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Ann Thorac Surg 1995;60:635-639
© 1995 The Society of Thoracic Surgeons

Original Articles: General Thoracic

Minimal Dose of Cyclosporin A for Tracheal Allografts

Ryoichi Nakanishi, MD, Kosei Yasumoto, MD

Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan

Accepted for publication April 13, 1995.


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Minimizing immunosuppression after allotransplantation is desirable.

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.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Tracheal allotransplantation has been studied by several investigators. With few exceptions, these experiments have been unsuccessful because graft ischemia was the major cause of allograft failure [1, 2]. We have previously demonstrated that omentopexy improves revascularization of devascularized tracheal autografts [3, 4]. This leaves the difficulties encountered with the use of immunosuppression for tracheal allotransplantation to be studied. Tracheal allografts are generally believed to induce only a weak rejection response by the host in the presence of major histoincompatibility [5]. Therefore, there have been few studies that have addressed immunosuppression for tracheal allografts. However, tracheal allograft rejection has been demonstrated [6, 7]. The viability of tracheal allografts in the setting of immunosuppression needs to be carefully examined.

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.


   Material and Methods
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
The heterotopic tracheal transplant model with omentopexy [8] was employed using a technique modified for dogs [7]. Fifty tracheal segments consisting of six rings were harvested and implanted into the omentum. In 10 control dogs, each transplantation was performed to the donor's omentum. The other 40 animals were randomly allocated into four experimental groups (Table 1Go). 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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  		Table 1. . Experimental Design
 
Donor Procedure
Fifty adult mongrel dogs weighing from 8.8 to 16.5 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). Ventilation was maintained via an endotracheal tube using a pressure-limiting respirator. Through a midline cervical incision, six tracheal rings were identified, excised in continuity, and placed in a physiologic saline solution for the few minutes before implantation. A transtracheal tube was advanced into the lower trachea for ventilation after tracheal resection, and the tracheal defect was repaired by primary end-to-end anastomosis using running 3-0 Prolene sutures (Ethicon, Inc, Somerville, NJ). The anterior portion of the trachea was released for reduction of tension. The cervical wound was closed in the usual fashion.

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 1Go). 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.


   Results
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Gross Appearance
Grossly, all free tracheal allografts appeared normal. However, preservation of lumen rigidity was lost in group II. Dark green mucoid fluid filled the lumen of almost all grafts in groups I, IV, and V, but was not evident in groups II and III. Evidence of infection such as packed pus was observed in only transplants in group V.

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 1AGo). 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 1BGo). 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 1CGo). 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 1DGo). 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 1EGo). 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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  		Fig 1. . Histology of tracheal epithelium and submucosal tissue 28 days after transplantation. (A) Group I: the epithelium and submucosal tissue are restored to normal. (B) Group II: the mucosa is denuded and necrosis of submucosal tissue is moderate. (C) Group III: a single layer of metaplastic epithelium is seen, and hemorrhage in the submucosa is prominent. (D) Group IV: normal mucociliary epithelium is recognized extensively. Hemorrhage and slight mononuclear cell infiltration in the submucosa still remain. (E) Group V: inflammatory changes are severe. (Hematoxylin and eosin; magnification, x200.)

 
EPITHELIAL REGENERATION.
In group I, epithelial regeneration on posttransplantation day 28 was significantly better than on posttransplantation day 10. When compared with the control group, epithelial regeneration in groups II and III was worse at both time points. This was true in group V on posttransplantation day 28. In contrast, epithelial regeneration in group IV was significantly better than that in group I on posttransplantation day 10. Regeneration in groups IV and V was significantly better than that in group III at both time points (Fig 2Go).



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  		Fig 2. . Epithelial regeneration score (mean ± standard error of the mean) for the five groups studied. Open bars represent animals treated for 10 days, and solid bars represent animals treated for 28 days. (CsA = cyclosporin A; Tx = treatment; *,** = epithelial regeneration score in each group was significantly worse than that in the control group at that time point [p < 0.05]; {dagger} = epithelial regeneration score in group IV significantly better than that in the control group [p < 0.05].)

 
VESSEL NUMBER.
In group I, vessel number was significantly higher on posttransplantation day 28 than on posttransplantation day 10. In contrast, vessel number on the 28th day in group II was significantly lower than that on the 10th day. There were also fewer vessels at day 28 in group II than in group I. There was no difference in vessel number in any of the other groups (Fig 3Go).



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  		Fig 3. . Number of blood vessels in the tracheal graft (mean ± standard error of the mean) for the five groups studied. Open bars represent animals treated for 10 days, and solid bars represent animals treated for 28 days. (CsA = cyclosporin A; Tx = treatment; ** = epithelial regeneration score in each group was significantly worse than that in the control group [p < 0.05].)

 

   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Long-term administration of immunosuppressive agents is associated with significant complications including the development of malignant disease [10]. Therefore, minimizing immunosuppression after transplantation is desirable. We assessed the effectiveness of a short course of immunosuppression after tracheal allotransplantation in a preliminary study. It was concluded that an appropriate immunosuppressive agent, if used for only 3 weeks after transplantation, may maintain the viability of tracheal allografts over the long-term [11]. Minimal doses of immunosuppressive agents that are used in the clinical setting were thus studied. In this study, we selected CsA because it is an excellent immunosuppressant and has no adverse effects on wound healing [12]. The effect of minimal doses of CsA on the viability of heterotopic tracheal allografts was examined. The heterotopic tracheal allograft model offers the advantages of simplicity and low cost [9]. It allows the evaluation of tracheal viability because the model shows the same healing course as orthotopic transplantation model with omentopexy [3]. We adopted a canine model close to the clinical setting for the purpose of analysis of the tracheal morphology rather than immunology in this study. It has already been reported that heterotopic transplantation in dogs was an effective model in a previous experiment on the histopathology of tracheal allotransplant rejection [7].

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.


   Acknowledgments
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
We thank Ms Miki Kiyofuji for her expert technical assistance. Cyclosporin A was kindly supplied by Sandoz Pharma AG, Basle, Switzerland.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
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, Japan.


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Spinazzola AJ, Graziano JL, Neville WE. Experimental reconstruction of the tracheal carina. J Thorac Cardiovasc Surg 1969;58:1–13.[Medline]
  2. Neville WE, Bolanowski PJP, Soltanzadeh H. Homograft replacement of the trachea using immunosuppression. J Thorac Cardiovasc Surg 1976;72:596–601.[Abstract]
  3. Nakanishi R, Shirakusa T, Takachi T. Omentopexy for tracheal autografts. Ann Thorac Surg 1994;57:841–5.[Abstract]
  4. Nakanishi R, Shirakusa T, Mitsudomi T. Maximum length of tracheal autografts in dogs. J Thorac Cardiovasc Surg 1993;106:1081–7.[Abstract]
  5. Rose KG, Sesterhenn K, Wustrow F. Tracheal allotransplantation in man [Letter]. Lancet 1979;24:433.
  6. Beigel A, Muller-Ruchholtz W. Tracheal transplantation. I. The immunogenic effect of rat tracheal transplants. Arch Otorhinolaryngol 1984;240:185–92.[Medline]
  7. Nakanishi R, Shirakusa T, Hanagiri T. Early histopathologic features of tracheal allotransplantation rejection. A study in nonimmunosuppressed dogs. Transplant Proc 1994;26:3715–8.[Medline]
  8. Olech VM, Keshavjee SH, Chamberlain D, Slutsky AS, Patterson GA. The role of basic fibroblast growth factor in revascularization of rabbit tracheal autografts. Ann Thorac Surg 1991;52:258–64.[Abstract]
  9. Davreux CJ, Chu NH, Waddell TK, Mayer E, Patterson GA. Improved tracheal allograft viability in immunosuppressed rats. Ann Thorac Surg 1993;55:131–4.[Abstract]
  10. Veith FJ, Norin AJ, Montefusco CM, et al. Cyclosporin-A in experimental lung transplantation. Transplantation 1981;32:474–81.[Medline]
  11. Nakanishi R, Yasumoto K, Shirakusa T. Short course immunosuppression after tracheal allotransplantation in dogs. J Thorac Cardiovasc Surg 1995;109:910–7.[Abstract]
  12. Goldberg M, Lima O, Morgan E, et al. A comparison between cyclosporin A and methylprednisolone plus azathioprine on bronchial healing following canine lung autotransplantation. J Thorac Cardiovasc Surg 1983;85:821–6.[Abstract]
  13. Mayer E, Cardoso PFG, Puskas JD, et al. The effect of basic fibroblast growth factor and omentopexy on revascularization and epithelial regeneration of heterotopic rat tracheal isografts. J Thorac Cardiovasc Surg 1992;104:180–8.[Abstract]
  14. Kalb TH, Chuang MT, Marom Z, Mayer L. Evidence for accessory cell function by class II MHC antigen-expressing airway epithelial cells. Am J Respir Cell Mol Biol 1991;4:320–9.



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