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Ann Thorac Surg 1996;62:1480-1484
© 1996 The Society of Thoracic Surgeons

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Original Article: General Thoracic

Reconstruction of Trachea and Carina With Immediate or Cryopreserved Allografts in Dogs

Second Department of Surgery, Fukuoka University School of Medicine, Fukuoka, Japan

Accepted for publication May 28, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Background. Grafting is required when primary reconstruction of a tracheocarinal defect is not feasible. To determine the viability of and the nature of the healing process occurring in the cryopreserved graft, we performed tracheocarinal transplantation in dogs.

Methods. We performed 32 tracheocarinal reconstructions in dogs using autotransplanted, immediately transplanted, or cryopreserved allografts. The viability of each graft was evaluated serially by fiberoptic macroexamination and by measurement of the tracheal mucosal blood flow using a hydrogen clearance method. In group A (n = 8), the tracheal carina was removed and reimplanted immediately. In group B (n = 8), the tracheocarina was allotransplanted immediately after harvest. In group C (n = 8), allotransplantations were performed using grafts cryopreserved for 1 to 3 weeks by freeze-drying. In group D (n = 8), we attempted to achieve immunosuppression-free transplantation with the cryopreserved allografts.

Results. Sufficient viability and good healing (6/8, 75%) occurred in the dogs with cryopreserved tracheocarinal allotransplants. Three of 8 dogs (38%) with cryopreserved allotransplants survived for 25 to 57 days without immunosuppression.

Conclusions. The cryopreservation of tracheocarinal allografts for 3 weeks without the use of a preservative solution was shown to be feasible. Cryopreservation prolonged the survival of nonimmunosuppressed allotransplants in dogs.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Tracheocarinal allotransplantation is best performed immediately after the carina is resected. Patients in whom major anatomical defects are created by resection of the carinal lesion are at risk for such postoperative complications as ischemia, stenosis, and disruption of the anastomosis as a result of tension [1]. Therefore, the length of the tracheal resection must be less than six to seven rings without grafting [2]. Although autogenous tissue [3] and artificial prostheses [4] have not proved clinically useful for carinal reconstruction, experimental tracheal allotransplantation has been successfully performed in dogs [5, 6]. Lima and colleagues [7] reported that omentopexy markedly improves the healing of bronchial anastomoses. We previously reported on our successful results from carinal allotransplantation that included the administration of FK506 and omentopexy in dogs [8]. In the present experimental study, we evaluated a tracheocarinal allograft that was three rings longer than the one we previously described and cryopreserved the graft at -80°C for 1 to 3 weeks.

The trachea is a relatively simple structure that consists of cartilage, epithelium, and connective tissue. Because its blood supply is limited, regardless of the nonvascularized procedure used for implantation, a simple tracheal transplant can survive without immediate neovascularization from the surrounding tissue [2, 5, 6]. This situation resembles that seen for transplantation of a cardiac valve, in which the cryopreservation of human aortic valves has been successful [9, 10]. Cryopreservation may also be feasible for tracheal grafts [11, 12]. If this technique of preservation could be used, prolonged storage of various tracheal grafts would be feasible. We attempted to reconstruct the tracheal carina using an immunosuppression-free allotransplantation model to investigate the utility of this technique.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Experimental Design
Thirty-two adult mongrel dogs were used. In group A (autotransplant control; n = 8), a section of the tracheal carina, consisting of five rings of the trachea along with one ring of the right main bronchi and two rings of the left, was removed and immediately reimplanted. In group B (n = 8), a section of the tracheal carina of the same length was allotransplanted immediately after harvest. In group C (n = 8), allotransplantation was performed with grafts that had been cryopreserved for 1 to 3 weeks. In group D (n = 8), immunosuppression-free transplantation was performed with cryopreserved allografts.

Donor Operation
Each adult mongrel dog (n = 24; weight, 9.2–16 kg) was anesthetized with an intravenous injection of 25 mg/kg of pentobarbital sodium. It was then orally intubated and connected to a pressure-limiting respirator. A right thoracotomy was performed, and the trachea and carina were isolated. The section of the tracheocarina to be transplanted was then harvested.

Cryopreservation
Each carinal specimen was placed in a sterile bottle without a preservative solution and stored at -80°C for 1 to 3 weeks. Before transplantation, specimens were warmed to 35°C for 15 minutes.

Transplantation Procedure
A weight-matched dog was paired with each of the donor animals and anesthetized as described. This dog was then placed in the left semidecubitus position, and a right thoracotomy was performed. After the azygos vein was ligated and divided, the tracheal carina was isolated from the surrounding tissue. The right main bronchus was transected one ring distal to the carina and intubated to the distal bronchus through the pleural cavity. The right lung was ventilated, and five rings of the trachea, along with two rings of the left main bronchus, were transected and removed. Initially a telescope-type of anastomosis of the left main bronchus and trachea was performed using 4-0 monofilament absorbable sutures. An orotracheal tube was inserted into the left main bronchus, and the left lung was ventilated. A similar anastomosis was performed in the right main bronchus. After these anastomoses were completed, the graft was wrapped with the pedicled omentum that was previously drawn through the diaphragm. All animals were given 0.125 mg of cefoperazone (CPZ; Toyama Chemical Co, Tokyo, Japan) daily for 2 weeks postoperatively. Dogs in groups B and C also received the immunosuppressant agent FK506 (tacrolimus; Fujisawa Pharmaceutical Co, Osaka, Japan), 0.1 mg/kg injected intramuscularly, postoperatively.

Measurement of Tracheal Mucosal Blood Flow
Bronchoscopy was performed each week postoperatively until the animals died of natural causes or were sacrificed in order to evaluate the healing of the anastomosis and the viability of the graft. The tracheal mucosal blood flow (TMBF) in the donor grafts was measured preoperatively and on day 3, 7, 14, 28, 42, and 56 after operation using the hydrogen clearance technique [13], and this involved the placement of a hook-shape–tipped electrode in contact with the tracheal mucosa through a channel of the fiberoptic bronchoscope. Blood flow was measured in the carinal region, the proximal site, and the midportion of the grafts. To evaluate the restoration of blood flow in the grafts, postmortem microangiography was performed from 5 to 365 days postoperatively in 2 dogs from each group by injecting a 20% gelatin-barium solution through the right gastroepiploic artery.

Histologic Assessment
Specimens were evaluated by light microscopy. Graft status was assessed according to the semiquantitive rating scale described by Nakanishi and associates [14] for evaluating the histologic characteristics of the epithelium, the damage to the tracheal glands, the development of a granulation tissue–like reaction with fibrosis, the degeneration of cartilaginous rings, and other characteristics (Table 1). Infiltration by mononuclear cells was also assessed, as this is closely associated with rejection of the allograft [15]. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
The dogs in groups A, B, C, and D survived for 7 to 935 days. The causes of death were pneumonia, respiratory failure, wasting, or unknown causes (Table 2). Fiberoptic bronchoscopy showed that anastomotic healing of the graft was excellent in 6 of the 8 dogs (75%) in group A, 5 of the 8 dogs (63%) in group B, 6 of the 8 dogs (75%) in group C, and 3 of the 8 dogs (38%) in group D, including those that were sacrificed during the early postoperative period to assess graft viability. Edema, stenosis, or malacia of the graft was observed in the remaining animals. One dog in group B and 2 in group C survived for 204, 258, and 147 days, respectively, without anastomotic complications, despite discontinuance of the FK506, on day 731, 92, and 58, respectively, after transplantation.

View larger version (114K): [in this window] [in a new window]   Fig 1. . Histologic findings in a representative dog (no. 7) from group C that received a cryopreserved allograft and was sacrificed 25 days postoperatively. Normal mucociliary epithelium covers the graft, and no damage to the tracheal glands or cartilage is observed. Mononuclear cell infiltration and a granulation tissue–like reaction with fibrosis are absent. (Hematoxylin and eosin; x25.)

View larger version (45K): [in this window] [in a new window]   Fig 2. . Measurement of tracheal mucosal blood flow (TMBF) in grafts by the hydrogen clearance method. Three sites on each graft were selected as measuring points. Open circles represent the flow in the carinal area of the graft; filled circles represent the flow at a proximal site on the graft; and triangles represent the flow at the midportion of the graft. Tracheocarinal mucosal blood flow in autografts (A), in fresh allografts in animals treated with FK506 (B), and in cryopreserved allografts in animals treated with FK506 (C). (Data are expressed as the mean ± standard deviation.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

Endoscopic and histologic findings confirmed the sufficient viability and good healing in 6 of the 8 dogs (75%) with autotransplants and in 5 of the 8 dogs (63%) with fresh allografts. These results resemble those obtained in our previous study [7], in which we performed carinal allotransplantations with two tracheal rings. We also evaluated the effectiveness of the cryopreservation of the grafts at -80°C for 1 to 3 weeks without the use of a preservative solution. Deschamps and colleagues [11] successfully preserved canine tracheas for 1 week at -196°C. Yokomise and associates [12] reported that five rings of trachea could be cryopreserved for 1 month. However, only allografts were evaluated in these experiments. In the present series, cryopreservation was done for 1 to 3 weeks, because in our preliminary study we found that grafts preserved for more than 4 weeks showed a loss of epithelium. We observed sufficient viability and good healing in canine cryopreserved tracheocarinal allografts in 75% of our experimental animals.

Nakanishi and colleagues [14] assessed graft status by evaluating the histologic characteristics of the epithelium, the damage to tracheal glands, the granulation tissue–like reaction with fibrosis, and the degeneration of the cartilaginous ring. Lene and coworkers [15] reported that infiltration by mononuclear cells is closely associated with allograft rejection. We therefore evaluated the viability of the graft according to the semiquantitive rating scale of Nakanishi and colleagues [14]. We observed sufficient viability and good healing in 6 of 8 dogs (75%) with autotransplants (the control group) and in 6 of 8 dogs (75%) with cryopreserved allografts. Thus, cryopreservation of the graft at -80°C for 1 to 3 weeks without a preservative solution proved to be feasible in our study. In general, it is widely accepted that the degree of healing of a graft is related to the blood supply to the organ [2, 5, 6]. The TMBF could therefore be used to gauge graft healing. The present study showed that the TMBF was lower in the midportion of the graft than in either the proximal sites of the trachea or in the distal sites of the main bronchus. These findings indicate that blood was supplied directly from the omentum via the anastomosis. This conclusion is supported by the findings from the morphologic examination of grafts in rabbits reported by Inayama and colleagues [16]; they observed that the mitotic index values at the anastomotic sites of the graft were higher than those at the midportion. The TMBF in the grafts in the present study returned to normal by 4 weeks after operation. There was no significant difference in the TMBF among the animals with autografts and allografts, with or without cryopreservation. It is therefore conceivable that 4 weeks were needed to accomplish histologic healing. In addition, postmortem microangiography showed that graft revascularization, arising from the omentum, occurred on postoperative day 5 in the cryopreserved allografts. Hence, we presume that omentopexy is needed to reestablish blood flow in a tracheocarinal allotransplant that consists of five tracheal rings.

Grafts without immunosuppression were rejected in previous studies of canine tracheal allotransplantation [6, 17]. However, tracheal allotransplantation, in contrast to the transplantation of other organs, reportedly induces only mild graft rejection, even in the presence of major histoincompatibility [18]. Because most of the diseases for which tracheocarinal transplantation is indicated are malignant, minimal immunosuppression is desirable [17]. In our study, 1 dog in which immediate allotransplantation was performed and 2 dogs that received a cryopreserved graft survived for 204, 258, and 147 days, respectively, without anastomotic complications, despite the discontinuance of FK506 on days 731, 92, and 58, respectively, after transplantation. We found that 38% of the cryopreserved allografts in the dogs survived for 25 to 57 days after transplantation without immunosuppressive treatment. However, observation of these grafts for longer periods is necessary to determine whether immunosuppressive treatment is essential to prevent the rejection of cryopreserved tracheal allotransplants. Further study is also required to determine whether cryopreservation reduces alloantigenicity by modulating the expression of major histocompatibility complex class II antigen in the grafts.

We conclude that cryopreservation of tracheocarinal allografts without preservative for 3 weeks is feasible and that this technique is very simple and feasible for clinical use. Cryopreservation prolonged the survival of tracheal allograft in nonimmunosuppressed dogs.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Address reprint requests to Dr Inutsuka, Second Department of Surgery, Fukuoka University School of Medicine, 7-45-1 Nanakuma Jyohnan-ku, Fukuoka City, 814-01 Japan.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 

1. Grillo HC. Carinal reconstruction. Ann Thorac Surg 1982;34:356–73.[Abstract]
2. Nakanishi R, Shirakusa T, Mitsudomi T. Maximum length of tracheal autograft in dogs. J Thorac Cardiovasc Surg 1993;106:1081–5.[Abstract]
3. Letang E, Sanchez-Lloret J, Gimferrer JM, Ramirez J, Vicens A. Experimental reconstruction of canine trachea with a free revascularized small bowel graft. Ann Thorac Surg 1990;49:955–8.[Abstract]
4. Neville WE, Bolanowski PJP, Soltanzadeh H. Prosthetic reconstruction of the trachea and carina. J Thorac Cardiovasc Surg 1976;72:525–38.[Abstract]
5. Shirakusa T, Ueda H, Saito T. The experimental allotransplantation of trachea in canines. J Jpn Surg Soc 1990;91:524–8.
6. Moriyama S, Shimizu N, Teramoto S. Experimental tracheal allo-transplantation using omentopexy. Transplant Proc 1989;21:2596–600.[Medline]
7. Lima O, Goldberg M, Peters WJ, Ayabe H, Townsend E, Cooper JD. Bronchial omentopexy in canine lung transplantation. J Thorac Cardiovasc Surg 1982;83:418–21.[Medline]
8. Takachi T, Shirakusa T, Shiraishi T, et al. Experimental carinal autotransplantation and allotransplantation. J Thorac Cardiovasc Surg 1995;110:762–7.[Abstract/Free Full Text]
9. O'Brien MF, Stafford EG, Gardner MAH, Pohlner PG, McGiffin DC. A comparison of aortic valve replacement with viable cryopreserved and fresh allograft valves, with a note on chromosomal studies. J Thorac Cardiovasc Surg 1987;94:812–23.[Abstract]
10. Kirklin JK, Smith D, Novick W, et al. Long-term function of cryopreserved aortic homograft: a ten-year study. J Thorac Cardiovasc Surg 1993;106:154–66.[Abstract]
11. Deschamps C, Trastek VF, Ferguson JL, et al. Cryopreservation of canine trachea: functional and histological changes. Ann Thorac Surg 1989;47:208–12.[Abstract]
12. Yokomise H, Inui K, Wada H, Hasegawa S, Ohno N, Hitomi S. Reliable cryopreservation of trachea for one month in a new trehalose solution. J Thorac Cardiovasc Surg 1995;110:382–5.[Abstract/Free Full Text]
13. Aukland K, Bower BF, Berliner RW. Measurement of local blood flow with hydrogen gas. Circ Res 1964;14:164–87.[Abstract/Free Full Text]
14. Nakanishi R, Shirakusa T, Hanagiri T. Early histopathologic features of tracheal allograft rejection: study in nonimmunosuppressed dogs. Transplant Proc 1994;26:3715–8.[Medline]
15. Lene BP, Habicht GS, Jasper GS. Lymphocyte-epithelium interaction during resection of nonisogenic rat tracheal grafts. Am J Pathol 1977;86:71–7.[Abstract]
16. Inayama Y, Tomiyama I, Akaike M, et al. Morphologic alteration and cytokinetic studies of tracheal autograft epithelium in rabbits. Ann Thorac Surg 1995;60:952–7.[Abstract/Free Full Text]
17. Nakanishi R, Yasumoto K, Shirakusa T. Short-course immunosuppression after tracheal allotransplantation in dogs. J Thorac Cardiovasc Surg 1995;109:910–7.[Abstract]
18. Rose KG, Sesterhenn K, Wustrow FT. Tracheal allotransplantation in man [Letter]. Lancet 1979;1:433.[Medline]

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