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Ann Thorac Surg 1995;60:646-650
© 1995 The Society of Thoracic Surgeons
Laboratoire de Chirurgie Expérimentale and Centre National de Recherche Scientifique, Unité de Recherche Associée 1159, Centre Chirurgical Marie Lannelongue, Paris-Sud University, Le Plessis Robinson, France
Accepted for publication April 15, 1995.
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Abstract |
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Methods. Using an isolated rabbit lung preparation perfused with whole blood, we studied the effects of cold ischemia followed by a warm reperfusion on pulmonary vascular responses to reproduce experimentally the conditions encountered during lung transplantation.
Results. Pulmonary vascular responses to acetylcholine were rapidly altered by warm ischemia (relaxation of 7% versus 60% in controls). Conversely, relaxation was maintained even after a prolonged cold ischemic storage (maximal relaxation of 57% at 48 hours). Warm reperfusion in ischemic lungs induced major alteration of endothelium-dependent relaxation (maximal relaxation of 13% at 4 hours). The addition of L-arginine or pentoxifylline during reperfusion prevented the pulmonary endothelial alteration resulting from warm reperfusion.
Conclusion. These data suggest that treatments aimed at maintaining intact functional endothelium reduce ischemia-reperfusion injury in transplanted lungs.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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In addition, during reperfusion, the lung perfusate was supplemented with either L-arginine (L-arg), a precursor of the vascular relaxant cascade [2], or pentoxifylline (PTX) an agent known to reduce lung neutrophil sequestration, which has been demonstrated to play an important role in the IR syndrome [3, 4].
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Protocols
In group 1 (control; n = 30), after median sternotomy, the heart and lung were removed immediately and segments of PA were dissected free of surrounding tissue and prepared for in vitro studies.
In group 2 (warm ischemia; n = 20), immediately after removal, with lungs inflated at end-tidal volume, the heart-lung block was immersed in normal saline solution and stored at 37°C. After 1 to 8 hours of ischemia, segments of PA were collected for organ bath studies.
In group 3 (cold ischemia; n = 20), after the chest was opened and the thymus resected, the pericardium was opened and the ascending aorta, main PA, and right PA were mobilized and encircled by ligature at their origin. The PA was cannulated with a soft silicone tube (internal diameter, 3 mm) through the right ventricular outflow tract. The right main PA and ascending aorta were ligated. Subsequent to ligation of the PA trunk around the cannula, lungs were flushed with a cold crystalloid solution of Ringer's lactate (60 mL/kg) at 10°C. Both pleurae were then opened and the distal esophagus and descending aorta were transected at the diaphragm. The trachea and cervical esophagus were then divided together proximal to the tracheotomy and lung removal was completed with posterior mediastinal dissection. Lungs were continuously ventilated throughout the procedure. At end-tidal volume the trachea was clamped before storage (in normal saline solution at 10°C) for an ischemic period ranging from 4 to 48 hours.
Groups 4 through 6 had cold ischemia and reperfusion (group 4, cold ischemia and simple reperfusion, n = 15; group 5, cold ischemia and L-arg–supplemented reperfusion, n = 10; group 6, cold ischemia and PTX-supplemented reperfusion, n = 10). After exposure and cannulation of the abdominal aorta, 75 mL of blood was collected and used during the reperfusion step. The PA cannulation and perfusion were as described above. A drainage tube was placed and secured with a pursestring suture at the apex of the left ventricle. The heart-lung block and blood reperfusate were kept at 10°C for 5 hours before reperfusion. The heart-lung block was then mounted in a thermostated chamber at 37°C. Lungs were ventilated with a gas mixture (O2, 20%; CO2 and N2, 75%) at 25 breaths per minute, with a tidal volume of 30 mL and 0.5 cm H2O end-expiratory pressure. Reperfusion was initiated with a peristaltic pump (Ismatec; Bioblock, Strasbourg, France), and perfusion flow rate was adjusted to maintain pulmonary perfusion pressure less than 20 mm Hg. In group 5, at the onset of reperfusion L-arg (20 mg/kg body weight) was added to the perfusate. Then during reperfusion repeated doses of L-arg (10 mg • kg-1 • h-1) or normal saline solution were administered. In group 6, PTX (50 mg) was added to the reservoir before the initiation of reperfusion. At the completion of reperfusion, PA segments were dissected and prepared for pharmacologic assessment.
Organ Bath Studies on Pulmonary Artery Rings
Pulmonary artery segments from right and left lungs were prepared as follows: short segments (1.5 cm) were dissected and gently cleaned of fat and connective tissue. The vessel rings (outer diameter, 4 mm; length, 3 mm) were set up with an isometric force transducer and a fixed wire support in a 10-mL isolated organ chamber containing Krebs solution at 37°C gassed with 95% O2 and 5% CO2. Composition of the Krebs-Henseleit solution in the organ bath was (in millimoles per liter): NaCl, 118; KCl, 4.7; CaCl2, 1.5; NaHCO3, 25; MgSO4, 1.1; KH2PO4, 1.2; and glucose, 5.6. The preparations were washed every 15 minutes by exchanging Krebs solution in the bath. Isometric tension was continuously recorded on a physiograph (Kipp and Zonen). During the equilibration period (90 minutes), resting force was maintained at 1.5 g. Subsequently a cumulative concentration contractile response curve to phenylephrine (0.005 to 30 µmol/L final concentration), a selective 
1 contractile agonist, was produced. Acetylcholine was then added cumulatively (0.1 to 100 µmol/L final concentration) to produce endothelium-dependent relaxation. At maximal relaxation with ACh, sodium nitroprusside (10-5 final concentration) was added to evaluate the endothelium-independent vascular smooth muscle relaxation.
Drugs
L-Arginine hydrochloride, acetylcholine hydrochloride, and indomethacin were purchased from Sigma Chemical Co, St. Louis, MO. Phenylephrine and sodium nitroprussiate were provided by Boehringer Ingelheim, Paris, France, and Hoffmann-LaRoche Ltd, Basel, Switzerland, respectively. Pentoxifylline was obtained from Hoechst Laboratories, Paris, France.
Statistical Analysis
Changes in force were measured from isometric recordings and expressed in grams. The maximal responses to PE or ACh (Emax values) and the concentration of agonist yielding 50% of maximal response (EC50 values) were interpolated from the individual concentration response curves. These latter values were transformed to pD2 values (negative logarithm of EC50 values). Relaxation was expressed as the percent of decrease in tension of the PE-elicited contraction. Data are presented as means ± standard error of the mean. Statistical analysis was performed using two-way analysis of variance and the post hoc Newman-Keuls test for multiple comparison when the overall F was significant. A p value less than 0.05 was considered to be statistically significant.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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), the relaxant response to ACh was reduced (39% ± 3%) when compared with values (60% ± 2%) observed in freshly collected vessels. After 2 hours of warm ischemia only 29% ± 2% relaxation to ACh was obtained, whereas at 4 hours responses were strongly attenuated (7% ± 3%). Similarly, contractile responses to PE were significantly diminished at different times (Fig 2). A contractile response was observed at 2 hours (1.9 ± 0.2 g) but at 4 hours the response was reduced to 0.6 ± 0.1 g and at 8 hours of warm ischemia both contractile and relaxant responses of pulmonary vessels were abolished.
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) were not significantly different from those observed at 24 hours (Emax, 49% ± 2% versus 45% ± 3%; pD2, 7.24 ± 0.14 versus 7.23 ± 0.21) and sodium nitroprusside further relaxed pulmonary artery rings. Responses to PE (Fig 4) did not decrease or were slightly increased with time (Emax, 2.7 ± 0.2 g at 4 hours versus 3.5 ± 0.2 g at 48 hours).
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show the vascular relaxation to ACh obtained in pulmonary vessels derived from reperfused lungs. In the control group (simple reperfusion, group 4), the relaxant response was maintained at 3 hours and significantly reduced at 4 hours. Administration of L-arg during reperfusion attenuated the deleterious effects of prolonged reperfusion (4 hours) on pulmonary vascular bed. Similarly, after administration of PTX during reperfusion (4 hours) relaxant responses of PA rings were not significantly different from controls (group 4, 3 hours). These results demonstrate the protective action of L-arg and PTX against lung IR injury.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The isolated blood perfused lung preparation is a widely used model. This model was selected in accordance with the expressed necessity for standardization of experimental lung reperfusion models [5]. Early postpreservation lung function can be evaluated by measuring lung gas exchanges [6], but more specific points were addressed in this study. The present work was undertaken to study pulmonary vascular damage by focusing attention on vascular smooth muscle reactivity and determining Emax and pD2 for contractile and relaxant responses. Although the results obtained from conduit pulmonary arteries may or may not reflect a parallel alteration of lung microcirculation, a recent report demonstrated that responses of isolated conduit pulmonary artery rings were closely related to an increased pulmonary vascular resistance [7].
The adverse effect of warm ischemia in lungs has been recognized for some time [8]. Results from the present study provide further support that endothelial damage (relaxation) was detected before smooth muscle dysfunction (contraction) was observed. Therefore, endothelium-dependent relaxation appeared to be a sensitive index of lung vascular alteration.
Even though pulmonary endothelium seems to require little oxidative phosphorylation for NO production in the rat [9], warm ischemia nevertheless depleted endothelial metabolic reserve as agonist-induced vasodilatation was completely abolished at 4 hours. Although lungs exhibit a marked sensitivity to normothermic ischemia, adenosine triphosphate catabolism in warm ischemic lung tissue has been, until recently, poorly investigated. DeLeyn and associates [10], measuring adenosine triphosphate levels in normal and ischemic deflated lung grafts stored at 37°C, showed that the normal lung metabolism entails a highly energized state and described precocious adenosine triphosphate degradation during warm ischemia. However, the same group [11] reported that, in lung grafts stored inflated at end-tidal volume with room air (as in the present study), adenosine triphosphate degradation and lactate increase were inhibited, thereby supporting the notion that lung inflation, by itself, could increase tolerable ischemic time.
Currently employed preservation techniques rely on hypothermia both at organ retrieval (topical cooling or PA perfusion) and storage. Hypothermia at 10°C was employed in the present study because previous works suggested that this condition was optimal [12]. Moreover, a recent report demonstrated that flushing with solution at lower temperature may have adverse effects on lung preservation [13].
Ischemia followed by reperfusion, which is inherent to the conditions prevailing in lung transplantation, triggers numerous pathophysiologic processes including endothelial damage and eventually vascular dysfunction. Endothelial homeostasis is associated with the synthesis and release of endothelium-dependent relaxing factor/NO, which is a metabolically active process that is rapidly abrogated by the ill effects of warm ischemia. Reperfusion experiments from the present report clearly demonstrate that reperfusion had a detrimental action in lungs that had normal contractile and relaxant responses after cold ischemia. Endothelium-dependent relaxation was affected, whereas vascular contractile responses were less altered. Endothelial dysfunction associated with ex vivo rabbit lung reperfusion may derive from decreased NO synthesis or increased NO degradation. We therefore investigated whether NO decreased synthesis could be prevented by providing exogenous L-arg during reperfusion. Because L-arg has been shown to be the natural precursor of NO [2], NO decreased synthesis may be impaired by depletion of endogenous L-arg or possibly by blocking the recycling of L-citrulline to L-arg [14]. This depletion can be a consequence of both cellular efflux and metabolic utilization of endothelium-derived relaxing factor/NO [15]. Results of the present study indirectly indicate that the L-arg endogenous endothelial reserve was adequate at onset of reperfusion. However, after 4 hours of reperfusion an alteration of the L-arg/NO pathway was unmasked. Moreover, the data from lungs reperfused with L-arg, in which a normal relaxant response was observed, support the notion that a latent alteration of the endothelial function was present at the end of the ischemic period and was reversed by supplementing the perfusate with a precursor of the relaxant cascade. This observation eliminates two possible mechanisms, namely, an alteration of endothelial receptors involved in vasorelaxation and an impaired transmembrane transduction mechanism for these receptors [16]. In addition, two other mechanisms, (1) the increased formation of an inhibitor of the L-arg pathway such as asymmetric dimethyl arginine [17] and (2) the production of an endothelium-derived contracting factor known to impair relaxation to ACh even in the presence of a functionally intact L-arg/NO pathway [18], may not explain the endothelium dysfunction.
Recent investigations have shown that IR events invoke free radical mechanisms [19]. These studies showed that addition of free radical scavenger to preservation solutions may reduce the effects of IR injury. Free radicals produced during IR could induce an increased breakdown of endothelium-derived relaxing factor/NO, which may explain the depressed endothelium-dependent relaxation observed after reperfusion of ischemic lungs. Conversely, the normal relaxant responses observed after reperfusion using L-arg–supplemented perfusate may reflect the beneficial effect of facilitating endothelial cells' NO production because NO also has a cytoprotective effect [20]. Recent evidence suggests that NO may reduce leukocyte–endothelial cell interaction [21]. Neutrophil activation and sequestration are key factors to endothelial microvascular damage that can be induced by free radicals. Factors involved in neutrophil activation include changes in temperature and circulation that occur after cold preservation and during warm lung reperfusion. In fact, neutrophil activation and sequestration could be crucial factors for the altered endothelial function we observed after 3 hours of reperfusion. An augmented myeloperoxidase activity, a specific marker for neutrophils, has been reported in rabbit lung tissue 2 hours after reperfusion of pulmonary artery [22]. That study also demonstrated tumor necrosis factor-alpha generation during reperfusion. Results from another group showed that PTX was able to attenuate the leukocyte-mediated injury induced by tumor necrosis factor-alpha [23]. The addition of PTX to the perfusate in the present study preserved the endothelium-dependent relaxation in pulmonary vessels. Moreover, our previous study in isolated, blood-perfused rat lungs demonstrated a protective action of PTX against IR microvascular injury [4]. The mechanisms underlying this beneficial effect of PTX are multiple. Among the recognized actions of PTX is the ability to reduce neutrophil adhesion to the endothelium and the subsequent sequestration of these cells in lungs. Although white blood cell counts in perfusate have not been presented in this study, these results have been previously documented in rat lungs [4]. These data suggest that the attenuated relaxant response observed with pulmonary artery rings after reperfusion may reflect an alteration of the delicate endothelium lining induced by oxygen free radicals produced by neutrophils trapped in the pulmonary circulation. Conversely, the unaltered relaxant responses observed in the PTX-treated group could indirectly reflect reduced neutrophil sequestration in lung tissue.
In conclusion, considerable strides have been made in recent years in experimental research to optimize conditions for harvesting, storage, and reperfusion of lung grafts. Moreover, the need for additional research to develop procedures that allow extended preservation time has been recently emphasized. In parallel a growing knowledge has accumulated on the mechanisms underlying lung alterations during ischemia. Recently the notion that reperfusion injury may increase or unmask a latent lung ischemic injury and its associated endothelial damage has emerged. As demonstrated in this study, metabolic support of NO synthesis with L-arg and maintenance of NO availability with PTX preserve the endothelium functional integrity, which plays a key role in the initiation of IR injury.
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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