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Ann Thorac Surg 1996;61:963-968
© 1996 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

ET-Kyoto Solution for 48-Hour Canine Lung Preservation

Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, Kyoto, Japan

Accepted for publication November 1, 1995.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Background. ET-Kyoto (ET-K) solution, proven safe for 20-hour lung preservation, was modified to achieve longer preservation: ET-K2 solution with more buffer capacity and ET-K3 solution with less potassium.

Methods. Lungs were preserved with one of the three solutions (with prostaglandin E₁) at 4°C for 48 hours (n = 5 for each). Left lung transplantation was performed and evaluated for 6 hours.

Results. Each solution became acidic after preservation (p < 0.01), though the change was lowest in the ET-K2 solution. All animals in the ET-K and ET-K3 groups survived for 6 hours after reperfusion, but only 1 survived in the ET-K2 group (p < 0.05). In all groups, partial pressure of oxygen in arterial blood decreased gradually after reperfusion. Pulmonary vascular resistance after reperfusion was significantly lower in the ET-K group than in the ET-K3 group (p < 0.01). Scanning electron microscopic examination showed that endothelial cell swelling and disruption were milder in the ET-K group (with solution containing potassium of 44 mEq/L) than in the ET-K3 group.

Conclusion. Lung preservation can be achieved for 48 hours in ET-K and ET-K3 solutions. Enhancement of buffer capacity provides no advantage. Potassium at 44 mEq/L does not cause deterioration of endothelial cells.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Lung transplantation has been established recently as a therapeutic option for patients with end-stage lung disease, but the shortage of donors remains the biggest problem. In clinical lung transplantation, donor lungs have been preserved mostly in Euro-Collins solution with prostaglandins [1], and the safe limit of preservation by this method is less than 10 hours [2]. Many experimental studies have evaluated lung preservation for more than 24 hours with other solutions, such as University of Wisconsin or low-potassium dextran solutions, but these have not yet been used for a longer period of clinical lung preservation [3–6].

Trehalose is a nonreducing disaccharide (1--D-glucopyranosyl-1--D-glucopyranoside) consisting of two glucose moieties connected by a 1,1,-linkage, which is extremely stable chemically. It exists in some plants, honey, fungi, red algae, and insect body fluid. In the human body, trehalose yields glucose after hydrolysis by trehalase. Trehalose is reported to have stabilizing and protective effects on cell membrane structures under various forms of stress, such as desiccation, freezing, and high temperatures [7, 8]. Previously we proved the effectiveness of trehalose in 12-hour canine lung preservation [9, 10]. Next we developed ET-Kyoto (ET-K) solution, which is a low-potassium extracellular-type lung preservation solution containing 4.1% trehalose, hydroxyethyl starch, and gluconate; it preserves canine lungs perfectly for at least 20 hours [11, 12].

For more reliable elective operations, a method of preservation for more than 24 hours will be necessary. Safe preservation for at least 30 to 40 hours is preferable for large animals. In this study, we stored lungs for 48 hours and evaluated pulmonary function for 6 hours after left lung transplantation. We modified our ET-K solution as follows. (1) Because solutions in the pulmonary vessels have caused severe acidosis after 20 hours of preservation (unpublished data), it seemed probable that excessive acidity might lead to cell injury after longer periods of preservation. Therefore, a preservation solution with greater buffering capacity might be desirable. (2) As shown in the study by Kimblad and associates [13], the appropriate potassium concentration might be less than 30 mEq/L to avoid pulmonary vasoconstriction. Hence we prepared ET-K2 solution with improved buffering capacity to suppress excessive acidosis and ET-K3 solution with a potassium concentration of 20 mEq/L to prevent vasoconstriction. In this trial of preservation for 48 hours, we compared these two ET-K solutions with the one that we had developed previously.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Preservation Solutions
Each solution was an extracellular-type solution with a low potassium content and identical doses of trehalose and hydroxyethyl starch. The ET-K2 solution contained phosphate of 75 mEq/L, three times as much as in ET-K solution, and gluconate limited to 8 mEq/L. The ET-K3 solution contained less potassium than ET-K solution, and phosphate was increased to 36 mEq/L (Table 1).

Donor Operation
Induction and maintenance of anesthesia were performed as described previously [9–12]. The lungs were ventilated with an inspired oxygen fraction of 0.5, a tidal volume of 20 mL/kg, a respiration rate of 15 breaths/min, and a positive end-expiratory pressure of 5 cm H₂O. After the induction of anesthesia, a 20-gauge arterial catheter was inserted in the right femoral artery, and a 7F Swan-Ganz catheter (Baxter Healthcare Corp, Irvine, CA) was introduced in the main pulmonary artery through the right femoral vein. Before operation, we recorded arterial blood gas analysis, peak inspiratory pressure (PIP), mean pulmonary artery pressure, pulmonary capillary wedge pressure, cardiac output, systemic blood pressure, and heart rate. After median sternotomy, the azygos vein was ligated and divided, and the superior and inferior vena cava, aorta, and pulmonary artery were encircled. Heparin (200 U/kg) was administered through the right ventricular outflow tract. A 5-mm cannula was inserted in the main pulmonary artery and fixed with a pursestring suture of 3-0 Prolene (Ethicon Inc, Somerville, NJ). Prostaglandin E₁ (25 µg/kg) was injected through the right ventricular outflow tract, and the superior and inferior vena cava and aorta were ligated and divided when systemic blood pressure declined by 60%.

After amputation of the left atrial appendage, we flushed the pulmonary artery by gravity from a height of 50 cm with ET-K, ET-K2, or ET-K3 solution at 4°C (70 mL/kg). The donor lungs were inflated once to the maximum inspiratory pressure just before flushing. During flushing, the ventilating condition (inspired oxygen fraction 0.5, tidal volume 20 mL/kg, respiratory rate 15 breaths/min, and positive end-expiratory pressure 5 cm H₂O) was maintained and the flushing time was recorded. After the pulmonary arterial flushing, the left atrial appendage was ligated to keep the preservation solution inside. Donor lungs were kept at a maximum inspiratory pressure of 30 cm H₂O for a while; then the trachea was clamped at an inspiratory pressure of 20 cm H₂O. The heart and lung block was excised with minimal handling, placed in 1,000 mL of the identical solution, and stored at 4°C for 48 hours. The pH, sodium and potassium concentrations, and O₂ and CO₂ tensions (PaO₂ and PaCO₂) of the flushing solution were measured before and after preservation.

After 20 hours of preservation, the right lateral basal segment of the donor lung was removed and examined in a scanning electron microscope.

Recipient Operation
The recipient dogs were anesthetized in the same way as the donors. A 20-gauge arterial catheter was inserted in the right femoral artery, and a 7F Swan-Ganz catheter was introduced in the main pulmonary artery through the right femoral vein. We measured arterial blood gas analysis, PIP, pulmonary artery pressure, systemic blood pressure and, heart rate. After left pneumonectomy, single left lung transplantation was performed as described previously [9–12]. Before anastomoses, we measured pH, sodium and potassium concentrations, and PaO₂ and PaCO₂ of the preservation solution in the left atrium of the stored heart and lung block. Anastomoses were performed in the order of left atrium, bronchus, and pulmonary artery. After the left atrium and the bronchus, the left pulmonary artery was anastomosed with both lungs ventilated. After transplantation, reperfusion was carried out for 6 hours. At 1, 2, 4, and 6 hours after reperfusion, the right main pulmonary artery was clamped for 10 minutes, and arterial blood gas analysis, PIP, pulmonary artery pressure, systemic blood pressure, and heart rate were measured. At 6 hours after reperfusion, left atrial pressure and heart rate were measured with the right main pulmonary artery clamped. During these procedures, ventilation was performed as described. Pulmonary vascular resistance (PVR) was calculated as follows: PVR = [(PAP - PCWP)/CO] x 80 dynes•s•cm^-5, in which PAP = pulmonary artery pressure; PCWP = pulmonary capillary wedge pressure; and CO = cardiac output. After the measurement, the recipient dog was sacrificed for evaluation of the preserved lung; specimens from two segments (S¹⁺² and S⁹) were excised and examined histologically. After S¹⁺² and S⁸ had been removed and weighed, they were dried at 70°C for 72 hours and weighed again, and the wet/dry weight ratio was calculated.

Statistical Analysis
All data were expressed as mean ± standard error of the mean. The results in the three groups were analyzed statistically with analysis of variance and Scheffe's multiple comparison test. The survival ratio was calculated with the Kaplan-Meier method, and the log rank test was used for analysis. Differences between the pretransplantation and posttransplantation data and between the data of the two groups were analyzed with the Student's t test. A p value less than 0.05 was considered significant.

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'' prepared by the National Academy of Science (NIH publication 85-23, revised 1985).

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Donor Data
Cold ischemic time, warm ischemic time, and flush time were as follows in the ET-K group, the ET-K2 group, and the ET-K3 group, respectively: cold ischemic time 2,820.8 ± 13.9, 2,859.2 ± 28.2, and 2,867.0 ± 23.6 minutes; warm ischemic time 75.0 ± 1.6, 71.4 ± 2.3, and 69.6 ± 6.1 minutes; and flush time 109.0 ± 6.2, 133.4 ± 11.6, and 117.0 ± 6.6 seconds. The differences among the three groups were not significant. In addition, PaO₂, PaCO₂, PIP, and PVR of the donors before harvest were similar in all groups.

pH Before and After Preservation
The pH of the solutions before preservation was between 7.30 and 7.36, with no significant difference among the three groups (ET-K 7.38 ± 0.05, ET-K2 7.34 ± 0.00, and ET-K3 7.30 ± 0.00). The pH of the solutions obtained from the left atria of heart and lung blocks after 48-hour preservation dropped significantly in all groups (p < 0.01). The pH was highest in the ET-K2 group (6.68 ± 0.03) and lowest in the ET-K group (6.27 ± 0.09), the reflecting buffering capacity of each solution. The ET-K3 group had a pH of 6.35 ± 0.06. The differences between the ET-K2 group and the ET-K group and between the ET-K2 group and the ET-K3 group were significant (p < 0.01).

Survival Rate
All animals in the ET-K and ET-K3 groups withstood clamping of the right pulmonary artery for 10 minutes and survived 6 hours after reperfusion. In the ET-K2 group, 1 animal died within 2 hours after reperfusion, 2 more within 4 hours, and 1 more within 6 hours, so only 1 of the 5 animals survived the clamp test to 6 hours after reperfusion. The survival rates in the ET-K group and the ET-K3 group were significantly higher than that of the ET-K2 group (p < 0.05) (Fig 1).

View larger version (14K): [in this window] [in a new window]   Fig 1. . Survival rate. All animals in the ET-K and ET-K3 groups, but only 1 animal in the ET-K2 group, survived for 6 hours after reperfusion. The survival ratios of the ET-K and ET-K3 groups were significantly higher than that of the ET-K2 group (*p < 0.05). (ET-K = ET-Kyoto.)

View larger version (16K): [in this window] [in a new window]   Fig 2. . Arterial blood gas analysis. Oxygen tension (PO2) at 1, 2, 4, and 6 hours after reperfusion dropped gradually. There was no significant difference among PO2 data in the three groups, and only the PO2 at 1 hour after reperfusion in the ET-K group tended to be higher than that in the ET-K2 group (p = 0.0997). (ET-K = ET-Kyoto.)

Peak Inspiratory Pressure
Peak inspiratory pressure at 1, 2, 4, and 6 hours after reperfusion was, respectively, 20.2 ± 3.8, 22.6 ± 4.1, 24.0 ± 2.5, and 24.4 ± 3.1 cm H₂O in the ET-K group; 24.2 ± 3.5, 19.5 ± 1.3, 20.5 ± 0.5, and 21.0 cm H₂O in the ET-K2 group; and 15.0 ± 0.8, 17.0 ± 1.3, 17.4 ± 1.5, and 19.6 ± 1.9 cm H₂O in the ET-K3 group. In all groups, PIP increased gradually. There was no significant difference among the three groups, but PIP in the ET-K3 group tended to be lower than that in the ET-K2 group at 1 hour after reperfusion, and PIP in the ET-K3 group tended to be lower than that in the ET-K group at 4 hours after reperfusion (p = 0.0648 and p = 0.0519, respectively).

Pulmonary Vascular Resistance
During right pulmonary artery clamping at 6 hours after reperfusion, PVR was 1,954.4 ± 152.7 dynes•s•cm^-5 in the ET-K group and 3,227.7 ± 202.3 dynes•s•cm^-5 in the ET-K3 group; the values in the ET-K group were significantly lower than those in the ET-K3 group (p < 0.01) (Fig 3). In the ET-K2 group, only 1 animal could be evaluated (PVR = 2705.3 dynes•s•cm^-5), so statistical analysis was not possible.

View larger version (11K): [in this window] [in a new window]   Fig 3. . Pulmonary vascular resistance (PVR) values in each group during clamping of the right pulmonary artery at 6 hours after reperfusion were 1954.4 ± 152.7 dynes•s•cm-5 in the ET-K group and 3227.7 ± 202.3 dynes•s•cm-5 in the ET-K3 group; the values in the ET-K group were significantly lower than those in the ET-K3 group (*p < 0.01). (ET-K = ET-Kyoto.)

Light Microscopic Examination
In the ET-K group, severe pulmonary edema was observed in 4 of the 5 lungs and moderate edema in 1; severe edema was found in all 5 lungs in the ET-K2 group. In the ET-K3 group, severe edema was observed in 2 of 5 lungs and moderate edema in 3 lungs.

Scanning Electron Microscopic Examination
Endothelial swelling observed in the ET-K group suggested moderate injury. Furthermore, disruption of endothelial cells was distinct in the ET-K3 group, so the ET-K3 group was considered to be more injured than the ET-K group (Fig 4).

View larger version (397K): [in this window] [in a new window]   Fig 4. . Scanning electron microscopic examination. Endothelial swelling observed in the ET-K group suggested moderate injury (A). Distinct disruption and swelling of endothelial cells in the ET-K3 group showed severe injury (B).

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

Another factor in the poor preservation with ET-K2 solution may be the lack of gluconate. Belzer and Southard [18] proposed that raffinose (MW 504) and lactobionate anion (MW 358) in University of Wisconsin solution act as impermeants and prevent cell swelling. Indeed, Jamieson and co-workers [19] and Sumimoto and colleagues [20] demonstrated that postpreservation liver function worsened after the replacement of lactobionate or gluconate anion with chloride (MW 35) in the preservation solution. In the present study, gluconate may have contributed to the reduction of the cell swelling during preservation and may have been more effective than phosphate (MW 95) and chloride.

Another strategy to prolong safe preservation time is to change the ion composition. Solutions of high-potassium intracellular-type composition such as Euro-Collins and University of Wisconsin are favorable for organs other than the lung in maintaining intracellular homeostasis during preservation [20]. In the 20-hour canine lung preservation model, we compared ET-K solution of extracellular-type ion composition (Na, 100 mEq/L; K, 44 mEq/L) and IT-K solution of intracellular-type ion composition (Na, 20 mEq/L; K, 130 mEq/L). The difference between these solutions was only the ion composition, and the ET-K solution was superior [11, 12]. Kimblad and associates [13] examined the contractile response of porcine pulmonary arteries placed in buffer solutions with various concentrations of potassium and reported that contraction occurred in a solution with potassium greater than 30 mEq/L; prostaglandin E₁ or nifedipine could not suppress this contraction completely. Accordingly, the optimal potassium concentration in a preservation solution may be less than 30 mEq/L for avoiding pulmonary vasoconstriction. With this information, we developed ET-K3 solution with an ion composition of Na at 145 mEq/L and K at 20 mEq/L. As a result, ET-K3 solution did not show any significant difference as to oxygenation, PIP, or wet/dry weight ratio compared with the ET-K group (Na, 100 mEq/L; K, 44 mEq/L). Moreover, PVR was significantly lower and endothelial cell injury seen by scanning electron microscopic examination was milder in the ET-K group. Therefore, a potassium concentration of 44 mEq/L might be better for endothelial cells than one of 20 mEq/L. Further examinations are necessary to determine the optimal ion composition.

Of course, our model has limitations, such as the use of only 10 minutes of temporary clamping of the right main pulmonary artery and only a 6-hour observation time. To make the recipient 100% dependent on the transplanted donor lung, pneumonectomy of the recipient lung or bilateral sequential transplantation would be optimal. To confirm safe preservation, a 24-hour or longer observation period would be necessary. These experiments should be performed using large animals such as dogs or baboons before clinical use.

In conclusion, it seems that gluconate is important in preservation solutions, whereas enhancement of the buffer capacity may not be. Dogs receiving transplants of left lungs preserved for 48 hours with ET-K or ET-K3 solution could survive for 6 hours. With modification of the components of ET-K solution, reliable 48-hour preservation can be achieved. It is expected that clinical application will be possible and that elective lung transplantation can be achieved.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Address reprint requests to Dr Wada, Chest Disease Research Institute, Kyoto University, Shogoin Kawahara-cho 53, Sakyo-ku, Kyoto 606, Japan.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Hiromi Wada Toru Bando Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wada, H. Articles by Hitomi, S. Search for Related Content PubMed PubMed Citation Articles by Wada, H. Articles by Hitomi, S.