Ann Thorac Surg 2000;69:887-891
© 2000 The Society of Thoracic Surgeons
Original Articles
Significance of cyclic adenosine monophosphate and nitroglycerin in ET-Kyoto solution for lung preservation
Toru Bando, MDa,b,
Johannes M. Albes, MDa,
Jörg Schöne, BSa,
Hiromi Wada, MDb,
Shigeki Hitomi, MDb,
Thorsten Wahlers, MDa,
Hans-Joachim Schäfers, MDa
a Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany
b Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan
 |
Abstract
|
|---|
Background. We previously demonstrated that the supplement of both dibutyryl cyclic adenosine monophosphate (db-cAMP) and nitroglycerin to the conventional ET-Kyoto solution improved lung preservation significantly. However, the significance of each component in lung preservation remained unclear. We examined the efficacy of the two components on lung preservation in the current study.
Methods. Rat lung grafts (eight per group) were studied in an isolated lung perfusion model. Group 1 grafts were flushed and preserved with ET-Kyoto solution containing 2 mmol/L of db-cAMP. Group 2 grafts were flushed and preserved with ET-Kyoto solution containing 100 mg/L of nitroglycerin. In group 3, the grafts were flushed and preserved with ET-Kyoto solution containing neither db-cAMP nor nitroglycerin as control group. After 4-hour cold storage, the lung grafts were reperfused for 50 minutes.
Results. The lung grafts in groups 1 and 2 showed significantly better lung function after reperfusion than those in group 3 with regard to arterial oxygen tension, shunt fraction, peak inspiratory airway pressure, mean pulmonary arterial pressure, and pulmonary vascular resistance. The supplementation of db-cAMP improved especially the pulmonary arterial pressure and pulmonary vascular resistance, while the supplementation of nitroglycerin improved especially the oxygenation and airway pressure of the grafts.
Conclusions. Both of db-cAMP and nitroglycerin had beneficial effects on lung preservation and are essential to the ET-Kyoto solution. There was a difference between the two components in the effects on preserved lungs.
|
Introduction
|
|---|
One of the most important strategies to increase the supply of transplantable lungs is the development of effective and highly reliable methods for lung preservation. Current clinical lung preservation with Euro-Collins (EC) or University of Wisconsin solution provides satisfactory graft function, but these methods are generally believed to be limited to periods of ischemia up to 6 hours [1]. Previously, we developed an extracellular-type preservation solution, ET-Kyoto (ET-K) solution, and revealed it to be quite effective in 20-hour canine lung preservation [2]. Our subsequent study with scanning and transmission electron microscopy demonstrated a significant correlation between the postischemic graft function and the structural changes of pulmonary endothelial cells [3]. A number of recent studies have demonstrated that ischemia-reperfusion causes pulmonary endothelial damage [4–6]. In addition, ischemia-reperfusion reduces endothelial nitric oxide (NO) and cyclic adenosine monophosphate (cAMP) levels [7, 8],both of which play critical roles in maintaining the vascular endothelial barrier function [8, 9], in relaxation of vascular smooth muscle [10], and in preventing the adherence to vascular endothelial cells of neutrophils and platelets [7, 10, 11]. Focusing on the maintenance of graft vascular homeostasis during ischemia and reperfusion, we developed a modified ET-K solution by adding dibutyryl cyclic adenosine monophosphate (db-cAMP), nitroglycerin, and N-acetylcysteine (NAC) to the conventional ET-K solution. Db-cAMP is a membrane-permeable cAMP analogue and elevates intracellular cAMP levels. Nitroglycerin is a potential NO donor and elevates intracellular NO/cyclic guanosine monophosphate (cGMP) levels [12]. NAC protects endothelial cells from oxygen free radicals and attenuated reperfusion injury after lung transplantation in our previous experiment [13]. We have demonstrated that the modified ET-K solution provided excellent 30-hour canine lung preservation and was significantly superior to the previous ET-K solution [14]. However, the effect of each supplement has not been investigated. In the present study, the roles of db-cAMP and nitroglycerin in the new ET-Kyoto solution were examined in a rat isolated lung perfusion model.
|
Material and methods
|
|---|
Twenty-four male inbred Sprague-Dawley rats (405 to 550 g) were assigned randomly to three groups according to the type of preservation solutions: group 1 (n = 8), ET-K solution containing 2 mmol/L of dibutyryl cyclic adenosine monophosphate (db-cAMP); group 2 (n = 8), ET-K solution containing 100 mg/L of nitroglycerin (TNG), and group 3 (n = 8), ET-K solution containing neither db-cAMP nor TNG as control group (Table 1). The lungs in each group were stored for 4 hours with corresponding solutions at 4°C before reperfusion. All animals received humane care in compliance with the "Principals 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 Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).
Bovine blood was drawn directly from the jugular vein of living cows. The red cells were processed within a day, using sterile techniques to remove plasma, white cells, and platelets. The blood was spun at 3500 g for 10 minutes. The supernatant plasma was removed. The cells were diluted with 0.9% saline and spun in the same way as in the first process. The red blood cells were then diluted with Krebs-Henseleit solution to a hematocrit of 37% to 38%. Thereafter, leukocytes were removed through a leukocyte filter (leukocyte removal filter RC100E; PALL Europe Ltd, Portsmouth, UK).
Animals were anesthetized with intraperitoneal injection of 40 mg/body pentobarbital and intubated by tracheostomy. During the operation, animals were ventilated with room air at a tidal volume of 5 mL and a respiration rate of 40 breaths per minute with a positive end-expiratory pressure of 3 cm H2O. Through a laparotomy, 100 U heparin was given into the inferior vena cava. After a sternotomy, the right and left superior vena cavae and the left azygos vein were clipped. A no. 14 cannula was inserted into the pulmonary artery through the right ventricle and the inferior vena cava was clipped. The left atrial appendage was then cut and the lungs were flushed with 20 mL of cold perfusates under 20 cm of gravity pressure. Ventilation of the lungs was continued during the flush and the duration of flushing was recorded. The heart-lung block was carefully excised and immediately immersed in cold (4°C) Ringer solution. The pulmonary artery, trachea, and left atrium were cannulated (inner diameter of 1.8 mm). The lungs were allowed to deflate during the preparation. After lungs were reinflated with 10 mL of room air, the heart-lung block was then immersed in cold corresponding solution and stored at 4°C for 4 hours.
An extracorporeal circuit described previously [15] was used. The perfusion circuit was primed with 600 mL of Krebs-Henseleit solution containing washed red blood cells, and the perfusate was continuously deoxygenated by a deoxygenator (Monolyth-integrated membrane lung; SORIN Biomedica Ltd, Saluggia, Italy) gassed with 95% N2 and 5% CO2. The heart-lung block was suspended at a 45° angle in a humidification chamber. Mechanical ventilation was performed with a small animal respirator (animal respirator 4601; Rhema Labortechnik Ltd, Germany). All vessels were water-jacketed, and temperature was controlled by a warming pump (Water thermostat type VTS 13c; Radiometer Ltd, Copenhagen, Denmark) at 37°C.
After storage for 4 hours, the heart-lung block was connected to the extracorporeal circuit. The lungs were ventilated via tracheal cannula with room air at a tidal volume of 5 mL and a respiration rate of 40 breaths per minute, with a positive end-expiratory pressure of 3 cm H2O, and were reperfused via pulmonary arterial cannula with the deoxygenated perfusate by a roller pump (PA21-A; Cole Parmer Co, Chicago, IL). The perfusion rate was gradually increased from 1.0 to 8.0 mL/min during the first 9 minutes, and thereafter, the lung was perfused at the constant rate of 8.0 mL/min up to 50 minutes after reperfusion. If the tracheal cannula was entirely filled with fluid due to pulmonary edema, reperfusion was interrupted and the experiment was terminated. The pulmonary arterial pressure was assessed continuously with a transducer and a pressure monitor (Servomed; Hellige Ltd, Hamburg, Germany). The pulmonary flow rate was measured by collecting flow from the left atrial cannula. Peak inspiratory pressure was monitored continuously. Blood gas analysis, mean pulmonary arterial pressure, peak inspiratory pressure, and pulmonary flow rate were recorded every 10 minutes after reperfusion. Oxygen tension (PO2) of the perfusate collected from the left atrium was defined as arterial PO2, and PO2 of the perfusate after deoxygenation as venous PO2. Pulmonary vascular resistance (PVR) was calculated as follows: PVR = 80 x (mean pulmonary arterial pressure - left atrial pressure)/pulmonary flow rate (dyne/s/cm5). In addition, shunt fraction, which shows the fraction of arterial-venous shunts in the lungs, indicating the invalid respiration or gas exchanges, was calculated as follows: shunt fraction (%) = (Cc - Ca)/(Cc - Cv) x 100; C = (PO2 x 0.003) + (1.34 x Hb concentration x O2 saturation), where Cc, Ca, and Cv are the oxygen contents of the pulmonary capillary, arterial, and venous blood, respectively.
Statistical analysis of the data was performed by analysis of variance with posthoc comparison and paired, two-tailed t test. A p value less than 0.05 was considered significant. All results are expressed as means ± standard deviation unless stated otherwise.
|
Results
|
|---|
Pulmonary flush time was significantly shorter in group 2 (46.6 ± 5.5 seconds) than in control group (52.3 ± 4.1 seconds, p < 0.05), but no significant difference was detected between group 1 (49.3 ± 6.0 seconds) and control group. All lungs in the three groups were reperfused for 50 minutes, except one animal of group 2, where the reperfusion was interrupted due to severe lung edema after 39 minutes of reperfusion. The mean reperfusion time of groups 1, 2, and 3 were 50 minutes, 48.6 ± 3.9 minutes, and 50 minutes, respectively, and no significant difference was detected among the three groups.
The arterial PO2 of group 2 was higher than that of control group throughout reperfusion, and significant differences between the two groups were detected up to 40 minutes after reperfusion (p < 0.01, 10 minutes after reperfusion; p < 0.05, 30, and 40 minutes after reperfusion; Fig 1). The PO2 of group 1 was also higher than control group after reperfusion, but the differences did not reach significance. The shunt fractions of groups 1 and 2 remained lower than control group after reperfusion (Fig 2). The difference was significant throughout the reperfusion between group 2 and control (p < 0.01), and was also significant after 30 minutes of reperfusion between group 1 and control (p < 0.05).
View larger version (20K):
[in this window]
[in a new window]
|
Fig 1. Arterial oxygen tension (PO2) after reperfusion (mean ± standard error of the mean). *p < 0.05 versus control group (group 3); **p < 0.01 versus control group (group 3).
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Fig 2. Shunt fraction after reperfusion (mean ± standard error of the mean). *p < 0.05 versus control group (group 3); **p < 0.01 versus control group (group 3).
|
|
Mean pulmonary arterial pressure increased significantly after reperfusion in each group (p < 0.01 minutes vs 50 minutes after reperfusion) (Table 2). Mean pulmonary arterial pressures of groups 1 and 2 were lower than that of control group. Significant differences were detected between group 1 and control all through the reperfusion (p < 0.01), while the difference between group 2 and control was significant only at 20 and 30 minutes of reperfusion (p < 0.05), and did not reach significance after 40 minutes of reperfusion.
In groups 1 and 2, the PVRs remained lower than in control group after reperfusion (Fig 3). The differences were significant between control group and experimental groups throughout the reperfusion, except between group 2 and control at 50 minutes of reperfusion.
View larger version (21K):
[in this window]
[in a new window]
|
Fig 3. Pulmonary vascular resistance (PVR) after reperfusion (mean ± standard error of the mean). *p < 0.05 versus control group (group 3); **p < 0.01 versus control group (group 3).
|
|
Peak inspiratory pressures of group 1 and 2 were lower than that of control group throughout the reperfusion (Fig 4). In contrast to pulmonary arterial pressure, the difference between group 2 and control was significant all through the reperfusion (p < 0.05), while significant differences were detected between group 1 and control only for the first 30 minutes of reperfusion (p < 0.05), and the difference did not reach significance after 40 minutes of reperfusion.
View larger version (20K):
[in this window]
[in a new window]
|
Fig 4. Peak inspiratory pressure (PIP) after reperfusion (mean ± standard error of the mean). *p < 0.05 versus control group (group 3); **p < 0.01 versus control group (group 3).
|
|
|
Discussion
|
|---|
We have previously demonstrated that the supplement of both db-cAMP and TNG was beneficial to 30-hour canine lung preservation [14]. The significance of each component, however, remained unclear. We, therefore, examined the efficacy of each component on lung preservation in the present investigation. The current study revealed that both of the components improved postischemic lung function and were proved essential to ET-K solution.
Db-cAMP is deacylated after its entry into the cells [16] and elevates intracellular cAMP level. TNG is a potential NO donor and elevates intracellular NO/cGMP level [12]. cAMP and cGMP are considered to attenuate vascular permeability [8, 9] to relax pulmonary vascular smooth muscle [10], and to prevent adherence of neutrophils and platelets to the vascular wall [7, 10, 11]. The intracellular concentrations of both monophosphates were reported to be reduced during ischemia and reperfusion [7, 8], and supplementation of cAMP or NO/cGMP pathway enhanced cardiac [11, 17] and pulmonary [7, 18] preservation. Naka and associates [19] emphasized the antineutrophil and antiplatelet effects of the monophosphates for improved lung preservation. In the present ex vivo study, however, the antineutrophil effects of the monophosphates might not have much influence on the results because leukocytes were removed from the perfusate in our model. Therefore, the beneficial effects of the monophosphates seemed to be caused mainly by attenuation of vascular permeability and relaxation of pulmonary vascular smooth muscle. A recent investigation by Nakamura and associates [18] demonstrated that db-cAMP has a protective effect on the preserved pulmonary endothelium documented by electron microscopy.
We did not examine the effect of NAC, which is also a component of the new ET-Kyoto solution, because NAC is considered as a free radical scavenger and protects endothelial cells from oxygen free radicals during reperfusion. We did not think that the current leukocyte-depleted model was suited to investigate the effect of NAC. On the other hand, thiols, such as NAC, can react readily with NO to form the corresponding S-nitrosothiols (RS-NO). The formation of RS-NO is viewed as a means of stabilizing NO in bioactive form, potentially facilitating its transport in tissues [20], and may enhance the physiological activities of NO. In the present study, NAC in the preservation solution could possibly modify the effects of NO released from TNG. Further studies are needed to examine the interaction of NAC and TNG.
Interestingly, a difference between TNG and db-cAMP with respect to postischemic lung function was observed in the current study, although the two monophosphates generated from TNG and db-cAMP are quite similar in the physiological activities. Lungs preserved with the solution containing TNG showed better function, especially in oxygenation and low airway pressure compared with lungs of control group. Lungs preserved with db-cAMP exhibited in particular low pulmonary arterial pressure and pulmonary vascular resistance. These results could suggest a different mechanism of the beneficial effects of the two monophosphates. TNG in the preservation solution may act on bronchial smooth muscles in addition to vascular smooth muscles. Further investigations are required to elucidate a role of each monophosphate in lung preservation.
In conclusion, both of db-cAMP and TNG had beneficial effects on lung preservation in this model and are essential components to the ET-K solution. There was a difference between the two components in the effects on the preserved lungs.
|
Acknowledgments
|
|---|
Doctor Bando was supported by Fellowships from the Alexander von Humboldt Foundation.
|
References
|
|---|
-
Kirk A.J.B., Colquhoun I.W., Dark J.H. Lung preservation. Ann Thorac Surg 1993;56:990-1000.[Abstract]
-
Bando T., Kosaka S., Liu C.J., et al. Effects of newly developed solutions containing trehalose on twenty-hour canine lung preservation. J Thorac Cardiovasc Surg 1994;108:92-98.[Abstract/Free Full Text]
-
Kosaka S., Bando T., Liu C.J., Suzuki Y., Hitomi S., Wada H. Ultrastructural changes in canine lung preserved in newly developed solutions. J Surg Res 1996;63:467-473.[Medline]
-
Killinger W.A., Dorofi D.B., Tinsley E.A., Keagy B.A., Johnson G. Flow cytometric analysis of organ preservation-induced endothelial cell membrane damage. Ann Thorac Surg 1992;53:472-476.[Abstract]
-
Fullerton D.A., Mitchell M.B., McIntyre R.C., et al. Cold ischemia and reperfusion each produce pulmonary vasomotor dysfunction in the transplanted lung. J Thorac Cardiovasc Surg 1993;106:1213-1217.[Abstract]
-
Chan B.B.K., Kron I.L., Flanagan T.L., Kern J.A., Hobson C.E., Tribble C.G. Impairment of vascular endothelial function by high-potassium storage solutions. Ann Thorac Surg 1993;55:940-945.[Abstract]
-
Pinsky D.J., Naka Y., Chowdhury N.C., et al. The nitric oxide/cyclic GMP pathway in organ transplantation. Proc Natl Acad Sci USA 1994;91:12086-12090.[Abstract/Free Full Text]
-
Ogawa S., Koga K., Kuwabara J., et al. Hypoxya-induced increased permeability of endothelial monolayers occurs through lowering of cellular cAMP levels. Am J Physiol 1992;262:C546-C554.[Abstract/Free Full Text]
-
Kubes P., Granger N. Nitric oxide modulates microvascular permeability. Am J Physiol 1992;262:H611-H615.[Abstract/Free Full Text]
-
Vane J.R.,
nggard E.E., Botting R.M. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:27-36.[Medline]
-
Pinsky D., Oz M., Liao S., et al. Restoration of the cAMP second messenger pathway enhances cardiac preservation for transplantation in a heterotopic rat model. J Clin Invest 1993;92:2994-3002.
-
Ignarro L.J. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 1989;65:1-21.[Free Full Text]
-
Yagi K., Liu C.J., Bando T., et al. Inhibition of reperfusion injury by human thioredoxin (adult T-cell leukemia-derived factor) in canine lung transplantation. J Thorac Cardiovasc Surg 1994;108:913-921.[Abstract/Free Full Text]
-
Wada H., Liu C.J., Hirata T., Bando T., Kosaka S. Effective 30-hour preservation of canine lungs with modified ET-Kyoto solution. Ann Thorac Surg 1996;61:1099-1105.[Abstract/Free Full Text]
-
Bando T., Albes J.M., Nüsse T., et al. Comparison of Euro-Collins solutions, low-potassium dextran solution containing glucose, and ET-Kyoto solution for lung preservation in an extracorporeal rat lung perfusion model. Eur Surg Res 1998;30:297-304.[Medline]
-
Kaukel E., Hilz H. Permeation of dibutyryl cAMP into HeLa cells and its conversion to monobutyryl cAMP. Biochem Biophys Res Commun 1972;46:1011-1018.[Medline]
-
Pinsky D.J., Oz M.C., Koga S., et al. Cardiac preservation is enhanced in a heterotopic rat transplant model by supplementing the nitric oxide pathway. J Clin Invest 1994;93:2291-2297.
-
Nakamura T., Hirata T., Fukuse T., Ueda M., Hitomi S., Wada H. Dibutyryl cyclic adenosine monophosphate attenuates lung injury caused by cold preservation and ischemia-reperfusion. J Thorac Cardiovasc Surg 1997;114:635-642.[Abstract/Free Full Text]
-
Naka Y., Chowdhury N.C., Liao H., et al. Enhanced preservation of orthotopically transplanted rat lungs by nitroglycerin but hydralazine. Circ Res 1995;76:900-906.[Abstract/Free Full Text]
-
Gaston B., Drazen J.M., Loscalzo J., Stamler J.S. The biology of nitrogen oxides in the airways. Am J Crit Care Med 1994;149:538-551.[Abstract]
Accepted for publication August 3, 1999.
Related Article
-
- David J. Pinsky and Yoshifumi Naka
Ann. Thorac. Surg. 2000 69: 891-892.
[Extract]
[Full Text]
[PDF]
This article has been cited by other articles:
|
|
|
|
 
T. Wittwer, J. M. Albes, A. Fehrenbach, T. Pech, U. F. W. Franke, J. Richter, and T. Wahlers
Experimental lung preservation with Perfadex: Effect of the NO-donor nitroglycerin on postischemic outcome
J. Thorac. Cardiovasc. Surg.,
June 1, 2003;
125(6):
1208 - 1216.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
