Ann Thorac Surg 1995;60:952-957
© 1995 The Society of Thoracic Surgeons
Original Articles: General Thoracic
Morphologic Alterations and Cytokinetic Studies of Tracheal Autograft Epithelium in Rabbits
Yoshiaki Inayama, MD,
Izumi Tomiyama, MD,
Makoto Akaike, MD,
Masahiro Kase, MD,
Haruhiko Nakayama, MD,
Takao Morohoshi, MD,
Akihiko Matsumoto, MD,
Masayoshi Kanisawa, MD
Departments of Pathology and Surgery, Yokohama City University School of Medicine, Yokohama, Japan
Accepted for publication May 9, 1995.
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Abstract
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Background. Although tracheobronchoplasty has been used widely in the field of thoracic surgery, few details of the morphologic changes in and cytokinetics of the graft epithelium have been reported. The aim of this study was to focus on these aspects in autografted rabbit tracheas.
Methods. Resected cervical tracheas were anastomosed immediately after removal, retrieved on postoperative days 1 through 28, and examined morphologically. Mitotic and bromodeoxyuridine-labeling indices of the graft epithelium were analyzed.
Results. On postoperative days 1 to 4, the graft epithelium showed focal desquamation at the anastomoses. Ciliated cells disappeared during postoperative days 4 to 7 and then increased gradually. Nonciliated cells retained a somewhat columnar shape on postoperative days 4 to 7, except at denuded foci. Thereafter, the grafts were covered completely with pseudostratified mucociliary epithelium. On postoperative day 4, both indices were maximal and appeared higher at the anastomotic than midgraft sites.
Conclusions. Most of the graft epithelium was preserved during acute ischemia and then started to regenerate. The increased regenerative activity near the anastomoses may be attributable to mechanical damage or different nutritional conditions.
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Introduction
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Many experimental studies have been carried out to develop tracheal prostheses made of artificial materials [1]. However, artificial prostheses have not proved suitable for clinical use because of various unsolved problems, such as infection and graft stenosis. In humans, therefore, tracheobronchoplasty without the use of prostheses is performed widely. One such procedure is circumferential resection of part of the airway tract followed by end-to-end anastomosis. However, in such cases, few details of the resulting morphologic alterations in and cytokinetics of graft airway epithelium have been reported. Preliminary studies by us revealed that the epithelial lining of resected and reanastomosed tracheas in rabbits showed fairly good preservation of its columnar form with immature cell types and then began to grow and differentiate (unpublished data). In this study, we examined the morphologic alterations in and the cytokinetics of the epithelium in autografted tracheas over a period of 1 day to 28 days after operation.
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Material and Methods
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Twenty-one healthy male New Zealand white rabbits more than 29 weeks old (Nihon SLC, Shizuoka, Japan) were used. During the entire experimental period, they were housed in individual cages placed in an animal cabinet with front windows and a double-filtered laminar airflow system. 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).
Operative Procedure
The animals were anesthetized with intravenous sodium pentobarbital. After the fur was shaved from the anterior cervical region and local anesthesia was induced with lidocaine hydrochloride, a skin incision was made along the line of the trachea. The trachea was then exposed and released carefully from the surrounding tissue from the third tracheal ring to the distal cervical region by blunt dissection. One to 1.5 cm of the trachea, consisting of four to six rings, was resected.
After the trachea was removed from the host, it was reduced to about 70% of its original length, washed with saline solution containing an antibiotic (cefmetazol sodium), and then reanastomosed end-to-end with interrupted 6-0 Maxon sutures (Japan Lederle, Tokyo) with 12 knots at each anastomosis. It took 10 minutes or less from removal of the trachea to the start of the anastomosis, which required 1.5 to 2 hours to complete.
During the operation, the animals breathed spontaneously. Intraluminal mucus and coagulation tissue were gently aspirated, and lidocaine was sprayed into the airway lumen as required. On completion of the anastomosis, the cervical muscles and subcutaneous tissue were ligated tightly layer by layer, and the skin was closed with silk sutures. Antibiotics (cefmetazol sodium or cefodizime) were injected subcutaneously for 7 days postoperatively.
Removal and Examination of Grafts
The rabbits were sacrificed by injecting an overdose of pentobarbital intravenously on postoperative day (POD) 1, 4, 7, 10, 14, 21, or 28 (two to four grafts per POD). One hour before sacrifice, bromodeoxyuridine (Sigma-Aldrich Corp, Tokyo), 10 mg/kg of body weight, was injected intravenously. The trachea including the graft was removed and fixed with periodate-lysine-paraformaldehyde. Three longitudinal sections of each trachea, including proximal and distal regions of the host trachea adjacent to the graft as well as the graft itself, were dissected. Deparaffined sections 4 µm thick were stained with hematoxylin and eosin and alcian blue (pH 2.5)--periodic acid--Schiff.
The proportion of mitotic cells, or the mitotic index (MI), in the entire length of the graft was calculated by counting all the nucleated epithelial cells and the cells with mitotic figures in tangentially cut longitudinal sections under a microscope at x400 and dividing the latter by the former. The MI was also calculated at the following five different sites: proximal host trachea; proximal, middle, and distal portions of the graft (Prox-G, Mid-G, Dist-G, respectively); and distal host trachea. In each portion, the total number of nucleated epithelial cells and the number of mitotic cells per 750 µm of basement membrane length were determined with the aid of an ocular micrometer at x400, and the MI was calculated as just described.
For bromodeoxyuridine immunostaining, the streptavidin-biotin-peroxidase complex technique with an anti-bromodeoxyuridine antibody (Beckton Dickinson, Mountain View, CA) was used, and the bromodeoxyuridine-labeling index (LI) was determined in the same way as MI. In addition, select portions of the graft mucosa were examined ultrastructurally using an electron microscope.
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Results
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Morphologic Examination of Grafts
One day after the operation (POD 1) two grafts were examined. The gross changes, hyperemia and anemia, were virtually identical (Fig 1A
). Histologically, the epithelium lining the grafts was focally desquamated but well preserved across most of the mucosa, which was composed of apparently normal pseudostratified columnar epithelium (Fig 1B). The submucosa showed edema and vascular dilatation with blood retention. The host tracheal epithelium evidenced focal disappearance at the anastomoses. Ultrastructurally, intercellular edema and dilatation of intracellular organelles were observed (not shown).
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Fig 1. . Grafts removed on postoperative day 1. (A) Macroscopic appearance of graft and host trachea showing anemic, hyperemic, and hemorrhagic changes in graft (bar = 1 cm). (B) Representative view of graft mucosa showing pseudostratified columnar epithelium. (Alcian blue--periodic acid--Schiff; x100 before 25% reduction.)
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The mucosa of two of the three grafts examined on POD 4 was hyperemic and glossy. Histologically, focal disappearance of the mucosa in the graft and the host trachea at the anastomoses was seen (Fig 2A), and the posterior wall of the host trachea was partially denuded. The residual mucosa in each graft had columnar epithelium with extensive ciliary loss (Fig 2B), and mitotic figures were present (Fig 2C). Electron microscopic examination disclosed that the columnar cells were dissociated from each other because of intercellular edema and showed dilatation of intracytoplasmic organelles. There was also a decrease in the number of ciliated cells (Fig 2D). In the third rabbit sacrificed on POD 4, which had marked stridor during and after the operation, the graft was anemic and showed complete loss of its epithelial lining.
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Fig 2. . Grafts on postoperative day 4. (A) Low-power micrograph of distal part of graft (G), the mucosa of which is completely covered with epithelial cells, although the host trachea near the anastomosis is denuded. Occasional fresh thrombi are evident in submucosal vessels of graft (arrowheads). (B) High-power view of middle portion of graft mucosa. The mucosa has pseudostratified columnar epithelial cells and apparently lacks ciliated cells. (C) Mitotic figures (arrowheads) were often evident at this time. (D) Ultrastructurally, moderate intercellular and intracellular edema is present. There are fewer ciliated cells, and they have longer microvilli than on postoperative day 1 (A--C: alcian blue--periodic acid--Schiff; A, x5 before 25% reduction; B, x132 before 25% reduction; C, x160 before 25% reduction; D, x2,600 before 47% reduction.)
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On POD 7, three of the four grafts examined were markedly hyperemic (Fig 3A), and the fourth was necrotic as a result of infection. In each of the former three grafts, the epithelium was generally pseudostratified columnar with almost no cilia (Fig 3B), except for focal loss at both the proximal and the distal anastomotic portions. Near the anastomoses, the epithelium was rather flat and in parts resembled immature squamous epithelium (Fig 3C). At this time, mitotic and bromodeoxyuridine-positive epithelial cells were plentiful, even in the graft midportions (Fig 3D). Ultrastructurally, the graft epithelium resembled that on POD 4.
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Fig 3. . Grafts on postoperative day 7. (A) Gross view of graft showing hyperemia and hemorrhage (bar = 1 cm). (B) Most of middle portion of graft is covered with columnar epithelium, which shows no ciliated cells, except for areas with focal mucosal disappearance. (C) The epithelium near the anastomosis is flat and resembles immature squamous epithelium. (D) Abundant bromodeoxyuridine-labeled cells are present in graft epithelium. (B, C: alcian blue--periodic acid--Schiff; D, bromodeoxyuridine immunostaining; B, C: x160 before 25% reduction; D, x50 before 25% reduction.)
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Ten days after the operation (POD 10, n = 3), hyperemia of the graft mucosa was less marked. Histologically, an epithelial lining covered the entire mucosal surface of every graft (Fig 4A), and abundant mucous cells and fewer ciliated cells were observed (Fig 4B).
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Fig 4. . Grafts on postoperative day 10. (A) The graft is covered completely with pseudostratified columnar epithelium. (B) The number of ciliated cells has increased in comparison with that on postoperative day 7, although there are still apparently less than in normal trachea, and abundant mucous cells are evident. (Both: alcian blue--periodic acid--Schiff; A, x3.3 before 25% reduction; B, x80 before 25% reduction.)
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By POD 14 (n = 3), the mucosal surfaces of all the grafts were glossy and did not appear to differ from those of the host tracheas (Fig 5A), all being covered with pseudostratified columnar epithelium. There were more ciliated cells than on POD 10 (Fig 5B). By electron microscopy, immature columnar, nonciliated epithelial cells were rarely observed (Fig 5C).
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Fig 5. . Grafts on postoperative day 14. (A) The inner surface of this graft is glossy and does not differ from that of the host trachea (bar = 1 cm). (B) More ciliated cells are present than on postoperative day 10. (C) Ultrastructurally, sparse undifferentiated columnar cells (*) were scattered among ciliated and mucous cells. (B, alcian blue--periodic acid--Schiff; x80 before 27% reduction; C, x2,800 before 25% reduction.)
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The appearances of the tracheal grafts on PODs 21 and 28 (n = 3 each) were almost identical. Macroscopically, there was no difference in the histologic (Fig 6) or ultrastructural appearance between the graft and host tracheal mucosas, both of which were composed of mucociliary columnar epithelium.
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Fig 6. . Grafts on postoperative days 21 and 28. Abundant ciliated and mucous cells are present on postoperative day 28; the appearance on postoperative day 21 was virtually identical. (Alcian blue--periodic acid--Schiff; x50 before 25% reduction.)
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In the tracheal grafts with epithelial linings, chondrocytes were observed and considered viable on all PODs.
Temporal Changes in MI and LI of Entire Graft
The temporal changes in the MI and the LI of entire grafts were evaluated (Table 1). The MI was maximal on POD 4, followed by a slight decrease on POD 7. Both values were significantly higher than those on PODs 10, 14, 21, and 28 (p < 0.01 and p < 0.001 for POD 4 and POD 7, respectively). However, there was no significant difference between the values on PODs 4 and 7. The LI showed the same trend; maximal on POD 4 (p < 0.001 or p < 0.002 compared with the values for the other days) with a slight decrease on POD 7, when it was significantly higher than the values on PODs 1, 10, 28 (p < 0.04), 14, and 21 (p < 0.05).
MI and LI in Different Portions of Graft and Host Trachea
The temporal changes in the MI and the LI of the five different portions of the grafts and host tracheas were also evaluated (Table 2). Generally, the MI appeared to be higher at the proximal and distal anastomoses (Prox-G and Dist-G, respectively) compared with Mid-G. On POD 7, in particular, the MI at Prox-G was significantly higher than that at Mid-G (p < 0.05), as it was at Dist-G (p < 0.005). The LI showed the same trend on PODs 7 and 10: it was significantly higher at Dist-G on POD 10 than Mid-G (p < 0.02). Neither the MI nor the LI at Prox-G and proximal host trachea or at Dist-G and distal host trachea differed significantly.
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Table 2. . Temporal Changes in Mitotic and Labeling Indices For Five Different Portions of Graft and Host Tracheaa
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Comment
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In this experiment, morphology of the epithelium of the autografted trachea, which was resected completely and then reanastomosed immediately (within 10 minutes) to the host trachea, showed it had been fairly well preserved except for focal loss of its lining at the anastomoses and dorsal mucosal surface. Such focal epithelial loss appeared to be due to mucosal damage created by the forceps or suction tube used during the operation. The number of cilia on the remaining epithelium covering the graft declined markedly postoperatively, although the cells were still columnar in shape even on PODs 4 to 7 and maximally stratified on POD 7.
The usual pattern of morphologic changes in airway epithelium induced by injuries such as mechanical trauma [2] is as follows: (1) dedifferentiation of epithelial cells into flat cells, (2) their proliferation, migration, or both and (3) redifferentiation. This pattern was also observed for epithelialization of an artificial tracheal prosthesis [3].
The morphologic changes observed in the present study appeared to differ from the usual type of cellular reaction. This was probably due to differences in the type and degree of epithelial injury. Few details of epithelial changes in grafts used for autologous tracheal replacement have been described. The present results were similar to those of a histologic study of subcutaneously transplanted tracheal homografts in rats [4] in terms of ciliated cell loss in the membranous portions of some grafts on day 2, which was followed by epithelial regeneration, although epithelial denudation and squamous metaplasia appeared to occur readily in the rat model.
The immature columnar cells, observed at the early postoperative stages (PODs 4 to 7), appeared to be derived from degranulated secretory cells, and then these immature cells differentiated into mature secretory and ciliated cells during the first 10 days after grafting. Some of the newly formed ciliated cells may have derived from preexisting ciliated cells that had lost all their cilia because of ischemia but survived despite the injury.
Maximal epithelial cell regeneration, estimated in terms of MI and LI, was observed on PODs 4 and 7. The MI values at the anastomotic sites of the graft were higher than those at the midportion. Especially, those at Prox-G and Dist-G on POD 7 were significantly higher than those at Mid-G (p < 0.05 and p < 0.005, respectively). The LI appeared to show the same trend, although a significant difference was seen only between the Mid-G and Dist-G values on POD 10 (p < 0.02). This may have been due to the effects of regenerative stimulation of the epithelium in the form of unexpectedly induced mechanical injury. Alternatively, the nutritional conditions for epithelial cellular growth and regeneration may have been better around the anastomoses as a result of the blood supply from the host trachea, even though complete anastomosis of the blood vessels of the graft and host trachea had not been established.
In general, it is widely accepted that a good blood supply is an essential factor for rapid epithelialization of artificial tracheal grafts and minimization of postoperative complications. Therefore, to improve graft revascularization, wrapping the grafts with sternocleidomastoid muscle [5], thoracic fascia [6], or omentum [4, 7–9] has been reported. In fact, omentopexy is widely used experimentally [4, 7–9] and has also been used clinically in recent years [10]. In addition, blood supplied directly from the host trachea by way of the anastomoses appears to be an important factor for successful replacement, at least until a collateral circulation has been established through the surrounding connective tissue. Support for this is provided by the findings of an experimental study performed by Nakanishi and associates [11]. When autograft replacement of long (>4.0 cm) segments was performed in dogs, the middle part of the grafts became stenosed readily because of ischemia, even if omentopexy was performed.
Although greater regenerative activity of epithelial cells was observed at the anastomotic sites, epithelial cells at Mid-G also showed higher MI and LI values than those of tracheal epithelial cells in a normal steady state [12]. Further, epithelial migration from the host trachea is unlikely to be complete within a few days. Therefore, the majority of the epithelial cells responsible for establishing the graft epithelium were considered to have originated from cells of the grafts themselves; they had not migrated from the host trachea.
In conclusion, ischemic regenerative stimuli in rabbits that underwent autologous tracheal replacement induced epithelial morphologic changes that differed from those evoked by various injuries reported hitherto. In addition, epithelial cells near the anastomoses appeared to have more active growth potential than those further away. The results obtained in this study should prove useful for studying the cellular responses to acute ischemic injury in patients who undergo tracheobronchoplasty and lung transplantation.
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Footnotes
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Address reprint requests to Dr Inayama, Department of Pathology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan.
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References
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- Lane BP, Gordon R. Regeneration of rat tracheal epithelium after mechanical injury. I. The relationship between mitotic activity and cellular differentiation. Proc Soc Exp Biol Med 1974;145:1139–44.[Medline]
- Hirai K, Shimizu Y, Hino T. Epithelial regeneration in collagen-coated and uncoated patch grafts implanted into dog tracheas. J Exp Pathol 1990;71:51–62.
- Hirata T, Yamazaki F, Fukuse T, et al. Omentopexy for revascularization of free tracheal grafts in rats. Thorac Cardiovasc Surg 1992;40:178–81.[Medline]
- Rose KG, Sesterhenn K, Wustrow F. Tracheal allotransplantation in man. Lancet 1979;1:433.[Medline]
- Delaere PR, Ziying L, Pauwels P, Feenstra L. Experimental revascularization of airway segments. Laryngoscope 1994;104:736–40.[Medline]
- Lima O, Goldberg M, Peters WJ, et al. Bronchial omentopexy in canine lung transplantation. J Thorac Cardiovasc Surg 1982;83:418–21.[Medline]
- Morgan E, Lima O, Goldberg M, Ferdman A, Luk SK, Cooper JD. Successful revascularization of totally ischemic bronchial autografts with omental pedicle flaps in dogs. J Thorac Cardiovasc Surg 1982;84:204–10.[Abstract]
- Nakanishi R, Shirakusa T, Takachi T. Omentopexy for tracheal autografts. Ann Thorac Surg 1994;57:841–5.[Abstract]
- Müller LC, Abendstein B, Salzer GM. Use of the greater omentum for treatment and prophylaxis of anastomotic and stump dehiscence in major airway surgery. Thorac Cardiovasc Surg 1992;40:323–5.[Medline]
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- Keenan KP, Combs JW, McDowell EM. Regeneration of hamster tracheal epithelium after mechanical injury. II. Multifocal lesions: stathmokinetic and autoradiographic studies of cell proliferation. Virchows Arch [Cell Pathol] 1982;41:215–29.
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Abstract
Introduction
