Ann Thorac Surg 1999;67:203-207
© 1999 The Society of Thoracic Surgeons
Original Articles
University of Wisconsin solution with butanedione monoxime and calcium improves rat lung preservation
Serguei Y. Lopukhin, MDa,
David R. Onsager, MDa,
Robert L. Conhaim, PhDa,
James H. Southard, PhDa,
Robert B. Love, MDa
a Department of Surgery, University of Wisconsin-Madison, Madison, Wisconsin, USA
Accepted for publication June 16, 1998.
Address reprint requests to Dr Southard, Department of Surgery, University of Wisconsin-Madison, 600 Highland Ave, CSC H4/395, Madison, WI 53792
e-mail: southard{at}surgery.wisc.edu
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Abstract
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Background. A limitation to fully using lung transplantation for patients with end-stage lung diseases is short, safe preservation time (4 to 6 hours). Our goal is to extend this to 24 hours or more, which would greatly improve clinical lung transplantation.
Methods. We used the isolated perfused rat lung to test how two preservation solutions (low potassium dextran and University of Wisconsin solution) affected quality of lungs after 6, 12, and 24 hours of preservation. Also, we tested modifications of the University of Wisconsin solution, including reversing the ratio of Na/K, the addition of 1.5 mmol/L calcium, and the combination of calcium and butanedione monoxime, agents that improve cardiac preservation. After preservation at 4°C, lungs were reperfused at 37°C with a physiologically balanced solution. Pulmonary artery flow rate, airway peak inspiratory pressure, and tissue edema were used to assess degree of preservation and reperfusion injury.
Results. Low potassium dextran solution gave poor preservation (decreased pulmonary artery flow, tissue edema) after 12 hours of cold storage. There were no differences between regular and reversed Na/K ratio University of Wisconsin solutions at 12 or 24 hours of preservation. Addition of calcium had no beneficial effect on lung preservation. However, University of Wisconsin solution with calcium and butanedione monoxime gave excellent 24-hour cold storage, with pulmonary artery flow rate, tissue edema, and airway peak inspiratory pressure equal to control (0 hours of preservation) lungs.
Conclusions. The University of Wisconsin solution appears capable of lung preservation for up to 24 hours if modified to contain calcium and butanedione monoxime. The mechanism of action of butanedione monoxime may be related to the suppression of smooth muscle contraction resulting in vasodilation of the cold-stored lung on reperfusion.
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Introduction
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Although lung transplantation is an acceptable clinical method for treatment of patients with end-stage lung diseases, this therapeutic modality is limited by the lack of adequate numbers of donor lungs. This is partially owing to the short tolerance of the lung to hypothermic preservation, which is limited to 4 to 6 hours. Increasing safe preservation of the donor lung to 24 to 36 hours would greatly enhance the capabilities of using this life-saving treatment. Furthermore, longer preservation would allow tissue matching between donor and recipient and increase the likelihood for delaying acute and chronic rejection. Finally, development of a safe method to preserve the lung for greater than 24 hours could mean that lungs transplanted within shorter periods of time would show reduced reperfusion injury. In general, most organs can be safely placed within about 17 to 20 hours of harvesting, including kidneys [1], livers [2, 3], and the pancreas [4]. Thus, demonstrating safe preservation for 24 to 36 or more hours, in the laboratory, would provide a margin of safety that would give assurance that the organ is viable for preservation periods in the clinically relevant range of 12 to 20 hours.
The commonly used method for lung preservation is simple cold storage, which involves a vascular flush of blood with a preservative solution. The composition of the preservative is important for successful organ preservation. For the lung, the commonly used preservatives are those developed for the kidney (Eurocollins solution) [5], for liver, kidney, and pancreas (University of Wisconsin solution) [6], and one developed specifically for the lung (low potassium dextran) [7]. These have been used successfully for short-term clinical lung preservation (4 hours). They are effective because they are used at hypothermia (0° to 4°C), and because they all contain agents that suppress cell edema. University of Wisconsin solution also contains agents (adenosine, glutathione, allopurinol) that facilitate restoration of normal metabolism on reperfusion. However, none of these solutions have been shown to be ideal for long-term (>36 hours) lung preservation. We are interested in developing a better cold-storage solution for lung preservation, and this study was designed to compare the effects of these solutions on the limits to safe rat lung preservation. The solution that appears to give the best quality preservation (in terms of degree of lung edema on reperfusion, pulmonary artery resistance, and airway resistance) will be selected for modifications to obtain our goal of longer-term lung preservation.
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Material and methods
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Experimental preparation
White, male Sprague Dawley rats (400 to 580 g, n = 36) were anesthetized with intraperitoneal chloral hydrate (4 mg/g weight), intubated through a tracheostomy, and ventilated using a rodent ventilator (model 683, Harvard Apparatus, South Natick, MA, tidal volume, 3 mL; positive end-expiratory pressure, 3 cm H2O) with room air at a rate of 45 cycles/min. The chest was opened through a midsternal incision, and the animal was heparinized (1 U/g). The pulmonary artery was cannulated with a polyethylene catheter (2.4 mm internal diameter) through a stab incision of the right ventricle and secured with a suture placed around the great vessels. The left atrium was then decompressed with a stab incision, and the lungs were flushed with 30 mL (4°C) of one of five preservation solutions at a pulmonary artery pressure of 30 cm H2O. The heart–lung block was then excised from the chest with the lungs inflated, placed into a plastic storage container with 50 mL of the same preservative, and stored for a specified period of time at 4°C.
After storage the lungs were placed in a humidified chamber (37°C) on a recirculating reperfusion circuit primed with 150 mL of Krebs-Henseleit perfusate (KHB) with 4% bovine serum albumin (Table 1). The lungs were suspended by the tracheal cannula and ventilated at 45 cycles/min (tidal volume, 6 mL/kg) using 95% O2, 5% CO2 with positive end-expiratory pressure set at 3 cm H2O. The perfusate was suspended above the lung. Perfusate was delivered through the pulmonary artery at a constant pressure of 20 cm H2O for 45 minutes at 37°C. This model is similar to others previously described [8]. Pulmonary artery flow was measured with a flowmeter (Carolina Medical Electronics, King, NC). Airway pressure was measured with a pressure transducer (Ohmeda P23XL, Madison, WI). Pulmonary artery flow and airway pressure were continuously recorded on a multichannel chart recorder (Gilson, Middleton, WI). At the end of the reperfusion period, the left lung was tied off at the hilum to prevent vasculature collapse, separated from the heart–lung block, and weighed to determine wet weight (WW), then dried overnight at 50°C and reweighed to determine dry weight (DW). Total tissue water (TTW) was calculated as 
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 Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).
Study groups
Five preservation solutions were studied (Table 1): (1) University of Wisconsin solution (UW-K), (2) low potassium dextran solution (LPD), (3) UW solution with reversed Na/K ratio (UW-Na), (4) UW solution with 1.5 mmol/L Ca2+ (UW-Ca), and (5) UW-Ca solution plus butanedione monoxime (UW-BDM). In each preservative group, three subgroups were stored for 6, 12, and 24 hours (6 lungs each). The lungs immediately reperfused after the harvest served as a control reperfusion group (preservation time 0 hours, n = 6).
Results are expressed as mean ± SEM. Statistical analysis of the results were performed using analysis of variance, and the Newman-Keuls post hoc test was performed. Differences were considered to be significant at p less than 0.05.
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Results
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Pulmonary artery flow rate
The effects of preservation time and solution composition on pulmonary flow rates after 45 minutes of reperfusion at 37°C are shown in Figure 1. Freshly harvested lungs (control) had a flow rate of 20.6 ± 0.5 mL/min after 45 minutes of normothermic perfusion. The effect of preservation time on flow rate was identical in lungs preserved in UW-K or UW-Na solutions. Flow rates were similar to nonpreserved lungs after 6 and 12 hours of cold storage but declined sharply to 16.0 ± 0.8 and 17.3 ± 1.1 mL/min, respectively, after 24 hours of cold storage (about a 20% decrease). Low potassium dextran solution had no effect on flow rate in lungs preserved 6 hours, but there was a large decrease in flow to 17.0 ± 0.6 mL/min after 12 hours of storage (p < 0.05). After 24 hours of storage in LPD, airways of all lungs filled with fluid during the first few minutes of reperfusion. Hence, flow rates are not shown.
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Fig 1. Effect of cold storage with different preservation solutions on the pulmonary artery flow rate at the end of 45 minutes of normothermic reperfusion. (UW-K = University of Wisconsin solution; UW-Na = high-sodium University of Wisconsin solution; LPD = low potassium dextran solution; UW-Ca = University of Wisconsin solution plus 1.5 mmol/L Ca2+; UW-BDM = University of Wisconsin solution plus calcium and 30 mmol/L butanedione monoxime; *p < 0.05 versus others in column;  p < 0.05 versus control.)
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The additional 1.5 mmol/L of Ca2+ to UW solution caused an approximate 20% decrease in pulmonary artery flow after 6 hours of preservation (17.0 ± 1.0 mL/min). Thereafter, flow remained constant. The addition of BDM to the UW solution containing calcium resulted in stable pulmonary artery flows for all cold-storage periods. After 24 hours of cold storage, pulmonary artery flow was similar to that of nonpreserved lungs (20.7 ± 0.9 mL/min).
Total tissue water (edema)
Freshly harvested lungs (control) had TTW values of 6.5 ± 0.4 g water/g DW after 45 minutes of perfusion (Fig 2). Lungs preserved in UW-K or UW-Na solutions showed similar increases in TTW with time, although the magnitude of tissue water gain was slightly less in lungs preserved in UW-K solution. Lungs preserved in LPD gained water similarly to lungs preserved in the other solutions after 6 hours of preservation. However, after 12 hours of cold storage, lungs preserved in LPD became severely edematous and showed nearly a twofold gain in TTW, to 12.5 ± 1.0, (p < 0.05 versus other groups).
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Fig 2. Effect of cold storage with different preservation solutions on the amount of total tissue water (TTW) at the end of 45 minutes of normothermic reperfusion. (UW-K = University of Wisconsin solution; UW-Na = high-sodium University of Wisconsin solution; LPD = low potassium dextran solution; UW-Ca = University of Wisconsin solution plus 1.5 mmol/L Ca2+; UW-BDM = University of Wisconsin solution plus calcium and 30 mmol/L butanedione monoxime; *p < 0.05 versus others in column; p < 0.05 versus control.)
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Lungs preserved in UW-Ca gained water similarly to those preserved in UW-K after 6, 12, and 24 hours of cold storage. The addition of BDM to the UW-Ca resulted in minimal increases in TTW compared with the other groups (p < 0.05). After 24 hours of cold storage and 45 minutes of reperfusion, lung water increased only by about 13% compared with nonpreserved lungs perfused for 45 minutes (p > 0.05).
Airway pressure
Ventilation of nonpreserved (control) lungs at a tidal volume of 6 mL/kg resulted in peak inspiratory pressures (PIP) of approximately 20 cm H2O after 45 minutes of perfusion (Fig 3). Although it did not reach statistical significance, airway pressures increased with cold-storage time in all groups of lungs. Both UW-K and UW-Na solutions showed similar effects of preservation on airway pressure. The addition of calcium to UW solution had practically no effect on airway pressure when compared with lungs preserved in UW-Na or UW-K solutions. The PIP in the LPD group did not differ from other groups after 6 and 12 hours of storage. After 24 hours, all LPD-preserved lungs failed early in the reperfusion period; hence PIP at this time point is not shown. After 6, 12, and 24 hours of cold storage, the addition of BDM to UW-Ca significantly suppressed preservation-related increases in PIP. After 6 hours of preservation, PIP in this group was lower than in controls (17 ± 0.3 cm H2O, p < 0.05); after 12 to 24 hours of cold storage, PIP in the reperfused lungs reached 18.8 ± 0.5 and 19.4 ± 0.5 cm H2O, respectively, similar to that of nonpreserved lungs (19.8 ± 0.5 cm H2O).
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Fig 3. Effect of cold storage using different preservation solutions on the airway peak inspiratory pressures (PIP) at the end of 45 minutes of normothermic reperfusion. (UW-K = University of Wisconsin solution; UW-Na = high-sodium University of Wisconsin solution; LPD = low potassium dextran solution; UW-Ca = University of Wisconsin solution plus 1.5 mmol/L Ca2+; UW-BDM = University of Wisconsin solution plus calcium and 30 mmol/L butanedione monoxime; *p < 0.05 versus others in column.)
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Comment
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The goal of our study was to establish a simple, reproducible model of lung preservation and reperfusion injury that would allow screening of lung preservation solutions. From the results of this study the next step would be to modify the solution that yields the best quality of preservation in an attempt to further improve the longevity and quality of preservation using a blood-reperfused transplant model.
In our rat lung model we used as criteria for successful preservation the integrity of the microcirculation, as reflected in pulmonary artery flow rates and lung edema, and the compliance of the lung, as measured by airway PIP. Each of these has been used to characterize lung ischemia-reperfusion injury in the past [8–12]. Increased pulmonary vascular resistance and an increase in microvascular permeability are two characteristics of lung reperfusion injury. Our study shows that the UW-K solution (high potassium) gives equivalent preservation of these parameters when compared with high-sodium UW solution, and superior preservation when compared with LPD or UW-Ca. Calcium was studied because our previous work in kidney [13], liver [14, 15], and heart [16] preservation suggested that the addition of calcium to a preservation solution can be beneficial. Our results do not agree with Oka and colleagues [17], who found that lung function was significantly better after 30 hours of cold storage in LPD and low-potassium UW solutions than with UW-K solution. In our study LPD preservation was comparable to UW only after 6 hours of cold storage. When preservation time in LPD was extended to 12 hours, pulmonary artery flow rate was significantly reduced and lungs gained twice as much water on reperfusion as compared with UW preservation. None of the lungs in the LPD group survived reperfusion after 24 hours storage. Our reperfusion results using UW-K and UW-Na suggest that the electrolyte ratio may not be as important as other components of the UW solution. This difference in the results of our study versus those of Oka and coworkers [17] may be related to their use of a blood-reperfused model, prostaglandin E1 pretreatment of the lungs, storage temperature of 10°C, slow rewarming of lungs, and a short reperfusion time (10 minutes).
Our results suggest that successful lung preservation using the current UW solution can be achieved for about 12 hours. At this time period we found little change in pulmonary artery flow, airway pressure, or tissue edema when compared with nonpreserved rat lungs. This has been confirmed in our clinics in which human lungs have been successfully preserved for 8 to 13 hours. However, to confidently extend clinical lung preservation beyond 13 hours requires demonstrating experimentally successful lung preservation for at least twice the period to be used clinically. This rule-of-thumb would give surgeons confidence that the preservation time used clinically is probably safe. Hence, our goal is to preserve the lung successfully for 30 hours or more.
As a first step in improving lung preservation, we investigated the effects of adding BDM to UW solution. Lungs preserved for 24 hours in UW solution with BDM and calcium showed excellent preservation as judged by pulmonary artery flow, airway respiratory compliance, and degree of tissue edema. Pulmonary artery flow and airway PIP were similar to nonpreserved lungs. Butanedione monoxime with calcium was shown by Stringham and associates [18, 19] and Lopukhin and coworkers [20] to extend experimental heart preservation to 24 to 30 hours as tested in an isolated rabbit heart model. A proposed mechanism for this effect may be the suppression of ischemia-induced contraction (actin–myosin coupling) caused by the loss of ATP during cold ischemic storage, and prevention of the calcium paradox. Why BDM improved lung preservation is unclear. Butanedione monoxime is a potent, reversible muscle relaxant [21, 22]. In our study significantly better pulmonary artery flow rates after 24 hours of cold ischemia suggest better vasorelaxation, and lower PIP suggests better lung compliance. The beneficial effect of BDM in long-term lung preservation may be because of the contractile inhibition it provides in opposing factors that favor vasoconstriction. Such factors include decreased vasorelaxation during cold storage because of high-potassium-stimulated vasoconstriction [23]. Prospastic influences during reperfusion include decreased endogenous nitric oxide production by damaged endothelium and increased intracellular calcium flux [24].
Our results suggest that (1) high-potassium and low-potassium UW solutions provide similar qualities of rat lung preservation at 4°C, (2) LPD is less effective than UW solution beyond 12 hours, (3) addition of calcium to UW solution is not beneficial for rat lung preservation, and (4) BDM appears to improve preservation for up to 24 hours.
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Acknowledgments
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Research support from University Surgical Associates, Department of Surgery, University of Wisconsin, Madison, WI.
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References
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Abstract
Introduction
