The role of dextran 40 and potassium in extended hypothermic lung preservation for transplantation

The role of dextran 40 and potassium in extended hypothermic lung preservation for transplantation We have previously demonstrated that a low-potassium dextran solution provides superior and more reliable preservation of lungs for 12 hours than that provided by the commonly used Euro-Collins solution. This study was designed to examine the individual contributions of dextran 40 and a low (extraceUular) potassium concentration to lung preservation. In a randomized, blinded study using an in vivo canine single-lung transplant model, lungs preserved with low-potassium dextran solution (K+, 4 mmol/L; dextran 40, 20 gm/L) were compared to lungs preserved with low-potassium, no-dextran solution (K+, 4 mmol/L) and high-potassium dextran solution (K+, 123 mmol/L; dextran 40, 20 gm/L), The lungs were assessed immediately and 3 days after transplantation. The low-potassium dextran solution provided exceUent immediate pulmonary function with little variability (arterial oxygen tension, 519 ± 12 mm Hg, measured on the transplanted lung alone, inspired oxygen fraction = 1.0, n = 6). Removing the dextran 40 from the flush solution (low-potassium group) led to a significant deterioration in pulmonary function (arterial oxygen tension, 243 ± 78 mm Hg, n = 6, p < 0.01). The high-potassium dextran solution provided extremely poor preservation (arterial oxygen tension, 176 ± 79 mm Hg; n = 6; p < 0.01). Two animals in this group died within 6 hours of operation. Viability of the transplanted bronchus was significantly improved with the two solutions containing dextran 40. These results indicate that dextran 40 and low potassium concentration both contribute significantly to the uniformly exceUent 12-hour lung preservation seen with the low-potassium dextran solution. (J THORAC CARDIOVASC SURG 1992;103:314-25)

S. H. Keshavjee, MD, MSc,a F. Yamazaki, MD,a H. Yokomise, MD,a P. F. Cardoso, MD,a J. B. M. Mullen, MD,b A. S. Slutsky, MD,a and G. A. Patterson, MD, FRCS(C), FACS,a Toronto, Ontario, Canada

h e shortage of donor organs remains a major limiting factor in the widespread application of lung transplantation for patients with end-stage pulmonary disease. A more reliable method of preservation, providing a longer available ischemic period, would permit the safe procurement oflungs from greater distances. It might also permit the use of lungs currently judged inadequate because of the limitations of current preservation capabilities. From the Division of Thoracic Surgery; Department of Surgery and Department of Pathology," University of Toronto, Toronto, Ontario, Canada. Supported by Medical Research Council Grant No. 10142. Received for publication April 13, 1990. Accepted for publication Oct. 9, 1990. Address for reprints: G. Alexander Patterson, MD, c/o Dr. Joel D. Cooper, Professor of Surgery, Head, Section of Thoracic Surgery, Division of Cardiothoracic Surgery, Suite 3107, Queeny Tower, 4989 Barnes Hospital Plaza, St. Louis, MO 63110.

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These factors would improve the supply oflungs available for transplantation. High-potassium C'intracellular") solutions, such as Euro-Collins, are commonly used in lung preservation. Their popularity derives primarily from the proven success of such solutions in extending renal preservation times.' High potassium concentrations, however, are known to cause intense pulmonary vasospasm.l' Furthermore, although high potassium levels were initially thought beneficial for organ preservation, after the work of Collins and coworkers,' more recent studies have demonstrated that they can actually lead to increased cellular injury under hypothermic conditions. 5-12 Specifically with relation to the lung, work by Fujimura and associates':' has shown that "extracellular" solutions may provide improved preservation. We hypothesized that a low-potassium solution would provide improved pulmonary preservation by minimizing the pulmonary vasospasm, leading to more uniform and

Volume 103 Number 2 February 1992

Dextran 40 and potassium in lung preservation

3 15

PULMONARY ARTERY OCCLUSION CUFF

ANASTOMOSES: PULMONARYARTERY BRONCHUS LEFT ATRIUM

Fig. 1. Canine single-lung allotransplant model. Pulmonary artery occlusion cuffs enable independent study of native and transplanted lung function.

effective cooling by pulmonary artery flush. Furthermore, any potassium-related cellular injury during the storage phase would also be minimized. Impaired flow in the microcirculation after reperfusion is a potential problem after lung transplantation. In severe cases, this is referred to as the no-reflow phenomenon's where no perfusion of the implanted organ occurs despite widely patent large-vessel anastomoses. It has been suggested that this phenomenon may be due to the plugging of the microcirculation by red blood cells. 14 Hypothermic, energy-depleted erythrocytes (as would be found in an organ stored for transplantation) aggregate and their membranes become stiff. 15 This leads to sludging and plugging in the microcirculation. We therefore hypothesized that an agent that improves flow in the microcirculation could potentially contribute to improved posttransplant pulmonary function. Dextran 40 (low-molecular weight dextrans, average molecular weight 40 kD) has been shown to improve microvascular flow under various conditions. 16- 21 It has been shown to coat the surface of red blood cells, causing disaggregation of already aggregated erythrocytes.P It also increases the

deformability of red blood cells,17, 19 thus improving their ability to traverse capillary beds. Further benefit may be gained from the added antithrombotic effect resulting from the surface coating of platelets-' and endothelial cells.24 Improved posttransplantation reflow in the pulmonary microcirculation should lead to improved function of the transplanted lung. It could also potentially lead to an improvement in viability of the transplanted bronchus by improving flow in the microcirculatory collaterals between the pulmonary and bronchial circulations. Since the transplanted bronchus does not have its systemic blood supply restored within the first few days of transplantation, its viability is critically dependent on blood flow from the pulmonary circulation through these collaterals. On the basis of these hypotheses, in a previous study we demonstrated that a low-potassium dextran (LPD) solution provided superior and more reliable preservation of lungs for 12 hours compared with the commonly used Euro-Collins solution." This present randomized, blinded study was designed to examine the individual contri-

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Table I. Composition offlush solutions studied Na+ (mmol/L) K+ (rnmol/L) cr (mrnol/L) HCO J - (mmol/L) Mg2+ (mmol /L)

P0 4 (rnmol/L) pH Osmolarity (mOsm/L) Dextran 40 (gm/L)

LPD

LP

HPD

168 4 103 0 2 37 7.45 280-290 20

168 4 103 0 2 37 7.45 280-290 0

50 123 105 0 2 37 7.45 280-290 20

Table II. Reagents used to manufacture the flush solutions Reagent

NaH 2P0 4 . H 20* Na2HP0 4 t K2HP0 4* NaCl* KCI* MgS04 • 7H 20 t Dextran 40*

LPD (gm/L)

LP (gm/L)

HPD (gm/L)

0.69 4.5

0.69 4.5

0.69

o

5.8

o

5.8

0.3

0.3

0.5 20

0.5

o

o

5.52 2.63 4.47 0.5 20

'Supplied by BDH Chemicals, Toronto, Ontario, Canada. tSupplied by Mallinckrodt Inc., Parkis, Ky. :j:Supplied by Sigma Chemical Co., St. Louis, Mo.

butions of dextran 40 and a low potassium concentration to the improved lung preservation that we have demonstrated with LPD.

Material and methods The function of lung allografts stored for 12 hours after pulmonary arterial flush was studied in a canine single lung transplant model.P Inflatable cuffs, attached to subcutaneous injection ports, were placed around each main pulmonary artery at operation (Fig, 1), Temporary occlusion of either pulmonary artery allowed independent study of the native or transplanted lung, immediately and 3 days after transplantation, This protocol enables the study of both acute preservation-related lung injury and delayed manifestations of ischemic and reperfusion injury on the lung and the airway, while minimizing the confounding effects of infectious or rejection episodes. Mongrel dogs were randomly allocated to one of three groups, according to the flush solution used (Table I). These solutions were designed to examine the individual roles of dextran 40 and potassium in lung preservation. The entire experimental team was blinded to the type of solution used in each experiment until the series of experiments had been completed, In the first group (n = 6), the recipients received lungs flushed with LPD, as in the previous study.25 In the second group (n = 6), the lungs were flushed with low-potassium solution without dextran (LP), and in the third (n = 6), they were flushed with high-potassium dextran solution (HPD), The flush solutions used in this study

were prepared according to the following protocol. All glassware was autoclaved before use. Reagents (Table II) were weighed and combined in a glass beaker. The required volume of double-distilled water (Barnstead glass still, model A I 040; Servomex Norwood, Mass.) was then added. The solution was then stirred until all the reagents had dissolved, without the application of heat. The flush solution was then sterilized by filtration through a 0,22 ~m cellulose acetate membrane (Corning 25952-1000; Corning Inc., Corning, N.Y.) under a laminarflow hood, The solutions were then stored at 4 0 C. Before use, the absence of endotoxins in the solutions was confirmed with the limulus amebocyte lysate (Pyrotell) test (Associates of Cape Cod Inc., Woods Hole, Mass.), Sterility was confirmed by plating the solution on nutrient agar and incubating it for 2 weeks at 37° C. The electrolyte composition (Na+, K+, 0-, and HC0 3- ) was assayed with an ion-selective electrode (Beckman system E4A; Beckman Instruments Inc., Fullerton, Calif.). A Hitachi 737 chemistry analyzer (Bering, Mannheim, Germany) was used to assay P0 4 and Mg2+. The pH was measured with a Radiometer ABL2 pH meter (Radiometer A/S, Copenhagen, Denmark), The osmolarity was measured with an Osmette A osmometer (Precision Systems, Inc., Natick, Mass.). In the preparation of the LPD solution, it is essential not to autoclave sterilize the solution in plastic containers, or to use water that has been heat-sterilized in plastic containers, We have found that this can cause severe pulmonary edema. Analysis by gel-permeation chromatography of the solutions causing pulmonary edema revealed the presence of a 500 kD contaminant. Presumably this represents clumped dextran molecules, since there are no other constituents of the LPD solution with a large enough molecular weight to account for this. Clumping of the dextrans may be due to an interaction with the plastic container or with a substance that leaches out of the plastic since this problem does not occur with water sterilized in glass containers, Donor procedure. Donor dogs weighing 20 to 25 kg were sedated with meperidine hydrochloride 50 mg intravenously and azepromazine maleate I mg intravenously, After they had been given atropine 0.6 mg intravenously and cefazolin I gm intravenously, the animals were anesthetized with thiopental sodium 10 rug/kg. They were intubated and their lungs ventilated (Ventimeter ventilator; Air-Shields, Hatboro, Pa.) with a tidal volume of 550 ml, a respiratory rate of 12 breaths/min, and an inspired oxygen fraction of 1.0. Baseline arterial blood gases and hematocrit value were determined. A median sternotomy was performed and the heart-lung block was dissected as previously described" After systemic heparinization (500 U /kg), an 8 mm aortic arch cannula (Sarns Inc./3M, Ann Arbor, Mich.) was inserted into the pulmonary artery. The pulmonary artery was flushed with 50 ml/kg cold (4 0 to 8 0 C) flush solution while the pressure was carefully maintained between 10 and 20 mm Hg by adjusting the flow rate. Ventilation was continued throughout the procedure. The left atrial effluent was allowed to collect in the chest cavity to provide additional topical cooling of the lungs, The trachea was clamped with the lungs maintained in the inflated state. The heart-lung block was then excised and the organs were placed in a sterile plastic bag containing 2 L cold (4 0 to 8 0 C) solution of the same kind used for flushing. The block was then placed in a refrigerator and stored for 12 hours at 4 0 C.

Volume 103 Number 2 February 1992

Dextran 40 and potassium in lung preservation

Pa02 (mmHg)

70

3 17

PaC02 (mmHg) ,-----------------------~

600

60

500

400

..

50

*

300 40

200

100

---- LPD --A- LP

30 ---- LPD --A- LP ....-- HPD

---- HPD

oL-----+----------t----------! IMMED. POST-OP DAY 3 Fig. 2. Assessment of transplanted lung function: arterial P02. LPD provided excellent immediate and postoperative day 3 pulmonary function, with little variability. Removing the dextran 40 from the solution (LP) caused a significant deterioration in lung function, with variable recovery by postoperative day 3. HPD provided extremely poor pulmonary function. Inspired oxygen fraction = I, positive end-expiratory pressure = O. All values mean ± the standard error of the mean; n = 6 for each group. *p < 0.01.

Recipient procedure. On the next day, a weight-matched dog was treated with oral cyclosporine (15 rug/kg) and azathioprine (I rug/kg). The animal was sedated and anesthetized by the same procedure described for the donor. On induction, the recipient received cefazolin I gm intravenously and methylprednisolone 500 mg intravenously. The animal was connected to an electrocardiographic recorder. An arterial line was percutaneously introduced into the femoral artery. A Swan-Ganz flow-directed thermal-dilution catheter (Baxter Healthcare Corp., Edwards Division, Santa Ana, Calif.) was inserted into the femoral vein and directed into the pulmonary artery. Arterial and venous lines were connected to transducers by pressure-monitoring lines. A bronchial blocker (model 62-080-8/14F, 10 cc; Fogarty Engineering, A California Corp., Portola Valley, Calif.) was used to occlude the left main-stem bronchus and to maintain right lung ventilation while the bronchial anastomosis was being performed. Baseline assessment of arterial blood gases, systemic hemodynamics, and pulmonary hemodynamics was performed before the operation. The left pneumonectomy and implantation was performed as previously described.P The atrial anastomosis was performed first with a 5-0 Prolene horizontal everting mattress suture

20

'---------t-------------j---------'

IMMED. POST-OP

DAY 3

Fig. 3. Assessment of transplanted lung function: arterial Pco-, Both the LP and HPD groups had a higher arterial Pco, than the LPD group. All values mean ± the standard error of the mean; n = 6 for each group. *p < 0.05; **p < 0.01.

(Ethicon, Inc., Somerville, N.J.). The pulmonary artery anastomosis was then performed with a running 6-0 Prolene suture, interrupted at opposite ends of the anastomosis to prevent narrowing of the pulmonary artery at the anastomotic site. The suture was left untied at this point. The bronchial anastomosis was performed one ring proximal to the first bronchial bifurcation with a running 4-0 Prolene suture interrupted at each membranocartilaginous junction. A bronchial omentopexy was not performed, since the experiments were designed for shortterm studies (3 days). After the lung had been reinflated (to ensure more uniform reperfusion), the atrial clamp was released to permit back bleeding and removal of air through the pulmonary circulation. On appearance of back bleeding through the open pulmonary artery anastomosis, the pulmonary artery clamp was released. So that anastomotic narrowing would be avoided, the pulmonary artery suture was tied after the arterial lumen had been dilated by the restoration of flow. An inflatable cuff was placed around the left pulmonary artery and the injection ports connected to each of the cuffs were then implanted subcutaneously. The animals' lungs were ventilated with 5 em H 20 positive end-expiratory pressure while the chest was being closed to reinflate any atelectatic areas of the lung. The dogs were then placed in the supine position for the combined and unilateral lung assessment detailed below. The dogs received cyclosporine 15 mg/kg by mouth, azathioprine I mg/kg by mouth, prednisone I mg/kg by mouth, and cefazolin I gm intramuscularly daily. They also received

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Table III. Assessment of lung function: Low-potassium dextran solution Preop.

2L

Day 3

Immediate TL

519 ± Arterial POz (mm Hg) 578 ± 16 Arterial Pco- (mm Hg) 39 ± 29 ± 2 PAP (mm Hg) 14 ± 1 32 ± PVR (dynes. sec . cm") 230 ± 22 880 ± CO (Lyrnin) 3.1 ± 0.2 2.4 ± PCWP (mm Hg) 6 ± I 5± CVP(mmHg) 4±1 7± SVR (dynes. sec . cm") 2054 ± 144 2097 ± Arterial BP (rnm Hg) 71 ± 83 ± 8 pH 7.435 ± 0.040 7.315 ± HC0 3 (mmol/L) 18.7 ± 0.8 19.7 ± 39 ± 3 Hct

2L

NL 12 470 ± 2 34 ± 3 21 ± 388 ± 83 3.2 ± 0.1 5± I 5± 2 120 1906 ± 5 78 ± 0.026 7.332 ± 18.3 ± 1.5 35 ±

46 497 ± 3 33 ± 16 ± 1 67 244 ± 0.4 3.1 ± I 5± I 4± 2116 ± 206 83 ± 3 0.038 7.373 ± 2.0 19.4 ± 2

NL

TL

555 ± 15 42 ± I 1 32 ± 17 835± 0.2 2.5 ± 5± I I 6± 138 2653 ± 2 86 ± 0.026 7.303 ± 1.1 21.2 ±

8 512 ± 2 32 ± 3 20 ± 439 ± 90 0.2 2.6 ± I 6 ± 5± I 275 2667 ± 7 85 ± 0.034 7.407 ± 1.7 20.2 ± 33 ±

2L

52 533 ± 2 30 ± I 16 ± 62 284 ± 0.3 2.7 ± 1 6± 1 5± 421 2584 ± 5 88 ± 0.043 7.442 ± 1.9 19.9 ± 2

23 2 I 30 0.2 1 I 210 5 0.028 0.9

Values are mean ± SEM. Preop, Before operation; 2L, both lungs; TL, transplanted lung; NL, native lung; Hct, hematocrit.

Table IV. Assessment of lung function: Low-potassium solution Preop.

2L Arterial POz (mm Hg) Arterial Pco- (mm Hg) PAP (mm Hg) PVR (dynes. sec . cm- S) CO (Lyrnin) PCWP (mm Hg) CVP (mm Hg) SVR (dynes· sec . cm") Arterial BP (rnm Hg) pH HC03 (mrnol/L) Hct

Day 3

Immediate TL

NL

2L

NL

TL

494 ± 23 442 ± 243 ± 78 537 ± 25 572 ± 21 56 ± 38 ± 3 37 ± 2 30 ± 2 53 ± 3 14 ± I 25 ± 30 ± 3 17 ± 2 13 ± I 205 ± 24 619 ± 964 ± 246 281 ± 34 193 ± 10 2.9 ± 0.2 2.5 ± 2.2 ± 0.3 2.8 ± 0.2 2.8 ± 0.1 5± 6± 1 6± 1 5± 1 5± 1 5± I 6± 5± I 8± I 5± I 2016 ± 90 1901 ± 2488 ± 112 1581 ± 283 2010 ± 156 76 ± 3 65 ± 90 ± 4 50 ± 5 75 ± 4 7.437 ± 0.015 7.213 ± 0.028 7.338 ± 0.028 7.348 ± 0.017 7.202 ± 20.4 ± 1.1 20.1 ± 0.3 21.1 ± 0.3 19.9 ± 0.3 23.3 ± 41 ± 3 38 ± 2

2L

85 605 ± 9 524 ± 22 5 36 ± 2 34 ± 2 3 17 ± 2 14 ± I 54 344 ± 39 222 ± 18 0.2 2.3 ± 0.1 2.7 ± 0.1 1 5± 1 5± 1 2 4±1 4±1 337 2597 ± 112 2506 ± 117 7 79 ± 1 87 ± 2 0.014 7.372 ± 0.017 7.393 ± 0.014 1.2 20.6 ± 0.9 20.5 ± 0.7 34 ± 2

Values are mean ± SEM. Preop, Before operation; 2L, both lungs; TL, transplanted lung; NL, native lung; Hct, hematocrit.

buprenorphine 0.3 mg intramuscularly twice daily for analgesia. The dogs were allowed to recover for 3 days. They were fed a full diet and allowed to exercise freely. On the third postoperative day, they were anesthetized and combined and unilateral lung function was assessed as before. The dogs were then killed and all anastomoses were examined for technical failures. The animals used in this study received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Assessment. After closure of the chest, with the dog in the supine position, arterial blood gases (pH, arterial carbon dioxide tension [Pco-], arterial oxygen tension [Po-], and H C0 3 concentration) were measured. Inspired oxygen concentration of 1.0and positive-endexpiratory pressure of 0 were maintained. The following systemic and pulmonary hemodynamics were measured: systolic, diastolic, and mean systemic blood pressure (BP), central venous pressure (CVP), cardiac output (CO),

systolic,diastolic, and mean pulmonary artery pressure (PAP), and pulmonary capillary wedge pressure (PCWP). The systemic vascular resistance (SVR) was calculated in dynes . second . centimeter- S by the following formula: SVR = ([mean BP - CVPl/CO) X 80 dynes. sec . cm- s. The pulmonary vascular resistance (PVR) was similarly calculated by the following formula: PVR = ([mean PAP - PCWPl/CO) X 80 dynes· sec . cm- s. The first (baseline) measurement of posttransplantation bilateral lung function was made approximately 1 hour after reperfusion of the transplanted lung. The complete assessment was repeated after a 10-minute occlusion of the left pulmonary artery and again after a 10-minute occlusion of the right pulmonary artery. After each occlusion, flow was restored to both lungs for 10 minutes for the two lung assessments.The dogs were then awakened and allowed to recover for 3 days. Arterial blood gases were determined with a Corning pll/blood gas analyzer (Ciba Corning Diagnostics Corp. Medfield, Mass.). Blood samples were anaerobically drawn into a 1 ml heparinized syringe and assayed within 1 minute. Blood gases were measured at 5, 7, and 10 minutes to ensure that the

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Dextran 40 and potassium in lung preservation

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Table V. Assessment of lung function: High-potassium dextran solution Preop.

2L

Day 3

Immediate TL

Arterial P02(mm Hg) 569 ± 12 176 ± Arterial PC02 (mm Hg) 32 ± 3 50 ± PAP (mm Hg) 15 ± 2 36 ± PVR (dynes. sec . cm- S) 176 ± 16 1184 ± CO (Lj'min) 3.3 ± 0.3 2.6 ± PCWP (rnm Hg) 6± I 3± CVP (rnm Hg) 4± I 6 ± SVR (dynes. sec . cmr") 2008 ± 187 2184 ± Arterial BP (mm Hg) 84 ± 4 63 ± pH 7.423 ± 0.032 7.197 ± HC0 3 (rnmol/L) 20.3 ± 0.6 18.7 ± Hct 41 ± I

NL

2L

TL

NL

2L

79 453 ± 65 403 ± 44 496 ± 31 568 ± II 586 ± 37 ± 3 46 ± 7 7 35 ± 4 29 ± 3 30 ± 5 20 ± 3 16 ± I 24 ± 3 17 ± 4 II ± 477 303 ± 46 196 ± 23 533 ± 67 384 ± 98 240 ± 0.5 3.1 ± 0.5 3.3 ± 0.4 2.9 ± 0.4 2.7 ± 0.3 2.5 ± I 5± I 4± I 3± I 4±2 3± I 5± I 4±1 4±1 4±1 4 ± 633 1886 ± 291 1749 ± 188 1994 ± 67 2742 ± 279 2809 ± 68 ± 3 69 ± 3 66 ± 6 94 ± 2 6 89 ± 0.033 7.325 ± 0.044 7.324 ± 0.030 7.260 ± 0.024 7.398 ± 0.018 7.414 ± 1.8 17.8±1.l 18.6 ± 0.7 21.0 ± 3.1 18.1 ± 2.5 19.0 ± 39 ± 3 35 ± I

10 2 I 10 0.1 I I 168 3 0.018 1.6

All values mean ± SEM. Preop, Before operation; 2L, both lungs; TL, transplanted lung; NL, native lung; Hct, hematocrit.

animal had indeed reached equilibrium by 10 minutes. Most animals had reached a steady state by 7 minutes. The value at 10 minutes was used for all animals to ensure that each had an equal length of time to reach equilibrium while also having equal exposure to any detrimental effects resulting from the diversion of the total CO through one lung. The entire assessment was repeated on the third postoperative day. After the arlimals were killed on the third postoperative day, the bronchial anastomoses were resected and fixed by immersion in 10% neutral buffered formalin. Representative longitudinal sections of the anastomotic sites were processed for histologic examination in the usual manner, cut at 5 tLm thickness, and stained with hematoxylin and eosin. The specimens were examined by a pathologist, who was blinded to the flush solutions used. A modification of the pictoral grading method of Mullen and coworkers-" was used to assess the transplanted bronchus. Segments extending 5 mm proximal and distalto the anastomotic site were graded from 0 to 3 for the following: mucosal ulceration, inflammation involving the mucosa and submucosa, and mural injury as assessed by mural inflammation, hemorrhage, and necrosis affecting muscle, cartilage, and interstitial tissue. A single pathologic score (degree of histopathologic abnormality) for each airway was then determined by summing the segment scores and assessing this total as a percentage of the maximum possible score. Statistical analysis. All values are expressed as mean ± the standard error of the mean. Statistical analysis was performed with analysis of variance (ANOVA) and the Newman-Keuls multiple-comparison procedure. Further analysis of the data was carried out only if the p value of the ANOVA was less than 0.05. Since the design of the experiment is essentially a 2 X 2 factorial design with one missing cell (we did not study a highpotassium, no-dextran solution), the data were also analyzed with the SAS general linear model procedure (SAS, Inc., Cary, N.C.) for 2 X 2 factorial designs.I? The same conclusions with respect to significant differences were reached in all cases with both tests. The bronchial gross viability data were analyzed with Fisher's exact test. The histopathologic data were analyzed with Fisher's exact test. The histopathologic scores were analyzed with ANOVA and Fisher's protected t test. Two animals were omitted because of surgical technical failures: one at harvesting

(failure to ligate the azygos vein, resulting in poor flushing) and one at implantation (irreparable right pulmonary artery laceration during placement of the right pulmonary artery occlusion cuff).

Results The mean weights of the animals were 20.7 ± 0.5 kg, 22 ± 1 kg, and 22.7 ± 1.3 kg in the LPD, LP, and HPD groups, respectively. The mean total ischemic times were as follows: LPD, 13.4 ± 0.1 hours; LP, 13.6 ± 0.2 hours; and HPD, 13.7 ± 0.1 hours (p not significant according to ANOVA). The mean warm ischemic times (the time between removal of the lung from the cold storage and reperfusion, that is, trimming on the side table plus implantation time) were as follows: LPD, 95.7 ± 4.3 minutes; LP, 97.8 ± 5 minutes; and HPD, 101.3 ± 3.4 minutes (p not significant according to ANOVA). The mean anastomotic times (time for the implantation procedure) were as follows: LPD, 68 ± 3.3 minutes; LP, 69 ± 2.9 minutes; and HPD, 75.5 ± 2.6 minutes (p not significant according to ANOVA). The mean times for delivery of the lung flush were as follows: LPD, 74 ± 13 seconds; LP, 57 ± 6 seconds; and HPD, 64 ± 10 seconds (p not significant according to ANOVA). At immediate assessment, with perfusion to the transplanted lung alone, the arterial P0 2 in the LPD group (519 ± 12 mm Hg) was significantly greater than that in theLPgroup(243 ± 78 mmHg,p < 0.01) and theHPD group (I 76 ± 79mmHg,p < O.Ol,ANOVAp <0.005) (Fig. 2). Two animals in the HPD group died within 6 hours of operation. At 3 days after operation, with perfusion to the transplanted lung alone, the arterial P0 2 did not differ significantly among the groups (LPD, 555 ± 8 mmHg;LP,442 ± 85 mm Hg; HPD, 496 ± 31 mmHg; ANOVApnotsignificant) (Fig. 2). One animal in theLP

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Table VI. Bronchial viability of transplanted lung With dextran Without dextran

Group

Viable

LPD HPD LP

4

o

2

4*

5

Necrotic 1

'Compared with groups preserved in flush solutions containing dextran 40. p < 0.05.

group could not tolerate the complete assessment on the third postoperative day because of severe hypotension several minutes after occlusion of the right pulmonary artery. The rest of the animals recovered well. The pulmonary artery pressure and PVR did not differ significantly among the groups, either immediately or 3 days after operation (Tables III, IV, and V). The arterial Peo2 ' r~th perfusion to the transplanted lung alone tended to be hi~ht;'j in the LP and HPD groups at both assessment timtS(Fig. 3). The arterial Pcoj in the LP group was significantly higher than that in the LPD group at both assessment times: 53 ± 3 mm Hg versus 38 ± 2 mm Hg on day 0, p < 0.05; and 56.5 ± 5 mm Hg versus 42 ± 2 mm Hgon day 3,p < 0.01; ANOVAp < 0.05. (Fig. 3). On examination of the transplanted bronchi by the pathologist, nine of lOin the dextran-eontaining group (LPD, five of six; HPD, four of four) were graded as clearly viable, whereas only two of six airways in the LP group were graded as clearly viable (p < 0.05, Fisher's exact test, Table VI). A clearly necrotic bronchus (LP) is

shown in Fig. 4. A well-preserved bronchus (LPD) is shown in Fig. 5. A normal canine bronchus is shown in Fig. 6 for comparison. The mean pathologic score of the LP group (36.1 ± 5.6) was significantly higher than the mean scores of the dextran-eontaining groups (LPD, 23.0 ± 1.9, p < 0.05; HPD, 17.9 ± 4.3, p < 0.05; ANOVA p < 0.05) (Fig. 7) . The LPD and HPD groups did not differ significantly in mean pathologic score. The LP group also had a significant (p < 0.05) increase in submucosal inflammation proximal to the anastomotic site, as compared with the LPD and HPD groups (66.7 ± 14.9 versus 22.3 ± 7.0 and 19.2 ± 9.6, respectively, ANOVA P < 0.05). Discussion We have previously demonstrated that the LPD solution provides significantly superior and more reliable immediate function of the transplanted lung after a 12-hour period of ischemia than does the Euro-Collins solution.P In the present study. we have demonstrated that a low (extracellular) potassium concentration contributes significantly to this improved preservation and that a high (intracellular) potassium concentration is deleterious to lung preservation. We have also shown that dextran 40 contributes not only to the improved transplanted lung function but also to a significant improvement in bronchial viability . The LPD solution mimics the extracellular milieu in

Volume 103 Number 2 February 1992

Dextran 40 and potassium in lung preservation

321

Fig. 5. A well-preserved (LPD) bronchus at the anastomotic site, with a pathologic score of 9. The transplanted bronchus (right) shows intact mucosa and a mild inflammatory infiltrate. The recipient bronchus is on the left. (Hematoxylin and eosin stain. Original magnification X300.)

Fig. 6. A normal canine bronchus, with a pathologic score of O. (Hematoxylin and eosin stain. Original magnification X300.)

that it has a low potassium concentration and a high sodium concentration. High potassium concentrations induce significant pulmonary vasospasm.v 3, 4, 28 In an in vitro rabbit model of lung preservation, we found that solutions containing high potassium concentrations induced significant pulmonary vasospasm, leading to high pulmonary arterial perfusion pressures and poor flush-

ing. 29 A similarly poor result was found with the Belzer (UW) solution, which also has a high potassium concentration (unpublished data). The LPD solution, however, flushed effectively with good results in the rabbit model. Furthermore, high potassium concentrations, initially thought essential to hypothermic organ preservation because of the success of intracellular-type solutions in

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HISTOPATHOLOGY SCORE

50 r - - - - - - - - - - - - - - - - - - - - - - ,

LPD

HPD

LP

FLUSH SOLUTION Fig. 7. The groups with the solutions containing dextran (LPD and HPD) had significantly lower histopathologic scores than had the group without dextran (LP). All values mean ± the standard error of the mean; n = 6 for LPD, LP; n = 4 for HPD. *p < 0.05 compared with LP group.

renal preservation,1,30 have since been shown to have potentially harmful effects on cells preserved under hypothermic conditions.t '? Thus the LPD solution, with its low potassium concentration, likely flushes more effectively (and thus cools more effectively), without inducing further potassiumrelated damage during storage. Our finding that the LPD solution provides superior preservation to that of the HPD solution (Fig. 2, Tables III and V) supports this hypothesis. Further evidence to support this hypothesis is derived from another series of experiments recently reported by our group. 29 In that study, the flushing and storage phases of lung preservation were studied separately in an in vitro rabbit model to determine in which phase of preservation the damage is incurred by lungs preserved with the EuroCollins solution. We found that the intracellular-type Euro-Collins solution causes damage during both the flushing and storage phases oflung preservation, whereas the extracellular-type LPD solution provides better preservation during both phases. The presence of dextran 40 in a lung preservation solution significantly improved transplanted lung function

(Figs. 2 and 3, Tables III and IV) and bronchial viability (Table VI, Fig. 7). There are three possible mechanisms by which dextran 40 could lead to these improvements. First, dextran functions as an oncotic agent, tending to keep water in the intravascular compartment and thereby decreasing interstitial edema formation. A 2% solution of dextran 40, however, represents 0.5 mmol/L, which contributes only 0.5 rrrOsm/L to the solution and exerts a calculated oncotic pressure of about 15to 19mm Hg.3l Therefore, the improvement in lung preservation seen with the addition of dextran 40 in this concentration is greater than one might expect from this agent's oncotic effects alone. A second hypothesis with respect to the function of dextran 40 relates to its potential role as a scavenger of oxygen-derived free radicals. In the in vivo canine single lung transplantation model, we have shown that the dextran 40 in the lung preservation solution contributes significantly to improved posttransplantation pulmonary function (Fig. 2, Tables III and IV). We therefore wondered whether some of the beneficial effects of this agent could be attributed to a superoxide radical-scavenging action. Given the size of the molecules, it is not likely that dextran 40 is able to scavenge superoxide radicals intracellularly. It is, however, present intravascularly and thus available to perform this function at the endothelial cell surface. Furthermore, it is also present throughout the storage phase. More important, it is still present at the reperfusion phase, when the major part of free radicalmediated injury is thought to occur.V However, in another study,32a we failed to demonstrate any superoxide radical-scavenging activity of dextran 40 at the concentrations used in the LPD lung preservation solution. This does not completely rule out a possible scavenging action related to other oxygen radicals. A third, and more attractive, hypothesis relates to the effects of dextran 40 on the microcirculation. Trapped, hypothermic, energy-depleted erythrocytes (such as would be found in an organ stored under hypothermic conditions), exhibit decreased membrane deformability and increased sludging in the microcirculation. I 0, 15 Dextran 40 improves erythrocyte deformability,17, 19 prevents erythrocyte aggregation.P: 24 and induces disaggregation of already aggregated cells. 16, 17,22,33 These effects, which have been attributed to an alteration of surface charge'? and to a conformational change in the erythrocyte surface glycocalyx.r' decrease sludging and improve microvascular flow.35 Improved renal allograft function has been attributed to this phenomenon in studies where dextrans were used in renal preservation. 14, 36-38 Dextran 40 exerts an additional potentially beneficial antithrombotic effect by coating endothelial surfaces, especially at

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sites of injury,24 and coating platelets, thereby interfering with platelet activation.P: 33, 39 Through these actions, this agent could conceivably improve the clearance of red cells and platelets at flushing. Dextran 40 would also be present to exert similar effects on the cells remaining in the vasculature throughout the storage phase and thus could improve reflow at reperfusion. Furthermore, since the dextran 40 is present for some time before being metabolized and excreted, it may exert beneficial antithrombotic and rheologic activity for some time after reperfusion. Despite innovative techniques developed to protect the bronchial anastomosis and to hasten neovascular ingrowth,40,41 donor-airway ischemia continues to be a major problem in clinical lung transplantation.f In vivo, the tracheobronchial tree has a dual blood supply.43-46 Both the systemic bronchial and the pulmonary arterial circulations normally supply the airway. The transplanted bronchus is unique in that it has not had its systemic circulation restored. Its viability therefore depends totally on its blood flow from the pulmonary circulation. Theoretically, improved preservation of the pulmonary parenchymal microcirculation and improved posttransplantation reflow in the microvascular collaterals between the pulmonary and bronchial circulations should lead to improved bronchial viability after transplantation. The improved bronchial viability seen with the addition of dextran 40 (Table VI, Fig. 7) probably reflects both the improved pulmonary parenchymal preservation and improved reflow through the critical microvascular connections between the pulmonary and bronchial circulations. This is likely attributable to the dextran 40, and not simply to the improved oxygenation, since the lungs in the HPD group that had the poorest oxygenation (arterial Po 2, 176 ± 79 mm Hg) (Fig. 2, Table V) still exhibited as significant an improvement in bronchial viability as did lungs in the LPD group. The limitations, however, of histopathologic examination in making inferences on viability must be taken into consideration. Further study is required to prove that the improvement seen is indeed the result of improved posttransplantation microcirculatory blood flow. It has been demonstrated that the provision of oxygen, either in the gaseous state or in the dissolved state, to cells stored under hypothermic conditions is beneficial to preservation.lv 47-49 The exact mechanism for this improvement has not been elucidated, though it seems likely that the presence of oxygen enables the more efficient aerobic metabolism to continue during the ischemic phase, 14,49 albeit at a significantly reduced rate.50 The unique structure of the lung may be ideally suited to take advantage of this phenomenon. By virtue of the short diffusion dis-

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tances between the alveolar space and the epithelial and endothelial cells, the lung is structurally suited to provide direct oxygenation of the ischemic cells throughout the storage phase. This would, in effect, provide a state of aerobic hypothermic storage for the lung, rather than the usual state of anaerobic hypothermia experienced by other ischemic organs. To ensure adequate and comparable oxygenation in the study groups, the donors were ventilated with an inspired oxygen fraction of 1 before removal of the lungs and the lungs were stored in the inflated state at 4 0 C for 12 hours. This should provide an ample supply of oxygen throughout the storage phase, considering the lowered metabolic rate.50 Hypothermia may decrease the rate of chemical reactions thought to be involved in oxygen toxicity (spontaneous and neutrophil- and macrophage-related formation of oxygen-derived free radicalsj'" to the extent that the potentially detrimental effects of oxygen are outweighed by the beneficial effects of maintaining aerobic metabolism. Studies have implicated oxygen in lung injury under conditions of warm ischemia,52 but the role of oxygen in hypothermic lung preservation is not yet clear. 53-55 Further study is required to determine the optimal level of oxygenation. Histopathologic changes within a transplanted lung have been found to be extremely heterogeneous.P with areas of normal lung adjacent to severely damaged areas. This limits the usefulness of lung morphologic examination in assessing adequacy of lung preservation. We therefore did not examine lung histopathologic status in this study. We did however, examine bronchial pathologic status, since in this 3-day survival model the viability of the transplanted bronchus depends totally on blood flow from the pulmonary circulation of the transplanted lung. Experimental studies have shown that bronchial neovascularization from local tissue sources does not occur during this short postoperative period. 40,41 Better preservation of the pulmonary parenchymal microcirculation and improved collateral microvascular reflow could therefore be expected to result in improved bronchial viability. The results of this study indicate that the presence of dextran 40 in a lung preservation solution leads to a significant decrease in the histopathologic abnormalities seen in the transplanted bronchus. These findings indicate that bronchial viability should be included as an additional parameter in the assessment of the adequacy of preservation of the transplanted lung, since the preservation technique can have a significant impact on the viability of the transplanted airway. In summary, we have shown that reliable, reproducible l2-hour preservation, with excellent immediate pulmonary function, is possible with the LPD solution. This

3 24

Keshavjee et al.

approach represents a marked advance over currently available methods of lung preservation. It provides significant improvements in lung preservation and airway ischemia, currently two of the most significant problems in lung transplantation. We have demonstrated that the low potassium concentration is an important component of the LPD solution and that a high potassium concentration is deleterious to hypothermic lung preservation. Further, we have demonstrated that dextran 40 contributes to the excellent pulmonary function and improved bronchial viability observed after a l2-hour period of preservation with the LPD solution. We acknowledge the expert technical assistance provided by J. Mates and S. Diamant. We also thank Ethicon, Inc., Somerville, New Jersey, for providing the suture material. Assistance with statistical analysis was received from K. O'Rourke, Department of Clinical Epidemiology,Toronto General Hospital. REFERENCES 1. Collins GM, Bravo-Shugarman M, Terasaki PI. Kidney preservation for transportation. Lancet 1969;2:1219-22. 2. Devine CE, Somylo AV, Somylo AP. Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles. J Cell Bioi 1972;52:690-718. 3. Van Nueten JM. Calcium entry blockers and vascular smooth muscle reactivity. In: Godfraind T, Vanhoutte PM, Govoni S, Paoletti R, eds. Calcium entry blockers and tissue protection. New York: Raven Press, 1985:6977. 4. Haeusler G. Contraction, membrane potential, and calcium fluxes in rabbit pulmonary artery muscle. Fed Proc 1983;42:263-8. 5. Pegg DE. An approach to hypothermic renal preservation. Cryobiology 1978;15:1-17. 6. Trump BF, Ginn FL. Studies of cellular injury in isolated flounder tubules. II. Cellular swelling in high potassium media. Lab Invest 1968;18:341-51. 7. Gordon EE, Maeir DM. Effect of ionic environment on metabolism and structure of rat kidney slices.Am J Physiol 1964;207:71-6. 8. Tyers GFO, Todd GJ, Niebauer 1M, Manley NJ, Waldhausen JA. The mechanism of myocardial damage following potassium citrate (Melrose) cardioplegia. Surgery 1975;78:45-53. 9. Green CJ, Pegg DE. Mechanism of action of "intracellular" renal preservation solutions. World J Surg 1979;3:115-20. 10. Fuller BJ, Pegg DE. The assessment of renal preservation by normothermic bloodless perfusion. Cryobiology 1976; 13:177-84. 11. Collins GM, Hartley LCJ, Clunie GJA. Kidney preservation for transportation. Experimental analysis of optimal perfusate composition. Br J Surg 1972;59:187-9.

The Journal of Thoracic and Cardiovascular Surgery

12. Moen J, Claesson K, Pienaar H, et al. Preservation of dog liver, kidney, and pancreas using the Belzer-UW solution with a high-sodium and low-potassiumcontent. Transplantation 1989;47:940-5. 13. Fujimura S, Handa M, Kondo T, Ichinose T, Shiraishi Y, Nakada T. Successful 48-hour simple hypothermic preservation of canine lung transplants. Transplant Proc 1987;19: 1334-6. 14. Pegg DE. Organ preservation. Surg Clin North Am 1986;66:617-32. 15. Weed RI, LaCelle PL, Merrill EW. Metabolic dependence of red cell deformability. J Clin Invest 1969;48: 795-809. 16. Atik M. Dextrans, their use in surgery and medicine. Anesthesiology 1966;27:425-38. 1966;27:425-38. 17. Eisenberg S. The effect oflow molecular weight dextran on the viscosityand suspension characteristics of blood. Am J Med Sci 1969;257:336-43. 18. Barnes AD, Dawson-Edwards P, PowisSJA, Thomas DR. Preservation of kidneys for transplantation. Lancet 1972;I: 199-200. 19. Winfrey EW III, Foster JH. Low molecular weight dextran in small artery surgery: antithrombogenic effect.Arch Surg 1964;88:100-4. 20. Bergentz SE, Bergqvist D. Dextran in vascular surgery. Vase Surg 1984;18:51-6. 21. Schatz RA. A view of vascular stents. Circulation 1989;79:445-57. 22. Thorsen G, Hint H. Aggregation, sedimentation and intravascular sludging of erythrocytes. Acta Chir Scand 1950;154:(suppl)I-51. 23. Harker LA, Fuster V. Pharmacology of platelet inhibitors. J Am Coli Cardiol 1986;8:21 B-32B. 24. Bloom WL, Harmer DS, Bryant MF, Brewer SS. Coating of vascular surfaces and cells: a new concept in prevention of intravascular thrombosis. Proc Soc Exp Bioi Med 1964;115:384-6. 25. Keshavjee SH, Yamazaki F, Cardoso P, McRitchie 01, Patterson GA, Cooper JD. A method for safe twelve-hour pulmonary preservation. J THORAC CARDIOVASC SURG 1989;98:529-34. 26. Mullen JBM, Wright JL, Wiggs BR, Pare PD, Hogg rc. Reassessment of inflammation of airways in chronic bronchitis. Br Med J 1985;291:1235-9. 27. SAS/STAT user's guide. Release 6.03 edition. North Carolina: SAS Institute Incorporated, 1988:549. 28. Somylo AV, Vinall P, Somylo AP. Excitation contraction coupling and electrical events in two types of vascular smooth muscle. Microvasc Res 1969;1:354-73. 29. Yamazaki F, Yokomise H, Keshavjee SH, et al. An extracellular type solution is superior to Euro-Collins solutionfor pulmonary preservation. Transplantation. 1990;49:690-4. 30. Sacks SA, Petritsch PH, Kaufman JJ. Canine kidney preservation using a new perfusate. Lancet 1973;1:1024-222. 31. Hint H. Relationships between the chemical and physiochemical properties of dextran and its pharmacological

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effects. In: Derrick JR, Guest MM, eds Dextrans: current concepts of basic actions and clinical applications. Springfield, Illinois: Charles C Thomas, 1971:3-26. 32. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159-63. 32a. Keshavjee SH, McRitchie DI, Vittorini T, Rotstein OD, Slutsky AS, Patterson GA. Improved lung preservation with dextran 40 is not mediated by a superoxide radical scavenging mechanism. J THORAC CARDIOVASC SURG 1992;103:326-8). 33. Bergqvist D. Pharmacologic prevention of thrombotic occlusion in arterial reconstructive surgery. Acta Chir Scand 1983;149:721-7. 34. Baumer H, Halbhuber KH, Stibenz D, Lerche D. Topooptical investigations of human erythrocyte glycocalyx conformational changes induced by dextrans. Biochem Biophys Acta 1987;923:22-8. 35. Atik M. Dextran-40 and dextran-70: a review. Arch Surg 1967;94:664-72. 36. Dempster WJ, Kountz SL, Jovanovic M. Simple kidney storage technique. Br Med J 1964;1:407-10. 37. Manax WG, Block JH, Longerbeam JK, Lillehei RC. Successful 24 hour in vitro preservation of canine kidneys by the combined use of hyperbaric oxygen and hypothermia. Surgery 1964;56:275-82. 38. W usteman MC, Jacobsen lA, Pegg DE. A new solution for initial perfusion of transplant kidneys. Scand J Urol Nephrol 1978;12:281-6. 39. Shoenfeld NA, Yeager A, ConnollyR, eta!' A new primate model for the study of intravenous thrombotic potential and its modification. J Vasc Surg 1988;8:49-54. 40. Lima 0, Goldberg M, Peters W, Ayabe H, Townsend E, Cooper JD. Bronchial omentopexy in canine lung transplantation. J THORAC CARDIOVASC SURG 1982;83:418-21. 41. Morgan E, Lima 0, Goldberg M, Ayabe H, Ferdman A, Cooper JD. Improved bronchial healing in canine left lung reimplantation using omental pedicle wrap. J THORAC CARDIOVASC SURG 1983;85:134-9. 42. Cooper JD. Lung transplantation. Ann Thorac Surg 1989;47:28-44.

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43. Ladowski JS, Hardesty RL, Griffith BP. The pulmonary artery blood supply to the supracarinal trachea. J Heart Transplant 1984;4:40-2. 44. Deffebach ME, Charan NB, Lakshimnarayan S, Butler J. The bronchial circulation. Am Rev Respir Dis 1987;135: 463-81. 45. Charan NB, Albert RK, Lakshimnarayan S, Kirk W, Butler J. Factors affecting bronchial blood flow through bronchopulmonary anastomoses in dogs. Am Rev Respir Dis 1986;134:85-8. 46. Barman SA, Ardell JL, Parker JC, Perry ML, Taylor AE. Pulmonary and systemic blood flow contributions to upper airways in canine lung. Am J PhysioI1988;255:HI130-5. 47. Fischer JH, Czerniak A, Hauer U, Isselhard W. A new simple method for optimal storage of ischaemically damaged kidneys. Transplantation 1974;25:43-9. 48. Bodenhammer RM, DeBoer LMV, Geffin GA, et al. Enhanced myocardial protection during ischemic arrest. J THORAC CARDIOVASC SURG 1983;85:769-80. 49. Buckberg GD. Strategies and logic of cardioplegic delivery to prevent, avoid, and reverse ischemic and reperfusion damage. J THORAC CARDIOVASC SURG 1987;93:127-39. 50. Levy MN. Oxygen consumption and blood flow in the hypothermic, perfused kidney. Am J Physiol 1959;197: 111-4. 51. Said SI, Foda HD. Pharmacologic modulation of lung injury. Am Rev Respir Dis 1989;139:1553-64. 52. Koyama I, Toung TJK, Rogers MC, Gurtner GH, Traystrnan RJ. 02 radicals mediate reperfusion lung injury in ischemic 02 ventilated canine pulmonary lobe. J Appl Physiol 1987;63:111-5. 53. Haverich A, Scott WC, Jamieson SW. Twenty years of lung preservation-a review. J Heart Transplant 1985;4: 234-40. 54. Veith FJ, Montefusco CM. Lung. In: Karrow AM, Pegg DE, eds. Organ preservation for transplantation. 2nd ed. New York: Marcel Dekker Inc, 1981:599-616. 55. Veith FJ, Sinha SBP, Graves JS, Boley SJ, Dougherty rc Ischemic tolerance of the lung: the effect of ventilation and inflation. J THORAC CARDIOVASC SURG 1971;61:804-10.