H tation are now established therapeutic options for

Denervation of Transplanted Porcine Lung Causes Airway Obstruction Charles E. Hobson, MD, W. Gerald Teague, MD, Curtis G. Tribble, MD, Stacey E. Mills, MD, Barry Chan, MD, Jon Agee, MD, Terry L. Flanagan, MPH, and Irving L. Kron, MD Departments of Surgery, Pediatrics, and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia Lung transplantation can be complicated by a form of small airway obstruction known as bronchiolitis obliterans. We tested the hypothesis that lung denervation causes small airway obstruction in young pigs (10 f 1 weeks). Control pigs had an innervated native lobe, and study pigs had either a denervated native lobe or a denervated transplant lobe. Transplanted pigs received standard immunosuppression. At 10 weeks we measured isolated left lobe pulmonary mechanics. Dynamic resistance in both study groups was significantly higher than in the lobectomy group, whereas dynamic compliance in both study groups was significantly lower than in the lobectomy group. No significant difference in resistance or compliance was noted between the transplant and reimplant groups. Histologic changes consistent with rejection were noted in the transplant lobes. We conclude that the small airway obstruction noted in this model is due to operative denervation rather than to immunosuppression or rejection. (Ann Thorac Surg 1991;52:1295-9) eart-lung and unilateral or bilateral lung transplan- H tation are now established therapeutic options for patients with end-stage pulmonary disease. Unfortunately, the long-term survival of graft recipients may be reduced significantly by progressive small airway obstruction [l]. This complication, which has the histopathologic features of bronchiolitis obliterans, is of unknown etiology but is thought to result from chronic immunemediated injury to the bronchiolar epithelium [2]. Pulmonary denervation has also been identified as a potential cause of small airway obstruction in the transplanted lung through bronchial hyperreactivity to cholinergic stimulation [>6]. In previous studies, denervation of a single porcine lobe through excision and reimplantation led to small airway obstruction in the absence of immune rejection [7]. We hypothesized that neither immunosuppression nor rejection would potentiate the small airway obstruction seen in denervated porcine allograft lobes. To test this hypothesis we studied respiratory system mechanics and pulmonary histology in immunosuppressed pigs with denervated left lower lobe allografts, in pigs with denervated left lower lobe autografts, and in pigs receiving only a left upper lobectomy leaving an innervated native left lower lobe. Material and Methods Surgical Preparation Twenty-two pigs, aged 10? 1 weeks, underwent surgical preparation and survived to complete the study protocol. Animals were not littermates and were not Accepted for publication July 16, 1991 Address reprint requests to Dr Kron, Department of Surgery, University of Virginia Health Sciences Center, Box 181, Charlottesville, VA 22908. matched for blood type. All animals were sedated with intramuscular ketamine (20 mg/kg) and xylazine (2 mgkg), intubated with a No. 5 endotracheal tube, and ventilated with oxygen and halothane (1% to 1.5%). Each pig underwent a left lateral thoracotomy through the fifth intercostal space. A control group of 8 pigs had a left upper lobectomy alone. The reimplant group of 9 pigs had a left pneumonectomy after administration of sodium heparin for anticoagulation (200 Ukg). The excised lung was flushed through the pulmonary artery with heparinized normal saline solution at 4" C until the venous effluent was clear. The upper lobe was then dissected free and removed. The lower lobe was reimplanted by anastomosing first the pulmonary vein with running 6-0 absorbable monofilament suture, then the bronchus with running 4-0 absorbable monofilament suture, and finally the pulmonary artery with running 6-0 absorbable monofilament suture. A pedicle of greater omentum was brought through the diaphragm and wrapped around the bronchial anastomosis to protect the bronchus during healing. The transplant group of 5 pigs was operated on in pairs. Two animals simultaneously had a left pneumonectomy and isolation of the left lower lobe. Each animal then underwent transplantation of the allograft left lower lobe using a technique identical to that of the reimplant group. In all animals the chest was closed in layers. A chest tube was placed and was removed the following day. lrnmunosuppression All animals in the transplant group received cyclosporine (15 mg * kg-' day-') orally starting the day before operation. These animals also received azathioprine (2 mg - kg-' * day-') orally starting immediately postoperatively and prednisone (1 mg - kg-' * day-') orally starting 0 1991 by The Society of Thoracic Surgeons 0003-4975/91/$3.50

1296 HOBSON ET AL Ann Thorac Surg 1991;521295-9 on the 15th postoperative day [8]. Acute rejection was diagnosed on the basis of an elevated rectal temperature of greater than 39.4 C and symptoms of respiratory distress. Episodes of acute rejection were treated with intramuscular methylprednisolone (500 mglday for 3 days). Study Protocol Ten weeks after operation each pig was sedated with intramuscular ketamine (20 mg/kg) and xylazine (2 mgkg), intubated with a No. 8 endotracheal tube, and ventilated with 100% oxygen at a tidal volume of 12 mg/kg and a rate of 15 breathdmin. Intravenous pentobarbital (10 mg/kg) and metocurine (0.4 mg/kg) were given for anesthesia and paralysis as needed. A Teflon catheter was inserted into the right carotid artery, and a balloon-tipped thermodilution catheter was inserted into the right internal jugular vein and advanced into the pulmonary artery. A tracheostomy with placement of a No. 8 cuffed tube was then performed. To isolate the left lower lobe for measurement of respiratory mechanics the right main bronchus and eparterial accessory bronchus to the right upper lobe were occluded with 6F balloon catheters placed under direct vision using a flexible bronchoscope. Each pig recovered for 15 minutes after isolation of the left lower lobe. Measurements Electrocardiogram, heart rate, carotid artery pressure, and pulmonary artery pressure were monitored continuously throughout each experiment. Arterial blood gas levels, pulmonary capillary wedge pressures, cardiac output, and end-tidal CO, level were recorded before and after every experiment. Airflow and transrespiratory pressure were measured at the tracheostomy tube using a Fleisch pneumotachometer (size No. 2, linear to 300 ml/s with a dead space of 40 ml; OEM Corp, Richmond, VA) and two differential pressure transducers (MP45-1; Validyne Engineering Corp, Northridge, CA). The signals were digitized at a rate of 15 ms with a data acquisition system (A11 Devices, San Rafael, CA) and stored on diskettes (Apple IIE; Apple Computers, Cupertino, CA). These instruments were calibrated immediately before every experiment. A standard volume history of 1,000 ml was administered with a calibrated syringe before each set of measurements. Gas flow and transrespiratory pressure were then recorded over at least five ventilated breaths. Pressurevolume loops were constructed later for each inflation by hand editing the three points of zero flow with a manual cursor and text editor program. Using the method of multiple linear regression the dynamic compliance was calculated as the slope of the line of best fit through the origin and apex of the pressure-volume loop, and the dynamic resistance was calculated from the total pressure change at 50% tidal volume divided by the flow at that volume. Functional residual capacity of the left lower lobe was then measured using the helium dilution method as previously described [7]. His tology After measurement of left lobe mechanics the chest was opened and the heart and lungs dissected free. The pig then received a lethal dose of pentobarbital. The heartlung block was then removed and the trachea instilled with 10% formalin at 25 cm H,O pressure for 1 to 2 hours. Tissue slices in a sagittal plane were prepared for paraffin embedding and staining with hematoxylin and eosin. Statistics Measurements are reported as the mean 5 standard error of the mean. Student s t test for unpaired samples was used to compare the two study groups, and to compare each study group with the control group. A p value of 0.05 or less was used to indicate a significant difference between measurements. Humane Animal Care All animals received care in facilities accredited by the American Association for Accreditation of Laboratory Animal Care and in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1985). Results Two pigs in the transplant group had an episode of acute rejection within the first 10 days after operation that responded to intramuscular methylprednisolone. All pigs appeared healthy at the time of study. At bronchoscopy no evidence of bronchial anastomotic strictures or pneumonia was evident in any of the animals. At autopsy the only apparent difference between the lobectomy lobes, the reimplanted lobes, and the transplanted lobes was the finding of somewhat denser pleural-parenchymal adhesions in the transplanted animals. lsolated Left Lower Lobe Mechanics Dynamic resistance in the isolated left lower lobe was significantly higher in both the transplant and reimplant groups than that observed in the control lobectomy group. No significant difference in resistance was noted between the transplant and reimplant groups (Fig 1). Dynamic compliance in the isolated left lower lobe was significantly lower in both the transplant and reimplant groups than that observed in the lobectomy group. No difference in compliance was noted between the transplant and reimplant groups (Fig 2). No significant differences were noted in the functional residual capacity of the left lower lobes in the three groups (Fig 3). Histology Grossly all lobes appeared normal. All vascular and bronchial anastomoses were widely patent. On hematoxylin and eosin stained sections the reimplant and lobectomy lobes had occasional areas of very mild peribronchiolar lymphoid aggregates, but there were no acute or chronic inflammatory changes, and these lobes were indistinguishable from normal porcine lung. Microscopic exami-

Ann Thorac Surg 1991 :5212959 HOBSON ET AL 1297 20 1s P<0.05 VS LOBECTOMY 1 SO0 I 1000 8 i 10 6 T d so0 0 LOBECTOMY REMPUNT TRANSPLANT Fig I. Dynamic resistance of the isolated left lower lobe. 0 LOBECTOMY RE- TRANSPLANT Fig 3. Functional residual capacity of the isolated left lower lobe. nation of all left lower lobes from the transplant group showed focal, patchy, mild to moderate penbronchial and bronchiolar lymphoid infiltrates (Fig 4). These infiltrates would occasionally focally elevate the bronchial mucosa but were not associated with epithelial erosion or necrosis. The lymphoid infiltrate was mitotically active with immunoblasts and plasma cells noted, and occasionally formed nodules resembling germinal centers (Fig 5). One animal in the transplant group had, in addition to the previously mentioned changes, multiple areas of severe mixed penbronchial and peribronchiolar inflammation with mucosal erosion and necrosis (Fig 6). In this animal there were occasional areas of frank small airway destruction with replacement by chronic inflammatory cells consistent with the histologic picture of bronchiolitis obliterans (Fig 7) [9]. Despite these histologic findings the pulmonary mechanics for this animal were better than the mean in all measured categories. Comment The salient finding in this study is that reimplantation of an autologous lobe and transplantation of an allograft lobe result in similar changes in respiratory resistance and compliance. No significant difference in isolated left lower lobe mechanics was noted between the reimplant and transplant groups, even though histologic changes con- sistent with rejection were noted in the transplant group. We conclude that the changes in pulmonary mechanics noted in both groups are due to denervation and not to postoperative factors such as chronic immunosuppression or rejection. Operative factors that could conceivably cause airway obstruction in a transplanted lung include bronchial anastomotic stricture, pulmonary edema secondary to lymphatic disruption, bronchial and bronchiolar smooth muscle hypertrophy, and increased bronchial and bronchiolar muscle tone. In this study all vascular and bronchial anastomoses were sewn with absorbable suture and remained intact and widely patent even given the somatic growth of the animal. Edema has not been evident in the reimplanted porcine lobe at 10 weeks, and in fact other investigators have demonstrated lymphatic regeneration at 3 weeks in the canine reimplanted lobe [lo]. Bronchial smooth muscle hypertrophy in the reimplanted porcine lobe has not been demonstrated by immunostaining of the smooth muscle actin of the lobe (111. We believe that operative denervation of the lung results in decreased airway diameter secondary to increased smooth muscle tone in the bronchi and bronchioles. Respiratory resistance is a direct measure of airflow 80 70 60 Pe0.05 VS LOBECTOMY 50 I LOBECTOMY REWRLAHT TRANSPLANT Fig 2. Dynamic compliance of the isolated left lower lobe. Fig 4. Mild peribronchial infiltrate in transplanted pig left lower lobe. (Hematoxylin and eosin; x ZOO before 50% reduction.)

1298 HOBSON ET AL Ann Thorac Surg 1991;521295-9 Fig 5. Lymphoid infiltrate in transplanted pig left lower lobe. (Hematoxylin and eosin; XI00 before 50% reduction.) Fig 7. Small airway destruction in transplanted pig left lower lobe. (Hematoxylin and eosin; x 100 before 50% reduction.) obstruction and is largely determined by the diameter of the lobar, segmental, and subsegmental airways. An increase in bronchial smooth muscle tone would decrease the caliber of these airways and result in increased resistance as noted in our animals. Similarly, respiratory compliance is a measure of the elastic recoil properties of the lung and depends on the physical properties of the lung tissues, the surface tension of the alveoli, and the mechanical properties of the chest wall. An increase in bronchial and bronchiolar smooth muscle tone would stiffen the lung and result in the decreased compliance noted in our animals. In humans, nervous control of airway smooth muscle is mediated through cholinergic parasympathetic pathways that promote smooth muscle contraction and through padrenergic sympathetic pathways that promote smooth muscle relaxation. Stimulation of a-adrenergic receptors also results in smooth muscle contraction, but a-receptors are numerically insignificant compared with preceptors. Possible causes of denervation-induced increased smooth muscle tone in humans include up-regulation of acetyl- choline receptors, up-regulation of a-adrenergic receptors, or loss of pstimulation. Clinically the most important manifestation of small airway obstruction in human lung transplant recipients is the progressive lesion of bronchiolitis obliterans. Although bronchiolitis obliterans in transplant recipients is of unknown etiology, there are some data to suggest that it is related to rejection. The reported decrease in the incidence of bronchiolitis obliterans in an era of improved monitoring for rejection with transbronchial biopsies is thought to support the theory that rejection is the cause [12]. Furthermore, the predictive value of the primed lymphocyte test for bronchiolitis obliterans and the presence of Leu-7 positive T lymphocytes in 2 patients with lung allograft rejection and bronchiolitis obliterans support an immunologic cause for this process [13, 141. However, the failure of most patients with biopsy-proven bronchiolitis obliterans to respond to augmented immunosuppression argues against rejection as a primary factor. Regardless of the contribution that rejection may play in the development of progressive airway obstruction, this study may have implications for the care of lung transplant patients. Most importantly, the airway dysfunction seen in patients with bronchiolitis obliterans may be partly due to the denervation of the lung and not solely to a rejection phenomenon. The practice of using pulmonary mechanics measurements, especially forced expiratory volume in 1 second, to assess for rejection may be complicated by the patient s underlying fixed airway changes. Finally, although current bronchodilator therapy has been almost uniformly ineffective in treating lung transplant patients with established bronchiolitis obliterans, pharmacologic manipulation of airway tone may be possible to prevent the development or progression of bronchiolitis obliterans in these patients. Fig 6. Severe peribronchial inflammation in transplanted pig left lower lobe. (Hematoxylin and eosin; XI00 before 50% reduction.) This work was supported in part by a grant from the Virginia Affiliate of the American Heart Association.

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