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Ann Thorac Surg 1996;62:1448-1453
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Department of Surgery, and Department of Cell Biology and Anatomy, University of North Carolina School of Medicine, Chapel Hill, North Carolina, and Division of Cardiac Surgery, University of Pavia, and IRCCS Policlinico San Matteo, Pavia, Italy
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. Seventy-two Sprague-Dawley rats were sacrificed and then ventilated with 100% oxygen for 4 hours. The lungs were then flushed with modified Euro-Collins, University of Wisconsin, or Carolina rinse solution, either alone, with prostaglandin E1, or with prostaglandin E1 plus the free radical scavenger dimethylthiourea. After an additional 4-hour cold storage, the left lung was flushed with trypan blue solution to quantify cell viability, whereas the right lung was used to determine wet-to-dry weight ratios and to measure the levels of the adenine nucleotides adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate by high-performance liquid chromatography.
Results. Viability was consistently better in the lungs flushed with Carolina rinse solution; these differences were statistically significant compared with those in the corresponding modified Euro-Collins subgroups (p < 0.005). The addition of prostaglandin E1 to all three preservation solutions improved the total adenine nucleotide levels; this increase was statistically significant for the modified Euro-Collins subgroup (p < 0.005). The total adenine nucleotide levels for the University of Wisconsin subgroups were higher than those for the corresponding modified Euro-Collins subgroups. The highest total adenine nucleotide levels were obtained in lungs flushed with Carolina rinse plus prostaglandin E1. Wet-to-dry weight ratios were always significantly lower in the lungs preserved with University of Wisconsin solution (p < 0.05), with a value similar to that of fresh tissue.
Conclusions. The characteristics of the solution used to flush and to store rat cadaveric lungs have an impact on lung viability and adenine nucleotide metabolism. The ideal preservation strategy may allow for lung retrieval from cadavers for safe transplantation.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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In our laboratory, we have demonstrated the feasibility of lung transplantation using cadaver donors in dogs [1, 2]. Subsequently we performed experiments using a rat model to determine the time course of pulmonary cell death after circulatory arrest [3]. Oxygen ventilation of cadaver lungs was found to delay significant cell death and ultrastructural damage [4] and to maintain adenine nucleotide levels in nonperfused lungs [5]. Recently we evaluated the impact of hypothermic storage on rat lungs retrieved 4 hours after death and flushed with either modified Euro-Collins or University of Wisconsin solution [6]. Because we had previously shown a beneficial effect from the use of the free radical scavenger dimethylthiourea (DMTU) in canine lung preservation [7, 8], we chose to study the effects of the addition of this agent and prostaglandin E1 (PGE1) to perfusate solutions of rat lungs retrieved from cadavers.
The current study was undertaken to evaluate three preservation solutions used alone, with PGE1, or with PGE1 and DMTU. A perfusate solution has been developed at this institution for use as a "rinse" of liver grafts after a period of ischemia [9, 10], and we elected to evaluate it as a preservation solution for lungs retrieved from cadavers.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Animal Preparation
Seventy-two Sprague-Dawley rats weighing 300 to 500 g were sacrificed with an intraperitoneal injection of 1 mL of pentobarbital (Anpro Pharmaceuticals, Arcadia, CA) and then ventilated with 100% oxygen for 4 hours.
Immediately after the sacrifice of each of the animals, a cervical tracheotomy was performed, an 8F cannula was inserted, and mechanical ventilation was established using a Harvard rodent ventilator (model 683; Harvard Apparatus Company, Inc, Millis, MA) that delivered 100% oxygen with a tidal volume of 0.01 mL/g at a rate of 30 breaths/min. After a median sternotomy was performed, 300 units of heparin (Elkins-Sinn, Cherry Hill, NJ) was injected into the pulmonary artery and delivered into the lungs by compression of the heart, then the chest was closed with skin staples (Davis and Geck, Danbury, CT).
Specimen Preparation and Experimental Groups
At 4 hours postmortem, the chest was reopened and the venae cavae (left superior, right superior, and right inferior) were ligated. A 20-gauge catheter was inserted into the main pulmonary artery, and a left atriotomy was made. The animals were divided into three primary study groups of 24 animals each: (1) modified Euro-Collins (EC) group, (2) University of Wisconsin (UW) group, and (3) Carolina rinse (CR) group. Through the catheter, each rat received a 50-mL infusion of EC (Fresenius USA, Inc, Benicia, CA), UW (DuPont Merck Pharmaceutical Co, Wilmington, DE), or CR (University of North Carolina, John Lemasters) solution over 5 to 10 minutes. For each flush solution, three interventions were investigated: plain solution (EC-1, UW-1, CR-1), plain solution with 250-µg/L PGE1 (Upjohn Co, Kalamazoo, MI) (EC-2, UW-2, CR-2), or plain solution with 250-µg/L PGE1 and 50-mmol/L DMTU (Aldrich Chemical Co, Milwaukee, WI) (EC-3, UW-3, CR-3) (Table 1
). This produced nine subgroups with 8 animals in each subgroup.
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At the end of a 4-hour hypothermic storage period, the heart-lung block was removed from the solution. The right hilum was clamped, and the right lung was excised, cut into three parts, flash frozen in liquid nitrogen, and stored at -70°C until subsequent high-performance liquid chromotography (HPLC) analysis. A 20-gauge catheter was then reinserted into the main pulmonary artery. Trypan blue (Sigma Chemical Co, St. Louis, MO) was dissolved in Krebs-Ringers-HEPES buffer (pH, 7.4), and 50 mL of 500-µmol/L solution was infused over 10 minutes, followed by 50 mL of 2 + 2 fixative (2% glutaraldehyde + 2% paraformaldehyde in 0.1-mol/L Sorenson's buffer), infused over another 10 minutes. The infusion reservoirs were positioned 25 cm above the heart. During the two successive infusions, mechanical ventilation was reestablished briefly with the same parameters, except for the tidal volume, which was halved. The midportion of the left lung was then excised and stored at 4°C in the same fixative.
Histology and Viability Assessment
One histologic section of the midportion of the blue-stained left lung from each rat was prepared, using previously described standard techniques [3]. Briefly, the tissue was dehydrated in ethanol, washed in xylene, and embedded in paraffin. Sections 5 µm in thickness were cut on a microtome, mounted on glass slides, and counterstained with eosin only. Hematoxylin was not used because its blue color would have interfered with the interpretation of the trypan blue-staining.
Each glass slide was delineated into four quadrants and then viewed through a light microscope (Nikon Labophot-2) at high (x1,000) magnification with oil immersion. The assessing microscopist was unaware of the group identity for each slide. Twenty-five parenchymal cell nuclei were identified and counted in each of the four quadrants, and the color of each nucleus was recorded. A deliberate attempt was made to exclude white cells or macrophages from analysis. Cell nuclei were identified as either pink (viable) or blue (nonviable), the latter color imparted by the trypan blue, which stains the nuclei of nonviable cells. Each section was counted on a second occasion, and the average of the two counts was recorded for each histologic section. If the difference in the percentage of trypan blue–positive cells between counts was greater than 10%, a third assessment was done and the average determined. No attempt was made to establish whether cells were epithelial or endothelial.
Thus, the percentage of viable cells was determined for each animal in a blinded fashion. The percentage of viable cells in each rat lung specimen was used to calculate the mean percentage viability in each subgroup. By studying each of four quadrants of each histologic section, and by counting the same slide two or three times, possible variations in trypan blue staining resulting from nonhomogeneous dye distribution were minimized.
High-Performance Liquid Chromatography
One of three right lung portions was weighed (wet weight), stored in an oven at 80°C for 48 hours, and then reweighed (dry weight) using an analytical balance (Fischer Scientific, Pittsburgh, PA). The wet-to-dry weight ratio was calculated. The two remaining lung portions were used for adenosine triphosphate (ATP), adenosine diphosphate (ADP), or adenosine monophosphate (AMP) analysis.
The HPLC measurement of adenine nucleotides was performed as described previously [5]. The tissue samples for HPLC were pulverized using a liquid nitrogen–cooled Bessman pulverizer, and then homogenized in an ice-cooled test tube with ice-cooled 0.6-mol/L perchloric acid (5 mL/g tissue), using a tissue tearor (Biospec Products, Bartlesville, OK) set at high speed (30,000 rpm) for 30 seconds. After 2 minutes of centrifuging at 10,000 rpm, the supernatant was transferred into another ice-cooled test tube and buffered with 1-mol/L potassium phosphate dibasic (pH, 12) to achieve a pH of 6.8. The supernatant was separated from precipitated salt by repeat centrifuging for 2 minutes at 10,000 rpm. The remaining solution was passed through a 0.45-µm acro disc filter (Gelman Sciences, Ann Arbor, MI).
The ATP, ADP, and AMP concentrations were determined by HPLC using an LKB-Bromma apparatus (LKB-Produkter AB, Bromma, Sweden). To measure the ATP and ADP levels, a partisil 10 SAX Whatman column (Whatman, Inc, Clifton, NJ) in 0.25-mol/L potassium phosphate monobasic (pH, 6.5) was used at a flow rate of 1.5 mL/min. To measure the AMP level, an LC-18 Supelco column (Supelco, Inc, Bellafonte, PA) in 0.25-mol/L ammonium phosphate monobasic (pH, 4.5) was used at a flow rate of 2.0 mL/min. For each assessment, 50 µL of solution was injected into the HPLC system. Standard curves were constructed using serial dilutions (Sigma Chemical Co). An IBM 486 DX 33 MHz computer with Peaksimple II software (SRI Instruments, Torrance, CA) was used to record and process the data from the HPLC system.
Statistical Analysis
Differences between subgroups were analyzed statistically using analysis of variance. Data are presented as the mean ± standard errors of the mean. The differences were considered significant at a p value of less than 0.05. Total adenine nucleotide (TAN) levels were calculated according to the simple formula TAN = ATP + ADP + AMP, to document preservation of adenosine phosphate levels in lung tissue.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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. After 4 hours of 100% oxygen ventilation of circulation-arrested lungs plus 4 hours of hypothermic storage in EC, UW, or CR solutions, viability was found to vary between 72% ± 3% and 84% ± 2%. Within each group, the addition of PGE1 or PGE1 and DMTU had no significant effect on viability. However, the percentage of viable cells was always higher in the CR-flushed subgroups than it was in the UW- or EC-flushed subgroups. The differences between the CR and EC subgroups were statistically significant (p < 0.005). Discounting the addition of PGE1 or DMTU, lungs perfused with CR solution showed significantly higher viability than lungs flushed with EC or UW solution (p < 0.005). The cell viability observed in the EC and UW subgroups without additives was similar to that noted in the previous study [6].
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. The ATP levels were somewhat higher in lungs perfused with CR solution; this difference was statistically significant for lungs flushed with CR solution as compared with lungs flushed with EC solution. No statistically significant differences were observed among subgroups, however. The ATP levels for all subgroups are presented in Figure 2.
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. The TAN levels were favorably affected by the addition of PGE1 in each group. This was statistically significant for lungs flushed with EC solution. The addition of DMTU with PGE1 improved the TAN levels only in lungs flushed with EC or UW solution. Discounting the effects of the perfusate solution, lungs flushed with solutions containing PGE1 showed significantly higher TAN levels than those flushed with solutions not containing PGE1 (p < 0.05).
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. Lungs flushed with UW solution showed significantly lower wet-to-dry weight ratios than did lungs in the other groups (p < 0.0001). The addition of PGE1 to the CR solution appeared to have a modest benefit with respect to the wet-to-dry weight ratio. In our experience, a wet-to-dry weight ratio of normal rat lung freshly retrieved without flushing is 5.8 ± 0.5 (standard error). Thus flushing with UW solution resulted in normal wet-to-dry weight ratios, whereas flushing with EC or CR solution resulted in mild edema, as shown by elevation of this ratio.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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In a canine single-lung transplant model using cadaveric donors, we noted that the addition of DMTU to the EC flush solution lengthened the survival and lowered the increase in the amount of extravascular lung water during the 8-hour observation period [11]. In another experiment, we assessed parenchymal lung viability, using the trypan blue dye exclusion technique, in canine lungs ventilated for 2 hours with 100% oxygen after cardiocirculatory arrest and flushed with EC solution, with or without DMTU. The percentage of viable cells was slightly higher in the DMTU group (87% ± 5% versus 80% ± 7%). Also observed in this experiment was the finding that the amount of extravascular lung water at the end of the 8-hour observation period was lower in the DMTU group [12].
The current study was undertaken to ascertain whether the addition of PGE1 alone or with DMTU into two different preservation solutions (EC and UW) could improve the viability of or the adenine nucleotide levels in rat cadaveric lungs. Another aim of this study was to evaluate the CR solution as a pulmonary preservation solution.
A variety of studies have shown a beneficial effect from PGE1 in lung preservation using different types of solutions. Mulvin and associates [13] used Wallwork's solution for their experiment and concluded that the positive action of PGE1 correlated with the solution's vasodilative property rather than with other characteristics, such as antiplatelet aggregation, anti–white blood cell adherence, and cytoprotection. Patterson's group [14, 15] came to the same conclusion from two experiments in which PGE1 was used before lung perfusion with EC or low-potassium dextran solution. More recently, Lin and colleagues [16] utilized UW solution and concluded that the addition of PGE1 increased the safe lung preservation time.
The CR solution has been used successfully in experimental and clinical liver transplantation to rinse the grafts after the storage period and has been observed to decrease the amount of ischemic-reperfusion injury [9, 10]. Because this solution was designed as a "rinse solution," we evaluated its efficacy as a pulmonary rinse solution in a previous study [6]. In half of the experimental rat lungs, trypan blue was dissolved in this solution, whereas the other lungs were perfused with trypan blue dissolved in the usual Krebs-Ringers-HEPES buffer. In this model, CR solution was found to have no beneficial effect on viability. We reasoned that this failure may be due to the lack of time for this solution to effect recovery of the lungs. Because lungs in the current study were retrieved from non–heart beating donors and had already been subjected to a warm ischemic period, we decided to use CR solution as a preservation solution to flush and store cadaveric lungs. We chose to compare its effects with those of EC and UW solution under similar conditions.
In this study, viability was relatively well maintained in all subgroups. However, lungs flushed with CR solution fared better, and the percentage of viable cells was significantly higher in these subgroups. The highest percentage of viable parenchymal lung cells was seen for lungs flushed with CR solution with PGE1 and DMTU. Glycine, a component of the solution, has been shown to reduce ischemic-reperfusion injury and to improve function in and the survival of rat liver grafts after transplantation [17].
The TAN levels were consistently higher in lungs flushed with solutions containing PGE1. We speculate that this is due to a better distribution of perfusate, which provides a more uniform distribution of substrate for glycolysis. Although some investigators have evaluated the energy charge or ATP levels as end-points, we found in a prior study that the TAN levels correlated best with viability [5].
The addition of DMTU appeared to have little effect in this model. Because free radical–mediated processes generally require a period of reperfusion, it is possible that the potential benefit of a free radical scavenger could not be adequately assessed in this model. Because solutions containing DMTU and PGE1 together had effects similar to those found for solutions containing only PGE1, we decided not to pursue further studies in this model using DMTU alone.
Wet-to-dry weight ratios were better in lungs perfused with the UW solution. Perhaps this is related to the high-molecular-weight osmotic-impermeant components, which theoretically prevent cellular expansion during ischemia [18]. Hopkinson and colleagues [19] pointed out that the impermeant trisaccharide raffinose appears to be the major factor responsible for the efficacy of the UW solution in lung graft preservation.
In an earlier experiment [6], we employed a larger volume of flush solution (100 mL) and found substantial increases in the wet-to-dry weight ratios for cadaveric lungs. We chose to use a smaller volume of flush solution for the current experiments (50 mL), because this volume is more consistent with volumes used in clinical lung preservation for human transplantation. Even this volume, however, corresponds to a perfusion volume of 100 to 150 mL/kg, which should exaggerate any tendency for pulmonary edema to develop.
These studies lend support to the hypothesis that lungs retrieved from oxygen-ventilated cadavers may be suitable for transplantation. Further studies are necessary to elucidate the tolerable period of in situ ischemia and the tolerable period of hypothermic storage after retrieval from cadavers. The CR solution may be a useful pulmonary preservation strategy and merits further study. If lungs can maintain viability and provide good gas exchange function after retrieval from circulation-arrested cadavers, then the scarcity of suitable lung donors may be minimized.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We express appreciation to David T. Currin and Kimberlie Burns for their excellent technical assistance; to DuPont Merck Pharmaceutical Company, Fresenius USA, Inc., and Upjohn Co for generous gifts of UW solution, EC solution, and PGE1, respectively; and to Betsy L. Mann for editorial 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 Egan, University of North Carolina at Chapel Hill, 108 Burnett-Womack Bldg, CB 7065, Chapel Hill, NC 27599-7065.
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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D. E. M. Van Raemdonck, N. C. P. Jannis, F. R. L. Rega, P. R. J. De Leyn, W. J. Flameng, and T. E. Lerut Extended Preservation of Ischemic Pulmonary Graft by Postmortem Alveolar Expansion Ann. Thorac. Surg., September 1, 1997; 64(3): 801 - 808. [Abstract] [Full Text]
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