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Canine pulmonary homografts with uncontrolled cross-circulation
Canine pulmonary homografts with uncontrolled cross-circulation O. Gago, M.D. (by invitation), R. Zajtchuk, M.D. (by invitation), S. L. Nigro, M.D., and W. E. Adams, M.D., Chicago, III.
JLn 1945, Owen1 observed and described the phenomenon of red cell chimerism in twin cattle, where the vascular systems be fore birth are not sharply distinct from each other, as they are in other twins. Instead, there are anastomoses with each other, re sulting in a prolonged intrauterine exchange of blood, and possible red cell precursors. He also discovered that most twin cattle may retain through life a stable mixture of each other's red cells. A few years later, Anderson, Billingham, Lampkin, and Medawar2 showed that most dizygotic cattle twins would accept skin grafts from each other, and that this mutual tolerance was specific, since skin transplants from third parties were rejected in the expected manner. For some time, it was believed that the induction of tolerance to living tissue was limited by the so-called "adaptive period" which in most species ends around the time of birth. The first indication that artificial tolerance could be artificially induced came from the radiobiologists in lethally x-irradiated adult animals treated with homologous or heterologous bone marrow, respectively. Nakic and associates3 have been investi gating the possibility of inducing specific From the Department of Surgery, University of Chicago, Chicago, 111. This work was supported by the Douglas Smith Founda tion for Medical Research, the Danforth Foundation Grant in Surgery, and U. S. Public Health Service Fellowship 1-F2-AI-24.235-01. Read at the Forty-fifth Annual Meeting of The American Association for Thoracic Surgery, New Orleans, La., March 29-31, 1965.
tolerance to skin homografts in immunologically mature rats by the method of parabiosis, finding that homografts exchanged between homologous rat parabionts sepa rated after 5 to 6 days survived longer than in control animals. Intermittent uncontrolled cross-circulation is a form of parabiosis which has been used as an investigative pro cedure in many physiological problems since L. Frederiq in 18904 studied the chemical factors affecting respiration. In 1955, Egdahl5 described the physiological basis of uncontrolled cross-circulation in dogs, and, in 1957, Roberson and Horvath 6 studied the cardiovascular hemodynamics related with the procedure. In 1956, Egdahl and Hume 7 investigated the effect of cross-circulation on kidney homotransplantation. They concluded that cross-circulating 2 animals prior to kidney transplantation for as short an interval as 5 minutes results in an immune state in the animals so that future renal homografts behave as secondary transplants. They also suggested that cross-circulating 2 animals continuously after transplantation stabilizes the transplant in some manner so that the homograft reaction is either delayed or in hibited. This work was planned to study the effect of postoperative intermittent cross-circula tion upon the survival of pulmonary tissue homografts. In one group, the cross-circula tion was carried out between the host and the donor, whereas, in the other group, the 775
Journal of
776
Gago et al.
Thoracic and Cardiovascular Surgery
host was cross-circulated with indifferent dogs. Method A total of 60 homografts was performed in nonrelated mongrel dogs weighing 20 to 30 kilograms. Left lower lobe homotransplants were made in 39 of these, of which 25 were cross-circulated and 14 served as control. The remaining 21 were right lung homografts out of which 12 were cross-cir culated and 9 used as control. No blood typing techniques were per formed previous to or after the graft. Oxytetracycline was given intravenously the day of grafting and of cross-circulation. All dogs received atropine, 0.4 to 0.8 mg., and heparin, 40 mg., intravenously before the procedure. The techniques of grafting the left lower lobe have been described previ ously by us 8 and essentially were unchanged. The cross-circulation was intermittent and uncontrolled, and was made by connecting artery to vein with a polyethylene tubing (P.E. 280) of 120 cm., with the use of either the femoral vessels or the carotid artery and the external jugular vein. At the end of each procedure, the vessels were either ligated or repaired. This procedure required frequent anesthesia. In order to obviate this, arteriovenous fistulas between the carotid artery and external jugular vein were made with two 10-cm. cannulas of Teflon No. 7 as intravascular segments joined with a loop of Silastic that could be clamped and reunited as needed. If the dog was not anticoagulated, the fistulas tended to clot within 3 to 4 days; however, they were easy to open and reuse. The blood flow in these fistulas was around 200 c.c. per minute and was usually higher at the beginning of the pro cedure. The cross-circulations were started on the first postoperative day and continued daily for 2 to 3 hours during the first 5 postoperative days, after which time the in tervals were increased gradually up to 4 days. Protocol of Dog Z-7 August 3, 1965. A mongrel dog weighing 25 kilograms was anesthetized with Surital in saline
by continuous drip method, as needed, and atro pine, 0.8 mg., intravenously. A left lower lobe homotransplant from an unrelated dog was made according to the technique formerly described by us. 8 Following surgery, the dog regained con sciousness and had normal reflex activity. (See Table I.)
Postoperative studies It has been our experience that the most accurate way to study the function and histological status of the graft is by open thoracotomies with the animal under general anesthesia, endotracheal intubation, and the respirator connected to 100 per cent oxygen. Biopsies of the graft and host upper lobe were taken at this time. Partial pressure of oxygen measurements were performed by drawing blood samples from the pulmonary artery, superior pulmonary vein, and in ferior pulmonary vein after occlusion at the atrial level of the latter two vessels. The first studies were made on the third postTable I Day
1 2 3 4 5 6
The dog was cross-circulated daily with unrelated dogs, as described previously, for a period of 2 to 3 hours; no untoward reaction was noted First open thoracotomy: Blood from the left lower lobe vein showed normal oxygen values; a biopsy of the transplanted lobe showed nor mal histologie pattern
9
Cross-circulation with unrelated dog
10
Second open thoracotomy: Normal lung function in transplanted lobe; histologie examination showed min imal chronic rejection changes
12 15 20
}
Cross-circulation with unrelated dogs; no reaction
21
Third open thoracotomy; normal function continues; mild chronic re jection (see Fig. 2)
24
Dog died from pneumonia; weight loss and pneumonia were likely the result of repeated anesthetizations and thoracotomies
Volume 50
Canine pulmonary homografts
Number 6
777
December, 1965
operative day in the control group and usually on the sixth or seventh day in the dogs that were cross-circulated. Results Homotransplantation of the left lower lobe. The control group of 14 dogs showed equal findings to those obtained by us pre
viously8 (Table II). Two of these prepara tions were performed in littermates, and thoracotomy on the fifth and sixth day showed gross rejection of the graft. As an ticipated in this group, there was a signifi cant uniformity in the functional and histological data which showed a complete loss of function on the third postoperative day.
Table II. Homotransplantation of left lower lobe (untreated) pOi mm. Hg Dog 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
No.
Day posttransplantation 3 3 3 3 3 3 3 3 3 3 4 4 6 5
(100) (117) (Π6) (120) (160) (Z-27) (Z-30) (Z-31) (Z-32) (Z-34) (Z-29) (Z-35) (G-8A) (Z-13)
Pulmonary Pulmonary
artery
veins
Inferior
Superior
31.6 34.0 55.0 65.2 55.2 35.0 49.0 56.0 59.0 59.0 64.0
365.0 143.0 223.0 432.0 350.0 400.0 270.0 560.0 475.0 415.0 460.0
22.5 37.8 44.7 71.5 50.6 51.0 36.0 40.0 43.0 45.0 43.0 Gross rejection* Gross rejectiont Gross rejectiont
*Hepatization of the graft. t Donor and host were litte rmates.
Table III. Homotransplantation with donor dogs)
of left lower lobe (uncontrolled cross-circulation pOt mm.
Dog 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
No.
Day posttransplantation
(Z-4) (Z-ll) (Z-21) (Z-47) (Z-24) (Z-l) (Z-44) (Z-49) (Z-5) (Z-3) (Z-2) (Z-6) (Z-8) (Z-19)
6 6 5 5 5 7 6 5 7 6 5 6 2 2
15. (Z-48)
5
Hg Pulmonary
Pulmonary
artery
67 68 22 40 64 Gross rejection Gross rejection Gross rejection 60 40 Thrombosis of vein Pneumonia Hemorrhage Hemorrhagic lungs on autopsy 42
Inferior
\
veins Superior
470 340 385 105 75
425 365 330 120 240
350 65
490 430
420
430
Journal of
778
Gago et al.
Thoracic and Cardiovascular Surgery
With these consistent findings, preoperative histocompatibility studies are probably of no value in planning pulmonary homografts in dogs. The cross-circulated group of 25 dogs was subdivided into 15 dogs that were crosscirculated with the donor and 10 dogs crosscirculated with indifferent dogs (a different dog was used each day for cross-circulation with the host). Of the first sub-group (Table III), 6 preparations exhibited normal func tion of the graft on the fifth, sixth, and sev enth postoperative days, 3 dogs had gross rejection of the graft at the same postopera tive period, 2 dogs had minimal function, 1 had thrombosis of the venous anastomosis, 1 had pneumonia, 1 died with postoperative bleeding, and 1 died on the second postop erative day with a possible anaphylactic re action, showing hemorrhagic lungs on au topsy. Table IV summarizes the results of cross-circulation with indifferent dogs; 4 ani mals showed normal function of the graft on the sixth, seventh, fourteenth, and twentyfirst postoperative days, 3 dogs had de creased function on the fourth, sixth, and seventh days, 1 had no function on the sixth day, in 1 the preparation was grossly re jected on the sixth day, and 1 animal had thrombosis of the venous anastomosis on the sixth day. Homotransplantation of the right lung. The control group of 9 dogs (Table V)
showed no function or insignificant oxygéna tion on the third postoperative day, and 5 of them died between the fifth and seventh postoperative days with rejection (only 1 survived for 30 days with a rejected right lung). In the cross-circulated group of 12 dogs, 10 animals (Table VI) were cross-circulated with the donor and 2 with indifferent dogs. In the former group, two preparations were rejected on the fifth postoperative days, 2 dogs had a possible anaphylactic reaction during cross-circulation on the fourth day, 3 dogs died with aspiration during crosscirculation on the fourth day, and 3 were considered as technical failures. In the latter group of 2 dogs cross-circulated with indif ferent dogs, one preparation was rejected on the sixth day and the other animal died on the second postoperative day. Histologie studies supported the func tional data, showing normal histology in those animals with normal p 0 2 determi nations and moderate or severe rejection in the others (Fig. 1). Dog Z-24 showed severe diffuse pneumonia and minimal function. Dogs Z-7 and Z-20, at 10, 21, and 14 days, showed mild chronic rejection changes, al though the function was normal (Fig. 2). The findings in the group with right lung homografts were equivocal and will be a matter of further study.
Table IV. Homotransplantation of the left lower lobe, (uncontrolled cross-circulation with indifferent dogs) pOi mm. Hg Dog 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
No.
Day posttransplantation
(Z-7) (Z-12) (Z-16) (Z-20) (Z-22) (Z-38) (Z-41) (Z-43) (Z-40) (Z-42)
•Hepatization of the graft.
21 6 7 14 6 4 7 6 6 6
Pulmonary Pulmonary
artery
39 51 40 42 33 62 55 64 Gross rejection* Thrombosis inf. pul. vein
Inferior 358 34 440 380 320 78 69 74
\
veins Superior 500 410 495 520 490 410 370 450
Volume 50 Number 6
Canine pulmonary homografts
December, 1965
779
Table V. Homotransplantation of right lung (untreated)
Dog No. 1. 2. 3. 4. 5. 6. 7. 8. 9.
(Z-44) (Z-45) (Z-46) (Z-39) (2A) (3B) (4A)
(5A) (1A)
Day post-trans plantation
pOt (Rt. pul monary veins)
3 4 3 5
75 92 69 Died from rejection
7
Died from rejection
30
Died from rejection
J
Table VI. Homotransplantation of right lung (uncontrolled cross-circulation with donor dogs)
Dog No. 1 (Z-9) 2. (Z-10)
Day post-trans plantation 5 5
3. (Z-23) 4. 5. 6. 7. 8. 9. 10.
(Z-26) (Z-18) (Z-19B) (Z-27B) (Z-25) (Z-39B) (Z-4 IB)
4 4
Status Rejected Sacrificed—func tional? Anaphylaxis (?) during crosscirculation Died from aspiration during crosscirculation
Fig. 1. Open chest biopsy of the homografted lobe in Dog Z-4, 6 days after grafting, shows normal histologie pattern, (x 40; reduced ·%.)
Technical failures
Discussion In the control groups of 24 dogs with left lower lobe homografts and right lung homografts, there is a constant loss of function on the third postoperative day. The main difference between these two groups is that the animals with left lower lobe homografts tolerated the surgical procedure bet ter than those with grafts of the right lung. In 25 dogs with left lower lobe homotransplants, intermittent cross-circulation alone was shown to be effective, although inconstantly, in modifying the immune re sponse, inducing temporary tolerance to the grafted pulmonary lobe as expressed by the
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Fig. 2. Biopsy of the homotransplanted lobe in Dog Z-7, 21 days after procedure, shows mild peribronchial and perivascular mononuclear cell infiltration, (x 40; reduced ψΊ.)
Journal of
780
Gago et al.
Thoracic and Cardiovascular Surgery
normal function and satisfactory histology as long as 21 days post-grafting. No longterm survival was obtained since frequent thoracotomies were necessary for perform ing biopsies and making functional studies. In addition, the great majority of these animals developed a syndrome of "parabiotic intoxication," characterized by anorexia, weight loss, diarrhea, and weakness, in creasing the mortality rate of additional thoracotomies. In those animals with retained normal function, in which the cross-circulations were discontinued following lung biopsy and p 0 2 determinations, rejection of the graft occurred within a few days. These findings could be explained accord ing to the concept of "competitive replace ment" where both nonspecific depression of the immune reaction and specific tolerance observed in separated parabionts result from the transfer of immunologically competent cells from one animal to another. The for eign cells may develop antihost antibodies and suppress the lymphopoietic structures of the recipient. This would account for the depression of the immune response in the group of dogs cross-circulated with the donor in which the state of non-reactivity is maintained through a constant immunological pressure of donor cells against immunologically active host cells. In the group of dogs cross-circulated with different animals following left lower lobe homografts, the dilution factor of the antibody level of the host was the most important mechanism of the temporary tolerance. A third possible mechanism of temporary tolerance may have been adsorption of anti bodies by the donor tissues during crosscirculation, and thus a decrease in the level of circulating antibodies in the host, delay ing in this way the host versus graft reac tion. This latter theory could be applied to either one of the discussed groups of dogs. In 12 dogs with right lung homotransplants, cross-circulation alone was unable to induce tolerance, although only 2 dogs re ceived cross-circulation with different dogs. The unequivocal negative results in the
group cross-circulated with the donor makes us assume that the quantity of antigen (lung in this case) plays a significant role in de termining its subsequent fate. Summary Intermittent uncontrolled cross-circulation is an effective procedure for inducing tem porary tolerance in homotransplants of pul monary tissue in adult dogs (as long as 21 days post-grafting). Rejection occurred a few days after the cross-circulations were discontinued. The control groups of left lower lobe and right lung homografts showed a constant loss of function at 72 hours post-grafting. We wish to acknowledge the assistance of Dr. Francis L. Archer, Department of Pathology, in the interpretation of the histological preparations. REFERENCES 1 Owen, R. D.: Immunogenetic Consequences of Vascular Anastomoses Between Bovine Twins, Science 102: 400, No. 2651, 1945. 2 Anderson, D., Billingham, R. E., Lampkin, G. H., and Medawar, P. B.: The Use of Skin Graft ing to Distinguish Between Monozygotic and Dizygotic Twins in Cattle, Heredity 5: 379, 1951. 3 Nakic, B., Kastelan, A., and Audalovic, N.: In duction of Specific Tolerance in Adult Rats by the Method of Parabiosis, Ciba Foundation Symposium on Transplantation, Boston, 1962, Little, Brown & Company. 4 Frederiq, L.: Sur la circulation cephalique croisée, on échange de sang carotidien entre deux animaux, Arch. Biol. 10: 127, 1890. 5 Egdahl, R. H.: Physiological Basis of Uncon trolled Cross-Circulation in Dogs, Am. J. Physiol. 182: 454, 1955. 6 Roberson, W. J., and Horvath, S. M.: Cardio vascular Hemodynamics Consequent to Crossed Circulation in Dogs, Am. J. Physiol. 192: 345, 1958. 7 Egdahl, R. H., and Hume, D . M.: Immunologie Studies in Renal Homotransplantation, Surg., Gynec. & Obst. 102: 450, 1956. 8 Gago, O., Delgado, E., Archer, F . L., Schoenfeld, F . G., Ranniger, K., Nigro, S. L., and Adams, W. E.: Homotransplantation and Autotransplantation of a Pulmonary Lobe, J. THORACIC & CARDIOVAS. SURG. 48: 726,
(For Discussion,
see page 789)
1964.
JLn 1945, Owen1 observed and described the phenomenon of red cell chimerism in twin cattle, where the vascular systems be fore birth are not sharply distinct from each other, as they are in other twins. Instead, there are anastomoses with each other, re sulting in a prolonged intrauterine exchange of blood, and possible red cell precursors. He also discovered that most twin cattle may retain through life a stable mixture of each other's red cells. A few years later, Anderson, Billingham, Lampkin, and Medawar2 showed that most dizygotic cattle twins would accept skin grafts from each other, and that this mutual tolerance was specific, since skin transplants from third parties were rejected in the expected manner. For some time, it was believed that the induction of tolerance to living tissue was limited by the so-called "adaptive period" which in most species ends around the time of birth. The first indication that artificial tolerance could be artificially induced came from the radiobiologists in lethally x-irradiated adult animals treated with homologous or heterologous bone marrow, respectively. Nakic and associates3 have been investi gating the possibility of inducing specific From the Department of Surgery, University of Chicago, Chicago, 111. This work was supported by the Douglas Smith Founda tion for Medical Research, the Danforth Foundation Grant in Surgery, and U. S. Public Health Service Fellowship 1-F2-AI-24.235-01. Read at the Forty-fifth Annual Meeting of The American Association for Thoracic Surgery, New Orleans, La., March 29-31, 1965.
tolerance to skin homografts in immunologically mature rats by the method of parabiosis, finding that homografts exchanged between homologous rat parabionts sepa rated after 5 to 6 days survived longer than in control animals. Intermittent uncontrolled cross-circulation is a form of parabiosis which has been used as an investigative pro cedure in many physiological problems since L. Frederiq in 18904 studied the chemical factors affecting respiration. In 1955, Egdahl5 described the physiological basis of uncontrolled cross-circulation in dogs, and, in 1957, Roberson and Horvath 6 studied the cardiovascular hemodynamics related with the procedure. In 1956, Egdahl and Hume 7 investigated the effect of cross-circulation on kidney homotransplantation. They concluded that cross-circulating 2 animals prior to kidney transplantation for as short an interval as 5 minutes results in an immune state in the animals so that future renal homografts behave as secondary transplants. They also suggested that cross-circulating 2 animals continuously after transplantation stabilizes the transplant in some manner so that the homograft reaction is either delayed or in hibited. This work was planned to study the effect of postoperative intermittent cross-circula tion upon the survival of pulmonary tissue homografts. In one group, the cross-circula tion was carried out between the host and the donor, whereas, in the other group, the 775
Journal of
776
Gago et al.
Thoracic and Cardiovascular Surgery
host was cross-circulated with indifferent dogs. Method A total of 60 homografts was performed in nonrelated mongrel dogs weighing 20 to 30 kilograms. Left lower lobe homotransplants were made in 39 of these, of which 25 were cross-circulated and 14 served as control. The remaining 21 were right lung homografts out of which 12 were cross-cir culated and 9 used as control. No blood typing techniques were per formed previous to or after the graft. Oxytetracycline was given intravenously the day of grafting and of cross-circulation. All dogs received atropine, 0.4 to 0.8 mg., and heparin, 40 mg., intravenously before the procedure. The techniques of grafting the left lower lobe have been described previ ously by us 8 and essentially were unchanged. The cross-circulation was intermittent and uncontrolled, and was made by connecting artery to vein with a polyethylene tubing (P.E. 280) of 120 cm., with the use of either the femoral vessels or the carotid artery and the external jugular vein. At the end of each procedure, the vessels were either ligated or repaired. This procedure required frequent anesthesia. In order to obviate this, arteriovenous fistulas between the carotid artery and external jugular vein were made with two 10-cm. cannulas of Teflon No. 7 as intravascular segments joined with a loop of Silastic that could be clamped and reunited as needed. If the dog was not anticoagulated, the fistulas tended to clot within 3 to 4 days; however, they were easy to open and reuse. The blood flow in these fistulas was around 200 c.c. per minute and was usually higher at the beginning of the pro cedure. The cross-circulations were started on the first postoperative day and continued daily for 2 to 3 hours during the first 5 postoperative days, after which time the in tervals were increased gradually up to 4 days. Protocol of Dog Z-7 August 3, 1965. A mongrel dog weighing 25 kilograms was anesthetized with Surital in saline
by continuous drip method, as needed, and atro pine, 0.8 mg., intravenously. A left lower lobe homotransplant from an unrelated dog was made according to the technique formerly described by us. 8 Following surgery, the dog regained con sciousness and had normal reflex activity. (See Table I.)
Postoperative studies It has been our experience that the most accurate way to study the function and histological status of the graft is by open thoracotomies with the animal under general anesthesia, endotracheal intubation, and the respirator connected to 100 per cent oxygen. Biopsies of the graft and host upper lobe were taken at this time. Partial pressure of oxygen measurements were performed by drawing blood samples from the pulmonary artery, superior pulmonary vein, and in ferior pulmonary vein after occlusion at the atrial level of the latter two vessels. The first studies were made on the third postTable I Day
1 2 3 4 5 6
The dog was cross-circulated daily with unrelated dogs, as described previously, for a period of 2 to 3 hours; no untoward reaction was noted First open thoracotomy: Blood from the left lower lobe vein showed normal oxygen values; a biopsy of the transplanted lobe showed nor mal histologie pattern
9
Cross-circulation with unrelated dog
10
Second open thoracotomy: Normal lung function in transplanted lobe; histologie examination showed min imal chronic rejection changes
12 15 20
}
Cross-circulation with unrelated dogs; no reaction
21
Third open thoracotomy; normal function continues; mild chronic re jection (see Fig. 2)
24
Dog died from pneumonia; weight loss and pneumonia were likely the result of repeated anesthetizations and thoracotomies
Volume 50
Canine pulmonary homografts
Number 6
777
December, 1965
operative day in the control group and usually on the sixth or seventh day in the dogs that were cross-circulated. Results Homotransplantation of the left lower lobe. The control group of 14 dogs showed equal findings to those obtained by us pre
viously8 (Table II). Two of these prepara tions were performed in littermates, and thoracotomy on the fifth and sixth day showed gross rejection of the graft. As an ticipated in this group, there was a signifi cant uniformity in the functional and histological data which showed a complete loss of function on the third postoperative day.
Table II. Homotransplantation of left lower lobe (untreated) pOi mm. Hg Dog 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
No.
Day posttransplantation 3 3 3 3 3 3 3 3 3 3 4 4 6 5
(100) (117) (Π6) (120) (160) (Z-27) (Z-30) (Z-31) (Z-32) (Z-34) (Z-29) (Z-35) (G-8A) (Z-13)
Pulmonary Pulmonary
artery
veins
Inferior
Superior
31.6 34.0 55.0 65.2 55.2 35.0 49.0 56.0 59.0 59.0 64.0
365.0 143.0 223.0 432.0 350.0 400.0 270.0 560.0 475.0 415.0 460.0
22.5 37.8 44.7 71.5 50.6 51.0 36.0 40.0 43.0 45.0 43.0 Gross rejection* Gross rejectiont Gross rejectiont
*Hepatization of the graft. t Donor and host were litte rmates.
Table III. Homotransplantation with donor dogs)
of left lower lobe (uncontrolled cross-circulation pOt mm.
Dog 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
No.
Day posttransplantation
(Z-4) (Z-ll) (Z-21) (Z-47) (Z-24) (Z-l) (Z-44) (Z-49) (Z-5) (Z-3) (Z-2) (Z-6) (Z-8) (Z-19)
6 6 5 5 5 7 6 5 7 6 5 6 2 2
15. (Z-48)
5
Hg Pulmonary
Pulmonary
artery
67 68 22 40 64 Gross rejection Gross rejection Gross rejection 60 40 Thrombosis of vein Pneumonia Hemorrhage Hemorrhagic lungs on autopsy 42
Inferior
\
veins Superior
470 340 385 105 75
425 365 330 120 240
350 65
490 430
420
430
Journal of
778
Gago et al.
Thoracic and Cardiovascular Surgery
With these consistent findings, preoperative histocompatibility studies are probably of no value in planning pulmonary homografts in dogs. The cross-circulated group of 25 dogs was subdivided into 15 dogs that were crosscirculated with the donor and 10 dogs crosscirculated with indifferent dogs (a different dog was used each day for cross-circulation with the host). Of the first sub-group (Table III), 6 preparations exhibited normal func tion of the graft on the fifth, sixth, and sev enth postoperative days, 3 dogs had gross rejection of the graft at the same postopera tive period, 2 dogs had minimal function, 1 had thrombosis of the venous anastomosis, 1 had pneumonia, 1 died with postoperative bleeding, and 1 died on the second postop erative day with a possible anaphylactic re action, showing hemorrhagic lungs on au topsy. Table IV summarizes the results of cross-circulation with indifferent dogs; 4 ani mals showed normal function of the graft on the sixth, seventh, fourteenth, and twentyfirst postoperative days, 3 dogs had de creased function on the fourth, sixth, and seventh days, 1 had no function on the sixth day, in 1 the preparation was grossly re jected on the sixth day, and 1 animal had thrombosis of the venous anastomosis on the sixth day. Homotransplantation of the right lung. The control group of 9 dogs (Table V)
showed no function or insignificant oxygéna tion on the third postoperative day, and 5 of them died between the fifth and seventh postoperative days with rejection (only 1 survived for 30 days with a rejected right lung). In the cross-circulated group of 12 dogs, 10 animals (Table VI) were cross-circulated with the donor and 2 with indifferent dogs. In the former group, two preparations were rejected on the fifth postoperative days, 2 dogs had a possible anaphylactic reaction during cross-circulation on the fourth day, 3 dogs died with aspiration during crosscirculation on the fourth day, and 3 were considered as technical failures. In the latter group of 2 dogs cross-circulated with indif ferent dogs, one preparation was rejected on the sixth day and the other animal died on the second postoperative day. Histologie studies supported the func tional data, showing normal histology in those animals with normal p 0 2 determi nations and moderate or severe rejection in the others (Fig. 1). Dog Z-24 showed severe diffuse pneumonia and minimal function. Dogs Z-7 and Z-20, at 10, 21, and 14 days, showed mild chronic rejection changes, al though the function was normal (Fig. 2). The findings in the group with right lung homografts were equivocal and will be a matter of further study.
Table IV. Homotransplantation of the left lower lobe, (uncontrolled cross-circulation with indifferent dogs) pOi mm. Hg Dog 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
No.
Day posttransplantation
(Z-7) (Z-12) (Z-16) (Z-20) (Z-22) (Z-38) (Z-41) (Z-43) (Z-40) (Z-42)
•Hepatization of the graft.
21 6 7 14 6 4 7 6 6 6
Pulmonary Pulmonary
artery
39 51 40 42 33 62 55 64 Gross rejection* Thrombosis inf. pul. vein
Inferior 358 34 440 380 320 78 69 74
\
veins Superior 500 410 495 520 490 410 370 450
Volume 50 Number 6
Canine pulmonary homografts
December, 1965
779
Table V. Homotransplantation of right lung (untreated)
Dog No. 1. 2. 3. 4. 5. 6. 7. 8. 9.
(Z-44) (Z-45) (Z-46) (Z-39) (2A) (3B) (4A)
(5A) (1A)
Day post-trans plantation
pOt (Rt. pul monary veins)
3 4 3 5
75 92 69 Died from rejection
7
Died from rejection
30
Died from rejection
J
Table VI. Homotransplantation of right lung (uncontrolled cross-circulation with donor dogs)
Dog No. 1 (Z-9) 2. (Z-10)
Day post-trans plantation 5 5
3. (Z-23) 4. 5. 6. 7. 8. 9. 10.
(Z-26) (Z-18) (Z-19B) (Z-27B) (Z-25) (Z-39B) (Z-4 IB)
4 4
Status Rejected Sacrificed—func tional? Anaphylaxis (?) during crosscirculation Died from aspiration during crosscirculation
Fig. 1. Open chest biopsy of the homografted lobe in Dog Z-4, 6 days after grafting, shows normal histologie pattern, (x 40; reduced ·%.)
Technical failures
Discussion In the control groups of 24 dogs with left lower lobe homografts and right lung homografts, there is a constant loss of function on the third postoperative day. The main difference between these two groups is that the animals with left lower lobe homografts tolerated the surgical procedure bet ter than those with grafts of the right lung. In 25 dogs with left lower lobe homotransplants, intermittent cross-circulation alone was shown to be effective, although inconstantly, in modifying the immune re sponse, inducing temporary tolerance to the grafted pulmonary lobe as expressed by the
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, " . . - ■ ■*■■
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ϊ.·„
,
,
Fig. 2. Biopsy of the homotransplanted lobe in Dog Z-7, 21 days after procedure, shows mild peribronchial and perivascular mononuclear cell infiltration, (x 40; reduced ψΊ.)
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normal function and satisfactory histology as long as 21 days post-grafting. No longterm survival was obtained since frequent thoracotomies were necessary for perform ing biopsies and making functional studies. In addition, the great majority of these animals developed a syndrome of "parabiotic intoxication," characterized by anorexia, weight loss, diarrhea, and weakness, in creasing the mortality rate of additional thoracotomies. In those animals with retained normal function, in which the cross-circulations were discontinued following lung biopsy and p 0 2 determinations, rejection of the graft occurred within a few days. These findings could be explained accord ing to the concept of "competitive replace ment" where both nonspecific depression of the immune reaction and specific tolerance observed in separated parabionts result from the transfer of immunologically competent cells from one animal to another. The for eign cells may develop antihost antibodies and suppress the lymphopoietic structures of the recipient. This would account for the depression of the immune response in the group of dogs cross-circulated with the donor in which the state of non-reactivity is maintained through a constant immunological pressure of donor cells against immunologically active host cells. In the group of dogs cross-circulated with different animals following left lower lobe homografts, the dilution factor of the antibody level of the host was the most important mechanism of the temporary tolerance. A third possible mechanism of temporary tolerance may have been adsorption of anti bodies by the donor tissues during crosscirculation, and thus a decrease in the level of circulating antibodies in the host, delay ing in this way the host versus graft reac tion. This latter theory could be applied to either one of the discussed groups of dogs. In 12 dogs with right lung homotransplants, cross-circulation alone was unable to induce tolerance, although only 2 dogs re ceived cross-circulation with different dogs. The unequivocal negative results in the
group cross-circulated with the donor makes us assume that the quantity of antigen (lung in this case) plays a significant role in de termining its subsequent fate. Summary Intermittent uncontrolled cross-circulation is an effective procedure for inducing tem porary tolerance in homotransplants of pul monary tissue in adult dogs (as long as 21 days post-grafting). Rejection occurred a few days after the cross-circulations were discontinued. The control groups of left lower lobe and right lung homografts showed a constant loss of function at 72 hours post-grafting. We wish to acknowledge the assistance of Dr. Francis L. Archer, Department of Pathology, in the interpretation of the histological preparations. REFERENCES 1 Owen, R. D.: Immunogenetic Consequences of Vascular Anastomoses Between Bovine Twins, Science 102: 400, No. 2651, 1945. 2 Anderson, D., Billingham, R. E., Lampkin, G. H., and Medawar, P. B.: The Use of Skin Graft ing to Distinguish Between Monozygotic and Dizygotic Twins in Cattle, Heredity 5: 379, 1951. 3 Nakic, B., Kastelan, A., and Audalovic, N.: In duction of Specific Tolerance in Adult Rats by the Method of Parabiosis, Ciba Foundation Symposium on Transplantation, Boston, 1962, Little, Brown & Company. 4 Frederiq, L.: Sur la circulation cephalique croisée, on échange de sang carotidien entre deux animaux, Arch. Biol. 10: 127, 1890. 5 Egdahl, R. H.: Physiological Basis of Uncon trolled Cross-Circulation in Dogs, Am. J. Physiol. 182: 454, 1955. 6 Roberson, W. J., and Horvath, S. M.: Cardio vascular Hemodynamics Consequent to Crossed Circulation in Dogs, Am. J. Physiol. 192: 345, 1958. 7 Egdahl, R. H., and Hume, D . M.: Immunologie Studies in Renal Homotransplantation, Surg., Gynec. & Obst. 102: 450, 1956. 8 Gago, O., Delgado, E., Archer, F . L., Schoenfeld, F . G., Ranniger, K., Nigro, S. L., and Adams, W. E.: Homotransplantation and Autotransplantation of a Pulmonary Lobe, J. THORACIC & CARDIOVAS. SURG. 48: 726,
(For Discussion,
see page 789)
1964.