Experimental Homotransplantation of the Lungs with Cytoxic Agents

Experimental Homotransplantation of the Lungs with Cytoxic Agents* PARVIZ PARSA., M.D ..,

L.

PENFIELD FABER., M.D..,

E. WILSON STAUB., M.D. AND EDWARD J. BEATTIE., JR.., M.D.

Chicago., Illinois

T

HE

TRANSPLANTATION

OF

survival of only six days. There has been a considerable amount of experimental work with cytotoxic agents in homotransplantation, a complete review of which is beyond the scope of this paper.

TISSUES

and organs is of vital importance to surgeons. Replacement of the lung would be of value whenever the lungs are irreversibly diseased of damaged. Homotransplantation of the lung in man appears technically feasible; and since the lung is a paired organ, potential donors are available. It has been demonstrated in our laboratory and by other investigators, 1.2 that 21 days was the maximal survival period for untreated dogs with homologously transplanted lungs, with an average

Blumenstock and colleagues3 have reported the use of methotrexate (4-amino10 N -methyl-pteroglutamic acid) in homografted canine lungs with some success. l\lexandre and Murray,4 in Boston, by utilizing Imuran** (l-methyl-4-nitro-5-imidozolyl thiopurine), have shown that the homotransplanted kidney can survive for more than three months in over 20 per cent of dogs. The effect of Imuran has been disappointing in prolonging skin homograft survival,5 but this may be due to the difference in the ability of different tissues to survive homotransplantation. The amount of antigen produced by the homotransplanted organ appears to be of im-

*Supported by grant-in aid from Research and Education Committee, Presbyterian-St. Luke's Hospital. From the Department of Surgery, PresbyterianSt. Luke's Hospital, affiliated with the U niversity of Illinois. Presented at the 29th Annual Meeting, American College of Chest Physicians, Atlantic City, June 13-17, 1963. **Supplied to us as BW-57-322 by Burroughs-Wellcome Co., Tuckahoe, New York.

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(C~ AORTA FIGURE 1: Diagram of the technic of homotransplantation of lung. Note that the atrial cuff is anastomosed rather than individual pulmonary veins.

365

Diseases of the Chest

PARSA, FABER, STAUB AND BEATTIE

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portance. Billingham and co-workers6 have suggested that a large amount of circulating antigen will suppress antibody formation by the host against the donor. It is believed that the kidney has a longer survival time than skin because of the larger amount of antigen which the kidney produces. It might also be expected that the lung would produce more circulating antigen than skin and be a reasonable organ for homotransplantation. The purpose of this experiment was to investigate the effect of Imuran and methotrexate on homologously transplanted canine lungs. MATERIALS AND METHODS

Pairs of mongrel dogs were selected

weighing approximately 12 to 20 kg. Homotransplantation of the lung was performed in 16 dogs by anastomosing the atrial cuff (Fig. 1). The average time required for re-establishing the circulation in the transplanted lung was 50 minutes. Blood transfusion was given from the donor to the recipient, if needed. The operative procedure was completed on every dog, and no dog was lost during the operation. The dogs were divided into two treatment groups. Group I consisted of five dogs. They received methotrexate 0.1 mg./ kg., intramuscularly, every other day for one week, followed by 0.02 mg',/kg. three times per week thereafter as employed by

TABLE I-TRANSPLANTED LUNGS IN DOGS TREATED WITH METHOTREXATE

Dog No.

Survival (No. of Days)

2

I

17 7

3

24

4

14

5

3

Cause of Death (Gross) Mediastinal Emphysema Gastric Hemorrhage Both lungs aerated Dense L pleural adhesions L Lung not aerated Firm with dense adhesions Empyema and lung adhesions Pulmonary art. thrombosis

Microscopic Findings Mild rejection reaction Moderate rejection no necrosis Severe rejection with necrosis Severe rejection with loss of architecture Severe necrosis

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EXPERIMENTAL HOMOTRANSPLANTATION OF THE LUNGS

Blumenstock and associates.' The first dose was started immediately following transplantation. Group II consisted of 11 dogs which received Imuran. Each dog was given 6-mercaptopurine, 10 mg.lkg., intramuscularly, at the time of the operation. The first day following surgery, Imuran, 10 mg.lkg. was started orally for three days, followed by 6 mg/kg. daily thereafter, as used by Alexandre and Murray' in renal homotransplantation. In addition, each dog in both groups received penicillin and streptomycin intramuscularly for five to seven days.

of 27 days. Four of 11 dogs (37 per cent) survived over 21 days, i.e., 26, 28, 64, and 32 days.

Follow-up studies of the dogs included frequent examination of the chest by auscultation, blood counts, chest x-ray films, bronchoscopy, and bronchospirometry. as indicated. RESULTS

Table 1 summarizes the results of methotrexate-treated dogs. In the four technically successful homotransplanted lungs treated with methotrexate, the survival time ranged from seven to 24 days, with an average of 15 days. One animal survived more than 21 days. Table 2 summarizes the results of Imuran-treated dogs. In the 11 dogs treated with Imuran, the survival varied from seven to 64 days, with an average survival

LL HT•• 5wks. Posf·op. FIGURE 3: Both lung fields of dog No. 13 are clear five weeks following surgery.

TABLE 2-TRANSPLANTED LUNGS IN DOGS TREATED WITH IMURAN

Dog No.

Survival (No. of Days)

Cause of Death (Gross)

Microscopic Findings

7

20

Empyema and lung abscess

8

20

Lung abscess

9 10 11

21 62 28

12

13

Pneumonia Aspiration pneumonia Firm meaty lung with dense adhesions Hemorrhagic pneumonia

Severe necrosis of lung parenchyma Moderate rejection with no necrosis Severe rejection and necrosis No rejection Severe rejection

13

64

Hemorrhagic pneumonia

14

32 7

Bilateral pneumonia Pneumonia Finn heavy lung Pneumonia

15 16 17

11

17

Hemorrhagic pneumonia with mild rejection Hemorrhagic pneumonia with no rejection Pneumonia with severe rejection Pneumonia with mild rejection Pneumonia with mild rejection Pneumonia with moderate rejection

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PARSA, FABER, STAUB AND BEATTIE

TABLE 3-UNTREATED DOGS REPORTED PREVIOUSLY! Source of Variation

Analysis of Variance Degrees of Freedom

Treated vs. non-treated Methotrexate vs. Imuran Individuals within Groups Total

1 1 19 21

Mean Square F ._----'-----------

663.76 656.36 152.33

4.36 4.31

The two F values are almost equal to F at the 5 per cent significanc elevel (4.38) and suggest that: (1) both Methotrexate and Imuran treated dogs live longer than non-treated dogs; and (2) dogs treated with Imuran live significantly longer than those treated with Methotrexate.

All the dogs on the first postoperative day were alert and were allowed to leave their cages for 15 to 30 minutes. They would eat their meals zealously for a period of four to five days. The group of dogs on methotrexate usually became anorexic about the fifth day postoperatively and re-

fused all feedings. The dogs on Imuran did not manifest anorexia until shortly before death. Bronchospirometry was performed In three of the methotrexate-treated dogs as follows: No. 1 at ten and 17 days, No.2 at five days, and No. 3 at nine days post-

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FIOURE 4: Microscopic section of the homotransplanted left lung in dog No. 10 62 days post-homotransplantation reveals no evidence of rejection (xlOO). FIOVRE 5: Microscopic section of the left lung in dog No. 13 64 days post-homotransplantation also shows no evidence of rejection (xlOO).

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EXPERIMENTAL HOMOTRANSPLANTATION OF THE LUNGS

Flo.

6

FlO. 7

FIG. 8

FIOURE 6: Microscopic section of the left lung in dog No. 1 17 days post-homotransplantation demonstrates mild perivascular infiltration with round cells and slight thickening of septal wall (xl00). FIOURE 7: Microscopic section of the left lung in dog No. 2 seven days post-homotransplantation shows a more intense perivascular and peribronchial round cell infiltrations (xIOO). FlOUR£. 8: Microscopic section of the left lung in dog No. 11 28 days post-homotransplantation shows severe loss of architecture of the lung (xl00).

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Diseases of the Chest

PARSA, FABER, STAUB AND BEATTIE

transplantation. Ventilation was present, but there was no oxygen uptake by the transplanted lung. Dog No. 1 suffered bronchial disruption during final bronchospirometry. In one dog treated with Imuran, there was significant oxygen consumption two months after surgery. Bronchospirometry on this dog (No. 10), performed on two occasions, revealed 32 per cent oxygen uptake for the left lung, as compared to 62 per cent for the right lung (Fig. 2). This dog died of aspiration pneumonia following the second bronchospirometry. We limited the number of bronchoscopy and bronchospirometry examinations to eliminate the complications of bronchial disruption and introduction of bacteria in an animal in which the immune response is suppressed. In another dog (No. 13) treated with Imuran, we have reason to believe that the homografted lung was functioning. A chest x-ray film of this dog (No. 13) was normal (Fig. 3). There were breath sounds in the chest, noted by auscultation; and the microscopic examination of the lung, at necropsy, revealed no evidence of rejection. At necropsy two of the homotransplanted lungs treated with methotrexate were crepitant and collapsible. In the homotransplanted lungs treated with Imuran, seven of 11 dogs had crepitant and collapsible lungs. Microscopic section of all transplanted lungs were reviewed. We classified the findings as normal or mild, moderate and severe rejections. Two Imurantreated dogs revealed no evidence of rejection (Fig. 4 and 5). One methotrexatetreated and three Imuran-treated dogs showed mild perivascular and peribronchial round-cell infiltration, with slight thickening of the septal wall (Fig. 6). This reaction appeared to be the mildest type of rejection. One methotrexate-treated and two Imuran-treated dogs revealed perivascular and peri-bronchial infiltration. The septal wall was also infiltrated with round cells (Fig. 7). This was considered a moderate type of rejection. Lastly, in three methotrexate and four Imuran-treated

dogs, the lung parenchyma was destroyed by round cell infiltration and necrosis (Fig. 8) . COMMENTS

Pneumonia and lung abscess were the main contributing factors to death. However, these cannot be explained due to transplantation per se because animals on cytotoxic agents are more prone to infection. It is difficult to obtain good nursing care in dogs during the postoperative period. Loss of cilia as seen in microscopic sections leads to stagnation of mucus in the bronchi, providing a good culture medium for bacteria introduced during bronchoscopy and intubation. A breakdown of the bronchial anastomosis was observed in four dogs, in spite of precautionary measures not to strip the bronchial wall during surgery. The vascular anastomoses were almost completely satisfactory. Only one dog was lost due to pulmonary artery thrombosis. In all other dogs, anastomosis of the left atrial cuff and the left pulmonary artery were well healed and re-endothelialized. Statistical analysis (Table 3) revealed that both Imuran and methotrexate dogs live longer than non-treated dogs, and dogs treated with Imuran live significantly longer than those treated with methotrexate. SUMMARY

Lung homotransplantation was performed in 16 paired dogs. Methotrexate and Imuran were used to prolong the survival time. In the group of dogs treated with methotrexate, the average survival was 15 days, and no dog revealed evidence of function of the transplanted lung by bronchospirometry. In the group of dogs treated with Imuran, the average survival was 27 days, with evidence of relatively normal pulmonary function in at least one dog, two months following surgery. In this dog, there was no evidence of graft injection. Death occurred from aspiration pneumonitis in the normal lung. RESU,.fEN

Se llev6 a cabo el homotransplante del pulm6n en 16 pares de perros. Se usaron Metotrexate e

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EXPERIMENTAL HOMOTRANSPLANTATION OF THE LUNGS

Imuran para prolongar el tiempo de sobrevida. En el grupo de perros tratados con Metotrexate la media de sobrevida fue de 15 dias y ningun perro mostro evidencia de trastorno funcional par la espirometria. En el grupo de perros tratados con Imuran la sobrevida media fue de 28 dias con evidencia de funci6n pulmonar relativamente normal, por 10 menos en un perro, dos meses despues de la cirugia. En este perro no hubo evidencias de rechazo del injerto. La muerte sobrevino a consecuencia de neumonitos por aspiraci6n en el pulm6n normal.

schnittliche Oberlebenszeit 15 Tage, ohne daf3 ein Hund Beweis fiir eine Loogenfunktion ergab, wie durch Bronchospirometrie nachgewiesen wurde. Bei der mit Imuran behandelten Gruppe von Hunden lag die durchschnittliche Oberlebenszeit bei 28 Tagen mit Anhaltspunkten einer relativ normalen Loogenfunktion bei wenigstens einem Tier 2 Monate nach dem Eingriff. Bei diesem Hund fand sich kein Anhalt fiir eine Abstof3ung des Transplantates. Der Tod trat infolge einer Aspirationspneumonie in der normalen Lunge ein.

RESUME

L'homogrefIe du poumon fut pratiquee chez 16 couples de chiens. Le methotrexate et l'Imuran furent utilises pour prolonger Ie temps de survie. Dans Ie groupe de chiens traites par Ie methotrexate, la survie moyenne fut de 15 jours, et aucun chien ne montra la reprise de la fonction pulmonaire par bronchospirometrie. Dans Ie groupe de chiens traite par l'Imuran, la survie moyenne fut de 28 jours, avec mise en evidence d'un fonction pulmonaire relativement normaIe chez au moins un chien, deux mois apres l'intervention. Pour ce chien, il n'y eut aucune preuve du rejet de la greffe. La mort survint apres pneumonie par aspiration du poumon normal. ZUSAMMENFASSUNG

Eine Homotransplantation wurde an 16 gepaarten Hooden vorgenommen. Zur Verlangerung der Oberlebenszeit wurden Methotrexat und Imuran verwandt. In der mit Methotrexat behandelten Gruppe von Hunden betrug die durch-

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REFERENCES FABER, L. P. AND BEATTIE, E. J., JR.: "Respiration Following Lung Denervation," Surge Forum, 9: 383, 1959. FABER, L. P., KEN WELL, J. M. AND BEATTIE, E. J., JR.: "Homologous Lung Transplantation," Arch. Surg., 83:491, 1961. BLUMENSTOCK, D. A., COLLINS, J. A., THOMAS, E. D. AND FERREBEE, J. W.: "Homotransplantation of the Lung in Dogs Treated with Methotrexate," Surge Forum, 12: 121, 1961. ALEXANDRE, G. P. AND MURRAY, J. E.: "Further Studies of Renal Homotransplantation in Dogs Treated by Combined Imuran Therapy," Surge Forum, 13 :64, 1962. HECHTMAN, H. B., BLUMENSTOCK, A. A., THOMAS, E. D. AND FERREBEE, J. W.: "Prolongation of Canine Skin Homografts by Antimetabolites," Surge Forum, 13: 55, 1962. BILLINGHAM, R. E., BRENT, L. AND MEDAWAR, P. B.: "Enhancement in Nonnal Homografts with a Note on the Possible Mechanism," Trans. Bull., 3: 84, 1956.

For reprints, please write Dr. Parsa, 1753 West Congress Street, Chicago 12.

CONCEALED VENTRICULAR EXTRASYSTOLES Evidence Is presented for the new concept that ventricular extrasystoles can be "concealed," I.e., that the extrasystole focus may be discharged without Its Impulse Invading the surrounding myocardium, as in the "exit block" of parasystole. Concealed bigeminy Is recognIzed when only odd numbers of sinus beats Intervene between consecutive

extrasystoles. Concealed trigeminy is recognizable when a multiple of 3 plus 2, I.e., 5, 8, 11, 14, etc., sinus beats Invariably occupy the Interectoplc Intervals. These concealed extrasystollc rhythms are illustrated and discussed. SCHAMROTH, L. AND MARRIOTT, H. J. L.: ··Concealed Ventricular Extrasystoles," Circulation, 27:1043. 1963.

SURGICAL TREATMENT OF ACQUIRED ANEURYSM OF THORACIC AORTA Four patients with aneurysms of the ascending aorta and four with aneurysms of the descending aorta, probably all syphllltlc, were treated by surgery with complete cure In five and death in three. Advanced disease was present In all, the patients presenting with superior mediastinal compression, arteriovenous fistula, erosion of the sternum, ribs, and vertebrae, massive hemoptyses, or obstruction to the pulmonary artery. Aortic incompetence was

present in four, in one of whom it was gross. The three deaths occurred In patients submitted to emergency or semi-emergency surgery. Retrograde angiography was most helpful In locallzlng the lesion and outllnlng the disturbed anatomy. BARNARD, C. N. AND SCHRIRE, V.: ··The Surgical Treatment of Acquired Aneurysm of the Thoracic Aorta.' • Thorax,

18: 101. 1963.