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J Thorac Cardiovasc Surg 1994;107:1391-1397
© 1994 Mosby, Inc.

GENERAL THORACIC SURGERY

High-dose irradiation prevents rejection of canine tracheal allografts

Kyoto, Japan

Received for publication June 1, 1993. Accepted for publication Sept. 24, 1993. Address for reprints: H. Yokomise, MD, Thoracic Surgery, Chest Disease Research Institute, Kyoto University, 53 Shogoin-Kawaharamachi, Sakyo-ku, Kyoto 606, Japan.

Abstract

We investigated the possibility of immunosuppressant-free transplantation of the trachea using high doses of ⁶⁰ Co irradiation of the graft before transplantation. Twenty mongrel dogs were used. Five rings of the trachea were removed from the donors and irradiated with ⁶⁰ Co rays. Five corresponding rings were removed from the thoracic trachea of the recipient dogs, and the irradiated trachea was transplanted. Five animals were placed in each of four dosage groups: group A, no irradiation; group B, 20,000 cGy; group C, 50,000 cGy; and group D, 100,000 cGy. The anastomotic site and graft were covered with a pedicled greater omentum graft. No immunosuppressants were given. In group A, all the animals died within 1 month of tracheal stenosis caused by graft rejection. In groups B and C, one animal in each group survived for a long period, but all the others died of tracheal stenosis caused by graft rejection. In group D (100,000 cGy), the graft became incorporated into the recipient tissue in four of the five animals, and three are still alive (more than 1 year later). These findings indicate that allotransplantation of the trachea without the use of immunosuppressants is possible with pretransplantation irradiation of the graft at the dose of 100,000 cGy. (J T HORAC C ARDIOVASC S URG 1994;107: 1391-7)

When tracheal tumors have a wide range of infiltration, there are many difficulties in end-to-end anastomosis after circumferential resection of the trachea. Therefore conservative approaches such as laser therapy ¹ and stent therapy ² are often used. To treat patients with this kind of tumor, tracheal transplantation has been investigated, but the results of the studies have been unfavorable. ^3,4 Ischemia and rejection of the graft must be overcome for successful tracheal transplantation. ⁵

Because most of the diseases for which tracheal transplantation is indicated are malignant, immunosuppressants cannot be used. Radiation therapy for the transplantation of kidneys without the use of immunosuppressants has been investigated clinically as a method of preventing rejection. ^6-8

There have been reports of significant prolongation of graft survival by ⁶⁰Co irradiation before transplanta tion in experiments in skin, kidney, and heart trans plantation. ^9,10 Because the viability of cartilageis not lost even after high-dose irradiation, ¹¹ we investigated the possibility of immunosuppressant-free tracheal transplantation with preoperative high-dose radiation with ⁶⁰Co .

MATERIALS AND METHODS

Experimental animals and anesthesia
Twenty adult mongrel dogs weighing 10 to 15 kg were used. After an intramuscular injection of ketamine hydrochloride (10 mg/kg), anesthesia was induced with intravenous sodium thiopental (15 mg/kg). After insertion of a tracheal tube, lungs were ventilated mechanically at a tidal volume of 20 ml/kg and a frequency of 20 breaths/min with a Harvard pump (Harvard Apparatus, Inc., S. Natick, Mass.). The anesthesia was maintained with 50% oxygen, 50% nitrous oxide, and 1% halothane.

Irradiation of grafts
The right chest was opened and five rings of the thoracic trachea were removed, immersed in Euro-Collins (EC) solution at 8° to 10° C, and preserved for 8 to 10 hours. The optimal dose for this situation was unknown. In a few preliminary experiments, 50,000 cGy was found to be promising, but still unreliable. To find the optimal dose for this experiment, doses of 20,000, 50,000, or 100,000 cGy of ⁶⁰C rays were applied to the graft with a radiation unit (Theratron Atomic Energy of Canada, Canada). The irradiation was given at the rate of 214.4 cGy/min and the source-to-graft distance was 50 cm. During the irradiation period, the graft was put in a clean plastic bag containing EC solution and cooled with ice slush.

Preservation
As previously mentioned, the graft was immersed in EC solution at 8° to 10° C and preserved for 16 to 17 hours (including the irradiation period). Grafts from control animals were preserved in the same way.

Operation of the recipient
Recipient dogs were anesthetized in the same way as donor animals. After abdominal median incision, the spleen was held, the gastroepiploic artery was ablated from the greater curvature side, and a pedicled omental graft was prepared. After the peritoneum was closed temporarily, the right chest was opened. The azygos vein was ligated and cut and the trachea exposed. Five rings of trachea were removed five rings above the carina. Tracheal continuity was restored by insertion of the preserved graft with continuous suturing with 4-0 Prolene Sutures (Ethicon, Inc., Somerville, N.J.).

The grafts were sutured by the telescope method so that both the central and peripheral sides could be arranged externally. A small hole was prepared at the attachment of the right anterior diaphragm, and a pedicled omental graft was introduced into the right thoracic cavity to cover the anastomotic site and graft. The chest and abdomen were closed and the operation was completed. One gram of cefazolin was given intramuscularly 1 week after operation.

Groups and radiation doses
The animals were divided into the following four groups: group A, no irradiation (n = 5); group B, 20,000 cGy (n = 5); group C, 50,000 cGy (n = 5); and group D, 100,000 cGy (n = 5).

Observation
The grafts were observed by bronchoscopy 1, 2, and 3 weeks after operation and thereafter at 1-month intervals.

Histologic examination and immunohistochemical staining
At the time of an animal's death or when the animal was killed, the grafts were removed and fixed in formalin, stained with hematoxylin and eosin, and examined histopathologically. Cells were collected from the grafts transbronchoscopically with a brush before operation and 1, 2, 3, and 4 weeks after operation, smeared on glass slides, and fixed in 10% formalin. The samples were stained with anti-HLA-DR-Ab (mouse monoclonal Ab: DAKO M746, Dako, AS, Denmark) ¹² by thealkaline phosphatase—antialkaline phosphatase method. The samples were lightly counterstained with hematoxylin. From a few preliminary studies, including immunosuppressant-free canine tracheal allotransplantation and canine lung transplantation, we found that this anti-human HLA-DR antibody crossreacts with dog tissues.

All animals received humane care in compliance with the "Principles of Laboratory Animal Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health.

RESULTS

Surviving animals
(Table I) In group A (nonirradiated), all the animals died within 1 month because of stenosis and necrosis of the grafts caused by rejection. In groups B (20,000 cGy) and C (50,000 cGy), the graft became incorporated into the recipient tissue in one animal in each group (these animals are still alive with no evidence of stenosis or atrophy of the grafts). However, the other eight animals died of tracheal stenosis considered to be due mostly to rejection. In group D (100,000 cGy), one animal died of weakness caused by anorexia 1 week after operation. The other four survived for more than 3 months. One of these was killed for histopathologic examination. The grafts became incorporated into the recipient tissue in all four survivors in group 4.

View larger version (90K): [in this window] [in a new window]   Fig. 1. Animals that received grafts without irradiation before transplantation. A, Flare and edema were observed in the grafts 7 days after the operation. B and C, Marked shrinkage and stenosis were observed in grafts 15 days after the operation. D, Many mononuclear cells have infiltrated mucosa, and resorption of cartilage can be observed (original magnification x100). POD, Postoperative day.

View larger version (94K): [in this window] [in a new window]   Fig. 2. Animals that received grafts with irradiation of 100,000 cGy before transplantation. Grafts were pale up to 2 weeks after the operation butwere covered with mucosa by 4 weeks. Complete incorporation of graft is evident, and no atrophy or stenosis can be seen 300 days after the operation. POD, Postoperative day.

View larger version (100K): [in this window] [in a new window]   Fig. 3. Endoscopy showed that the graft became covered with the recipient's epithelium over the proximal anastomotic site 3 weeks after the operation (100,000 cGy irradiation before transplantation).

View larger version (93K): [in this window] [in a new window]   Fig. 4. Animal that received graft with 100,000 cGy irradiation before transplantation (killed 3 months after the operation). A, B,and C, Complete incorporation can be observed, and no atrophy or stenosis in the graft was seen 3 months after the operation. D, The graft is covered with normal stratified ciliate depithelium, and there is no MNC infiltration or resorption of cartilage.

Immunohistochemical staining
Most cells collected from the grafts were epithelial cells morphologically. Major histocompatibility complex class II (MHC-II) antigens of the trachea were not expressed in any of the donors. After transplantation, MHC-II of the grafts were expressed in every animal with rejection in groups A, B, and C (Fig. 5). In group D, expression of MHC-II after transplantation was suppressed.

View larger version (130K): [in this window] [in a new window]   Fig. 5. MHC-II staining of the cells of the graft without irradiation beforetransplantation (original magnification x 400). MHC-II was notobserved before the transplantation, but there were several cells inwhich MHC-II developed by postoperative day (POD) 7.

Operation is impossible at present when end-to-end anastomosis of the trachea cannot be done after wide resection. Although Neville, Bolanowski, and Hooshang ¹³ and others ¹⁴ introduced a silicon artificial trachea for transplantation and obtained favorable clinical results, complications, consisting of dehiscence of the anastomosis, occurred frequently and an artificial trachea has rarely been used. Successful human tracheal transplantation was reported in 1979, ¹⁵ but there have been no reports since then. Although experiments on allotransplantation of the trachea have been conducted for a long time in several animal species, ^8,9,16 the results have not been satisfactory, often because of complications, such as dehiscence of the anastomotic site and graft necrosis caused by rejection or ischemia. Moriyama, Shimizu, and Teramoto ⁵ performed allotransplantation of the trachea in dogs successfully with the use of FK 506. Previously we performed canine lung transplantation with the use of FK 506 and obtained favorable results. ¹⁷ However, it has been reported that when immunosuppressants are given to patients who have a malignant disease and who have received transplants, tumors may recur at an early stage. ¹⁸ Therefore immunosuppressants cannot be given to patients who undergo transplantation of the trachea because of malignant tumors.

Radiation therapy has long been used for immunosuppression in kidney transplantation. ^6-8 There have been favorable reports onirradiation of the grafts after transplantation. ⁶ However, because high doses of irradiation to suppress the antigenicity of organs cause severe damage, ^19-21 radiation therapy is rarely used at present.

The trachea is generally believed to have weak antigenicity because it is a comparatively simple organ. ¹⁵ Recently, antigenicity of the trachea was investigated for the purpose of transplantation of the trachea in rats. ^22-24 Beigel andMuller-Ruchholtz ^22,23 performed tracheal transplantation in inbred rat strains and demonstrated the development of rejection as in the case of other organs. Bujia and colleagues ²⁵ found that the human tracheal epithelium develops HLA-DR antigens and suggested that the epithelium may play an important role in graft rejection after transplantation of the trachea. In the same experiment, it was found that the tracheal cartilage does not seem to develop HLA-DR antigens consistently. Kalb and associates ²⁶ noted that the tracheal epithelium develops MHC-II, which activates T lymphocytes as antigen-presenting cells. In our experiment, the epithelium was ablated, but the viability of the tracheal cartilage was maintained in one animal (which died of weakness 1 week after operation) of group D. The bronchoscopic findings in the three surviving animals indicated that the internal surface of the grafts was pale 1 and 2 weeks after operation, and the presence of normal epithelium was in doubt. MHC-II of the grafts was not expressed at this time. From 3 weeks after transplantation, the epithelium of the recipient began to cover the graft. The trachea was hyperemic and edematous at 1 week, and necrosis was observed in part of the trachea at 2 weeks in the majority of the animals in groups A, B, and C, and graft rejection was suggested histopathologically. At this stage, the collected cells developed MHC-II strongly. These findings suggest that high doses of irradiation before transplantation suppress the development of MHC-II of the tracheal epithelium by necrosis or removal of the tracheal epithelium in which immunologic responses mainly occur after transplantation, or by elimination of its function. As a result, radiation might suppress graft rejection. Tracheal cartilage is considered to have low immunogenicity as does other cartilagenous tissue, ²⁵ and it is considered to play an important role in the support of the tracheal structure after transplantation.

Irradiation of grafts of cartilage is done clinically in the field of plastic surgery. It has been reported that grafts remain viable even after irradiation with doses far higher than those we used. ¹¹ In the present experiment, no adverse effects ascribed to the irradiation were observed, even in group D.

An important factor for successful tracheal transplantation is blood flow in the grafts. Balderman and associates ²⁷ reported that omentopexy was ineffective in autotransplantation of eight tracheal cartilage rings. Moriyama, Shimizu, and Teramoto ⁵ stated that omentopexy caused no problems in five rings of tracheal transplantation. In our experiment, omentopexy was effective in the transplantation of five rings, and the grafts became incorporated. From these results, five rings of tracheal transplantation appear to be possible with omentopexy. Even if 10 rings of trachea are resected clinically, 5 rings of transplantation may be sufficient to restore tracheal continuity. Therefore blood flow is not considered to be a problem.

In summary, canine tracheal allotransplantation was successfully done without the use of any immunosuppressants when ⁶⁰Co irradiation (100,000 cGy) was applied to the grafts (five rings of trachea) before transplantation. These findings suggest that this method may be used clinically to transplant the trachea and that further investigations regarding this approach should be done.

Footnotes

From the Department of Thoracic Surgery and the Department of Oncology,^a Chest Disease Research Institute, Kyoto University, Kyoto, Japan.

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