Cyclosporine and bronchial healing in canine lung transplantation

J

THORAC CARDIOVASC SURG

88:993-999, 1984

Cyclosporine and bronchial healing in canine lung transplantation Long-term bronchial anastomotic healing has been assessed in the canine hmg tran.
Nigel R. Saunders, M.A., M.B., F.R.C.S.(Eng.),* Thomas M. Egan, M.D., Dean Chamberlain, M.D., F.R.C.P.(C), and Joel D. Cooper, M.D., F.A.C.S., F.R.C.S.(C),

Toronto. Ontario. Canada

Bronchial healing remains a major problem in human lung transplantation. Fifty percent of recipients have bronchial anastomotic problems, and these have contributed directly to the deaths of the majority of patients surviving for longer than 10 days.' These complications have ranged from mucosal ulceration to full-thickness necrosis of the bronchial wall with anastomotic disruption and have included peribronchial infection with bronchial and pulmonary artery hemorrhage and late bronchostenosis.v' Ischemia of the donor bronchus and high-dose steroid therapy in the early postoperative period are undoubtedly major etiologic factors in the pathogenesis of these complications. Cyclosporine has been shown to have powerful immu-

nosuppressant properties in a variety of animal models'? and in man."!' Unlike steroids, it does not interfere significantly with tissue healing and it spares polymorphonuclear leukocyte function.' These features should make cyclosporine especially suitable for immunosuppression in lung transplantation, where bronchial healing and pulmonary infection are major problems. The present study was undertaken to assess cyclosporine as an immunosuppressant in canine lung transplantation and specifically to determine whether cyclosporinebased immunosuppression and early donor bronchial revascularization via an omental pedicle would prevent bronchial complications with particular reference to late bronchostenosis.

Materials and methods From the Division of Thoracic Surgery, Department of Surgery, • University of Toronto, Toronto, Ontario, Canada. Supported in part by a grant from the Medical Research Council of Canada (MA-6909). Received for publication Sept. 23, 1983. Accepted for publication Jan. 21, 1984. Address for reprints: Joel D. Cooper, M.D., 10-226 Eaton Building, Toronto General Hospital, Toronto, Ontario, Canada M5G 1L7. *Present address: Department of Cardiothoracic Surgery, Killingbeck Hospital, York Road, Leeds, England.

Left lung allotransplantation was performed in 10 mongrel dogs weighing between 12 and 19 kg. Recipient and donor were matched for size only. After premedication with intramuscular droperidol and fentanyl citrate (Innovar-Vet) 1 ml/IO kg, general anesthesia was induced with intravenous thiopental sodium (Pentothal) and maintained with a halothane/ oxygen mixture via an endotracheal tube. Atropine 0.05 mg/kg and penicillin and streptomycin 1 ml were administered intramuscularly prior to induction. 993

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9 9 4 Saunders et al.

Table I. Results

Dog No.

Survival (days)

1 2

100 120

3

19

4

121

5 6 7 9 12

13 116 110 8 140

13

24

Cause of death Put to death Put to death (respiratory failure) Put to death (encephalitis) Put to death (respiratory failure) Bronchopneumonia Put to death Put to death Bronchopneumonia Surviving Intussusception of small intestine

No. of antemortem rejection episodes

Rejection at autopsy

Bronchial anastomotic patency (%)

RPA ligation

2 I

+

64.7 75.0

+

0

0 0 0 0 0

Survival for 48 hr after RPA ligation

No significant stenosis

+

91.5

+

No significant stenosis 91.0 96.8 No significant stenosis 85.0*

No rejection at open lung biopsy (140 days)

0

+ + +

+ +

+

+

77.0

Legend: RPA. Right pulmonary artery. 140 days.

*Assessed endoscopically at

Left lung allotransplantation. The left lung transplant was performed through a left lateral thoracotomy in the fifth intercostal space. The recipient left main bronchus was divided just proximal to the bifurcation into lobar bronchi and the donor bronchus at the midpoint of the left main bronchus. An everting horizontal mattress suture of 5-0 Prolene was used for the left atrial anastomosis and a continuous through-andthrough suture of 5-0 Prolene for the pulmonary artery and bronchial anastomoses. Great care was taken to assure good mucosal apposition at the bronchial anastomosis. The mean duration of warm ischemia was 44 minutes. The donor animal was heparinized before lung removal, but the donor lung was not perfused or cooled. After completion of the transplant procedure, the omentum was mobilized via a midline upper abdominal incision and delivered to the left hemithorax via a subxiphoid tunnel. The omentum was wrapped around the bronchial anastomosis and anchored in position with interrupted 5-0 Prolene sutures. At the time of harvesting of the donor lung, a large flap of donor pericardium was left attached anteriorly to the hilum of the lung. Its inferior free edge was sutured to the omentum within the left hemithorax of the recipient to determine whether or not it would become systemically revascularized and provide additional systemic blood flow to the transplanted lung. The distal end of the pericardial flap was sutured in a subcutaneous pocket at the anterior end of the thoracotomy for ease of

identification and biopsy when the dog was put to death. Immunosuppression. All dogs received cyclosporine 20 mg/kgyday and azathioprine (Imuran) 1.5 tug] kg/day orally for the first 14 days after transplantation. At 14 days the azathioprine was discontinued. Two animals (Dogs 2 and 4) were then begun on a program of low-dose prednisone (5 mg/day orally). The remainder were maintained on the cyclosporine regimen alone. Low-dose prednisone was started in Dog 1 after 48 days following two rejection episodes. Beginning at 1 month, the cyclosporine dose in all animals was reduced by 2 mg/kg/wk to a minimum of 10 mg/kg/day. Rejection episodes were managed with pulse doses of prednisolone (l gm) given intravenously daily for 3 to 5 days. All dogs received intramuscular penicillin and streptomycin daily for 5 days after transplantation. Investigations. The dogs were monitored with biweekly chest x-ray films and blood counts and weekly estimations of blood urea nitrogen (BUN), creatinine, and liver function (total bilirubin, alkaline phosphatase, serum glutamic oxaloacetic transaminase [SOOT], and serum glutamic pyruvic transaminase [SGPT]. Fiberoptic bronchoscopy (Olympus B.F. Type 5) was performed regularly with the dog under general anesthesia to assess bronchial mucosal changes and anastomotic healing. When rejection was suspected radiologically, an open lung biopsy was performed with immediate frozensection examination of the tissue.

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Cyclosporine in canine lung transplantation

Assessment of lung function. Five of the six dogs that survived to 100 days underwent right pulmonary artery ligation via a right lateral thoracotomy. Survival for 48 hours after this procedure was considered to indicate satisfactory transplant function. Arterial blood gases were monitored during this period. Assessment of bronchial anastomosis and donor pericardial flap. Five dogs were put to death between 100 and 121 days after transplantation for assessment of the bronchial anastomosis and donor pericardial flap. All animals were fully heparinized before death, and three animals were subjected to postmortem cannulation of the celiac axis and injection of 100 m1 of silicone rubber (Microfil, Canton Biomedical Products, Inc., Boulder, Colo.) into this vessel at a pressure of 100 mm Hg. The thoracic contents were then removed en-bloc with the omentum, and dissection of the tracheobronchial tree and donor pericardial flap was undertaken. The bronchial anastomosis was excised, photographed, measured, and fmally fixed in formalin for subsequent light microscopy. The donor pericardial flap was inspected for the presence of the silicone rubber dye within its vasculature. Biopsyspecimens were taken from the liver, kidney, each lobe of the transplanted lung, each lobe of the recipient's own right lung, and donor pericardium for histologic examination. All dogs underwent a full postmortem examination. The cross-sectional area (CSA) of the bronchial anastomosis was calculated by the formula: a

b

CSA = -x-xTI 2 2 where a = sagittal diameter and b = coronal diameter. Internal diameters were measured with a No. 274 Staret spring caliper. The cross-sectional area of the bronchial anastomosis was then expressed as a percentage of the cross-sectional area of the left main bronchus 5 mm proximal to the anastomosis.

Results (Table I) Survival. There were no operative deaths, and six dogs survived longer than 100 days. Of the remaining four dogs, one died at 8 days (bronchopneumonia), one at 13 days (bronchopneumonia), one at 24 days (intussusception of small intestine), and one was put to death at 19 days (encephalitis). All dogs surviving more than 100 days were healthy when killed. One dog underwent contralateral pulmonary artery ligation at 100 days and was not subsequently put to death. It remains alive and well at 6 months. Morbidity. There was little morbidity with the cyclosporine-based immunosuppressive regimen, in con-

995

Fig. 1. Bronchial anastomotic healing in four representative dogs surviving 100 days after left lung transplantation. All anastomoses were well healed with minimal, if any, narrowing at the anastomotic site.

trast to previous steroid-based protocols. ,Only one wound infection occurred, and this was after a second thoracotomy for open lung biopsy. There were no other significant infective complications and, in general, the dogs were noteworthy for their sustained appetite, weight gain, and good health. In one dog, the left lobe of the liver herniated through the diaphragm in association with the omental pull up, but this complication was asymptomatic. Serum BUN and creatinine levels were not consistently elevated in any animal, and renal biopsy specimens obtained at autopsy were normal in all animals. Liver function tests were often mildly deranged, with some elevation of SOOT, SGPT, and particularly alkaline phosphatase, but the total bilirubin value was never elevated and liver biopsy specimens were always normal. Full blood counts were obtained regularly, and in no case were there signs of marrow depression. Rejection. Four biopsy-proved rejection episodes occurred in three animals, and in each case rejection was easily reversed with pu1se-dose steroid therapy. These three dogs were put to death between 100 and 121 days postoperatively. Arterial blood gases, measured at the time of right pulmonary artery ligation in two of these animals, showed very poor gas exchange across the transplanted lung. By contrast, the three animals alive at 100 days without evidence of previous rejection survived contralateral pulmonary artery ligation with normal

The Journal of Thoracic and Cardiovascular Surgery

9 9 6 Saunders et al.

.:

...,

..- ;-: ... ., . ... "

lung. At autopsy the donor pericardium appeared viable in all animals (8 to 121 days after transplantation). Histologic study of the donor pericardium showed viable fibrous and connective tissue with no significant ischemic necrosis. Histology. Histologically, rejection was characterized by varying degrees of perivascular mononuclear cell infiltration. The mononuclear cells were predominantly lymphocytic, but plasma cells and occasional macrophages were also seen. The infiltrate was maximal in relation to small arterioles and venules rather than large vessels and was often not uniform throughout the transplanted lung, with considerable variation between different lobes. There was no consistent distribution of histologic changes between the lobes in different animals. When rejection was present, the donor pericardium usually showed perivascular cuffing similar to that of the transplanted lung (Fig. 3). No such fmdings were detected in the donor bronchus, however. This latter observation confirms the unsuitability of endobronchial biopsy as a monitor of lung rejection." Discussion

Fig. 2. A, Gross appearance showing well-healed bronchial anastomosis 100 days after left lung transplantation. B, Site of bronchial wall anastomosis showing disruption of mucosa and cartilage, 'mild nonspecific inflammatory changes, and some fibrosis. (Original magnification X63.)

arterial blood gases. At autopsy, the transplanted lung of one of these animals showed histologic evidence of mild rejection. It should be noted that serum levels of cyclosporine were not monitored in any of the 10 animals; thus, the maintenance of therapeutic levels of cyclosporine cannot be presumed. Bronchial healing. No significant bronchial anastomotic complications occurred, in particular, no bronchial dehiscence or early bronchostenosis. The mean percent cross-sectional area of the six dogs surviving to 100 days was 84% (range 64.7% to 96.8%), and no serious late bronchostenosis was identified (Fig. 1), despite histologic evidence of rejection in the transplanted lung of three dogs. All bronchial anastomoses were well healed macroscopically, as confirmed histologically (Fig. 2). Donor pericardium. The celiac axial injection studies in three dogs demonstrated systemic revascularization of the donor pericardial flap from the omental pedicle, with the pericardial vessels filling vessels at the hilum of the

As an organ graft, the lung has two unique features that predispose to complications after transplantation. First, one of its major connections with the recipient, the bronchial anastomosis, remains relatively ischemic after transplantation, as it is not routinely surgically revascularized from the systemic circulation. Second, the lung is continuously exposed to the risk of airborne infection from the atmosphere and from the contralateral lung. For successful clinical transplantation, it is obvious that any other factors that further prejudice tissue healing or host defense must be minimized . Until recently, azathioprine and steroids were the mainstays of transplant immunosuppression. The latter, which significantly retards bronchial healing" and renders the recipient more prone to infective complications, should obviously be avoided. Cyclosporine is not myelotoxic and has little effect on tissue healing." It is a potent immunosuppressant in many species, including man, acting predominantly against the T-Iymphocyte population." In the canine allograft model it has been more effective than conventional immunosuppression in prolonging survival and reducing morbidity. 8.17.18 In this series, 60% of dogs survived past 100 days , at which time they were electively put to death. This result compares favorably with our, and other authors', previous experience using conventional immunosuppression (azathioprine, prednisone), when survival past 6 weeks was unusual .P " Of the six dogs surviving past 100 days, two never showed

Volume 88 Number 6 December, 1984

Cyc/osporine in canine lung transplantation

997

Fig. 3. Microscopic appearance following rejection episode. Perivascular aggregates composed of lymphocytes and occasional plasma cells are seen in both the attached donor pericardium (Aj and the transplanted lung (Bj. (Original magnification X160),

any evidence of rejection even on low-dose maintenance cyclosporine therapy (10 rug/kg/day). In a third animal, mild rejection was diagnosed only after autopsy. Rejection was reversed with steroid therapy in the other three animals, but subsequent lung function was poor. The transplanted lungs of two of these animals showed histologic evidence of rejection at autopsy, despite the addition of low-dosesteroids to their immunosuppressive regimen when azathioprine was stopped. It seems likely that a low-grade rejection process continued and eventually caused enough organ damage to significantly prejudice function. The failure to monitor serum levels of cyclosporine in these animals may have contributed to the incidence of rejection problems. On the other hand, the primary purpose of these experiments was to study long-term bronchial healing, including the response to rejection episodes. Although bronchial complications are not as common in the canine lung transplant model as in human lung transplantation, a significant percentage of experimental animals have bronchial dehiscence or bronchostenosis after transplantation. A degree of bronchostenosis resulting from mucosal edema secondary to surgical trauma and interruption of vascular and lymphatic connections inevitably occurs in the first few days after lung transplantation. This usually resolves after 7 to 10 days as the edema subsides. In some animals, progressive stricture formation occurs; in others, bronchostenosis develops after several weeks. Poor surgical technique, contraction of scar tissue, continuing ischemia, infection, and rejection have been implicated by various authors.i":" In this series of 10 dogs, no significant bronchial complications occurred. The mean percent cross-sectional area of 84% for the six dogs surviving

past !OO days is a significant improvement from previous studies." :25 Rejection was documented in three of these animals, but this did not prejudice bronchial healing or result in stricture formation. In any case, bronchostenosis is not uncommon after lung autotransplantation, in which rejection is not a factor. 15, 2! Continuing ischemia and disordered wound healirig associated with conventional immunosuppression, particularly high-dose steroid therapy, appear to be more important factors affecting bronchial healing. All dogs in this series had cyclosporine immunosuppression, which has little effect on tissue healing and , unlike steroids, does not alter the ultrastructural pattern of collagen at healing sites. An omental pedicle was placed around the bronchial anastomosis in .each animal. This technique reliably restores the distal bronchial circulation to the transplanted lung within 4 days," whereas restoration of the bronchial circulation by direct growth of vessels across the anastomosis has not been demonstrated earlier than the twelfth day after lung reimplantation or transplantation in dogs.26-30 We 31 have previously demonstrated that wrapping the bronchial anastomosis with an omental pedicle improves bronchial healing after lung reimplantation. We 32 have demonstrated that the early revascularization of the bronchus from the omentum occurs after canine lung transplantation as well. We believe that early bronchial revascularization via the omental pedicle helps to limit ischemia at the anastomosis and thus contributes to sound bronchial healing. Furthermore, the omentopexy should prevent a major bronchial disruption in the event that anastomotic healing is compromised. In this study, cyclosporine immunosuppression and bronchial omentopexy appear to have been a reliable combination for minimizing

9 9 8 Saunders et al.

bronchial complications, including stenosis. Other authors have demonstrated satisfactory bronchial healing in a similar model in which cyclosporine immunosuppression was used without an omental wrap. IS Bronchial omentopexy may add little benefit in the canine model when high-dose steroid therapy is avoided, but we would certainly urge its use in human transplantation, in which bronchial complications have been a frequent cause of death. In the canine model, we have demonstrated that the donor pericardial flap can be revascularized via the omental pedicle by 8 days. The pericardial tissue remains viable and, although not as vascular as the omentum, it provides a systemic circulation to the hilum of the transplanted lung and could certainly be used to reinforce the bronchial anastomosis. We therefore recommend that an anterior flap of donor pericardium be left attached to the donor lung in human transplantation, as it may be useful in supporting bronchial or vascular anastomoses and in providing an additional route for systemic revascularization of the lung. Histologically,vessels in the donor pericardium usually showed changes similar to those of the transplanted lung when rejection occurred. This fact raised the possibility that a flap of the donor pericardium, anchored in a suitable extrapleural position, might be useful in monitoring rejection in the lung. We gratefully acknowledge the skilled assistance of Mr. John Mates and Ms. Elaine Murphy. We are grateful to Mrs. Ann Botelho for typing the manuscript. REFERENCES

2

3 4

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6 7

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21 Duvoisin GE, Payne WS, Ellis FH Jr: Influence of surgical technique on the results of pulmonary reimplantation. Chest 58:96-102, 1970 22 Andrews MJ, Pearson FG: Relation of bronchial arterial circulation, and other factors, to the transient defect in oxygen uptake following autotransplantation of the canine lung. Can J Surg 16:97-109, 1973 23 Lima 0, Goldberg M, Peters WJ, Ayabe H, Townsend ER, Cooper JD: Bronchial omentopexy in canine lung transplantation. J THORAC CARDIOVASC SURG 83:418-421, 1982 24 Shimada K, Gondos B, Benfield JR: Fibreoptic bronchoscopic findings during lung transplant rejection. Arch Surg 106:773-778, 1973 25 Sinha SB, Dougherty JC, Boley SJ, Veith FJ: Elimination of bronchial complications in lung transplantation. Surg Forum 22:225-226, 1971 26 Siegelman SS, Hagstrom JW, Koerner SK, Veith FJ: Restoration of bronchial artery circulation after canine lung allotransplantation. J THORAC CARDIOVASC SURG 73:792-795, 1977

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27 Rabinovich 11: Re-establishment of bronchial arteries after experimental lung lobe autotransplantation. J THORAC CARDIOVASC SURG 64:119-126, 1972 28 Pearson FG, Goldberg M, Stone RM, Colapinto RF: Bronchial artery circulation restored after reimplantation of canine lung. Can J Surg 13:243-250, 1970 29 Stone RM, Ginsberg RJ, Colapinto RF, Pearson FG: Bronchial artery regeneration after radical hilar stripping. Surg Forum 17:109-110, 1966 30 Fisher AB, Kollmeier H, Brody JS: Restoration of systemic blood flow to the lung after division of bronchial arteries. J Appl Physiol 29:839-846, 1970 31 Morgan E, Lima 0, Goldberg M, Ayabe H, Ferdman A, Cooper JD: Improved bronchial healing in canine left lung reimplantation using omental pedicle wrap. J THORAC CARDIOVASC SURG 85:134-139, 1983 32 Dubois P, Choiniere L, Cooper JD: Bronchial omentopexy in canine lung allotransplantation. Ann Thorac Surg (in press)