Relative Importance of Prostaglandin/Cyclic Adenosine Monophosphate and Nitric Oxide/Cyclic Guanosine Monophosphate Pathways in Lung Preservation

Transcription

1 Relative Importance of Prostaglandin/Cyclic Adenosine Monophosphate and Nitric Oxide/Cyclic Guanosine Monophosphate Pathways in Lung Preservation Moninder S. Bhabra, FRCS, David N. Hopkinson, MD, Trudi E. Shaw, and Timothy L. Hooper, MD Department of Cardiothoracic Surgery, Wythenshawe Hospital, Manchester, United Kingdom Background. Modulation of vascular tone and platelet and neutrophil function through the prostaglandin/cyclic adenosine monophosphate or nitric oxide/cyclic guanosine monophosphate pathway can benefit lung graft function. The relative importance of these pathways is unclear. Methods. Rat lung grafts (5 per group) were studied in an ex vivo reperfusion model. Group I grafts were pretreated with prostacyclin (20 ng" kg -1. min-1), flushed with cold Euro-Collins solution containing prostacyclin (200 /~g/l), and reperfused immediately for 1 hour. Group II grafts were similarly procured but were stored at 4 C for 6 hours before reperfusion. In group III, no prostacyclin therapy was used; instead, the nitric oxide donor glyceryl trinitrate (0.1 mg/ml) was added to the flush/storage solution, and the grafts were stored for 6 hours. Results. Group II grafts performed poorly compared with those in group I, with substantial deterioration of oxygenation and blood flow and elevation of pulmonary artery pressure, peak airway pressure, and wet to dry weight ratio. In contrast, graft function in group III was similar to that in controls. Conclusions. Lung graft integrity after storage in Euro- Collins solution was better preserved by glyceryl trinitrate than by prostacyclin in this model. (Ann Thorac Surg 1996;62:1494-9) ung and heart-lung transplantation have become L established as successful clinical procedures, but the numbers carried out worldwide are no longer increasing [1]. Prolonged lung preservation has been achieved in laboratory models, but clinically the incidence of early graft dysfunction has restricted available storage periods and prejudiced the distribution of scarce organs and donor-recipient matching. Early graft dysfunction with hypoxia and noncardiogenic pulmonary edema is a manifestation of ischemia-reperfusion injury and is related to inadequate preservation. Single-flush perfusion with modified Euro-Collins (EC) solution followed by hypothermic storage is the most widely used technique of clinical lung graft procurement [2]. Concern about vasoconstriction caused by cold EC solution led to the concomitant use of prostaglandins. Prostaglandin E 1 (PGE1) is popular in North American centers, whereas prostacyclin (PGI2) is favored in Europe [2]. Administered to the donor before flushing or in the flush solution, their vasodilatory effect on the pulmonary vasculature is deemed to improve distribution of the flush and to confer more rapid and even cooling. These prostaglandins also have other potentially Accepted for publication June 10, Address reprint requests to Dr Hooper, Department of Cardiothoracic Surgery, Wythenshawe Hospital, Southmoor Rd, Manchester, United Kingdom M23 9LT. protective actions, such as inhibition of platelet aggregation and leukocyte sequestration and stabilization of membranes [3]. A beneficial effect of the use of prostaglandins in lung preservation has been demonstrated in some animal studies [4-8]. However, others have found little [9, 10] or no [11-14] such benefit. In recent years, considerable interest has been aroused by the identification of nitric oxide (NO) as a mediator of endothelial homeostatic mechanisms. Nitric oxide is a potent vasodilator, reduces platelet aggregation and leukocyte adhesion [15], and quenches oxygen-derived free radicals [16]. The actions of NO are mediated by increasing levels of cyclic guanosine monophosphate in target cells, in contrast to PGE~ and PGI2, which act by increasing intracellular cyclic adenosine monophosphate. Naka and colleagues [17, 18] have shown recently using a rat lung transplant model that lung preservation is enhanced by the addition of an NO donor, nitroglycerin (also known as glyceryl trinitrate, GTN), to the flush solution. In these studies, supplementation with GTN was compared either with no additive or with the vasodilator hydralazine. In the current study, we set out to compare directly the effects of supplementation of the NO/cyclic guanosine monophosphate and PGIz/cyclic adenosine monophosphate pathways on pulmonary function after hypothermic storage using an isolated rat lung reperfusion model by The Society of Thoracic Surgeons /96/$15.00 Published by Elsevier Science Inc PII S (96)

2 Ann Thorac Surg BHABRA ET AL ;62: LUNG PRESERVATION: PROSTACYCLIN VERSUS GTN Material and Methods An ex vivo lung reperfusion model incorporating a support animal was used in these studies; it has been described in detail previously [19, 20]. All animals received humane care in compliance with the United Kingdom Government's Animals (Scientific Procedures) Act of 1986, and all procedures were carried out under terminal general anesthesia. Male Sprague-Dawley rats (Charles River Laboratories, Kent, UK) weighing 350 to 420 g were used as both lung donors and support animals. Anesthesia was induced using halothane inhalation and maintained with 100 mg/kg intraperitoneal pentobarbitone sodium. Animals and isolated grafts were ventilated using a Harvard rodent ventilator (Harvard Apparatus, Kent, UK). Donor Procedure Lung donors were anesthetized, intubated through a tracheostomy, and ventilated with room air. Methylprednisolone 30 mg/kg was given to all donors through the femoral vein 45 minutes before lung explantation. Median sternotomy was performed, and a ligature was passed around the aorta and main pulmonary artery. After administration of heparin, 500 U intravenously, the inferior vena cava was clamped and the left atrial appendage was amputated. A primed, olive-tipped cannula was inserted into the pulmonary artery (PA) trunk through a ventriculotomy and secured with the previously placed ligature. The lungs were flushed through this cannula with filtered, modified EC solution precooled to 4 C and delivered by 25 cm hydrostatic pressure at a volume of 60 ml/kg. The flush time was recorded. The tracheal cannula was then clamped with the lungs fully inflated, and the heart and lungs were excised en bloc. Donors were divided into three groups (n = 5 each). Group I and II donors were pretreated with PGI 2 (Flolan; Wellcome Foundation, London, UK) given through the inferior vena cava at a rate of 20 ng kg i. min 1 for 10 minutes immediately before flushing. Group III donors received no pretreatment. The EC flush solution (Fresenius AG, Bad Homburg, Germany) was modified by the addition of 5 mmol/l magnesium sulfate and was supplemented with PGI2, 200 ~g/l, in groups I and II and GTN (David Bull Laboratories, Warwick, UK), 0.1 mg/ml, in group III. The PGI 2 was prepared, filtered, and added just before use. Group I grafts were reperfused immediately after explantation for baseline data. Group II and III grafts were stored submerged in their respective flush solutions at 4 C for 6 hours before reperfusion. We used PGI 2 pretreatment and addition to the flush/ storage solution in the baseline group as well as in group II because the objective was to evaluate GTN supplementation against this clinically used combination. Methylprednisolone was also administered to donors (in all groups) because of its use in clinical practice. Reperfusion Support animals were anesthetized and ventilated with room air. Through a median sternotomy, mediastinal structures were exposed and the brachiocephalic artery was ligated. For connection to the reperfusion circuit, a cannula was inserted through the right superior vena cava and advanced through the right atrium until its tip lay in the inferior vena cava. Another cannula was passed through the left superior vena cava into the right atrium. After removal of the left lung and postcaval lobe, the grafts were suspended in an insulated chamber and ventilated with room air using 30 cycles/min, 10 ml/kg tidal volume, and 3 cm H20 positive end-expiratory pressure. Deoxygenated blood drawn from the inferior vena cava of the support animals was delivered into the PA of the grafts using hydrostatic pressure equivalent to the physiologic PA pressure of these rats (18 to 20 mm Hg). Graft effluent drained through the opened left atrium and was collected and returned to the right atrium of the support animals by a pump. Thus, the support animals functioned as physiologic deoxygenators. Heat losses were compensated for by waterlagging the circuit tubing and reperfusion chamber, and a warming blanket was used for the support animals. Blood obtained from a separate animal was used to prime the circuit and to replace losses together with 0.9% saline solution. All grafts were reperfused for 60 minutes. Measurements Gas tension and acid/base analysis was performed on blood samples taken from the reperfusion circuit proximal to the graft and from graft effluent every 5 minutes for the first 20 minutes of reperfusion, and every 10 minutes thereafter. This allowed monitoring of the stability of the support animal, consistency of (de)oxygenation of afferent blood, and reoxygenation by the graft. Graft blood flow was measured using an in-line ultrasonic flow probe (Transonic Systems, Ithaca, NY), and one of the two lumens of the reperfusion cannula was connected to a transducer for measurement of PA pressure. Flow and PA pressure were recorded and subsequently analyzed using a data aquisition package (Dataq Instruments, Akron, OH). Another transducer was used to monitor graft peak airway pressure. Lung tissue was weighed at the end of the reperfusion period and again after drying to constant weight at 120 C. Wet to dry weight ratio was calculated as (wet weight - dry weight)/ dry weight. Statistical Analysis All data are expressed as mean _+ standard error of the mean. Means were analyzed by one-way analysis of variance. If differences were found, the Bonferroni post hoc test was used to compare groups; p values less than 0.05 were considered significant. Results The average flush flow rate (total flush volume/flush time) was similar in the three groups: group I, ml/min; group II, 28 _+ 1 ml/min; group III, ml/min (p = not significant). All support animals re-

3 1496 BHABRA ET AL Ann Thorac Surg LUNG PRESERVATION: PROSTACYCLIN VERSUS GTN 1996;62: III i {/l'?-l-t-t-l-t-l-i-l-ltll-l-t-i-t-t-i-t-t~-t=l-'l==i I p02 (mmhg) I - ii Flow (ral/min) 6 il "r * 1o ;0 30 ~0 so 6o Time (min) Fig 1. Partial pressure of oxygen (po 2) in graft effluent during 1 hour of reperfusion. I - prostacyclin, no storag6" II prostacyclin, 6-hour storagtv III - glyceryl trinitrate, 6-hour storage. Data are shown as mean +_ standard error of the mean. (*p < versus group L) mained stable during reperfusion as assessed by venous blood gas tensions and acid/base balance, core temperature, and visual inspection of the mediastinum and lungs. The partial pressure of oxygen in the graft reperfusate was constant during the experiments and within and between groups. Reoxygenation Grafts flushed with PGI 2 treatment and stored for 6 hours (group II) performed poorly compared with controls (Fig 1). At I hour, graft effluent partial pressure of oxygen was 144 ± 8 rnm Hg in group I and mm Hg in group II (p < 0.001). In contrast, with the addition of GTN to the flush and storage solution (group IlI), oxygen tensions during reperfusion were at control levels and at 1 hour, the partial pressure of oxygen was mm Hg (p = not significant). Hemodynamic Indices Blood flow in the PGI2-treated group II grafts was poor (Fig 2), and mean PA pressure was elevated (Fig 3) compared with controls. At 1 hour, flow in group I was 11.3 ± 0.4 ml/min and in group II was 1.8 ± 1.0 ml/min (p < 0.001); PA pressure in group I was 14.6 ± 1.0 mrn Hg and in group II was 26.5 ~ 2.2 mm Hg (p < 0.001). In group III, on the other hand, graft flow and PA pressure were similar to values in controls; at 1 hour they were 11.3 ± 0.3 ml/min and 13.0 ± 0.4 mm Hg, respectively (p - not significant for both). Airway Pressure Peak airway pressure (Fig 4), which with fixed tidal volume ventilation is related to lung compliance, became significantly elevated in group II (47.0 ± 1.1 mm Hg at 1 hour, compared with 14.6 ± 0.2 mm Hg in group I; p < 0.001). In contrast, peak airway pressure remained at baseline levels in group III (14.0 +_ 0.3 mm Hg at 1 hour; p = not significant) Time (rain) Fig 2. Graft blood flow during 1 hour of reperfusion. I = prostacyclin, no storage; II = prostacyclin, 6-hour storage; III = glyceryl trinitrate, 6-hour storage. Data are shown as mean standard error of the mean. (*p < versus group I.) Wet to Dry Weight Ratio The wet to dry weight ratio was significantly higher in group II (9.7 _+ 0.2) than in group I ( ) (p < 0.001). Supplementation with GTN prevented this weight gain; the ratio in group III was (p not significant versus group I). Comment This study has shown that after 6 hours of hypothermic storage in modified EC solution, rat lung grafts perform significantly better in terms of reoxygenation, blood flow, PA pressure, peak airway pressure, and weight gain if the flush and storage solution is supplemented with the NO donor GTN than if conventional PGI 2 treatment is used. This beneficial effect is not accompanied by improved perfusate flow rate during flush perfusion. The rapid deterioration of function in group II is consistent with our previous experience with 6-hour rat PAP (mrnhg] III 5 IIIlI_!_;-I-!-I-LI_ -l-i-l-li~ ~-~-i=t=~-,-,-,-,-,-,-,-,-'-'-- - -!-T-I-!-I-I-I-!JII ~itliilliitlil-l~ v 1'o 20 3o 4' ~'o Time (rain) Fig 3. Graft mean puhnonary artery pressure (PAP) during 1 hour of reperfusion. 1 - prostacyclin, no storage; II - prostacyclin, 6-hour storag~ III - glyceryl trinitrate, 6-hour storage. Data are shown as mean + standard error of the mean. (*p < versus group L) - 6O

4 Ann Thorac Surg BHABRA ET AL ;62: LUNG PRESERVATION: PROSTACYCLIN VERSUS GTN AwP [mmhg) 50 4O 30 /1 20 S 10 T ] / I I ~ : i/l ~I,,. I HI 0 0 I '0 2'0 3'0 4'0 5'0 6'0 Time (mind Fig 4. Graft peak airway pressure (AwP) during 1 hour of reperfusion. I = prostacyclin, no storage; II - prostacyclin, 6-hour storage; III = glyceryl trinitrate, 6-hour storage. Data are shown as mean + standard error of the mean. (*p < versus group I.) lung preservation in EC solution [20]. Although a detrimental effect of PGI 2 on graft preservation cannot be excluded as an explanation for this deterioration, our objective was to compare GTN supplementation with current clinical practice, which frequently includes prostaglandin therapy. Further, even when EC solution is used without PGI2, transplanted rat lung grafts fail rapidly after 6-hour storage [18]. The introduction of prostaglandins into lung graft procurement techniques resulted from concerns that reflex vasoconstriction upon administration of cold EC solution may compromise the adequacy of the flush. Pulmonary vasodilatation achieved by prostaglandins given to the donor before flushing or in the flush solution was considered to improve its distribution, wash out potentially harmful blood constituents more thoroughly, and allow more rapid and uniform cooling of the graft. Most units now include the use of PGEa or PGI2 in their clinical lung harvesting procedure [2]. However, the few laboratory studies looking specifically at the effects of prostaglandins on lung preservation have not produced consistent results. Most of these studies have used cold EC solution, with or without the addition of magnesium, for flush perfusion of lungs. In a canine heart-lung transplant model with 6-hour storage, PGI 2 pretreatment and addition to the flush solution improved survival and oxygenation [4]. Similar application of PGI 2 to in situ canine lungs flushed with cold perfusate and then subjected to 60 minutes of warm ischemia improved oxygenation, pulmonary hemodynamic indices, weight gain, neutrophil influx, and ultrastructural changes [5]. Several groups have used canine single-lung transplant models. Novick and colleagues [9] found that after 12-hour cold storage, PGI 2 pretreatment improved oxygenation but not PA pressure, airway pressure, or weight gain, whereas PGI l pretreatment conferred no benefit at all. With 4-hour storage and Iloprost (a PGI 2 analogue) pretreatment and addition to the flush, improvement in oxygenation and PA pressure was observed, but was only statistically significant at one time point [10]. Similar Iloprost therapy with 6 hours of storage did not benefit survival oxygenation, or pulmonary hemodynamic indices [11]. Puskas and colleagues [12] found that donor lung hyperinflation gave better results than PGE 1 administration to the donor and flush. In a primate heart-lung transplant model, PGE 1, PGI2, and nitroprusside pretreatment were compared; PGE1 was found to improve ultrastructural preservation, but oxygenation was not significantly different among the groups [6]. A canine double-lung transplant model showed no benefit from PGE 1 pretreatment; in fact, survival and early gas exchange were significantly worse [13]. A recent study using a porcine single-lung transplant model found no improvement in gas exchange or pulmonary vascular resistance after the addition of PGE 1 or PGI 2 to the flush solution ]14[. Two studies using Wallwork's solution for flush perfusion in isolated rat lung reperfusion models have shown the addition of PGI 2 to be beneficial [7, 8]. With no flush at all pretreatment with PGI 2 in a canine model of in situ ischemia and reperfusion significantly improved pulmonary structure and function [21]. The conflicting results from these studies cannot be explained by differences in doses of prostaglandins used or in method of administration (ie, as pretreatment or to the perfusate). What is notable is that prostaglandin therapy does not improve the flush flow rate [10, 12], enhance cooling of the lung ]9-12], or significantly alter the distribution of EC flush solution [11, 22]. This is contrary to evidence from isolated canine lobe perfusion studies, which showed that the addition of PGI 2 prevents the elevation of vascular resistance elicited by cold (4 C) EC solution [23]. On the other hand, 37 C EC solution causes vasoconstriction as well, but this is not countered by PGI 2 or PGE 1 [24]. The explanation offered by the authors of the latter study was that there are two independent triggers of vasoconstriction involved: low temperature and, at temperatures above 20 C only, high potassium concentration; prostaglandins may counter the former but not the latter. Hence in the initial phase of flush perfusion, before lung temperature falls significantly, potassium-induced, prostaglandin-resistant vasoconstriction may still occur. Any beneficial effects of prostaglandin therapy may therefore be mediated by mechanisms other than vasodilatation. Prostacyclin is normally produced by endothelium and is known to inhibit platelet aggregation and leukocyte-endothelial interactions and to stabilize membranes [3]. Little is known about how these actions may improve the preservation of flushed lungs or attenuate reperfusion injury. It seems unlikely that exogenous prostaglandins (which have short half-lives), particularly when used for pretreatment alone, would still be available at reperfusion, during the early phase of which levels of endothelial production of PGI 2 are reduced [3]. Alteration of the behavior of any platelets and leukocytes still present in the lung after flushing or some other unidentified cytoprotective mechanism may play a role. The other main feature of postischemic endothelial

5 1498 BHABRA ET AL Ann Thorac Surg LUNG PRESERVATION: PROSTACYCL N VERSUS GTN 1996;62: dysfunction is reduced production of NO soon after the onset of reperfusion [25, 26]. Nitric oxide has a short half-life in vivo but is a potent vasodilator, inhibits platelet aggregation and neutrophil adhesion/activation [15], and quenches oxygen-derived free radicals [16]. There is accumulating experimental evidence suggesting that interventions aimed at increasing the availability of NO during reperfusion of ischemic myocardium are protective [27, 28]. In the context of interventions at an earlier stage, enhancement of myocardial preservation by the addition of L-arginine, the precursor of endothelial NO production, before [25, 29] or during [30] ischemia has been demonstrated recently. L-arginine added to Wallwork's flush solution also improved function after 6-hour storage in an isolated rat lung reperfusion model [8]. An alternative approach to supplementing endogenous production is to use NO donors such as GTN. Although more efficient NO donors are available, such as the sydnonimine compounds [27], GTN has the advantage of already being in widespread clinical usage, and it may stimulate cyclic guanosine monophosphate production through other mechanisms as well. In a rat lung transplant model, the addition of GTN to Ringer's solution for flush perfusion was found to significantly improve survival, oxygenation, and hernodynamic indices and to reduce neutrophil sequestration during 30 minutes of reperfusion after 4 hours of hypothermic storage [17[. Similar benefit was obtained with 6-hour storage in EC solution supplemented with GTN as compared with EC alone or EC plus hydralazine [181, and also when a cyclic guanosine monophosphate analogue was added to the preservation solution [31]. Our current study has confirmed, in a different model, that supplementation with GTN during 6-hour storage in EC solution yields excellent lung graft function. Further, we have demonstrated that this is markedly more effective than the currently used clinical technique of PGI 2 administration to the donor and perfusate. The mechanism of this benefit is not yet known. Improved flush efficiency is unlikely to be the main factor in view of the similarity of perfusate flow rates between experimental groups. The conclusion of Mulvin and colleagues [7] that PGI 2 supplementation during storage is beneficial because of its vasodilatory properties was based on their finding of similar benefit with the addition of GTN, chosen as a simple vasodilator; the other NO-mediated actions of GTN had not been discerned at that time. The same questions therefore arise as to whether NO modulates the behavior of residual platelets and leukocytes in the flushed lung or preserves endothelial integrity by some other mechanism. In relation to the observed superiority of GTN over PGI 2, it may be that NO/cyclic guanosine monophosphate pathways are more important than PGI2/cyclic adenosine monophosphate pathways in the endothelial milieu during ischemia, or that the longer half-life of GTN makes exogenous NO available for a greater proportion of the ischemic period and maybe even at reperfusion. Further investigation of an alternative to the current practice of prostaglandin therapy in lung graft procure- ment is warranted. Addition of GTN to the flush solution would be simple and relatively inexpensive and would eliminate the donor systemic hypotension sometimes caused by prostaglandin pretreatment. This work was supported by a grant from the National Heart Research Fund, United Kingdom. References 1. 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J Thorac Cardiovasc Surg 1993;105: Mulvin D, Jones K, Howard R, Grosso M, Repine J, Johnston M. The effect of prostacyclin as a constituent of a preservation solution in protecting lungs from ischemic injury because of its vasodilatory properties. Transplantation 1990;49: Xiong L, Mazmanian M, Chapefier AR, et al. Lung preservation with Euro-Collins, University of Wisconsin, Wallwork, and low-potassium-dextran solution. Universit6 Paris- Sud Lung Transplant Group. Ann Thorac Surg 1994;58: Novick RJ, Reid KR, Denning L, Duplan J, Menkis AH, McKenzie FN. Prolonged preservation of canine lung allografts: the role of prostaglandins. Ann Thorac Surg 1991;51: Klepetko W, Muller MR, Khunl-Brady G, et al. Beneficial effect of Iloprost on early pulmonary function after lung preservation with modified Eurocollins solution. Thorac Cardiovasc Surg 1989;37: Hooper TL, Fetherston GJ, Flecknell PA, Dark JH, McGregor CG. The use of a prostacyclin analog, Iloprost, as an adjunct to pulmonary preservation with Euro-Collins solution. Transplantation 1990;49: Puskas JD, Hirai T, Christie N, Mayer E, Slutsky AS, Patterson GA. Reliable thirty-hour lung preservation by donor lung hyperinflation. J Thorac Cardiovasc Surg 1992;104: Bonser RS, Fragomeni LS, Jamieson SW, et al. Effects of prostaglandin E1 in twelve-hour lung preservation. J Heart Lung Transplant 1991;10: Kukkonen S, Heikkila LJ, Verkkala K, Mattila SP, Toivonen H. Prostaglandin E~ or prostacyclin in Euro-Collins solution fails to improve lung preservation. Ann Thorac Surg 1995;60: Cooke JP, Tsao PS. Cytoprotective effects of nitric oxide. Circulation 1993;88: Gaboury J, Woodman RC, Granger DN, Reinhardt P, Kubes P. Nitric oxide prevents leukocyte adherence: role of superoxide. Am J Physiol 1993;265:H Naka Y, Chowdhury NC, Oz MC, et al. Nitroglycerin main-

6 Ann Thorac Surg BHABRA ET AL ;62: LUNG PRESERVATION: PROSTACYCLIN VERSUS GTN rains graft vascular homeostasis and enhances preservation in an orthotopic rat lung transplant model. J Thorac Cardiovasc Surg 1995;109: Naka Y, Chowdhury NC, Liao H, et al. Enhanced preservation of orthotopically transplanted rat lungs by nitroglycerin but not hydralazine. Requirement for graft vascular homeostasis beyond harvest vasodilation. Circ Res 1995;76: Hopkinson DN, Bhabra MS, Odom NJ, Bridgewater BJM, van Doorn CA, Hooper TL. Controlled pressure reperfusion of rat pulmonary grafts yields improved function after twenty-four-hours' cold storage in University of Wisconsin solution. J Heart Lung Transplant 1996;15: Bhabra MS, Hopkinson DN, Shaw TE, Hooper TL. Critical importance of the first 10 minutes of lung graft reperfusion after hypothermic storage. Ann Thorac Surg 1996;61: Yamashita C, Oobo H, Tsuji F, et al. Effect of prostaglandin 12 and superoxide dismutase on reperfusion injury of warm ischemic lung. Ann Thorac Surg 1992;54: Baretti R, Bitu-Moreno J, Beyersdorf F, Matheis G, Francischetti I, Kreitmayr B. Distribution of lung preservation solutions in parenchyma and airways: influence of atelectasis and route of delivery. J Heart Lung Transplant 1995;14: Unruh H, Hoppensack M, Oppenheimer L. Vascular properties of canine lungs perfused with Eurocollins solution and prostacyclin. Ann Thorac Surg 1990;49: Kimblad PO, Steen S. Eliminating the strong pulmonary vasoconstriction caused by Euro-Collins solution. Ann Thorac Surg 1994;58: Engelman DT, Watanabe M, Engelman RM, et al. Constitutive nitric oxide release is impaired after ischemia and reperfusion. J Thorac Cardiovasc Surg 1995;110: Ma XL, Weyrich AS, Lefer DJ, Lefer AM. Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. Circ Res 1993;72: Lefer AM. Attenuation of myocardial ischemia-reperfusion injury with nitric oxide replacement therapy. Ann Thorac Surg 1995;60: Vinten-Johansen J, Zhao Z, Sato H. Reduction in surgical ischemia-reperfusion injury with adenosine and nitric oxide therapy. Ann Thorac Surg 1995;60: Engelman DT, Watanabe M, Maulik N, et al. L-arginine reduces endothelial inflammation and myocardial stunning during ischemia/reperfusion. Ann Thorac Snrg 1995;60: Hiramatsu T, Forbess JM, Miura T, Mayer JE Jr. Effect of L-arginine cardioplegia on recovery of neonatal lamb hearts after 2 hours of cold ischemia. Ann Thorac Surg 1995;60: Pinsky DJ, Naka Y, Chowdhury NC, et al. The nitric oxide/ cyclic GMP pathway in organ transplantation: critical role in successful lung preservation. Proc Natl Acad Sci USA 1994;91: Notice From the American Board of Thoracic Surgery The part I (written) examination will be held at the Atlanta Airport Hilton and Towers, Atlanta Airport, Atlanta, GA, on February 1, The closing date for registration is August 1, To be admissible for the part II (oral) examination, a candidate must have successfully completed the part I (written) examination. A candidate applying for admission to the certifying examination must fulfill all the requirements of the board in force at the time the application is received. Please address all communications to the American Board of Thoracic Surgery, One Rotary Center, Suite 803, Evanston, IL