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Ann Thorac Surg 2002;74:811-818
© 2002 The Society of Thoracic Surgeons
a Cardiovascular Institute Dresden, Dresden, Germany
b Department of Obstetrics and Gynaecology, University of Dresden, Dresden, Germany
c Department of Cardiovascular Surgery, University Hospital Eppendorf, Hamburg, Germany
* Address reprint requests to Dr Wagner, Department of Cardiovascular Surgery, University Hospital Eppendorf, Martinistr 52, D-20246 Hamburg, Germany
e-mail: fl.wagner{at}uke.uni-hamburg.de
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Abstract |
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Methods. After left lateral thoracotomy in group I (n = 6), normothermic lung ischemia was maintained for 90 minutes followed by a 5-hour reperfusion period. In group II, animals (n = 6) underwent ischemia as in group I, but received vitamins (preoperative IV bolus C = 1 g, E = 0.75 g, then continuous infusion (125 mg/h) each throughout the study). In Group III, animals (n = 6) underwent sham surgery and served as controls. Hemodynamic variables and gas exchange were assessed. The CM was performed for injury quantification in blood samples and to determine activation of isolated PMNs. The Wilcox rank test was used for statistical analysis.
Results. During reperfusion, all animals in group I developed significant pulmonary edema with significant loss of pulmonary function. The addition of vitamins (group II) improved oxygenation and almost abolished pulmonary inflammatory cell infiltration; however, as in group I, pulmonary compliance still tended to decline and the number of circulating leucocytes increased. The CM showed that, compared with group I, vitamins reduced O2- basic release by PMNs significantly (460% to 170%, p < 0.05; control 165%), but could not prevent an increase of free ROS in whole blood similar to group I (443% to 270%, p = ns, control 207%). With regard to lipid peroxidation only a trend of reduction was observed (117% to 105%, p = ns, control 100%).
Conclusions. Differentiated analysis by CM demonstrated that vitamins C and E inhibited PMN activation but were not able to prevent radical production by other sources. This offers a potential explanation why radical scavengers like vitamins only attenuate but ultimately do not prevent reperfusion injury.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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Therefore we established an in vivo pig model of standardized lung ischemia followed by a 5-hour reperfusion period concentrating on the analysis of biochemical rather than functional aspects of the observed injury. The applied method of enhanced chemiluminescence measurement (CM) allows "on-line" quantification of free ROS, to evaluate activation status of isolated polymorphonucleated cells (PMN) by means of O2- release and of subsequent lipid peroxidation. In this model we determined the efficacy and mechanisms of vitamins C and E to prevent reperfusion injury.
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Material and methods |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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Study groups and measurements
Three groups were investigated: in group I (n = 6) after heparinization (400 IU/kg) left pulmonary artery as well as veins were clamped and a Fogarty ballon catheter (8F, Baxter, Unterschleissheim, Germany) was inflated in the left main bronchus to disconnect the left lung from ventilation. Normothermic ischemia was maintained for 90 minutes followed by a 5-hour reperfusion period. Animals in group II (n = 6) were treated as in group I, however, received an initial oral dose of vitamins 1 day before surgery, plus one IV loading dose immediately after start of anaesthesia (C = 1 g, E = 0.75 g, respectively), followed by a perioperative continuous IV infusion of both vitamins throughout the study (C = 125 mg/h, E = 125 mg/h). In group III (control; n = 6), animals underwent sham surgery, ie, thoracotomy, pulmonary hilum dissection with disruption of all bronchial arterial vascularization but no clampings. Thereafter, anesthesia and ventilation were maintained for an overall study period comparable to that of the other groups. In animals in groups I and II, an additional catheter was placed into the the left inferior pulmonary vein immediately before reperfusion. This catheter served to collect left lung effluent before mixing with the blood from the contralateral lung. Variables assessed included hemodynamics (systemic systolic/diastolic blood and central venous pressure), arterial and venous blood gases, airway pressures, pulmonary compliance, and leukocyte count in the pulmonary artery and veins. Representative tissue samples were collected after dissection of the hilum was completed and after 5 hours of reperfusion from the left and right lung. Histologic examination was performed by light microscopy on hematoxylin and eosin—stained sections. At indicated time points (baseline, after dissection of the hilum, during ischemia, as well as after 30, 60, 120, 180, 240, and 300 minutes of reperfusion), postpulmonary blood samples were taken for CM. Measurement of ROS and O2- were performed in whole blood samples and measurements of lipid peroxidation performed in serum. For detection of O2- basic release by isolated arterial PMNs, separate samples of heparinized blood were taken at baseline, after dissection of the hilum, and at 2 hours of reperfusion. Serum levels of vitamin E were determined in group II animals at baseline, after dissection, and at 2 hours of reperfusion. In all other animals (which had not received any vitamin substitution), vitamin serum levels were measured only at baseline.
Chemiluminometric measurements
Chemiluminometric measurements (CM) were performed as previously published [4, 5]. In brief, EDTA whole blood (10 µL) was diluted with Hanks solution (1,000 µL), Zymosan (50 µL) added and the mixture incubated for 30 minutes at 37° Celsius. Addition of luminol (250 µmol/L) or lucigenin (250 µmol/L) allowed to measure the stimulated activity of ROS (mainly OH- and H2O2) or of O2-, respectively. The emitted light units were taken as relative measure of substrate concentration/reactivity.
The peroxidation of lipid groups was quantified by addition of luminol (250 µmol/L) after immunoprecipitation of LDL and HDL from serum in specifical antibody-coated tubes.
To determine the release of O2- by PMNs, those cells were separated from blood by standardized gradient centrifugation, cell number adjusted to 106/mL; addition of Lucigenin (250 µmol/L) allowed us to measure the quantity of secreted free O2- by those pure and isolated cell populations (without stimulation, ie, baseline secretion). Luminol- and Lucigenin-enhanced chemiluminometric measurements were performed in two luminometers (whole blood and lipidperoxidation: LB9503; PMNs: LB9505, Berthold, Franfurt, Germany).
Animal care
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research, and the "Guide for the Use and Care of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institute of Health (NIH publication 86-23, revised 1985). All protocols were approved by the Animal Care Section of the Saxonian Government as well as the Animal Care Committee of the University of Dresden.
Statistical analysis
Group mean values with standard deviations were calculated for all measured parameters. Each chemiluminometric measurement was normalized to the respective value obtained at the starting point of surgical intervention (base line value). Analysis of variance, Wilcox rank test, and Mann-Whitney U test were used for statistical analysis; p less than 0.05 was accepted as significant.
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Results |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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However, all animals in the ischemic group (group I) developed significant pulmonary edema, a decreased p art O2 in the effluent of the ischemic lung during early reperfusion and after 5 hours of reperfusion, as well as reduced pulmonary compliance starting 3 hours after reperfusion (p < 0.05 vs control) (Fig 1). Those findings correlated with increased numbers of circulating leukocytes, their absorption into the lung (detected by the difference of pre- and postpulmonary leukocyte count), and typical signs of parenchymal reperfusion injury on light microscopic examination (marked cellular infiltration, swelling of the alveolar septa, and signs of intraalveolar hemorrhage) (Fig 2).
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In contrast, animals in group I who underwent unprotected ischemia showed, in all of these variables, statistically significant changes during reperfusion when compared with control animals: the basic release of O2- by isolated PMNs increased after 2 hours of reperfusion (460% vs control 165%, p < 0.05; Fig 3A). The CM of free radicals in whole blood samples demonstrated a continuous rise already starting during ischemia, with O2- reaching its peak value after 5 hours (558% vs control 154%, p < 0.05, Fig 3B) and ROS reaching its peak value after 4 hours (443% vs control 209%, p < 0.05, Fig 3C). Lipid peroxidation peaked significantly at 1 hour after reperfusion (117% vs control 100%, p < 0.05, Fig 3D) and decreased thereafter.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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Administration of well-known radical scavenger vitamins C and E did attenuate functional consequences of reperfusion injury such as reduced gas exchange, lipid peroxidation, and leukocyte infiltration with resulting tissue injury; but already the tremendous increase in circulating leukocytes, paralleled by lack of significant improvement in pulmonary compliance compared with unprotected ischemia, underline incomplete prevention. In general, these observations correlated well with the results of many other animal and clinical studies that tested similar substances to prevent injury. Baker and colleagues [9] showed that the addition of a water-soluble 
-tocopherol to a preservation solution prolonged ischemic viability of vascular endothelial cells, and improved but did not normalize postischemic lung function in an isolated perfused rat lung model. Others tested radical scavengers such as dimethylthiourea or N-acetylcysteine and found them to be beneficial, although protection still remained incomplete [10, 11].
The chemiluminometric results of this study clearly show one of theoretically possible reasons for this incomplete protection: the used vitamins blocked radical release by PMNs, certainly a major source during injury; however, they were not able to scavenge the remaining free ROS in whole blood, nor were they able to prevent their production from other sources. This reflects quite well the documented discrepancy in clinical variables: for example improvement of oxygenation but worsening of pulmonary compliance. It remains unclear, however, why PMNs from animals with elevated vitamin blood levels secrete fewer oxygen radicals. One potential explanation is that the administered vitamins do reduce injury-produced signal transduction, ie, inflammatory cascade activation, thereby preventing activation of aggressor cells such as PMNs. Engelhardt and colleagues [12], for example observed in a rat model of reperfusion injury that -tocopherol reduced lipid peroxidation, resulting in a protective effect on the vascular endothelium [12]. Another hypothesis could be that the liposoluble vitamin E is capable of penetrating the cellular membrane and exerting a stabilizing effect of intracellular protective enzymes against oxidation. Velsor and Postlethwait [13] showed, in their rat lung injury model, that a larger part of administered a-tocopherol is indeed bound to cells. Another indication for its potential intracellular effects is the observation that -tocopherol stabilizes posttransscriptional mRNA of gluthation-peroxidase, another important intrinsic protection enzyme against radical injury [14].
Other investigators have also observed that PMNs in healthy human volunteers who had previously taken vitamins C and E produce substantially fewer oxygen free radicals upon stimulation with arachidonic acid, and also showed lowered serum lipid peroxidation [5].
The chemiluminometric results of this study confirm that vitamins have the capacity to modulate the radical secretory activity of PMNs; however, the underlying mechanism remains unclear. The major limitation of the present study is that CM allows differentiated analysis of ongoing processes in blood or serum but does not permit any direct examination regarding the status of noncirculating parenchymal cells, such as endothelial or other signal- and radical-producing cells. This is particularily important, as there is increasing evidence that other pathways of the inflammatory cascade (such as cytokines, complement activation, adhesion and other important molecules in signal transduction) are also involved in the development of reperfusion injury after ischemia [3].
In summary, chemiluminometric measurements were able to highlight reasons for incomplete protection against reperfusion injury by scavenging substances such as the tested vitamins by differentiation of radical production from various sources (ie, PMNs from other, not yet identified cells). Although this method is still not able to identify exact sources, the significant differences of radical production observed indicate that a more sophisticated analysis of complex in vivo reperfusion mechanisms might be a successful approach for a better understanding of the underlying pathophysiology. To combine this model with analysis of factors involved in the inflammatory cascade (such as endothelial cell activation and other tissue factors) might finally help to develop more effective strategies of protection against reperfusion injury.
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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Discussion |
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DR WAGNER (Dresden, Germany): Sure. Studies about reperfusion injury generally produce enormous intragroup variation as also observed in our study. This is particularly true for functional parameters after pulmonary transplantation, indeed fair oxygenation capacity of a transplanted lung does not exclude significant reperfusion injury. And it is the latter that in the clinical situation often leads to prolonged ventilator dependence with all its sequelae. Therefore, it was no surprise that we observed this phenomenon also in our animal model, however, as shown in my talk despite large standard deviation mean value differences, were still large enough to reach statistical significant differences. On top of that, macroscopically lungs after unprotected ischemia were very heavy and had a much higher wet/dry ratio. But this is not the important message of this study. We all know from several studies that vitamins alone do not protect against reperfusion injury. The clue is that our study showed for the first time that vitamins still do suppress radical formation, but unfortunately only partially, a phenomenon that correlates nicely with the observed partial protection on the pulmonary functional level. So, the part of radical release that is not taken care of by the vitamins is responsible for the ongoing injury and needs to be a target of further studies.
DR ZWISCHENBERGER: So there are additional data that may be forthcoming?
DR WAGNER: They are included in the paper.
DR ZWISCHENBERGER: I would encourage you not to hold back if you have data that show functional improvement, such as wet-to-dry ratios. You said the compliance didn’t change, so you may have concluded that the mild reduction in reactive species by vitamins has very little functional impact on the lungs. I would challenge you to come forth with data that supports your hypothesis.
DR WAGNER: That is correct. The statistically significant decrease in pulmonary compliance after unprotected ischemia was not reversed by application of vitamins. However, oxygenation capacity and leukocyte migration were both influenced positively reflecting a mild protective effect of vitamins.
DR MARK I. BLOCK (San Francisco, CA): I have a very quick follow-up on the point you have been discussing. On your curves, are those standard errors or standard deviations that you were showing?
DR WAGNER: They were standard deviations.
DR BLOCK: You point out that the difference at many of those time points are statistically significant with a p value of less than 0.05. But if you’re going to compare differences at more than one time point in the same experiment, you need to correct your p value to a lower number, using the Bonferroni correction. If you’re looking at five different time points, your threshold is now a p of less than 0.01. So I would caution you that when you put together your manuscript, you should be careful about the statistics and what you say is significant and what’s not significant.
DR WAGNER: Sure. I know. Thanks for the comment; it’s certainly very valuable. In general, I think you can always argue about this problem with these kinds of studies, because you look at isolated time points and you try to compare them and see if there is a statistically significant difference. We did the variance test with ANOVA and the Wilcox rank. So I think it is fair enough to say there is a statistically significant difference, but it is, of course, reduced or eliminated to singular time points and not through the whole study.
DR WILLIAM A. COOK (North Andover, MA): Doctor Wagner, is it possible for you to bring back one of your slides, the ones that have the histologic pictures?
DR WAGNER: Sure.
DR COOK: Now, if you look at the picture in the upper right-hand corner, the ischemia slide, many years ago I did some work with various rejection problems and changes secondary to shock. What was happening when this appeared was that red cells were aggregating on some sort of a cell, which we never could define, and forming little microemboli. This gives you that picture you’re looking at there. When I was looking at it, it occurred to me that there was something about your vitamin treatment that must have altered that clumping phenomenon. I find this very interesting, because we never really could figure out why it happened, and it’s a common endpoint for all kinds of pulmonary injury.
DR WAGNER: Thank you very much for this comment. I absolutely agree, and indeed I think the histological interpretation, to begin with, of these lung injuries always is very, very difficult because you have to pick good samples, representative tissue. And I think when we think about the presentation from Dr Vollmar before, I absolutely agree that obviously coagulation or some sort of a similar mechanism does play a role in these injuries. And I think other information in this paper that hints in the same direction is that the vitamins prevented the migration of the leukocytes into the tissue, at least not to the same amount as it was in the ischemic group. So this might go into the same direction (which is, of course, pure speculation at this point, but it might well be).
DR COOK: Very interesting. Thank you.
DR DIRK E. M. VAN RAEMDONCK (Leuven, Belgium): I may have missed it, but did you exclude the right lung after reperfusion of the left lung?
DR WAGNER: No, we didn’t.
DR VAN RAEMDONCK: If not, how do you know that the differences you have seen were coming from the left lung and not from the right lung?
DR WAGNER: You mean the difference in regard to the chemiluminescence?
DR VAN RAEMDONCK: The difference in functional assessment, the difference in compliance, and the difference in blood gases. Was it related to the function of the left lung only?
DR WAGNER: No, no, no. What we did is, basically we clamped the lung, we took it off the ventilation by a balloon that was inflated into the main bronchus, and then after 90 minutes of warm ischemia, the lung was ventilated again. So there were both lungs ventilated, and all together you found a systemic effect that overall the pulmonary compliance of both lungs did decrease. So we look at an effect of the overall lung function. But if you look at other papers, recently you find proof that if you have a significant reperfusion injury probably due to cytokines and other mediators, it does affect your healthy or your nonischemic lung as well. So if you have a significant injury, it spreads to the whole lung, so to speak, at least in inflammatory parameters, et cetera. There is not a good explanation for why this happens. We just know it does happen. All I can tell you is basically that histologically there is very severe damage in the previously ischemic lung. We also looked at the control lung, which looks pretty normal, and you see the functional differences and you find the differences in the radical release, the radical release measured in the effluent by a catheter that was put up at the pulmonary vein of the inferior lobe just to make sure that we really collect blood out of the left lung and not mixed blood.
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References |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentDiscussionReferences |
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