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J Thorac Cardiovasc Surg 1994;108:1066-1075
© 1994 Mosby, Inc.
CARDIAC AND PULMONARY REPLACEMENT |
Le Plessis Robinson, France
Supported by grants from the CRAMIF and Fondation de l'Avenir.
Received for publication April 27, 1994. Accepted for publication July 12, 1994. Address for reprints: Paolo Macchiarini, MD, Department of Thoracic and Vascular Surgery and Heart-Lung Transplantation, Hôpital Marie-Lannelongue (Paris-Sud University), 133, Avenue de la Resistance, 92350 Le Plessis Robinson, France.
Abstract
A macrosurgical technique of thyrotracheal harvesting and direct revascularization with and without venous drainage in a heterotopic thyrotracheal and immunosuppressed allograft in the pig model is described. Harvesting included en bloc cervicothoracic exenteration of the aortic arch and its supraortic trunks, anterior vena cava, jugular veins, subclavian vessels, thyroid gland, cervicothoracic trachea, and esophagus. This technique conserves the tracheal arterial supply provided by either the right or left subclavian artery, directly or indirectly via the inferior thyroid artery, and venous return provided by the anterior vena cava, directly or indirectly via the descending cervical vein. In recipients, implantation included (1) arterial end-to-end anastomoses of the proximal and postscalenic stumps of donor's subclavian artery to the proximal and prescalenic stumps of recipient's subclavian artery; (2) end-to-side venous anastomosis of the donor's anterior vena cava to the recipient's brachiocephalic venous trunk; and (3) heterotopic implantation of the proximal and distal orifices of the grafted trachea into the neck. Ten adult Large White pigs underwent direct revascularization of a thyrotracheal allograft with (n = 6, group 1) and without (n = 4, group 2) venous drainage. All grafts of group 2 exhibited a venous infarction, extensive inferior thyroid artery thrombosis, and ischemic and suppurative thyrotracheal necrosis 1 to 2 days after transplantation. In group 1, the length of the grafted trachea and number of rings were 9.75 ± 1.5 cm and 22.1 ± 3.3, respectively; ischemic time was 236.3 ± 338.3 minutes. Group 1 pigs were put to death 4 (n = 4) and 3 (n = 2) weeks after transplantation. All tracheal grafts had histologically normal airway epithelium; isolated areas of necrotic ischemia of the chorion and submucosa lasted for the first 7 days after transplantation but disappeared after epithelial regeneration. Premortem angiograms showed that all vascular anastomoses were patent. Grafts were histologically normal at postmortem examinations and all but one had no rejection. This large animal model demonstrates that long tracheal allografts might be transplanted by means of this direct revascularization and venous drainage technique. (J THORAC CARDIOVASC SURG 1994;108:1066-75)
Unlike other major organs, the trachea has not been widely allotransplanted in human beings. 
1 The vascular pedicle of the upper trachea, derived in mammals 2,3 and human beings 4,5 from a delicate blood supply of the related thyroid gland, cannot reasonably be obtained to provide revascularization of the graft by direct microvascular suture. 6 Not surprisingly, the vast majority of experimental efforts have attempted to replace the trachea with prosthetic, 7-11 autogenous, 12-16 fresh and pretreated, 17-20 or indirectly revascularized 21-24 allogeneic grafts. Despite four decades of intensive search, results have been dismal because of ischemia of the unvascularized tracheal grafts leading to injury to airway epithelium, necrosis caused by bacterial invasion, and inspiratory collapse.
Similarly, airway ischemic necrosis remains a serious obstacle to successful lung transplantation. Several solutions have been proposed. One of them has focused on direct bronchial artery revascularization at the time of transplantation. Experimental 25 andclinical 26,27 studies have both demonstrated that this technique increases airway perfusion and reduces the occurrence of bronchial ischemia and necrosis. On the basis of these observations, we developed a technique for direct revascularization and venous drainage of a thyrotracheal heterotopic allograft in the pig, and the preliminary technical feasibility, histologic data, and immunologic data are presented.
MATERIALS AND METHODS
This study was conducted in 30 young Large White pigs (15 to 26 kg) provided by Lebeau Inc., Gambais, France. Twenty were assigned to the anatomic (n = 15) and hemodynamic (n = 5) evaluation of the tracheal blood supply and 10 to the development of the operative technique of heterotopic thyrotracheal transplantation. All received care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" formulated by the National Academy of Sciences and published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).
Experimental protocol.
Animals were premedicated with intramuscular ketamine hydrochloride (25 mg/kg) and anesthetized with intravenous sodium pentobarbital (25 mg/kg). After orotracheal intubation, anesthesia was maintained with inhaled halothane and the animals' lungs were ventilated (Laboz Inc. ventilator, Pau, France) with an equal gas mixture of oxygen and protoxide at a tidal volume of 250 ml and a rate of 20 breaths/min. Peripheral vascular access was accomplished by placing 16- or 18-gauge angiocatheters into an ear vein and either cephalic vein. Adequacy of ventilation and oxygenation was assessed by arterial blood gas analysis and a pulse oximeter.
Donor organ harvest and preparation.
Under sterile techniques, the median and lateral aspects of the cervicothoracic junction were exposed via a median vertical cervicotomy and total sternotomy, working from cranial to caudal. The superior and inferior aspects of the platysma muscle, both cervical thymus glands with the underlying omohyoid muscles, and the sternohyoid and sternothyroid muscles were excised bilaterally. The brachiocephalic venous confluence was exposed after removal of the inferior cervical lymph nodes and division of the triangular retrosternal ligament. After excision of the mediastinal thymus gland, the anterior pericardium was opened, the anterior and posterior venae cavae were isolated, and the aortocaval and aortopulmonary sinuses were dissected. The right and left azygos veins were dissected and ligated at their confluence into the anterior vena cava after both pleural cavities were opened. The lateral cervicothoracic junction was then approached. The internal mammary artery and vein and the first intercostal artery and vein were dissected and ligated. The external and internal jugular veins were isolated at their origin and encircled. The vertebral artery and vein were ligated. The subclavian vein was dissected and encircled at its axillary origin. The postscalenic portion of the subclavian artery and the preintracranial portion of the common carotid artery were isolated and encircled. This part of the operation was done first on the right and then on the left side. Dissection was done carefully to preserve the entire thyrolaryngotracheal blood supply.
After intravenous heparin (3 mg/kg), the anterior and posterior venae cavae were ligated, the aortic arch was crossclamped before the origin of the right brachiocephalic artery, and the heart was arrested with a high potassium chloride solution (20 mEq) injected into the right appendage. All previously isolated cervical vessels were then ligated and divided between ligatures, and cold (4° C) saline solution was applied for topical cooling. The anterior vena cava was transected at the level of its confluence into the right atrium, and the aortic arch was divided proximally at the level of the ascending aorta and distally after the ostium of the left subclavian artery. Thereafter, the endotracheal tube was withdrawn and an en bloc cervicomediastinal exenteration was made. This included caudally the previously divided aortic arch with its supraortic branches, the esophagus, and the distal trachea. These last two organs were transected one cartilaginous ring below the origin of the right upper bronchus and separated from the prevertebral ligamentum with sharp dissection and cautery up to the posterior arch of the first ribs. Cranially, excision is continued laterally on the right side by an "out-in" side division of the clavicular attachment of the inferoanterior scalenus, superior humoral-mastoid, rectus anterior, and longus colli prevertebral muscles. Arteries and venules emerging from these muscular structures were ligated with fine sutures. Following the lateral aspect of the longus collis muscle, the esophagus, trachea, supraortic branches, and anterior vena cava were separated from the posterior mediastinal fatty tissue immediately anterior to the cervical and dorsal vertebrae with the use of an electrocautery device. The same procedure was then made on the left side. Extraction was completed by dividing the laryngopharyngeal junction at the level of the thyrohyoid membrane.
Anatomic evaluation of the tracheal vascular supply.
Ex situ, continuous, pressure-controlled perfusion of the graft with cineangiographic Hartmann's solution, methylene blue, barium sulfate, or a combination, was done by retrogradely injecting cold (4° C) Hartmann's solution into the postscalenic subclavian and common carotid arteries after crossclamping of their origins. All interrupted vessels were identified and ligated. Particular care was paid to observe which artery vascularized the thyroid gland and trachea and was associated with a venous return into the anterior vena cava. This procedure was done first on the right and then on the left side. After identification of the predominant blood supply of the graft, those vessels providing no blood supply to the graft, for example, ipsilateral or contralateral common carotid or contralateral subclavian arteries, were ligated but not excised.
Regional blood flow and hemodynamic measurements.
Regional flow through the distal segment of the subclavian and inferior thyroid arteries was determined in vivo with a Statham SP2202 electromagnetic flowmeter (Viggo-Spectramed Inc., Critical Care Div., Oxnard, Calif.). The systemic blood pressure was recorded by inserting a catheter into the left carotid artery. The functional status of the tracheal vasculature was assessed ex vivo by the pressure-flow curve and analyzed with a microcomputer driving a digital-to-analog converter.
Heterotopic thyrotracheal transplantation.
Donors and recipients were matched for ABO and weight and underwent, in pairs, heterotopic thyrotracheal transplantation with a revascularized allograft. Grafts were harvested with the previously described technique and preserved in cold (4° C) Hartmann's solution until implantation. After ex situ evaluation of the blood supply of the graft, the larynx was divided from the cervical trachea at the level of the second ring below the cricoid cartilage; the first ring above the right upper lobe takeoff was the lowermost resected. The esophagus was then peeled off over its entire length as close as possible to its muscular layer.
Recipients were sedated with ketamine (10 mg/kg intramuscularly) and anesthetized and monitored in the manner described earlier. After a vertical cervical incision and an upper third sternotomy, the cervical thymus glands with the underlying sternohyoid and sternothyroid muscles were excised bilaterally. The confluence of brachiocephalic veins and origin of the anterior vena cava were exposed after division of the triangular retrosternal ligament and excision of the mediastinal thymus gland and inferior cervical nodes. Care was paid, at this stage of the operation, to avoid opening the pleural cavities.
Thereafter, the subclavian artery was dissected first at its origin behind the anterior vena cava and then distally at its prescalenic portion, just before the origin of the internal mammary artery. During this portion of the operation, great care was taken to identify and avoid injury to the ipsilateral phrenic and vagus nerves lying in the anterior aspect of the subclavian artery. All prescalenic branches of the recipient's subclavian artery, including the internal mammary, first intercostal, and vertebral arteries, were divided between ligatures. After intravenous heparinization (8 mg/kg), the recipient's subclavian artery was crossclamped proximally at its origin and distally before the origin of the recipient's inferior thyroid artery with small bulldog clamps, and it was then divided. The proximal and distal lumina of the donor's subclavian artery were then anastomosed with the recipient's stumps by an end-to-end 6-0 polypropylene continuous suture (Prolene, Ethicon, Inc., Somerville, N.J.). The clamps on the recipient's subclavian artery were released, allowing reperfusion of the graft, after injection of 240 mg of intravenous methylprednisolone acetate. A strong flow signal by macroscopy, palpation, and/or Doppler examinations was necessary to establish the perfect revascularization of the graft and the venous return from the anterior vena cava. Thereafter, the recipient's brachiocephalic venous confluence was crossclamped with a vascular clamp and anastomosed to the donor's anterior vena cava by an end-to-side 7-0 Prolene suture. In grafts receiving no venous anastomosis, the stump of the anterior vena cava was closed with a continuous 6-0 Prolene suture. During implantation, allograft hypothermia was further maintained by topical application of cold saline solution.
Depending on the site and geometry of the vascular sutures, the proximal and distal orifices of the donor's trachea were then anastomosed to the skin in either a transverse or perpendicular position by 2-0 polyglactin interrupted sutures (Vicryl, Ethicon). A central venous catheter was placed through either external jugular vein. The sternotomy was closed with interrupted 1-0 Vicryl sutures and the cervical incision with a two-layer 2-0 Vicryl suture after single drainage of the cervicomediastinal region.
Postoperative course and immunosuppression.
Animals received intravenous antibiotics (cephalothin, 500 mg/daily), oral acetylsalicylic acid (100 mg/daily), prednisolone (40 mg, days 1 and 3 after the operation), and low-molecular-weight heparin (0.2 ml subcutaneously) for the first 10 postoperative days. They were immunosuppressed with intramuscular cyclosporine (5 to 10 mg/kg per day) to maintain plasma concentrations of 250 to 350 µg/ml and oral azathioprine (2.5 mg/kg daily). The first dose of cyclosporine was given immediately after induction of anesthesia. Animals were placed in cages and fed standard laboratory pig food and water ad libitum. Cineangiographic studies and fiberoptic tracheal examinations with tracheal biopsies were performed weekly until the animals died or were put to death at the set time of 4 or 3 weeks after transplantation or in the presence of extensive necrosis of the grafts.
Histologic studies.
Biopsy and postmortem specimens were immediately fixed in Bouin's fixative and in 10% buffered formalin, respectively. Four tracheal and two thyroid cross sections were taken from postmorten specimens. After being embedded in paraffin, 5 µm thick sections of the trachea and thyroid gland were cut transversly, stained with hematoxylin and eosin, and assessed histologically in a blind fashion for the status of the tracheal wall and thyroid gland (presence or absence of follicular atrophy for ischemia and lymphoid infiltrates for rejection). Nonspecific tracheal epithelial lesions were defined as alterations of epithelial differentiation and/or presence of squamous metaplasia, ulceration, and necrosis and were interpreted according to (1) the absence of inflammation (ischemia); (2) presence of lymphocytic infiltrates into the chorion (rejection), and/or (3) polynuclear cell infiltrates into the chorion, mucupurulent and/or polyleukocyte debris, and/or germs (primary or secondary infection). The trophic condition of the tracheal allograft wall was assessed according to the following arbitrary semiquantitative 0 to 4 scale:
Immunocytologic and immunohistologic studies.
To investigate the phenotypic modification of lymphocyte T cell subsets in peripheral blood and tracheal specimens after transplantation, we conducted immunocytometric and immunohistologic analyses of peripheral blood lymphocytes and tracheal biopsy specimens. For immunocytometric phenotypic analysis, consecutive blood samples, taken on ethylenediaminetetraacetic acid, were obtained. The unlabeled monoclonal antibody specific for the membrane antigens expressed on T cells in pigs used in this study are presented in Table I. In brief, 100 µl blood was incubated with 5 µl of the appropriate dilution of monoclonal antibodies for 20 minutes in the dark at room temperature. Cells were washed with phosphate-buffered saline solution and incubated with the appropriate dilution of goat antimouse F(ab')2 immunoglobulin G fragment conjugated with fluorescein isothiocyanate (GAM-FITC, Immunotech Corp., Boston, Mass.). Red blood cells were lysed and peripheral blood mononuclear cells were then fixed with 1% paraformaldehyde and analyzed within 24 hours. Cells incubated with no antibody and with only the GAM-FITC in the second step were considered as controls of autofluorescence and unspecific staining, respectively. Cell phenotype was analyzed with FACScan and Lysis 2 software (Becton Dickinson & Co., Mountain View, Calif.). Results were expressed as the percentage of positively stained lymphocytes. For immunohistologic analysis of lymphocytes infiltrating tracheal biopsy specimens, the same monoclonal antibodies were used. In brief, paraffin-embedded biopsy specimens were deparaffinated and, after rehydration, slides were incubated, first with the specific monoclonal antibodies (30 minutes at room temperature in a humid chamber) and then with FITC-conjugated GAM secondary antibody.
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RESULTS
Anatomic studies of 15 pigs showed that the blood supply of the cervical and thoracic trachea up to the right upper lobe takeoff was provided by a superior, middle, and inferior branch of the inferior thyroid artery, which originates directly or indirectly from the subclavian artery. The superior branch is the uppermost, originates close to the thyroid gland, and vascularizes the thyroid and upper third of the cervical trachea. The middle and inferior branches originate at the middle and at the beginning of the inferior thyroid artery and supply the middle third of the cervical trachea and the thoracic trachea up to the right upper lobe. These branches are interconnected along the lateral tracheal surface by longitudinal vascular anastomoses, which provide, through transverse vessels, the blood supply to the thyroid gland, anterolateral wall of the trachea, esophagus, and pretracheal tissues. The posterior tracheal wall is vascularized by small esophageal arteries. Venous blood returns by a descending cervical vein, satellite of the ipsilateral arterial supply, into the anterior vena cava (Fig. 1). In 14 specimens (93%) the inferior thyroid artery arose from the right and once from the left (7%) subclavian artery. In vivo and ex vivo studies were made on five pigs; in vivo, the average subclavian artery and inferior thyroid artery blood flows were 100 ± 37 and 10 ± 4 ml/min (Fig. 2), demonstrating that the tracheal vascular bed is a low-flow high-pressure system. The ex vivo inferior thyroid artery pressure-flow relationship was linear and the resistances of the tracheal arterial microvasculature were elevated (average, 4.46 mm Hg/ml per minute).
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Table II). Seromucosal glands immediately produced biochemically normal mucous secretions. At postmortem examinations, tracheal grafts were histologically normal in five pigs whereas the remaining had isolated lesions of the epithelium located exclusively in the narrow confines of tracheal orifices; thyroid grafts were histologically normal in all but one pig, which had isolated areas of hemorrhage (Fig. 4). None of the transplanted allografts had bacterial colonization by air-borne bacteria; reversible bacterial contamination from Escherichia coli and Enterobacter cloacae strains developed in one pig each (Table II). The mean daily dose of administered cyclosporine was 9.1 ± 3.3 mg/kg and the average residual cyclosporine level was 268.6 ± 132.8 µg/ml. Only one pig (No. 31896) had a mild episode of histologic allograft rejection. In the remaining, cyclosporine-induced immunosuppression avoided, in a dose-dependent fashion, the immunologic alloresponses by lowering the CD25+ cell population and CD4/CD8 ratio as depicted in a representative pig follow-up in Table III.
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Table IV, all pigs were put to death within 3 days after transplantation because of venous infarction and necrotic purulent ischemia of the composite grafts surrounding soft tissues. At macroscopic postmortem examinations, the venous bed and the inferior thyroid artery of all four grafts were extensively thrombosed.
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This study describes a model of heterotopic tracheal allotransplantation in pigs and reports its preliminary technical feasibility and histologic and immunosuppressive results. Its results are best assessed in a historical context. During the past four decades, an encyclopedic number of experiments have investigated how to replace extensive (
6.4 cm) 28 tracheal defects but, unfortunately, there were more failures than successes. 6 Prosthetic grafts are unable to reepithelialize the tracheal surface and, more important, their use is limited by the development of serious complications. 7-11 Autogenous grafts like free periosteal, 12 jejunal, 13 muscular, 14 esophageal, 15 and smallintestinal 16 grafts require multistaged operations, the complexity and risks of which dictate caution before clinical use. Hence our previous experience 17 and that ofothers 18-20 have clearly demonstrated that fresh or pretreated and unrevascularized tracheal allografts are unsuitable for long-segment replacement because they ultimately do not heal appropriately, become necrotic, and are the milieu of chronic infections.
The common pathogenic factor of all these failures is tracheal ischemia related to the deprivation of the graft's physiologic arterial blood supply, which so far has been considered technically impossible to harvest. 6 Given this and the initially enthusiastic reportsof Dubois, Choiniere, and Cooper 29 and Morgan and associates 30 on the revascularization of avascular bronchial autografts by the use of omentum, several authors have recently attempted to revascularize tracheal autografts indirectly with omentum 21,24 or muscle. 31 Unfortunately, results were promising at best for tracheal allografts no longer than 4 cm 24 but very discouraging for longer segments because chondrolysis and cartilage resorption could not be reversed. 21
In 1993, Khalil-Marzouk and Cooper 32 described a technique for direct revascularization without venous drainage of a composite thyrotracheal allograft in six adult beagle dogs by establishing anastomoses of the cranial thyroid arteries to the ipsilateral common carotid arteries. The results appeared promising because five of six vascularized and immunosuppressed grafts showed preservation of tracheal cartilages and surrounding soft tissues. Apart from the absence of details about the exact length and survival of the grafts and cause of deaths, for example, airway collapse, one might wonder why the authors did not observe graft failure resulting from venous infarction and arterial thrombosis. This was a common scenario in our group of grafts without venous drainage in which a venous infarction occurred similar to that observable in phlegmasia cerulea dolens. In this sense, their study mirrors that performed by Boles 33 in 1966, who successfully revascularized the larynx by using a single craniothyroid artery without venous return. Unfortunately, this last experience has not been reproduced. Furthermore, Strome and coworkers 34 definitively demonstrated that directly revascularized laryngeal allografts, even with both superior thyroid arteries without a venous drainage, ultimately thrombose at the arterial anastomosis as a result of the lack of a venous return and endothelial injury induced by a high-flow system channeled into a small superior thyroid vessel. These findings coupled with our results provide evidence that venous drainage is an essential prerequisite for successful tracheal transplantation, and they weaken the validity of the model proposed by Khalil-Marzouk and Cooper. 32
Our model uses for the first time a macrosurgical technique for direct revascularization and venous drainage of long (up to 11 cm) tracheal allografts. The pig model was selected because the vascularization of the thyroid gland and the cervical trachea and part of the upper thoracic trachea has several similarities to that of human beings: It is provided by the inferior thyroid artery, a branch of the subclavian artery; the venous return is indirectly or directly through the anterior vena cava via the descending cervical vein; and the size and topographic distribution of the inferior thyroid artery and descending cervical vein are similar to those of human beings. Moreover, after removal of both cervical thymus glands, the pig neck accommodates the graft without excessive compression, as compared with an intraabdominal or inguinal position; the graft is exposed to air contamination, thus remaining in a physiologic environment; the readily available and constant access to the heterotopically located trachea by routine use of direct or fiberoptic biopsies offers unique histologic, functional, rejection, and infection surveillance; the pig is an established model for the investigation of transplant immunology 35,36; and the pig is inexpensive.
The key success of this model was the identification of the inferior thyroid artery and descending cervical vein as the targets of thyrotracheal vascularization. The inferior thyroid artery is a low-flow (5 to 10 ml/min) high-pressure system that supplies the trachea from the first ring below the cricoid plate just to the orifice of the right upper lobe. Its presence was constant in more the 90% of the animals studied, making revascularization technically predictable. One of its major advantages is that microsurgical sutures at its origin might be avoided by directly anastomosing the respective subclavian arteries, thereby facilitating the inflow and tracheal distal perfusion of high-pressure oxygenated arterial blood ejected from the recipient's right innominate artery. Hence it also permits animals of disparate size to be used as donors and recipients. Because the loading blood returns to the recipient's anterior vena cava via a large end-to-side anastomosis, the potential consequences of increased venous outflow resistances and the recipient's venous backflow pressure on the donor's venous return, and thus the risk of venous infarction and arterial thrombosis, are avoided. Routine follow-up with angiography showed graft patency in all animals, confirming the validity of the arterial and venous anastomoses and the efficacy of our anticoagulant regimen. Although the hemostatic habitus of pigs is more thrombogenic than that of human beings, we believe that maintaining anticoagulation for more than 10 days after transplantation is unnecessary because tracheal revascularization is most critical within the first 7 days. Taken together, these observations underscore the need for preserving all peritracheal tissues and supraaortic vessels during harvesting and performing a perfect venous anastomosis, because this last might be the factor inducing arterial thrombosis.
The validity of our tracheal revascularization network is also supported by the fact that none of the long-segment tracheal grafts become necrotic. Tracheal grafts had normal airway epithelium associated with isolated areas of ischemic necrosis of the chorion and submucosa chiefly located in the posterior tracheal wall. This ischemic status, expressed mainly during the first 7 days after transplantation, disappeared thereafter because epithelial regeneration occurred. At postmortem examinations all layers of all tracheal grafts, including cartilaginous and intercartilaginous tissues, were morphologically normal. This time-course phenomenon compares favorably with the evolution of experimental in vivo regeneration of airway epithelium after nonischemic 37 and ischemic 23,38 mechanical injuries. Similar evolutions have also been observed in clinical practice with bronchial revascularization for double lung transplantation. 26,27 Cold ischemia and reperfusion each contribute to increased vascular resistance that might result in early allograft dysfunction in laboratory animal models. 39 The fact that the observed morphologic changes were isolated, transient, and reversible and that our ischemic time was approximately 4 hours might suggest that a similar vasomotor imbalance also occurred in our animals. It is not hazardous to postulate that allograft perfusion with preservation solutions might overcome these ischemic phenomena. However, the importance of the observed epithelial injury was negligible because the tracheal function was well preserved in terms of ingrowth of tracheal epithelium, mucous secretion, and bacterial invasion. Only two pigs had short-lasting bacterial contamination from Escherichia coli and Enterobacter cloacae strains. However, these are two bacteria that usually belong to the normal intestinal flora and that might have contaminated the graft by direct contact. Despite the graft's exposure to air-borne bacteria, none of them had chronic aerial infection, reinforcing the concept that only a revascularized graft with its own perfusion has all the functions of the trachea.
It has been postulated that tracheal transplants, in contrast to other human organ transplants, induce only a weak graft rejection, reflecting a weak organ-specific antigenicity. 1 However, previous experience by Khalil-Marzouk and Cooper 32 showed that only immunosuppressed and revascularized animals do not have allograft rejection. The immunosuppressive regimen in our study was similar to that applied in our clinical transplantation program, 40 but the dose of cyclosporine was adapted according to its intramuscular pharmacokinetic profile in pigs. 41 This route of administration was selected because it has several practical advantages over the intravenous or oral routes, 41 especially in the first days after transplantation. Because the pigs received daily cyclosporine doses of 5 to 10 mg/kg, none of the pigs died as a direct consequence of graft rejection. Only one pig had a histologically and immunologically confirmed episode of rejection, successfully treated with corticosteroids. In pigs, the normal CD4/CD8 cell ratio is 0.60, and CD25 + cells are usually not expressed in normal conditions but elevated during allograft rejection. 33 The phenotypical profile of peripheral lymphocyte T cells showed a steady low percentage of CD25+ cells (activated T cells presenting interleukin-2 receptors) and a CD4+/CD8+ ratio usually lower than 0.60. This provides evidence that immunoresponses were absent and inhibited in a dose-dependent fashion by cyclosporine during the entire posttransplantation period.
In conclusion, the presented pig model provides evidence that tracheal grafts measuring up to 11 cm might be allotransplanted. The fundamental prerequisite for successful application is the restoration of the original vascularization of the trachea by performing the described harvesting and implantation macrosurgical technique. The study also provides definitive evidence that the inferior tracheal artery is the target of this direct revascularization and that this model works only when the graft's venous drainage is reestablished. The histologic and immunologic data demonstrate that only directly revascularized grafts maintain their own tracheal functions and that the trachea, like other organs, has a specific antigenicity controllable with immunosuppression. Although the presented harvesting technique was successfully reproduced in cadaveric studies (unpublished data), further questions need to be answered before its application in patients with extensive tracheal defects not manageable with the currently available treatment options.
Acknowledgments
We wish to express our gratitude to Dr. B. Charley (INRA, Jouy-en-Josas, France) for providing the unlabeled monoclonal antibodies and to Laboratoire Sandoz, Inc. (Paris) for providing cyclosporine. The excellent technical contributions of Chantal Verriest, Michele Gaillard, and Gerard Allain are also acknowledged.
Footnotes
From the Departments of Thoracic and Vascular Surgery and Heart-Lung Transplantation a and Surgical Research Laboratory, b Hôpital Marie-Lannelongue, Le Plessis Robinson, Paris-Sud University, and Technology University of Compiègne, c France. 
References
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P. Macchiarini, E. Dulmet, V. d. Montpreville, G.-M. Mazmanian, A. Chapelier, and P. Dartevelle TRACHEAL GROWTH AFTER SLIDE TRACHEOPLASTY J. Thorac. Cardiovasc. Surg., March 1, 1997; 113(3): 558 - 566. [Abstract] [Full Text]
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H. Yokomise, K. Inui, H. Wada, M. Ueda, S. Hitomi, and H. Itoh SPLIT TRANSPLANTATION OF THE TRACHEA: A NEW OPERATIVE PROCEDURE FOR EXTENDED TRACHEAL RESECTION J. Thorac. Cardiovasc. Surg., August 1, 1996; 112(2): 314 - 318. [Abstract] [Full Text]
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P. Macchiarini, G.-M. Mazmanian, V. T. de Montpreville, E. M. Dulmet, A. R. Chapelier, P. G. Dartevelle, and Paris-Sud University Lung Transplantation Group Maximal preservation time of tracheal allografts Ann. Thorac. Surg., December 1, 1995; 60(6): 1597 - 1604. [Abstract] [PDF]
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P. Macchiarini, G.-M. Mazmanian, V. de Montpreville, E. Dulmet, M. Fattal, B. Lenot, A. Chapelier, P. Dartevelle, and f. t. P.-S. U. L. T. Group EXPERIMENTAL TRACHEAL AND TRACHEOESOPHAGEAL ALLOTRANSPLANTATION J. Thorac. Cardiovasc. Surg., October 1, 1995; 110(4): 1037 - 1046. [Abstract] [Full Text]
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