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Ann Thorac Surg 2002;73:220-225
© 2002 The Society of Thoracic Surgeons
a Division of Thoracic Surgery, University Hospital, Zurich, Switzerland
b Division of Pulmonary Medicine, University Hospital, Zurich, Switzerland
c Children’s Hospital, University of Zurich, Zurich, Switzerland
Accepted for publication July 6, 2001.
* Address reprint requests to Dr Weder, Division of Thoracic Surgery, University Hospital, Rämistrasse 100, Zurich 8091, Switzerland
e-mail: walter.weder{at}chi.usz.ch
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Abstract |
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Methods. Rat single-lung transplantation was performed in two (n = 10) experimental groups after 18 hours of cold (4°C) ischemia. Group I animals consisted of the ischemic control group. In group II, donor and recipient animals were treated with intraperitoneal injection of 10 mg/kg melatonin 10 minutes before harvest and reperfusion, respectively. After 2 hours of reperfusion, oxygenation, plasma, and bronchoalveolar lavage nitrite levels were measured. Lung tissue was assessed for thiobarbituric acid reactive substances and myeloperoxidase activity. Peak airway pressure was recorded throughout the reperfusion period.
Results. The melatonin-treated group showed significantly better oxygenation (321.8 ± 33.8 mm Hg versus 86.1 ± 17.4 mm Hg; p < 0.001), reduced lipid peroxidation (0.65 ± 0.3 nmol/g versus 1.63 ± 0.8 nmol/g; p = 0.032), and reduced myeloperoxidase activity (0.56 ± 0.1 
OD · mg-1 · min-1 versus 1.01 ± 0.2 OD · mg-1 · min-1; p = 0.032). Bronchoalveolar lavage nitrite levels in the transplanted lungs were significantly lower in group II than in group I (0.34 ± 0.06 µmol/L versus 1.65 ± 0.6 µmol/L; p = 0.016). In group II significant reduction in peak airway pressure was noted compared with group I (p = 0.002).
Conclusions. In this model, exogenously administered melatonin effectively protected lungs from reperfusion injury after prolonged ischemia.
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Introduction |
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Melatonin (5-methoxy N-acetylserotonin), the main secretory product of the pineal gland, was recently found to be a free radical scavenger and antioxidant [8]. Melatonin is believed to work through electron donation to directly detoxify free radicals such as the highly toxic hydroxyl radical [8]. In both in vitro and in vivo experiments, melatonin has been found to protect cells, tissues, and organs against oxidative damage induced by a variety of free radical-generating agents and processes, for example, the carcinogen safrole, lipopolysaccharide, kainic acid, Fenton reagents, potassium cyanide, L-cysteine, excessive exercise, glutathione depletion, carbon tetrachloride, paraquate, amyloid ß protein, ionizing radiation, and ischemia-reperfusion injury [9–17].
The purpose of this study was to determine the effect of melatonin on posttransplant lung ischemia-reperfusion injury.
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Material and methods |
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Melatonin (Sigma Chemicals, Buchs, Switzerland) was dissolved in absolute ethanol and then diluted in saline (the final concentration of ethanol was 10%).
All animals received humane care in accordance with the "Guide 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 volume-controlled ventilator (Harvard Rodent Ventilator, model 683; Harvard Apparatus, South Natick, MA) and ventilated with a fraction of inspired oxygen of 1, a tidal volume of 10 mL/kg at 75 breaths/min, and a positive end-expiratory pressure of 2 cmH2O. After this, a median laparosternotomy was performed and 1,000 IU/kg of heparin (Liquemin; Roche Pharma [Schweiz] AG, Reinach, Switzerland) was injected into the inferior vena cava. For the 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 (Perfadex; Xvivo Transplantation Systems AB, Gøteborg, Sweden) at 4°C which also contained 500 µg/L prostaglandin E1 (Prostin VR, Pharmacia & Upjohn, Peapack, NJ). After the lungs had been flushed, the intratracheal tube was clamped to keep the lungs inflated during the storage. Hypothermic condition was maintained during the cuff (16-gauge) placement into the pulmonary artery, pulmonary vein, and main bronchus. The vessels or bronchus was 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 measurements were the same as in donor animals. For measuring the airway pressure during the procedure, a three-way stopcock was inserted between the intratracheal tube and the ventilator circuit and connected to a pressure transducer. 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 the left main 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/min, and a positive end-expiratory pressure of 2 cmH2O) for 2 hours.
Experimental setting
Animals were randomized into two groups (n = 10, each). The ischemic control (group I) group underwent 18 hours cold (4°C) ischemia followed by transplantation, and intraperitoneal saline injection (1 mL) 10 minutes before harvest and reperfusion. The melatonin-treated group (group II) underwent 18 hours cold (4°C) ischemia followed by transplantation, and donor and recipient treatment with an intraperitoneal injection of 10 mg/kg melatonin 10 minutes before harvest and reperfusion, respectively. In each group 5 transplantations were performed for metabolic analysis such as for myeloperoxidase (MPO) and thiobarbituric acid reactive substances (TBARS) with an additional 5 transplantations for the measurement of bronchoalveolar lavage (BAL) nitrite level of the transplanted lung and plasma nitrite concentration (total 10 transplantations in each group). After all transplantations, blood gas analysis was performed and peak airway pressures were measured during the 2-hour reperfusion period. Right donor lungs (n = 5) were assessed for MPO and TBARS to obtain base line values in normal lung. In an additional 5 rats, selective left lung BAL and plasma nitrite levels were measured to obtain normal levels.
Myeloperoxidase assay
Frozen lung tissue (100 mg) was homogenized in 1 mL of 0.5% hexadecyltrimethylammonium bromide, 5 mmol/L EDTA, and 50 mmol/L potassium phosphate buffer (pH 6.2) with a tissue grinder [18]. The homogenate was centrifuged at 4,000 rpm for 15 minutes at 4°C. The supernatant was assayed for total soluble protein and for MPO activity. Enzyme activity was measured spectrophotometrically. Ten mg of fivefold supernatant was combined with 0.6 mL Hanks’ BSA, 0.5 mL 100 mmol/L potassium phosphate buffer (pH 6.2), 0.1 mL 0.05% H2O2, and 0.1 mL 1.25 mg/mL o-dianisidine. The reaction was stopped by addition of 1% NaN3 after 5 and 20 minutes at room temperature, respectively. The optical density was measured at 460 nm with a spectrophotometer (Kadas 100, Dr. Lange AG Zurich, Zurich, Switzerland). Color development from 5 to 20 minutes was linear. Enzyme activity is expressed as change in optical density unit per milligram of tissue protein per minute (OD · mg-1 · min-1).
Thiobarbituric acid reactive substances
Thiobarbituric acid reactive substances was measured 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 dodecyl sulfate, 1.5 mL of 20% acetic acid solution adjusted to 3.5 pH 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) was added (all chemical by Fluka AG, Buchs, Switzerland). The solution was centrifuged at 4,000 rpm for 10 minutes. The absorbance of upper layer was measured at 532 nm with a spectrophotometer (Kadas 100, Dr Lange, AG Zurich, Zurich, Switzerland). The TBARS levels were determined by reference to a standard curve of 1,1,3,3-tetramethoxypropane (Sigma Chemicals, Buchs, Switzerland), and the results were expressed as nanomoles of malondialdehyde (MDA) per gram of wet lung.
Measurement of bronchoalveolar lavage nitrite concentration
The heart-lung block was harvested immediately after 2-hour reperfusion period (as in the donor procedure). Under direct vision with the use of microscope, right main bronchus was occluded by a clamp. Two milliliters of saline solution was instilled selectively into the left (transplanted) lung and withdrawn in turn. The returned fluid was stored on ice and processed immediately. It was centrifuged at 2,000 rpm for 10 minutes. The BAL supernatant was freshly analyzed for nitrite level by a modified Griess reagent (Sigma Chemicals, Buchs, Switzerland) [20]. The absorbances were measured at 550 nm using a microplate reader (MRX microplate reader, Dynatech Medical Products, Guernsey, United Kingdom). Nitrite concentrations (µmol/L) were calculated by comparison with optical density at 550 nm of standard solutions of sodium nitrite.
Measurement of plasma nitrite concentration
Heparinized arterial blood (3 mL) was obtained from the thoracic aorta. It was centrifuged at 2,000 rpm for 10 minutes. Plasma was freshly evaluated for its nitrite concentration (µmol/L) with the same method described above.
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 hours of reperfusion, oxygenation of the graft was evaluated by 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. The transplanted lung was excised and put into the liquid nitrogen and stored at -80°C for further evaluation of TBARS and MPO.
Statistical analysis
Data analysis was performed with the SPSS for Windows 8.0 (SPSS Inc, Chicago, IL). All data were expressed as mean values ± standard deviation. As it was not possible to check for the normality of the variables with only five observations per group, we used nonparametric procedures. We performed the Mann–Whitney test for comparing between two groups. To evaluate the statistical difference between the groups regarding the peak airway pressure over the 2-hour reperfusion (which consisted of 14 measurements), analysis of variance for repeated measures was used. A p value less than 0.05 was considered significant. The normal values were just given for comparison purpose, but were not used in any testing procedure.
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Results |
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Peak airway pressures
The analysis of variance for repeated measures using all measurements made during the reperfusion period differed significantly between the groups (p = 0.002). At the end of 2-hour reperfusion, PawP was significantly less in group II than that of group I (10.2 ± 0.4 mm Hg, versus 15.4 ± 0.5 mm Hg, respectively) (p = 0.008) (Fig 1).
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Myeloperoxidase activity
Myeloperoxidase activity in the normal lungs was 0.4 ± 0.12 OD · mg-1 · min-1. Myeloperoxidase activity in group II was significantly lower compared with group I (0.56 ± 0.1 OD · mg-1 · min-1 versus 1.01 ± 0.2 OD · mg-1 · min-1, respectively) (p = 0.032).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Oxidative stress is described as cellular, tissue, and organ damage inflicted because of toxic molecules that are persistently generated in organisms. Many of the free radicals that are produced are a consequence of the utilization of oxygen. Because of their unpaired electron, free radicals are highly toxic and lead to damage of lipids, proteins, and DNA. Considering the role of oxidative damage in a wide variety of disease processes, interest in molecules that neutralize free radicals has increased substantially in past few years.
Recently it was reported that melatonin is a free radical scavenger with the ability to neutralize the toxicity of the hydroxyl radical, singlet oxygen, and possibly, the peroxyl radical and the superoxide anion [8]. Melatonin as an antioxidant is effective in protecting nuclear DNA, membrane lipids and, presumably, cytosolic proteins from oxidative damage [8]. Besides its direct free radical scavenging and membrane stabilization, melatonin has been reported to alter activities of enzymes that improve the total antioxidative defense capacity of the organism, that is, superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase [21], and nitric oxide (NO) synthase [22]. Other than being a highly effective direct free radical scavenger and antioxidant, melatonin is readily absorbed when it is administered by any route, crosses all morphophysiologic barriers (eg, the blood–brain barrier and placenta) with ease, seems to enter all parts of every cell where it prevents oxidative damage, and preserves mitochondrial function [23].
The effect of melatonin on ischemia-reperfusion-induced arrhythmias in the isolated rat heart model has been investigated and compared with the well-known antioxidant, vitamin C [10]. It has been found that melatonin was more potent than vitamin C in protecting against ischemia-reperfusion-induced arrhythmias, including decreasing the severity and reducing their incidence and duration [10]. In isolated rat heart models, melatonin has been found to be effective for protection of postischemic reperfusion injury as a result of its hydroxyl radical scavenging effect and reducing the extent of lipid peroxidation [15, 17].
Melatonin administration prevented the postischemic gastric mucosal injury induced by ischemia-reperfusion, as shown by reduced lipid peroxidation, MPO activity, and limitation of decreased glutathione peroxidase activity [11].
The protective effect of melatonin against oxidative stress during reperfusion of the liver after induced ischemia was examined using both biochemical and morphologic measurements [12]. The authors reported that exogenously administered melatonin effectively protected the liver against oxidative damage, an effect that was shown by reduced lipid peroxidation, lowered polymorphonuclear leukocyte infiltration, reduced glutathione and oxidized glutathione levels, and elevated glutathione reductase activity.
In a model of rat intestinal ischemia-reperfusion injury, melatonin has been reported to have a strong antioxidant effect in preventing intestinal damage, as shown by light microscopy and reduced MDA levels [16].
Using hamster cheek pouch microcirculation, during ischemia-reperfusion injury, melatonin was found to completely inhibit the microvascular edema formation and reduce the number of leukocytes sticking to venules after reperfusion [14]. Additionally, melatonin prevented the marked decrease in perfused capillary length, preserving microvascular perfusion. Melatonin protected the endothelial barrier integrity and preserved microvascular blood perfusion after ischemia-reperfusion [14].
The peroxyl radical scavenger ability of melatonin was compared with that of vitamins E, C, and glutathione. The scavenging ability of melatonin was about two times higher than that of vitamin E and C and about three times higher than that of glutathione [24].
The effect of exogenous melatonin administration on NO-induced changes during brain ischemia-reperfusion has been studied recently [12]. Nitrite/nitrate levels and cyclic guanine monophosphate were used as an indicator of cerebral cortical and cerebellar NO production. It has been shown that melatonin prevented the increases in NO and cyclic guanine monophosphate production after transient ischemia-reperfusion in the frontal cerebral cortex and cerebellum of Mongolian gerbils. The inhibitory effect of melatonin on NO production and its ability to scavenge free radicals and the peroxynitrite anion might be responsible for the protective effect of melatonin [12]. The peroxynitrite anion is formed after the interaction of NO and the superoxide anion, which is highly toxic to cells and has also been shown to contribute to the oxidative injury of the lung [25].
Inhibition of NO synthesis with NO synthase inhibitors has been shown to decrease lung injury in various reports [26]. Nitric oxide activity in biologic fluids can be indirectly assayed by measuring its stable metabolic byproducts such as nitrite and nitrate [27]. In a canine unilateral hypochloric acid-induced lung injury model, it has been reported that increased NO production is compartmentalized to the injured lung, which was shown by increased BAL nitrite and nitrate levels [27]. The increase in BAL nitrite levels support the notion that lung injury results in alveolar production of NO. Nitric oxide may contribute to the pathogenesis of lung injury by secondary formation of peroxynitrite, which may reflect an effect of lung injury [27]. Recently, peroxynitrite production in human acute lung injury has been shown by staining for nitrotyrosine residues [28]. In our study, we observed high BAL nitrite levels in the nontreated ischemic group that was lowered by melatonin treatment nearly to normal levels. However, plasma nitrite levels were close to normal levels in both groups, a finding that may demonstrate the localized production of NO in the injured lung.
We observed high levels of MDA and increased MPO activity, an index of neutrophil infiltration, in the nontreated ischemic control group, supporting the notion that lipid peroxidation and neutrophil infiltration occur during ischemia-reperfusion injury. In the present study lipid peroxidation and neutrophil infiltration in the melatonin-treated group was significantly less than that of nontreated group. Endotoxic shock, which is stimulated by high doses of lipopolysaccharide, and ischemia-reperfusion injury have in common a generation of free radicals and accumulation of neutrophils in the damaged tissues [9]. Melatonin has been shown to abolish lipopolysaccharide-induced lipid peroxidation in liver, brain, and lung homogenates [9]. Besides its efficacy as a direct free radical scavenger, melatonin has other features that might be beneficial during and after lung transplantation. For example, melatonin has been shown to limit both viral and bacterial infections in experimental studies [29].
In conclusion, in this experimental model, donor and recipient treatment with melatonin protected the lungs against ischemia-reperfusion injury, which could be due to melatonin’s free radical scavenging activity and its ability to reduce neutrophil infiltration.
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Acknowledgments |
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We thank Dr Valentin Rousson who reviewed the statistical analysis of this study as a biostatistician, and Vlasta Strohmeier for preparing the animals.
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References |
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