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Ann Thorac Surg 1996;61:1055-1061
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri
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Abstract |
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Methods. Fourteen canine left lung allotransplantations were performed. Donor lungs were flushed with modified Euro-Collins solution and stored for 24 hours at 1°C. Immediately after transplantation, the contralateral right main pulmonary artery and bronchus were ligated to assess isolated allograft function. Hemodynamic indices and arterial blood gas analysis (inspired oxygen fraction 1.0) were assessed for 6 hours before sacrifice. Allograft myeloperoxidase activity was assessed. Bronchoalveolar lavage fluid was obtained from the allograft middle lobe for neutrophil counts. The animals were divided into three groups based on the timing of PTX administration. Group 1 (n = 5) animals received no PTX. Group 2 (n = 4) animals received PTX (20 mg/kg) just before reperfusion as well as continuous infusion (0.1 mg•kg-1 • min-1) during the assessment period. In group 3 (n = 5), donor lungs received PTX (200 mg/L) in the flush solution only.
Results. Superior gas exchange was noted in the lungs receiving PTX only in the flush solution (group 3). Myeloperoxidase activity in group-3 allografts was significantly reduced. In addition, protein levels and neutrophil counts in the bronchoalveolar lavage fluid were significantly reduced in group-3 allografts.
Conclusions. Pentoxifylline ameliorates lung allograft reperfusion injury when administered in the flush solution. Our data suggest that PTX will prevent graft endothelial dysfunction during 24-hour cold ischemic storage and consequently will prevent neutrophil activation and migration into lung tissue.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Lung transplantation has become an effective treatment for a variety of patients with end-stage lung disease [1]. Although early postoperative morbidity has been improved, early graft dysfunction is still an unpredictable and major clinical problem. A variety of factors have been implicated in the genesis of reperfusion injury, including activated neutrophils [2], cytokines [3], oxygen free radicals [4, 5], and platelets [6]. However, the precise mechanism of injury is not completely understood.
Pentoxifylline (PTX), a methylxanthine derivative, has been shown to reduce ischemic injury in several organs [7, 8]. In a previous study, we showed that administration of PTX in the flush solution and its administration during the reperfusion period significantly reduced early dysfunction of preserved canine lung allografts [9]. However, it is not clear whether PTX given in the flush solution or its administration during the reperfusion period is more effective. The aim of the present study was to investigate whether PTX administered in the flush solution or during reperfusion would reduce ischemia-reperfusion lung injury in preserved canine lung allografts. In addition, we sought to determine the effect of PTX on neutrophil activation and migration.
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Material and Methods |
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Donor Procedure
Harvest and left lung transplantation were performed as described previously [9]. Briefly, donor animals were anesthetized with thiopental sodium intravenously (10 mg/kg) followed by atropine (0.5 mg), and were intubated with a 9F endotracheal tube. The lungs were ventilated (Bennet MA1; Puritan Bennet, Inc, Overland Park, KS) with 100% oxygen at a tidal volume of 550 mL at a rate of 10 breaths/min and with 5 cm H2O of positive end-expiratory pressure. After a median sternotomy, the superior and inferior vena cava, the ascending aorta, the trunks of the pulmonary artery (PA), and the trachea were isolated. The animals were administered heparin (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 prostaglandin E1 (PGE1) (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 vena cavae 20 seconds after the infusion of PGE1. 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 50 mL/kg of cold (4°C) modified Euro-Collins solution (MgSO4 4 mEq/L, glucose 3%). 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) and the heart-lung block was excised. The harvested organs were stored in modified Euro-Collins solution (1°C) for 24 hours before implantation.
Recipient Procedure
Left single-lung transplantation was performed as described previously [9]. Recipient animals were anesthetized in the same manner as the donor animals and were 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 arterial line and a Swan-Ganz catheter were placed and continuously transduced (Hewlett-Packard 1290A, Andover, MA). After left pneumonectomy, the contralateral main PA and 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 [9]. The allograft was cooled topically with ice slush during implantation. 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 bronchi and PA were ligated. At this point, ventilation was changed to 15 breaths/min at a tidal volume of 550 mL and 5 cm H2O positive end-expiratory pressure (Bennet MA1; Puritan Bennet, Inc). 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. The animals were turned to the supine position for the 6-hour assessment period.
Study Groups
In group 1, the donor lungs were flushed as described earlier, and the PTX vehicle (saline) was administered (5.0 x 10-3 mL • kg-1& bull; min-1) with a syringe pump (model 355; Orion Research Inc, Boston, MA) during the assessment period. In group 2, the donor lungs were flushed as described earlier, and the recipients were given a PTX bolus (20 mg/kg) immediately before reperfusion and then PTX continuously (5.0 x 10 -3 mL • kg-1 • min-1, PTX concentration 2%) during the 6-hour reperfusion period. In group 3, PTX (200 mg/L) was added to the flush solution described earlier. During reperfusion, group-3 animals received saline vehicle (5.0 x 10-3 mL • kg-1 •min-1) as given in group 1.
Assessment of Lung Function
Anesthesia was maintained with intravenous administration of sodium thiopental. Cardiac output was measured hourly (model 9520; Edwards Laboratories Inc, Santa Ana, CA). Aortic, PA, central venous, and left atrial pressures were monitored continuously, and arterial and mixed venous blood gases were assessed every 15 minutes during the 6-hour assessment period. Sodium bicarbonate was infused intravenously as necessary to maintain the pH level. Intravenous Ringer's lactate solution was administered to keep the central venous pressure within baseline ± 2 mm Hg. At the end of the assessment period, the animals were sacrificed by overdose of sodium thiopental and intravenous administration of KCl 20 mEq. Samples of transplanted lungs were obtained for tissue myeloperoxidase (MPO) assay, bronchoalveolar lavage fluid (BALF) study, and wet/dry ratio.
Bronchoalveolar Lavage Fluid Analysis
Immediately after the 6-hour assessment, the animals were sacrificed and left lingular segments were obtained for use in the BALF study. Fifty milliliters of saline solution was injected slowly, and BALF was collected by gravity. This procedure was repeated twice, so that the segment was washed with a total of 100 mL saline solution. The BALF was centrifuged at 400 g to separate the supernatant and cell pellet. One milliliter of the supernatant was reserved to measure the concentration of protein using the method of Pierce Laboratories [10]. After the erythrocytes were removed by hypotonic hemolysis, the cell pellet was resuspended in 10 mL of phosphate buffer solution, and leukocytes were counted with a hemocytometer (Fisher Scientific, Pittsburgh, PA) to assess the differential cell counts in the BALF. The smear was prepared by using cytospin (Cytospin 2; Shandon Southern Inst, Cheshire, England) and stained with a modified Wright stain. Differential leukocyte counts were obtained from the smears. Absolute neutrophil count (polymorphonuclear leukocytes [PMN]) was obtained using the formula: PMN = total leukocyte count x percentage of neutrophils.
Myeloperoxidase Assay
Recipient lung samples were frozen immediately by immersion in dichlorodifluoromethane (CCl2F2) that had been precooled to the freezing point and stored at -70°C until assay. Quantitative MPO activity was determined as described previously [9, 11]. Frozen lung tissue (100 mg) was homogenized in 1 mL of 0.5% hexadecyl-trimethyl-ammonium bromide, 5 mmol/L ethylenediamine tetraacetic 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 PMN. 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 [10]. Enzyme activity was measured spectrophotometrically: 10 µL of tenfold dilute supernatant was combined with 0.6 mL Hanks' BSA (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 0.05% H2O2, and 0.1 mL of 1.25 mg/mL o-dianisidine. Color development was stopped by the addition of 0.1 mL of 1% NaN3 after 5 and 20 minutes at room temperature, respectively. The optical density was measured at 460 nm with a spectrophotometer (PMQ II; Carl Zeiss, Oberkochen-Wuett, Germany). 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 ± standard error of the mean. Comparisons among groups were made by one-way analysis of variance followed by Scheffe's test for multiple comparisons. In addition, two-way analysis of variance with repeated measures was used to compare an overall difference of hemodynamic indices and blood gas data between groups. Differences were considered significant at p less than 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). Flushing times were 87 ±8, 92 ±4, and 85 ±3 seconds, and flushing pressures were 19.9 ±0.6, 19.0 ±0.8, and 17.2 ±1.2 mm Hg in groups 1, 2, and 3, respectively. Preservation times in groups 1, 2, and 3 were 24:28 ±0:17, 24:07 ±0:13, and 24:04 ±0:09 hours, respectively (not statistically significant).
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). During the first 45 minutes of reperfusion, there was no statistical difference among the three groups, but gas exchange deteriorated rapidly in groups 1 and 2. Mean arterial oxygen tension (PaO2) showed significant differences after 60 minutes of assessment. Values of PaO2 in groups 1, 2, and 3 at 360 minutes of assessment were 96.3 ±27.3, 97.2 ±20.5, and 351.9 ±51.5 mm Hg (p < 0.05 versus group 3), respectively. Arterial carbon dioxide tension (PaCO2) in group 3 was lower than that in groups 1 and 2 (p < 0.05), and significant differences at each time point were observed after 45 minutes of assessment between group 3 and groups 1 and 2 (PaCO2 in groups 1, 2, and 3 at 360 minutes of assessment: 58.2 ±4.1, 64.6 ±8.5, and 42.2 ±3.4 mm Hg [p < 0.05, group 2 versus group 3]). It is interesting that there was no significant difference between group 1 and group 2 with respect to PaO2 and PaCO2 levels throughout the entire study period (see Fig 1).
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OD•mg-1 • min-1 in groups 1, 2, and 3, respectively [p < 0.01 versus group 3]), but there was no significant difference between groups 1 and 2 (Fig 4).
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).
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We have demonstrated previously that PTX administration in the flush solution and during the reperfusion period was effective in reducing reperfusion injury in a canine lung allotransplantation model [8]. However, our previous work could not determine whether the beneficial effect was due to PTX administration at the time of flush or at the time of reperfusion. In the present study, PTX in the flush solution (group 3) provided excellent lung allograft function during the 6-hour reperfusion period after a prolonged storage of 24 hours. Surprisingly, PTX administration during the reperfusion period (group 2) did not ameliorate reperfusion injury.
Pentoxifylline has been used for the treatment of peripheral vascular disease and has a number of biologic effects [15, 16]. It has been shown to reduce ischemia-reperfusion injury in a variety of organs, including the heart [17], liver [18], kidney [7], and lung [8, 19]. Although the present study did not examine the specific mechanism of the beneficial effect of PTX on reperfusion injury, we can make some speculations.
First, PTX is known to prevent endothelial dysfunction [20] by increasing intracellular 3'-5'-cyclic monophosphate (cAMP) [15] and promoting the production of prostacyclin in the endothelium [16]. Mizus and co-workers [21] reported the important role of cAMP in the modification of pulmonary vascular injury in an isolated lung perfusion model. It has been suggested that increased cAMP prevents or reverses membrane gap formation, and the constriction of these gap junctions is one possible mechanism for the attenuation of increased permeability [22]. On the other hand, prostacyclin itself is known to prevent lung injury and platelet aggregation. Pohanka and Sinzinger [16] reported that PTX increased prostacyclin formation in various tissues. Their study demonstrated induction of prostacyclin in the endothelium within 30 minutes after PTX bolus injection, reaching peak levels within 60 minutes. Synthesis of prostacyclin in the endothelium may be reduced at low temperatures. However, it could be synthesized to certain threshold levels during a 24-hour preservation period as used in our study and could contribute to the prevention of endothelial dysfunction [20]. Kimblad and associates [23] demonstrated in a pig model that flushing and 12-hour cold storage significantly impaired endothelial-dependent relaxation in the PA. Others reported damage to the endothelial cell membrane after 24-hour cold storage [24]. Pentoxifylline is also reported to prevent cyclosporine A–induced vascular endothelial dysfunction [20]. Preadministration of PTX could maintain endothelial homeostasis and the integrity of the cell membrane. A loss of endothelial homeostasis and cell membrane integrity might further cause fluid edema after transplantation and subsequent pulmonary dysfunction. In the present study, the endothelial integrity in groups 1 and 2 could have been already damaged after the 24-hour preservation period, as PaO2 levels fell rapidly after the initial assessment, and statistically significant differences could be seen in PaO2 and PaCO2 after reperfusion. Two animals died in groups 1 and 2, respectively, because of graft failure, but all animals receiving PTX in the flush solution (group 3) survived for the 6-hour assessment. Our data suggest that the addition of PTX to the flush solution limits graft endothelial damage during the 24-hour cold ischemic period. These results are consistent with those in McDonald's isolated rat lung study [25], which showed that PTX prevented lung injury when it was present in the perfusate buffer, regardless of whether neutrophils were preincubated in PTX before infusion into the lung. Wet to dry weight ratios were elevated in all groups. It is surprising that there was no difference between the groups, especially because there was such a striking difference in PaO2. This lack of difference is likely due to the small sample size, particularly in groups 1 and 2.
Second, PTX has been shown to inhibit the production of tumor necrosis factor-alpha [26] and neutrophil and monocyte activation [19], which should prevent tissue injury. Neutrophils and neutrophil-derived oxidants appear to play a central role in ischemia-reperfusion lung injury [2]. In vivo studies with PTX have shown a decrease in neutrophil adherence to endothelial cells, neutrophil aggregation, and superoxide production by activated neutrophils [19, 25]. Studies in several animal models also have demonstrated that PTX attenuates acute lung injury in response to endotoxin and tumor necrosis factor-alpha [26]. The present study showed that PTX administration in the flush solution improved allograft function and that allograft MPO activity, PMN in BALF, and protein levels in BALF were significantly lower than in group 1 (control). Myeloperoxidase activity in group 3 was similar to that of the normal lung. Interpretation of the BALF neutrophil counts and protein levels is also hampered by small sample sizes. Nonetheless, the BALF neutrophil count in group 3 was dramatically reduced and indeed approached that of the normal lung, in which BALF contains no neutrophils. However, PTX administration during the reperfusion period (group 2) did not improve allograft function or decrease MPO activity in comparison with controls (group 1). Lambert and Egan [14] showed that dimethylthiourea, an oxygen free radical scavenger, attenuated lung allograft injury when it was administered only in the flush solution. Haniuda and colleagues [13] also demonstrated that dimethylthiourea reduced endothelial permeability during storage. Therefore, it is reasonable to suspect that PTX exerts its antineutrophil effects, including reduction in subsequent oxygen free radical injury, during the storage phase before reperfusion.
Another possible mechanism of the effects of PTX administration relates to its hemorrheologic properties. It is known that PTX improves the microcirculation by increasing the deformability of circulating blood cells, lowering blood viscosity, and decreasing platelet and neutrophil aggregation [7, 15]. It also has a vasodilatory effect, but this effect is apparently seen at relatively high concentration [27]. Pentoxifylline was administered in the flush solution at high concentration (200 mg/L) or at about 20 mg/kg of bolus injection just before reperfusion, but we could not detect any statistical differences in flush pressures or in flush times. In clinical lung transplantation, PGE1, a strong vasodilatory agent, is used at the time of donor lung harvest to avoid vasoconstriction during cold modified Euro-Collins flush. In these experiments, we used PGE1 to mimic the clinical situation. It is possible that the vasodilatory effects of PGE1 may have masked any hemodynamic effects of PTX that we might otherwise have observed.
We evaluated the use of PTX in an acute animal model of lung transplantation. Ligation of the contralateral PA and bronchus directed the total blood flow and ventilation volume to the transplanted lung. This represents a severe test of preserved lung allograft that is similar to the clinical situation of single-lung transplantation for pulmonary hypertension and analogous to the situation in the first allograft while the second lung is being extracted and implanted during sequential bilateral single-lung transplantation. The model permits lengthy assessment of allograft function alone. The 6-hour assessment period was shown previously to be sufficient to demonstrate severe allograft dysfunction [4, 5, 8]. Perhaps because of the severity of injury, no statistical difference in the wet/dry ratio could be seen among the three groups. Not surprisingly, the wet/dry ratio in these experiments was higher than that observed in a previous study in which we used an 18-hour preservation period [8]. Twenty-four–hour preservation in modified Euro-Collins solution might cause too much injury for detection of any difference in the wet/dry ratio among the three groups.
We conclude that PTX administration in the flush solution before ischemic storage significantly improves early postoperative graft function. Our data suggest that PTX decreases graft endothelial dysfunction during 24-hour cold ischemic storage and consequently reduces postreperfusion neutrophil activation and migration into lung tissue.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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This study was supported by National Institutes of Health grant 1 R01 HL41281.
Pentoxifylline was kindly supplied by Hoechst-Roussel Pharmaceutical Inc, Somerville, NJ.
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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, One Barnes Hospital Plaza, Suite 3108 Queeny Tower, St. Louis, MO 63110.
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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