MDA levels in myocardial tissue (nmol/g) plotted against time are presented in Figure 2. Measurements did not change significantly over time (P ¼ 0.22), and there was no time group interaction (P ¼ 0.71). Myocardial MDA levels were not significantly different between the AT-III and placebo groups at 0 minutes, 30 minutes, and 4 hours based on Tukey post hoc test (Table II).
DISCUSSION The potential protective effect of AT-III as an antioxidant has not previously been investigated in remote ischemiaereperfusion injury. This study is the first to provide evidence that pretreatment with AT-III in an experimental animal model of hind limb ischemiaereperfusion may attenuate the harmful effects of remote ischemia in the lungs and, less so, the myocardium through the reduction of oxygen free radicals. We used MDA levels, the biochemical marker of the lipid hyperoxidation, to determine whether there is any effect on oxygen free radicals in the lungs and the heart during ischemiaereperfusion syndrome. In the lung tissue, MDA levels were found to be significantly lower when AT-III had been administered. Because the lower the MDA levels are, the less extensive the oxidant damage to the tissues is, the aforementioned finding indicates a protective effect of AT-III against the ischemia/reperfusion-induced lung injury. This protective effect was present at 0 and 30 minutes after reperfusion, but not at 4 hours. The half time of AT III is 2.3e4.8 days under normal circumstances in healthy individuals, but could decrease down to a few hours in acute inflammation or severe sepsis.13,14 This possible reduction in the AT-III half time as a result of the pronounced inflammatory response induced by the ischemiae reperfusion may explain the lack of statistically significant differences at 4 hours after reperfusion. As the present experimental protocol included no
Fig. 1. Malondialdehyde levels in lung tissue (nmol/g) plotted against time from reperfusion (0 minutes, 30 minutes, and 4 hours) after administration of either antithrombin III (group A) or placebo (group B). Values are presented as means with 95% confidence intervals.
other subgroups between 30 minutes and 4 hours, it is impossible to ascertain at which time point AT-III is losing its protective action. Another important parameter that may have an impact on the findings is the dose of AT-III that was administered as a pretreatment in this study, that is, a single dose of 250 mg/kg. Studies investigating the anti-inflammatory action of AT-III in septic patients suggested that AT-III was effective in a subgroup of patients in septic shock, but not in all septic patients. However, the concentrations were different when the treatment course lasted for 5 days. Higher plasma levels of AT-III should be reached to achieve the expected concentration and anti-inflammatory action.15 Whether the protective effect against ischemiaereperfusion at 0 and 30 minutes after reperfusion could be prolonged by higher and repeated doses of AT-III remains to be investigated. One unique feature of our study is that it is the first to investigate the systemic effect of AT-III in lower-limb ischemiaereperfusion by focusing on two remote organs, the lungs and the heart. Regarding the lungs, a previous experimental study suggested that pretreatment with AT-III in lowerlimb ischemiaereperfusion syndrome may not only reduce the local harmful effects but also protect from remote injury to the lungs owing to its antiinflammatory action.16 In another experimental study using a lung transplantation model, AT-III had been found to protect against injury to the lung by increasing the levels of prostaglandin I2.17 Finally, AT-III had been investigated in colonic anastomosis after ischemiaereperfusion of the bowel produced by occlusion of the superior mesenteric artery for 30 minutes. The investigators found that AT-III appears to attenuate the deleterious
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Table I. Malondialdehyde levels in lung tissue (nmol/g) at 0-minute, 30-minute, and 4-hour intervals after reperfusion in groups A and B
0 min 30 min 4 hr
Antithrombin III (group A)
Placebo (group B)
P
1.47 ± 0.32 (0.80e2.15) 2.17 ± 0.41 (1.31e3.03) 3.48 ± 0.37 (2.70e4.27)
3.23 ± 0.32 (2.56e3.91) 3.63 ± 0.41 (2.77e4.49) 3.55 ± 0.37 (2.77e4.34)
0.001* 0.02* 0.9
Results are expressed as means ± standard errors followed by 95% confidence intervals. Tukey post hoc test results are shown.
Fig. 2. Malondialdehyde levels in myocardial tissue (nmol/g) plotted against time from reperfusion (0 minutes, 30 minutes, and 4 hours) in the two groups. Values are presented as means with 95% confidence intervals.
effects of ischemiaereperfusion injury on healing of the colonic anastomosis.18 With respect to the myocardium, the potential protective effect of AT-III was not definitively proven because although there was a trend for reduced MDA levels in the AT-III group, this difference did not achieve statistical significance. Possible explanations are that the cardiac tissue is not affected as much as the lungs from the harmful effects of the lower-limb ischemiaereperfusion or that the neutrophil recruitment in the cardiac tissue is not as pronounced as in the lungs. Finally, it may be simply that AT-III is less protective of the myocardium than it is of the lungs. To date, there are no published studies in the literature that had investigated the protective role of AT-III against remote injury to the heart after ischemiaereperfusion. Therefore, the therapeutic potential of AT-III in the myocardial tissue should be further investigated by larger studies using different dose regimens. Another crucial factor that may influence the present study results is the duration of ischemia of 6 hours. It is well known that permanent damage to the muscle tissue may occur after 6 hours of complete ischemia and that the longer the ischemic time to the limb, the more pronounced and harmful
the local and systemic effects of the ischemiaereperfusion are. Perhaps, the protective effect of AT-III in lungs and myocardium would have been more evident if the duration of ischemia was longer. This relatively short ischemic time may also provide an explanation for the lack of statistical significance and the weak protective effect of AT-III on the heart. A follow-up project should clarify this question by testing the effect of AT-III on the lungs and heart using longer occlusion times. Previous studies, using similar experimental animal models of ischemiaereperfusion, investigated the protective effect of certain pharmacological agents other than AT-III, such as clopidogrel and simvastatin. Kanko et al. tested the potential therapeutic benefit of pretreatment with clopidogrel (in a dose of 0.2 mg/kg/d through nasogastric tube) for 10 days before ischemiaereperfusion.5 Apart from estimating MDA levels, investigators also measured the endogenous antioxidants glutathione and superoxide dismutase. The latter two are among the first line of defense mechanisms that cells are armed with to defend tissues against free radicals. Clopidogrel was shown to prevent the increase in MDA levels and the decrease in glutathione level and superoxide dismutase activity caused by ischemiaereperfusion. These findings suggest that clopidogrel is beneficial in prevention of ischemiaereperfusion injury, probably through its effects on inflammatory cells, platelets, and endothelial cells. In another study, Sun et al. suggested that simvastatin might have a protective effect against the deleterious effects of ischemiaereperfusion. They used a model of bilateral limb ischemia for 2 hours and reperfusion time of 3 hours.19 Given these positive results, it would be interesting to test in the future whether the beneficial effects of AT-III could be potentiated when administered in combination with clopidogrel, simvastatin, and other known drugs in lower-limb ischemiaereperfusion syndrome. Finally, one might argue that after 6 hours of external occlusion of the hind limb, the possibility of thrombus formation and of pulmonary embolism
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Table II. Malondialdehyde levels in myocardial tissue (nmol/g) at 0-minute, 30-minute, and 4-hour intervals after reperfusion in groups A and B
0 min 30 min 4 hr
Antithrombin III (group A)
Placebo (group B)
P
1.22 ± 0.28 (0.62e1.81) 1.54 ± 0.18 (1.15e1.93) 1.84 ± 0.23 (1.36e2.32)
2.01 ± 0.28 (1.41e2.60) 2.05 ± 0.18 (1.66e2.44) 2.24 ± 0.23 (1.76e2.72)
0.07 0.07 0.2
Results are expressed as means ± standard errors followed by 95% confidence intervals. Tukey post hoc test results are shown.
has to be excluded. AT-III is a well-known inhibitor in the coagulation cascade, and it may be that the group receiving AT-III was more protected in that respect compared with the placebo group. Although we did not look specifically for venous thrombosis or pulmonary embolism, there was no evidence of the latter in any of the lung specimens that were prepared for optical and electron microscopy (either in the AT-III or the placebo group). In conclusion, the potential protective effect of AT-III as an antioxidant through the decrease of oxygen free radicals has not been investigated previously in remote ischemiaereperfusion injury. The results of this study suggest that pretreatment with AT-III 30 minutes before the reperfusion of an acutely ischemic limb can reduce the oxidant stress to lung and, less so, to myocardial tissue owing to its anti-inflammatory and antioxidant action.
REFERENCES 1. Lindsay T, Hill J, Wiles M, et al. Antamadine reduces hindlimb ischemiaeinduced PMN sequestration and lung permeability. Surg Forum 1991;42:32e3. 2. Zhou W, Zeng D, Chen R, et al. Limb ischemic preconditioning reduces heart and lung injury after an open heart operation in infants. Pediatr Cardiol 2010;31:22e9. 3. Plitas G, Gange JP, Muhs EB, et al. Experimental hind limb ischemia increases neutrophil-mediated matrix metalloproteinase activity: a potential mechanism for lung injury after limb ischemia. J Am Coll Surg 2003;196:761e7. 4. Draper HH, Hardley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186: 421e31. 5. Kanko M, Maral H, Akbas HA, et al. Protective effects of Clopidogrel on oxidant damage in a rat model of acute ischemia. Tohoku J Exp Med 2005;205:133e9. 6. Koca U, Olguner G, Ozkardesler S, et al. Antithrombin III pretreatment reduces neutrophil recruitment into the lung in a rat model of abdominal sepsis. Acta Anaesthesiol Scand 2005;49:203e8.
7. Ostrovsky L, Woodman RC, Payne D, et al. Antithrombin III prevents and rapidly reverses leukocyte recruitment in ischemia/reperfusion. Circulation 1997;96:2302e10. 8. Dunzendorfer S, Kaneider N, Rabensteiner A, et al. Cellsurface heparan sulfate proteoglycanemediated regulation of human neutrophil migration by the serpin antithrombin III. Blood 2001;97:1079e85. 9. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Guide for the care and use of laboratory animals. Washington, DC: National Academic Press, 1996. 1e124. 10. Mathru M, Dries DJ, Barnes L, et al. Tourniquet induced exsanguination in patients requiring lower limb surgery: an ischemia reperfusion model of oxidant and antioxidant metabolism. Anesthesiology 1996;84:14e22. 11. Babuccu O, Peksoy I, Hoknuter M, et al. Evaluation by scintigraphy of hindlimb ischemia in a rat model. J Reconstr Microsurg 2004;20:405e10. 12. Botsoglou NA, Fletouris DJ, Papageorgiou GE, et al. Rapid, sensitive and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food and feedstuff samples. J Agric Food Chem 1994;42:1931e6. 13. Ilias W, List W, Decruyenaere J, et al. Antithrombin III in patients with severe sepsis: a pharmacokinetic study. Intensive Care Med 2000;26:704e15. 14. Baudo F, Caimi TM, de Cataldo F, et al. Antithrombin III (ATIII) replacement therapy in patients with sepsis and/or postsurgical complications: a controlled double-blind, randomized, multicenter study. Intensive Care Med 1998;24:336e42. 15. Eisele B, Lamy M, Thijs LG, et al. Antithrombin III in patients with severe sepsis. Intensive Care Med 1998;24:663e72. 16. Duru S, Koca U, Oztekin S, et al. Antithrombin III pretreatment reduces neutrophil recruitment into the lung and skeletal muscle tissues in the rat model of bilateral lower limb ischemia and reperfusion: a pilot study. Acta Anaesthesiol Scand 2005;49:1142e8. 17. Salvatierra A, Guerrero R, Rodriguez M, et al. Antithrombin III prevents early pulmonary dysfunction after lung transplantation in the dog. Circulation 2001;104:2975e80. 18. Tekin K, Aytekin OF, Ozden A, et al. Antithrombin III prevents deleterious effects of remote ischemiadreperfusion injury on healing of colonic anastomosis. Am J Surg 2002;184:160e5. 19. Sun XF, Wang LL, Wang JK, et al. Effects of Simvastatin on lung injury induced by ischaemiadreperfusion of the hind limbs in rats. J Int Med Res 2007;35:523e33.
Background: Restoration of blood flow to an acutely ischemic limb can trigger systemic inflammation. We investigated whether antithrombin III (AT-III) exerts a protective action against remote lung and myocardial injury in an experimental animal model of lower-limb ischemiae reperfusion. Methods: Ischemia was induced by lower-limb arterial occlusion for 6 hours in 60 male Wistar rats. Animals were divided into those receiving AT-III (dose, 250 mg/kg) 30 minutes before the reperfusion (group A, n ¼ 30) and those receiving placebo (group B, n ¼ 30). Animals were then sacrificed, and lung and myocardial tissue samples were taken at baseline, 30 minutes, and 4 hours after reperfusion. Levels of malondialdehyde (MDA), a compound used as indirect index of oxygen free radicals, were estimated in lung and myocardium, and the two groups were compared at different time points using the independent sample t test. Results: Animals administered AT-III had significantly lower levels of lung MDA compared with the placebo group at baseline and at 30 minutes, but not at 4 hours (P ¼ 0.001, P ¼ 0.01, and P ¼ 0.9, respectively), indicating a protective action of AT-III against remote lung injury early in the reperfusion phase. With regard to myocardial MDA levels, no statistically significant differences existed between the AT-III and placebo groups at baseline, at 30 minutes, and at 4 hours (P ¼ 0.07, P ¼ 0.07, and P ¼ 0.2, respectively) after reperfusion. Conclusions: In this experimental animal model, AT-III appears to exert a protective effect against remote ischemiaereperfusion injury in the lung tissue, but not in the myocardium.
INTRODUCTION Acute lower-limb ischemia followed by reperfusion may be encountered in several clinical circumstances, such as abdominal aortic aneurysm repair, acute thromboembolism of the lower-limb arteries, trauma surgery, and in crushed victims being brought to the
Presented as an abstract at CHEST 2009, Annual Meeting of the American College of Chest Physicians, San Diego, CA, October 31 to November 5, 2009. 5th Department of Surgery, Medical School, Aristotle University of Thessaloniki, Hippocratio Hospital, Thessaloniki, Greece. Correspondence to: Christos D. Karkos, MD, FRCS, PhD, EBIR, 5th Department of Surgery, Medical School, Aristotle University of Thessaloniki, Hippocratio Hospital, Konstantinoupoleos 49, Thessaloniki 546 42, Greece; E-mail: [email protected] Ann Vasc Surg 2012; 26: 566–570 DOI: 10.1016/j.avsg.2012.01.004 Ó Annals of Vascular Surgery Inc.
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emergency room. Such clinical scenarios are associated with high mortality and morbidity rates owing to a systemic inflammatory response and multiple organ dysfunction occurring during the reperfusion phase. In particular, remote injury to the lungs and the myocardium after lower-limb ischemiaereperfusion is believed to represent one of the major causes for this high mortality.1,2 The pathogenesis of remote ischemiaereperfusion injury is complex; to date, no treatment has been proven to attenuate these deleterious effects. Systemic inflammatory response to ischemiae reperfusion involves polymorphonuclear leukocyte sequestration in the lungs and increased pulmonary microvascular permeability.3 Ischemiaereperfusion also triggers a chain of events that may result in cellular death caused by oxygen free radicals released in response to the exposure of oxygen.4,5
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In specific, oxygen free radicals are believed to cause lipid peroxidation, resulting in damage to the biological membranes. End products of lipid peroxidation include aldehydes, hydrocarbon gases, and malondialdehyde (MDA).4 Because quantifying oxygen free radicals is difficult because of their reactive nature and short lives, it has been suggested that MDA can be used instead to estimate the extent of oxidant damage to tissues.4,5 Previous investigators have used MDA levels to estimate the protective effects of pharmacological agents, such as clopidogrel, in ischemiaereperfusion of the muscle and remote ischemiaereperfusion of the lungs and liver.5 Antithrombin III (AT-III) is a serine protease inhibitor, which, in addition to its anticoagulant function, is believed to exhibit an anti-inflammatory activity in sepsis and in ischemiaereperfusion.6e8 However, the mechanism of its action is unclear. It has been suggested that pretreatment with AT-III may attenuate acute lung injury in intra-abdominal sepsis, probably by inhibiting leukocyte recruitment into the lung.6 Similarly, a study of a feline mesentery ischemiaereperfusion model demonstrated that AT-III can be used therapeutically after reperfusion to attenuate the leukocyte recruitment response in inflammation.7 The potential protective effect of AT-III as an antioxidant by decreasing the free radicals has not previously been investigated in remote ischemiaereperfusion injury. In this study, we investigated the possible anti-inflammatory action of AT-III on remote lung and myocardial injury by assessing MDA levels in an experimental animal model of hind limb ischemiaereperfusion.
METHODS Sixty male Wistar rats weighing between 250 and 300 g were included in this study. The rats were cared for in accordance with the Guide for the Care and Use of Laboratory Animals,9 kept under a 12-hour light/dark cycle, and permitted ad libitum access to standard laboratory rodent chow and tap water for 2 weeks before the beginning of the experimental procedure. The rats were anesthetized with ether (diethyl ether, Panreac Quimica SAU, Barcelona, Spain) and then weighted. Ischemia was induced by the application of a polypropylene suture (Prolene 0, Ethicon, Inc, a Johnson & Johnson company, Gargrave, England) on the right hind limb, and the absence of flow was checked by Doppler ultrasound (Huntleigh Diagnostics Ltd, South Glamorgan, UK). A previous in-house study, using both Doppler ultrasound and arteriography,
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567
verified that the tourniquet-like application of a nylon suture produces almost complete limb ischemia. Similarly, previous studies by other investigators had shown that this method reduces blood flow by 98% and leads to acute ischemia.5,10,11 AT-III (or placebo) was administered intravenously through the penile vein 5.5 hours after the induction of ischemia and 30 minutes before the reperfusion. The animals were divided in two groups of 30. Group A received AT-III (dose, 250 mg/kg) and group B received placebo (NaCl, 0.9%). Reperfusion was begun after 6 hours of ischemia by removing the nylon suture. After the rats had been anesthetized, a midline sternotomy was performed. The lungs and heart were removed, and MDA levels were determined. The two groups were further divided into three subgroups of 10, depending on the time point when lung and myocardium tissue samples had been taken for determination of MDA levels after blood flow restoration: subgroups A1 and B1 immediately after reperfusion (‘‘zero’’ time), subgroups A2 and B2 at 30 minutes, and subgroups A3 and B3 at 4 hours after restoration of blood flow. Determination of MDA levels was performed using a selective third-order derivative method.12 In brief, 1-g samples were thoroughly homogenized (Polytron homogenizer, PCU, Kinematica, Lucerne, Switzerland) with 5 mL of 5% aqueous trichloroacetic acid, and 2 mL of 0.8% butylated hydroxytoluene in hexane was added and the mixture was centrifuged. The top layer was discarded, and a 2.5-mL aliquot from the bottom layer was mixed with 1.5 mL of 0.8% aqueous two-thiobarbituric acid to be further incubated at 70 C for 30 minutes. After incubation, the mixture was cooled down to room temperature, and conventional spectrophotometric analysis (Shimadzu, Model UV-160A, Tokyo, Japan) was performed in the range of 400 to 650 nm, with a scanning speed of 480 nm/min. Thirdorder derivative spectra were obtained by electronic differentiation (derivative difference setting, 21 nm) of the conventional absorption spectra of samples from both groups. MDA concentration (nmol/g wet tissue) was calculated on the basis of the third-order derivative peak height at 532 nm by referring to the slope and intercept data of the computed least-squares fit of standard calibration curve. Repeated measures analysis of variance with Tukey post hoc tests was used to study the change in MDA levels during the study in the two groups. Two-sided P values less than 0.05 were considered statistically significant. Statistical analysis was performed using Statistica 8 (StatSoft Inc., Tulsa, OK).
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RESULTS MDA levels in lung tissue (nmol/g) at the different study points are depicted in Figure 1. Comparison of the AT-III versus the collapsed placebo groups (group A and B) produced statistically significant differences (P ¼ 0.005). Measurements changed significantly over time (P ¼ 0.007). There was a significant time group interaction (P ¼ 0.04). Tukey post hoc tests for the comparison of the two groups at different time points revealed that lung MDA levels in the AT-III group were significantly reduced compared with the placebo group at 0 and at 30 minutes, but not at 4 hours (Table I). MDA levels in myocardial tissue (nmol/g) plotted against time are presented in Figure 2. Measurements did not change significantly over time (P ¼ 0.22), and there was no time group interaction (P ¼ 0.71). Myocardial MDA levels were not significantly different between the AT-III and placebo groups at 0 minutes, 30 minutes, and 4 hours based on Tukey post hoc test (Table II).
DISCUSSION The potential protective effect of AT-III as an antioxidant has not previously been investigated in remote ischemiaereperfusion injury. This study is the first to provide evidence that pretreatment with AT-III in an experimental animal model of hind limb ischemiaereperfusion may attenuate the harmful effects of remote ischemia in the lungs and, less so, the myocardium through the reduction of oxygen free radicals. We used MDA levels, the biochemical marker of the lipid hyperoxidation, to determine whether there is any effect on oxygen free radicals in the lungs and the heart during ischemiaereperfusion syndrome. In the lung tissue, MDA levels were found to be significantly lower when AT-III had been administered. Because the lower the MDA levels are, the less extensive the oxidant damage to the tissues is, the aforementioned finding indicates a protective effect of AT-III against the ischemia/reperfusion-induced lung injury. This protective effect was present at 0 and 30 minutes after reperfusion, but not at 4 hours. The half time of AT III is 2.3e4.8 days under normal circumstances in healthy individuals, but could decrease down to a few hours in acute inflammation or severe sepsis.13,14 This possible reduction in the AT-III half time as a result of the pronounced inflammatory response induced by the ischemiae reperfusion may explain the lack of statistically significant differences at 4 hours after reperfusion. As the present experimental protocol included no
Fig. 1. Malondialdehyde levels in lung tissue (nmol/g) plotted against time from reperfusion (0 minutes, 30 minutes, and 4 hours) after administration of either antithrombin III (group A) or placebo (group B). Values are presented as means with 95% confidence intervals.
other subgroups between 30 minutes and 4 hours, it is impossible to ascertain at which time point AT-III is losing its protective action. Another important parameter that may have an impact on the findings is the dose of AT-III that was administered as a pretreatment in this study, that is, a single dose of 250 mg/kg. Studies investigating the anti-inflammatory action of AT-III in septic patients suggested that AT-III was effective in a subgroup of patients in septic shock, but not in all septic patients. However, the concentrations were different when the treatment course lasted for 5 days. Higher plasma levels of AT-III should be reached to achieve the expected concentration and anti-inflammatory action.15 Whether the protective effect against ischemiaereperfusion at 0 and 30 minutes after reperfusion could be prolonged by higher and repeated doses of AT-III remains to be investigated. One unique feature of our study is that it is the first to investigate the systemic effect of AT-III in lower-limb ischemiaereperfusion by focusing on two remote organs, the lungs and the heart. Regarding the lungs, a previous experimental study suggested that pretreatment with AT-III in lowerlimb ischemiaereperfusion syndrome may not only reduce the local harmful effects but also protect from remote injury to the lungs owing to its antiinflammatory action.16 In another experimental study using a lung transplantation model, AT-III had been found to protect against injury to the lung by increasing the levels of prostaglandin I2.17 Finally, AT-III had been investigated in colonic anastomosis after ischemiaereperfusion of the bowel produced by occlusion of the superior mesenteric artery for 30 minutes. The investigators found that AT-III appears to attenuate the deleterious
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Table I. Malondialdehyde levels in lung tissue (nmol/g) at 0-minute, 30-minute, and 4-hour intervals after reperfusion in groups A and B
0 min 30 min 4 hr
Antithrombin III (group A)
Placebo (group B)
P
1.47 ± 0.32 (0.80e2.15) 2.17 ± 0.41 (1.31e3.03) 3.48 ± 0.37 (2.70e4.27)
3.23 ± 0.32 (2.56e3.91) 3.63 ± 0.41 (2.77e4.49) 3.55 ± 0.37 (2.77e4.34)
0.001* 0.02* 0.9
Results are expressed as means ± standard errors followed by 95% confidence intervals. Tukey post hoc test results are shown.
Fig. 2. Malondialdehyde levels in myocardial tissue (nmol/g) plotted against time from reperfusion (0 minutes, 30 minutes, and 4 hours) in the two groups. Values are presented as means with 95% confidence intervals.
effects of ischemiaereperfusion injury on healing of the colonic anastomosis.18 With respect to the myocardium, the potential protective effect of AT-III was not definitively proven because although there was a trend for reduced MDA levels in the AT-III group, this difference did not achieve statistical significance. Possible explanations are that the cardiac tissue is not affected as much as the lungs from the harmful effects of the lower-limb ischemiaereperfusion or that the neutrophil recruitment in the cardiac tissue is not as pronounced as in the lungs. Finally, it may be simply that AT-III is less protective of the myocardium than it is of the lungs. To date, there are no published studies in the literature that had investigated the protective role of AT-III against remote injury to the heart after ischemiaereperfusion. Therefore, the therapeutic potential of AT-III in the myocardial tissue should be further investigated by larger studies using different dose regimens. Another crucial factor that may influence the present study results is the duration of ischemia of 6 hours. It is well known that permanent damage to the muscle tissue may occur after 6 hours of complete ischemia and that the longer the ischemic time to the limb, the more pronounced and harmful
the local and systemic effects of the ischemiaereperfusion are. Perhaps, the protective effect of AT-III in lungs and myocardium would have been more evident if the duration of ischemia was longer. This relatively short ischemic time may also provide an explanation for the lack of statistical significance and the weak protective effect of AT-III on the heart. A follow-up project should clarify this question by testing the effect of AT-III on the lungs and heart using longer occlusion times. Previous studies, using similar experimental animal models of ischemiaereperfusion, investigated the protective effect of certain pharmacological agents other than AT-III, such as clopidogrel and simvastatin. Kanko et al. tested the potential therapeutic benefit of pretreatment with clopidogrel (in a dose of 0.2 mg/kg/d through nasogastric tube) for 10 days before ischemiaereperfusion.5 Apart from estimating MDA levels, investigators also measured the endogenous antioxidants glutathione and superoxide dismutase. The latter two are among the first line of defense mechanisms that cells are armed with to defend tissues against free radicals. Clopidogrel was shown to prevent the increase in MDA levels and the decrease in glutathione level and superoxide dismutase activity caused by ischemiaereperfusion. These findings suggest that clopidogrel is beneficial in prevention of ischemiaereperfusion injury, probably through its effects on inflammatory cells, platelets, and endothelial cells. In another study, Sun et al. suggested that simvastatin might have a protective effect against the deleterious effects of ischemiaereperfusion. They used a model of bilateral limb ischemia for 2 hours and reperfusion time of 3 hours.19 Given these positive results, it would be interesting to test in the future whether the beneficial effects of AT-III could be potentiated when administered in combination with clopidogrel, simvastatin, and other known drugs in lower-limb ischemiaereperfusion syndrome. Finally, one might argue that after 6 hours of external occlusion of the hind limb, the possibility of thrombus formation and of pulmonary embolism
570 Zambas et al.
Annals of Vascular Surgery
Table II. Malondialdehyde levels in myocardial tissue (nmol/g) at 0-minute, 30-minute, and 4-hour intervals after reperfusion in groups A and B
0 min 30 min 4 hr
Antithrombin III (group A)
Placebo (group B)
P
1.22 ± 0.28 (0.62e1.81) 1.54 ± 0.18 (1.15e1.93) 1.84 ± 0.23 (1.36e2.32)
2.01 ± 0.28 (1.41e2.60) 2.05 ± 0.18 (1.66e2.44) 2.24 ± 0.23 (1.76e2.72)
0.07 0.07 0.2
Results are expressed as means ± standard errors followed by 95% confidence intervals. Tukey post hoc test results are shown.
has to be excluded. AT-III is a well-known inhibitor in the coagulation cascade, and it may be that the group receiving AT-III was more protected in that respect compared with the placebo group. Although we did not look specifically for venous thrombosis or pulmonary embolism, there was no evidence of the latter in any of the lung specimens that were prepared for optical and electron microscopy (either in the AT-III or the placebo group). In conclusion, the potential protective effect of AT-III as an antioxidant through the decrease of oxygen free radicals has not been investigated previously in remote ischemiaereperfusion injury. The results of this study suggest that pretreatment with AT-III 30 minutes before the reperfusion of an acutely ischemic limb can reduce the oxidant stress to lung and, less so, to myocardial tissue owing to its anti-inflammatory and antioxidant action.
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