Ann Thorac Surg 1998;66:362-366
© 1998 The Society of Thoracic Surgeons
Original articles: general thoracic
Evaluation of 18-hour lung preservation with oxygenated blood for optimal oxygen delivery
Kou Takigami, MDa,
Shigeyuki Sasaki, MDa,
Norihiko Shiiya, MDa,
Masakazu Kawasaki, MDa,
Eriho Takeuchi, MDa,
Keishu Yasuda, MDa
a Department of Cardiovascular Surgery, Hokkaido University Hospital, Sapporo, Japan
Accepted for publication March 12, 1998.
Address reprint requests to Dr Sasaki, Department of Cardiovascular Surgery, Hokkaido University School of Medicine, Kita-14, Nishi-5, Kita-ku, Sapporo, Japan 060
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Abstract
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Background. Previous studies have shown that availability of oxygen during lung preservation to maintain aerobic metabolism may be essential for the optimal viability of preserved lung tissue. The purpose of this study was to evaluate lung preservation with oxygenated blood for optimal oxygen delivery to the lung graft in a rabbit model.
Methods. Eighteen excised rabbit lungs were flushed and stored for 18 hours at 10°C with one of the following: Euro-Collins solution (EC; n = 6), oxygenated homologous blood (OB; n = 6), or low-potassium dextran solution (LPD; n = 6). Poststorage lung functions were evaluated with isolated, blood-perfused lung model for 10 minutes.
Results. The mean oxygen tensions after reperfusion for the EC, OB, and LPD groups (47.0 ± 2.8, 76.9 ± 13.1, 96.2 ± 10.9 mm Hg at 10 minutes, respectively) were significantly different throughout the perfusion period (EC < OB < LPD, p < 0.05; EC < LPD, p < 0.01). Pulmonary artery pressure during the reperfusion period in the EC group (35.8 ± 4.4 mm Hg at 10 minutes) was higher than that in the OB and LPD groups (29.8 ± 4.3 and 22.4 ± 2.2 mm Hg, respectively) (EC > OB, EC > LPD, p < 0.05). However, the E-selectin level in the reperfused blood in the OB group (5.04 ± 0.24 ng/mL) was significantly elevated compared with that in other groups (EC, 3.56 ± 0.54; LPD, 2.92 ± 0.35 ng/mL, p < 0.05), which indicated enhanced neutrophil recruitment in the OB group. Comparisons of thrombomodulin and endothelin among the three groups did not reach statistical significance.
Conclusions. We conclude that OB may enhance lung preservation as compared with EC solution, probably through its enriched oxygen delivery during storage and extracellular composition. However, the availability of oxygenated blood does not exceed that of LPD solution because of augmented neutrophil recruitment, which may activate neutrophil–endothelial interactions.
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Introduction
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Lung transplantation is a promising therapeutic option for patients with respiratory failure and limited life expectancy. One-year survival of 70% to 90% for single-lung transplant recipients for the treatment of end-stage interstitial lung disease and obstructive lung disease has been achieved [1, 2]. Current preservation techniques of donor lungs for subsequent transplantation include core-cooling and single-flush perfusion; however, lung storage intervals for transplantation remain at 6 hours or less, which restrict the pulmonary donor pool clinically available for lung transplantation [3]. The lungs remain vulnerable to the ischemia and reperfusion that accompany the transplantation procedure in spite of every attempt to optimize organ preservation techniques. The wide variety of lung graft vasculature may result in vasoconstriction, neutrophil sequestration, and edema in the reperfused lung graft after prolonged preservation.
Some recent reports have shown that availability of oxygen during lung preservation to maintain aerobic metabolism may be essential for the optimal viability of preserved lung tissue [4, 5]. These findings prompted us to undertake a current study on the use of oxygenated homologous blood (OB) for initial flush and storage in lung preservation, because hemoglobin in oxygenated blood may optimize oxygen delivery to the lung graft as compared with dissolved oxygen in Euro-Collins solution (EC) or low-potassium dextran solution (LPD). The purpose of this study, therefore, was to evaluate lung preservation with oxygenated blood for an optimal oxygen delivery to the lung graft.
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Material and methods
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New Zealand white rabbits weighing 2.5 to 3.7 kg were used in this study and obtained from Hokudoh Animal, Inc. (Hokkaido, Japan). All experiments were approved by the Hokkaido University School of Medicine Animal Care and Use Committee, and conformed to the United States National Institutes of Health guidelines regulating the care and use of laboratory animals (NIH publication 85-23, revised 1985). Unless otherwise stated, all reagents were purchased from Takeyama, Inc (Hokkaido, Japan).
Experimental procedures and protocol
An ex vivo rabbit model described by Wang and associates [6] was used to reperfuse the lung graft and evaluate poststorage lung function in this study. Briefly, the animals were premedicated with subcutaneous atropine sulfate (0.25 mg) and ketamine hydrochloride (15 mg/kg) and anesthetized with intravenous sodium pentobarbital (20 mg/kg). Heparin (700 IU/kg) was administered intravenously into the ear vein. An endotracheal tube was introduced through a tracheostomy, and animals were ventilated with room air (tidal volume, 25 mL; respiratory rate, 35 breaths/min; positive end-expiratory pressure, 0.5 cm H2O) with a Harvard rodent ventilator (model 665; Harvard Apparatus Inc, Natick, MA). A venous cannula for blood drainage was introduced from the internal jugular vein to the right atrium. After median sternotomy, the thymus was resected and the ascending aorta and the main pulmonary artery were isolated. The main pulmonary artery was cannulated with a 14-gauge catheter through the right ventricular outflow tract and the pulmonary artery pressure was continuously recorded. A bolus injection of 10 µg prostaglandin E1 (Ono Pharmaceutical, Inc, Osaka, Japan) was given into the pulmonary arterial cannula before the initial flush of the lungs. Subsequently, both lungs were flushed gravitationally with 70 mL/kg of cold (10°C) solution from a height of 30 cm. The ventilation was continued during this procedure. The trachea was then clamped at half-inflation of the lungs with room air, and the lungs and heart were harvested en bloc with utmost care to prevent lung injury. The heart-lung block was immersed immediately and stored for 18 hours at 10°C in the same solution as was used for the initial flush.
Eighteen excised rabbit lungs were divided into the following three groups according to the solution used for flush and storage: group I, EC (n = 6); group II, OB (n = 6); and group III, LPD (n = 6). The composition of EC was as follows (in mmol/L): Na+, 10; K+, 115; Cl-, 15; phosphate, 50; bicarbonate, 10; glucose, 3.5%; osmolarity, 355 mOsm/L. The composition of LPD solution was the same as previously reported [7, 8] (in mmol/L): Na+, 168; K+, 4; Mg2+, 2; Cl-, 103; phosphate, 37; sulfate, 2; 2% low-molecular dextran 40; osmolarity, 282 mOsm/L. Oxygenated blood for flush and storage was prepared by exsanguination from blood donor rabbits (n = 18) followed by oxygenation using a rodent membranous oxygenator with a 95% O2/5% CO2 gas mixture, which we have previously reported [9]. Blood gas tensions after oxygenation were adjusted to 30 to 35 mm Hg CO2 and 250 to 350 mm Hg O2 in all experiments of group II. Heparin (700 IU/kg) was administered intravenously to blood donor rabbits before exsanguination, and was supplemented to blood during storage every 6 hours with a dose of 1000 IU/L blood for anticoagulation.
After cold storage for 18 hours, all preserved lungs were reperfused with fresh homologous venous blood to evaluate lung functions by means of an ex vivo model previously reported by Wang and associates [6]. A drainage catheter for the pulmonary effluent was placed into the left atrium through the left ventricular apex. An endotracheal tube was inserted into the trachea. The right hilum was ligated with 2-0 silk, which allowed isolated perfusion of the left lung. The lung-heart block was then mounted in a prewarmed (37° to 38°C) and humidified acrylic plastic chamber. The left lung alone was ventilated with a Harvard rodent ventilator (model 665) with room air at a tidal volume of 15 mL, respiratory rate of 25/min, and positive end-expiratory pressure of 0.5 cm H2O. The left lung was reperfused through the pulmonary arterial cannula with 400 mL of fresh venous blood at a rate of 40 mL/min for 10 minutes using a peristaltic roller pump (Terumo BP-100, Terumo Inc, Osaka, Japan). The mean pulmonary artery pressure was continuously monitored. Blood gas tensions in reperfusate taken from the left atrial catheter were measured with a Radiometer ABL 300 pH/blood gas analyzer (Copenhagen, Denmark) at 1, 5, and 10 minutes after the onset of reperfusion.
To assess neutrophil recruitment and endothelial cell injury in the lung graft [10, 11], we measured levels of thrombomodulin, E-selectin, and endothelin by the enzyme-linked immunosorbent assay method. The serum was separated from blood samples taken at 1 minute after the onset of reperfusion with an ordinary centrifugal method and stored in the freezer at -70°C for later determination. At completion of the reperfusion, the left lung was removed from the lung-heart block. The upper lobe was used for the measurement of wet-to-dry weight ratio by the traditional method previously described [12]. The lower lobe was stored in formalin for light microscopic histologic analysis.
Statistical analysis
All values are presented as mean ± standard error of the mean. Statistical analysis for comparisons of wet-to-dry weight ratio and markers of endothelial cell injury between the three groups was performed using one-way analysis of variance with post-hoc pairwise comparisons (Tukey-Kramer). Statistical analysis for comparisons of oxygen tensions and the pulmonary artery pressure between the groups was performed using repeated measures analysis of variance. A p value less than 0.05 was considered statistically significant.
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Results
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Oxygenation ability of the preserved lung graft, evaluated as arterial oxygen tension values in the left atrial effluent during reperfusion, is shown in Figure 1. The arterial oxygen tension values in the EC group at 1, 5, and 10 minutes after the onset of reperfusion were 50.2 ± 3.8, 42.6 ± 2.1, and 47.0 ± 2.8 mm Hg, respectively, which were significantly deteriorated compared with those in the OB group (83.7 ± 6.7, 76.6 ± 11.8, and 76.9 ± 13.1 mm Hg at 1, 5, and 10 minutes, respectively; p < 0.05) and those in the LPD group (133.4 ± 2.1, 104.7 ± 11.6, and 96.2 ± 10.9 mm Hg at 1, 5, and 10 minutes, respectively; p < 0.01) throughout the reperfusion period. The arterial oxygen tension values in the OB group were also significantly deteriorated compared with those in the LPD group throughout the reperfusion period (p < 0.05).
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Fig 1. Oxygen tension (PaO2) in the left atrial effluent at 1, 5, and 10 minutes after the onset of reperfusion. (EC = Euro-Collins; LPD = low-potassium dextran; OB = oxygenated blood.)
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The mean pulmonary artery pressure during the reperfusion period, which reflects pulmonary vascular resistance during constant flow, are shown in Figure 2. Pulmonary arterial pressures in the EC group at 1, 5, and 10 minutes after the onset of reperfusion were 43.3 ± 2.7, 40.2 ± 3.9, and 35.8 ± 4.4 mm Hg, respectively, which were significantly elevated compared with those in the OB group (28.7 ± 4.2, 27.0 ± 3.6, and 29.8 ± 4.3 mm Hg at 1, 5, and 10 minutes, respectively; p < 0.05) and those in the LPD group (19.8 ± 1.9, 20.0 ± 1.8, and 22.4 ± 2.2 mm Hg at 1, 5, and 10 minutes, respectively; p < 0.05) throughout the reperfusion period.
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Fig 2. Mean pulmonary artery pressure (P ) at 1, 5, and 10 minutes after the onset of reperfusion. (EC = Euro-Collins; LPD = low-potassium dextran; OB = oxygenated blood.)
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The wet-to-dry weight ratios of the EC, OB, and LPD groups were 7.94 ± 1.17, 5.53 ± 0.32, and 5.49 ± 0.09, respectively. Significant differences were noted between the EC and OB groups (p < 0.05) and the EC and LPD groups (p < 0.05).
Figure 3 summarizes results of thrombomodulin, E-selectin, and endothelin measured by enzyme-linked immunosorbent assay. No significant differences were found among the three groups in terms of thrombomodulin and endothelin-1 levels. However, the level of E-selectin in the OB group (5.04 ± 0.24 ng/mL) was significantly higher than that in the EC (3.56 ± 0.54 ng/mL) and LPD (2.92 ± 0.35 ng/mL) groups (p < 0.05). In the histologic analysis with a light microscope, no remarkable differences were found among the three groups.
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Fig 3. Thrombomodulin, E-selectin, and endothelin levels measured from blood samples taken at 1 minute after the onset of reperfusion by the enzyme-linked immunosorbent assay method. (E-C = Euro-Collins; LPD = low-potassium dextran; O-B = oxygenated blood.
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Comment
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In spite of recent advances in all areas of lung transplantation, extended preservation of the lung graft has not been achieved. Experimental studies have shown that lungs were successfully preserved for at least 12 hours maintaining lung function after storage; however, storage intervals are limited to 6 hours in the clinical setting [3, 8]. In the current standard protocol for lung transplantation, the intracellular type EC solution is most commonly used [3, 13]. However, there has been a controversy over which type of preservation solution, extracellular or intracellular, would provide optimal lung protection. A number of studies have been made in an attempt to determine the ultimate composition of solutions for extended lung preservation, but it remains to be elucidated.
The present study was designed to determine whether OB enhanced lung preservation. Date and associates [5] have shown that lung cells were able to maintain aerobic metabolism with oxygen during preservation and that the maintenance of aerobic metabolism may be essential for optimal viability of preserved lung tissue. Availability of oxygen to the lungs during the preservation period has also been reported to be important for optimal lung preservation by Weder and associates [4]. Although hemoglobin can deliver a greater amount of oxygen to the lung graft as compared with dissolved oxygen in crystalloid-based solution, homologous venous blood has not been reported to enhance lung preservation. Because we speculated that the low oxygen concentration in the venous blood would be one of the major reasons for the unfavorable results, we tested oxygenated blood instead of venous blood so that the lung grafts could receive optimal oxygen delivery during the harvest and storage. With the use of an ex vivo blood-perfused rabbit model, lung functions after a single flush and storage using EC and LPD solution were tested and compared with those after use of OB. Although the short perfusion time is one of the limitations of this model [6, 8], it was selected for this study as a standard model for the assessment of poststorage lung function. In addition, we considered it important to avoid the adverse effects of recirculation of the blood perfusate in this study. Euro-Collins solution was chosen as a representative intracellular solution and as a clinical standard solution for lung preservation. Low-potassium dextran solution was chosen as a representative extracellular solution, which has been reported by several investigators to be much more effective for lung preservation than EC in this model [7, 8]. The OB solution was expected to enhance lung preservation compared with EC through its extracellular composition, but we were not certain about possible detrimental effects from the blood components.
In our study using an 18-hour ischemia model, the LPD solution was more beneficial for preservation than clinical standard EC solution. An intracellular EC has been used for lung preservation because it may protect lung parenchymal cells from unfavorable cation exchange and cell edema; however, the detrimental effects of high potassium content have been documented in a number of reports. Potassium-induced vasoconstriction not only causes elevation of pulmonary vascular resistance during the early reperfusion period as shown in this study, but also causes nonhomogeneous flushing of the lung graft, which may produce poorly preserved areas [14, 15]. In addition, high potassium content may have an adverse effect on endothelial cell function [16]. The oxygenation ability and the pulmonary vascular resistance of lung grafts in the EC group were inferior to those in the LPD group in this study, which was consistent with previous reports [7, 8, 17].
Lung grafts in the OB group showed improved lung functions as compared with those in the EC group; however, they did not demonstrate any advantage, but rather showed impaired oxygenation as compared with the LPD group. Recently, Binns and associates [18] compared lung preservation with EC solution, LPD solution, and 20% blood–LPD solution. They also found no beneficial effects with an addition of 20% whole heparinized blood to the LPD solution. Theoretically, the blood components benefit organ preservation by keeping buffering mechanisms intact and by provision of substrates for aerobic metabolism [19]. In addition, OB may provide a sufficient amount of oxygen for aerobic metabolism. However, there appears to be significant detrimental effects of leukocytes on lung preservation with OB, as was shown in recent reports [20].
To assess neutrophil recruitment and endothelial cell injury in the preserved lung grafts [10, 11], we measured levels of thrombomodulin, E-selectin, and endothelin in this study. In general, endothelial cell activation can be differentiated into two types [21]. In one type, reactive oxygen species and activated complement fragments may induce the release, within seconds to minutes, of preformed proteins within the endothelium, which promote leukocyte–endothelial cell interactions and coagulation, in response to the abrupt restoration of blood flow to ischemic tissues. Alternatively, in response to tumor necrosis factor, interleukin-1, interleukin-6, and transcriptional activation of several genes and translation of specific transcripts into protein products on the endothelial surface are completed during the course of several hours. Thrombomodulin was measured mainly as a marker of the first type of endothelial cell activation, and E-selectin and endothelin-1 were measured as markers of the second type [21]. Using this experimental model, we could evaluate, at least in part, the type 1 endothelial cell activation evoked immediately after reperfusion, and possibly the type 2 endothelial cell activation initiated during lung storage. A limitation of this model is that the second type of endothelial cell activation evoked by reperfusion of the lung graft cannot be assessed because it takes several hours to complete; a different transplant model that may allow for reliable measurements for a longer period is required to assess the type 2 activation induced by reperfusion.
Because the production of E-selectin is mediated with the second type of endothelial cell activation, the increase in E-selectin level in the OB group might result from either hypoxic endothelial cell activation initiated during the storage interval or leukocyte–endothelial cell interactions evoked during the storage interval. However, the lack of an increase in endothelin-1 level may indicate the absence of hypoxia during lung storage, because hypoxia followed by reoxygenation has been reported to result in a 198% increase in endothelial cell release of endothelin-1 [22]. In addition, hypoxic conditions in lung tissues are less likely to occur in oxygenated blood during the preservation period. Therefore, leukocyte–endothelial cell interactions would be responsible for the increase in E-selectin level in the OB group, possibly through the presence of leukocytes during the preservation period. Augmented neutrophil recruitment may activate neutrophil–endothelial interactions, which can lead to deterioration of poststorage lung functions in the OB group.
Thrombomodulin is an endothelial cell membrane glycoprotein that contributes to the regulation of the coagulation system by binding thrombin and activating protein C, and it serves as a reliable marker of endothelial cell dysfunction [23]. The interactions between leukocytes and endothelial cells that are mediated by cellular adhesion molecules are central to the development of ischemia–reperfusion injury as occurs when blood flow is restored after an ischemic preservation period [24]. In this study, thrombomodulin was measured as a marker of endothelial cell dysfunction that would occur shortly after reperfusion, but its measurements were not significantly different among the three groups, which was a rather unexpected result. One reason for this may be the relatively short duration between onset of reperfusion and measurements. Another possibility is that the total amount of thrombomodulin might not be completely washed out from the lung graft at the time of reperfusion because of vasoconstriction that induced pulmonary hypertension in the EC group. Further investigation would be required to elucidate these kinetics with measurements of markers of endothelial cell injury at different times or at the later period in different transplant models.
In conclusion, OB may enhance lung preservation as compared with EC, probably through its enriched oxygen delivery during storage and extracellular composition. However, the availability of oxygen does not exceed that of LPD solution because of augmented neutrophil recruitment, which activates neutrophil–endothelial interactions.
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References
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Abstract
Introduction
