Recipient treatment with trimetazidine improves graft function and protects energy status after lung transplantation

Recipient Treatment With Trimetazidine Improves Graft Function and Protects Energy Status After Lung Transplantation Ilhan Inci, MD,a Andre´ Dutly, MD,a Demet Inci, MD,b Annette Boehler, MDc and Walter Weder, MDa Background: Ischemia–reperfusion injury remains an important obstacle to successful lung transplantation. Trimetazidine is an anti-ischemic drug that restores the ability of ischemic cells to produce energy and reduces the generation of oxygen-derived free radicals. The aim of this study was to assess the protective effect of trimetazidine after prolonged ischemia in lung transplantation. Methods: Rat single-lung transplantation was performed in 4 experimental groups (n ⫽ 5 each). In all groups, transplantation was performed after 18 hours of cold (4° C) ischemia. All donor lungs were flushed with low-potassium dextran– glucose (LPDG) solution that also contained 500 ␮g/liter prostaglandin estradiol (E1). Groups studied included: Group I: flush solution was administered containing 10⫺6 mol/liter trimetazidine (TMZ), neither donor nor recipient treatment given; Group II: donors were treated with 5 mg/kg intravenous TMZ 10 minutes prior to harvest, but the flush solution did not contain TMZ; Group III: recipients treated with 5 mg/kg intravenous TMZ 10 minutes before reperfusion, and flush solution contained 10⫺6 mol/liter trimetazidine; Group IV: ischemic control group. After 2 hours of reperfusion, oxygenation was measured and lung tissue was frozen and assessed for adenosine triphosphate (ATP) content, myeloperoxidase (MPO) activity and thiobarbituric acid–reactive substances (TBARS). Peak airway pressure (PawP) was recorded throughout the reperfusion period. Results: Group III showed significantly higher levels of ATP content (11.1 ⫾ 5.01 pmol vs Group I, 3.36 ⫾ 1.8 pmol, p ⫽ 0.008; vs Group II, 4.7 ⫾ 1.9 pmol, p ⫽ 0.03; vs Group IV, 0.7 ⫾ 0.2 pmol, p ⫽ 0.008), better oxygenation (442.5 ⫾ 26.5 mm Hg, vs Group I, 161.06 ⫾ 54.5 mm Hg; vs Group II, 266.02 ⫾ 76.9 mm Hg; vs Group IV, 89.4 ⫾ 14.7 mm Hg, p ⫽ 0.008) and reduced lipid peroxidation (TBARS) (0.15 ⫾ 0.03 nmol/g; vs Group I, 1.04 ⫾ 0.76 nmol/g; vs Group II, 0.69 ⫾ 0.4 nmol/g; vs Group IV, 2.29 ⫾ 0.4 nmol/g, p ⫽ 0.008). PawP and MPO activity were comparable in the 4 study groups. Conclusion: Recipient treatment with TMZ provided significant protection of energy status, better oxygenation and reduced lipid peroxidation. Our data suggest that TMZ may be an important adjunct in the prevention of post-transplant lung ischemia–reperfusion injury. J Heart Lung Transplant 2001;20:1115–1122. From the aDivision of Thoracic Surgery, University Hospital; Zurich, Switzerland; bChildren’s Hospital, University of Zurich, Zurich, Switzerland; and cDivision of Pulmonary Medicine, Zurich, Switzerland. Submitted February 28, 2001; accepted May 2, 2001. Supported by a grant from the Olga Mayenfisch Foundation, Zurich. Annette Boehler is a recipient of a grant from the Silva Casa Foundation, Switzerland. The abstract to this article was presented at the 21st annual meeting of the International Society for Heart and Lung

Transplantation, April 25–28, 2001, Vancouver, British Columbia, Canada. Reprint requests: Dr Walter Weder, University Hospital, Division of Thoracic Surgery, Ra¨mistrasse 100, Zurich 8091, Switzerland. Telephone: 00-411-255-88-02. Fax: 00-411-255-88-05. E-mail: [email protected] Copyright © 2001 by the International Society for Heart and Lung Transplantation. 1053-2498/01/$–see front matter PII S1053-2498(01)00312-6

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schemia–reperfusion lung injury involves a multitude of pathways, including stimulation of leukocytes and platelets, formation of reactive oxygen species, activation of the complement cascade and generation of other inflammatory mediators and arachidonic acid metabolites leading to epithelial, endothelial and alveolar type II cell dysfunction.1– 4 Several agents have been shown to be protective, such as prostaglandins, oxygen free-radical scavengers (superoxide dismutase, catalase, glutathione, allopurinol, dimethylthiourea desferoxamine), verapamil, platelet-activating factor antagonists, complement inhibitor, pentoxyfylline, inhaled nitric oxide, nitroglycerin, nitroprusside, exogenous surfactant and angiotensin-converting enzyme inhibition by captopril.2– 6 Trimetazidine [1-(2,3,4-trimethoxybenzyl) piperazine dihydrochloride] (TMZ) is an anti-ischemic agent known to improve exercise tolerance and cardiac function in patients with ischemic heart disease.7 Its anti-ischemic effect has been assessed experimentally in various models including cell cultures, isolated and perfused organs and in vivo.8 –15 It is ineffective under physiologic conditions.8 TMZ acts mostly on mitochondria by restoring adenosine triphosphate (ATP) synthesis, which was previously blocked by the Ca2⫹ overload, by releasing Ca2⫹ accumulated in the matrix, and by restoring mitochondrial membrane impermeability and its affinity for protons.9 In a previous study we showed that combined treatment of donor and recipient with TMZ resulted in improved graft function, protection of energy status and reduced lipid peroxidation after 18 hours of cold storage and 2 hours of reperfusion (data for publication accepted in The Journal of Thoracic and Cardiovascular Surgery). The aims of this study were to: (1) determine the protective effect of recipient treatment alone with TMZ on post-transplant lung ischemia–reperfusion injury; and (2) compare the effects of recipient treatment alone with donor treatment and TMZ added to flush solution alone. Assessment was performed by blood oxygenation, peak airway pressure, lung tissue ATP content, lipid peroxidation and neutrophil accumulation.

MATERIALS AND METHODS Orthotopic single left lung transplantation was performed in male Fischer (F344) rats, weighing 280 to 300 g, using a cuff technique for anastomoses.16 Trimetazidine was generously provided by Servier Research Institute (Paris, France). All animals received humane care in accordance with the Guide

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for the Care and Use of Laboratory Animals (National Institutes of Health, Publication 85-23, revised 1985). The study protocol was approved by the local animal study committee.

Donor Procedure Animals were anesthetized by intraperitoneal injection of 50 mg/kg sodium thiopental (Pentothal; Abbott AG, Baar, Switzerland) and intubated through a tracheostomy with a 16-gauge intravenous catheter. Animals were connected to a volumecontrolled ventilator (Model 683, Harvard Rodent Ventilator Harvard Apparatus Co, Inc, S Natick, MA) and ventilated with a fraction of inspired oxygen of 1, a tidal volume of 10 ml/kg at 75 breaths/minute, and a positive end-expiratory pressure of 3 cm H2O. Median laparosternotomy was then performed and 1000 IU/kg of heparin (Liquemin; Roche Pharma [Schweiz] AG, Switzerland) was injected into the inferior vena cava. For harvest of the heart–lung block, the inferior vena cava was incised, the left atrial appendage was cut, and a 14-gauge cannula was placed into the main pulmonary artery. The lungs were flushed through this cannula with 20 ml of low-potassium dextran– glucose (LPDG; Perfadex; Xvivo Transplantation Systems AB, Gøteborg, Sweden) at 4° C, which also contained 500 ␮g/liter prostaglandin estradiol (E1) (Prostin VR, Pharmacia & Upjohn). After the lungs had been flushed, the intratracheal tube was clamped to keep the lungs inflated during storage. Hypothermic condition was maintained during cuff (16 gauge) placement into the pulmonary artery, pulmonary vein and main bronchus. The vessels or bronchus were drawn through the center of the cuff, everted circumferentially around it and secured with a 7-0 silk ligature.

Recipient Procedure Recipient animals were anesthetized and intubated as described for donor animals. Anesthesia was maintained with 0.5% halothane during the operation and reperfusion period. Ventilation parameters were the same as in donor animals. For measuring the airway pressure during the procedure, a threeway tap was inserted between the intratracheal tube and the ventilator circuit and connected to a pressure transducer (Benjamin Type AVAD, Belwa AG, Zu ¨rich, Switzerland). A left thoracotomy was performed through the fifth intercostal space. The left lung was mobilized by dividing the pulmonary ligament. The hilum of the left lung was dissected, and the pulmonary artery, pulmonary vein and left main

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bronchus were isolated. All three structures were clamped by using microsurgical aneurysm clamps. They were incised on their anterior aspect, and the cuffs of the donor lung were placed into the equivalent recipient structures and fixed with a 6-0 polypropylene suture. The transplanted lung was inflated and pulmonary vein and arterial clamps were released, respectively. The thoracotomy was closed loosely. The recipient animal was ventilated (with 99.5% oxygen, 0.5% halothane, a tidal volume of 10 ml/kg at 75 breaths/minute, and a positive end-expiratory pressure of 3 cm H2O) for 2 hours.

Experimental Setting Animals were randomized into 4 groups (n ⫽ 5 transplantations each). In all groups, transplantation was performed after 18 hours of cold (4° C) ischemia. All donor lungs were flushed with LPDG solution that also contained 500 ␮g/liter prostaglandin E1. Group I received flush solution containing 10⫺6 mol/liter trimetazidine (TMZ), but neither donor nor recipient treatment; Group II was given donor treatment with 5 mg/kg intravenous TMZ 10 minutes prior to harvest by injection into the inferior vena cava; Group III was given recipient treatment with 5 mg/kg intravenous TMZ 10 minutes before reperfusion by injection into the left superior vena cava, and the flush solution contained 10⫺6 mol/liter trimetazidine; and Group IV was the ischemic control (IC) group. In Groups I and IV, animals received the same amount (0.2 ml) of saline solution via the same route as in Groups II and III. Right donor lungs (n ⫽ 5) were assessed for ATP, MPO and TBARS to obtain baseline values in the normal lung.

MPO Assay Quantitative MPO activity, as measured for neutrophil migration to the graft was determined as previously described.17 Frozen lung tissue (100 mg) was homogenized in 1 ml of 0.5% hexadecyltrimethylammonium bromide, 5 mmol/liter ethylene-diamine tetraacetic acid (EDTA) and 50 mmol/liter potassium phosphate buffer (pH 6.2) with a tissue grinder. The homogenate was centrifuged at 10,000g for 15 minutes at 4° C. The supernatant was assayed for total soluble protein by the method of Pierce Laboratories18 and for MPO activity. Enzyme activity was measured spectrophotometrically. Ten milligrams of 5-fold supernatant was combined with 0.6 ml Hanks bovine serum albumin (BSA), 0.5 ml 100 mmol/liter potassium phosphate buffer (pH 6.2), 0.1 ml 0.05% H2O2 and 0.1 ml 1.25 mg/ml o-dianisidine.

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The reaction was stopped by addition of 1% NaN3 after 5 and 20 minutes at room temperature, respectively. Optical density was measured at 460 nm with a spectrophotometer (Kadas 100, Dr Lange AG Zurich, Switzerland). Color development from 5 to 20 minutes was linear. Enzyme activity was expressed as change in optical density unit per milligram of tissue protein per minute (⌬OD/mg per minute).

TBARS TBARS was measured according to the method of Okhawa and co-workers in 10% wet weight per volume homogenate to determine the lipid peroxidation in the graft tissue.19 Aliquots (0.2 ml) of this homogenate were added to tubes containing 0.2 ml of 8.1% sodium dodecylsulfate, 1.5 ml of 20% acetic acid solution adjusted to pH 3.5 with NaOH and 1.5 ml of 0.8% solution of thiobarbituric acid. The mixture was brought to a volume of 4 ml by addition of distilled water, heated at 95° C for 60 minutes and then cooled with tap water. One milliliter of distilled water and 5 ml of butanol/pyridine (15:1) were added (all chemicals from Fluka AG, Switzerland). The solution was centrifuged at 4000 rpm for 10 minutes. Absorbance of the upper layer was measured at 532 nm with a spectrophotometer (Kadas 100, Dr Lange AG). The TBARS levels were determined by reference to a standard curve of 1,1,3,3tetramethoxypropane (Sigma Chemicals, Switzerland), and the results expressed as nanomoles of malondialdehyde per gram of wet lung.

Lung ATP Content Frozen lung tissue (100 mg) was homogenized in 0.9 ml of HEPES buffer. As extractant, 200 ␮l of trichloroacetic acid (1% final concentration, Fluka AG, Switzerland) was used and 100 ␮l of releasing agent was added to this homogenate. The homogenate was centrifuged at 3000 rpm for 15 minutes at 4° C. The supernatant was removed and adjusted to pH 7 by addition of 0.1N NaOH. ATP concentration in the supernatant was determined enzymatically using an ATP assay kit (Calbiochem ATP Assay Kit, Calbiochem-Novobiochem Corp, San Diego, CA). The results were expressed as picomoles of ATP.

Graft Assessment Peak airway pressure was recorded after intubation; after entering the chest; before reperfusion; at 1, 5, 10 and 15 minutes after reperfusion; and then every 15 minutes thereafter. At the end of 2-hour reperfusion, oxygenation of the graft was evaluated by

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FIGURE 1 ABG analysis of the study groups. For

Group I (only flush solution contains TMZ) (GI) vs Group II (only donor treatment with TMZ, flush solution does not contain TMZ) (GII), p ⫽ 0.03; among other groups (GIII) (recipient treated with TMZ, flush solution contains TMZ); (GIV) (ischemic control, no treatment), p ⫽ 0.008.

sampling the blood directly from the pulmonary vein of the transplanted lung by means of heparinized needle (29-gauge) aspiration inserted distal to the anastomotic cuff.20 The donor lung was excised, divided into 3 pieces and placed into liquid nitrogen and stored at ⫺80° C for further evaluation of ATP, TBARS and MPO.

Statistical Analysis Data analysis was performed using SPSS software for WINDOWS 8.0 (SPSS Inc, Chicago, IL). All data are expressed as mean values ⫾ standard deviation. We performed the Kruskal–Wallis test for comparison among groups. When a significant difference was found, we then performed a Mann–Whitney U-test for comparison between the two groups with regard to blood gas, ATP, TBARS and MPO data. To evaluate the statistical difference between the groups regarding the peak airway pressures over the 2-hour reperfusion period, analysis of variance (ANOVA) was used. p ⬍ 0.05 was considered significant.

RESULTS Blood Gas Analysis Oxygenation 2 hours after graft reperfusion was significantly higher in Group III (442.5 ⫾ 26.5 mm Hg, vs Group I, 161.06 ⫾ 54.5 mm Hg; vs Group II, 266.02 ⫾ 76.9 mm Hg; vs Group IV, 89.4 ⫾ 14.7 mm Hg, p ⫽ 0.008) (Figure 1).

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FIGURE 2 Lung ATP content of the study groups. For GI vs GII, p ⫽ 0.2; GII vs GIII (recipient treatment with 5 mg/kg TMZ and flush solution contains TMZ), p ⫽ 0.03; among other groups, p ⫽ 0.008.

Peak Airway Pressures Peak airway pressures did not differ significantly among the groups (p ⫽ 0.68) as determined using analysis of variance (ANOVA). At the end of 2-hour reperfusion PawP in Group I (GI) was 14.4 ⫾ 1.5 mm Hg, 13.8 ⫾ 0.8 mm Hg in Group II (GII), 13.2 ⫾ 0.8 mm Hg in Group III (GIII) and 14.2 ⫾ 0.4 mm Hg in Group IV (GIV) (GI vs GII, p ⫽ 0.69; GI vs GIII, p ⫽ 0.42; GI vs GIV, p ⫽ 0.42; GII vs GIII, p ⫽ 0.69; GII vs GIV, p ⫽ 0.69; GIII vs GIV, p ⫽ 0.09).

Lung ATP Content GIII showed significantly higher levels of ATP content (11.1 ⫾ 5.01 pmol vs GI, 3.36 ⫾ 1.8 pmol, p ⫽ 0.008; vs GII, 4.7 ⫾ 1.9 pmol, p ⫽ 0.03; vs GIV, 0.7 ⫾ 0.2 pmol, p ⫽ 0.008) (Figure 2). Lung ATP content in the normal lung was 13.4 ⫾ 2.2 pmol ATP.

TBARS The amount of lipid peroxidation was significantly lower in GIII (0.15 ⫾ 0.03 nmol/g; vs GI, 1.04 ⫾ 0.76 nmol/g; vs GII, 0.69 ⫾ 0.4 nmol/g; vs GIV, 2.29 ⫾ 0.4 nmol/g, p ⫽ 0.008) (Figure 3). The normal lungs had a mean TBARS level of 0.12 ⫾ 0.01 nmol of malondialdehyde per gram of wet lung.

MPO Activity MPO activity was not significantly reduced among the groups (Kruskal–Wallis test, p ⫽ 0.075): GI, 1.32 ⫾ 0.5 ⌬OD/mg/min; GII, 0.87 ⫾ 0.4 ⌬OD/mg per minute; GIII, 0.53 ⫾ 0.3 ⌬OD/mg per minute;

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FIGURE 3 TBARS levels of the study groups. For GI

vs GII, p ⫽ 0.42; GI vs GIV (untreated ischemic control group), p ⫽ 0.009; among other groups, p ⫽ 0.008.

GIV, 2.8 ⫾ 1.8 ⌬OD/mg per minute (Figure 4). MPO activity in the normal lungs was 0.36 ⫾ 0.05 ⌬OD/mg per minute.

DISCUSSION In this study, we evaluated the protective effect of TMZ in a rat single-lung transplant model of ischemia–reperfusion injury. The purpose of this study was to determine the protective effect of recipient treatment alone with TMZ on post-transplant lung ischemia–reperfusion injury and to compare the effects of recipient treatment alone with donor treatment and TMZ added to flush solution alone. We found that recipient treatment with TMZ resulted in significantly improved graft function, protection of energy status and reduced lipid peroxida-

FIGURE 4 MPO activity of the study groups.

Comparison among the groups not statistically significant (Kruskal–Wallis test, p ⫽ 0.075).

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tion after 18 hours of cold storage and 2 hours of reperfusion. MPO activity, an index of leukocyte infiltration, and peak airway pressure did not differ among the groups. We used LPDG solution as a preservation and flush solution because we use it both in our clinical lung transplant program and experimental studies. LPDG solution has been demonstrated to be superior to Euro-Collins solution in a model of 12-hour pulmonary preservation.21 In addition, LPDG solution does not contain any pharmacologic additions, unlike other standard preservation solutions such as Euro-Collins and University of Wisconsin.9 Therefore, only the effect of TMZ when added to the LPDG solution was assessed. The concentration of TMZ used in flush and preservation solution was 10⫺6 mol/liter, because this concentration has been shown to be effective for restoring ATP synthesis of isolated mitochondria previously decreased by Ca2⫹ overload.8 Higher concentrations of TMZ exerted no protective effect in a previous study.22 The toxicity of oxygen-derived free radicals is due to oxidation of membrane lipids, proteins and nucleic acids.8 It is now well established that peroxidation of polyunsaturated fatty acids of the membranes by free radicals leads to severe functional distortions, including alterations in membrane permeability and fluidity and inhibition of enzyme activities.8 These dysfunctions are combined with the effects of acidosis that develop during ischemia, and potentiate the decrease in ATP synthesis and intracellular calcium accumulation, blocking all vital enzymatic functions, leading to necrosis.8 TMZ has been shown to be an effective antiischemic drug in cultured cells, isolated organs or animal models of ischemia.8 Although it has been reported that it effects both metabolic functions and ion permeabilities in mitochondria, its mechanism of action is not fully understood.23 TMZ restored the ATP synthesis in isolated mitochondria previously exposed to a Ca2⫹ overload.24 Recently, it has been reported that TMZ counteracts the mitochondrial permeability transition induced by Ca2⫹ overload associated with the pro-oxidant tert-butylhydroperoxide.25 The biochemical events mediating this transition are not fully understood but probably involve the formation of a giant pore, called the mitochondrial transition pore, allowing the exchange of small solutes (⬍1,500 Da) across the inner membrane.26 TMZ acts on mitochondrial function in 2 different ways: as a mitochondrial Ca2⫹ releaser when the giant pore is closed, and by inducing its closure when it is open.22

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Various experimental studies have shown that TMZ preserves the intracellular concentrations of ATP and inhibits extracellular leakage of potassium during cellular ischemia. It prevents excessive release of free radicals, which are particularly toxic to phospholipid membranes and are responsible for both the fall in intracellular ATP concentration and extracellular leakage of potassium that occur during ischemia.27,28 TMZ also reduces intracellular accumulation of sodium and calcium.27 In an isolated rat kidney model, it was shown that TMZ improved ATP reconstitution in the kidneys.29 The investigators concluded that TMZ provides significant protection in terms of energy status, cellular acidosis and tubular morphometry.29 It has been shown that, under anoxic conditions, in isolated rat cardiac quiescent myocytes, TMZ improves resistance to the effects of high concentrations of Ca2⫹ in which their ATP content was maintained at almost the control value and the K⫹ leakage was reduced.10,30 –32 Also, TMZ was shown to lowered the increase in liver enzymes and maintain higher concentrations of hepatic ATP in a rat liver model of ischemia–reperfusion injury.33 TMZ decreased tissue water content and malondialdehyde (MDA) levels in an isolated perfused rat kidney model.9 It sustained the normal functions of mitochondria in isolated rat liver mitochondria by inhibiting mitochondrial swelling, the decrease in NAD(P)H level and the decrease in ATP synthesis.34 In the present study, a marked decrease in lung ATP levels was observed in the ischemic control group (Group IV) after 18 hours of cold ischemia confirming that this index of function is rapidly and seriously affected by ischemia–reperfusion.18 Recipient treatment with TMZ and addition of the drug into the flush and preservation solution significantly preserved lung ATP content compared with other study groups. These results are comparable to data from the previous studies showing that TMZ is able to preserve the post-ischemic tissue ATP content. Free radical oxygen species can be measured indirectly using MDA as a marker.19 The high MDA levels observed in this and earlier studies support the notion that lipid peroxidation processes occur during ischemia–reperfusion injury. We have shown that recipient treatment with TMZ decreases lipid peroxidation significantly, which reflects the dominant anti-oxidative effect of this regimen as compared with donor-only treatment or only adding this drug into the flush solution. In a rat model of renal ischemia followed by

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24-hour reperfusion, TMZ treatment resulted in significantly lowered lipid peroxidation and an increase in glutathione peroxidase activity.35 Treatment with TMZ did not significantly modify enzymatic activities or biochemical parameters, except for MDA content in isolated perfused rat hearts.36 TMZ, in the isolated perfused pig kidney model, minimized cell swelling after 48 hours of cold ischemia and improved perfusion flow rate during normothermic reperfusion.37 This effect was related to both reduction of intracellular acidosis and efficient restoration of oxidative phosphorylation.37 They also found an efficient limitation of lipid peroxidation in groups treated with TMZ. Efficient limitation of lipid peroxidation has also been reported by other investigators.12,37,38 After myocardial ischemia–reperfusion in rabbits, it was found that, in the TMZ-treated group, the number of neutrophils in the area at risk was significantly lower than in the control group. This effect did not result from a direct action on the neutrophil.14 We were not able to show a difference of MPO activity, an index of neutrophil accumulation, among our 4 study groups. The problem of experimental studies concerning ischemia–reperfusion injury is the transfer of these agents into clinical lung transplantation. However, TMZ has been used by cardiologists for many years in the treatment of ischemic heart disease without any side effects.7 Thus, the transfer of TMZ to clinical lung transplantation might be even simpler. In conclusion, recipient treatment with TMZ and adding it into the flush solution decreased the severity of ischemia–reperfusion injury, as shown by the significant protection of energy status, better oxygenation and reduced lipid peroxidation. In contrast, neutrophil accumulation was not different among the groups. Our data suggest that TMZ may be an important adjunct for safely prolonging ischemic time and preventing ischemia–reperfusion injury after lung transplantation. Based on our results, recipient treatment with TMZ and addition of this drug to the flush solution may be an option to prevent ischemia–reperfusion injury in clinical lung transplantation. The authors thank Servier Research Institute (Paris, France) for generously providing the trimetazidine for this study; Dr Valentin Rousson who reviewed statistical analysis of this study as a biostatistician; Dr Stefan Fischer, Dr Mingyao Liu and Dr Shaf Keshavjee (Toronto, Canada) for their technical support in establishing the animal model; Dr Gloria Perewusnyk for her technical

The Journal of Heart and Lung Transplantation Volume 20, Number 10 support for ATP measurement; and Vlasta Strohmeier for preparing the animals.

REFERENCES 1. Trulock EP. Lung transplantation. Am J Respir Crit Care Med 1997;155:789 – 818. 2. Novick RJ, Menkins AH, McKenzie FN. New trends in lung preservation: a collective review. J Heart Lung Transplant 1992;11:377–92. 3. Christie N, Waddell TK. Lung preservation. Chest Surg Clin N Am 1993;3(1):29 – 47. 4. Novick RJ, Gehman KE, Ali IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996;62:302–14. 5. Kim JD, Baker CJ, Roberts RF, et al. Platelet activating factor acetylhydrolase decreases lung reperfusion injury. Ann Thorac Surg 2000;70:423– 8. 6. Fischer S, MacLean AA, Liu M, Kalirai B, Keshavjee S. Inhibition of angiotensin-converting enzyme by captopril: a novel approach to reduce ischemia–reperfusion injury after lung transplantation. J Thorac Cardiovasc Surg 2000;120: 573– 80. 7. Brottier L, Barat JL, Combe C, et al. Therapeutic value of a cardioprotective agent in patients with severe ischaemic cardiomyopathy. Eur Heart J 1990;11:207–12. 8. Harpey C, Clauser P, Labrid C, Freyria JL, Porier JP. Trimetazidine, a cellular anti-ischemic agent. Cardiovasc Drug Rev 1989;6(4):292–312. 9. Hauet T, Bauza G, Goujon JM, et al. Effects of trimetazidine on lipid peroxidation and phosphorus metabolites during cold storage and reperfusion of isolated perfused rat kidneys. J Pharmacol Exp Ther 1998;285(3):1061–7. 10. Fantini E, Demaison L, Sentex E, Grynberg A, Athias P. Some biochemical aspects of the protective effect of trimetazidine on rat cardiomyocytes during hypoxia and reoxygenation. J Mol Cell Cardiol 1994;26:949 –58. 11. Rossi A, Lavanchy N, Martin J. Antiischemic effect of trimetazidine: 31P-NMR spectroscopy study in the isolated rat heart. Cardiovasc Drug Ther 1990;4:812–3. 12. Tetik C, Ozden A, Calli N, et al. Cytoprotective effect of trimetazidine on 60 minutes of intestinal ischemia–reperfusion injury in rats. Transplant Int 1999;12:108 –12. 13. Tsimoyiannis EC, Moutesidou KJ, Moschos CM, et al. Trimetazidine for prevention of hepatic injury induced by ischemia and reperfusion in rats. Eur J Surg 1993;159:89 –93. 14. Williams FM, Tanda K, Kus M, Williams TJ. Trimetazidine inhibits neutrophil accumulation after myocardial ischemia and reperfusion in rabbits. J Cardiovasc Pharmacol 22:828 – 33. 15. Baumert H, Goujon JM, Richer JP, et al. Renoprotective effects of trimetazidine againts ischemia–reperfusion injury and cold storage preservation: a preliminary study. Transplantation 1999;68(2):300 –3. 16. Mizuta T, Kawaguchi, Nakahara K, Kawashima Y. Simplified rat lung transplantation using a cuff technique. J Thorac Cardiovasc Surg 1989;97:578 – 81. 17. Goldblum SE, Wu KM, Jay M. Lung myelperoxidase as a measure of pulmonary leukostasis in rabbits. J Appl Physiol 1985;59:1978 – 85. 18. Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150: 768 – 85.

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19. Okhawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979, 95:351– 8. 20. Fischer S, Hopkinson D, Liu M, Keshavjee S. Raffinose improves the function of rat pulmonary grafts stored for twenty-four hours in low-potassium dextran solution. J Thorac Cardiovasc Surg 2000;119:488 –92. 21. Keshavjee S, Yamazaki F, Cardoso PF, McRitchie DI, Patterson GA, Cooper JD. A method of for safe twelve-hour pulmonary preservation. J Thorac Cardiovasc Surg 1989;98: 529 –34. 22. Boucher FR, Hearse DJ, Opie LH. Effects of trimetazidine on ischemic contracture in isolated perfused rat hearts. J Cardiovasc Pharmacol 1994;24:45–9. 23. Morin D, Elimadi A, Sepana R, Crevat A, Carrupt PA, Testa B, Tillement JP. Evidence for the existence of [3H]-trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore. Br J Pharmacol 1998; 123:1385–94. 24. Salducci MD, Chauvet-Monges AM, Tillement JP, Albengres E, Testa B, Carrupt P, Crevat A. Trimetazidine reverses calcium accumulation and impairment of phosphorylation induced by cyclosporin A in isolated rat liver mitochondria. J Pharmacol Exp Ther 1996;277(1):417–22. 25. Elimadi A, Morin D, Sepana R, Chauvet AM, Crevat A, Tillement JP. Comparison of the effects of cyclosporin A and trimetazidine on Ca⫹2-dependent mitochondrial swelling. Fundam Clin Pharmacol 1997;11:440 –7. 26. Haworth RA, Hunter DR. The Ca⫹2-induced membrane transition in mitochondria. II. Nature of the Ca⫹2 trigger site. Arch Biochem Biophys 1979;195:460 –7. 27. Labrid C. Cellular disorders induced by ischemia: the action of trimetazidine. Presse Med 1986;15:1754 –7. 28. Moridonneau-Parini I, Harpey C. Effects of trimetazidine on membrane damage induced by oxygen free radicals in human red cells. Br J Clin Pharmacol 1985;20:148 –51. 29. Hauet T, Bauza G, Mothes D, Moyec L, Goujon JM, Dore B, Caritez JC, Carretier JC, Eugene M, Tillement JP. Beneficial effect on rat kidney preservation of the antiischemic agent trimetazidine during cold storage and reperfusion: assessment by 31P nuclear magnetic resonance spectroscopy. Transplant Proc 1997;29:2343– 4. 30. Cruz C, Zaoui A, Ayoub S, Harpey C, Goupit P, Younes A. Alte´rations des myocetes isole´s des ventricules de coeur de rat adulte: protection par la trime´tazidine. Concours Med 1987;36(suppl):3470 –5. 31. Rochette L, Fitoussi, M, Bralet J. Effets du pre´-traitement par la trime´tazidine sur le me´tabolisme ´energe´tique du coeur isole´ de rat soumis `a une ligature coronaire et perfuse´ en normoxie ou en hypoxie. Gaz Med Fr 1984;91(suppl):17–21. 32. Lavancy N, Martin JR, Rossi A. Antiischemic effects of trimetazidine: 31P-NMR spectroscopy in the isolated rat heart. Arch Int Pharmacodyn Ther 1987;286:97–110. 33. Settaf A, Morin D, Lamchouri F, Elimadi A, Cherrah Y, Tillement JP. Trimetazidine ameliorates the hepatic injury associated with ischemia–reperfusion in rats. Pharmacol Res 1999;39(3):211– 6. 34. Elimadi A, Settaf A, Morin D, Sapena R, Lamchouri F, Cherrah Y, Tillement JP. Trimetazidine counteracts the hepatic injury associated with ischemia–reperfusion by preserving mitochondrial function. J Pharmacol Exp Ther 1998; 286(1):23– 8. 35. Ozden A, Aybek Z, Calli N, Saydam O, Du ¨zcan E, Gu ¨ner G.

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Inci et al.

Cytoprotective effect of trimetazidine on 75 min warm renal ischemia-reperfusion injury in rats. Eur Surg Res 1998;30: 227–34. 36. Charlon V, Boucher C, Clauser P, Harpey C, Favier A, Koukay N, Mouhieddine S, Leiris J. Effect of a 5 day trimetazidine pretreatment in a model of ischemic and reperfused rat heart: spin trapping experiments. Adv Exp Med Biol 1990;264:377– 82. 37. Hauet T, Mothes D, Goujon JM, Germonville T, Caritez

The Journal of Heart and Lung Transplantation October 2001 JC, Carretier M, Eugene M, Tillement JP. Trimetazidine reverses deleterious effects of ischemia-reperfusion in the isolated perfused pig kidney. Nephron 1998;80:296 – 304. 38. Richer JP, Gibelin H, Tallineau C, Amor IB, Hebrard W, Carretier M, Hauet T. Limitation of lipid peroxidation and renal medullary cell injury of the kidney after 48- and 72-hour cold storage in University of Wisconsin solution: effect of trimetazidine. Transplant Proc 2000;32:46.