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Ann Thorac Surg 1995;60:1597-1604
© 1995 The Society of Thoracic Surgeons
Department of Thoracic and Vascular Surgery and Heart-Lung Transplantation, Hôpital Marie-Lannelongue, Le Plessis Robinson, Paris-Sud University, Paris, France
Accepted for publication August 8, 1995.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. Donor grafts were flushed with Euro-Collins solution (65 mL/kg at 4°C) by simultaneous inferior thyroid artery and bronchial artery perfusion through a 15-cm aortic segment and heterotopically implanted on their own vascular pedicle after 3 (group 1), 6 (group 2), 15 (group 3), and 24 (group 4) hours of static storage in Euro-Collins solution at 4°C (n = 5 each). The animals were observed for 4 weeks after transplantation and then sacrificed. Histologic evaluation of the tracheal allografts was routinely done using specimens from open biopsies.
Results. The overall length of tracheal grafts was 12.4 ± 0.6 cm, and this variable was not significantly different between the four groups. Graft exocrine (mucous secretion) function began 1.3 ± 0.5 days after transplantation in groups 1 through 3 but was absent in all group 4 grafts (p < 0.0001). All grafts in groups 1 through 3 were viable at the time of sacrifice and showed little discernible intergroup and intragroup histologic and functional (tracheal smooth muscle contraction and relaxation) variations except for a significantly higher (p < 0.001) incidence of rejection in group 3 allografts. In contrast, all grafts in group 4 became completely necrotic 4 days after transplantation (p < 0.001) despite full patency of all the vascular anastomoses.
Conclusions. These results demonstrate that tracheal allografts may be safely preserved for as long as 15 hours and that longer periods of ischemia are likely to result in irreversible allograft damage.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Previous work by our laboratory [1, 2] has shown that perfusion of an inferior thyroid artery (ITA) followed by static cold Euro-Collins solution (ECS) storage minimizes ischemia-reperfusion injury after tracheal transplantation and that the most discriminating indicators of early and late viability of the graft are the morphology and the function of the respiratory epithelium and smooth muscle, respectively. In those studies, reimplantation was accomplished after 3 to 4 hours of ECS preservation without knowledge of the tolerable limit of ischemia of the allografts. In this study, we investigated the ischemic tolerance of tracheal allografts in a heterotopic, immunosuppressed piglet model.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Animals were premedicated with intramuscular administration of ketamine hydrochloride (25 mg/kg) and atropine sulfate (1 mg/kg) and anesthetized with intravenous sodium pentobarbital (25 mg/kg). After orotracheal intubation, anesthesia was maintained with inhaled halothane, and ventilation with an equal gas mixture of oxygen and protoxide was provided by a Labaz ventilator (Pau, France). Jugular and femoral venous catheters were placed for infusion of crystalloid solutions; adequacy of ventilation and oxygenation was assessed by arterial blood gas analysis and pulse oximetry.
Donor Operation
The techniques for harvesting and implanting long-segment tracheal grafts have been described previously [1, 2]. Briefly, after completion of a median cervicosternotomy, the subclavian, jugular, vertebral, first intercostal, and mammary veins were isolated and ligated distally on both sides, as were the right and left azygos veins at their confluence with the anterior vena cava (AVC); the AVC and posterior vena cava were then isolated and encircled. The first intercostal, vertebral, and mammary arteries were isolated and divided between ligatures, and the axillary and carotid arteries on both sides were isolated and encircled. The ascending aorta and the pulmonary artery trunk were separated from one another.
After systemic heparinization (5 mg/kg), a 4-0 polypropylene (Prolene; Ethicon, Somerville, NJ) pursestring suture was placed on the anterior surface of the ascending aorta, and an aortic root cannula (DLP, Inc, Grand Rapids, MI) was inserted for infusion of ECS for tracheoplegia. After cannulation, the thoracic aorta was isolated 10 cm distal to the origin of the left subclavian artery to obtain a closed aortic segment incorporating the brachiocephalic and left subclavian arteries (branching the ITAs) and origin of the bronchial artery or arteries.
The subsequent steps included: (1) inflow occlusion (clamping of the AVC and ligation of the posterior vena cava); (2) cross-clamping of the aorta before the origin of the brachiocephalic artery; (3) administration of a high-potassium chloride solution into the aortic root; (4) division between ligatures of all previously isolated cervical arteries; (5) cross-clamping of the previously isolated distal thoracic aorta; (6) perfusion through the closed aortic segment of the thyrotracheal block with ECS (total volume, 65 ml/kg at 4°C at a perfusion pressure of 80 mm Hg); and (7) transection of the AVC at its confluence with the right atrium and amputation of the tip of the left atrial appendage. After completion of perfusion, the thyrotracheal grafts were harvested en bloc and placed in two sterile plastic bags containing cold (4°C) ECS.
Recipient Operation
Recipient piglets were sedated and anesthetized as described for the donors. After a median vertical cervicotomy, the right cervical region was exposed, and the right carotid artery and the brachiocephalic vein were dissected. After systemic heparinization (5 mg/kg), the recipient right carotid artery was cross-clamped with small bulldog clamps at its origin and distally before its entrance into the cranium and divided, the donor subclavian artery (furnishing the predominant ITA) was then interposed between the proximal and distal recipient right carotid stumps by an end-to-end anastomosis using 6-0 polypropylene (Prolene) continuous suture. After intravenous injection of 240 mg of methylprednisolone acetate, the clamps were released, allowing reperfusion of the graft.
The recipient right brachiocephalic vein confluence was clamped laterally and anastomosed end-to-side to the donor AVC with a 7-0 Prolene suture. During implantation, allograft hypothermia was maintained by topical application of cold saline solution. A modification of the original technique was to leave the entire esophageal muscular wall that had been denuded of its mucosa attached to the tracheal allografts; to this end, the esophageal mucosa was stripped away as close as possible to and over the entire length of the esophageal muscular wall. The proximal and distal orifices of the tracheal graft were then anastomosed to the surrounding skin with 2-0 polyglactin (Vicryl, Ethicon) interrupted sutures. Through these two tracheostomies, the tracheal segments were constantly exposed to aerial contamination.
A central venous catheter was placed through the left external jugular vein, and the cervical incision was closed with a two-layer 2-0 Vicryl suture after single-tube drainage of the cervical region.
Posttransplantation Immunosuppression
Animals received an intravenous antibiotic (cephalothin sodium, 500 mg/day), oral acetylsalicylic acid (100 mg/day) and prednisolone (40 mg, days 1 and 3 postoperatively), and low-molecular weight heparin sodium (0.2 mL subcutaneously) for the first 10 postoperative days. Immunosuppression included oral cyclosporin A (10 to 15 mg/kg per day) to maintain the trough plasma levels between 200 and 250 ng/mL and oral azathioprine (2.5 mg/kg per day) [1, 2]. Rejections were intentionally not treated. The first dose of cyclosporin A was given intramuscularly (5 mg/kg) 3 hours before induction of anesthesia. Animals were placed in cages and given standard laboratory pig food and water ad libitum.
Posttransplantation Monitoring
In each group, a tracheal and ITA ring was obtained after cold storage and 2 hours of blood flow recirculation. Exocrine allograft function was assessed by determining the presence of mucous secretion. Fiberoptic examinations and tracheal biopsies were routinely performed after transplantation. Animals were sacrified using intravenous 26% pentobarbital (0.5 mL/kg) at the onset of graft failure or at the set time of 30 days after transplantation. Grafts were removed for final histologic study and assessment of tracheal smooth muscle function. Vascular patency was evaluated by means of premortem angiography.
Histopathologic Studies
Biopsy and postmortem specimens were immediately fixed in Bouin's solution and in 10% buffered formalin, respectively. After the specimens were embedded in paraffin, 5-µm thick sections were stained with hematoxylin and eosin and assessed histologically in a blind fashion. The status of the thyroid gland was evaluated using the presence or absence of follicular atrophy as a sign of ischemia and lymphoid infiltrates as a sign of rejection.
Nonspecific tracheal epithelial lesions were defined as alterations in differentiation, presence of squamous metaplasia, ulceration, and necrosis, or both. These lesions were ranked according to (1) the absence of inflammation (ischemia) or (2) the presence of lymphocytic infiltrates in the lamina propria (rejection) or (3) the presence of polynuclear cell infiltrates in the lamina propria, mucopurulent or polyleukocyte debris, or germs (primary or secondary infection).
The histologic condition of the tracheal allograft walls was scored according to the following arbitrary semiquantitative scale: 0 = normal wall; 1 = isolated lesions of the epithelium; 2 = ischemic necrosis with or without hemorrhage of the lamina propria; 3 = ischemic necrosis of the submucosa; and 4 = ischemic necrosis of the cartilage [1]. The following scoring system for grading the severity of allograft rejection [2] was used.
For electron microscopic studies, specimens were fixed in 2% glutaraldehyde, postfixed in 2% osmic acid, and embedded in Epon. Sections were stained with uranyl acetate and lead citrate and examined under a Philips EM301 electron microscope.
Tracheal Smooth Muscle Studies
Tracheal rings from transplanted and native (nontransplanted) tracheas were obtained at the time of sacrifice. Smooth muscle was gently cleaned from all other tracheal layers and maintained between an isometric force transducer and a fixed wire support in a 30-mL isolated organ chamber containing Krebs solution (NaCl, 118; KCL, 4.7; CaCl2, 1.5; NaHCO3, 25; MgSO4, 1.1; KH2PO4, 1.2; and glucose, 5.6 in mmol/L) at 37°C. During a 90-minute equilibration period, resting force was kept at 1.5 g. At equilibration, a cumulative concentration-response curve to carbachol (10-8 to 10-4 mol/L final concentration) was produced. Isoproterenol hydrochloride was then added cumulatively (10-9 to 1.5 x 10-5 mol/L) to promote muscle relaxation. Changes in force were measured from isometric recordings and expressed in grams. The maximal response to carbachol or isoproterenol and the concentration of agonist yielding 50% of maximal response were interpolated from the individual concentration-effect curves. Relaxation was expressed as the percent decrease in tension of carbachol-elicited constriction.
Statistical Analysis
Data are expressed as the mean ± the standard deviation or the standard error of a given number of observations. Ischemic and contraction-relaxation data were analyzed by one-way analysis of variance (repeated measures) with Fisher's protected least significant difference, Scheffé's F test, and Bonferroni's or Dunnett's t test for multiple comparisons. Data were analyzed using a software package (STATISTICA, StatSoft, Paris, France). The a priori level of significance was set at a p value of less than 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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). In all 20 procedures, the right subclavian artery incorporated the origin of the predominant ITA and was anastomosed to the recipient right carotid artery. All grafts in each group were histologically normal after cold ECS storage and 2 hours of blood flow restoration (Fig 1); mononucleated cells infiltrating the trachea and ITA were observed in a graft reimplanted after 15 hours. All animals survived the operation and were in good condition during the entire study period. All vascular anastomoses were patent before the animals were sacrificed.
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details the histologic status of the allografts after transplantation. All grafts in groups 1, 2, and 3 were histologically viable 30 days after transplantation (Fig 2). There was little discernible intergroup or intragroup variation in incidence and severity of ischemic lesions; none of them showed chondrolysis or cartilage resorption. Conversely, all layers of the tracheal grafts in group 4 became completely necrotic 4 days after reimplantation (p = 0.001) (Fig 3).
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) and were significantly lower (p = 0.01) than those observed in the preservation-matched native tracheas (Fig 4B). In contrast, the relaxation capacity of grafted tracheal smooth muscles was not affected by the length of the preservation time (Fig 5A) and was similar (p = not significant) to that of native tracheas (Fig 5B).
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). Overall, the incidence of rejections was higher (p = 0.157) in grafts with (6/21 biopsy specimens, 29%) than in those without (10/63 specimens, 16%) ischemic lesions. None of the thyroid glands showed rejection at the time of sacrifice, nor did the grafted thyroid glands.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Our allotransplantation model investigates the maximal ischemic tolerance of directly revascularized tracheal allografts. By permitting a realistic assessment of allograft morphology, function, and survival after reimplantation, reflecting closely what the clinical situation would be [1], the present experience provides evidence that tracheal allografts might be safely preserved after simultaneous ITA and bronchial artery perfusion and 15-hour storage in ECS at 4°C and that longer periods of cold ischemia are likely to result in irreversible deterioration of the tracheal allografts. These findings suggest that the metabolic requirements of the trachea during storage differ from those of the lung, which must be reimplanted within 6 hours of harvest using current preservation techniques. If so, preservation of the trachea, unlike that of the lung, might be as successful as that of other intraabdominal organs [4, 5], and this may reflect the absence in the trachea of the delicate alveolar-capillary membrane network.
All grafts were histologically normal after cold storage and 2 hours of blood flow recirculation, a finding indicating that any graft deterioration occurs primarily after this reperfusion period. These data may imply that a 2-hour reperfusion injury model is inadequate for screening tracheal preservation solutions other than ECS. There is experimental and clinical evidence that extracellular-type perfusates, ie, low-potassium-dextran and Wallwork solutions, may be superior to intracellular-type perfusates, ie, modified ECS and University of Wisconsin solution, for lung preservation [4, 5]. However, we selected ECS because it is still the most used solution in clinical lung transplantation [5], we wanted no bias toward a particular solution, and it is the least expensive of the commercially available perfusates.
The harvesting and preservation techniques used should be regarded as key factors for the results obtained. The trachea has a fragile microvascular anatomy and a complex physiologic role, thus rendering it particularly susceptible to ischemic, immunologic, and mechanical injuries. Its blood supply in humans [7, 8] and in piglets [1], is provided by the right ITA, a branch of the subclavian artery, and the venous blood enters the cervical descending veins and the pulmonary circulation through the AVC. This vascular network supplies all layers (cartilaginous rings to airway epithelium) of the cervical and proximal thoracic trachea [1], whereas the distal thoracic trachea is vascularized by the bronchial arteries originating from the aortic arch [9].
Our working hypothesis was that adequate diffusion of the preservation solution throughout the thyrotracheal microcirculation could be provided only by a simultaneous dual ITA and bronchial artery perfusion. In effect, after the clamping of the ascending and distal thoracic aorta beyond the origin of the bronchial artery or arteries and ligation of all distal cervical vessels, the perfusate enters the closed aortic segment incorporating the origin of the innominate and left subclavian arteries and circulates selectively through the right and left carotid and subclavian arteries, ITAs, thyroid gland, right anterolateral surface of the cervicothoracic trachea up to the carina, esophageal arteries vascularizing the posterior tracheal wall and esophagus, and left anterolateral surface of the tracheal allograft and finally returns through the descending cervical veins into the AVC. This dual flush technique has an obvious advantage over the single ITA perfusion employed before [2] in that it ensures a physiologic flow of the perfusate to the microcirculation of the cervical and distal thoracic trachea, thus allowing longer (up to 13 cm) segments of tracheal allografts to be successfully preserved and allotransplanted. These findings mirror those of LoCicero and colleagues [10], who demonstrated that simultaneous pulmonary and bronchial artery perfusion is superior to pulmonary or bronchial artery perfusion alone in preserving the lungs for extended storage before transplantation.
In this study, the trachea was transplanted along with the muscular wall of the esophagus denuded of its mucosa. In our previous experience [2], the blood supply to the posterior tracheal wall was better preserved by performing a combined tracheoesophageal rather than a single tracheal transplantation. However, the question arose whether the hazards of adding two esophageal anastomoses to the tracheal transplantation could be obviated by simply transplanting the trachea and esophageal muscular wall denuded of its submucosa. This question was addressed here. The absence of ischemic lesions on the posterior tracheal wall in surviving grafts suggests that the muscular wall of the esophagus alone provides equivalent blood supply to the posterior tracheal wall as does transplantation of the entire esophagus. This situation relates to the fact that the muscular esophageal wall upregulates the vascular network of the trachea by supplying important nutrients to the posterior tracheal wall, intratracheal airway epithelium, and walls of the tracheal arteries and veins [2]. By transplanting the entire muscular esophageal wall alone, this tracheal vascular supply was maintained; this evidence has clinical relevance.
Another advantage of using the same preservation solution and technique throughout the study was that the only variable influencing graft outcome was the length of the ischemic interval before implantation. It is well known that periods of ischemia are followed by reperfusion injuries once blood flow is restored. These injuries are linked to a complex cascade resulting in the generation of oxygen free radicals and an excessive immune response, and can do as much or more damage as ischemia. Others events, such as surgical manipulation, denervation, and lymphatic interruption, may also play a role but are more likely to occur after lung [11] rather than tracheal transplantation.
Although there was a trend toward a higher severity and incidence of airway ischemia as the preservation time increased, the difference was not significant between groups 1, 2, and 3, and all grafts demonstrated uniform survival after transplantation. Potentially influential factors for the higher incidence and severity of ischemic lesions observed in grafts reimplanted after 15 hours may include the prolonged ischemia per se [12] and, in part, the higher incidence of acute rejections. Tanabe [13], Takao [14], and their associates, using a canine single-lung allotransplantation model, clearly demonstrated that bronchial blood flow at the graft bronchi is much lower in animals with acute rejection than in animals with no rejection. We might indirectly speculate that a similar relationship may have reduced the perfusion of the tracheal respiratory epithelium during rejection. However, the association between airway ischemia and rejection was not constant in our experience, and this suggests that other alloantigen-independent stimuli may have triggered the genesis of the ischemic lesions.
All ischemic lesions in group 3 (15-hour storage) were reversible once epithelial generation occurred. Conversely, the degree of ischemia was very marked in the early postoperative period in almost all grafts reimplanted after 24-hour ECS storage (group 4) and led, despite the viability of the arterial and venous blood supply, to irreversible ischemic necrosis of all thyrotracheal grafts 4 days after transplantation. This primary graft failure is probably not due to an excessive alloimmune response, as graft rejection was observed only in the 2 piglets whose tracheal wall was histologically intact 2 days after transplantation. The remaining allografts experienced no rejection, and a plausible explanation might be that the complete graft ischemia destroyed the expression of the major histocompatibility complex class II antigens, the trigger cells of alloimmune response, on the tracheal mucosa and submucosa [15, 16]. More likely, this primary graft failure can be attributed to an excessive ischemia-reperfusion injury. Its pathophysiologic mechanism remains obscure at present, but we postulate that in the absence of endothelial injury at the level of the ITA, it might have been triggered by an irreversible deterioration of the tracheal epithelium. This means that the permissible limit of ischemia for safe preservation of tracheal allografts is somewhere between 15 and 24 hours; that the trachea, like other thoracic organs [11], needs to be preserved at a higher level of function than other intraabdominal organs; and that it must function immediately at nearly maximal capacity.
Studies of porcine tracheal smooth muscle have been limited. The only report known to us is that of Munoz and coauthors [17], who provided evidence that although physiologic innervation to the cervical trachea always is bilateral, substantial smooth muscle contraction might be provided by unilateral electric stimulation of either vagus nerve. Our results show that muscle contractions declined as preservation time lengthened, and this may be the result of both the increased incidence of tracheal epithelial ischemia and the rejection episodes, factors that have been reported to modify the tone of the underlying smooth muscle in dogs [18].
Another finding was the reduced contraction of the grafted as opposed to the native smooth muscles. This could be related to the vagal denervation, and the question arises whether some form of neuronal anastomosis might improve the contractile response of allograft smooth muscle. Previous experience [19] in laryngeal transplantation has demonstrated that direct anastomosis of the recurrent laryngeal nerves or separate neurorrhaphy often results in neuroma formation and misdirection of regenerating fibers and offers little, if any, benefit; moreover, the trachea differs from the larynx in that a selective neuromuscular anastomosis is not available. However, this and the fact that the smooth muscle contractibility of the allografts, although reduced, was still higher than its physiologic threshold, suggest that tracheal allografts do not require direct vagal reinnervation from the recipient.
In conclusion, our results indicate that donor graft flushing with ECS by simultaneous ITA and bronchial artery perfusion safely preserves long segments of tracheal allografts for as long as 15 hours and that longer periods of ischemia are likely to result in irreversible allograft damage.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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This work was supported by grants from the CRAMIF and Foundation de l'Avenir.
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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