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Ann Thorac Surg 1997;63:1383-1389
© 1997 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Department of Surgery, and Division of Cardiothoracic Anesthesiology, Washington University School of Medicine, Barnes Hospital, St. Louis, Missouri
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Abstract |
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Methods. Ten dogs underwent left lung allotransplantation. Donor lungs were flushed with modified Euro-Collins solution and stored for 21 hours at 1°C. Immediately after transplantation, the contralateral main pulmonary artery and bronchus were ligated to assess isolated allograft function. Hemodynamics and arterial blood gases (inspired oxygen fraction, 1.0) were assessed intermittently for 6 hours prior to sacrifice. Allograft myeloperoxidase activity and wet to dry weight ratio were assessed. Donor animals were divided into two groups. Group I animals (n = 5) received no NO. In group II (n = 5), donors received inhaled NO (60 ppm) at the time of harvest.
Results. Pulmonary vascular resistance decreased to 79.6% of baseline because of inhalation of 60 ppm NO in group II donor animals. Thiobarbituric acid-reactive materials were reduced during the storage period in group II, a finding suggesting less oxidant injury during storage in donor lungs treated with NO. Throughout the 6-hour assessment, oxygenation in group II was superior to that in group I (p < 0.05). At 360 minutes of assessment, mean arterial oxygen tension in groups I and II was 88.9 ± 11.4 mm Hg and 169.1 ± 33.0 mm Hg, respectively. Myeloperoxidase activity was significantly decreased in group II (p < 0.05), data indicating reduced neutrophil sequestration. Wet to dry weight ratio was significantly lower in group II.
Conclusions. These data suggest that inhaled NO at the time of harvest improves early function of preserved lung allografts by attenuating oxidant injury during storage and subsequent neutrophil sequestration.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Lung transplantation has become a successful strategy for the treatment of a variety of end-stage lung diseases. The results of lung transplantation now equal those of other solid-organ transplantations [1]. However, primary graft failure as a result of ischemia-reperfusion injury remains a major problem in lung transplantation. Mild to severe reperfusion injury occurs in 40% to 50% of lung recipients and is the second most frequent cause of deaths occurring less than 90 days after transplantation [2].
We have previously demonstrated that inhaled nitric oxide (NO) can improve lung allograft function. In canine lung allografts subjected to prolonged ischemia, postimplantation function was improved when NO was administered during the reperfusion period [3]. In human lung allografts with severe reperfusion injury, function was immediately improved by NO [4]. Nitric oxide has a number of properties that are of specific interest in pulmonary allograft reperfusion injury. It is a potent pulmonary vasodilator. In addition, NO has been demonstrated to play a critical role in the maintenance of vascular integrity through its interaction with neutrophils [5, 6], platelets [7, 8], and vascular endothelial cells [9].
This study was undertaken to determine whether inhaled NO administered to the donor lung prior to harvest would affect subsequent reperfusion injury after a prolonged period of storage.
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Material and Methods |
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Donor Procedure
Harvest and left lung transplantation were performed as previously described [10]. Briefly, donor animals were anesthetized with sodium thiopental intravenously (10 mg/kg) followed by atropine sulfate (0.5 mg) and were intubated with a 9F endotracheal tube. The lungs were ventilated (Bennett MA1; Puritan Bennett, Inc, Overland Park, KS) with 100% oxygen at a tidal volume of 550 mL at a rate of 15 breaths per minute and 5 cm H2O of positive end-expiratory pressure. After a median sternotomy, the superior and inferior venae cavae, the ascending aorta, the trunk of the pulmonary artery (PA), and the trachea were isolated. Animals were heparinized (400 U/kg) before insertion of a curved metal-tipped cannula (Sarns, Inc, Ann Arbor, MI) through a pursestring suture in the main PA just distal to the pulmonary valve. Before administration of the flush solution, 250 µg of prostaglandin E1 (Prostin VR Pediatric; The Upjohn Company, Kalamazoo, MI) was injected directly into the PA. Cardiac inflow was occluded by ligation of the superior and inferior venae cavae 20 seconds after the infusion of prostaglandin E1. The proximal inferior vena cava was cut, and the left atrial appendage was amputated for decompression of the PA flush.
The lungs were perfused immediately, at a pressure of 40 cm H2O, with 1,500 mL of cold (4°C) modified Euro-Collins solution. During the flush, the lungs were cooled topically by flooding the thoracic cavity with cold (1°C) saline solution. The flushing pressure was monitored through a transducer between the flushing tube and the PA cannula. When the flushing was completed, the trachea was clamped at end-inspiration (tidal volume, 550 mL; positive end-expiratory pressure, 5 cm H2O), and the heart-lung block was excised. The harvested organs were stored in modified Euro-Collins solution (1°C) for 21 hours before implantation. In this model, 21 hours' storage produces a uniformly reliable reperfusion injury in control allografts.
Recipient Procedure
Left single-lung transplantation was performed as previously described [10]. Recipient animals were anesthetized in the same manner as the donor animals and ventilated with an adjustable-rate Harvard pump respirator (model 613; Harvard Apparatus, South Natick, MA) with 98.5% oxygen and 1.5% halothane. A femoral artery line and a Swan-Ganz catheter were placed, and pressures were continuously recorded (model 1290A; Hewlett-Packard, Andover, MA). After left pneumonectomy, the contralateral main PA and the upper and intermediate bronchi were mobilized and encircled separately. The donor left lung was separated from the heart-lung block, and left single-lung allotransplantation was performed using standard techniques [10].
The allograft was topically cooled with ice slush during implantation. The left atrial anastomosis was performed first using a continuous everting mattress suture. The PA and the bronchus were anastomosed by a continuous over-and-over suture. After reperfusion of the allograft, a Millar pressure transducer was placed in the left atrium, and two chest tubes were inserted. The contralateral main pulmonary artery and bronchus were ligated. At this point, ventilation was changed to 15 breaths per minute at a tidal volume of 550 mL and 5 cm H2O positive end-expiratory pressure (Bennett MA1). This ventilator change was required to maintain precise inspired oxygen fraction and positive end-expiratory pressure levels during the subsequent assessment period. The chest was closed in layers with absorbable sutures. Animals were turned to the supine position for the 6-hour assessment period.
Study Groups
Donor animals were allocated randomly to two groups. Group I (n = 5) received no NO. In group II (n = 5), after induction of anesthesia, a femoral artery line and a Swan-Ganz catheter were placed. Aortic, central venous, and PA pressures were recorded continuously (Hewlett-Packard model 1290 A). In group II, NO was administered in sequential concentrations of 20, 40, 60, and 80 (ppm) for 10 minutes per sequence. Cardiac output and pulmonary capillary wedge pressure were measured (model 9520; Edwards Laboratories, Santa Ana, CA) at the end of every 10-minute sequence. Harvest was accomplished after the NO concentration was returned to 60 ppm. The concentration of NO was kept at 60 ppm from sternotomy to excision of the donor heart-lung block.
Gas Administration
The NO/N2 gas mixture (2,070 ppm NO in pure N2) (Nellcor Puritan Bennett, St. Louis, MO) was administered directly into the inspiratory limb of the respirator circuit, about 50 cm proximal to the endotracheal tube. Inspired NO and nitrous dioxide (NO2) concentrations were continuously measured just proximal to the endotracheal tube using electrochemical analyzers (model AR-PRN-1; B&W Technologies, Calgary, AL, Canada).
Measurements
Immediately after implantation and skin closure, recipients were repositioned to the supine position for the 6-hour assessment period. During the assessment interval, anesthesia was maintained with intravenous administration of thiopental. Aortic, PA, central venous, and left atrial pressures were continuously recorded throughout the assessment period. Cardiac output was determined hourly (Edwards Laboratories model 9520). Arterial and mixed venous blood gas analysis was performed every 15 minutes. Sodium bicarbonate was infused intravenously as necessary to maintain pH level. Intravenous Ringer's lactate solution was administered to keep central venous pressure within baseline ± 2 mm Hg. Pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) were determined as follows: PVR (dynes • s • cm-5) = [mean pulmonary artery pressure - left atrial pressure (or pulmonary capillary wedge pressure)]/cardiac output x 79.9 and SVR (dynes•s•cm-5) = (mean aortic pressure - central venous pressure)/cardiac output x 79.9.
After the final measurement, recipients were sacrificed, and samples of transplanted lungs were submitted for tissue myeloperoxidase (MPO) assay and determination of wet to dry weight ratio.
Thiobarbituric Acid-Reactive Materials Assay
Samples of donor right lungs before preservation (just after flush) and after 21-hour preservation (before implantation) were submitted for tissue thiobarbituric acid-reactive materials (TBARM) assay. The level of TBARM was determined using the method of Fleming [11]. Briefly, 2.0 mL of phthalate buffer (0.35 mol/L at pH 3.4) and 0.4 mL of the homogenized test sample were added to a 15-mL screw-topped centrifuge tube. To this, 0.4 mL of a 1% wt/vol solution of thiobarbituric acid in 0.05 mol/L NaOH, prepared daily for the assay, and 0.1 mL of 2% wt/vol solution of butylated hydroxytoluene in 100% ethanol were added. The tubes were capped tightly and heated at 90°C for 1 hour in a water bath. After cooling, 0.1 mL of concentrated HCl was added, followed by 2.5 mL of n-butanol. Extraction of the colored complex was achieved by mixing for 1 minute, centrifuging at 3,000 g for 10 minutes, and transferring the organic layer by pipette into a 5-mL glass culture tube. A small amount of anhydrous Na2SO4 was added to dehydrate the organic layer, and the absorbance was then read at 560 and 535 nm on a spectrophotometer (PMQ II, Carl Zeiss, Oberkochen, Germany). Background interference was subtracted to obtain a corrected absorbance (Abscor) by subtracting 560-nm absorbance from 535-nm absorbance using the equation provided by Fleming [11]: (Abscor) = (Abstest535 - Absblank535) - (Abstest560 - Absblank560). Deionized water was used for blanks, and a standard curve was constructed using solutions of zero to 10 nmol/mL of malondialdehyde (Sigma, St. Louis, MO), first diluted 1:5 in 0.5 mol/L Na2SO4 and then diluted to final concentration in deionized water. Results were expressed as nanomoles per gram of weight (nmol/gW).
Myeloperoxidase Assay
Each lung sample was frozen immediately by immersion in dichlorodifluoromethane (CCl2F2) that had been precooled to the freezing point and was stored at -70°C until assay. Quantitative MPO activity was determined as previously described [12]. Frozen lung tissue (100 mg) was homogenized in 1 mL of 0.5% hexadecyl-trimethyl-ammonium bromide, 5 mmol/L EDTA (ethylene diaminetetraacetic acid), and 50 mmol/L potassium phosphate buffer (pH 6.2) with a Broeck tissue grinder (Kontes Glass Co, Vineland, NJ) to release MPO from the primary granules of the polymorphonuclear leukocytes. The homogenate was centrifuged at 10,000 g for 15 minutes at 4°C. The supernatant was assayed for MPO activity and total soluble protein by the method of Pierce Laboratories [13]. Enzyme activity was measured spectrophotometrically: 10 µL of tenfold dilute supernatant was combined with 0.6 mL Hanks' bovine serum albumin (0.25% bovine serum albumin added to Hanks' solution), 0.5 mL of 100 mmol/L potassium phosphate buffer (pH 6.2), 0.1 mL of 0.05% H2O2, and 0.1 mL of 1.25 mg/mL o-dianisidine. Color development was stopped by addition of 0.1 mL of 1% NaN3 after 5 minutes and after 20 minutes at room temperature. The optical density was measured at 460 nm with a spectrophotometer (PMQ II). The color development from 5 minutes to 20 minutes was linear. Enzyme activity was defined as the amount of MPO that produced an absorbance change of 1.0 optical density unit per minute per milligram of tissue protein at room temperature (
OD•min-1•mg-1).
Statistical Analysis
All data are presented as the mean ± the standard error of the mean. Comparisons between groups were made by one-way analysis of variance followed by the Scheffé test for multiple comparisons. In addition, analysis of variance with repeated measures was used to compare an overall difference in hemodynamics and blood gas data between groups. Differences were considered significant when the p value was less than 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). Although flush pressure in group II was lower than in group I and flush time in group II was shorter than in group I, the differences did not reach significance.
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). However, PVR decreased significantly as the concentration of NO was increased.
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).
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). At the first assessment, there was no significant difference between the two groups, but gas exchange deteriorated rapidly in group I. There was a significant difference in mean arterial oxygen tension after the first 15 minutes of assessment. However, arterial carbon dioxide tension was not significantly different between the two groups over time. Factorial analysis of variance showed significant arterial carbon dioxide tension differences between the two groups at the 45-minute and 75-minute assessments (both, p < 0.05) (see Fig 3).
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).
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OD•mg-1•min-1) was significantly higher than the activity in group II (0.310 ± 0.029 OD•mg-1•min-1; p < 0.05) (Fig 5).
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).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Among numerous factors influencing vascular function, NO serves as a key modulator of normal pulmonary vascular physiology. In vascular endothelial cells, NO is synthesized from the terminal guanidine nitrogen of L-arginine and diffuses rapidly into subjacent vascular smooth muscle, where it binds to the heme-iron complex of soluble guanylate cyclase. The resulting nitrosyl-heme activates guanylate cyclase, stimulating the production of cyclic guanosine 3,5'-monophosphate and producing relaxation of vascular smooth muscle [18]. In addition to its vasodilator action, NO prevents neutrophil adherence to the endothelium, maintains endothelial barrier properties, and inhibits platelet aggregation.
Inhalation of NO gas in concentrations of 5 to 80 ppm has been shown, in animals and humans, to produce pulmonary vasodilation without causing systemic vasodilation [19], even in the presence of pulmonary endothelial injury [20]. Nitric oxide is inactivated rapidly by binding to hemoglobin. Inhaled NO results in preferential pulmonary vasodilation in regions of ventilated lung. In patients with various lung injuries such as adult respiratory distress syndrome [21] and after cardiopulmonary bypass [22] and in newborns with persistent pulmonary hypertension [23], inhalation of low-dose NO selectively improves perfusion to ventilated alveoli, resulting in an improvement in pulmonary gas exchange [21] and decreased PA pressure. There are few reports describing the effects of NO on hemodynamics of anesthetized humans or animals without pulmonary hypertension or lung injury [19]. In the present study, NO was administered in increasing concentrations to anesthetized healthy donor dogs. We observed very little change in PA pressure, an increase in cardiac output, and a decrease in SVR, none of which reached significance. Yet a consistent and significant reduction in donor PVR was observed. We think it reasonable to speculate that inhaled NO administered to suitable lung donors would have no adverse effects on other organs being harvested.
We have previously reported that inhaled NO during the reperfusion period [3] or administration of an NO donor in the flush solution [17] was effective in reducing lung allograft reperfusion injury. Our data indicate that inhaled NO administered before harvest effectively reduces reperfusion injury after prolonged lung allograft storage. There are a number of possible mechanisms to explain this effect.
First, NO as a potent vasodilator may improve the distribution of hypothermic flush solution. Although the differences between the two groups did not reach significance, the flush pressure and time in group II were lower and shorter, respectively, than those in group I. In a previous study, we [24] showed that administration of sodium nitroprusside (an exogenous NO donor) significantly reduced early lung allograft dysfunction. In that study, flush pressure in treated donor lungs was significantly lower than in the control group.
Second, NO may act as a free radical scavenger during the period of harvest and storage. In an in vitro rabbit model, we [25] have previously demonstrated that superoxide-induced permeability injury occurs during the ischemic phase. In the present study, lung oxidant injury during the storage period was estimated on the basis of TBARM levels in donor right lungs before (after flush) and after (before implantation) storage. Many studies [26, 27] have demonstrated that lung TBARM levels increase after ischemia plus reperfusion. However, attempts to establish the relationship between TBARM levels and ischemia have led to conflicting results. Fisher and associates [28] demonstrated that ischemia increased TBARM values in rat lung in relation to inspired oxygen fraction and ischemic time, whereas Aeba and colleagues [29] reported that TBARM levels decreased during the ischemic period in rat lung. These incompatible results reflect an unstable balance between the progress of lipid peroxidation and metabolism of malondialdehyde because malondialdehyde is metabolized in vivo by a mitochondrial aldehyde oxidase [30]. In the present study, the mean TBARM level of poststorage lungs was significantly higher than that of prestorage lungs in the control group, whereas the increase in TBARM levels during the storage period was well controlled in donor lungs treated with NO. We believe that inhaled NO has a beneficial effect through its oxygen radical-scavenging properties during the period of harvest and storage.
Finally, NO has been shown to be an endogenous inhibitor of leukocyte chemotaxis, adherence, and activation [6, 31]. In the present study, MPO levels in group II were significantly lower than in the control group, a finding indicating reduced neutrophil sequestration. It is most probable that NO prevents subsequent neutrophil activation by scavenging superoxide radicals during storage [9, 31].
It is interesting to speculate why the impressive differences in oxygenation during the early reperfusion period are reduced after 6 hours of reperfusion. The effects of inhaled NO are very short lasting. Further, these 21-hour stored lungs have a severe ischemic injury as evidenced by the serious injury in control animals. It is possible that the progressive reduction in gas exchange in group II might be lessened by administration of NO during the reperfusion period, as we [3] have previously demonstrated.
To avoid the generation of excessive NO2, a known air pollutant and hazardous chemical, we connected the NO gas to the circuit close to the endotracheal tube, did not use a reservoir bag, and monitored NO2 concentration continuously. The NO2 concentrations at the time of NO inhalation in concentrations of 20, 40, 60, and 80 ppm were 0.73 ± 0.045 ppm, 2.10 ± 0.07, 4.60 ± 0.122, and 7.75 ± 0.065 ppm, respectively. Because NO2 can be transformed into nitric and nitrous acids, causing severe pulmonary edema, acid pneumonitis, and death [10], recommendations for occupational safety and health standards put the upper limit for NO2 inhalation at 5 ppm per 8-hour shift [32]. Foubert and co-workers [33] suggested in their safety guidelines that great care should be taken when NO concentrations greater than 80 ppm are administered. In the present study, therefore, we used NO in a concentration of 60 ppm and were able to control the concentration of NO2 at less than 5 ppm during harvest.
In conclusion, our findings suggest the following: (1) Nitric oxide inhalation in anesthetized healthy dogs caused a significant decrease in PVR as a result of increased cardiac output. Pulmonary artery pressure and systemic hemodynamics did not show any change. (2) Inhaled NO at the time of harvest improved oxygenation of preserved canine lung allografts by attenuating oxidant injury during the storage period and subsequent neutrophil sequestration in the early postoperative period.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We express our appreciation to Dennis Gordon, Duane Probst, Donna Marquart, and Jill Manchester for their expert technical assistance and to Dawn Schuessler for secretarial support. We thank Mary Ann Kelly for her assistance in the preparation of the manuscript.
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Patterson, Division of Cardiothoracic Surgery, Washington University School of Medicine, 3108 Queeny Tower, One Barnes Hospital Plaza, St. Louis, MO 63110.
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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