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Ann Thorac Surg 2001;71:458-461
© 2001 The Society of Thoracic Surgeons

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Original article: general thoracic

Successful retrieval and function of lungs from non-heart-beating donors

^a Departments of Pathology, Medicine and Surgery, The Montreal General Hospital and McGill University, Montreal, Quebec, Canada

Accepted for publication May 6, 2000.

Address reprint requests to Dr Giaid, The Montreal General Hospital, 1650 Cedar Ave, Suite L3-314, Montreal, PQ, Canada H3G 1A4
e-mail: mdga{at}musica.mcgill.ca

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Lung transplantation has been used effectively as a therapeutic tool in end-stage pulmonary diseases, but organ shortages have restricted its use. There is growing interest in alternative organ sources such as organs from circulation-arrested cadavers, so called non-heart-beating donors.

Methods. We examined the effects of postmortem rapid in situ cadaver lung cooling by bilateral chest cavity flushing (group 2) and by pulmonary artery flush through right heart catheterization followed by pleural cavity flushing (group 3) on pulmonary function and morphology in a rabbit non-heart-beating donor model. The results were compared with those in a control group of heart-beating donors (group 1).

Results. At the end of a 2-hour reperfusion period, there were no significant differences in mean pulmonary artery pressure, pulmonary vascular resistance, pulmonary compliance, arteriovenous oxygen, pulmonary wet to dry weight ratio, and lung morphology between the three groups.

Conclusions. Our study demonstrates that using bilateral chest cavity flushing with or without pulmonary flush protects the function and morphology of cadaver lungs and renders them suitable for lung transplantation.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
Lung transplantation is limited by the scarcity of good donor organs. It is estimated that less than 10% of available multiorgan donors have lungs suitable for transplantation [1]. Currently, the majority of donor organs are retrieved from brain-dead patients who still possess physiologic function (ie, a beating heart). These patients are referred to as heart-beating donors. Another source of potential donors that to date remains untapped is the large number of individuals who died as a result of irreversible cardiac arrest. Use of organs from non-heart-beating donors (NHBDs) could increase the number of transplantations performed. However, obtaining consent for organ donation and organizing organ retrieval consume precious time. During this interval, organs could be protected against ongoing destruction by preservation inside the cadaver.

Rapid cooling of perfused organs by in situ flush with cold crystalloid solutions forms the basis of any solid-organ preservation before transplantation [1]. Lungs preserved in this way can be transplanted safely after up to 8 hours of cold ischemia. Minimally invasive techniques to salvage and properly preserve these organs quickly are required to reduce the period of warm ischemia as much as possible and to allow optimal preservation during the time required to obtain consent.

We hypothesized that metabolic inhibition in preservation of the lung could be achieved almost exclusively by hypothermia. We therefore investigated whether lung preservation in situ using continuous cold flush through bilateral pleural cavities with or without lung flush by way of right heart catheterization would be a simple and reliable method to prolong lung viability after circulatory arrest.

	Material and methods

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Study groups
Forty-five New Zealand White male rabbits weighing 3 to 3.5 kg were used. Fifteen animals served as organ donors and 30 as blood donors. Three experimental groups (n = 5 in each) were set up. In group 1, normal control lungs from heart-beating donors were flushed with 50 mL/kg of cold modified Krebs-Henseleit bicarbonate-buffered (KHBB) solution through the pulmonary artery (PA) and stored semi-inflated in 4°C saline solution for 4 hours. In group 2, after circulatory arrest, the pleural cavities were infused continuously with cold saline solution for 2 hours prior to PA flush with 50 mL/kg of KHBB solution. Lungs were subsequently stored semi-inflated for 2 additional hours in cold saline solution. In group 3, after circulatory arrest, the PA was flushed in situ with 50 mL/kg of KHBB solution using a right heart catheter inserted peripherally and the pleural cavities were cooled with saline solution for 2 hours. Circulation-arrested rabbits (groups 2 and 3) were not ventilated during the 2 hours before lung procurement. Lungs were harvested and stored for 2 additional hours in cold saline solution.

All animals were given humane care in compliance with the Animal Care Committee regulations of The Montreal General Hospital and McGill University, 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" published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Surgical procedure
The rabbits were premedicated and anesthetized with an intramuscular injection of ketamine hydrochloride (25 mg/kg), xylazine hydrochloride (4 mg/kg), and acepromazine maleate (1.25 mg/kg). The animals were intubated through a cervical tracheostomy with a cannula whose inner diameter was 3.5 mm, and the lungs were ventilated using a Harvard rodent ventilator (model 683) with room air (respiratory rate, 30 breaths/min; tidal volume, 10 mL/kg of body weight; positive end-expiratory pressure, 2 cm H₂O). In group 1, the lungs were harvested as described previously [2]. Topical cooling was done with cold (4°C) saline solution slush. After the PA flush, the inflow and outflow cannulas were clamped. The tracheal tube was clamped semi-inflated, and the lung-heart block was excised and stored in 4°C normal saline solution for 4 hours.

In group 2, after intravenous heparinization, the rabbits were killed by ligating the ascending aorta and caval veins, thus resulting in cardiac arrest. Both the endotracheal cannula and the PA catheter remained in place. The chest was closed layer by layer. After circulatory arrest, the pleural cavities were infused continuously with 4°C saline solution for 2 hours from a height of 50 cm by way of chest tubes inserted through the second intercostal space at the midclavicular line into the pleural cavities bilaterally. The drainage tubes were placed through the seventh intercostal space at the posterior axillary line. After the 2-hour period, the chest was reopened, and 50 mL/kg of modified KHBB solution at 4°C with 30 µg of prostaglandin E₁ (alprostadil; The Upjohn Company, Don Mills, ON, Canada) was flushed into the PA through the PA catheter from a height of 30 cm. The lung-heart blocks were harvested and subsequently stored semi-inflated in 4°C cold saline solution for 2 additional hours.

In group 3, the rabbits were heparinized and killed by clamping the ascending aorta and caval veins. An 18-gauge intravenous Teflon catheter was introduced through the right internal jugular vein into the right atrium. After the inferior vena cava was ligated and the descending aorta cut at the phrenic level, 50 mL/kg of modified KHBB solution at 4°C was infused into the lung through the right heart catheter from a height of 50 cm. The chest was then closed layer by layer, and a pleural cavity flush was done for 2 hours as group 2. Thereafter, the chest was reopened, and the PA and the left atrium were cannulated through the right and left ventricles, respectively. The lung-heart block was excised and stored semi-inflated in 4°C cold saline solution for 2 more hours.

Thirty rabbits, 2 for each rabbit in the three experimental groups, served as blood donors. After induction of anesthesia and heparinization (700 IU/kg), the animals were exsanguinated by means of a 14-gauge intravenous Teflon catheter inserted into the right ventricular apex immediately after a sternotomy was made. About 250 mL of blood was collected and stored in a blood transfusion bag. The modified KHBB solution was added to each aliquot of blood to achieve a hematocrit of 15% at 4°C until needed for perfusion. After 4 hours of ischemia, the lung-heart blocks were set up in a reperfusion-ventilation circuit described elsewhere [2].

Measurements and analyses
Blood samples were collected simultaneously from the pulmonary venous and arterial sides at 5, 15, 30, 60, 90, and 120 minutes after the start of reperfusion for blood gas analyses. Pulmonary artery, left atrial, and mean airway pressures were recorded continuously. At the end of the 2-hour reperfusion period, tissue samples were obtained for determination of wet to dry weight ratios and histologic analysis. Small pieces of representative lung tissue (1 cm²) from the three lobes were cut, fixed in paraformaldehyde, and embedded in paraffin. Sections were stained with hematoxylin and eosin.

All values are expressed as the mean ± the standard error. Differences between groups were assessed by analysis of variance. A p value of less than 0.05 was considered significant.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
There were no significant differences in donor weight, total ischemia time, and perfusate hematocrit between groups. Mean hematocrit for all groups combined was 15.3% ± 1.2%. Values for mean PA pressure (mPAP), pulmonary vascular resistance (PVR), pulmonary compliance, and arteriovenous oxygen for the three groups are presented in Figure 1. In all three groups, adequate arteriovenous oxygen differences were maintained throughout the 2 hours of reperfusion with diluted blood. At the end of the 2-hour period, the functional results in every group were within normal ranges: (mPAP—I 14.6 ± 2.0 mm Hg in group 1, 16.0 ± 1.7 mm Hg in group 2, and 13.6 ± 3.2 mm Hg in group 3; PVR—12.5 ± 1.6 10³ dyne · s · cm^-5 in group 1, 16.0 ± 2.3 10³ dyne · s · cm^-5 in group 2, and 11.5 ± 4.5 10³ dyne · s · cm^-5 in group 3; pulmonary compliance—3.8 ± 0.5 mL/mm Hg in group 1, 3.9 ± 0.4 mL/mm Hg in group 2, and 4.7 ± 0.4 mL/mm Hg in group 3; and arteriovenous oxygen—126.5 ± 7.1 mm Hg in group 1, 100 ± 20.5 mm Hg in group 2, and 127 ± 19.8 mm Hg in group 3. Wet to dry weight ratios measured at the end of the study were not significantly different between the three groups: 6.8 ± 0.4 in group 1, 6.4 ± 0.3 in group 2, 7.3 ± 0.4 in group 3 (P = 0.26).

View larger version (32K): [in this window] [in a new window]   Fig 1. Pulmonary hemodynamics and function in rabbit lungs from beating-heart donors (group 1) and from non-heart-beating donors with bilateral chest cavity flush (group 2) and pulmonary artery flush (group 3). The effects on (A) mean pulmonary artery pressure (mPAP) (B) pulmonary vascular resistance (PVR) (C) pulmonary compliance, and (D) arteriovenous oxygen (V-A O2) are shown.

Histologic analyses for all three groups showed normal parenchymal architecture with normal-appearing airways and vessels. There were only a few scattered inflammatory cells, with no signs of interstitial edema or hemorrhage.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
Lungs from NHBDs have not been used in the clinical setting of lung transplantation. However, transplantation of lungs retrieved from animal cadavers after cardiac arrest has been the focus of investigation by many lung transplant groups. We have demonstrated in the present study that in situ cooling of lungs from NHBD allows the maintenance of normal pulmonary function and morphology 2 hours after reperfusion. This method of preservation, although different from the model of exsanguination, may open new sources and thus provide a larger pool of tissue available for lung transplantation, thereby benefiting a large number of patients awaiting lung transplantation.

Previous studies conducted by the North Carolina Lung Transplant Group [3] have indicated that in a canine lung transplantation model, lungs harvested 1 hour after cessation of circulation without any cooling of the animal show adequate gas exchange function. Subsequently, this group [4] found that oxygen ventilation of cadaver lungs during in situ ischemia may be beneficial for lung viability, maintenance of tissue levels of adenosine triphosphate, and gas exchange function after transplantation. However, lungs harvested after more than 2 hours of warm in situ ischemia demonstrate significantly poor pulmonary function [3]. Moreover, lungs from NHBDs subjected to 60 minutes of warm ischemia before harvest have impaired bronchial healing on the basis of both bronchoscopic and histologic findings but adequate pulmonary function at 7 days after transplantation [5]. Thus, the deleterious effects of an obligatory period of warm in situ ischemia seem to be the major limiting factor in NHBD lung transplantation.

Rapid cooling to accomplish reversible metabolic inhibition forms the basis of any solid-organ preservation before transplantation. Although hypothermia prolongs the tolerance of the lung for ischemia, the ideal storage temperature has not been defined. Recent evidence suggests that the optimal temperature for prolonged storage is in the range of 7° to 10°C [6]. However, the natural postmortem decline in lung temperature inside the warm cadaver is slow. The core temperature of the lung in the rabbit cadaver was still more than 30°C after 2 hours of circulatory arrest. In contrast, when cadavers were submerged in an ice bath (1°C) after initial topical lung cooling with cold (1°C) saline solution, the core temperature of the lung decreased to less than 20°C after 2 hours of cooling. In lungs flushed in situ with a cold (4°C) preservative solution through the PA immediately after cardiac arrest, lung temperature decreased to less than 10°C within 5 minutes [7]. At this temperature, enzymatic metabolic rate is suppressed about tenfold [1]. Using this method, our data showed normal values for mPAP, PVR, blood gas, and pulmonary compliance 2 hours after reperfusion. As such, in situ single flush of the PA should be used in the retrieval of lung from NHBDs, as this results not only in immediate homogeneous cooling of the lung, but also in clearance of harmful blood constituents such as fibrin, complement, platelets, and leukocytes.

Steen and coworkers [8] suggested in 1997 that in situ topical cooling of deflated lungs from NHBDs seems to permit a period of at least 6 hours before the lungs need to be harvested. This group applied systemic anticoagulation, cooled the lungs for 6 hours with saline slush placed in both pleural cavities through a small left-sided anterior thoracotomy after cardiac arrest, and then transplanted the lungs. All transplant recipients (pigs) remained in excellent condition with good lung function throughout the 24-hour observation period. In the present experimental setting, we compared results in rabbit lungs from heart-beating donors (control group), with results in rabbit cadaver lungs harvested after in situ cooling by continuous infusion of the pleural cavities with cold (4°C) saline solution for 2 hours (group 2) or PA flush with cold (4°C) preservative solution through a right heart catheter before cold infusion of the pleural cavities (group 3). Groups 2 and 3 had good hemodynamic and physiologic function after 2 hours of reperfusion with diluted deoxygenated blood (hematocrit 15%) in an ex vivo closed-circuit ventilation-perfusion apparatus. Right heart catheterization and cold infusion of the pleural cavity not only achieve rapid cooling inside the lung after circulatory arrest without a sternotomy or a thoracotomy before family consent is obtained for organ donation, but also provide an easy way to accomplish pulmonary circulatory anticoagulation immediately after resuscitation stops. When a cadaver lung that was not infused was treated with anticoagulation, the percentage of pulmonary nonviable cells was about 18.1% in the deflated lung and 7.9% in the lung inflated with oxygen after 24 hours of cold (4°C) storage [9].

From this study, we conclude that the technique of lung preservation in situ using continuous cold infusion through bilateral pleural cavities with or without PA flush by way of right heart catheterization may be a simple and reliable method to mitigate ischemia-reperfusion injury in NHBDs. We plan to extend our findings with long-term follow-up of transplanted lungs from NHBDs to investigate lung allograft injury by ischemia plus reperfusion.

	References

Top Abstract Introduction Material and methods Results Comment References

 

1. Belzer F.O., Southard J.H. Principles of solid-organ preservation by cold storage. Transplantation 1988;45:673-676.[Medline]
2. Shennib H., Kuang J.-Q., Ohlstein E.H., Giaid A. Endothelin receptor antagonist improves pulmonary hemodynamics during lung ischemia/reperfusion injury. Transplantation 1998;66:917-920.[Medline]
3. Egan T.M., Lambert C.J., Jr, Reddick R., Ulicny K.S., Jr, Keagy B.A., Wilcox B.R. A strategy to increase the donor pool: use of cadaver lungs for transplantation. Ann Thorac Surg 1991;52:1113-1121.[Abstract]
4. D’Armini A.M., Tom E.J., Roberts C.S., Henke D.C., Lemasters J.J., Egan T.M. When does the lung die? Time course of high energy phosphate depletion and relationship to lung viability after "death". J Surg Res 1995;59:468-474.[Medline]
5. Binns O.A., DeLima N.F., Buchanan S.A., et al. Impaired bronchial healing after lung donation from non-heart-beating donors. J Heart Lung Transplant 1996;15:1084-1092.[Medline]
6. Cooper J.D., Vreim C.E. NHLBI workshop summary. Biology of lung preservation for transplantation. Am Rev Respir Dis 1992;146:803-807.[Medline]
7. Van Raemdonck D.E.M., Jannis N.C.P., Rega F.R.L., De Leyn P.R.J., Flameng W.J., Lerut T.E. External cooling of warm ischemic rabbit lungs after death. Ann Thorac Surg 1996;62:331-337.[Abstract/Free Full Text]
8. Steen S., Ingemansson R., Budrikis A., Bolys R., Roscher R., Sjöberg T. Successful transplantation of lungs topically cooled in the non–heart-beating donor for 6 hours. Ann Thorac Surg 1997;63:345-351.[Abstract/Free Full Text]
9. Kuang J.-Q., Van Raemdonck D.E.M., Jannis N.C.P., et al. Pulmonary grafts should be inflated with 100% oxygen prior to hypothermic storage. J Heart Lung Transplant 1997;16:99.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Hani Shennib Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shennib, H. Articles by Giaid, A. Search for Related Content PubMed PubMed Citation Articles by Shennib, H. Articles by Giaid, A. Related Collections Lung - transplantation