Experimental tracheal transplantation for possible clinical application

ELSEVIER

Experimental Tracheal Transplantation for Possible Clinical Application M. Ueda, H. Yokomise,

H. Wada, and S. Hitomi

T

RACHEAL TRANSPLANTATION has been one of the most challenging procedures for thoracic surgeons, and at least three problems must be solved before it is feasible clinically: immunosuppression, preservation of the tracheal graft, and vascularization of the graft. As concerns immunosuppression, most candidates for tracheal transplantation have malignant diseases, and immunosuppressive agents must not be used. There have been many reports that the allogenicity of preserved allograft is decreased after cryopreservation.‘g We have reported that reliable l-month cryopreservation of the trachea is possible in a trehalose cryopreservation solution (TC solution).6 Therefore, we tried immunosuppressant-free tracheal allotransplantation after long-term cryopreservation in TC solution. Next, we studied the remaining problem: blood supply to the tracheal graft. In a preliminary experiment, we found that transplantation of a lo-ring length of trachea is not possible even with omentopexy,7 and we proved that the blood supply to tracheal graft is reestablished around the suture lines and that revascularization is insufficient at the midportion of the graft if it contains 10 or more rings of trachea. Therefore, we developed a new operative procedure known as split transplantation (STx), in which grafts are divided at the midportion and an omental pedicle is introduced to improve local blood flow at three suture lines. MATERIALS

AND

METHODS

Adult mongrel dogs were used for these procedures. Experiment

1

Six to 10 rings of the trachea were resected and immersed in TC

solution containing Dulbecco’s modified Eagle’s medium (DMEM), 10% dimethyl sulfoxide (DMSO), 20% fetal calf serum (FCS) and 0.1 mol/L trehalose or Euro-Collins (EC) solution. In the cryopreservation group (n = 12), the harvested tracheas were cryopreserved in TC solution and stored in a deep freezer at -85°C for 285 2 28 days. In the control group (n = 6) the harvested tracheas were preserved in EC solution at 10°C for 16 to 17 hours. In the cryopreservation group, five rings of the mediastinal tracheas of recipients were resected and cryopresetved tracheas were allotransplanted. Before transplantation, the graft was thawed at 37°C in an incubator and rinsed with physiolosic saline 10 times. In the control group, the same procedure was performed as for the cryopreserved group. Tracheal transplantation was per-

0 1997 by Elsevier Science inc. 655 Avenue of the Americas, New York, NY 10010

formed by a technique described previous1y.s The anastomotic site and graft were covered with omental pedicle graft. In both groups, immunosuppressants were never used. Experiment

2

A lo-ring length of the mediastinal trachea was allotransplanted. In the STx group (n = lo), the grafts were divided at mitportion and omentum was introduced between the halves. The anastomotic sites and the grafts were covered with omental pedicles. Then the midportion of the graft was resutured. In the control group (n = 4), a lo-ring length of trachea was autotransplanted and covered with pedicled omentum as in the STx group. In both experiments, intramuscular administration of CEZ (1 g) was continued daily for 1 week after transplantation. In all groups, the graft was examined by bronchoscopy 1, 2, 3, and 4 weeks postoperatively. Thereafter, bronchoscopic examination was conducted every month. When the experimental animals either died or were killed, transplanted grafts were removed and fixed in 10% formalin for histologic examination. In experiment 2, microangiograms of the bronchial circulation of three animals were taken (STx group, postoperative day 152 and postoperative day 158; control group, postoperative day 158). The experimental dogs were treated using humane care according to the guidelines outlined in National Institutes of Health Publication No. 86-23. RESULTS Experiment

1

In the cryopreservation group, all animals, except one which was killed for pathologic examination 7 days after the operation, survived for more than 2 months (Table 1). In the control group, most animals died within 1 month mainly because of tracheal stenosis. In the cryopreservation group, grafts were incorporated in all cases. Stenosis or tracheomalacia was not observed in any cases. In the control group, severe stenosis was observed in most cases. Histologic findings of tracheal graft cryopreserved for 9 months showed the depletion of epithelium. However, after transplantation, the graft was covered with normal epithelium, and the viability of the tracheal cartilages was maintained. Experiment

2

In the STx group, all grafts remained viable and all 10 animals survived for 2 months or longer (Table 2). In the From the Department of Thoracic Surgery, Chest Research Institute, Kyoto University, Kyoto, Japan.

Disease

0041-l 345/97/$17.00 PII SO041 -1345(96)00182-O

871

Transplantation Proceedings, 29, 871-873

(1997)

UEDA,

872

Table 1. Results of the Experiment 1

Cryopreservation

(Days) group

CP-1

Sacrifice

(64)

Incorporation

CP-2

Sacrifice

(293)

incorporation

CP-3

Sacrifice

(251)

Incorporation

CP-4

Sacrifice

(259)

Incorporation

CP-5

Sacrifice

(238)

Incorporation

CP-6

Sacrifice

(231)

Incorporation

CP-7

Sacrifice

(231)

Incorporation

CP-8

Sacrifice

(132)

Incorporation

CP-9

Sacrifice

(21.5)

Incorporation

CP-10

Sacrifice

(7)

Incorporation

CP-11

Alive (247)

Incorporation

CP-12

Alive (240)

Incorporation

Control

group

C-l

Sacrifice

(14)

Severe stenosis

c-2

Sacrifice

(37)

Severe

c-3

Sacrifice

(15)

Severe

stenosis

c-4

Sacrifice

(14)

Severe

stenosis

c-5

Sacrifice

(15)

Severe stenosis

C-6

Sacrifice

(7)

Edematous

stenosis

control group, three of four grafts lost viability. In the STx group, all grafts were incorporated without stenosis, necrosis, or malasia. Macroscopic findings 7 days after the transplantation showed ischemic changes of the graft in the control group. In the STx group, the viability of the epithelium and cartilage was observed. At midportion of the graft, neovascularization was facilitated by the inserted omentum. In the control group, the epithelium of the graft was lost and the tracheal cartilage was almost absorbed. Microangiograms in the STx group showed abundant neovascularization in the midportion of the graft. In the control group, neovascularization was poor at the midportion of the graft.

Table 2. Results of the Experiment 2 Groups (Animal No.)

Survival (Davsl

WADA ET AL

DISCUSSION status of Grafts

Survival

Groups (Animal No.)

YOKOMISE,

Status of Grafts

STx group ST-l

Sacrifice

(60)

ST-2

Sacrifice

(175)

Incorporation Incorporation

ST-3

Sacrifice

(217)

Incorporation

ST-4

Alive (168)

Incorporation

ST-5

Alive (174)

Incorporation

ST-6

Alive (168)

Incorporation

ST-7

Alive (162)

Incorporation

ST-8

Alive (156)

Incorporation

ST-9

Alive (155)

Incorporation

ST-10

Alive (152)

Incorporation

C-l

Sacrifice

(7)

Necrosis

c-2

Sacrifice

(24)

Malacia

c-3

Sacrifice

(31)

Stenosis

c-4

Alive (152)

Control group

Incorporation

Most candidates for tracheal transplantation have malignant diseases, and immunosuppressants cannot be given after the operation. In addition, it is difficult to get a suitable and fresh tracheal graft when required. To solve these basic problems, we tried immunosuppressant-free tracheal allotransplantation after a 9-month cryopreservation of the trachea. There have been many reports that the allogenicity of preserved allografts can be decreased by freezing.lm5 We have already reported that the epithelium of the trachea plays an important role in graft rejection after transplantation.’ Thus, we hypothesized that if either the structure or the function of the tracheal epithelium is depleted after cryopreservation, immunosuppressant-free allotransplantation after long-term cryopreservation might be possible. In fact, after 9 months of cryopreservation in TC solution, the epithelium of the trachea was depleted, and these graft survived more than 2 months with preservation of graft viability. In this experiment, depletion of the epithelium must have played the most important role in the success of tracheal transplantation without immunosuppression. TC solution consists of DMEM, 10% DMSO, 20% FCS and 0.1 mol/L trehalose. DMSO is a cryoprotectant that can penetrate cell membranes, whereas trehalose is a cryoprotectant that cannot penetrate cell membranes. The combined use of these different types of cryoprotectants has been reported to exhibit a synergistic effect in cryopreservation.‘.‘” The current study revealed that 9-month cryopreservation is possible with the use of TC solution. In experiment 2, we studied the third major problem: graft ischemia. We found that omentopexy did not maintain the viability of the graft after lo-ring tracheal transplantation. In these cases, ischemia was most pronounced at the midportion of the graft and there were no ischemic changes at the anastomotic sites as previously reported.” These findings indicated that early restoration of blood supply to the grafts probably occurs around the suture lines, so some additional procedure is required when a long tracheal graft is transplanted with omentopexy. It is obvious that pedicled omentum is useful in improving the local microcirculation,12*13but it cannot preserve the viability of long tracheal grafts. To meet this need, we developed a new operative procedure, STx, in which grafts are divided at the midportion and omentum is inserted. Thus, revascularization is facilitated at three anastomotic sites. In fact, this procedure worked well and preserved the viability of transplanted grafts. Microscopic and microangiographic examination showed that revascularization of the anterior wall was enhanced by the inserted omental pedicle. In conclusion immunosuppressant-free tracheal allotransplantation is possible after 9 months of cryopreservation with TC solution. Ten-ring length of tracheal transplantation is possible with STx. The combination of these hvo simple methods may make possible clinical tracheal allotransplantation.

TRACHEAL TRANSPLANTATION REFERENCES 1. Friedlaender GE: Transplant Proc 8:195, 1976 (suppl) 2. Hirase Y, Kojima T, Uchida M, et al: J Reconstr Microsurg 8437, 1992 3. Ingraham E, Matthews JB, Kearney JN, et al: Cryobiology 30:443, 1993 4. Takeishi M, Hirase Y, Kojima T: Microsurgery 15:55, 1994 5. Lupinetti FM, Tsai ‘IT, Kneebone JM, et al: J Thorac Cardiovasc Surg 106:912, 1993 6. Yokomise H, Inui K, Wada H, et al: J Thorac Cardiovasc Surg 110:382, 1995 7. Yokomise H, Inui K, Wada H, et al: Surg Today 26:427,1996

873 8. Yokomise H, Inui K, Wada H, et al: J Thorac Cardiovasc Surg 107:1391, 1994 9. Honadel TE, Killian GJ: Cryology 25:331, 1988 10. Terada T, Ashizawa K, Maeda T, et al: Jpn J Animal Reprod 35:20, 1989 11. Nakanishi R, Shirakusa T, Mitsudomi T: J Thorac Cardiovast Surg 106:1081, 1993 12. Inui K, Wada H, Yokomise H, et al: J Thorac Cardiovasc Surg 99:614, 1990 13. Hirata T, Yamazaki F, Fukuse T, et al: J Thorac Cardiovasc Surg 40:178, 1992