Ann Thorac Surg 2000;70:1880-1884
© 2000 The Society of Thoracic Surgeons
Original article: general thoracic
Limit of warm ischemia time before cryopreservation in rat tracheal isografts
Ryoichi Nakanishi, MDa,
Masayoshi Umesue, MDa,
Mitsunori Hashimoto, MDa,
Hiroyuki Muranaka, MDa,
Mitsuhiro Hachida, MDa,
Kosei Yasumoto, MDa
a Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
Accepted for publication April 6, 2000.
Address reprint requests to Dr Nakanishi, Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807–8555, Japan
e-mail: ryoichi{at}med.uoeh-u.ac.jp
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Abstract
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Background. The viability of cadaveric tracheal grafts undergoing cryopreservation is still unclear. We evaluated the limit of warm ischemia time before cryopreservation in rat tracheal isografts.
Methods. Each isograft was harvested from donor rats 0 to 48 hours (0, 6, 12, 18, 24, and 48 hours) after circulatory arrest, immersed in the preservative solution, and stored in a deep freezer until reaching -80°C and then was kept in liquid nitrogen for 3 months. Heterotopic transplantation into the omentum was performed after the isografts were thawed. Graft morphology 3 months after transplantation was assessed.
Results. The stepwise increase of warm ischemia time significantly reduced graft survival. A prolonged period of warm ischemia had a degenerative effect on both the epithelium and cartilage. The morphology of the epithelium and cartilage in isografts undergoing warm ischemia for less than 18 hours was better preserved, whereas it deteriorated in isografts undergoing warm ischemia for more than 24 hours.
Conclusions. We thus conclude that the permissible period of warm ischemia before 3-month cryopreservation to maintain tracheal isograft viability is 18 hours in rats.
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Introduction
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Although the transplantation of the trachea is considered to be the preferred method for the reconstruction of extensive tracheal defects in the future, the deficit of donor trachea still remains a major obstacle. We have recently investigated the maximal period of cryopreservation for viable tracheal isografts in order to solve this problem [1]. As a result, the effect of cryopreservation on the trachea has been recognized as the degeneration of both the cartilage and the epithelium. The cartilage showed irreversible damage while the epithelium partially remained after cryopreservation for 6 months. The permissible period of cryopreservation for tracheal isografts is thus considered to be 3 months [1]. This period of cryopreservation could therefore help us to resolve the shortage of donors.
The trachea of less than 4 cm in length may tolerate warm ischemia because this tissue is exposed to devascularization after transplantation [2, 3]. If the trachea can be retrieved for transplantation late after circulatory arrest, the shortage of donors may be further alleviated. The combined use of nonheartbeating cadaver donors and a long period of cryopreservation for tracheal grafting offers great hope for their eventual clinical use in humans. We thus tried to elucidate the permissible period of warm ischemia before cryopreservation in rat tracheal isografts. We used isografts to avoid the immunologic complexity of allografts. The cryopreservation of cadaveric tracheal grafts could further help us to resolve the shortage of donors.
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Material and methods
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Animals and anesthesia
The heterotopic tracheal transplant model with omental wrapping has been employed using a technique modified for rats [4, 5]. Thirty-six male Brown Norway rats between 10 and 12 weeks of age and weighing approximately 250 to 300 g were premedicated with anesthetic ether. The animals were placed in the supine position and anesthetized with the intraperitoneal administration of sodium pentobarbital (10 mg/kg). Under spontaneous ventilation without an endotracheal tube, either harvesting or transplantation was performed. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (National Institutes of Health Publication No. 85 to 23, revised 1985).
Experimental design and harvesting
Six donor rats were sacrificed with an intravenous overdose of pentobarbital and left at room temperature (24°C). The animals were randomly assigned to 6 groups according to a stepwise increase in warm ischemia time in the donor from 0 to 48 hours (0, 6, 12, 18, 24, and 48 hours, respectively). After the assigned warm ischemia time, through a midline cervical to sternal incision, the whole trachea was identified and excised in continuity. The harvested trachea was trimmed off in 5-ring segments. Thus, 5 tracheal segments consisting of 5 rings were harvested from a donor rat after the assigned warm ischemia time. We did not use multiple donors for each of the groups because the donor-dependent problems were considered to be minimal from our rich experience.
Cryopreservation and grafting
The tracheal segments were then immediately stored in a plastic sterile tube filled with the freezing solution. The preservative solution in which the grafts were immersed contained a balanced buffered salt solution with L-glutamine (RPMI-1640 medium; JRM Biosciences, Lenexa, KS) with a final concentration of 20% fetal calf serum and 10% dimethyl sulfoxide (DMSO) as the cryoprotectant. The plastic tube containing the specimen was then stored in a Bicell biofreezing vessel (Nihon Freezer Co., Ltd, Tokyo, Japan) after cooling at 4°C for an hour, and then was subsequently stored in a deep freezer at -80°C. The Bicell biofreezing vessel has the ability to cool down at a rate of approximately 1°C per minute in a deep freezer until reaching -80°C [1]. The plastic tube containing the specimen was then stored in liquid nitrogen (at -196°C) for 3 months.
Cryopreserved isografts were thawed in an incubator for 15 minutes at 37°C for grafting. All isografts were stented over a silicone rod 1.35 mm in diameter (ATOM, Inc, Tokyo, Japan), and then were heterotopically implanted into the omentum of 30 recipient rats. A silicone rod was used to prevent the omentum from intruding into the graft. The recipient animals were placed in a supine position. Through a small upper midline laparotomy, the greater omentum was delivered into the wound. The anterior layer of the omentum was opened, and the tracheal transplant was completely enveloped. The omentum and the enclosed transplant were then returned to the peritoneal cavity, and the wound was closed in the usual fashion. After 3 months, a second laparotomy was performed to retrieve the transplants for the histopathologic analysis.
Histopathologic assessment
All tissue specimens were fixed in 10% formalin. Microscopic slides were made from cross-sections of the midportion of the tracheal segment and adherent omentum, and were stained routinely with hematoxylin and eosin. Thereafter, all specimens were examined by light microscopy. We attempted to quantify the viability of the heterotopically grafted trachea by subjectively evaluating the epithelial morphology and objectively calculating the ratio of chondrocytes possessing a viable nucleus in the cartilage. All assessments were performed in a blinded fashion.
Epithelial viability
Epithelial viability was evaluated according to the following grading system: 0, no epithelium; 1, single layer nonciliated epithelium; 2, multilayer nonciliated epithelium; and 3, normal mucociliary epithelium. The epithelial score of the graft was calculated from the following formula: Epithelial score = an occupation rate of grade 0 x 0 + an occupation rate of grade 1 x 1 + an occupation rate of grade 2 x 2 + an occupation rate of grade 3 x 3, after the ratio of occupation of a morphologically different epithelium was estimated in a cross-section of the graft [1, 4, 5, 6].
Cartilage viability
The ratio of chondrocytes possessing viable nuclei in the cartilage of each transplant was calculated after carefully counting the number of all chondrocytes in each high-powered field on a microscopic slide. A viable chondrocyte nucleus demonstrated rich staining and possessed a clear nucleus membrane [1, 7, 8].
Statistical analysis
All data are presented as the mean ± standard deviation of the mean. A statistical analysis was performed using the paired Student’s t test. A p value of less than 0.05 was considered to be statistically significant.
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Results
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Gross appearance
The omentum was wrapped firmly around the tracheal isografts in all rats. Grossly, all free tracheal isografts appeared normal and also preserved their lumen rigidity.
Histopathology
Longer periods of warm ischemia were likely to result in an irreversible deterioration of the tracheal isografts. All free isografts except for those of group 6 (warm ischemia time = 48 hours) had little infiltration of inflammatory cells or submucosal thickening by fibrous tissue.
Epithelial viability
Many isografts undergoing an immediate cryopreservation in group 1 (warm ischemia time = 0 hour) had normal mucociliary epithelium (Fig 1). The epithelium of the isografts undergoing warm ischemia for 6 to 18 hours (groups 2 to 4) showed low-grade degeneration and consisted mainly of single layer and multilayer nonciliated epithelium (Fig 2). The incidence of normal mucociliary epithelium seemed to decrease according to increasing warm ischemia time, and no such epithelium was found in groups 5 to 6. The epithelium of the isografts in group 6 was almost denuded (Fig 3). There was no difference in the epithelial score between group 1 (control group) and groups 2 to 4. The epithelial score of the isografts in group 5 was significantly worse than that of the control isografts in group 1. Furthermore, the score of the isografts undergoing warm ischemia for 48 hours (group 6) was significantly worse than that of all other groups (Table 1).
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Fig 1. Histology of tracheal isografts in group 1 (warm ischemia time = 0 hour). A normal mucociliary epithelium is extensively recognized. The cartilage has viable chondrocyte nuclei and cytoplasm filled with viscous liquid in the lacunae. (Hematoxylin and eosin; original magnification x 200.)
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Fig 2. Histology of tracheal isografts in group 4 (warm ischemia time = 18 hours). A single layer and multilayer nonciliated epithelium is extensively recognized while a mucociliary epithelium is partially observed. The cartilage has viable chondrocyte nuclei for the most part but some necrotic chondrocytes with a nonviable nucleus can also be observed. (Hematoxylin and eosin; original magnification x 200.)
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Fig 3. Histology of tracheal isografts in group 6 (warm ischemia time = 48 hours). Inflammatory cells infiltrate into thickened submucosal fibrous tissue and the cartilage. The cartilage is necrotic. (Hematoxylin and eosin; original magnification x 200.)
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Cartilage viability
The cartilage viability of the isografts showed an exacerbating trend as the period of warm ischemia increased. Tracheal isografts undergoing warm ischemia for less than 18 hours had some viable cartilage islands and also showed new chondrocytes in the adventitial area (Fig 2). In contrast, the isografts undergoing warm ischemia for more than 24 hours showed a exacerbating trend and had nonviable chondrocyte nuclei in the lacunae. Above all, the isografts undergoing warm ischemia for 48 hours had partially necrotic cartilage and also showed massive cellular infiltration (Fig 3). The ratio of viable chondrocytes of the isografts in group 5 was significantly worse than that of the control isografts in group 1, whereas no difference was seen in the ratio of viable chondrocytes between group 1 (control group) and groups 2 to 4. Furthermore, the ratio of viable chondrocytes of the isografts undergoing warm ischemia for 48 hours (group 6) was significantly worse than that of all other groups (Table 2).
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Comment
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A rising need for donor organs has increased studies on the potential use of nonheartbeating cadaver donors as an additional source to the current supply of brain-dead cadaver donors. Several studies on the nonheartbeating cadaver donors showed that the maximal warm ischemia times of donor organs are 45, 140, and 240 minutes in kidney [9], liver [10], and lung transplantation [11], respectively. On the other hand, no study on maximal warm ischemia time of the trachea is reported as yet, despite verifying the feasibility of tracheal transplantation [6, 7, 12]. In regard to the cold ischemia time of the trachea, Macchiarini and associates [13] reported that the trachea may be safely preserved for as long as 15 hours of static storage in Euro-Collins solution at 4°C and that longer periods of cold ischemia are likely to result in irreversible graft damage. However, their experiment on the cold preservation of the trachea is no longer practical because the potential of a longer period of cryopreservation for the tracheal grafts has since been verified, and the cryopreservation is more applicable to clinical use [1]. Assessment of the maximal warm ischemia time before cryopreservation for the tracheal graft is required to expand the human tracheal donor pool.
The trachea remains exposed to the warm ischemia until it is revascularized after transplantation [2, 3, 7, 8]. The trachea may tolerate warm ischemia because the metabolic mechanisms of the trachea differ from those of such solid organs as kidney and liver. The trachea and also the lung do not rely on vascular perfusion for cellular respiration because airway epithelial tissue can be successfully cultured after retrieval from morgue specimens [14]. Respiration can occur directly across the airway gas interface, and the gas change processes are entirely passive. Therefore, the metabolic requirements of airway organs may be low and airway organs may thus tolerate considerable periods of ischemia.
The metabolic requirements of the trachea may be lower than that of the lung because the trachea does not require a greater blood flow than the lung and has only a simple anatomical structure including epithelium, cartilage, and few interstitium. Therefore, the nonheartbeating cadaver is potentially qualified to be a candidate for the donor in tracheal transplantation. The maximal warm ischemia time for the tracheal grafts has to be determined for this reason, and the study of cryopreservation is thus considered to be extremely important. Similar investigations using large-size tracheal grafts may be required for clinical use, since oxygenation of small grafts can be easily obtained by passive diffusion from adjacent tissues. Moreover, an investigation dealing with many variables such as allograft may be required.
Our findings suggest that a stepwise increase of warm ischemia time, of more than 24 hours, significantly reduces graft survival. These results support the concept that the use of the trachea from human cadavers within 18 hours after cardiac death might be practical. The tolerable warm ischemia time of the trachea was significantly longer than those of previously described donor organs. This phenomenon brings some merits to the tracheal transplantation. Even if it is impossible to procure viable solid organs from the nonheartbeating cadaver because of longer periods of warm ischemia, the trachea can be potentially used as a donor organ. When the organ procurement is started from donor patients who are pronounced dead in emergency rooms, important solid organs such as heart, lung, liver, and kidney are normally harvested first. As the trachea has enough time to tolerate warm ischemia after procurement, the tracheal graft can then be prepared well and sufficiently stored in antibiotics for a while because the airway is unclean.
In conclusion, the morphology of the epithelium and cartilage in isografts undergoing warm ischemia for less than 18 hours was better preserved, and the morphology of both deteriorated in isografts undergoing warm ischemia for more than 24 hours. The permissible period of warm ischemia before 3-month cryopreservation to maintain tracheal isograft viability in this simple system using Bicell biofreezing vessel was determined to be 18 hours in rats. Within 18 hours after cardiac death, therefore, cadavers potentially qualify as viable candidates to be donors for tracheal transplantation.
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Acknowledgments
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This study was supported by a Research Grant for Immunology, Allergy and Organ Transplant, Ministry of Health and Welfare, and a Grant-in-Aid (08671555) from the Ministry of Education, Science, and Culture of Japan. The authors thank Mrs. Miki Shimada for her skillful technical assistance.
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References
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Abstract
Introduction
