Tracheal allotransplantation is one option for airway

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1 Tracheal Allotransplantation in Beagle Dogs Without Immunosuppressants Yu Liu, MD, Tatsuo Nakamura, MD, Yasuhiko Shimizu, MD, Hiroki Ueda, MD, Makoto Yoshitani, MD, Toshinari Toba, MD, and Seijun Fukuda, MD Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Background. The antigenicity of tracheal grafts is still unclear. We investigated the possibility of performing tracheal allotransplantation without immunosuppressants. Methods. Intrathoracic five-ring tracheal replacements were performed in beagle dogs without immunosuppressants (n 18). The dogs were divided into 9 pairs, and grafts were exchanged within the pairs. In group 1 (n 6), the paired dogs were blood relatives, whereas in group 2 (n 12), the paired dogs were not related. Full-thickness skin transplantation was also performed in both groups. Results. In group 1, 5 animals survived uneventfully for more than 3 months. No stenosis was observed in any of the dogs. In group 2, the grafts were incorporated by the host trachea in 2 dogs. Four animals died of airway obstruction within 3 months. Moderate or slight airway stenosis was observed in 6 dogs. Rejection was confirmed by histologic examination. In both groups, all of the skin allografts were destroyed within 2 weeks. Conclusions. After tracheal allotransplantation, longterm survival was achieved, especially in recipient dogs that were blood relatives of donors. We conclude that it is possible to perform tracheal allotransplantation using histocompatible matched grafts without immunosuppressants. (Ann Thorac Surg 2001;72:1190 4) 2001 by The Society of Thoracic Surgeons Tracheal allotransplantation is one option for airway reconstruction when a long segment of trachea must be excised. Tracheal allografts have been stored in tissue banks; however, the antigenicity of tracheal grafts is still unclear. In 1979, Rose and colleagues [1] reported successful tracheal allotransplantation in a human. The recipient had suffered extensive tracheal stenosis that affected eight segments after resuscitation, whereas the donor had died of severe brain trauma after an accident. Both were 21-year-old men. The results of HLA typing were as follows: The recipient s HLA-A locus was A1, 24 and his HLA-B locus was B8, X, W. The donor s HLA-A locus was A1, 28 and his HLA-B locus was B5, 8, W4, W6. In other words, HLA typing showed one A and one B match. The combined heteroorthotopic transplantation was performed in Germany, but no data on the donor s or recipient s ethnic origins were provided. The trachea was removed from the donor while blood was still circulating. Nine weeks after operation, the tracheal graft had become integrated and showed no evidence of rejection. Therefore, the authors concluded that the trachea has weak antigenicity, in contrast to other organs. However, experience gained from animal experiments conflicts with the above clinical results. Beigel and Muller-Ruchholtz [2, 3] performed tracheal transplantation in two combinations of inbred strains of rats and Accepted for publication June 23, Address reprint requests to Dr Liu, Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto , Japan; lyu@frontier.kyoto-u.ac.jp. stated that the trachea was subject to the same immunologic laws for transplantation as all other tissues. Yokomise and colleagues [4], Mukaida and associates [5], and Tojo and coworkers [6] performed fresh tracheal allotransplants in mongrel dogs. All the animals died of graft collapse between 14 and 78 days after transplantation. Histologic examination revealed epithelial defects, granulation tissue with fibrosis, monocyte infiltration into the sub, and absorption of tracheal cartilage. This suggested that graft rejection did indeed occur after fresh tracheal allotransplantation, and accordingly the use of immunosuppressants is necessary to control the immune reaction [7, 8]. Why did these differences between the clinical report and animal experiments arise? Is it possible to perform fresh tracheal allotransplantation without immunosuppressants? To resolve these questions, we designed the following experiments. Material and Methods Animal Selection Eighteen beagle dogs weighing 7.5 to 16.5 kg were used in this study. In group 1 (n 6, 3 pairs), the dogs were selected from a farm that kept strict control over breeding lines and detailed records of blood relationships. The paired dogs were related for three or four previous generations. The grafts were exchanged between paired dogs, which were, therefore, mutual donors and recipients. In group 2 (n 12, 6 pairs), the paired dogs were 2001 by The Society of Thoracic Surgeons /01/$20.00 Published by Elsevier Science Inc PII S (01)

2 Ann Thorac Surg LIU ET AL 2001;72: TRACHEAL ALLOTRANSPLANTATION IN BEAGLE DOGS 1191 Table 1. Results in Group 1 No. Survival (days) Cause of Death Status of Graft Epithelium of Implanted Graft Mononuclear Cell Infiltration Absorption of Graft Cartilage Alive No stenosis Alive No stenosis Killed No stenosis Ciliated columnar Scattered No 4 91 Killed No stenosis Ciliated columnar Scattered No 5 5 Unknown No stenosis Defective Scattered No Killed No stenosis, ulcerated mucosa (2 6 mm) Ciliated columnar and squamous Massive Slight selected from different farms with no business relationships. Therefore, we assumed that there was no blood relationship between the recipient and donor dogs. All the dogs received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised 1985). Tracheal Allotransplantation The tracheal replacements were performed at the same time in both dogs of each pair, under general anesthesia. The surgical procedure was similar to that reported by Yokomise and associates [4]. Briefly, through a midline epigastric incision, an omental pedicle graft was prepared and brought into the right chest through the diaphragm. A right thoracotomy was performed at the fourth intercostal space, and a seven-ring segment of the intrathoracic trachea was removed. One ring was taken off each end of the graft for trimming. The defect was immediately reconstructed using the five-ring graft harvested from the other dog of the pair. A telescopic anastomosis was performed using 4-0 Prolene (Ethicon, Inc, Somerville, NJ) sutures. In both groups, the anastomotic site and the graft were wrapped in the omental pedicle. Skin Transplantation Except for one pair of dogs in group 1, a full-thickness skin allotransplant was also performed in both paired dogs. The grafts were removed from the left back of the donor and resited on the left back of the recipient. Two flaps of full-thickness skin measuring 2 2 cm were harvested from each of the dogs and transplanted to the other member of the pair (32 flaps in total). Briefly, the four corners of each graft were sutured separately with 6-0 nylon sutures (Ethicon, Inc), and a continuous suture was run on each side. As a control, autotransplantation of two further flaps of full-thickness skin was performed (32 flaps in total). Postoperative Care and Observation No immunosuppressants were administered during this study. An antibiotic (a mixture of ampicillin sodium and cloxacillin sodium, each at a dose of 250 mg/d) was given intramuscularly for the first week and then orally until the end of the fourth week. Bronchoscopic examinations were performed once a week for 3 months and monthly thereafter in both groups. A skin autograft and an allograft were removed for histologic examination at 1 and 2 weeks after transplantation. Results In group 1 (Table 1), 1 animal died 5 days after transplantation. The reason for death was unclear as the airway was open and neither intrathoracic infection, bleeding, nor air leakage was observed during a postmortem examination. Four of these dogs survived uneventfully for more than 10 months. Bronchoscopically, the inner surface of the graft was moist and appeared dark red during the first 2 weeks. Around the third week, it became dark yellow, and after 1 month it gradually became pink. After 2 months, the lumen was shiny. No stenosis of the implanted grafts occurred in any of the 5 dogs during the observation period (Fig 1). Macroscopically, the inner surfaces of the grafts were shiny but paler than the host trachea (Fig 2A). The outlines of the cartilage rings were clearly discernible. Histologic examination revealed monocyte infiltration but no obvious absorption of the graft cartilage in these animals. The inner surface of the implanted graft was covered with ciliated columnar (Fig 2B). In group 2 (Table 2), 4 dogs died within 3 months of operation because of graft stenosis. The other 8 dogs survived without dyspnea, and for 4 dogs, the observation periods were longer than 9 months. Any abnormal Fig 1. Bronchoscopic findings in group 1 (left, 7 days after transplantation; right, 314 days after transplantation). No stenosis of the implanted graft was observed.

3 1192 LIU ET AL Ann Thorac Surg TRACHEAL ALLOTRANSPLANTATION IN BEAGLE DOGS 2001;72: destroyed within 2 weeks in both groups (Fig 4). Severe monocyte infiltration into the dermis was confirmed. Fig 2. Macroscopic (A) and histologic findings (B, hematoxylin and eosin staining) in a tracheal graft 314 days after transplantation in group 1. The graft had been incorporated into the host trachea. The outlines of the cartilage rings of the graft are clearly discernible. No absorption of the graft cartilage was observed. The surface of the graft is covered by ciliated columnar. signs seen during the bronchoscopic examination were classified as described below and the results are shown in Table 2. We defined severe stenosis as a reduction of the tracheal lumen by more than two thirds (Fig 3, left). Moderate stenosis was defined as a reduction of more than one third but less than two thirds, and slight stenosis was defined as a reduction of less than one third in combination with an irregular inner surface (Fig 3, right). In 2 dogs, no stenosis was confirmed bronchoscopically. Macroscopically, stenosis and sputum retention were observed in the grafts. Histologic examination revealed severe mononuclear cell infiltration and absorption of the graft cartilage. Simple squamous covered the surface of the absorbed cartilage in some places. After skin transplantation, 23 of the 32 autograft flaps remained soft and yellowish pink throughout the observation period. The other 9 skin autografts dropped off, probably owing to scratching or rubbing against the cage. Necrosis was observed in all 32 skin allografts within 1 week of transplantation, and all the allografts had been Comment The prognosis of an allograft transplant can be predicted on the basis of a skin transplantation test [9]. Skin grafts between genetically identical animals are accepted, whereas grafts between genetically nonidentical animals are rejected. Therefore, we used skin transplantation as an indicator of the immune reaction. In the present study, all the skin allografts in both groups were destroyed by rejection. However, the tracheal grafts were accepted in group 1. Even in group 2, 8 dogs survived the observation period. Although monocyte infiltration was confirmed, the cartilage was not damaged seriously. The lumen of the grafts was open. Because the tracheal grafts acted as the pathway for lung ventilation without immunosuppressants, we conclude that the antigenicity of tracheal grafts is weaker than that of other organs. Outcomes in group 1 were much better than in group 2. This strongly suggests that it is possible to perform tracheal allotransplants using histocompatible matched grafts without immunosuppressants. No stenosis was observed in 2 dogs of group 2. We suspect that these grafts were histocompatibility matched by chance. This may explain the successful tracheal transplantation performed by Rose and coworkers [1]. On the other hand, 4 dogs in group 2 died because of airway stenosis. Moreover, moderate or slight stenosis was observed in the other 6 dogs, although the stenosis was not fatal. Mononuclear cells infiltrated the sub, and absorption of the tracheal cartilage occurred. These findings are consistent with those of others [4 6]. Before transplantation the antigenicity of a tracheal graft should be taken into consideration. Inasmuch as mongrel dogs were used in the cited studies, great variety in major histocompatibility complex antigens was introduced, and serious rejection occurred when tracheal transplantation was performed. All dogs died within 3 months [4 6]. Therefore, in the present Fig 3. Bronchoscopic findings in group 2. Severe stenosis (left, dog 8, 70 days after transplantation) and slight stenosis (right, dog 4, 284 days after transplantation) were observed.

4 Ann Thorac Surg LIU ET AL 2001;72: TRACHEAL ALLOTRANSPLANTATION IN BEAGLE DOGS 1193 Table 2. Results in Group 2 No. Survival (Days) Cause of Death Status of Graft Epithelium of Implanted Graft Mononuclear Cell Infiltration Absorption of Graft Cartilage Alive No stenosis Alive No stenosis Killed Slight stenosis Ciliated and squamous Massive Killed Slight stenosis Ciliated and squamous Massive 5 35 Killed Severe stenosis Defective Massive 6 42 Killed Severe stenosis Defective Massive 7 91 Killed Moderate stenosis Ciliated and squamous Massive 8 70 Killed Severe stenosis Defective Massive 9 56 Killed Slight stenosis Ciliated and squamous Massive Killed Severe stenosis Squamous Massive Killed Slight stenosis Ciliated and squamous Massive Killed Moderate stenosis Ciliated and squamous Massive study, we used beagles, which have been widely used for studies of kidney and liver transplantation [10, 11]. We conclude that allotransplantation of the trachea is different from that of other organs. First, the blood supply to a tracheal transplant is different from that to a kidney or liver transplant. During organ transplantation, the major blood vessels belonging to the graft are anastomosed to those of the recipient, so the transplanted organ presents a large surface area of antigenic donor to the blood cells of the host. The host lymphocytes enter the graft through the blood vessels. This pathway is known as the central pathway of sensitization [12]. Rejection not only destroys the parenchyma of the graft, but also damages the graft vessels to promote further necrosis of the implanted organ. However, tracheal vessels are too small to be anastomosed easily [13]. We overcame this problem by performing an omentopexy. Immediately after transplantation, the graft did not contain any functioning blood vessels and there was no host blood entering the vessels of the graft. The graft was just wrapped in the omentum, and new vessels gradually revascularized the graft [14, 15]. This method of establishing a blood supply is similar to that used during tissue transplantation (eg, skin grafting). The host lymphocytes were sensitized by the so-called peripheral pathway of sensitization. However, the cartilage of a tracheal graft is located closer to the lymphatic capillaries of the host than the mucosa. Major histocompatibility complex antigens are expressed in the tracheal mucosa, but not in the cartilage [16 19]. Thus, a much smaller area of antigenic material is exposed to the host than with kidney, liver, or skin transplants. This may explain why the antigenicity of the trachea is weaker than that of these other organs. Second, the physiologic function of the trachea is simpler than that of other organs. Briefly, the cartilage supports the airway in an open position, and the ciliated removes inhaled dust, secretions, and so forth. Moderate or slight stenosis of the graft was observed in 6 dogs in group 2. Although rejection occurred, the cartilage was not damaged seriously enough to cause collapse of the graft, so the airway remained open, to allow the dogs to survive for a long time without dyspnea. Thus, unlike damage to nephrons or hepatic lobules, rejection did not have fatal consequences. However, squamous was substituted for ciliary in some places, and sputum retention was observed. For five-ring tracheal replacements, this is unlikely to cause serious problems. If a longer tracheal graft is needed, such sputum retention may encourage respiratory infection. Because donors are in short supply, it is sometimes difficult to find suitable major histocompatibility complex matched donors for tracheal transplantation. For such occasions, detergent-treated [19] or cryopreserved Fig 4. Macroscopic (left, autograft; right, allograft) findings in skin grafts 2 weeks after transplantation. Necrosis of the skin allograft was observed.

5 1194 LIU ET AL Ann Thorac Surg TRACHEAL ALLOTRANSPLANTATION IN BEAGLE DOGS 2001;72: [20] grafts can be used for immunosuppressant-free allotransplantation. References 1. Rose KG, Sesterhenn K, Wustrow F. Tracheal allotransplantation in man. Lancet 1979;1: Beigel A, Muller-Ruchholtz W. Tracheal transplantation. I. The immunogenic effect of rat tracheal transplants. Arch Otorhinolaryngol 1984;240: Beigel A, Muller-Ruchholtz W. Tracheal transplantation. II. Influence of genetic difference and degree of sensitization on reactions to the tracheal transplant. Arch Otorhinolaryngol 1984;240: Yokomise H, Inui K, Wada H, et al. High-dose irradiation prevents rejection of canine tracheal allografts. J Thorac Cardiovasc Surg 1994;107: Mukaida T, Shimizu N, Aoe M, et al. Experimental study of tracheal allotransplantation with cryopreserved grafts. J Thorac Cardiovasc Surg 1998;116: Tojo T, Niwaya K, Sawabata N, Nezu K, Kawachi K, Kitamura S. Tracheal allogenic immunoresponse is reduced by cryopreservation: canine experiment. Transplant Proc 1996; 28: Davreux CJ, Chu NH, Waddell TK, Mayer E, Patterson GA. Improved tracheal allograft viability in immunosuppressed rats. Ann Thorac Surg 1993;55: Delaere PR, Liu Z, Sciot R, Welvaart W. The role of immunosuppression in the long-term survival of tracheal allografts. Arch Otolaryngol Head Neck Surg 1996;122: Hutchinson I. Transplantation and rejection. In: Roitt I, Brostoff J, Male D, eds. Immunology, 5th ed. London: Mosby, 1998: Ochiai T, Nagata M, Nakajima K, et al. Studies of the effects of FK506 on renal allografting in the beagle dog. Transplantation 1987;44: Ko S, Nakajima Y, Kanehiro H, et al. The enhanced immunosuppressive efficacy of newly developed liposomal FK506 in canine liver transplantation. Transplantation 1995;59: Klein J. Allograft reaction. In: Klein J, ed. Immunology. Boston: Blackwell Scientific, 1990: Salassa JR, Pearson BW, Payne WS. Gross and microscopical blood supply of the trachea. Ann Thorac Surg 1977;24: Nakanishi R, Shirakusa T, Takachi T. Omentopexy for tracheal autografts. Ann Thorac Surg 1994;57: Messineo A, Filler RM, Bahoric B, Smith C, Bahoric A. Successful tracheal autotransplantation with a vascularized omental flap. J Pediatr Surg 1991;26: Daar AS, Fuggle SV, Fabre JW, Ting A, Morris PJ. The detailed distribution of MHC class II antigens in normal human organs. Transplantation 1984;38: Daar AS, Fuggle SV, Fabre JW, Ting A, Morris PJ. The detailed distribution of HLA-A, B, C antigens in normal human organs. Transplantation 1984;38: 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: Liu Y, Nakamura T, Yamamoto Y, et al. Immunosuppressant-free allotransplantation of the trachea: the antigenicity of tracheal grafts can be reduced by removing the and mixed glands from the graft by detergent treatment. J Thorac Cardiovasc Surg 2000;120: Yokomise H, Inui K, Wada H, Ueda M, Hitomi S. Long-term cryopreservation can prevent rejection of canine tracheal allografts with preservation of graft viability. J Thorac Cardiovasc Surg 1996;111:930 4.