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Cold ischemia and reperfusion each produce pulmonary vasomotor dysfunction in the transplanted lung
Cold ischemia and reperfusion each produce pulmonary vasomotor dysfunction in the transplanted lung Pulmonary vascular resistance is significantly increased in the transplanted lung. We hypothesized that the ischemic or reperfusion injuries incurred by the transplanted lung may produce pulmonary vasomotor dysfunction, which in turn may produce increased pulmonary vascular resistance. In a dog model of autologous lung transplantation, the purpose of this study was to examine the following mechanisms of pulmonary vasomotor control and to relate each of them to cold ischemia and to reperfusion: (1) endothelium-dependent cyclic guanosine monophosphate-mediated vasorelaxation (response to acetylcholine 10-6 moljL), (2) endothelium-independent cyclic guanosine monophosphate-mediated vasorelaxation (response to sodium nitroprusside 10-6 moIjL), and .a-adrenergic cyclic adenosine monophosphate-mediated vasorelaxation (response to isoproterenol 10-6 mol/L), Autologous right lung transplantation was performed in five dogs. At each of three times, two third-order pulmonary arteries were dissected from each transplanted lung and studied: control (immediately after harvest), cold ischemia (3 hours in 4° C saline solution), and cold ischemia plus reperfusion (1 hour after lung reimplantation). The vasorelaxing effects of acetylcholine, sodium nitroprusside, and isoproterenol were studied in isolated pulmonary arterial rings, suspended on fine wire tensiometers in individual organ chambers. Statistical analysis was by analysis of variance. Results demonstrated significant dysfunction of .a-adrenergic cyclic adenosine monophosphate-mediated relaxation after cold ischemia alone, and this dysfunction was exacerbated by reperfusion. Endothelium-dependent cyclic guanosine monophosphate-mediated relaxation was not impaired by cold ischemia alone but was significantly impaired by reperfusion. Endothelium-independent cyclic guanosine monophosphate-mediated relaxation was not impaired by cold ischemia or reperfusion. We conclude that cold ischemia and reperfusion each produce different patterns of pulmonary vasomotor dysfunction. Cumulatively, such dysfunction may contribute to increased pulmonary vascular resistance in the transplanted lung. (J THoRAe CARDIOV ASC SURG 1993;106:1213-7)
David A. Fullerton, MD (by invitation), Max B. Mitchell, MD (by invitation), Robert C. McIntyre, Jr., MD (by invitation), Anirban Banerjee, PhD (by invitation), David N. Campbell, MD (by invitation), Alden H. Harken, MD, and Frederick L. Grover, MD, Denver, Colo.
h e processesoflung transplantation require the transplanted lung to sustain both ischemic and reperfusion injuries. Clinically, the culmination of these injuries is From the University of Colorado Health Sciences Center, Denver, Colo. Read at the Seventy-Third Annual Meeting of The American Association for Thoracic Surgery, Chicago, Ill., April 25-28, 1993. Address for reprints: David A. Fullerton, MD, Cardiothoracic Surgery, C-310. University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. Copyright
1993 by Mosby-Year Book, Inc.
0022-5223/93 $1.00
+ .10
12/6/50046
referred to as the "reimplantation response," which is characterized by increased pulmonary capillary permeability, pulmonary edema, and respiratory insufficiency.1 However, an important pathophysiologic feature of lung transplantation that has been overshadowed by the "reimplantation response" is an acute rise in pulmonary vascular resistance (PVR) in the transplanted lung. 2- 12 This increased PVR is largely due to avid constriction of the pulmonary vascular smooth muscle, even in the absence of hypoxia. 10, 12 Furthermore, it has been noted to persist well beyond the acute "reimplantation response." In fact, PVR may remain chronically elevated
12 13
The Journal of Thoracic and Cardiovascular Surgery December 1993
I 2 I 4 Fullerton et al.
Cold Ischemia
Explant Right Lung
t
Cold Ischemia + Reperfusion
Reperfusion X 1 Hr
Reimplant Right Lung
Heparin (400 U/kg)
PGE t (10 Wkg) Modified Euro-Collins WC, 30crlkg)
Fig. 1. Experimental protocol. Pulmonary vascular control mechanisms were studied inisolated pulmonary artery rings, taken from each lung at each ofthreepoints intime: control (immediately afterharvest), cold ischemia (after 3 hours in 4° C saline solution), and reperfusion (after 1 hour of reperfusion). in laboratory animal models of lung transplantation." PVR remains elevated in canine autologous lung transplantation and is therefore not attributable to rejection.9• 12 We hypothesized that the obligatory ischemic or reperfusion injuries incurred by the transplanted lung may produce pulmonary vasomotordysfunction.In turn, this pulmonary vasomotordysfunction may contribute to the increased PVR in the transplanted lung.Therefore the first purpose of this study was to examine the following pulmonary vascular smooth muscle control mechanisms in a canine model of autologous lung transplantation: (I) endothelium-dependentcyclicguanosinemonophosphate (cGMP)-mediated relaxation (response to acetylcholine), (2) endothelium-independent cGMP-mediated relaxation (response to sodium nitroprusside), and (3) ,6-adrenergic cyclicadenosine monophosphate (cAMP)mediated relaxation (responseto isoproterenol). Our second purpose was to relate each of these mechanisms of pulmonary vascular smooth muscle control to (a) cold ischemia and (b) reperfusion. Methods Surgical protocol. All animals receivedhumane 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 Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23,revised 1985). After endotracheal intubation and mechanical ventilation, five mongrel dogs (25 to 35 kg) underwent right autologous single lung transplantation through a right thoracotomy incision under pentobarbital and halothane anesthesia. Blood pressure was continuously monitored with an intraarterial catheter and remained greater than
90 mm Hg throughout the experimental protocol. After systemicheparinization (heparin 250 U/kg), prostaglandin E 1 (l 0 ~gjkg) was infuseddistally into the right main pulmonary artery. Then, with the lung inflated and the pulmonary veinsventedinto the right sideof the chest,the right main pulmonary artery was occludedproximallyas modified Euro-Collins solution (4 C, 30 mljkg) was infuseddistally into the right pulmonary artery. With the aid of a balloon bronchial occluder, the right lung was then explanted, kept inflated by clamping the right mainstem bronchus, and stored in cold saline (4 0 C) for 3 hours. Thereafter the right lung wassurgicallyreimplanted by means of a running monofilamentsuture technique for the bronchial, the arterial, and the venous anastomoses. The pulmonary arterial clamp was removed and the lung reperfused and ventilated for I hour. Pulmonary artery ring preparation. With the use of a dissectingmicroscope, twothird-order pulmonaryarteries (approximately I mm diameter) were dissectedfrom each lung and studied at each of three pointsin time (Fig. I): control (immediately after harvest), at the end of cold ischemia (after 3 hours in 4 0 C salinesolution),and at the end of cold ischemia plus reperfusion (I hour after lung reimplantation). With the helpof a dissectingmicroscope, the surrounding tissue was dissectedfrom the pulmonary arteries. The pulmonary arteries were then each cut into rings, each ring being 3 to 4 mm wide. Great care was taken during this processto avoid endothelialinjury. The pulmonary arterial rings were suspended on fine wire tensiometers in individual 10 cc organ chambers. The organ chambers were surrounded by water jackets and continually warmed to 370 C. Ring tension was determined by use of a force-displacement transducer (Grass FT03, Grass Instruments Co., Quincy, Mass.) attached to each tensiometer apparatus. Ring tension was thereby recorded on a Grass four-channel recorder. The organ chambers were filled with Earle's balanced salt SOlution 0
The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 6
Fullerton et ai.
12 15
100 80 % Relaxation
*
60 40 20 0
* p
Cold Ischemia
Reperfusion
n=10 pulmonary artery rings in each group
Fig. 2. Endothelium-dependent cGMP-mediatedvasorelaxation. The relaxation by acetylcholine wasnot impaired by cold ischemia alone but was dysfunctional after reperfusion. Valuesare percentageof relaxationof phenylephrine-induced ring tension by acetylcholine. Valueare mean ± standard deviation. *p < 0.05 comparedwithcontrol. and continuously bubbled with gas comprised of 21% oxygen, 5% carbon dioxide, and balanced nitrogen. Earle's balanced salt solution is a standard physiologic salt solution and contains CaCI 2 1.80 mmol/L, MgS0 4 (anhydrous) 0.83 mmol/L, KCI 5.36 mrnol/L, NaCI 116.34 mmol/L, NaP0 4 0.40 mrnol/L (dibasic), D-glucose 5.50 mmol/L, NaHC0 3 19.04 mmol/L, and phenol red Na 0.03 mmol/L (as pH indicator). The optimal resting tension of pulmonary arterial rings of this size was determined to be 750 mg in a separate series of experiments. Once the rings had been allowed to reach a steady state (approximately 90 minutes) at a baseline tension of 750 mg, the vasorelaxing effects to acetylcholine 10-6 mol/L, sodium nitroprusside 10-6 mol/L, and isoproterenol 10-6 mol/L were determined in each ring in a random order. The vasorelaxing effects of each agent were studied after preconstriction of the pulmonary arterial ring with phenylephrine 10-6 moljL. Once ring tension reached a steady state in response to phenylephrine, a given vasorelaxing agent was added to the organ chamber. After each agent was tested, the organ chambers were flushed several times and the rings allowed to again reach a steady state before being preconstricted once again with phenylephrine to test the next vasorelaxing agent. Ten pulmonary arterial rings (two rings from each lung) were studied at each time of data collection. Data are presented as the percent relaxation of phenylephrineinduced ring tension produced by the given vasorelaxing agent. Values are expressed as mean ± one standard deviation. Statistical analysis used analysis of variance (Scheffe's F test). A value of p < 0.05 was considered statistically significant. Results Significant pulmonary vasomotor dysfunction was produced by the processes of transplantation. In control pul-
monary arterial rings, acetylcholine 10-6 mol/L produced 95% ± 5% relaxation, sodium nitroprusside 10-6 mol/Lproduced 98% ± 4% relaxation, and isoproterenol 10-6 mol/L produced 92% ± 8% relaxation. At the end of cold ischemia, neither endothelium-dependent (response to acetylcholine) (Fig. 2) nor endothelium-independent cGMP-mediated (response to sodium nitroprusside) (Fig. 3) pulmonary vascular smooth muscle relaxation were dysfunctional. At the end of cold ischemia, however, iJ-adrenergic cAMP-mediated vasorelaxation (response to isoproterenol) was significantly impaired inasmuch as isoproterenol 10-6 mol/L produced only 62% ± 8% relaxation (p < 0.05) (Fig. 4). After reperfusion, however, there was significant dysfunction of endothelium-dependent cGMP-mediated pulmonary vascular smooth muscle relaxation; acetylcholine produced only 43% ± 10% relaxation (p < 0.05) (Fig. 2). In addition, the dysfunction of iJ-adrenergic cAMP-mediated vasorelaxation was significantly exacerbated by reperfusion inasmuch as isoproterenol produced only 37% ± 6% relaxation (p < 0.05) (Fig. 4). Of interest, endothelium-independent cGMP-mediated pulmonary vascular smooth muscle relaxation (response to sodium nitroprusside) was not impaired by reperfusion (Fig. 3). Discussion The principal intracellular mechanisms of pulmonary vascular smooth muscle relaxation are ultimately mediated through either cGMP or cAMP.'3 Pulmonary vascular smooth muscle adenylate cyclase produces cAMP in response to agents such as isoproterenol, which activate iJ-adrenergic receptors on the vascular smooth muscle cell membrane. The increased cAMP in turn effects pulmonary vasorelaxation.l" On the other hand, cGMP-mediated relaxation may be
The Journal of Thoracic and
12 16
Fullerton et al.
Cardiovascular Surgery December 1993
100
80 % Relaxation
60 40
20 0
Cold Ischemia n=10 pulmonary artery rings in each group
Fig. 3. Endothelium-independentcGMP-mediated vasorelaxation. The relaxationby sodium nitroprussidewas not impaired by either cold ischemiaalone or reperfusion. Valuesare percentageof relaxation of phenylephrine-induced ring tension by sodium nitroprusside. Values are mean ± standard deviation.
100
*
80 % Relaxation
60 40
*
20 0
* p
Cold Ischemia
Reperfusion
n=10 pulmonary artery rings in each group
Fig. 4. iJ-Adrenergic cAMP-mediated vasorelaxation. The relaxation by isoproterenol was significantly impaired by coldischemiaalone and exacerbated by reperfusion. Valuesare percentageof relaxationof phenylephrine-induced ring tension by isoproterenol. Values are mean ± standard deviation. *p < 0.05 compared to control. either endothelium dependent or endothelium independent.l ' Agents such as acetylcholine produce pulmonary vascular smooth muscle relaxation by binding to muscarinic receptors on pulmonary vascular endothelium.P In response, the pulmonary vascular endothelium releases endothelium-derived relaxing factor, which is thought to be nitric oxide." Endothelium-derived relaxing factor in turn activates guanylate cyclase within the pulmonary vascular smooth muscle cell. IS Activated guanylate cyclase then produces cGMP, which effects pulmonary vascular smooth muscle relaxation. On the other hand, sodium nitroprusside is a functional analog of endotheliumderived relaxing factor and directly activates pulmonary vascular smooth muscle guanylate cyclase to produce cGMP independently of the endothelium.P Pulmonary vascular tone is in large part determined by the mechanistic balance of pulmonary vascular smooth muscle constriction and relaxation. If the mechanisms of relaxation are dysfunctional, this balance is shifted toward a net constriction. In addition, dysfunction of the
mechanisms of relaxation allow for an exaggerated response to vasoconstricting agents such as hypoxia. 17. 18 Lung transplantation obligates the transplanted lung to undergo the injurious processes of cold ischemia and reperfusion. The results of the present study demonstrate that these processes culminate in significant vasomotor dysfunction in the transplanted lung and further demonstrate that cold ischemia and reperfusion each produce different patterns of injury. Laboratory animal models of lung transplantation have shown PVR to rise acutely and to remain elevated in the transplanted lung. 2- 12 We hypothesized that this increased PVR may derive from impaired pulmonary vasomotor control mechanisms. The present study suggests significant dysfunction in several principal mechanisms of pulmonary vascular smooth muscle relaxation. {3-Adrenergic cAMP-mediated relaxation (response to isoproterenol) was impaired by cold ischemia alone, and this impairment was significantly exacerbated by reperfusion. Endothelium-dependent cGMP-mediated relaxation (response to acetylcholine)
The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 6
was not impaired by cold ischemia alone but was significantly impaired after reperfusion. Of interest, endothelium-independent cGMP-mediated relaxation (response to sodium nitroprusside) was not impaired by either cold ischemia alone or reperfusion. We conclude that this dysfunction of several principal mechanisms of pulmonary vascular smooth muscle relaxation implies a mechanistic imbalance of pulmonary vascular smooth muscle constriction and relaxation in the transplanted lung. This mechanistic imbalance may in turn contribute to the increased PVR found in the setting of lung transplantation. REFERENCES I. Stevens JH, Raffin TA, Baldwin J'C, The status of transplantation of the human lung. Surg Gynecol Obstet 1989; 169:179-85. 2. Corris PA, Odom N J, Jackson G, McGregor CGA. Reimplantation injury after lung transplantation in a rat model. J Heart Transplant 1987;6:234-7. 3. Jones MT, Hsieh C, Yoshikawa K, Patterson GA, Cooper JD. A new model for assessment of lung preservation. J THORAC CARDIOVASC SURG 1988;96:608-14. 4. Hachida M, Morton DL. A new solution (UCLA formula) for lung preservation. J THORAC CARDIOVASC SURG 1989;97:513-20. 5. Hachida M, Morton DL. Lung function after prolonged lung preservation. J THORAC CARDIOVASC SURG 1989;97: 911-9. 6. Wang L-S, Yoshikawa K, Miyoshi S, et al. The effect of ischemic time and temperature on lung preservation in a simple ex vivo rabbit model used for functional assessment. J THORAC CARDIOVASC SURG 1989;98:333-42. 7. Downing TP, Sadeghi AM, Baumgartner W A, et al. Acute physiological changes following heart-lung allotransplantation in dogs. Ann Thorac Surg 1984;37:479-83. 8. Paull DE, Keagy BA, Kron EJ, Wilcox BR. Reperfusion injury in the lung preserved for 24 hours. Ann Thorac Surg 1989;47:187-92. 9. Halasz NA, Catanzaro A, Trummer MJ, et al. Transplantation of the lung: correlation of physiologic, immunologic, and histologic findings. J THORAC CARDIOVASC SURG 1973;66:581-7. 10. Lynch MJ, Grum CM, Gallagher KP, Bolling SF, Deeb GM, Morganroth ML. Xanthine oxidase inhibition attenuates ischemic-reperfusion lung injury. J Surg Res 1988; 44:538-44. II. Hall TS, Breda MA, Baumgartner W A, et al. The role of leukocyte depletion in reducing injury to the lung after hypothermic ischemia. Curr Surg 1987;44:137-9. 12. Detterbeck FC, Keagy BA, Paull DE, Wilcox BR. Oxygen free radical scavengers decrease reperfusion injury in lung transplantation. Ann Thorac Surg 1990;50:204-10. 13. Epstein FH. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:27-36.
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14. Fishman AP. Pulmonary circulation. In: Fishman AP. Handbook of physiology: the respiratory system. vol. I. Bethesda: American Physiological Society, 1985:93-165. 15. Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 1989;65:1-21. 16. Palmer RMJ, Ferrige AG, Monacada S. Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature 1987;327:524-6. 17. Rodman DM, Yamaguchi T, Hasunuma K, O'Brien RF, McMurtry IF. Effects of hypoxia on endothelium-dependent relaxation of rat pulmonary artery. Am J Physiol I 990;258:L207-14. 18. Brashers VL, Peach MJ, Rose CEo Augmentation of hypoxic pulmonary vasoconstriction in the isolated perfused rat lung by in vitro antagonists of endothelium-dependent relaxation. J Clin Invest 1988;82:1495-1502. Discussion Dr. Severi P. Mattila (Helsinki, Finland). The contemporary preservation method that we use, Euro-Collins solution, is good enough for clinical purposes but is not entirely harmless. We studied the immediate postoperative recovery of the lung after left lung transplantation in pigs. We evaluated the arterial oxygen tension after transplantation-after preservation with Euro-Collins solution and after use of Fluosol DA, an artificial blood. After Euro-Collins preservation, an alveolar-capillary block exists, which lasts some hours after transplantation. After preservation with Fluosol DA, the oxygenation is normal immediately after transplantation. A surface electron microgram of the alveolar-capillary endothelium after Fluosol DA preservation shows that the endothelial cells are pretty well preserved, whereas after preservation with Euro-Collins solution the endothelium is almost gone. Thus there seems to be a morphologic correlation between the reactivity and the alveolar-capillary block that was seen immediately after the transplantation. Did you do any morphologic studies to determine the altered response to various endothelium-dependent agents? Dr. Martin F. McKneally (Toronto, Ontario, Canada). What is the implication in terms of management in the early postoperative period, particularly for the very vasoresponsive patient, such as the patient with pulmonary hypertension patient who receives a lung that has some reperfusion injury? Dr. Fullerton. To answer Dr. McKneally's question first: To effectively relax pulmonary vascular smooth muscle in the transplanted lung, we have found that the GMP mechanism independent of the endothelium was preserved in this process. Nitric oxide is a clinically available agent that could be used effectively, and in fact we have used it. We have not compared nitric oxide with any other agent in particular, but we have had success modulating PVR after lung transplantation with inhalation of nitric oxide, which of course works through this mecharnsm. Dr. Mattila's work is widely known among those of us interested in lung preservation. To answer his question: No, we did not do any morphologic correlations in this study. We were interested strictly in a functional parameter of vasomotor control, but his work is certainly enticing.
David A. Fullerton, MD (by invitation), Max B. Mitchell, MD (by invitation), Robert C. McIntyre, Jr., MD (by invitation), Anirban Banerjee, PhD (by invitation), David N. Campbell, MD (by invitation), Alden H. Harken, MD, and Frederick L. Grover, MD, Denver, Colo.
h e processesoflung transplantation require the transplanted lung to sustain both ischemic and reperfusion injuries. Clinically, the culmination of these injuries is From the University of Colorado Health Sciences Center, Denver, Colo. Read at the Seventy-Third Annual Meeting of The American Association for Thoracic Surgery, Chicago, Ill., April 25-28, 1993. Address for reprints: David A. Fullerton, MD, Cardiothoracic Surgery, C-310. University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. Copyright
1993 by Mosby-Year Book, Inc.
0022-5223/93 $1.00
+ .10
12/6/50046
referred to as the "reimplantation response," which is characterized by increased pulmonary capillary permeability, pulmonary edema, and respiratory insufficiency.1 However, an important pathophysiologic feature of lung transplantation that has been overshadowed by the "reimplantation response" is an acute rise in pulmonary vascular resistance (PVR) in the transplanted lung. 2- 12 This increased PVR is largely due to avid constriction of the pulmonary vascular smooth muscle, even in the absence of hypoxia. 10, 12 Furthermore, it has been noted to persist well beyond the acute "reimplantation response." In fact, PVR may remain chronically elevated
12 13
The Journal of Thoracic and Cardiovascular Surgery December 1993
I 2 I 4 Fullerton et al.
Cold Ischemia
Explant Right Lung
t
Cold Ischemia + Reperfusion
Reperfusion X 1 Hr
Reimplant Right Lung
Heparin (400 U/kg)
PGE t (10 Wkg) Modified Euro-Collins WC, 30crlkg)
Fig. 1. Experimental protocol. Pulmonary vascular control mechanisms were studied inisolated pulmonary artery rings, taken from each lung at each ofthreepoints intime: control (immediately afterharvest), cold ischemia (after 3 hours in 4° C saline solution), and reperfusion (after 1 hour of reperfusion). in laboratory animal models of lung transplantation." PVR remains elevated in canine autologous lung transplantation and is therefore not attributable to rejection.9• 12 We hypothesized that the obligatory ischemic or reperfusion injuries incurred by the transplanted lung may produce pulmonary vasomotordysfunction.In turn, this pulmonary vasomotordysfunction may contribute to the increased PVR in the transplanted lung.Therefore the first purpose of this study was to examine the following pulmonary vascular smooth muscle control mechanisms in a canine model of autologous lung transplantation: (I) endothelium-dependentcyclicguanosinemonophosphate (cGMP)-mediated relaxation (response to acetylcholine), (2) endothelium-independent cGMP-mediated relaxation (response to sodium nitroprusside), and (3) ,6-adrenergic cyclicadenosine monophosphate (cAMP)mediated relaxation (responseto isoproterenol). Our second purpose was to relate each of these mechanisms of pulmonary vascular smooth muscle control to (a) cold ischemia and (b) reperfusion. Methods Surgical protocol. All animals receivedhumane 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 Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23,revised 1985). After endotracheal intubation and mechanical ventilation, five mongrel dogs (25 to 35 kg) underwent right autologous single lung transplantation through a right thoracotomy incision under pentobarbital and halothane anesthesia. Blood pressure was continuously monitored with an intraarterial catheter and remained greater than
90 mm Hg throughout the experimental protocol. After systemicheparinization (heparin 250 U/kg), prostaglandin E 1 (l 0 ~gjkg) was infuseddistally into the right main pulmonary artery. Then, with the lung inflated and the pulmonary veinsventedinto the right sideof the chest,the right main pulmonary artery was occludedproximallyas modified Euro-Collins solution (4 C, 30 mljkg) was infuseddistally into the right pulmonary artery. With the aid of a balloon bronchial occluder, the right lung was then explanted, kept inflated by clamping the right mainstem bronchus, and stored in cold saline (4 0 C) for 3 hours. Thereafter the right lung wassurgicallyreimplanted by means of a running monofilamentsuture technique for the bronchial, the arterial, and the venous anastomoses. The pulmonary arterial clamp was removed and the lung reperfused and ventilated for I hour. Pulmonary artery ring preparation. With the use of a dissectingmicroscope, twothird-order pulmonaryarteries (approximately I mm diameter) were dissectedfrom each lung and studied at each of three pointsin time (Fig. I): control (immediately after harvest), at the end of cold ischemia (after 3 hours in 4 0 C salinesolution),and at the end of cold ischemia plus reperfusion (I hour after lung reimplantation). With the helpof a dissectingmicroscope, the surrounding tissue was dissectedfrom the pulmonary arteries. The pulmonary arteries were then each cut into rings, each ring being 3 to 4 mm wide. Great care was taken during this processto avoid endothelialinjury. The pulmonary arterial rings were suspended on fine wire tensiometers in individual 10 cc organ chambers. The organ chambers were surrounded by water jackets and continually warmed to 370 C. Ring tension was determined by use of a force-displacement transducer (Grass FT03, Grass Instruments Co., Quincy, Mass.) attached to each tensiometer apparatus. Ring tension was thereby recorded on a Grass four-channel recorder. The organ chambers were filled with Earle's balanced salt SOlution 0
The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 6
Fullerton et ai.
12 15
100 80 % Relaxation
*
60 40 20 0
* p
Cold Ischemia
Reperfusion
n=10 pulmonary artery rings in each group
Fig. 2. Endothelium-dependent cGMP-mediatedvasorelaxation. The relaxation by acetylcholine wasnot impaired by cold ischemia alone but was dysfunctional after reperfusion. Valuesare percentageof relaxationof phenylephrine-induced ring tension by acetylcholine. Valueare mean ± standard deviation. *p < 0.05 comparedwithcontrol. and continuously bubbled with gas comprised of 21% oxygen, 5% carbon dioxide, and balanced nitrogen. Earle's balanced salt solution is a standard physiologic salt solution and contains CaCI 2 1.80 mmol/L, MgS0 4 (anhydrous) 0.83 mmol/L, KCI 5.36 mrnol/L, NaCI 116.34 mmol/L, NaP0 4 0.40 mrnol/L (dibasic), D-glucose 5.50 mmol/L, NaHC0 3 19.04 mmol/L, and phenol red Na 0.03 mmol/L (as pH indicator). The optimal resting tension of pulmonary arterial rings of this size was determined to be 750 mg in a separate series of experiments. Once the rings had been allowed to reach a steady state (approximately 90 minutes) at a baseline tension of 750 mg, the vasorelaxing effects to acetylcholine 10-6 mol/L, sodium nitroprusside 10-6 mol/L, and isoproterenol 10-6 mol/L were determined in each ring in a random order. The vasorelaxing effects of each agent were studied after preconstriction of the pulmonary arterial ring with phenylephrine 10-6 moljL. Once ring tension reached a steady state in response to phenylephrine, a given vasorelaxing agent was added to the organ chamber. After each agent was tested, the organ chambers were flushed several times and the rings allowed to again reach a steady state before being preconstricted once again with phenylephrine to test the next vasorelaxing agent. Ten pulmonary arterial rings (two rings from each lung) were studied at each time of data collection. Data are presented as the percent relaxation of phenylephrineinduced ring tension produced by the given vasorelaxing agent. Values are expressed as mean ± one standard deviation. Statistical analysis used analysis of variance (Scheffe's F test). A value of p < 0.05 was considered statistically significant. Results Significant pulmonary vasomotor dysfunction was produced by the processes of transplantation. In control pul-
monary arterial rings, acetylcholine 10-6 mol/L produced 95% ± 5% relaxation, sodium nitroprusside 10-6 mol/Lproduced 98% ± 4% relaxation, and isoproterenol 10-6 mol/L produced 92% ± 8% relaxation. At the end of cold ischemia, neither endothelium-dependent (response to acetylcholine) (Fig. 2) nor endothelium-independent cGMP-mediated (response to sodium nitroprusside) (Fig. 3) pulmonary vascular smooth muscle relaxation were dysfunctional. At the end of cold ischemia, however, iJ-adrenergic cAMP-mediated vasorelaxation (response to isoproterenol) was significantly impaired inasmuch as isoproterenol 10-6 mol/L produced only 62% ± 8% relaxation (p < 0.05) (Fig. 4). After reperfusion, however, there was significant dysfunction of endothelium-dependent cGMP-mediated pulmonary vascular smooth muscle relaxation; acetylcholine produced only 43% ± 10% relaxation (p < 0.05) (Fig. 2). In addition, the dysfunction of iJ-adrenergic cAMP-mediated vasorelaxation was significantly exacerbated by reperfusion inasmuch as isoproterenol produced only 37% ± 6% relaxation (p < 0.05) (Fig. 4). Of interest, endothelium-independent cGMP-mediated pulmonary vascular smooth muscle relaxation (response to sodium nitroprusside) was not impaired by reperfusion (Fig. 3). Discussion The principal intracellular mechanisms of pulmonary vascular smooth muscle relaxation are ultimately mediated through either cGMP or cAMP.'3 Pulmonary vascular smooth muscle adenylate cyclase produces cAMP in response to agents such as isoproterenol, which activate iJ-adrenergic receptors on the vascular smooth muscle cell membrane. The increased cAMP in turn effects pulmonary vasorelaxation.l" On the other hand, cGMP-mediated relaxation may be
The Journal of Thoracic and
12 16
Fullerton et al.
Cardiovascular Surgery December 1993
100
80 % Relaxation
60 40
20 0
Cold Ischemia n=10 pulmonary artery rings in each group
Fig. 3. Endothelium-independentcGMP-mediated vasorelaxation. The relaxationby sodium nitroprussidewas not impaired by either cold ischemiaalone or reperfusion. Valuesare percentageof relaxation of phenylephrine-induced ring tension by sodium nitroprusside. Values are mean ± standard deviation.
100
*
80 % Relaxation
60 40
*
20 0
* p
Cold Ischemia
Reperfusion
n=10 pulmonary artery rings in each group
Fig. 4. iJ-Adrenergic cAMP-mediated vasorelaxation. The relaxation by isoproterenol was significantly impaired by coldischemiaalone and exacerbated by reperfusion. Valuesare percentageof relaxationof phenylephrine-induced ring tension by isoproterenol. Values are mean ± standard deviation. *p < 0.05 compared to control. either endothelium dependent or endothelium independent.l ' Agents such as acetylcholine produce pulmonary vascular smooth muscle relaxation by binding to muscarinic receptors on pulmonary vascular endothelium.P In response, the pulmonary vascular endothelium releases endothelium-derived relaxing factor, which is thought to be nitric oxide." Endothelium-derived relaxing factor in turn activates guanylate cyclase within the pulmonary vascular smooth muscle cell. IS Activated guanylate cyclase then produces cGMP, which effects pulmonary vascular smooth muscle relaxation. On the other hand, sodium nitroprusside is a functional analog of endotheliumderived relaxing factor and directly activates pulmonary vascular smooth muscle guanylate cyclase to produce cGMP independently of the endothelium.P Pulmonary vascular tone is in large part determined by the mechanistic balance of pulmonary vascular smooth muscle constriction and relaxation. If the mechanisms of relaxation are dysfunctional, this balance is shifted toward a net constriction. In addition, dysfunction of the
mechanisms of relaxation allow for an exaggerated response to vasoconstricting agents such as hypoxia. 17. 18 Lung transplantation obligates the transplanted lung to undergo the injurious processes of cold ischemia and reperfusion. The results of the present study demonstrate that these processes culminate in significant vasomotor dysfunction in the transplanted lung and further demonstrate that cold ischemia and reperfusion each produce different patterns of injury. Laboratory animal models of lung transplantation have shown PVR to rise acutely and to remain elevated in the transplanted lung. 2- 12 We hypothesized that this increased PVR may derive from impaired pulmonary vasomotor control mechanisms. The present study suggests significant dysfunction in several principal mechanisms of pulmonary vascular smooth muscle relaxation. {3-Adrenergic cAMP-mediated relaxation (response to isoproterenol) was impaired by cold ischemia alone, and this impairment was significantly exacerbated by reperfusion. Endothelium-dependent cGMP-mediated relaxation (response to acetylcholine)
The Journal of Thoracic and Cardiovascular Surgery Volume 106, Number 6
was not impaired by cold ischemia alone but was significantly impaired after reperfusion. Of interest, endothelium-independent cGMP-mediated relaxation (response to sodium nitroprusside) was not impaired by either cold ischemia alone or reperfusion. We conclude that this dysfunction of several principal mechanisms of pulmonary vascular smooth muscle relaxation implies a mechanistic imbalance of pulmonary vascular smooth muscle constriction and relaxation in the transplanted lung. This mechanistic imbalance may in turn contribute to the increased PVR found in the setting of lung transplantation. REFERENCES I. Stevens JH, Raffin TA, Baldwin J'C, The status of transplantation of the human lung. Surg Gynecol Obstet 1989; 169:179-85. 2. Corris PA, Odom N J, Jackson G, McGregor CGA. Reimplantation injury after lung transplantation in a rat model. J Heart Transplant 1987;6:234-7. 3. Jones MT, Hsieh C, Yoshikawa K, Patterson GA, Cooper JD. A new model for assessment of lung preservation. J THORAC CARDIOVASC SURG 1988;96:608-14. 4. Hachida M, Morton DL. A new solution (UCLA formula) for lung preservation. J THORAC CARDIOVASC SURG 1989;97:513-20. 5. Hachida M, Morton DL. Lung function after prolonged lung preservation. J THORAC CARDIOVASC SURG 1989;97: 911-9. 6. Wang L-S, Yoshikawa K, Miyoshi S, et al. The effect of ischemic time and temperature on lung preservation in a simple ex vivo rabbit model used for functional assessment. J THORAC CARDIOVASC SURG 1989;98:333-42. 7. Downing TP, Sadeghi AM, Baumgartner W A, et al. Acute physiological changes following heart-lung allotransplantation in dogs. Ann Thorac Surg 1984;37:479-83. 8. Paull DE, Keagy BA, Kron EJ, Wilcox BR. Reperfusion injury in the lung preserved for 24 hours. Ann Thorac Surg 1989;47:187-92. 9. Halasz NA, Catanzaro A, Trummer MJ, et al. Transplantation of the lung: correlation of physiologic, immunologic, and histologic findings. J THORAC CARDIOVASC SURG 1973;66:581-7. 10. Lynch MJ, Grum CM, Gallagher KP, Bolling SF, Deeb GM, Morganroth ML. Xanthine oxidase inhibition attenuates ischemic-reperfusion lung injury. J Surg Res 1988; 44:538-44. II. Hall TS, Breda MA, Baumgartner W A, et al. The role of leukocyte depletion in reducing injury to the lung after hypothermic ischemia. Curr Surg 1987;44:137-9. 12. Detterbeck FC, Keagy BA, Paull DE, Wilcox BR. Oxygen free radical scavengers decrease reperfusion injury in lung transplantation. Ann Thorac Surg 1990;50:204-10. 13. Epstein FH. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:27-36.
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14. Fishman AP. Pulmonary circulation. In: Fishman AP. Handbook of physiology: the respiratory system. vol. I. Bethesda: American Physiological Society, 1985:93-165. 15. Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 1989;65:1-21. 16. Palmer RMJ, Ferrige AG, Monacada S. Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature 1987;327:524-6. 17. Rodman DM, Yamaguchi T, Hasunuma K, O'Brien RF, McMurtry IF. Effects of hypoxia on endothelium-dependent relaxation of rat pulmonary artery. Am J Physiol I 990;258:L207-14. 18. Brashers VL, Peach MJ, Rose CEo Augmentation of hypoxic pulmonary vasoconstriction in the isolated perfused rat lung by in vitro antagonists of endothelium-dependent relaxation. J Clin Invest 1988;82:1495-1502. Discussion Dr. Severi P. Mattila (Helsinki, Finland). The contemporary preservation method that we use, Euro-Collins solution, is good enough for clinical purposes but is not entirely harmless. We studied the immediate postoperative recovery of the lung after left lung transplantation in pigs. We evaluated the arterial oxygen tension after transplantation-after preservation with Euro-Collins solution and after use of Fluosol DA, an artificial blood. After Euro-Collins preservation, an alveolar-capillary block exists, which lasts some hours after transplantation. After preservation with Fluosol DA, the oxygenation is normal immediately after transplantation. A surface electron microgram of the alveolar-capillary endothelium after Fluosol DA preservation shows that the endothelial cells are pretty well preserved, whereas after preservation with Euro-Collins solution the endothelium is almost gone. Thus there seems to be a morphologic correlation between the reactivity and the alveolar-capillary block that was seen immediately after the transplantation. Did you do any morphologic studies to determine the altered response to various endothelium-dependent agents? Dr. Martin F. McKneally (Toronto, Ontario, Canada). What is the implication in terms of management in the early postoperative period, particularly for the very vasoresponsive patient, such as the patient with pulmonary hypertension patient who receives a lung that has some reperfusion injury? Dr. Fullerton. To answer Dr. McKneally's question first: To effectively relax pulmonary vascular smooth muscle in the transplanted lung, we have found that the GMP mechanism independent of the endothelium was preserved in this process. Nitric oxide is a clinically available agent that could be used effectively, and in fact we have used it. We have not compared nitric oxide with any other agent in particular, but we have had success modulating PVR after lung transplantation with inhalation of nitric oxide, which of course works through this mecharnsm. Dr. Mattila's work is widely known among those of us interested in lung preservation. To answer his question: No, we did not do any morphologic correlations in this study. We were interested strictly in a functional parameter of vasomotor control, but his work is certainly enticing.