Compensatory Growth of Porcine Right Lungs After Chronic Rejection of Transplanted Left Lungs

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1 Compensatory Growth of Porcine Right Lungs After Chronic Rejection of Transplanted Left Lungs Scott E. Langenburg, MD, Lorne H. Blackbourne, MD, Scott A. Buchanan, MD, Michael C. Mauney, MD, Stephen S. Kim, BA, Kimberly N. Sinclair, MS, John A. Kern, MD, Sandeep S. Teja, BA, Curtis G. Tribble, MD, and Irving L. Kron, MD Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Virginia Health Sciences Center, Charlottesville, Virginia Neonatal lung hypoplasia is frequently a fatal condition often associated with congenital diaphragmatic hernia. Unilateral lung transplantation rarely has been performed for this indication, although it is a potential solution. It is not known whether the transplant needs to function permanently or to act as a bridge until the native lung develops. It is also not known whether the native lung will grow in the face of an immunosuppressed state and chronic rejection of the transplanted lung. We therefore developed a porcine model of left lung rejection to study this. Infant swine underwent left lung transplantation. Chronic rejection occurred in all, resulting in nonfunction of the transplanted lung. The right lungs of these animals were compared with the right lungs of size-matched and age-matched control animals not given immunosuppressive treatment and not undergoing trans- plantation. There were no differences in terms of the functional residual capacity, airway compliance, and airway resistance among the groups. There was a significant increase in the pulmonary vascular resistance in the animals with transplanted lungs. There was also a significant increase in the lung weight in these animals. Unilateral pneumonectomies were done in 4 infant pigs to serve as controls. Three of the 4 did not survive the operation because of acute pulmonary failure. In conclusion, the study group exhibited evidence of compensatory growth that was not seen in the control animals, as shown by the increase in lung weight. This suggests that contralateral lung growth occurs in a growing animal, despite the effects of immunosuppression therapy and chronic rejection of the transplanted lung. (Ann Thorac Surg 1995;59:28-32) ung transplantation is emerging around the world as a L treatment option for children with end-stage lung disease. Of particular interest is the performance of lung transplantation in infants with severe pulmonary hypoplasia [1]. Severe pulmonary hypoplasia in the neonatal period is secondary to congenital diaphragmatic hernia, oligohydramnios-induced pulmonary hypoplasia, or congenital cystic adenomatoid malformation of the lung. In infants with these malformations that result in severe pulmonary hypoplasia, there are currently no viable treatment options other than lung transplantation [2]. It has been speculated that lung transplantation may only be needed to serve as a temporary measure in infants with severe pulmonary hypoplasia due to congenital diaphragmatic hernia [3]. In these patients, the transplant would need to function only until the hypoplastic native lung has developed sufficiently to support respiratory function. Rejection continues to be a major problem in patients who undergo lung transplantation, due to the high antigenicity of lung tissue. Lung transplants have been shown to be associated with more episodes of rejection than any other transplanted organ [4]. It is thought, Presented at the Thirtieth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 31-Feb 2, Address reprint requests to Dr Kron, Department of SurgeD,, University of Virginia Health Sciences Center, Box , Charlottesville, VA however, that neonates who undergo transplantation have a distinct advantage over older patients in that they are victims of fewer rejection episodes and require less immunosuppression therapy [3]. Despite this immune privilege, pediatric patients with lung transplants are still subject to suffering acute and chronic rejection [5, 6]. A long-term immunosuppression regimen also has deleterious effects on the growing child. It has been shown that survivors of diaphragmatic hernia experience nearly normal pulmonary function over time [7-10]. In the ideal scenario, the transplanted lung would function until the native lung grows, thus averting the adverse effects of long-term immunosuppression therapy. If a patient with severe pulmonary hypoplasia due to congenital diaphragmatic hernia undergoes transplantation, will the native lung develop normal function in the face of a immunosuppressed state and chronic rejection? This study was designed to answer this question by evaluating the development and function of the contralateral, native lung of an infant swine that has undergone lung transplantation and subsequent rejection of the transplanted lung. Material and Methods Animal Model and Animal Care Immature Hanford minipigs (8 weeks old; Charles River, Wilmington, MA) were chosen for this study because of 1995 by The Society of Thoracic Surgeons /95/$ (94)00826-S

2 Ann Thorac Surg LANGENBURG ET AL ;59:28-32 EXPERIMENTAL LUNG TRANSPLANTATION their rapid postnatal lung development [11-13] and their smaller size at maturity, and also because of our familiarity with their tolerance of thoracotomy and lung transplantation. All experimental protocols were reviewed and approved by an institutional animal use committee. All animals have received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication no , revised 1985). Three animal groups were studied. The first was the control group (n = 4). These animals did not undergo transplantation and did not receive any imrnunosuppressive drugs. They were matched with the second group by age and size. The second group was the study group (n = 5). All study animals underwent left whole lung transplantation according to the method described previously by our group [14, 15]. The third group underwent unilateral pneumonectomy without transplantation. All transplantations were performed by the same surgeons using a consistent surgical technique and method of preservation (ice-cold, heparinized physiologic normal saline solution). All animals, both recipients and donors, were of similar age ( weeks) and body weight ( kg). Immunosuppression and Postoperative Care On the day before transplantation, recipient animals received cyclosporine (18 mg" kg 1. day 1, given orally; Sandoz Pharmaceutical, East Hanover, NJ), which was continued daily until sacrifice. The animals also received methylprednisolone (500 mg intravenously) at the time of transplantation and daily for 5 days, as well as azathioprine (1 mg/kg intravenously) at the time of transplantation and daily until sacrifice. Acute rejection was diagnosed when tachypnea, fever, lethargy, and dry cough occurred. These episodes were treated empirically with intramuscularly administered methylprednisolone (500 mg daily) for 3 days. Chest roentgenograms were obtained at 7 days and monthly thereafter. The 5 study animals all went on to suffer chronic rejection of the transplanted lung. At 10 months of age, both the control and study groups underwent evaluation of pulmonary hemodynamics, functional residual capacity, and airway mechanics under the same anesthetic. At the completion of these measurements, the lungs were harvested through a median sternotomy and weighed. A small piece of lung was excised from the posterior lower lobe in all the animals for calculation of the wet-to-dry lung weight ratio. The rest of the lung was preserved with 10% buffered formalin solution. The animals were euthanized with a lethal dose of pentobarbital. Calculation of Pulmonary Vascular Resistance The control and study group animals were anesthetized with ketamine (10 mg/kg) and intubated, and final chest roentgenograrns were obtained. The animals were maintained with intermittently administered intravenous pentobarbital and ventilated mechanically (15 ml/kg tidal volume, 12 breaths/min, inspired oxygen fraction of 1.0). Carotid artery and pulmonary artery catheters were placed through a cutdown access to the carotid artery and internal jugular vein after a tracheostomy tube was inserted. Arterial and pulmonary arterial pressures were continuously transduced and cardiac outputs were measured in triplicate using a thermistor-tipped Swan-Ganz catheter. The pulmonary capillary wedge and left atrial pressures were also measured. The pulmonary vascular resistance (PVR) was calculated using the following formula: PVR = (rnpap - PCWP) 80/CO, where CO is the cardiac output, PCWP is the pulmonary capillary wedge pressure, and mpap is the mean pulmonary artery pressure. Calculation of Functional Residual Capacity The left lungs in the control animals were isolated in vivo by placing intrabronchial balloon catheters, under bronchoscopic control, to occlude the left mainstem bronchus. The functional residual capacity of the right lung was then calculated using the helium dilution technique [16]. The functional residual capacities of the right lung were obtained in the study animals using the same technique, but without the need to isolate the left lung because of its absence, which was confirmed by bronchoscopic evaluation. Calculation of Airway Mechanics With the animals anesthetized and mechanically ventilated, as already described, airflow and transrespiratory pressure were measured at the tracheostomy tube using a Fleisch pneumotachometer (no. 2, linear to 3,000 ml/s with a dead space of 40 ml; OEM, Richmond, VA) and two differential pressure transducers (MP45-1; Validyne Engineering, Northridge, CA). The signals were digitized at a rate of 15 ms with a data acquisition system (AII Devices, San Rafael, CA) and stored on diskettes (Apple IIE; Apple Computers, Cupertino, CA). These instruments were calibrated immediately before every experiment. Gas flow and transrespiratory pressure were recorded over ten ventilated breaths. Pressure-volume loops were constructed for each breath by hand-editing the three points of zero flow with a manual cursor and text editor. Dynamic pulmonary compliance was calculated as the slope of the line of best fit through the origin and apex of the pressure-volume relationship. In every case, this was reported as the mean of at least ten breaths with a breathto-breath coefficient of variability of less than 10%. Dynamic airway resistance was calculated from measurements of the total (inspiratory and expiratory) pressure change at 50% tidal volume, divided by the absolute value of the flow at that volume. Studies of Gas Exchange Arterial blood for determination of the blood gas values was drawn from controls with the left hilar structures clamped. Arterial blood specimens were drawn from the study animals without the need for left hilar clamping because of the left pulmonary artery obliteration. Samples were analyzed by a blood gas instrument (model 158; Dow Corning).

3 30 LANGENBURG ET AL Ann Thorac Surg EXPERIMENTAL LUNG TRANSPLANTATION 1995;59:28-32 Table 1. Functional Residual Capacity and Airway Mechanics" Functional Dynamic Dynamic Residual Airway Airway Capacity Compliance Resistance Group (ml) (ml/cm) (cm. L 1. s 1) Study (n ~ 5) 779 _ Control (n = 4) 709 +_ a No significant differences existed between the study and control groups. All values are reported as the average mean +- standard error of the mean. Morphologic Analysis At the end of the study, the right lungs of the control and study group animals were harvested after systemic heparinization had been carried out. The lungs were placed so as to allow dependent drainage of all intravascular fluid. The lungs were fixed with 10% buffered formalin that was instilled through the airways at a constant pressure of 25 cm H20 for 1 hour. The lungs were then immersed in the buffered formalin and tissue blocks were taken from the periphery. The most peripheral lung tissue was studied in all lungs because there the airway dimensions are more uniform and not subject to the effects of dichotomous branching. The tissue blocks were embedded in paraffin, and 5-/xm sections were cut and stained with hernatoxylin-eosin. The cross-sectional surface areas of at least 50 alveoli and 25 noncartilaginous airways per lung were measured by tracing the alveoli and airway perimeters on a video monitor with a manual cursor (Zeiss Orthoplan; Carl Zeiss, Thornwood, NY). The values were then calculated by a software program on a computer (International Business Machines). These measurements were obtained by a person blinded as to whether the lung specimen was from a control or a study animal. Mean cross-sectional airway and alveolar areas were determined for each lung, and consequently for each group. Statistical Analysis All values are reported as the mean + standard error of the mean. Student's t test was used to compare paired samples. A p value of less than or equal to 0.05 was taken to indicate significant difference between the values. Results All five animals that received lung transplants suffered chronic rejection, defined as aggressive episodes of acute rejection resulting in fibrotic infarction, and served as the study group, versus 4 controls that did not undergo transplantation. Both control and study pigs had similar average weights at the end of the study (control animals, 74.5 kg; study animals, 73.7 kg). Three of the 4 pigs (75%) undergoing pneumonectomy without transplantation died within 30 minutes of lung removal, so long-term measurements of pulmonary hemodynamics and airway mechanics were not possible in this group. There were no technical problems or significant blood loss in the pneu- Table 2. Pulmonary Vascular Resistance and Oxygen Tension a Pulmonary Vascular Resistance Oxygen Tension Group (dynes- s/cm -s) (mm Hg) Study (n = 5) 117 _+ 7.9 a 270 _+ 13 a Control (n = 4) 78 _ _+ 23 Values reported are the average mean -+ standard error of the mean. b Study versus control, p < monectomy animals that died. Episodes of acute rejection, defined as periods of tachypnea, fever, lethargy, and dry cough, occurred, and averaged 0.8 times per animal during the 10-month study period. An animal was considered to have responded to the empiric therapy consisting of intramuscularly administered methylprednisolone (500 rag) when these signs and symptoms resolved. Lung Volumes and Airway Mechanics The functional residual capacity and dynamic airway compliance and resistance values are shown in Table 1. There was no statistically significant difference between the study group animals and the control animals in terms of these values. Pulmonary Hemodynamics and Blood Gas Findings The pulmonary vascular resistance and arterial oxygen tension values are given in Table 2. There was a statistically significant increase in the pulmonary vascular resistance in the study animals. The arterial oxygen tension in the study animals was also significantly higher than that in the control animals. However, there were no significant differences in the cardiac outputs between the study and control animals throughout the terminal period of the study. Grossly, the native right lungs of the study animals did not appear to differ from the right lungs of controls. The wet weight of the study group was, however, statistically greater (p < 0.05) than that of the controls ( g versus 177 _+ 3 g). The wet-to-dry weight ratios did not differ between the study and control lungs (p = 0.8). Morphologic Findings The cross-sectional areas of alveoli and noncartilaginous airways are given in Table 3. The sample size of the study group in this table is 4 because one of the lungs was found to be inadequately preserved. The lungs had no gross evidence of emphysema. The histologic specimens Table 3. Cross-sectional Alveolar and Airway Areas Alveolar Area Airway Area Groups (ram 2 x 103) (mm 2 x 103) Study (n = 4 a) _ b b Control (n = 4) 7.33 _ a One lung was inadequately preserved, b Study versus control, p <

4 Ann Thorac Surg LANGENBURG ET AL ;59:28-32 EXPERIMENTAL LUNG TRANSPLANTATION also did not exhibit signs of emphysema. Histologic examination of the chronically rejected lung remnants revealed evidence of fibrotic tissue with no recognizable alveoli, airways, or vasculature. There was also no evidence of infection in the rejected lungs. Comment Neonatal patients with severe pulmonary hypoplasia have little chance of survival, even when treated with extracorporal membrane oxygenation or high-frequency jet ventilation. It has been estimated that 18% of children with congenital diaphragmatic hernia evaluated as possible candidates for extracorporeal membrane oxygenation will die regardless of the treatment chosen [17]. For this subset of patients with severe bilateral pulmonary hypoplasia, lung transplantation may constitute their only chance of survival. Optimally, the candidate for transplantation would be placed on extracorporeal membrane oxygenation while awaiting either a neonatal donor, a cadaveric lobe, or a living-related donor lobe. This transplant may then only have to function until the native lung develops, at which time the transplanted lobe or lung could be removed and immunosuppression therapy stopped. Clearly, the infant swine undergoing pneumonectomy alone could not survive without a left lung transplant, as seen in the study group. Our study animals were aided by the left lung transplant until the native lung experienced compensatory growth. It is not known whether the native lung will develop in the setting of immunosuppression treatment. Our data suggest that the native lung of an 8-week-old swine with a lung transplant undergoes compensatory growth despite the effects of immunosuppressive agents and the presence of chronic rejection of the transplanted lung. Functional residual capacity (the volume of the lung at the end of normal respiration) is a gross measure of functional lung volume, and it tended to be higher in the study animals. Although this difference was not statistically significant, it does suggest a degree of compensatory functional growth. Although the peripheral vascular resistance was significantly greater in the study animals, the airway resistance was not, nor was the native lungs' ability to oxygenate blood impaired. The native right lung of the study animals was clearly able to oxygenate the blood better than the right lung of the controls with the left hilar structures clamped. The significant increase in the dry lung weight of the study animals represents convincing evidence that the native left lung experienced compensatory growth. This increase in weight was not due to an increase in the extravascular water content, given that the wet-to-dry weight ratios did not differ between the study and control lungs. Another indication of compensatory growth in our study animals was the significantly greater crosssectional surface areas of the alveoli and terminal air- ways. Shortcomings of this model need to be mentioned. First, the findings yielded by animal studies are difficult to extrapolate to the human setting. Therefore, correla- tive clinical studies must be conducted for this purpose. Second, the native right lung of the animals receiving transplants has no degree of pulmonary hypoplasia. Because of this, it was thought that this lung at the time of transplantation could support the animal by itself. For this reason, we performed left lung pneumonectomies in four 8-week-old pigs, resulting in a mortality of 75% shortly after extubation. The death that occurred in the pneumonectomy controls was prevented by left lung transplantation in the study animals. An additional pitfall of our study is the small sample size permitted in this resource intensive experiment. A small sample size limits our certainty regarding the magnitude of the difference seen between groups and values. In addition, the absence of significance may not accurately indicate that there are no clinically important differences. Lung transplantation in neonates with severe lung hypoplasia is an option for infants who otherwise would have no chance at survival. A lung transplant may only be needed as a bridge to allow the native lung time to develop and function by itself. Our data support the hypothesis that the development and function of the native contralateral lung may be nearly normal even in the face of chronic rejection of the transplanted lung and an immunosuppressed state. This project was supported by NIH grants HL and HL We also acknowledge Sandoz Pharmaceutical for providing the cyclosporine. References 1. Butler MW, Stolar CJH, Altman RP. Contemporary management of congenital diaphragmatic hernia. World J Surg 1993;17: Crombleholme TM, Adzick NS, Hardy K, et al. Pulmonary lobar transplantation in neonatal swine: a model for treatment of congenital diaphragmatic hernia. J Pediatr Surg 1990;25: MacGillivray TE, Adzick SA. Lung transplantation in the pediatric patient. Chest Surg Clin North Am 1993;3: Trulock EP, Cooper JD, Kaiser LR, et al. The Washington University-Barnes Hospital experience with lung transplantation. JAMA 1991;266: Metras D, Kreitman B, Shennib H, et al. Lung transplantation in children. J Heart Lung Transplant 1992;11(suppl): Bolman RM, Shumway SJ, Estrin JA, et al. Lung and heartlung transplantation. Ann Surg 1991;214: Landau LI, Phelan PD, Gillam GL, et al. Respiratory function after repair of congenital diaphragmatic hernia. Arch Dis Child 1977;52: Reid IS, Hutcherson RJ. Long-term follow-up of patients with congenital diaphragmatic hernia. J Pediatr Surg 1976; 11: Thurlbeck WM, Kida K, Langston C, et al. Postnatal lung growth after repair of congenital diaphragmatic hernia. Thorax 1979;34: Wohl MEB, Griscom NT, Streider DJ, et al. The lung following repair of congenital diaphragmatic hernia. J Pediatr 1977;90: Rendas A, Branthwaite M, Reid L. Growth of pulmonary circulation in normal pig--structural analysis and cardiopulmonary function. J Appl Physiol 1978;45:

5 32 LANGENBURG ET AL Ann Thorac Surg EXPERIMENTAL LUNG TRANSPLANTATION 1995;59: Winkler GC, Cheville NF. The neonatal porcine lung: ultrastructural morphology and postnatal development of the terminal airways and alveolar region. Anat Rec 1984;210: Winkler GC, Cheville NF. Morphometry of postnatal development in the porcine lung. Anat Rec 1985;211: Kern JA, Tribble CG, Zografakis JG, Cassada DC, Chan BBK, Kron IL. Analysis of airway function of immature whole lung transplants versus mature lobar transplants. Ann Thorac Surg 1994;57: Langenburg SE, Tribble CG. Experimental reduced-size lung transplantation: models, techniques, and pitfalls. In: Kern JA, Kron IL. eds. Reduced-size lung transplantation. Landes, 1993: Levitzky MG. Alveolar ventilation. In: Pulmonary physiology. New York: McGraw-Hill, 1982: Heiss K, Manning P, Oldham KT, et al. Reversal of mortality for congenital diaphragmatic hernia with ECMO. Ann Surg 1989;209: DISCUSSION DR JOHN C. WAIN (Boston, MA): This is a very interesting presentation. This study certainly shows some of the troubles involved in supporting immature animals with lung allografts, and in obtaining a lung graft that remains functional over time. I am curious about several things. First, how old were your donor animals when the transplantation was performed? Second, what did you see histologically in the transplanted lung graft at 10 months? Third, what was the time course of the histologic and functional changes you observed in the graft? DR LANGENBURG: The study group consisted of age-matched donors and recipients, so the lungs came from 8-week-old immature pigs. At final study, the lungs were actually grossly fibrotic. Histologically, there was no evidence of alveoli or terminal bronchioles. We performed whole-lung transplantations in 10 immature animals, 5 of which went on to exhibit this chronic rejection pattern. Two with chronic rejection had relatively normal lungs and the other 3 died shortly after operation due to technical problems with the pulmonary vein anastomoses.