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Ann Thorac Surg 2006;82:1219-1225
© 2006 The Society of Thoracic Surgeons
a Department of Cancer and Thoracic Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
b Department of Surgery, University of North Carolina, Chapel Hill, North Carolina
c Department of Pathology, University of North Carolina, Chapel Hill, North Carolina
Accepted for publication May 4, 2006.
* Address correspondence to Dr Egan, Division of Cardiothoracic Surgery, University of North Carolina, 3040 Burnett-Womack Building, CB 7065, Chapel Hill, NC 27599-7065 (Email: ltxtme{at}med.unc.edu).
Presented at the Poster Session of the Forty-second Annual Meeting of The Society of Thoracic Surgeons, Chicago, IL, Jan 30–Feb 1, 2006.
| Dr Egan discloses that he has a financial relationship with Vitrolife.
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Abstract |
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METHODS: One hour after death, Sprague-Dawley rat heart–lung blocks were flushed with 20 mL of cold Perfadex, stored cold for 1 hour, then warmed to 37°C in a circuit perfused with Earle's crystalloid solution with or without washed porcine erythrocytes (hematocrit 12% to 15%). At 37°C, lungs were ventilated for 15 minutes with alveolar gas, perfusion-cooled to 20°C, flushed again with cold Perfadex, and then stored cold for 2.5 hours. The left lung was transplanted using a modified cuff technique with flow probes on the main and left pulmonary arteries. After 1 hour of reperfusion, arterial blood gases from the left pulmonary vein and wet/dry weight ratio (W/D) of both donor lungs were determined. Lungs transplanted after retrieval from heart-beating or non-heart-beating donors served as controls (n = 6 per group).
RESULTS: Lungs gained weight in the circuit but W/D and PO 2 were similar after transplantation for all groups. After transplantation, vascular resistance was higher and dynamic compliance was lower for lungs perfused in the circuit. Myeloperoxidase and conjugated diene levels were modestly elevated in lungs transplanted from non-heart-beating donors irrespective of perfusion in the circuit.
CONCLUSIONS: Rat lungs are suitable for transplant after ex-vivo perfusion and ventilation. This model closely mimics methods used to evaluate the function of lungs retrieved from human non-heart-beating donors and can economically evaluate ex-vivo therapies for lungs retrieved from non-heart-beating donors.
Lung transplantation has extended and improved the lives of thousands of patients with end-stage lung disease but is severely constrained by an inadequate donor supply from conventional brain-dead organ donors. If lungs could be retrieved for transplant from non-heart-beating donors (NHBDs) at intervals after circulatory arrest and death, then the lung donor shortage could be eliminated.
We and others have demonstrated the feasibility of lung transplantation from NHBDs in animal models [1–7]. Events before death may play a crucial role in suitability of lungs retrieved from NHBDs, however. Exsanguination preceding cardiac arrest was associated with poorer lung function in an isolated perfused rat lung model [8]. In the clinical scenario, variable warm ischemic times and other premorbid events may influence lung function after transplantation. Thus, there is a pressing need to develop a reliable method to predict function of lungs retrieved from NHBDs before widespread use of NHBD lungs for transplant can occur.
Steen [9] described a method to rewarm, perfuse, and ventilate porcine NHBD lungs ex-vivo, with excellent gas exchange after transplantation. He used an identical circuit to evaluate and transplant lungs from a human NHBD [10]. Aitchison and colleagues [11] used a similar circuit to assess porcine lungs retrieved 2 hours after circulatory arrest by perfusion with deoxygenated blood at 500 mL/min and demonstrated that gas exchange after lung transplantation from these NHBDs was identical to lungs retrieved immediately after arrest. Rega and colleagues [12] used an ex-vivo ventilation and perfusion circuit to show equivalent gas exchange in porcine lungs retrieved from NHBDs after 1 hour of in situ warm ischemia and 3 hours of cold atelectatic ischemia compared with lungs flushed with cold Perfadex (Vitrolife, Kungsbacka, Sweden) immediately after arrest. All of these systems use leukocyte-free reperfusate, which may help to reduce ischemia–reperfusion injury.
We have established an ex-vivo ventilation and perfusion circuit to rewarm human lungs and assess gas exchange [13]. We plan to use this circuit to evaluate human lungs retrieved from NHBDs for transplant suitability. It would be ideal to have a similar animal model to test therapeutic strategies to mitigate ischemia–reperfusion injury in lungs retrieved from NHBDs. Unfortunately, large-animal models are resource intensive and very costly. Because of our laboratory's extensive experience with an isolated perfused rat lung model [14–19] and rat lung transplantation [4, 20], we developed a model to rewarm, perfuse, and ventilate rat lungs retrieved from NHBDs before lung transplantation. This model closely mimics the ex-vivo perfusion and ventilation system we developed to evaluate human lungs from NHBDs for transplant suitability.
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Material and Methods |
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Donor Lung Retrieval
Sprague-Dawley rats were anesthetized with intraperitoneal pentobarbital sodium (7.5 mg/100 g; Abbott Laboratories, Chicago, IL). Heparin (600 U; Elkins-Sinn, Cherry Hill, NJ) was injected intrahepatically after laparotomy. After tracheotomy with a 14-gauge catheter, donor rats were sacrificed with intrahepatic pentobarbital sodium (15 mg/100 g). One hour after cardiac arrest, a median sternotomy was performed, the right ventricle was cannulated with p60 tubing, and the left atrial appendage was incised to vent the pulmonary circulation. The heart–lung blocks were flushed with 20 mL of cold Perfadex and stored at 4°C for 1 hour.
The Ex-Vivo Perfusion/Ventilation Circuit
The donor heart–lung blocks were removed from cold storage, and the left atrium was cannulated through the left ventricle for perfusion in the circuit. Left atrial appendages were closed with vascular clips. The heart–lung blocks were suspended from a Grass force-weight transducer (Grass-Telefactor, Astro-Med, Inc, West Warwick, RI) to continually record lung weight, and were perfused through the pulmonary artery by using a Minipuls 3 peristaltic pump (Gilson Medical Electronics, Middleton, WI) in a modification of our isolated perfused rat lung model (Fig 1). The perfusate was Earle's balanced salt solution (ICN Biomedical Inc, Aurora, OH) containing (in mmol) 2.4 CaCL2
.2H2O, 0.4 MgSO4 (anhydrous), 5.4 KCl, 116 NaCl, 0.88 NaH2PO4 (anhydrous), 5.5 D-glucose, and 0.3 phenol red containing 0.21% NaHCO3 and 4% bovine serum albumin (Sigma Chemical, St. Louis, MO) (NHBD-crys, n = 6), or Earle's solution supplemented with leuko-filtered saline-washed porcine red blood cells, reconstituted to a hematocrit of 12% to 15% (NHBD-blood, n = 6). We used porcine erythrocytes at this hematocrit to closely mimic the human circuit and as an oxygen carrier and a source of free radical scavengers. Porcine erythrocytes (6 µm dry diameter) are slightly smaller than rat erythrocytes (6.5 µm) [21].
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Pulmonary artery and airway pressure were continuously monitored with transducers attached to National Instruments (Austin, TX) hardware connected to a computer through National Instruments software with continuous real-time display. Perfusion flow rate was adjusted to maintain pulmonary artery pressure of less than 20 mm Hg. When the circuit temperature reached 37°C, the lungs were mechanically ventilated (Harvard rodent respirator model 681, Harvard Apparatus Co, Millis, MA) with alveolar gas (5% CO2, 20% O2, 75% N2) at 60 breaths/min and a positive end-expiratory pressure of 3 cm H2O. Airway pressure was maintained at less than 30 cm H2O, with a maximum tidal volume of 3 mL. Perfusate pH was continuously monitored with an Accumet pH probe (Fisher Scientific, Pittsburgh, PA) in the venous reservoir. The pH was maintained near 7.40 by adding dilute HCl or NaHCO3 as necessary.
Ventilation and perfusion continued for 15 minutes at 37°C to simulate ex-vivo gas exchange assessment (as performed in the human circuit). The lungs were then perfusion-cooled to 20°C, flushed with cold Perfadex and stored cold for 2.5 hours before left lung transplantation into a recipient.
Technique of Rat Lung Transplantation
We have used a modified cuff technique to perform left single-lung transplantation in Sprague-Dawley rats [4, 20]. At the time of left lung preparation, the trachea is harvested with the left lung, and the right mainstem bronchus is ligated, so that the allograft can be ventilated separately from the native right lung in the anesthetized recipient [22]. Thus, left lung airway pressure can reflect dynamic compliance. Recipients were anesthetized, intubated, and ventilated with a Harvard rodent ventilator (Model 683) that delivered 100% oxygen with a tidal volume of 0.75 mL/100 g at a rate of 60 breaths/min and a positive end-expiratory pressure of 2 cm H2O. After left lung transplantation, the lungs were ventilated separately, with right/left tidal volume ratio of 2:1. Transonic flow probes (Transonic Systems, Inc, Ithaca, NY) were placed around the recipient main pulmonary artery and the left pulmonary artery to measure total pulmonary artery flow and left pulmonary artery flow as a measure of relative vascular resistance of the graft. The unused donor right lung was assessed for wet/dry (W/D) weight ratio and a portion fixed in formalin.
After 1 hour of reperfusion after transplant, arterial blood gases were measured with an i-STAT device (Abbott Point of Care, East Windsor, NJ) from a blood sample drawn from the left pulmonary vein (the transplanted lung) and the left ventricle. The animals were sacrificed, transplanted lungs were retrieved for W/D measurements, and transplanted lung tissue was partitioned. A portion was placed in formalin, and the remainder was flash frozen in liquid nitrogen and stored at –80°C for later analysis of conjugated dienes (CD) and myeloperoxidase (MPO) activity.
Experimental Groups
Lungs transplanted after retrieval from 6 heart-beating donors (HBD) or from 6 NHBDs retrieved 1 hour after arrest (NHBD) with 6.5 or 5.5 hours' cold storage, respectively, served as controls. Lungs perfused in the ex-vivo circuit were retrieved from NHBDs 1 hour after arrest, flushed with cold Perfadex, and stored cold for 1 hour before perfusion in the circuit (approximate time, 2 hours). They were then stored cold for an additional 2.5 hours. Thus, the four groups had similar total ischemic time from cardiac arrest in the donor to reperfusion in the recipient. In the circuit, lungs were perfused with either Earle's solution (NHBD-crys, n = 6) or Earle's solution supplemented with washed porcine erythrocytes (NHBD-blood, n = 6).
Histologic Analysis
Pieces of the donor right and left (transplanted) lungs were fixed in 10% buffered formalin. Paraffin-embedded sections were prepared using standard techniques and stained with hematoxylin and eosin. Specimens were evaluated by one of us (WF) in a masked manner to assess the degree of reperfusion injury and lung architecture.
Analytic Assessments
WET/DRY ratio
A portion of each frozen biopsy was weighed, then dried in a 60°C oven for 48 hours and reweighed to determine the W/D ratio.
Myeloperoxidase activity
Frozen lung tissue was homogenized in 50 mM Hepes (pH 8.0) at 2% dry w/v with a Rotor-stator homogenizer (Fisher Scientific) in 2 mL cryovials. Samples were centrifuged at 10,000 rpm for 30 minutes at 4°C, and 75 µL of the supernatant was added to wells of a 96-well plate to which 75 µmL of substrate solution (3 mM tetramethylbenzidine, 120 µM resorcinol, and 2.2 mM H2O2 in deionized water) was added. The negative control was 75 µL of 4 N H2SO4. The reaction was stopped after 2 minutes by adding stop solution (cold 4 N H2SO4) to all wells. The optical density was read at 450 nm and results expressed as OD/mg dry weight of lung.
Conjugated dienes
Lung CDs were determined as a surrogate for free-radical-mediated tissue damage [23]. Frozen lung tissue (50 to 75 mg) was homogenized in distilled water (5 mL/g tissue) for 1 minute. The homogenate was extracted in a mix of chloroform/methanol 2:1 (v/v), vortexed, and centrifuged at 1000g for 10 minutes. The lower organic layer was washed twice with 0.003 M HCl and centrifuged again. The final organic layer was dried under rotary evaporation and resuspended in 1.5 mL heptane. CD concentration was detected spectrophotometrically at 234 nm using a Beckman DU-6 UV-Visible spectrophotometer (Beckman Coulter, Inc, Fullerton, CA) against a heptane blank. Results are expressed as units of optical density per milligram of dry weight of lung tissue.
Statistics
Comparisons were made between groups by analysis of variance with Tukey's post hoc test for multiple comparisons or unpaired t tests by using Statistica software (Tulsa, OK). Calculation of means and standard errors was performed with Excel 2000 (Microsoft, Redmond, WA). All values are reported as the mean ± standard error of the mean (SEM). Differences were considered significant when p < 0.05.
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Comment |
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We attribute the superior performance of HBD and NHBD lungs in the current experiments to Perfadex flush. After 1 hour of reperfusion, the W/D ratio of transplanted lungs was similar among all four groups, as was PO 2 from the left pulmonary vein. Ex-vivo perfusion and ventilation resulted in modest weight gain in the circuit, reflected by modestly higher W/D (Fig 3). CD and MPO were no higher in lungs perfused in the circuit compared with lungs transplanted from NHBDs not perfused in the circuit. The addition of porcine erythrocytes to the circuit perfusate had little impact on lung function measured 1 hour after transplantation, although weight gain in the circuit was less. Vascular congestion was equally apparent in crystalloid-perfused or porcine erythrocyte-perfused NHBD lungs. Thus, ex-vivo perfusion and rewarming in the rat is associated with a mild amount of additional injury after transplant.
This model was designed to closely mimic our human lung ex-vivo perfusion and ventilation circuit, which can evaluate lungs obtained from NHBDs for transplant suitability [13]. Because ex-vivo perfusion and ventilation provides a unique opportunity to treat the lung before transplantation to minimize ischemia–reperfusion injury, we can use this rodent model to evaluate the effect of a variety of interventions to improve lung function after transplantation. Examples of such interventions include using agents that reduce capillary leak upon reperfusion or selectively inhibiting pathways responsible for gene transcription. Aitchison and colleagues [24] used a porcine model to show that ex-vivo nitric oxide ventilation of a lung retrieved from a NHBD resulted in better lung function after transplant.
Ex-vivo perfusion of NHBD lungs before lung transplantation may be analogous to ischemic preconditioning, wherein some degree of protection from ischemia–reperfusion injury is conferred by brief periods of antecedent ischemia before a prolonged ischemic insult, first described in myocardium [25]. Ischemic preconditioning improves lung function in animal models of lung ischemia–reperfusion injury [26, 27].
Because of the persistent shortage of suitable lungs for transplantation from the pool of brain-dead cadaver donors, interest has been growing in using lungs retrieved from NHBDs. Love and colleagues [28] reported five lung transplantations from donors whose life support was electively withdrawn. However, respiratory failure is often a reason to terminate life support, making this population inadequate to meet the demand for acceptable lungs. Interest has also been increasing in our hypothesis that lungs might be successfully transplanted after interval retrieval from NHBDs. Steen [10] reported a successful lung transplantation from a NHBD after ex-vivo gas exchange assessment. Varela has reported 16 lung transplantations from NHBDs after in vivo assessment [29].
In recent years, several investigators have reported ex-vivo lung perfusion and ventilation systems in large animals to evaluate lung function before transplantation [9, 11, 30]. Large-animal systems have several advantages. By incorporating a membrane deoxygenator, the perfusate can be deoxygenated and CO2-loaded, and gas exchange across the donor lung(s) can be assessed. Experience with such a circuit can be readily translated into the clinical arena for ex-vivo perfusion and ventilation of human lungs [10, 30]. However, large-animal models are expensive, as are disposable oxygenators. The rodent model we describe cannot assess gas exchange ex-vivo, but it is relatively inexpensive. Gas exchange and other parameters of pulmonary function can be obtained after transplant, however. This model offers the possibility of investigating several therapeutic strategies, with agents delivered either through the pulmonary artery or into the airway, or both, in a relatively economical manner, with an option to test the most promising strategies in the more resource-intensive large animal or human model.
To simulate the human ex-vivo circuit as closely as possible and to add more colloid and free radical scavengers, we wanted to add erythrocytes to the perfusate at the same hematocrit that we and others have used in human or large-animal circuits. To use rat erythrocytes would require sacrifice of several animals for each experiment. We investigated the use of washed (outdated) human red cells, but vascular resistance was excessive when washed human cells were used. The advantages of porcine cells are that they are readily available, can be obtained fresh, and are relatively inexpensive. We attribute the failure to perfuse rat lungs with human red cells to their larger size compared with rat erythrocytes [21].
In summary, we developed a system to perfuse, rewarm, and ventilate rat lungs in an ex-vivo circuit and demonstrated feasibility of performing left single-lung transplantation after ex-vivo perfusion and ventilation. This system can be manipulated to closely mimic ex-vivo perfusion and ventilation systems currently used to evaluate human lungs for transplant suitability. This model is a valuable tool to evaluate practical strategies to reduce ischemia–reperfusion injury in the setting of lung transplantation from NHBDs.
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Requirements for Recertification/Maintenance of Certification in 2006 |
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TopAbstractIntroductionMaterial and MethodsResultsCommentRequirements for...AcknowledgmentsReferences |
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The American Board of Thoracic Surgery will no longer publish the names of individuals who have not recertified in the American Board of Medical Specialties directories. The Diplomate's name will be published upon successful completion of the recertification/maintenance of certification process.
The CME requirements are 70 Category I credits in either cardiothoracic surgery or general surgery earned during the 2 years prior to application. SESATS and SESAPS are the only self-instructional materials allowed for credit. Category II credits are not allowed. The Physicians Recognition Award for recertifying in general surgery is not allowed in fulfillment of the CME requirements. Interested individuals should refer to the Booklet of Information for a complete description of acceptable CME credits.
Diplomates should maintain a documented list of their major cases performed during the year prior to application for recertification. This practice review should consist of 1 year's consecutive major operative experiences. If more than 100 cases occur in 1 year, only 100 should be listed.
Candidates for recertification/maintenance of certification will be required to complete all sections of the SESATS self-assessment examination. It is not necessary for candidates to purchase SESATS individually because it will be sent to candidates after their application has been approved.
Diplomates may recertify the year their certificate expires, or if they wish to do so, they may recertify up to two years before it expires. However, the new certificate will be dated 10 years from the date of expiration of their original certificate or most recent recertification certificate. In other words, recertifying early does not alter the 10-year validation.
Recertification/maintenance of certification is also open to Diplomates with an unlimited certificate and will in no way affect the validity of their original certificate.
The deadline for submission of applications for the recertification/maintenance of certification process is May 10 each year. A brochure outlining the rules and requirements for recertification/maintenance of certification in thoracic surgery is available upon request from the American Board of Thoracic Surgery, 633 N St. Clair St, Suite 2320, Chicago, IL 60611; telephone: (312) 202-5900; fax: (312) 202-5960; e-mail: info{at}abts.org. This booklet is also published on the website: www.abts.org.
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  G. Zanotti, M. Casiraghi, J. B. Abano, J. R. Tatreau, M. Sevala, H. Berlin, S. Smyth, W. K. Funkhouser, K. Burridge, S. H. Randell, et al. Novel critical role of Toll-like receptor 4 in lung ischemia-reperfusion injury and edema Am J Physiol Lung Cell Mol Physiol, July 1, 2009; 297(1): L52 - L63. [Abstract] [Full Text] [PDF]
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 P. A. Corris and J. D. Christie Update in Transplantation 2006 Am. J. Respir. Crit. Care Med., March 1, 2007; 175(5): 432 - 435. [Full Text] [PDF]
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