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Ann Thorac Surg 1995;60:635-639
© 1995 The Society of Thoracic Surgeons
Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
Accepted for publication April 13, 1995.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. We assessed the minimal dose of cyclosporin A for viable tracheal allografts in 50 dogs. Each tracheal transplant, consisting of a six-ring segment of cervical trachea, was harvested and heterotopically implanted into the omentum. In group I (n = 10), transplantation into each dog's own omentum was performed as a control. The remaining 40 tracheal segments were randomly assigned to four recipient groups receiving either no treatment (group II, n = 10), 10 mg • kg-1 • day-1 of cyclosporin A (group III, n = 10), 15 mg • kg-1 • day-1 of cyclosporin A (group IV, n = 10), or 20 mg • kg-1 • day-1 of cyclosporin A (group V, n = 10). After 10 or 28 days, the tracheal segments were evaluated histologically.
Results. Epithelial regeneration in group IV was significantly better than that in groups I, II, or III on posttransplantation day 10. Only group IV showed no difference in epithelial viability from group I on posttransplantation day 28. In terms of vascularity, groups IV and V exhibited no differences from group I as evidenced by vascular endothelial morphology.
Conclusions. We conclude that an appropriate dose of 15 mg • kg-1 • day-1 of cyclosporin A may be used to maintain tracheal allograft viability.
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Introduction |
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The use of cyclosporin A (CsA) for immunosuppression has represented a great advance in various organ transplantations. The immunosuppressive effect of CsA was greater than that of any previously used agents. However, CsA often caused side effects such as nephrotoxicity, hypertension, and infectious complications. Therefore, it is desirable to minimize the dose of CsA in using this agent.
In this study, we examined the effect of CsA on the epithelial viability and vascularity of heterotopic tracheal allografts in mongrel dogs. Our objective was to determine the minimal dose of CsA for maintenance of tracheal allograft viability. This would help to minimize immunosuppression and to reduce the side effects.
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Material and Methods |
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). 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 (NIH publication 85-23, revised 1985).
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Recipient Procedure
Fifty adult mongrel dogs weighing from 8.5 to 17.5 kg were premedicated with an intramuscular injection of ketamine hydrochloride (20 mg/kg). The animals were placed in the supine position and anesthetized with an intravenous injection of pentobarbital sodium (10 mg/kg) and pancuronium bromide (0.1 mg/kg). Ventilation was maintained via an endotracheal tube using a pressure-limiting respirator. 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 enveloped completely. The omentum and the enclosed transplant were then returned to the peritoneal cavity, and the wound was closed. After 10 or 28 days, a second laparotomy was performed in all groups to retrieve the transplants for histopathologic study.
Drug Administration
Cyclosporin A (Sandoz Pharma AG, Basle, Switzerland) was administered orally on a daily basis beginning on the operative day (see Table 1
). All animals received antibiotics for the first 5 postoperative days.
Histologic Assessment
All tissues were fixed in 10% formalin. Microscopic slides were made from longitudinal sections of the trachea and adherent omentum and 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 counting the number of vessels. Assessment was performed in a blinded fashion.
EPITHELIAL REGENERATION.
Epithelial regeneration was evaluated according to the following grading system: 0 = no epithelium, 1 = single layer of nonciliated epithelium, 2 = multilayer nonciliated epithelium, and 3 = normal mucociliary epithelium [9]. The epithelium of the grafts was assessed as a ratio of the epithelial regeneration score on a microscopic slide.
VESSEL NUMBER.
The number of vessels in the submucosa of each transplant was carefully counted in each high-powered field on a microscopic slide. We examined the longitudinal section of the transplants to include both the middle part of the transplant and the site of anastomosis, because there is potential difference in blood flow between these sites [4]. No attempt was made to distinguish between arteries and veins. The data were presented as the average of three measurements for each transplant.
Statistical Analysis
All data were presented as the mean ± standard error. Statistical analysis was performed using the paired Student's t test. A p value less than 0.05 was considered statistically significant.
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Results |
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Histopathology
In group I, reepithelialization and partial regeneration of tracheal glands were seen on posttransplantation day 10. The histologic appearance of the autografts had returned to normal by posttransplantation day 28 (Fig 1A). The omentum in which the autografts had been wrapped showed no evidence of histologic injury. In contrast, the histology of the allografts in group II was quite different from that of the autografts. None of these allografts demonstrated restoration of epithelium or tracheal glands, and the perichondrium of the cartilaginous rings had been destroyed in some cases. Severe hemorrhage and edema in the submucosa were observed on posttransplantation day 10, and necrosis and infiltration by mononuclear cells were seen on posttransplantation day 28 (Fig 1B). Histologic examination of the allografts in group III revealed partial regeneration of metaplastic epithelial cells, severe hemorrhage in the submucosa, slight necrosis, and moderate mononuclear cell infiltration on posttransplantation day 10. A single layer of nonciliated epithelium was seen in places, and hemorrhage in the submucosa was decreased on posttransplantation day 28 (Fig 1C). In group IV, normal mucociliary epithelium covered the allograft extensively on posttransplantation day 10, and hemorrhage was moderate. Slight mononuclear cell infiltration remained for 28 days (Fig 1D). The histology of the allografts in group V resembled that of group IV, but hemorrhage in the grafts was less prominent in group V. Inflammatory changes were severe in 2 allografts in group V (Fig 1E). The histologic injury was gradually repaired in all groups, except group II. In contrast, the histology gradually worsened in group II. In groups III, IV, and V, the speed with which histologic restoration occurred was slower than that in group I. In addition, endothelial cells were observed in the vessels in groups I, IV, and V, but not in group II on posttransplantation days 10 and 28. In group III, the vessels exhibited simple lumens without endothelial cells on posttransplantation day 10. Those cells were first apparent in the vascular lumina of these grafts on posttransplantation day 28.
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Comment |
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It is very important to examine the epithelial morphology in the evaluation of graft viability of the trachea. Mayer and associates [13] have reported that all of the epithelium was destroyed 2 days after tracheal transplantation to the omentum in rat isografts. We have previously found that all of the epithelium of devascularized grafts was destroyed on the third postoperative day, and that partial epithelial regeneration was seen 7 days after orthotopic tracheal autotransplantation with omentopexy in dogs [3, 4]. We also have reported that the epithelium was lost on the fifth postoperative day, and was partially regenerated 10 days after heterotopic tracheal autotransplantation to the omentum in dogs [7]. Thus, epithelial regeneration of tracheal autografts covered with omentum may begin 7 to 10 days after transplantation. On the other hand, Davreux and associates [9] have performed heterotopic tracheal allotransplantations to the omentum in rats and found that there was virtually no epithelial regeneration in allografts to animals treated without immunosuppression 14 days after operation. In contrast, high-dose CsA and methylprednisolone improved epithelial viability of the tracheal allografts [9]. In our study, reepithelization was not seen in tracheal allografts in the animals treated without immunosuppression up to 28 days after transplantation. Thus, epithelial regeneration in tracheal allografts is closely associated with avoidance of rejection. Therefore, appropriate immunosuppression is required. In our experiments, which were designed to examine different doses of CsA, epithelial regeneration in groups II and III was significantly worse than that in groups I, IV, and V at both time points. The dose of CsA in group III thus does not overcome allograft rejection. Therefore, 10 mg • kg-1 • day-1 of CsA is inadequate immunosuppression for tracheal allografts. In contrast, there was no difference in epithelial regeneration between groups IV and I on day 28. Furthermore, regeneration in group IV was significantly better than that in group I 10 days after transplantation. Immunosuppression using 15 mg • kg-1 • day-1 of CsA may allow allograft epithelial regeneration in the early period after transplantation. On the other hand, regeneration in group V was significantly worse than that in group I on the 28th posttransplantation day. High-dose CsA may have predisposed to infection in group V, suppressing epithelial regeneration in that group, as evidenced by gross and histologic findings.
We investigated vascularity as well as epithelial regeneration because revascularization is closely associated with the process of epithelial regeneration [13] and rejection [9]. The number of vessels in the nonimmunosuppressed allografts (group II) was significantly lower than that in group I. This difference was more apparent over time. Rejection may destroy vascular endothelial cells and reduce vessel number as demonstrated by our histologic study. In groups III, IV, and V, there was no difference in vessel number compared with the control group. However, with low-dose CsA (group III), there were no endothelial cells in the vascular lumen on the tenth posttransplantation day. Immunosuppression improves vascularity in allografts, and shows a subtle dose-dependency, although there is a little doubt because the serum blood levels of the drug were not measured in this model. It is interesting to note that group III showed significant differences in epithelial regeneration when compared with groups IV and V. The epithelium of the trachea is very sensitive to graft rejection, and has antigenicity [14]. Inadequate immunosuppression may not control this antigenicity.
The viability of immunosuppressed allografts was not significantly better than that of the autografts. However, with 15 mg • kg-1 • day-1 of CsA, the macroscopic and microscopic structural integrity of the heterotopically transplanted tracheal allografts was maintained. We conclude that tracheal allograft viability is maintained with 15 mg • kg-1 • day-1 of CsA in the heterotopic transplant model. Such a dose of CsA seems to be adequate for minimal immunosuppression.
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Acknowledgments |
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= epithelial regeneration score in group IV significantly better than that in the control group [p < 0.05].)