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Ann Thorac Surg 2007;84:240-246
© 2007 The Society of Thoracic Surgeons
a Division of Thoracic Surgery, University of Zurich, Zurich, Switzerland
b Department of Pathology, University of Zurich, Zurich, Switzerland
Accepted for publication March 26, 2007.
* Address correspondence to Dr Weder, University Hospital, Division of Thoracic Surgery, Rämistrasse 100, Zurich, 8091, Switzerland (Email: walter.weder{at}usz.ch).
Presented at the Forty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 29–31 2007.
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Abstract |
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Methods: Rat single-lung transplantation was performed in two experimental groups (n = 5) after 18 hours of cold (4°C) ischemia. Group I was the ischemic control (IC) group. In group II (NAC), donor and recipient animals were treated with an intraperitoneal injection of 150 mg/kg NAC 15 minutes before harvest, and recipient animals were treated again before reperfusion. After 2 hours of reperfusion, oxygenation was measured. Lung tissue was assessed for lipid peroxidation, neutrophil infiltration, and reduced glutathione level. Peak airway pressure was recorded throughout the reperfusion period.
Results: Rats treated with NAC showed significantly better oxygenation (184.5 ± 83.3 mm Hg versus 67.3 ± 16.4 mm Hg, p = 0.016) and reduced lipid peroxidation (7.34 ± 1.9 µmol/g versus 17.46 ± 10.6 µmol/g, p = 0.016). Lung tissue reduced glutathione levels were 6.8 ± 0.9 µM in the IC group and 20.6 ± 2.4 µM in the NAC group (p = 0.004). Peak airway pressure at the end of the reperfusion period was 14.4 ± 1.6 cm H2O in the NAC group, and 19.2 ± 2.2 cm H2O in the IC group (p = 0.008). Myeloperoxidase activity and the ratio of wet-to-dry weight did not differ between the groups.
Conclusions: In this model, exogenously administered NAC effectively protected the lungs from reperfusion injury after prolonged ischemia.
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Introduction |
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Pulmonary ischemia–reperfusion injury is characterized by increased pulmonary vascular resistance, poor oxygenation, worsened compliance, and increased capillary permeability leading to edema formation. The ischemic insult to the lung results in cytokine production and increased expression of adhesion molecules by hypoxic lung cells. The injury cascade is mediated mostly by neutrophil-endothelial adherence and subsequent neutrophil-mediated organ injury. Activated neutrophils secrete reactive oxygen species and proteolytic enzymes that result in structural and functional injury to the lung parenchyma [2].
Several studies have shown that agents such as prostaglandins; the oxygen free radical scavengers superoxide dismutase, catalase, glutathione, allopurinol, dimethyl thiourea, lazaroids, and trimetazidine; aprotinin; platelet factor antagonists; and angiotensin-converting enzyme inhibitor, captopril, and melatonin to be effective in protecting lungs against ischemia–reperfusion injury [3–8].
N-Acetylcysteine (NAC) is a precursor of the most important physiologic antioxidant glutathione. Sulfhydryl-containing compounds, especially reduced glutathione (GSH), are important in the protection of cells against hydroperoxide damage. GSH is involved in maintaining the cellular oxidation-reduction balance and has been shown to protect cells from a wide variety of endogenous and exogenous insults. GSH can also scavenge free radicals produced by oxidative challenges [9]. There have been many suggestions that GSH may be useful therapeutically as an antioxidant and cytoprotective agent [10].
NAC is used successfully in the treatment of chronic bronchitis and fulminant liver failure after acetaminophen overdose [11, 12]. It has also been used successfully in experimental conditions to protect the lung against reactive oxygen species attack and in heart failure after warm ischemia and reperfusion [13, 14]. Although the precise mechanism of action of NAC is unclear, it is assumed to involve direct or indirect reactive oxygen species scavenging.
We conducted this experimental study in a rat single-lung transplant model to investigate whether donor and recipient treatment with NAC would reduce ischemia–reperfusion injury after lung transplantation after 18 hours of cold (4°C) ischemic storage.
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Material and Methods |
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Donor Procedure
Animals were anesthetized by intraperitoneal injection of sodium thiopental (50 mg/kg; 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 Co Inc, 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 3 cm H2O. After this, a median laparosternotomy was performed and heparin (1000 IU/kg; Liquemin, Roche Pharma [Schweiz] AG, Basel, 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, Göteborg, Sweden) at 4°C. 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 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% isoflurane during the operation and reperfusion period. Ventilation parameters were the same as in donor animals. For measuring the airway pressure during the procedure, a three-way tap was inserted between the intratracheal tube and the ventilator circuit and connected to a pressure transducer.
A left thoracotomy was performed through the fourth 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 the pulmonary vein and arterial clamps were released. The thoracotomy was closed loosely. The recipient animal was ventilated for 2 hours with 99.5% oxygen, 0.5% isoflurane, a tidal volume of 10 mL/kg at 75 breaths/min, and a positive end-expiratory pressure of 3 cm H2O.
Experimental Setting
Animals were randomized into two groups of 5 rats each. Group I was the ischemic control (IC) group, in which 18 hours of cold (4°C) ischemia was followed by transplantation and intraperitoneal saline injection (1 mL) 15 minutes before harvest and reperfusion, respectively, with no treatment. In group II (NAC group), transplantation occurred after 18 hours of cold ischemia (4°C), and donor and recipient were treated with an intraperitoneal injection of 150 mg/kg NAC (Fluimucil, 100 mg/mL; Zambon Schweiz AG, Cadempino, Switzerland) 15 minutes before harvest and reperfusion, respectively. Right donor lungs (n = 5) were assessed for reduced glutathione, myeloperoxidase, and thiobarbituric acid reactive substances (TBARS) to obtain baseline values in the normal lung.
Assays and Evaluations
Myeloperoxidase assay
Quantitative myeloperoxidase (MPO) activity, as measured for neutrophil migration to the graft, was determined by a ready-to-use kit (Myeloperoxidase Assay Kit, Cytostore, Alberta, Canada). Frozen lung tissue was homogenized with a tissue-to-buffer ratio of 50 mg/mL. Absorbance was measured at 450 nm immediately after adding the development reagent in 1-minute intervals. Enzymatic activity is expressed as unit per gram of tissue protein.
Reduced glutathione assay
Lung tissue GSH measurement was performed by a kit (Calbiochem, San Diego, CA), in which 50 mg of tissue was homogenized in 950 µL of metaphosphoric acid working solution. The homogenate was centrifuged at 3000g for 10 minutes at 4°C. The upper clear layer was collected for assay and read at 400 nm. The GSH concentration was expressed as micromolar GSH.
Thiobarbituric acid reactive substances
Quantitative measurement of lipid peroxidation as TBARS was measured according to the ready-to-use kit (Malondialdehyde Assay Kit, Northwest Life Science Specialties, Vancouver, WA). A 10% wet weight per volume homogenate was prepared to determine the lipid peroxidation in the graft tissue. The absorbance of the upper layer was measured at 532 nm with a spectrophotometer, and the results were expressed as micromoles of malondialdehyde per gram of wet lung tissue.
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 thereafter every 15 minutes. At the end of 2 hours reperfusion, oxygenation of the graft was evaluated by sampling the blood directly from the pulmonary vein of the transplanted lung by means of aspiration with a 29-guage heparinized needle inserted distal to the anastomotic cuff. The transplanted lung was excised, divided into three pieces, put into liquid nitrogen, and stored at –80°C for further evaluation of GSH, TBARS, and MPO.
Histologic evaluation
At the end of the 2-hour reperfusion, the basal part of the graft was harvested for histologic evaluation. The specimens were fixed in 4% formalin and embedded in paraffin. Cut sections were stained with hematoxylin and eosin, and evaluated by an experienced pathologist (PV) completely blinded to the study.
Statistical Analysis
Data analysis was performed with SPSS 11.5 software (SPSS Inc, Chicago, IL). All data are expressed as mean values ± standard deviation. Because it was not possible to check for the normality of the variables with only five observations per group, we used nonparametric procedures. We used the Mann-Whitney test to compare between the two groups. To evaluate the statistical difference between the groups for the peak airway pressure during the 2-hour reperfusion period, which consisted of 14 measurements, analysis of variance for repeated measures was used. A value of p 
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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Wet-to-Dry Weight Ratio
The wet-to-dry weight ratio in the IC group was 8.2 ± 1.05 and 6.7 ± 1.6 in NAC group. The ratio was not different statistically between the two groups (p = 0.2).
Histologic Evaluation
Histologic sections were compared between IC and NAC groups after 18 hours of cold ischemic storage and 2 hours of reperfusion. Grafts from the IC group showed considerable interstitial edema and hemorrhagic congestion with intraalveolar accumulation of erythrocytes. However, sections from NAC treated grafts showed less edema and mild congestion (Fig 5).
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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Tissue injury in lung transplantation results from the production of oxygen free radicals initiated by reperfusion after ischemia or by ischemia alone [15, 16]. Endothelial dysfunction is a critical event during reperfusion and can be triggered within 2 to 3 minutes by the generation of a large burst of superoxide radicals. The free radical species are highly reactive and primarily cause injury through membrane lipid peroxidation [15].
Glutathione is an essential metabolite present in high concentrations (3 to 5 mmol/g) in most mammalian cells [17]. Glutathione is synthesized enzymatically within the hepatocyte from cysteine, glycine, and glutamic acid. GSH, which is glutathione in its reduced form, has an important role in cellular defense against free radical species and also acts extracellularly, either directly or by glutathione peroxidase catalysis, to scavenge free radicals [17].
The thiol-containing compound NAC has been used as an antioxidant, which may also lead to an increased GSH synthesis. As intracellular GSH build-up requires cysteine to be supplied from the outside, NAC may protect the host by entering cells and being hydrolyzed to cysteine, which stimulates GSH synthesis [18]. Most of the studies have stated that NAC can scavenge reactive oxygen species, increase glutathione levels, and serve as a reducing agent [19]. Activation of nuclear factor 
B in response to variety of signals such as interleukin 1, tumor necrosis factor-
, and hydrogen peroxide, has been shown to be inhibited by NAC, suggesting reactive oxygen species have common signaling modulators [19]. NAC inhibits activation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, redox-sensitive activating protein-1, and nuclear factor B activation [20–23]. NAC was also shown to attenuate tumor necrosis factor- messenger RNA expression and secretion in macrophages from human lung transplant recipients, which may help against transplant rejection [24].
Tissue ischemia reduces intracellular GSH. By maintaining high cellular GSH levels or by replenishing them, the degree of the potential injury by free radicals could be reduced [17, 25]. In an isolated rat liver reperfusion model, preconditioning of lungs with NAC attenuated lung respiratory or vascular derangement after ischemia–reperfusion injury [17]. The same group with the same model recently reported that the combination of NAC and mannitol afforded a higher grade of protection against lung reperfusion injury when administered as reperfusion process started [26].
In experimental studies, NAC treatment resulted in improved tissue GSH levels [19, 27]. In the present study, lung tissue GSH levels were significantly decreased in the IC group after 18 hours of cold ischemic storage, followed by 2 hours of reperfusion. NAC treatment improved GSH levels, which led to a better graft function. This could be due to the effect of NAC to increase the synthesis of GSH or direct scavenging effect of free radicals, or both. Indeed, it has been shown that pretreatment with buthionine sulfoximine, a specific inhibitor of glutathione synthesis, resulted in decreased liver tissue GSH concentrations. However, livers in the NAC treated group still functioned well, which shows the direct antioxidant effect of NAC [27].
The high levels of malondialdehyde observed in this and previous studies, support the notion that lipid peroxidation occurs during ischemia–reperfusion injury. We have shown that lipid peroxidation of the NAC group was significantly less than that of the nontreated IC group. This reduction in malondialdehyde levels is consistent with the powerful antioxidant effect of NAC. Significant reduction of lipid peroxidation has also been reported by other investigators [28–30].
Activation and accumulation of polymorphonuclear cells is one of the initial events of tissue injury that triggers the release of oxygen free radicals [29]. Although significant reduction of MPO activity has been shown in many experimental studies [29, 31, 32], in some studies, including the present study, MPO activity was not significantly different in the NAC treated groups compared with controls [30].
Recently, nebulized NAC administration was reported to protect pulmonary grafts from non-heart-beating donors from ischemia–reperfusion injury in an ex vivo lung reperfusion model [33]. Their results support our data, although they used a different model. Conversely, treatment with NAC did not result in improved kidney function after renal ischemia and reperfusion in rats [34].
The present study has several limitations. First, the sample size is small; therefore, conclusions that may be drawn from this study must be done cautiously. Second, this is a small-animal model, which may make it difficult to translate it into clinical practice. Therefore a large-animal model is planned and will be performed soon. Third, the drug was given in only one dose with a relatively short-term (2-hour) observation. Although we found a lower wet-to-dry weight ratio in NAC treated group, this was not statistically significant. A prolonged observation period, therefore, might have resulted in decreased pulmonary edema as reflected by the wet-to-dry weight ratio.
There is not a consensus on the time, optimal dose, or to whom (donor/recipient) NAC should be administered. We administered 150 mg/kg NAC intraperitoneally to both donor and recipient animals. We chose this dose because it has been shown that pretreatment in lungs with 150 mg/kg NAC afforded preservation of most indicators compared with 225 mg/kg NAC [17]. GSH content in the lung tissue with this dose was found to be 11% higher than that of 225 mg/kg, but twofold that in 100 mg/kg [17].
The rationale behind treating both donor and recipient was that ischemia reduces tissue GSH levels and administration of NAC before reperfusion will replenish or maintain the GSH levels, or both. In fact at the end of 2 hours of reperfusion after 18 hours of cold ischemic storage, lung tissue GSH levels in the treated group were approximately threefold higher than in the IC group. In this study, NAC seemed to have acted indirectly as an antioxidant agent by replenishing lung GSH levels. In addition, tissue levels of malondialdehyde, another marker for oxidative damage, were significantly lower in NAC group, supporting its antioxidant effect.
In another series of experiments, we treated the recipients only (n = 6) with 150 mg/kg NAC (the results are not shown in this report). We did not find a difference between recipient-only compared with donor-plus-recipient treatment in any of the indicators. However, we obtained better oxygenation, lower lung tissue malondialdehyde level, and higher lung tissue GSH levels in recipient-only treatment compared with the IC group. Again, there was no difference in the wet-to-dry weight ratio and lung tissue MPO levels between the groups.
Keeping in mind the difficulty of transferring experimental data into clinical practice, we believe that treating both donor and recipient will result in better organ preservation and function during ischemic preservation. Reperfusion GSH levels are decreased and free radicals are increased, which can be, in part, prevented by NAC.
In conclusion, in this experimental model, donor and recipient treatment with NAC, a drug that is used in clinical practice as a mucolytic agent, protected the lungs against posttransplantation ischemia–reperfusion injury. Further studies are warranted to assess the impact of NAC in posttransplantation lung ischemia–reperfusion injury.
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Discussion |
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Most of your data are descriptive and correlative. Can you think of any other ways that you could look at this in perhaps a little more mechanistic fashion with some transgenic mice? The second question is, what proof do you have that intraperitoneal delivery of the N-acetylcysteine actually reaches the lungs, or is this just a systemic effect of the NAC? And third, there is no real change in the wet-to-dry ratio, which would be at least one gross measurement of change in the capillary permeability index. So how do you theorize that the NAC is actually effecting changes in the permeability of the lung, which is what we see when we have a significant ischemia–reperfusion injury.
DR INCI: Regarding the first question, for transgenic models, there are no papers regarding this.
The third question was about wet-to-dry weight ratio. In this model, we couldn’t show the difference; and actually it was low, but it didn’t reach significancy. But the peak airway pressure, this is also a way to show the lung edema. So when we measured the peak airway pressures, it was significantly less; but it did not correlate with the wet-to-dry weight ratio.
And the second question, N-acetylcysteine can be applied, nebulized, intraperitoneal, and intravenous. But intraperitoneal, it is absorbed very quickly like intravenously, and its concentration is very high in the blood. So it goes directly to the lungs and then it affects them also systemically.
DR DIRK VAN RAEMDONCK (Leuven, Belgium): I also enjoyed your presentation very much. Now, you have studied two groups. And in the treated group, you gave NAC to both the donor and the recipient. Can you tell us when the drug is exactly working? Is it working in the donor prior to ischemia, or is it working in the recipient after ischemia?
DR INCI: Actually, ischemia reduces cellular glutathione levels. This has been shown in liver models, renal models, and cardiac models. When we give it both to donor and to the recipient, this is the so-called combined administration. I also made this only with the recipient treatment, but I did not show the results. What I obtained was actually that oxygenation was less, higher than the control groups, but less than the combined treatment groups. I mean, it could be mostly in the ischemic period. It enhances the glutathione level and protects the lung.
DR VAN RAEMDONCK: Another question that I would like to ask is how do you see this coming into clinical application? How can we treat the donor with NAC? Are there any other ways to administer NAC to the donors?
DR INCI: Yes, there are other ways other than intraperitoneal administration. In the clinical practice, we can use it by mouth, intravenous, or in nebulized form directly for transtracheal administration.
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Acknowledgments |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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r O, Oruc A, Kaya S, et al. N-Acetylcysteine reduces lung reperfusion injury after deep hypothermia and total circulatory arrest J Card Surg 2004;19:221-225.[Medline]This article has been cited by other articles:
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  I. Rahman Review: Antioxidant therapeutic advances in COPD Therapeutic Advances in Respiratory Disease, December 1, 2008; 2(6): 351 - 374. [Abstract] [PDF]
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 N. Geudens, M. Van de Wouwer, B. M. Vanaudenaerde, R. Vos, C. Van De Wauwer, G. M. Verleden, E. Verbeken, T. Lerut, D. E. M. Van Raemdonck, and E. M. Conway The lectin-like domain of thrombomodulin protects against ischaemia-reperfusion lung injury Eur. Respir. J., October 1, 2008; 32(4): 862 - 870. [Abstract] [Full Text] [PDF]
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 P. A. Corris and J. D. Christie Update in Transplantation 2007 Am. J. Respir. Crit. Care Med., May 15, 2008; 177(10): 1062 - 1067. [Full Text] [PDF]
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