Lung preservation with low-potassium dextran flush in a primate bilateral transplant model

Lung Preservation With Low-Potassium Dextran Flush in a Primate Bilateral Transplant Model Sudhir Sundaresan, MD, Oriane Lima, MD, Hiroshi Date, MD, Akihide Matsumura, MD, Hiroharu Tsuji, MD, Hidefumi Obo, MD, Motoi Aoe, MD, Takatoshi Mizuta, MD, and Joel D. Cooper, MD Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, Barnes Hospital, St. Louis,

Missouri

We used a bilateral lung transplant model to confirm, in primates, the results of lung preservation studies previously obtained in a canine single-lung transplant model. The donor lungs were flushed with low-potassium dextran solution and maintained semiinflated with 100% oxygen at 10°C for a planned ischemic time of 12 hours for the lung implanted first. Of eight experiments performed, results in the 6 operative survivors form the basis of this report. After bilateral lung transplantation, animals were maintained on a ventilator for 6 hours; arterial oxygen tension, pulmonary artery pressure, and pulmonary vascular resistance were determined in the recipients at 2, 4, and 6 hours after transplantation and compared with donor values, which served as controls.

Arterial oxygen tension in the recipients did not differ from the controls ( p = not significant), whereas the pulmonary artery pressure and pulmonary vascular resistance showed significant elevation ( p < 0.05 versus control values). After the 6 hours of assessment, the animals were extubated and 3 survived for 48 to 72 hours with a mean arterial oxygen tension of 69 mm Hg on room air. These results demonstrate excellent lung function after a minimum of 12 hours of preservation in a primate model in which the animal is totally dependent on the function of transplanted lung tissue, and confirm the potential for prolonged clinical lung preservation.

A

taken to verify, in a primate bilateral allotransplantation model, the satisfactory results previously obtained in the canine single-lung transplant model. With this primate model, postoperative lung function (and hence, animal survival) depends totally on the satisfactory preservation of the lung allografts, and therefore attests to the adequacy of the preservation method in question. Our objective is to identify a preservation method that not only provides superior lung function after prolonged ischemic periods but also is simple and routinely applicable for clinical use.

s lung transplantation has become increasingly successful, the need for suitable donor organs has become correspondingly more acute. The currently accepted "safe" ischemic time of 6 to 8 hours imposes certain restrictions on the logistics of procuring and distributing donor lungs. Improvements in lung preservation would increase the number of usable donor lungs by permitting lung retrieval from greater distances, and by promoting the sharing of the two donor lungs between different centers. On the basis of recent experimental investigations, we have concluded that (1) the lung appears to be able to utilize the oxygen present in the alveoli to maintain aerobic metabolism during preservation [l, 21; (2) although cold ischemia is preferable to normothermic ischemia, excessive hypothermia may be injurious, either by producing direct cold injury or by eliminating the low level of metabolic activity that appears crucial in preserving homeostasis [3, 41; and (3) if a low level of metabolic activity is to be maintained during preservation with maintenance of cellular homeostasis, then the use of an extracellular preservation solution, such as low-potassium dextran (LPD) (as opposed to the currently used EuroCollins solution, an intracellular-type solution) might be more appropriate [5-71. The current study was underAccepted for publication Jan 18, 1993 Address reprint requests to Dr Sundaresan, Division of Cardiothoracic Surgery, Washington University School of Medicine, Suite 3107, Queeny Tower, One Barnes Hospital Plaza, St. Louis, MO 63110.

0 1993 by The Society of Thoracic Surgeons

(Ann Thoruc Surg 1993;56:1129-35)

Material and Methods General Eight bilateral lung transplant procedures were performed using weight-matched pairs (28 to 33 kg) of adult male chacma baboons (Pupio cynocephulis unubis).

Donor Procedure The donor animals were sedated with intramuscular ketamine (10 to 15 mg/kg), then anesthetized with sodium thiopental (10 mg/kg). The animals were intubated and placed on a mechanical ventilator using a tidal volume of 400 to 450 mL, a respiratory rate of 10 breaths per minute, an inspired oxygen fraction of 1.0, and 5 cm H,O of positive end-expiratory pressure. Femoral cannulation was employed to insert an arterial catheter and a pulmonary artery (PA) catheter. The systemic arterial, PA, and right atrial pressures were monitored by means of a model 0003-4975/93/$6.00

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1290A transducer (Hewlett-Packard, Chelmsford, MA) and recorded on a Gould eight-channel recorder. After median stemotomy, both venae cavae, the ascending aorta, main PA, and trachea were isolated, and the azygos vein was divided. Systemic heparinization (500 Ukg) was then performed, and a 6.5-mm curved metal-tipped perfusion cannula (Sarns, Inc, Ann Arbor, MI) was inserted into the main PA just distal to the valve, secured with a pursestring suture, and connected by means of tubing to the bag of cold (4°C) LPD solution, which was hung at a height of 40 cm above the chest. Finally, after dissection of the interatrial groove, inflow occlusion was achieved by ligation of both venae cavae. After division of the inferior vena cava near the right atrium and amputation of the tip of the left atrial appendage, the lungs were flushed with 1.5 L of LPD solution. Simultaneous topical cooling was achieved by flooding the chest with cold saline solution (1"to 4°C). Flush pressure was not deliberately monitored; the adequacy of the flush was gauged by the speed and uniformity of blanching of the lungs. At the completion of the flush, the trachea was stapled, leaving the lungs semiinflated with 100% oxygen, and the double-lung block was excised, placed in a sterile plastic bag containing cold LPD solution, then maintained at 10°C for 12 hours (overnight). The composition of LPD solution was as follows: Na K c1 Mg PO, Dextran 40 PH Osmolarity

168 mmol/L 4 mmoVL 103 mmol/L 2 mmoUL 37 mmol/L 20 g/L 7.45 280 mOsm/L

Recipient Procedure The recipient animals were given oral cyclosporine (15 mg/kg) and azathioprine (1.5 mg/kg) on the night before the operation. The following morning, they were sedated, anesthetized, intubated with a standard endotracheal tube, and ventilated as for the donors, with the exceptions that anesthesia was maintained during the subsequent procedure with 1.5% to 2.0% halothane and an inspired oxygen fraction of approximately 0.4 was used. Methylprednisolone, 500 mg, was given as an intravenous bolus on induction. Percutaneous insertion of a femoral artery catheter and PA catheter (via the femoral vein) was then performed for monitoring during the procedure. With the animal in the supine position, a transverse thoracosternotomy incision was made employing the fourth intercostal space. The right and left hila were then completely mobilized, including the pulmonary arteries, veins, and main bronchi. The right and left donor lungs were then separated and prepared for implantation in a basin containing cold saline solution while the recipient left lung was removed. The pulmonary artery and veins were divided between ligatures, whereas the bronchus was divided distal to a heavy ligature. Because the left lung was implanted first, the right lung was returned to

Ann Thorac Surg 1993;56112%35

10°C storage until it was required for implantation. Implantation was performed using previously described standard techniques [8], with the order of anastomoses as follows: bronchus, PA, and left atrium. During implantation, the donor lung was wrapped in cold gauze and periodically irrigated with cold saline solution. During the bronchial anastomosis (with the recipient airway open), the endotracheal tube was advanced into the proximal contralateral main bronchus and then returned to its midtracheal position after reperfusion of the graft. In the first 2 recipient animals of this study, partial cardiopulmonary bypass (CPB) at normothermia was deliberately employed to protect the newly implanted left lung (by diverting a portion of blood flow from it) during implantation of the second lung. After systemic heparinization (500 Ukg), CPB was established via a single right atrial venous cannula (28F Bard wire-wound; Bard, Tewksbury, MA) and an ascending aortic cannula (16F Bard), using a Scimed disposable pediatric membrane oxygenator (SciMed, Maple Grove, MN). In the remaining 6 recipients, this approach was discarded and CPB was used only as necessary during implantation of the right lung if there was an unacceptable increase in PA pressure (to greater than 50 mm Hg), decrease in systemic arterial pressure (to less than 80 mm Hg), or decrease in arterial oxygen tension (PaO,) (to less than 100 mm Hg). Such use of CPB was required in three of the last six experiments. The right lung was implanted using a technique similar to that for the left side, except that during the bronchial anastomosis, the endotracheal tube was advanced into the proximal left main bronchus. After implantation of both lungs, chest drains were inserted and the chest was closed. Intercostal nerve blocks were performed on both sides using 0.5% bupivicaine. The animals were then maintained anesthetized in the supine position and ventilated for 6 hours for the purpose of data collection (arterial blood gases, hemodynamics, and radiologic studies-see below). After this period, they were weaned from ventilatory support, and when stable, the chest drains and monitoring lines were gradually removed. Once the animal showed satisfactory spontaneous ventilation on room air, it was extubated and returned to its cage. In surviving recipients, an oral postoperative immunosuppression regimen consisting of cyclosporine A (15 mg * kg-' day-'), azathioprine (1.5 mg kg-' * day-'), and methylprednisolone (0.5 mg kg-' * day-') was instituted. Administration of intramuscular antibiotics (gentamicin, 40 mg/day; and penicillin, 900,000 U/day) was also continued until death of the recipient.

-

-

Assessment ARTERIAL BLOOD GASES AND HEMODYNAMIC ASSESSMENT.

Arterial blood gases were obtained using an inspired oxygen fraction of 1.0, 5 cm H,O of positive endexpiratory pressure, respiratory rate of 10 per minute, and tidal volume of 400 to 450 mL. Hemodynamic parameters were also measured (systemic, PA, right atrial, and pulmonary capillary wedge pressures). Cardiac output was determined in triplicate by the thermodilution method (cardiac output computer model 9520A; Baxter Edwards

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Ann Thorac Surg 1993;56:112935

Table 1. Primate Bilateral Lung Transplantation: Summary of Preservation, Use of Cardiopulmonary Bypass and Outcome of Six Overative Survivors Ischemic Time (h) Experiment

Left

Right

CPB Used

1

14:OO

16:07

yes

2

1200

14:OO

yes

3

13:OO

14:20

no

4 5 6 7

13:51 12:22 12:15 13:02

15:14 13:57 14:28 15:41

no yes yes no

8

11:49

15:03

yes

12:47 f 16

14:51 f 16

Mean 2 SEM

ABG = arterial blood gases; postoperative day.

CPB

=

Clinical Course

Postmortem Findings

Weaned easily from CPB; clinically well for first 3 days; satisfactory CXR, ABG on 3rd day (PaO,, 89 mm Hg on room air); died POD 5. Weaned easily from CPB; died POD 1.

Left lower lobe atelectasis; thrombus in atrial suture line of left lung

Well on POD 1; assessed and sacrificed on POD 2 due to impending respiratory failure; satisfactory ABG at sacrifice (PaO,, 63 mm Hg on room air). Died POD 1. Operative death Operative death Well for first 5 days; died POD 6; satisfactory CXR and ABG on POD 3 (PaO,, 55 mm Hg on room air). Weaned easily from CPB; died POD 1.

cardiopulmonary bypass;

CXR

Division, Irvine, CA). These measurements were obtained in all donor animals (just before lung extraction) and in all surviving recipients at 2, 4, and 6 hours after lung reperfusion. All recipient animals underwent chest roentgenography and quantitative perfusion lung scanning between the 4-hour and 6-hour assessments. Lung scanning was performed using a portable Technicare camera (with on-board 560 computer) (Johnson and Johnson, Cleveland, OH) after administration of 1 mCi of technetium 99m macroaggregated albumin. RADIOLOGIC ASSESSMENT.

”LATE” ASSESSMENTS. A follow-up arterial blood gas measurement (on room air) and chest roentgenogram were obtained at 48 hours in 1 animal and at 72 hours in 2 animals.

After the death of each recipient, the heart-lung block was carefully examined grossly and then fixed in formalin. Slides were prepared from each upper and lower lobe using hematoxylin and eosin stain, and reviewed with one pathologist to permit a uniform histologic evaluation of the lungs for evidence of acute lung injury. The following changes were sought and were rated as follows: vascular congestion and architectural disruption (0 = none, 1 = minimal, and 2 = severe), and edema and hemorrhage (0 = none, 1 = interstitial, 2 = alveolar-mild, and 3 = alveolar-severe). PATHOLOGIC ASSESSMENT.

All data are represented as mean 5 standard error of the mean. Values obtained in recipient animals during posttransplantation assessments were STATISTICAL METHODS.

=

chest roentgenogram;

Diffuse hemorrhagic areas throughout right lung Atelectasis and consolidation of left lower lobe

Negative

...

... Thrombus in atrial suture line of left lung Negative

PaO, = arterial oxygen tension;

POD

=

compared for statistical significance with those obtained in donor animals (considered as controls) using the Tukey HSD multiple comparisons test.

Animal Care All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research, the ”Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Science and published by the National Institutes of Health (NIH Publication #86-23, revised 1985).

Results Summary of Preservation, Conduct of Experiment, Survival, and Gross Pathologic Findings Low-potassium dextran flush produced rapid and uniform blanching of the lungs in all experiments. The 1.5 L of flush was generally delivered within 1 to 2 minutes. The ischemic time of the left and right lung, as well as the necessity for CPB in each experiment, are shown in Table 1. The mean ischemic time of the left lung was 12 hours 47 minutes 16 minutes, and for the right lung, 14 hours 51 minutes 16 minutes. Cardiopulmonary bypass was used electively in the initial two experiments and of necessity in three of the remaining six experiments. The duration of survival for the recipient animals as well as the clinical course and postmortem findings in the operative survivors are also shown in Table 1. In experiments 5 and 6, profound hypotension was noted during CPB and high doses of vasopressors were used in an effort to correct this. In both cases, the animal could only be

* *

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SUNDARESAN ET AL LPD IN PRIMATE BILATERAL LUNG TRANSPLANTATION

Table 2. Primate Bilateral Lung Transplantation:Results of Posttransplantation Assessments PaO, (mm Hg; Group Donors (n = 8) Recipients (n = 6) 2h 4h 6h a

FiO, = 1.0, 5 cm H,O

PEEP)

569

f 20

486 502 467

2

20

f 26 f

25

Mean PA Pressure (mm Hg)

PVR

(dyne-sl

cm

*

m')

13.2 f 1.9

121 f 25

25.3 23.7 23.0

430 471 505

f 2.9" f 2.7" f 4.6"

2

58"

* 55" f 39"

p < 0.05 versus donor value.

PaO, = PA = pulmonary artery; FiO, = inspired oxygen fraction; PEEP = positive end-expiratory pressure; arterial oxygen tension; PVR = pulmonary vascular resistance.

weaned from bypass using massive inotropic support, and subsequent hemodynamic assessment showed a very low cardiac output state. Hence, these animals were considered to have operative deaths, and data from the remaining 6 operative survivors were used for analysis. Recipients 2, 4, and 8 died on the first postoperative day despite having had satisfactory lung function at the 6-hour assessment (see below); animals 4 and 8 had no significant postmortem findings. Recipient 2 had hemorrhagc areas throughout the right lung diffusely. Recipients 1 and 7 appeared well clinically for several days postoperatively and were available for "late" assessments (see below); these animals died on the 5th and 6th postoperative day, respectively, of uncertain cause. In recipient 3, respiratory failure developed on the second postoperative day. The animal was intubated, assessed, and then sacrificed. The left lower lobe was found to be atelectatic and consolidated.

Ann Thorac Surg 1993;56:1129-35

Results of "Late" Assessments Three animals were amenable to a "late" assessment within 3 days of operation. The 2 longest survivors (animals 1 and 7, which survived for 5 and 6 days, respectively) each underwent chest roentgenography and arterial blood gas measurement (on room air) on the third postoperative day. Animal 3, as previously noted, was studied on the second postoperative day. The results of the room air blood gas measurements on these 3 animals are summarized in Table 1; the lowest PaO, obtained was 55 mm Hg, and the mean value was 69 mm Hg. Animals 1 and 7 also had satisfactory appearing chest roentgenograms, as demonstrated in Figure 2.

Histologic Assessment Review of the slides prepared from the operative survivors consistently showed features of interstitial and occasionally mild alveolar edema, mild vascular congestion, interstitial and occasionally alveolar hemorrhage, and only occasional architectural disruption of a minimal to severe degree. There was considerable variability in the histologic appearance even within the same slide. The well-preserved alveolar architecture from one specimen is exemplified in Figure 3.

Comment The purpose of this study was to verify, in a primate bilateral allotransplant model, the satisfactory results of lung preservation studies previously obtained from rabbit and canine models. In these experiments, all pulmonary gas exchange occurs in transplanted lungs that have been subjected to a minimum of 12 hours of ischemic preservation. Although there was no separate control or comparison group, we used the donor animals as an internal control against which the transplanted lung function could be compared. We wished to confirm the satisfactory

Arterial Blood Gas and Hemodynamic Assessments The PaO, (using an inspired oxygen fraction of 1.0 and 5 cm H,O of positive end-expiratory pressure), the mean PA pressure, and the calculated pulmonary vascular resistance of the donor animals and the recipients at 2, 4, and 6 hours after reperfusion are shown in Table 2. There was no significant difference in PaO, between donor and recipient animals. There was a significant elevation of the PA pressure and pulmonary vascular resistance (PVR) in the recipient animals at all three assessment times compared with the donor controls ( p < 0.05). Although there appeared to be an upward trend in the PVR values with time, these values were not significantly different from each other.

Radiologic Assessments All animals had a very satisfactory appearing initial chest roentgenogram, as exemplified in Figure 1. The quantitative perfusion lung scans demonstrated that the left lung (implanted first) received 35% f 1.3% of the flow, whereas 65% f 1.3% was directed to the right lung (implanted second).

Fig 1. Appearance of immediate posttransplantation chest roentgenogram.

Ann Thorac Surg 1993:56:1129-35

outcome of rabbit and canine experiments using this sequential bilateral lung transplant model (similar to the clinical procedure) before assuming that similarly satisfactory results could be anticipated in humans. The excellent gas exchange obtained in these experiments suggests the potential for achieving similar results in the human situation. Haverich and co-workers, in their comprehensive review of the status of lung preservation [9], pointed out the confusion ensuing from the conflicting results obtained over the years by various investigators. Much of the confusion has stemmed from the fact that numerous different models have been used to assess lung preservation, and thus there is no "standard" method. We have previously discussed the merits of the various animal models [6, 101. Ex vivo rabbit models have satisfied the basic requirement of permitting evaluation of lung function via oxygenation and measurement of PVR, have been simple to perform and inexpensive, and have provided reproducible data; however, their main value has been in preliminary screening of the many variables in lung preservation, and hence in vivo models are ultimately required. Large animal (ie, canine, primate) models become necessary to evaluate quantitatively the results of any preservation method; small animal lung transplantation (eg, rat) has been successfully performed, but yields considerably less information regarding preservation, because recipient survival is the only end point. Canine autotransplantation allows evaluation of the same pulmonary effects of ischemic preservation as an allograft model, without the superimposed effects of rejection; however, these are technically much more complex, and lead to logistic problems such as the dilemma of management of the recipient animal during prolonged autograft preservation. Canine single-lung allotransplantation (usually left lung) has become a frequently used model, but has some limitations. First, the recipient can survive on the native lung even if the allograft sustains a severe

Fig 2 . Appearance of chest roentgenogram on third postoperative day.

SUNDARESAN ET AL LPD IN PRIMATE BILATERAL LUNG TRANSPLANTATION

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Fig 3 . Histologic appearance of transplanted lung after death of animal I on fifth postoperative day. Specimen from right upper lobe. (Hematoxylin and eosin stain; original magnification x 100.)

ischemic injury. Second, evaluation of graft function requires exclusion of the native lung. This can be accomplished permanently (either concomitant to the transplantation or as an interval procedure) through contralateral pneumonectomy or PA ligation. However, these manipulations have historically been demonstrated to carry a very high mortality (which is unrelated to the adequacy of the preservation method), and also would create an unphysiologic situation in which the freshly transplanted lung handles the entire cardiac output, thereby placing an excessive burden on the graft. We therefore have described the use of an inflatable cuff implanted around the native pulmonary artery to allow temporary selective perfusion of the grafted lung in order to evaluate its function alone. The bilateral lung transplant model obviates some of these problems; there is distribution of the cardiac output between two transplanted lungs, and determination of lung function will reflect only graft function and therefore the adequacy of preservation. However, this model is not ideal either. The lung implanted first is required to handle the entire cardiac output while the second lung is being implanted. Furthermore, if CPB is used to "protect" the first lung, another variable is introduced that may affect posttransplantation lung function and may also diminish survival of the recipient. Preservation techniques for lung and heart have been evaluated using combined heart-lung transplantation in bovine and canine models [ll-151. However, that model adds the additional variables associated with myocardial preservation and posttransplantation cardiac function, which can influence lung function. This makes the combined heart-lung transplant model

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less suitable for the study of lung preservation per se. With our model, we had hoped to achieve the additional benefit of long-term survival, but found the postoperative management of these ferocious animals to be too complex to be mastered within a limited number of experiments. Given these limitations, plus the costs involved, the primate bilateral lung transplant model is probably impractical for routine use in experimental lung preservation. Conversely, the data yielded by this model are highly pertinent in influencing future changes in our clinical lung preservation strategy. The current preservation method was based on the results of several prior animal studies, which evaluated preservation temperature, inflation of the lungs, and composition of the flush solution. Although it is intuitively understood that cold ischemia is better tolerated than warm ischemia, the optimum temperature for lung preservation is yet to be determined. We systematically evaluated the effect of temperature on the ischemic tolerance of the lung using the simple rabbit ex vivo model [3], and demonstrated satisfactory function in lungs for 12 hours longer at 10°C preservation than at 4°C. Using the canine left lung transplant model, we confirmed this in vivo by demonstrating significantly better PaO, in lung allografts stored for 18 hours at 10°C compared with 4°C [4]. The documentation by Hendry and associates [16] of reversible myocardial cold injury in canine hearts at a preservation temperature of 0°C suggests that a similar mechanism may operate in the lung; the detrimental effect may be either direct cold injury or elimination of a low level of metabolism required to maintain cellular homeostasis. There is substantial evidence from previous reports that lung grafts tolerate ischemic preservation better when stored inflated rather than deflated [17]. We have previously demonstrated in a rabbit lung model the importance of lung inflation with oxygen during 24-hour preservation. Those studies demonstrated the ability of the lung to utilize the oxygen for maintenance of aerobic metabolism, without depletion of high-energy phosphate levels and without excess lactate production [l, 21. The essential features of flush solutions for use in solid organ preservation have been reviewed by Belzer and Southard [18]. One fundamental feature of their University of Wisconsin solution is its intracellular nature; this is thought to minimize the tendency toward cellular swelling during ischemia (a consequence of cessation of action of the adenosine triphosphate-dependent sodiumpotassium pump). Modified Euro-Collins solution (an intracellular solution) is still used almost uniformly in clinical lung preservation. Fujimura and colleagues [5] reported on successful canine lung preservation up to 48 hours using extracellular solutions that featured low molecular weight dextrans and a phosphate buffer as key components. These solutions were the basis of the current LPD solution, and systematic comparisons between EuroCollins solution and LPD have been carried out, both in vitro [7] and in vivo [6]. Yamazaki and co-workers [7] demonstrated significantly higher PaO, and lower flush pressures for LPD (compared with Euro-Collins) using the simple ex vivo rabbit model. We also demonstrated sig-

Ann Thorac Surg 1993;56:1129-35

nificantly higher immediate postreperfusion PaO, in lungs flushed with LPD in a canine in vivo study [6]. The exact basis for these apparent benefits of LPD solution are unknown, but possible explanations include (1)the lower potassium content is less likely to induce vasospasm during flushing leading to more uniform flushing and cooling as demonstrated by Kimblad and associates [19]; (2) the phosphate buffer is more efficient in minimizing tissue acidosis; and (3) the dextran may have an oncotic effect (minimizing the accumulation of extravascular lung water) as well as preventing red cell aggregation, thereby leading to greater uniformity of flushing. Keshavjee and colleagues [20] have systematically evaluated the components of LPD and reported that the dextran-40 contributes significantly to the lung preservation achieved by LPD. They pointed out that the concentration of dextran-40 (20 g/L) was insufficient to account for its beneficial effects solely on the basis of an oncotic pressure effect, implying that it works through other mechanisms. Several parameters were chosen to assess the adequacy of preservation in this study and, based on these, immediate postreperfusion lung function was excellent. This was evidenced by the PaO, in excess of 400 mm Hg (and not significantly different from the baseline donor values), as well as the satisfactory radiologic appearance of the lungs. The PVR did show significant elevation over the donor values; this elevation may be attributed to various factors, including restriction imposed by the PA anastomosis, hypoxic injury to the pulmonary vasculature with resultant perivascular edema, and possibly the effects of pulmonary denervation on pulmonary vascular reactivity. The perfusion lung scans consistently showed about 35% of perfusion to the left lung and 65% of perfusion to the right lung. This is compatible with the larger size of the right lung, and comparable with results obtained from similar scanning of normal dogs. (We did not obtain perfusion scans on the normal donor animals before harvest because the presence of the isotope in the lungs would have affected the results of the early postoperative lung scans). The phenomenon of elevated PVR has been demonstrated by Fujimura and associates [21] to be the primary hemodynamic derangement after canine bilateral lung autotransplantation. They showed, however, that the elevated PVR returned to baseline values in long-term survivors. Formal assessment to evaluate PVR on the third postoperative day was not performed in this study. However, in a canine bilateral lung allotransplant model, we too have found return of the PVR to normal levels at 3-day follow-up (unpublished data). Despite the fact that several parameters suggested excellent immediate graft function, survival of the animals was modest. In only 1 case did postmortem examination of the lungs reveal any overt signs of lung injury. In the remaining cases, the findings were not judged to be of a severity to have caused the death of the animal. The baboon, being an extremely ferocious animal, was found not to be amenable to the delivery of any ”postoperative care” in our hands. Thus, the inability to monitor the animals or to maintain adequate hydration, electrolyte balance, and delivery of antibiotics and immunosuppression, along with the extensive magnitude of the opera-

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tion, all likely contributed to the demise of the animals. Although a formal “3-day” cardiopulmonary assessment was not routinely obtained, the satisfactory PaO, (on room air) and chest roentgenograms obtained in 3 animals at the 48- to 72-hour interval attested further to the adequacy of the lung preservation. Histologic evaluation of the specimens showed varying degrees of pathologic change (usually mild), including edema, hemorrhage, congestion, and architectural disruption. However, as pointed out by Haverich and associates [9], the tremendous heterogeneity and variability of these changes documented previously [22] imply that such changes do not correlate with significant functional impairment, and hence morphology may be an unsuitable parameter to evaluate lung preservation unless extensive morphometric studies are performed. In this study, partial CPB was used to facilitate right lung implantation in certain cases. It is noteworthy that both operative deaths occurred when CPB was used. In these animals, the systemic perfusion pressure was profoundly depressed and refractory to numerous vasopressors; both animals could never be weaned adequately from CPB. One animal suffered ventricular fibrillation before going on CPB, and although defibrillated almost immediately, cardiac function suffered significantly. These experiments did not use bolus administration of prostaglandin El into the PA before flushing as is used in clinical practice. In subsequent animal experiments, the use of prostaglandin E, as an adjunct to PA flushing has been demonstrated to be of significant value (unpublished results). Although we did not accomplish the hoped-for longterm survival in these experiments, this study has shown that lungs flushed with LPD solution and preserved semiinflated with 100% oxygen at 10°C provided excellent early graft function in a primate bilateral lung transplant model after 12 hours of ischemia. Undoubtedly further improvements in lung preservation methods will be developed, but these primate experiments strongly support the notion that safe preservation of human lungs for a prolonged period is achievable.

Addendum Based on these experiments we have subsequently had the confidence to proceed with several bilateral human lung transplants when the anticipated ischemic time for the second lung exceeded 9.5 hours. In all cases, the outcome was successful and the function of the lungs was not adversely affected by the prolonged ischemic time. Supported by National Institutes of Health grant R01 HL41281. We wish to gratefully acknowledge the contributions of the following: technical assistance from Michael Lischko, Barbara Gehrin, Dennis Gordon, Timothy Morris, Donna Marquart, and Gary Crancer; pharmacologic assistance from Ed Casabar; secretarial support from Dawn Schuessler; and statistical advice and assistance from Richard B. Schuessler, PhD.

References 1. Weder W, Harper B, Shimokawa S, et al. Influence of intra-alveolar oxygen concentration on lung preservation in a rabbit model. J Thorac Cardiovasc Surg 1991;101:103743.

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