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High-dose irradiation prevents rejection of canine tracheal allografts
Volume 107,
Number 6
June 1994
THORACIC AND CARDIOVASCULAR SURGERY The Journal of
General Thoracic Surgery
High-dose irradiation prevents rejection of canine tracheal allografts We investigated the possibility of immunosuppressant-free transplantation of the trachea using high doses of 60Co 'Y irradiation of the graft before transplantation. Twenty mongrel dogs were used. Five rings of the trachea were removed from the donors and irradiated with 60Co 'Y 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, aU the animals died within 1 month of tracheal stenosis caused by graft rejection. In groups Band C, one animal in each group survived for a long period, but aU 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 stiU alive (more than 1 year later). These findings indicate that aUotransplantation of the trachea without the use of immunosuppressants is possible with pretransplantation irradiation of the graft at the dose of 100,000 cGy. (J THORAC CARDIOVASC SURG 1994;107: 1391-7)
H. Yokomise, MD, K. Inui, MD, H. Wada, MD, T. Goh, MD, K. Yagi, MD, S. Hitomi, MD, and M. Takahashi, MD,a Kyoto, Japan
W
hen tracheal tumors have a wide range of infiltration, there are many difficulties in end-to-end anastomoFrom the Department of Thoracic Surgery and the Department of Oncology; Chest Disease Research Institute, Kyoto University, 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. Copyright @ 1994 by Mosby-Year Book, Inc. 0022-5223/94 $3.00
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sis after circumferential resection of the trachea. Therefore conservative approaches such as laser therapy! and stent therapy- 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.l" Ischemia and rejection of the graft must be overcome for successful tracheal transplantati0l1. 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 immuno-
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Table I. Results of the present study Groups
Survival (days)
Control A-I
14 (dead)
Necrosis
A-2
37 (dead)
Stenosis
A-3
15 (dead)
Stenosis
A-4
14 (dead)
Necrosis
A-5
15 (dead)
Necrosis
20 (dead)
Stenosis
20,000cGy B-1
Status of grafts
B-2 B-3 B-4
5 (dead) 432 (alive) 21 (dead)
B-5
13 (dead)
Stenosis
25 (dead) 32 (dead)
Incorporation Edematous
12 (dead) 409 (alive) 30 (dead)
Edematous Incorporation Stenosis
402 (alive) 388 (alive) 381 (alive) 90 (dead) 7 (dead)
Incorporation Incorporation Incorporation Incorporation Edematous
50,000cGy C-I C-2 C-3 C-4
C-5 100,000 cGy D-I D-2 D-3 D-4 D-5
Edematous Incorporation Stenosis
Causes of death
Tracheal stenosis Tracheal stenosis Tracheal stenosis Tracheal stenosis Tracheal stenosis Tracheal stenosis Weakness Tracheal stenosis Tracheal stenosis Empyema Tracheal stenosis Weakness Tracheal stenosis
Killed Weakness
suppressants has been investigated clinically as a method of preventing reiection.v" There have been reports of significant prolongation of graft survival by 60Co irradiation before transplantation in experiments in skin, kidney, and heart transplantation.v 10 Because the viability of cartilage is not lost even after high-dose irradiation.'! we investigated the possibility of immunosuppressant-free tracheal transplantation with preoperative high-dose radiation with 60Co 'Y. 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
rng/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 I% halothane. Irradiation of grafts. The right chest was opened and five rings of the thoracic trachea were removed, immersed in EuroCollins (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 'Y 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 em. 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 I 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 I-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) I 2 by the alkaline 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
The Journal of Thoracic and Cardiovascular Surgery Volume 107, Number 6
Yokomise et al.
7POD
15POD
Without radiation Fig. I. Animals that received grafts without irradiation before transplantation. A, Flare and edema were observed in the grafts 7 days after the operation. Band 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 X 100). POD, Postoperative day .
7POD
14POD
28 POD
300 POD
100000 cGy radiation Fig. 2. Animals that received grafts with irradiation of 100,000 cGy before transplantation. Grafts were pale up to 2 weeks after the operation but were 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.
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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).
100000 cGy radiation 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 ciliated epithelium, and there is no MNC infiltration or resorption of cartilage.
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found that this anti-human HLA- DR antibody crossreacts with dog tissues. AU 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 I 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 I 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. Bronchoscopic and macroscopic findings. In the control group, edema was severe in the grafts I week after operation, and necrosis and stenosis were observed 2 weeks after operation (Fig. I, A, B, and C). In groups B and C, the graft became incorporated in one animal in each group, but severe stenosis and shrinkage of the grafts were observed at an early period in the other animals as in the controls . In group D (lOO,OOOcGy), the grafts were pale I week after operation (Fig . 2). At 3 weeks, the grafts became covered with epithelium of the recipient over the proximal anastomotic site (Fig . 3). The grafts were completely covered endoscopically by epithelium after 4 weeks (Fig. 2). In group D ( 100,000 cGy), one animal was killed 3 months after operation. No stenosis or atrophy was observed, and the graft had become incorporated (fig, 4, A, B, and C), Histopathologic investigation. In the control group, mononuclear cellular (MNC) infiltration into the subepithelium, marked fibrosis, and absorption of the tracheal cartilage were observed (Fig. I, D). In groups B (20,000 cGy) and C (50,000 cGy), MNC infiltration into the grafts, fibrosis, and absorption of the tracheal cartilage were observed in the animals in which the grafts did not become incorporated, as in group A. In group D, the epithelium of the grafts was completely ablated, but the tracheal cartilage was intact in the animal that died I week after operation . Normal epithelium covered the surface of the grafts and viability of the tracheal cartilage was preserved in the animal killed 3 months after operation (Fig. 4, D) .
before operation
i I?OD Fig. 5. MHC-II staining of the ceUsof the graft without irradiation before transplantation (original magnification X400). MHC-II was not observed before the transplantation, but there were several cells in which MHC-II developed by postoperative day (POD) 7.
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 ofthe 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 . Discussion Operation is impossible at present when end-to-end anastomosis of the trachea cannot be done after wide resection. Although Neville, Bolanowski, and Hooshang' :' and others'? 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 transplan-
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Yokomise et al.
tation 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.t-" 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'' 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.!? 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.v" There have been favorable reports on irradiation of the grafts after transplantationf 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.P Recently, antigenicity of the trachea was investigated for the purpose of transplantation of the trachea in rats. 22-24 Beigel and Muller-Ruchholtz-s P performed tracheal transplantation in inbred rat strains and demonstrated the development of rejection as in the case of other organs. Bujia and colleagues-' 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'? noted that the tracheal epithelium develops MHC-II, which activates T lymphocytes as antigenpresenting 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 col-
lected cells developed MHC-II strongly. These findings suggest that high doses of irradiation before transplantation suppress the development of MHC-II ofthe 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. II 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'' 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"y irradiation (100,000 cGy) was applied to the grafts (fiverings 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. REFERENCES 1. Cavaliere S, Foccoli P, Farina PL. Nd: YAG laser bronchoscopy. a five-year experience with 1396 applications in 1000 patients. Chest 1988;94:15-21.
2. Tsang Y, Williams AM, Goldstraw P. Sequential Silastic and expandable metal stentingfor tracheobronchial strictures. Ann Thorac Surg 1992;53:856-60. 3. JacksonTL, O'Brien EJ, Tuttle W, Meyer J. The experimental use of homologous tracheal transplants in the restorationof continuity of the tracheobronchial tree. J THORAC CARDIOVASC SURG 1950;20:598-612. 4. Ferguson DJ, Wild JJ, Wangensteen OH. Experimental resection ofthe thoracictrachea.Surgery 1950;28:597-619.
5. MoriyamaS, Shimizu N, TeramotoS. Experimental tra-
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6.
7.
8.
9.
10.
II.
cheal allotransplantation using omentopexy. Transplant Proc 1989;21:2596-600. Fidler JP, Alexander JW, Smith EJ, Miller HC, Muthiah V. Radiation reversal of acute rejection in patients with life-threatening infections. Arch Surg 1973;107:256-60. Abramson N, Ornitz R, Cavanaugh PJ. The effect of local irradiation on rejection of the transplanted kidney. Radiology 1974;112:413-6. Halperin EC, Delmonico FL, Nelson PW, Shipley WU, Cosimi AB. The use of local allograft irradiation following renal transplantation. Int J Radiat Oncol Bioi Phys 1984; 10:987-90. Sharma M, Shastri KD. Delay in rejection of irradiated skin homograft: report of an experiment on albino rats. Indian J Med Res 1975;63:787-92. Steinmuller D, Warden G, Coleman M, et al. Prolonged survival of rat heart and kidney allografts irradiated in vitro. Transplantation 1971;12:153-6. Dingman RD, Grabb We. Costal cartilage homografts preserved by radiation. Plast Reconstr Surg 1972;50:5167.
12. Adams TE, Bodmer JG, Bodmer WF. Production and characterization of monoclonal antibodies recognising the alpha-chain subunits of human Ia alloantigens. Immunology 1983;50:613-24. 13. Neville WE, Bolanowski PJP, Hooshang S. Prosthetic reconstruction of the trachea and carina. J THORAC CAR0I0VASC SURG 1976;72:525-38. 14. Vogt-MoykopfI, Mickisch GH. Prosthetic replacement of the trachea. In: Grillo HC, Eschapasse H, eds. International trends in general thoracic surgery. Vol. 2. Philadelphia: WB Saunders, 1987:147-51. 15. Rose KG, Sesterhenn K, Wustrow F. Tracheal allotransplantation in man [Letter]. Lancet 1979;1:433. 16. Farrington WT, Hung WC, Binns PM. Experimental tracheal homografting. J LaryngoI1977;91:101-1O.
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17. Hirai T, Wada H, Hasegawa S, et al. The immunosuppressive effect of FK 506 on canine lung transplantation. J THORAC CAROIOVASC SURG 1992;103:1127-35. 18. Tucker HM. Laryngeal transplantation: current status 1974. Laryngoscope 1975;85:787-96. 19. LevittSH, O'Foghludha rr, RoysterRL, Wolf JS, Lower RR, DeGiorgi LS. Radiation for immunosuppression in human organ transplantation. I. Experimental data. Acta Radiol 1971;10:248-56. 20. Foster RS Jr. Local graft irradiation as an adjunct to pharmacologic immunosuppression. J Surg Res 1974;16:45762. 21. Malek GH, Richards MJS, Burkholder PM, Caldwell WL. Effect of single dose irradiation on isolated perfused canine kidney. Surgery 1974;76:272-7. 22. Beigel A, Muller-Ruchholtz W. Tracheal transplantation. I. The immunogenic effect of rat tracheal transplants. Arch Otorhinolaryngol 1984;240:185-92. 23. Beigel A, Muller-Ruchholtz W. Tracheal transplantation. II. Influence of genetic difference and degree of sensitization on reaction to the tracheal transplant. Arch Otorhinolaryngol 1984;240:217-25. 24. Beigel A, Steffens-Knutzen R, Muller B, Schumacher U, Stein H. Tracheal transplantation. III. Demonstration of transplantation antigens on the tracheal mucosa of inbred rat strains. Arch OtorhinolaryngoI1984;241:1-8. 25. Bujia J, Wilmes E, Hammer C, Kastenbauer E. Tracheal transplantation: demonstration of HLA class II subregion gene products on human trachea. Acta Otolaryngol (Stockh) 1990;110:149-54. 26. Kalb TH, Chuang MT, Marom Z, Mayer L. Evidence for accessory cell function by class II MHC antigen-expressing airway epithelial cells. Am J Respir Cell Mol Bioi 1991; 4:320-9. 27. Balderman SC, Weinblatt G. Tracheal autograft revascularization. J THORAC CAROIOVASC SURG 1987;94:434-41.
Number 6
June 1994
THORACIC AND CARDIOVASCULAR SURGERY The Journal of
General Thoracic Surgery
High-dose irradiation prevents rejection of canine tracheal allografts We investigated the possibility of immunosuppressant-free transplantation of the trachea using high doses of 60Co 'Y irradiation of the graft before transplantation. Twenty mongrel dogs were used. Five rings of the trachea were removed from the donors and irradiated with 60Co 'Y 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, aU the animals died within 1 month of tracheal stenosis caused by graft rejection. In groups Band C, one animal in each group survived for a long period, but aU 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 stiU alive (more than 1 year later). These findings indicate that aUotransplantation of the trachea without the use of immunosuppressants is possible with pretransplantation irradiation of the graft at the dose of 100,000 cGy. (J THORAC CARDIOVASC SURG 1994;107: 1391-7)
H. Yokomise, MD, K. Inui, MD, H. Wada, MD, T. Goh, MD, K. Yagi, MD, S. Hitomi, MD, and M. Takahashi, MD,a Kyoto, Japan
W
hen tracheal tumors have a wide range of infiltration, there are many difficulties in end-to-end anastomoFrom the Department of Thoracic Surgery and the Department of Oncology; Chest Disease Research Institute, Kyoto University, 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. Copyright @ 1994 by Mosby-Year Book, Inc. 0022-5223/94 $3.00
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12/1/51779
sis after circumferential resection of the trachea. Therefore conservative approaches such as laser therapy! and stent therapy- 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.l" Ischemia and rejection of the graft must be overcome for successful tracheal transplantati0l1. 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 immuno-
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Table I. Results of the present study Groups
Survival (days)
Control A-I
14 (dead)
Necrosis
A-2
37 (dead)
Stenosis
A-3
15 (dead)
Stenosis
A-4
14 (dead)
Necrosis
A-5
15 (dead)
Necrosis
20 (dead)
Stenosis
20,000cGy B-1
Status of grafts
B-2 B-3 B-4
5 (dead) 432 (alive) 21 (dead)
B-5
13 (dead)
Stenosis
25 (dead) 32 (dead)
Incorporation Edematous
12 (dead) 409 (alive) 30 (dead)
Edematous Incorporation Stenosis
402 (alive) 388 (alive) 381 (alive) 90 (dead) 7 (dead)
Incorporation Incorporation Incorporation Incorporation Edematous
50,000cGy C-I C-2 C-3 C-4
C-5 100,000 cGy D-I D-2 D-3 D-4 D-5
Edematous Incorporation Stenosis
Causes of death
Tracheal stenosis Tracheal stenosis Tracheal stenosis Tracheal stenosis Tracheal stenosis Tracheal stenosis Weakness Tracheal stenosis Tracheal stenosis Empyema Tracheal stenosis Weakness Tracheal stenosis
Killed Weakness
suppressants has been investigated clinically as a method of preventing reiection.v" There have been reports of significant prolongation of graft survival by 60Co irradiation before transplantation in experiments in skin, kidney, and heart transplantation.v 10 Because the viability of cartilage is not lost even after high-dose irradiation.'! we investigated the possibility of immunosuppressant-free tracheal transplantation with preoperative high-dose radiation with 60Co 'Y. 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
rng/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 I% halothane. Irradiation of grafts. The right chest was opened and five rings of the thoracic trachea were removed, immersed in EuroCollins (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 'Y 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 em. 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 I 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 I-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) I 2 by the alkaline 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
The Journal of Thoracic and Cardiovascular Surgery Volume 107, Number 6
Yokomise et al.
7POD
15POD
Without radiation Fig. I. Animals that received grafts without irradiation before transplantation. A, Flare and edema were observed in the grafts 7 days after the operation. Band 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 X 100). POD, Postoperative day .
7POD
14POD
28 POD
300 POD
100000 cGy radiation Fig. 2. Animals that received grafts with irradiation of 100,000 cGy before transplantation. Grafts were pale up to 2 weeks after the operation but were 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.
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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).
100000 cGy radiation 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 ciliated epithelium, and there is no MNC infiltration or resorption of cartilage.
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found that this anti-human HLA- DR antibody crossreacts with dog tissues. AU 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 I 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 I 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. Bronchoscopic and macroscopic findings. In the control group, edema was severe in the grafts I week after operation, and necrosis and stenosis were observed 2 weeks after operation (Fig. I, A, B, and C). In groups B and C, the graft became incorporated in one animal in each group, but severe stenosis and shrinkage of the grafts were observed at an early period in the other animals as in the controls . In group D (lOO,OOOcGy), the grafts were pale I week after operation (Fig . 2). At 3 weeks, the grafts became covered with epithelium of the recipient over the proximal anastomotic site (Fig . 3). The grafts were completely covered endoscopically by epithelium after 4 weeks (Fig. 2). In group D ( 100,000 cGy), one animal was killed 3 months after operation. No stenosis or atrophy was observed, and the graft had become incorporated (fig, 4, A, B, and C), Histopathologic investigation. In the control group, mononuclear cellular (MNC) infiltration into the subepithelium, marked fibrosis, and absorption of the tracheal cartilage were observed (Fig. I, D). In groups B (20,000 cGy) and C (50,000 cGy), MNC infiltration into the grafts, fibrosis, and absorption of the tracheal cartilage were observed in the animals in which the grafts did not become incorporated, as in group A. In group D, the epithelium of the grafts was completely ablated, but the tracheal cartilage was intact in the animal that died I week after operation . Normal epithelium covered the surface of the grafts and viability of the tracheal cartilage was preserved in the animal killed 3 months after operation (Fig. 4, D) .
before operation
i I?OD Fig. 5. MHC-II staining of the ceUsof the graft without irradiation before transplantation (original magnification X400). MHC-II was not observed before the transplantation, but there were several cells in which MHC-II developed by postoperative day (POD) 7.
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 ofthe 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 . Discussion Operation is impossible at present when end-to-end anastomosis of the trachea cannot be done after wide resection. Although Neville, Bolanowski, and Hooshang' :' and others'? 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 transplan-
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tation 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.t-" 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'' 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.!? 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.v" There have been favorable reports on irradiation of the grafts after transplantationf 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.P Recently, antigenicity of the trachea was investigated for the purpose of transplantation of the trachea in rats. 22-24 Beigel and Muller-Ruchholtz-s P performed tracheal transplantation in inbred rat strains and demonstrated the development of rejection as in the case of other organs. Bujia and colleagues-' 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'? noted that the tracheal epithelium develops MHC-II, which activates T lymphocytes as antigenpresenting 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 col-
lected cells developed MHC-II strongly. These findings suggest that high doses of irradiation before transplantation suppress the development of MHC-II ofthe 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. II 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'' 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"y irradiation (100,000 cGy) was applied to the grafts (fiverings 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. REFERENCES 1. Cavaliere S, Foccoli P, Farina PL. Nd: YAG laser bronchoscopy. a five-year experience with 1396 applications in 1000 patients. Chest 1988;94:15-21.
2. Tsang Y, Williams AM, Goldstraw P. Sequential Silastic and expandable metal stentingfor tracheobronchial strictures. Ann Thorac Surg 1992;53:856-60. 3. JacksonTL, O'Brien EJ, Tuttle W, Meyer J. The experimental use of homologous tracheal transplants in the restorationof continuity of the tracheobronchial tree. J THORAC CARDIOVASC SURG 1950;20:598-612. 4. Ferguson DJ, Wild JJ, Wangensteen OH. Experimental resection ofthe thoracictrachea.Surgery 1950;28:597-619.
5. MoriyamaS, Shimizu N, TeramotoS. Experimental tra-
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6.
7.
8.
9.
10.
II.
cheal allotransplantation using omentopexy. Transplant Proc 1989;21:2596-600. Fidler JP, Alexander JW, Smith EJ, Miller HC, Muthiah V. Radiation reversal of acute rejection in patients with life-threatening infections. Arch Surg 1973;107:256-60. Abramson N, Ornitz R, Cavanaugh PJ. The effect of local irradiation on rejection of the transplanted kidney. Radiology 1974;112:413-6. Halperin EC, Delmonico FL, Nelson PW, Shipley WU, Cosimi AB. The use of local allograft irradiation following renal transplantation. Int J Radiat Oncol Bioi Phys 1984; 10:987-90. Sharma M, Shastri KD. Delay in rejection of irradiated skin homograft: report of an experiment on albino rats. Indian J Med Res 1975;63:787-92. Steinmuller D, Warden G, Coleman M, et al. Prolonged survival of rat heart and kidney allografts irradiated in vitro. Transplantation 1971;12:153-6. Dingman RD, Grabb We. Costal cartilage homografts preserved by radiation. Plast Reconstr Surg 1972;50:5167.
12. Adams TE, Bodmer JG, Bodmer WF. Production and characterization of monoclonal antibodies recognising the alpha-chain subunits of human Ia alloantigens. Immunology 1983;50:613-24. 13. Neville WE, Bolanowski PJP, Hooshang S. Prosthetic reconstruction of the trachea and carina. J THORAC CAR0I0VASC SURG 1976;72:525-38. 14. Vogt-MoykopfI, Mickisch GH. Prosthetic replacement of the trachea. In: Grillo HC, Eschapasse H, eds. International trends in general thoracic surgery. Vol. 2. Philadelphia: WB Saunders, 1987:147-51. 15. Rose KG, Sesterhenn K, Wustrow F. Tracheal allotransplantation in man [Letter]. Lancet 1979;1:433. 16. Farrington WT, Hung WC, Binns PM. Experimental tracheal homografting. J LaryngoI1977;91:101-1O.
Yokomise et al.
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17. Hirai T, Wada H, Hasegawa S, et al. The immunosuppressive effect of FK 506 on canine lung transplantation. J THORAC CAROIOVASC SURG 1992;103:1127-35. 18. Tucker HM. Laryngeal transplantation: current status 1974. Laryngoscope 1975;85:787-96. 19. LevittSH, O'Foghludha rr, RoysterRL, Wolf JS, Lower RR, DeGiorgi LS. Radiation for immunosuppression in human organ transplantation. I. Experimental data. Acta Radiol 1971;10:248-56. 20. Foster RS Jr. Local graft irradiation as an adjunct to pharmacologic immunosuppression. J Surg Res 1974;16:45762. 21. Malek GH, Richards MJS, Burkholder PM, Caldwell WL. Effect of single dose irradiation on isolated perfused canine kidney. Surgery 1974;76:272-7. 22. Beigel A, Muller-Ruchholtz W. Tracheal transplantation. I. The immunogenic effect of rat tracheal transplants. Arch Otorhinolaryngol 1984;240:185-92. 23. Beigel A, Muller-Ruchholtz W. Tracheal transplantation. II. Influence of genetic difference and degree of sensitization on reaction to the tracheal transplant. Arch Otorhinolaryngol 1984;240:217-25. 24. Beigel A, Steffens-Knutzen R, Muller B, Schumacher U, Stein H. Tracheal transplantation. III. Demonstration of transplantation antigens on the tracheal mucosa of inbred rat strains. Arch OtorhinolaryngoI1984;241:1-8. 25. Bujia J, Wilmes E, Hammer C, Kastenbauer E. Tracheal transplantation: demonstration of HLA class II subregion gene products on human trachea. Acta Otolaryngol (Stockh) 1990;110:149-54. 26. Kalb TH, Chuang MT, Marom Z, Mayer L. Evidence for accessory cell function by class II MHC antigen-expressing airway epithelial cells. Am J Respir Cell Mol Bioi 1991; 4:320-9. 27. Balderman SC, Weinblatt G. Tracheal autograft revascularization. J THORAC CAROIOVASC SURG 1987;94:434-41.