Cardiac Allograft Pathology in Rhesus Monkeys C. F. HOLLANDER Institute f o r Experimental Gerontology T N O , Rijswijk-ZH, T h e Netherlands ABSTRACT Orthotopic, allogeneic heart transplantations were performed in 26 randomly selected rhesus monkeys to study the effect of immunosuppressive treatment on survival and cardiac morphology. The histological picture of the acute and chronically rejected hearts was similar to that described for acute and chronic cardiac allograft rejection in man. As seems to be the case in man, an obliterative arteritis of the coronary arteries appears to be the limiting factor in cardiac allograft survival in the rhesus monkey. Immunosuppressive treatment with either Imuran and ALS or Imuran, ALS, and Prednisone could not prevent the occurrence of obliterative arteritis of the coronary arteries. Contrary to the observation in man, no atherosclerotic changes were found in the affected parts of the coronary arteries or in the non-diseased parts. Orthotopic, allogeneic heart transplantations were performed in 26 randomly selected rhesus monkeys of about 3 kg to study the effect of immunosuppressive treatment on survival and cardiac morphology. In this study special attention was given to lesions of the coronary arteries because in m a n obliterative arteritis seems to emerge as the limiting factor in cardiac allograft survival (Bieber et al., '70; Kennedy et al., '71). MATERIALS AND METHODS The well-known superficial immersion cooling technique without extracorporeal circulation was adapted for these transplantations. Grafting of the heart was performed at a n esophageal temperature of 24°C. The circulation was arrested for about 30 minutes. The interarterial septum, the right and left atrium, the pulmonary artery, and the aorta were joined by continuous suture. The following experimental groups were established : Group 1. Five animals receiving no immunosuppressive treatment. Group 2. Eleven animals treated postoperatively with Imuran i.m., 4 mg/kg/ day for 36 days, combined with antilymphocyte serum (ALS) s . ~ . 2 , ml/kg/ day for 60 days. Group 3 . Ten animals treated post-operatively with: ALS as in group 2; Imuran AM. 3. PHYS. ANTHROP., 38: 431-434. i m . , 4 mg/kg/day for 28 days, followed by 1 mg/kg/day until death; and from day 20, Prednisone i.m., 2 mg/kg/day, also until death. A complete autopsy was performed on all animals, which were either killed when moribund or were found dead. At the time of autopsy, nearly all monkeys showed gross morphological signs of severe myocardial insufficiency. Tissues submitted for histological examination were fixed in 10% buffered formalin, embedded in paraffin, and routinely stained with Hematoxylin-phloxin-saffron. Special stains were made when necessary. RESULTS The mean survival time of the five monkeys receiving n o immunosupressive treatment (Group 1 ) was 13.2 days with a range of 6 to 27 days. At autopsy, the hearts of these animals were dark red, edematous, and enlarged. Histological examination showed severe congestion of small vessels in the myocardium with platelet thrombi and focal rupture of the wall. Extensive hemorrhages and degeneration of myocardial muscle fibers were seen. Focal signs of arteritis were seen only in the longest survivors and were restricted to the main branches of the coronary artery. They consisted of endothelial swelling, slight intimal proliferation, infiltration with poly- and mononu431 432 C. F. HOLLANDER clear cells, and fibrinoid material adhering closely to the internal elastic membrane. The mean survival time of the 11 animals treated w i t h Imuran and ALS (Group 2) was 44.9 days with a range of 11 to 128 days. At the time of autopsy, the hearts were also enlarged, of firm consistency, and with fibrinous pericarditis or its fibrous residue and a mottled brownyellow myocardium. Microscopic examination showed a focal or more diffuse infiltration of the myocardium with lymphoblasts, small lymphocytes, plasmacells, and a n occasional polynuclear granulocyte. The myocardial fibers showed myccytolysis and small areas of myocardial necrosis. In some cases scar tissue and minute hemorrhages were also observed. In all animals surviving beyond day 14, the main branches of the coronary arteries showed severe focal, obliterative arteritis. The microscopic picture ranged from a n active arteritis to sections of arteries in which the lumen was nearly totally occluded by fibrous tissue, which had also destroyed and replaced the vessel wall and exhibited a scar-like appearance. In case of active arteritis, extensive intimal proliferation was seen. This was characterized by infiltration of all wall layers with predominantly mononuclear cells and destruction of the internal and external elastic membranes. Pictures intermediate between more active arteritis and almost totally occluded vessels in which the wall was replaced by fibrous tissue were frequently observed. In some instances, the internal elastic membrane was found to be intact in parts of arteries in which the wall layers were replaced by fibrous, scar-like tissue. No explanation can be given for this observation. Small, intramural branches of the coronary arteries were affected only in the longest survivors. In a few animals, eosinophilic granulocytes were observed in the infiltrate of the coronary artery wall. In these animals arterial lesions of arteries elsewhere in the body as well as lesions in the kidney were observed that were consistent with the picture of serum sickness. In these cases serum sickness was thought to be induced by the non-purified ALS which was used. In no instances were atheroslerotic changes found in the affected or intact parts of the coronary arteries. The mean survival time of the ten animals treated with Imuran, ALS and Prednisone (Group 3 ) , not greatly different from the animals of Group 2, was 45.1 days and ranged from ten to 104 days. Microscopic examination of the hearts showed a picture identical to that of animals treated with Imuran and ALS alone. DISCUSSION AND CONCLUSIONS The lesions observed in acute and chronically rejected hearts in the rhesus monkey are similar to those described for acute and chronically rejected hearts in man (Bieber et al., '70; Knieriem et al., '71; Milam et al., '70; Stinson et al., '69) and dogs (Kosek et al., '68; Wegmann, '70). As seems to be the case in man, a n obliterative arteritis of the coronary arteries appears to be the limiting factor in cardiac allografts survival in the rhesus monkey. A survey of the histological changes observed in the present series is shown in table 1. No influence of the immunosuppressive treatment regimens on the occurrence of obliterative arteritis has been observed. Further studies therefore seem necessary in order to devise a n immunosuppressive treatment regimen that will effectively prevent obliterative arteritis of the coronary arteries. Such studies may also be of benefit for other solid organ grafts, including the kidney in man (Kennedy et al., '71). Up to the present time, no atherosclerotic lesions have been observed in rhesus monkeys kept under standard conditions in the Primate Center TNO, not even in a few approximately 15 year old animals. Failure to observe atherosclerotic changes similar to those often found in man in the affected parts of the coronary arteries in rhesus monkeys receiving a cardiac transplant may indicate that in the case of man the underlying metabolic state of the recipient is of importance (Kosek et al., '71). ACKNOWLEDGMENTS These studies were performed in collaboration with H. Snijders, J. Vincent, H. J. Stol, Z. Aytug, and H. F. L. Brom, Department of Thoracic Surgery, Univer- 433 CARDIAC ALLOGRAFT PATHOLOGY IN MONKEYS TABLE 1 Orthotopic-homologous heart transplantation in 26 rhesus monfieys Postoperative treatment None None None None None Morphology Parenchymatous rejection Survival in days Capillary lesions Cellular infiltrate hemorrhage muscle degenerations 6 6 7 20 27 + Imuran + Imuran + Imuran + Imuran + Imuran + Imuran + Imuran + Imuran + Imuran + Imuran + Imuran ALS + Im + Pred. ALS + Im + Pred. ALS + Im + Pred. ALS + Im + Pred. ALS + Im + Pred. ALS + Im + Pred. ALS + Im + Pred. ALS + Im + Pred. ALS + Im + Pred. ALS + Im + Pred. ALS ALS ALS ALS ALS ALS ALS ALS ALS ALS ALS + - Arterial rejection Arteritis - 11 12 14 15 16 4s 56 60 61 76 128 10 12 15 20 36 46 61 65 82 104 -, nolesion. +, clearcut lesion. 2 , minimal lesion. sity of Leyden, and D. W. van Bekkum, H. Balner, R. L. Marquet, and G. A. Heystek, Radiobiological Institute TNO, Rij swijk-ZH. LITERATURE CITED Bieber, C. P., E. B. Stinson, N. E. Shumway, R. Payne and J. Kosek 1970 Cardiac transplantation i n man. VII. Cardiac allograft pathology. Circulation, 41 : 753-772. Kennedy, Jr., L. J., and I. L. Weissman 1971 Dual origin of intimal cells in cardiac-allograft arterioscelerosis. N. Engl. J. Med., 285: 884-887. Knieriem, H. J., H. Meessen and H. D. Schulte 1971 Morphologische Befunde bei Herztransplantationen. Klin. Wschz., 49: 837-852. f +- - - - f ++ + + + + & ALS, Antilymphocyte serum. Im, Imuran. Pred, Prednisone. Kosek, J. C., E. J. Hurley and R. R. Lower 1968 Histopathology of orthotopic canine cardiac homografts. Lab. Invest., 19: 97-112. Kosek, J. C., C. P. Bieber and R. R. Lower 1971 Heart graft arteriosclerosis. Transplant. Proc., 3: 512-514. Milam, J . D., F. H. Shipkey, Jr., C. J. Lind, Jr., J. J. Nora, R. D. Leachman, D. G. Rochelle, R. D. Bloodwell, G . L. Hallman and D. A. Cooley 1970 Morphologic findings in human cardiac allografts. 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