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reperfusion injury by N-acetylcysteine in a rat hind limb model
Journal of Surgical Research 111, 236 –239 (2003) doi:10.1016/S0022-4804(03)00094-5
Attenuation of Ischemia/Reperfusion Injury by N-Acetylcysteine in a Rat Hind Limb Model Cengiz Koksal, M.D.,* ,1 A. Kursat Bozkurt, Assos. Prof.,† Ugur Cangel, M.D.,‡ Nil Ustundag, M.D.,§ Dildar Konukogˇlu, Assos. Prof., ¶ Benan Musellim, M.D.,㛳 and Ayla Gurel Sayın, Prof.** *Sureyyapasa Thoracic and Cardiovascular Disease Hospital, Dept. of Cardiovascular Surgery, Istanbul, Turkey, †Istanbul University, Cerrahpasa Medical Faculty, Dept. of Cardiovascular Surgery, Istanbul, Turkey, ‡Istanbul Memorial Hospital, Dept. of Cardiovascular Surgery, Istanbul, Turkey, §Bolu Izzet Baysal University, Medical Faculty, Dept. of Pathology, Bolu, Turkey, ¶Istanbul University, Cerrahpasa Medical Faculty, Dept. of Biochemistry, Istanbul, Turkey, 㛳Istanbul University, Cerrahpasa Medical Faculty, Dept. of Chest Medicine, Istanbul, Turkey, and **Istanbul University, Cerrahpasa Medical Faculty, Dept. of Cardiovascular Surgery, Istanbul, Turkey Submitted for publication August 23, 2002
Background. Ischemia/reperfusion is a complex set of events with severe pathologic consequences. Reperfusion initiates both the local and systemic damage in part through rapid oxygen generation. N-acetylcysteine (NAC) is a scavenger of free radical species, inhibits neutrophil accumulation, acts as a vasodilator and also improves microcirculation. In present study, we examined the protective effect of NAC in a rat hind limb ischemia/ reperfusion model. Dimethyl-sulfoxide (DMSO), a well-known antioxidant was also tested for comparison. Materials and methods. Ischemia was induced for 4 h by vascular clamping and followed by 1 h of reperfusion. Muscle injury was evaluated in 3 groups as a saline group (control), DMSO group, and NAC group. Plasma levels of creatine kinase, lactate dehydrogenase, thiobarbituric acid reactive substances (TBARS), and blood HCO 3, as well as muscle tissue TBARS, were measured at the end of reperfusion. Muscle tissue samples were taken for histological evaluation. Results. DMSO and NAC group showed significant amelioration of plasma CPK (P < 0.05, P < 0.05), plasma TBARS (P < 0.05, P < 0.05), and muscle tissue TBARS (P < 0.05, P < 0.05) compared with the control group. Similarly, neutrophil infiltration in DMSO and NAC groups were significantly less prominent than the control group (P < 0.01, P < 0.01). Conclusions. These results show that NAC improved effectively ischemia reperfusion injury in a rat hind limb model. © 2003 Elsevier Inc. All rights reserved. 1 To whom correspondence should be addressed at P.O. Box 26, Cerrahpasa, 34301-Istanbul/Turkey. E-mail: cengizkoksal@ hotmail.com.
0022-4804/03 $35.00 © 2003 Elsevier Inc. All rights reserved.
Key Words: N-acetylcysteine; ischemia/reperfusion injury; dimethyl sulfoxide; thiobarbituric acid reactive substances; histological examination.
INTRODUCTION
Management of acute skeletal muscle ischemia continues to be a formidable challenge for the vascular surgeon. Despite improved efficiency of invasive and noninvasive treatment modalities, the overwhelming impact of reperfusion has still to be overcome. If the mass of the ischemic tissue is large enough, such as the legs, reperfusion results not only in local damage but also remote organ injury. It is known that the major part of the damage occurs during the reperfusion period and that reactive oxygen substances (ROS) are responsible for the tissue injury [1, 2]. Several antioxidants like superoxide dismutase, catalase, mannitol, dimethyl sulfoxide (DMSO), and iloprost (a long-acting prostacyclin analogue) have proven to be efficient in attenuating the changes in microvascular permeability, which is the final outcome of ischemia/reperfusion (I/R) injury [3, 4]. Belkin et al. demonstrated reduction in the size of skeletal muscle infarcts in a canine gracilis muscle model with the use of iloprost [5]. Karthuis et al. also, in their experimental I/R injury study in canine skeletal muscle, showed attenuation in the increase of vascular permeability when subjected to pretreatment with DMSO [3]. N-acetylcysteine (NAC) is not simply an antioxidant drug. It acts as a glutathione (GSH) precursor, as a chemical reductant of oxidized thiols, as a scavenger of radical oxygen species, as a vasodilator, and also im-
236
KOKSAL ET AL.: N-ACETYLCYSTEIN ATTENUATES ISCHEMIA/REPERFUSION INJURY
proves microcirculation by restoring the decreased activity of endothelium-derived relaxing factor (EDRF) and may have antiaggregan features [6 – 8]. There is growing evidence regarding its beneficial effects in ameliorating lung I/R injury [9]. However its role in reducing the damage in skeletal muscle tissue of the involved extremity has not been addressed yet. In this experimental study, we aimed to examine the protective effect of NAC against I/R injury in a rat skeletal muscle model. DMSO, which is a well-established antioxidant, is also tested to prove the effectiveness of the experimental model.
237
TABLE 1 Comparison of DMSO and NAC Groups with the Control Group (mean ⴞ standard deviation)
HCO 3 (mmol/L) LDH (U/L) CPK (U/L) Plasma TBARS (nmol/mL) Muscle TBARS (nmol/mL)
Control group
DMSO group
NAC group
9.5 ⫾ 3.2 638 ⫾ 425 1705 ⫾ 228 3.83 ⫾ 0.14
15.4 ⫾ 5.8* 312 ⫾ 81 817 ⫾ 272* 3.19 ⫾ 0.37*
14.4 ⫾ 4.3* 350 ⫾ 78 760 ⫾ 140* 3.30 ⫾ 0.89*
303.65 ⫾ 82.3
193.8 ⫾ 13.8*
185.1 ⫾ 12*
* P ⬍ 0.05.
MATERIALS AND METHODS This study was conducted according to the guidelines of the animal care review board of Istanbul University Cerrahpasa Medical Faculty with adherence to the guide for care and use of laboratory animals.
Ischemia/Reperfusion Model Eighteen mature male Sprague–Dawley rats weighing 250 to 350 g were used. All animals had free access to standard rat chow and water. They were anesthetized with intraperitoneal ketamine (100 mg/kg body weight; Abbott Laboratories, North Chicago, Ill) and additional doses of ketamine (50 mg/kg) were given to sustain anesthesia throughout the experiment. Using magnifying spectacles (⫻3.5) bilateral groin incisions were made and the iliac arteries were dissected distally. All tissues except artery and vein were transected to eliminate the collateral blood supply from the rats’ pelvis. Right iliac artery was spared for clamping and the left iliac vein was cannulated with 24-G cannula for blood sampling and drug administration. Ischemia was induced with a 4-h period of iliac artery occlusion with a nontraumatic vascular clamp and followed by 1 h of reperfusion.
Experimental Groups Rats were divided into three groups. Group 1 (n ⫽ 6, control group) received 2 mL/kg/h of continuous saline with the induction of anesthesia and used as controls. Group 2 (n ⫽ 6, DMSO group) received dimethyl sulfoxide (Sigma Chemical, St. Louis, Mo; 15 mg/kg/h) continuously in the same manner. Group 3 (n ⫽ 6, NAC group) received 20 mg/kg NAC (Zambon GmBH, Grafelfing, Germany) 5 min before reperfusion and followed by an infusion of 20 mg/kg/h. Plasma creatine kinase (CPK, IU/L), lactate dehydrogenase (LDH, IU/L), bicarbonate (HCO3, mmol/L) and thiobarbituric acid reactive substances (TBARS, nmol/mL, as a marker of lipid peroxidation) were determined at the end of the reperfusion period. After the reperfusion, rats were sacrificed. Samples were taken from gastrocnemius muscle to determine TBARS levels (nmol/g wet tissue). Muscle tissue samples were also taken for histological examination.
Tissue Peroxide Level Analysis Approximately 1 g of thawed tissue samples was homogenized in 10 mL of 10 nM sodium phosphate buffer (pH 7.4) using a homogenizer. One ml aliquot of tissue homogenate was used for the determination of lipid peroxide level by measuring spectrophotometrically the formation of TBARS. The intra-and interassay coefficients of variation for TBARS were 4.7% and 4.9%, respectively [10].
Plasma Analysis Blood HCO 3 value was determined on Ciba Corning 860 blood gas analyzer. (Ciba Corning Diagnostics Corporation, Medfiels, Mass). Plasma CPK and LDH assays were performed on the Hitachi system 717 automated analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan).
Histological Analysis Muscle biopsies were taken at the end of the experiment into 10% buffered formalin. Using standard techniques, paraffin sections were obtained, stained with hematoxylin and eosin, and studied under light microscopy by a pathologist in a blinded manner. Histological changes were scored on a scale from 0 to 3 where 0 ⫽ absence (⬍5% of maximum pathology), 1 ⫽ mild (⬍10%), 2 ⫽ moderate (15–20%), and 3 ⫽ severe (20 –25%) [11]. A total of four slides from each muscle sample were randomly screened, and the mean was accepted as the representative value of the sample. Severity of neutrophil infiltration was estimated and calculated for each experiment.
Data Analysis Apparent differences between control and experimental groups were analyzed for their statistical significance by the use of Kruskall–Wallis test. For multiple comparisons Bonferroni correction was used. Level of significance was decreased to 0.016 and results were analyzed by Mann–Whitney U Test. P ⬍ 0.05 considered statistically significant.
RESULTS
Biochemical variables at the end of the reperfusion period are shown in Table 1. No significant difference was encountered among control and treatment groups in terms of LDH values. Plasma CPK values were significantly higher and HCO 3 values were significantly lower in control group compared with DMSO and NAC groups (P ⬍ 0.05) and the results of NAC and DMSO groups were comparable. Plasma TBARS values and muscle TBARS values were found to be significantly higher in control group rats compared with both treatment group rats (P ⬍ 0.05, P ⬍ 0.05, respectively). Also, the results of both TBARS values (plasma and muscle) were comparable in DMSO and NAC groups.
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Median neutrophil infiltration scores for muscle injury of DMSO and NAC was 1, whereas it was 2 for the control group, which revealed significantly less prominent muscle injury in both DMSO and NAC groups, compared with the control group (P ⬍ 0.01). DISCUSSION
The establishment of reflow replenishes vital substances to the tissue but also releases into the circulation toxic metabolites that have been generated during the no-flow state. Damage to the involved extremity itself and remote organs can be induced with these compounds. Many mediators have been implicated in the process of inducing I/R injury. ROS are perhaps the most pertinent agents in this matter [12]. In their experimental brain I/R injury study in gerbils, Cuzzocrea et al. encountered an increase in MDA levels, which is a well-known product of lipid peroxidation [13]. Their observation was in agreement with Capali et al., who showed similar elevated levels of lipid peroxidation products in the same experimental model [14]. There is now a large body of evidence suggesting that postischemic oxidative injury is caused by an imbalance between a burst of ROS production and the inability of reoxygenated cells to handle this radical load, because of an ischemia induced depression of the naturally present defense mechanisms against oxygen toxicity [6]. Eventually oxidants cause injury and cell death by modifying and/or disturbing the structure and function of any cellular or noncellular component. The protective effect NAC in acute lung injury is well established in clinical and experimental models. In an experimental study Boerjesson et al. showed that treatment with NAC prevents intestinal IR induced over activation of pulmonary macrophages and decreases pulmonary content [15]. This observation was supported by Weinbraum et al., which demonstrated, pretreatment of the lungs with NAC during reperfusion period with ischemic/reperfused liver effluent prevents acute lung injury [9]. In a randomized clinical trial on patients with acute respiratory distress syndrome, NAC enhanced recovery from acute lung injury by GSH deficiency [16]. There are also several studies concerning beneficial role of NAC in ameliorating myocardial and brain injury [13, 17]. Several studies have pointed out the activation and infiltration of neutrophils as one of the initial events of tissue injury, which triggers the release of ROS with subsequent tissue injury [18]. In search of an ideal drug for attenuation of IR injury, the cause of neutrophil activation and accumulation, as well as the consequence of ROS mediated injury, should be taken into consideration. The ideal drug should not be solely an antioxidant. The rationale behind supplying an ischemic tissue with compounds that can replenish the intracellular stores of GSH, thereby allowing preserva-
tion of the thiol-disulfide equilibrium, seems to be more clinically relevant. NAC is particularly attractive for many reasons. The thiol NAC works in the extracellular environment and is a precursor of intracellular cysteine and GSH. Besides its activity as a GSH precursor, NAC is, per se, responsible for protective effects in the extracellular environment, mainly due to its nucleophilic and antioxidant properties [19]. NAC acts by either directly interfering with the oxidants or upregulating antioxidant systems such as superoxide dismutase or enhancing the catalytic activity of glutathione peroxidase [20]. Apart from antioxidant effect, NAC has vasodilator effect and may act as an antiagregan, by restoring the decreased activity of EDRF, thereby improving microcirculation and tissue oxygenation [8, 16, 21]. EDRF, synthesized by endothelial cells, relaxes vascular smooth muscle and inhibits the aggregation, adhesion of platelets, an action potentiated by NAC. NAC, by replenishing tissue sulfhydryl groups either directly or by increasing cystine levels, could restore the full activity of EDRF by a mechanism similar to its reversal of tolerance to nitrates [7]. Activation and accumulation of neutrophils’, which triggers the release of ROS was ameliorated by NAC in the ischemic brain injury model in gerbils by Cuzzocrea et al. [13]. Also, Inhibition of neutrophil infiltration by NAC during cardiopulmonary bypass was shown in patients undergoing cardiac surgery by Andersen et al. [22]. In the current study we aimed to search the role of NAC in skeletal muscle I/R injury. Muscle and plasma MDA levels (TBARS) were significantly lower in NAC group than the control group (P ⬍ 0.05, P ⬍ 0.05; respectively), which suggested the efficacy of NAC as an antioxidant in ameliorating I/R. Neutrophil infiltration was significantly less prominent histologically in NAC group than the control group (P ⬍ 0.01). Reduction of MDA in blood and skeletal muscle tissue correlated well with diminished neutrophil infiltration in the skeletal muscle at histological examination, in both NAC and DMSO groups. The results of NAC group were comparable with DMSO group. The results of the present study supports the view that NAC can exert a protective effect against skeletal muscle injury caused by IR in the rats. NAC is not solely an antioxidant, but also inhibits neutrophil infiltration and improves microcirculation and may be more beneficial than other antioxidants in case of I/R injury. These encouraging results suggest the possibility of clinical application of NAC following skeletal muscle I/R injury. REFERENCES 1.
McCard, J. M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312: 159, 1985.
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Granger, D. N. Role of xnathine oxidase and granulocytes in ischemia-reperfusion injury. Am. J. Physiol. 255: H1269, 1988. Karthuis, R. J., Granger, D. N., Townsley, M. I., and Taylor, A. E. The role of oxygen-derived free radicals in ischemiainduced increases in canine skeletal muscle vascular permeability. Circ. Res. 57: 599, 1985. Blebea, J., Cambria, R. A., Defouw, D., Feinberg, R. N., Hobson, R. W., and Duran, W. N. Iloprost attenuates the increased permeability in skeletal muscle after ischemia and reperfusion. J. Vasc. Surg. 12: 657, 1990. Belkin, M., Wright, G., and Hobson, R. W. Iloprost infusion decreases skeletal muscle ischemia-reperfusion injury. J. Vasc. Surg. 11: 77, 1990. Tredger, J. M. N-Acetylcysteine: Not a simply glutathione precursor. Transplantation 69: 703, 2000. Harrison, P. M., Wendon, J. A., Gimson, A. E., Alexander, G. J., and Williams, R. Improvement by Acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure. N. Engl. J. Med. 324: 1852, 1991. Arstall, M. A., Yang, J., Stafford, I., Betts, W. H., and Harowitz, J. D. N-acetylcysteine in combination with nitroglycerin and streptokinase for the treatment of evolving acute myocardial infarction. Circulation 92: 2855, 1995. Weinbraun, A. A., Rudick, V., Ben-Abraham, R., and Kachevski, E. N-acetyl-L-cysteine for preventing lung reperfusion injury after liver ischemia-reperfusion: A possible dual protective mechanism in a dose-response study. Transplantation 69: 853, 2000. Buege, A., and Aust, S. D. Microsomal lipid peroxidation. Methods Enzymol. 12: 302, 1978. Ulrich, R., Roeder, G., Lorber, C., Quezago, Z. M. N., Kneifel, W., and Gasser, H. Continuous venovenous hemofiltration improves arterial oxygenation in endotoxin-induced lung injury in pigs. Anesthesiology 95: 428, 2001. Weinbraum, A. A., Hachhauser, E., Valery, R., Kluger, Y., Karchevsky, E., Gral, E., and Vidne, B. Multiple organ dysfunction after remote circulatory arrest: Common pathway of radical oxygen species? J. Trauma 47: 691, 1999.
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Cuzzocrea, S., Mazzan, E., Costantino, G., Serraino, I., Dugo, L., Calabro, G., Cucinotta, G., DeSarro, A., and Caput, A. P. Beneficial effects of n-acetylcysteine on ischemic brain injury. Br. J. Pharmacol. 130: 1219, 2000. Calapai, G., Squadrito, F., Rizzo, A., Crisafulli, C., Campo, G. M., Marciano, M. C., Mazzoglia, G., and Scuri, R. A. New antioxidant drug limits brain damage induced by transient cerebral ischemia. Drugs Exp. Clin. Res. 19: 159, 1993. Borjesson, A., Wang, X., Sun, Z., Wallen, R., Deng, X., and Johnson, E. Effects of N-acetylcysteine on pulmonary macrophage activity after intestinal ischemia and reperfusion in rats. Dig. Surg. 17: 379, 2000. Suter, P. M., Domenighetti, G., Schaller, M., Laverriere, M., Ritz, R., and Perret, C. N-acetylcysteine enhances recovery from acute lung injury in man. Chest 105: 190, 1994. Menasche, P., Grausset, C., Gauduel, Y., Monas, C., and Piwnica, A. Maintenance of the myocardial thiol pool by N-acetylcysteine. J. Thoracic. Cardiovasc. Surg. 103: 936, 1992. Welbourn, C. R. B., Goldman, G., Paterson, I. S., Valeri, C. R., Shepro, D., and Hechtman, H. B. Pathophysiology of ischaemia reperfusion injury: Control role of the neutrophil. Br J Surg 78: 651, 1991. Flora, S., Izzetti, A., Agastini, F., and Balnscky, R. M. Mechanism of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points. Carcinogenesis 22: 999, 2001. Groenvald, A. B. J., Den Hollander, W., and Straub, J. Effects of n-acetylcysteine and terbutaline treatment on hemodynamics and regional albumin extravasation in porcine septic model. Circ. Shock 27: 292, 1990. Zhang, H., Spapen, H., Nguyen, D. N., Rogiers, P., Bokker, J., and Vincent, J. L. Effects of N-acetyl-L-cysteine on regional blood flow during endotoxic shock. Eur. Surg. Res. 27: 292, 1995. Andersen, L. W., Thiis, J., Kharazmi, A., and Rugg, I. The role of N-acetylcysteine administration on the oxidative response of neutrophils during cardiopulmonary bypass. Perfusion 10: 21, 1995.
Attenuation of Ischemia/Reperfusion Injury by N-Acetylcysteine in a Rat Hind Limb Model Cengiz Koksal, M.D.,* ,1 A. Kursat Bozkurt, Assos. Prof.,† Ugur Cangel, M.D.,‡ Nil Ustundag, M.D.,§ Dildar Konukogˇlu, Assos. Prof., ¶ Benan Musellim, M.D.,㛳 and Ayla Gurel Sayın, Prof.** *Sureyyapasa Thoracic and Cardiovascular Disease Hospital, Dept. of Cardiovascular Surgery, Istanbul, Turkey, †Istanbul University, Cerrahpasa Medical Faculty, Dept. of Cardiovascular Surgery, Istanbul, Turkey, ‡Istanbul Memorial Hospital, Dept. of Cardiovascular Surgery, Istanbul, Turkey, §Bolu Izzet Baysal University, Medical Faculty, Dept. of Pathology, Bolu, Turkey, ¶Istanbul University, Cerrahpasa Medical Faculty, Dept. of Biochemistry, Istanbul, Turkey, 㛳Istanbul University, Cerrahpasa Medical Faculty, Dept. of Chest Medicine, Istanbul, Turkey, and **Istanbul University, Cerrahpasa Medical Faculty, Dept. of Cardiovascular Surgery, Istanbul, Turkey Submitted for publication August 23, 2002
Background. Ischemia/reperfusion is a complex set of events with severe pathologic consequences. Reperfusion initiates both the local and systemic damage in part through rapid oxygen generation. N-acetylcysteine (NAC) is a scavenger of free radical species, inhibits neutrophil accumulation, acts as a vasodilator and also improves microcirculation. In present study, we examined the protective effect of NAC in a rat hind limb ischemia/ reperfusion model. Dimethyl-sulfoxide (DMSO), a well-known antioxidant was also tested for comparison. Materials and methods. Ischemia was induced for 4 h by vascular clamping and followed by 1 h of reperfusion. Muscle injury was evaluated in 3 groups as a saline group (control), DMSO group, and NAC group. Plasma levels of creatine kinase, lactate dehydrogenase, thiobarbituric acid reactive substances (TBARS), and blood HCO 3, as well as muscle tissue TBARS, were measured at the end of reperfusion. Muscle tissue samples were taken for histological evaluation. Results. DMSO and NAC group showed significant amelioration of plasma CPK (P < 0.05, P < 0.05), plasma TBARS (P < 0.05, P < 0.05), and muscle tissue TBARS (P < 0.05, P < 0.05) compared with the control group. Similarly, neutrophil infiltration in DMSO and NAC groups were significantly less prominent than the control group (P < 0.01, P < 0.01). Conclusions. These results show that NAC improved effectively ischemia reperfusion injury in a rat hind limb model. © 2003 Elsevier Inc. All rights reserved. 1 To whom correspondence should be addressed at P.O. Box 26, Cerrahpasa, 34301-Istanbul/Turkey. E-mail: cengizkoksal@ hotmail.com.
0022-4804/03 $35.00 © 2003 Elsevier Inc. All rights reserved.
Key Words: N-acetylcysteine; ischemia/reperfusion injury; dimethyl sulfoxide; thiobarbituric acid reactive substances; histological examination.
INTRODUCTION
Management of acute skeletal muscle ischemia continues to be a formidable challenge for the vascular surgeon. Despite improved efficiency of invasive and noninvasive treatment modalities, the overwhelming impact of reperfusion has still to be overcome. If the mass of the ischemic tissue is large enough, such as the legs, reperfusion results not only in local damage but also remote organ injury. It is known that the major part of the damage occurs during the reperfusion period and that reactive oxygen substances (ROS) are responsible for the tissue injury [1, 2]. Several antioxidants like superoxide dismutase, catalase, mannitol, dimethyl sulfoxide (DMSO), and iloprost (a long-acting prostacyclin analogue) have proven to be efficient in attenuating the changes in microvascular permeability, which is the final outcome of ischemia/reperfusion (I/R) injury [3, 4]. Belkin et al. demonstrated reduction in the size of skeletal muscle infarcts in a canine gracilis muscle model with the use of iloprost [5]. Karthuis et al. also, in their experimental I/R injury study in canine skeletal muscle, showed attenuation in the increase of vascular permeability when subjected to pretreatment with DMSO [3]. N-acetylcysteine (NAC) is not simply an antioxidant drug. It acts as a glutathione (GSH) precursor, as a chemical reductant of oxidized thiols, as a scavenger of radical oxygen species, as a vasodilator, and also im-
236
KOKSAL ET AL.: N-ACETYLCYSTEIN ATTENUATES ISCHEMIA/REPERFUSION INJURY
proves microcirculation by restoring the decreased activity of endothelium-derived relaxing factor (EDRF) and may have antiaggregan features [6 – 8]. There is growing evidence regarding its beneficial effects in ameliorating lung I/R injury [9]. However its role in reducing the damage in skeletal muscle tissue of the involved extremity has not been addressed yet. In this experimental study, we aimed to examine the protective effect of NAC against I/R injury in a rat skeletal muscle model. DMSO, which is a well-established antioxidant, is also tested to prove the effectiveness of the experimental model.
237
TABLE 1 Comparison of DMSO and NAC Groups with the Control Group (mean ⴞ standard deviation)
HCO 3 (mmol/L) LDH (U/L) CPK (U/L) Plasma TBARS (nmol/mL) Muscle TBARS (nmol/mL)
Control group
DMSO group
NAC group
9.5 ⫾ 3.2 638 ⫾ 425 1705 ⫾ 228 3.83 ⫾ 0.14
15.4 ⫾ 5.8* 312 ⫾ 81 817 ⫾ 272* 3.19 ⫾ 0.37*
14.4 ⫾ 4.3* 350 ⫾ 78 760 ⫾ 140* 3.30 ⫾ 0.89*
303.65 ⫾ 82.3
193.8 ⫾ 13.8*
185.1 ⫾ 12*
* P ⬍ 0.05.
MATERIALS AND METHODS This study was conducted according to the guidelines of the animal care review board of Istanbul University Cerrahpasa Medical Faculty with adherence to the guide for care and use of laboratory animals.
Ischemia/Reperfusion Model Eighteen mature male Sprague–Dawley rats weighing 250 to 350 g were used. All animals had free access to standard rat chow and water. They were anesthetized with intraperitoneal ketamine (100 mg/kg body weight; Abbott Laboratories, North Chicago, Ill) and additional doses of ketamine (50 mg/kg) were given to sustain anesthesia throughout the experiment. Using magnifying spectacles (⫻3.5) bilateral groin incisions were made and the iliac arteries were dissected distally. All tissues except artery and vein were transected to eliminate the collateral blood supply from the rats’ pelvis. Right iliac artery was spared for clamping and the left iliac vein was cannulated with 24-G cannula for blood sampling and drug administration. Ischemia was induced with a 4-h period of iliac artery occlusion with a nontraumatic vascular clamp and followed by 1 h of reperfusion.
Experimental Groups Rats were divided into three groups. Group 1 (n ⫽ 6, control group) received 2 mL/kg/h of continuous saline with the induction of anesthesia and used as controls. Group 2 (n ⫽ 6, DMSO group) received dimethyl sulfoxide (Sigma Chemical, St. Louis, Mo; 15 mg/kg/h) continuously in the same manner. Group 3 (n ⫽ 6, NAC group) received 20 mg/kg NAC (Zambon GmBH, Grafelfing, Germany) 5 min before reperfusion and followed by an infusion of 20 mg/kg/h. Plasma creatine kinase (CPK, IU/L), lactate dehydrogenase (LDH, IU/L), bicarbonate (HCO3, mmol/L) and thiobarbituric acid reactive substances (TBARS, nmol/mL, as a marker of lipid peroxidation) were determined at the end of the reperfusion period. After the reperfusion, rats were sacrificed. Samples were taken from gastrocnemius muscle to determine TBARS levels (nmol/g wet tissue). Muscle tissue samples were also taken for histological examination.
Tissue Peroxide Level Analysis Approximately 1 g of thawed tissue samples was homogenized in 10 mL of 10 nM sodium phosphate buffer (pH 7.4) using a homogenizer. One ml aliquot of tissue homogenate was used for the determination of lipid peroxide level by measuring spectrophotometrically the formation of TBARS. The intra-and interassay coefficients of variation for TBARS were 4.7% and 4.9%, respectively [10].
Plasma Analysis Blood HCO 3 value was determined on Ciba Corning 860 blood gas analyzer. (Ciba Corning Diagnostics Corporation, Medfiels, Mass). Plasma CPK and LDH assays were performed on the Hitachi system 717 automated analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan).
Histological Analysis Muscle biopsies were taken at the end of the experiment into 10% buffered formalin. Using standard techniques, paraffin sections were obtained, stained with hematoxylin and eosin, and studied under light microscopy by a pathologist in a blinded manner. Histological changes were scored on a scale from 0 to 3 where 0 ⫽ absence (⬍5% of maximum pathology), 1 ⫽ mild (⬍10%), 2 ⫽ moderate (15–20%), and 3 ⫽ severe (20 –25%) [11]. A total of four slides from each muscle sample were randomly screened, and the mean was accepted as the representative value of the sample. Severity of neutrophil infiltration was estimated and calculated for each experiment.
Data Analysis Apparent differences between control and experimental groups were analyzed for their statistical significance by the use of Kruskall–Wallis test. For multiple comparisons Bonferroni correction was used. Level of significance was decreased to 0.016 and results were analyzed by Mann–Whitney U Test. P ⬍ 0.05 considered statistically significant.
RESULTS
Biochemical variables at the end of the reperfusion period are shown in Table 1. No significant difference was encountered among control and treatment groups in terms of LDH values. Plasma CPK values were significantly higher and HCO 3 values were significantly lower in control group compared with DMSO and NAC groups (P ⬍ 0.05) and the results of NAC and DMSO groups were comparable. Plasma TBARS values and muscle TBARS values were found to be significantly higher in control group rats compared with both treatment group rats (P ⬍ 0.05, P ⬍ 0.05, respectively). Also, the results of both TBARS values (plasma and muscle) were comparable in DMSO and NAC groups.
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JOURNAL OF SURGICAL RESEARCH: VOL. 111, NO. 2, MAY 15, 2003
Median neutrophil infiltration scores for muscle injury of DMSO and NAC was 1, whereas it was 2 for the control group, which revealed significantly less prominent muscle injury in both DMSO and NAC groups, compared with the control group (P ⬍ 0.01). DISCUSSION
The establishment of reflow replenishes vital substances to the tissue but also releases into the circulation toxic metabolites that have been generated during the no-flow state. Damage to the involved extremity itself and remote organs can be induced with these compounds. Many mediators have been implicated in the process of inducing I/R injury. ROS are perhaps the most pertinent agents in this matter [12]. In their experimental brain I/R injury study in gerbils, Cuzzocrea et al. encountered an increase in MDA levels, which is a well-known product of lipid peroxidation [13]. Their observation was in agreement with Capali et al., who showed similar elevated levels of lipid peroxidation products in the same experimental model [14]. There is now a large body of evidence suggesting that postischemic oxidative injury is caused by an imbalance between a burst of ROS production and the inability of reoxygenated cells to handle this radical load, because of an ischemia induced depression of the naturally present defense mechanisms against oxygen toxicity [6]. Eventually oxidants cause injury and cell death by modifying and/or disturbing the structure and function of any cellular or noncellular component. The protective effect NAC in acute lung injury is well established in clinical and experimental models. In an experimental study Boerjesson et al. showed that treatment with NAC prevents intestinal IR induced over activation of pulmonary macrophages and decreases pulmonary content [15]. This observation was supported by Weinbraum et al., which demonstrated, pretreatment of the lungs with NAC during reperfusion period with ischemic/reperfused liver effluent prevents acute lung injury [9]. In a randomized clinical trial on patients with acute respiratory distress syndrome, NAC enhanced recovery from acute lung injury by GSH deficiency [16]. There are also several studies concerning beneficial role of NAC in ameliorating myocardial and brain injury [13, 17]. Several studies have pointed out the activation and infiltration of neutrophils as one of the initial events of tissue injury, which triggers the release of ROS with subsequent tissue injury [18]. In search of an ideal drug for attenuation of IR injury, the cause of neutrophil activation and accumulation, as well as the consequence of ROS mediated injury, should be taken into consideration. The ideal drug should not be solely an antioxidant. The rationale behind supplying an ischemic tissue with compounds that can replenish the intracellular stores of GSH, thereby allowing preserva-
tion of the thiol-disulfide equilibrium, seems to be more clinically relevant. NAC is particularly attractive for many reasons. The thiol NAC works in the extracellular environment and is a precursor of intracellular cysteine and GSH. Besides its activity as a GSH precursor, NAC is, per se, responsible for protective effects in the extracellular environment, mainly due to its nucleophilic and antioxidant properties [19]. NAC acts by either directly interfering with the oxidants or upregulating antioxidant systems such as superoxide dismutase or enhancing the catalytic activity of glutathione peroxidase [20]. Apart from antioxidant effect, NAC has vasodilator effect and may act as an antiagregan, by restoring the decreased activity of EDRF, thereby improving microcirculation and tissue oxygenation [8, 16, 21]. EDRF, synthesized by endothelial cells, relaxes vascular smooth muscle and inhibits the aggregation, adhesion of platelets, an action potentiated by NAC. NAC, by replenishing tissue sulfhydryl groups either directly or by increasing cystine levels, could restore the full activity of EDRF by a mechanism similar to its reversal of tolerance to nitrates [7]. Activation and accumulation of neutrophils’, which triggers the release of ROS was ameliorated by NAC in the ischemic brain injury model in gerbils by Cuzzocrea et al. [13]. Also, Inhibition of neutrophil infiltration by NAC during cardiopulmonary bypass was shown in patients undergoing cardiac surgery by Andersen et al. [22]. In the current study we aimed to search the role of NAC in skeletal muscle I/R injury. Muscle and plasma MDA levels (TBARS) were significantly lower in NAC group than the control group (P ⬍ 0.05, P ⬍ 0.05; respectively), which suggested the efficacy of NAC as an antioxidant in ameliorating I/R. Neutrophil infiltration was significantly less prominent histologically in NAC group than the control group (P ⬍ 0.01). Reduction of MDA in blood and skeletal muscle tissue correlated well with diminished neutrophil infiltration in the skeletal muscle at histological examination, in both NAC and DMSO groups. The results of NAC group were comparable with DMSO group. The results of the present study supports the view that NAC can exert a protective effect against skeletal muscle injury caused by IR in the rats. NAC is not solely an antioxidant, but also inhibits neutrophil infiltration and improves microcirculation and may be more beneficial than other antioxidants in case of I/R injury. These encouraging results suggest the possibility of clinical application of NAC following skeletal muscle I/R injury. REFERENCES 1.
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