Being associated with high morbidity and mortality, obliterative bronchiolitis (OB) is a devastating manifestation of chronic rejection in human lung recipients (1 , 2 3–6 7 , 8 9–13
Large-animal orthotopic tracheal transplantation models have been applied for studies of tracheal repair after trauma and stenosis. Despite complications such as tracheomalacia, necrosis, granulation formation, and anastomotic rupture, long-term success has been achieved in experimental studies and clinical trials by using omental wrapping, immunosuppressive treatment, graft irradiation, or cryopreservation/fixation (14–18 19 , 20
Heterotopic tracheal transplantation from a histoincompatible donor results into irreversible OAD within 28 days (4–6 , 21–25 23 , 24 , 26 , 27 28
Aims of our study were 1) to develop a technique for orthotopic tracheal transplants in rats and 2) to evaluate the progression of OAD in orthotopic tracheal allografts compared to heterotopic tracheal allografts and nontransplanted tracheas. We hypothesized that contact with host trachea might reduce the development of OAD in orthotopic allografts by supplying epithelial cells for the regeneration of the epithelium on the allograft luminal surface.
MATERIAL AND METHODS
Animals and study design.
Viral antibody-free male rats weighing 275 to 400 g were selected as donors and recipients from two histoincompatible strains. Brown Norway (RT-1n ) and Lewis (RT-1l ) rats were used as donors; all recipients were Lewis rats. For the orthotopic allograft transplants, 14 donors and 27 recipients were used. In addition, two Lewis rats served as recipients of orthotopic tracheal isografts. Furthermore, heterotopic tracheal allografts (n=6) and isografts (n=3) were transplanted into the omentum of Lewis rats and tracheas from six nontransplanted Lewis rats were removed for controls.
The results of the following groups were compared with each other: Nontransplanted tracheas (n=6); day 7 orthotopic allografts (n=6); day 30 heterotopic allografts (n=5); day 30 orthotopic allografts (n=4); day 60 orthotopic allografts (n=5). In addition, orthotopic allografts were compared with adjacent host tracheas.
All laboratory animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the Institute of Laboratory Animal Resources and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Science (NIH publication no. 86–23, revised 1985). All rats were purchased from Charles River Laboratories (Wilmington, MA), and were allowed free access to food and water. The rats were killed if they had any signs of compromised respiration during daily observations.
Donor procedure.
After induction of general anesthesia in the donor animals with methoxyflurane inhalation, a lethal dose of pentobarbital (120 mg/kg) was administered i.p. The trachea of the donor rat was exposed through a midline cervical incision advanced to a midline sternotomy. The whole length of the trachea was excised and stored in cold physiological saline solution. The trachea was divided into two or three segments of 5–10 rings, and each segment was transplanted to one recipient.
Recipient procedures.
The recipient animal was anesthetized with methoxyflurane inhalation and a combination of pentobarbital 40 mg/kg i.p. and ketamine hydrochloride 30 mg/kg i.p. The animal was placed supine, the head pointing away form the surgeon on an operative board and fixed with tapes to all extremities. A midline cervicotomy was performed with meticulous hemostasis. The trachea was exposed and dissected free between the 3rd and 8th rings. The blood vessels running parallel to the trachea were saved, if possible, or cauterized or ligated to avoid any bleeding. The recipient trachea was divided three to four rings caudally from the epiglottis, and two to four rings were removed with careful haemostasis. The donor graft was anastomosed end-to-end starting with the distal trachea. Any bleeding or clots were removed instantaneously from the recipient trachea to insure a clear airway. For anastomosis, the distal recipient trachea was first suspended by two stay sutures 180o apart. The tracheal graft was placed so that distal to proximal orientation was maintained and the distal anastomosis of the graft was begun behind the right stay suture with a continuous 8–0 Prolene suture. After finishing the distal anastomosis, secretions or clots were cleared from the proximal recipient trachea, and the recipient trachea was anastomosed end-to-end with the proximal end of the graft in the same way as the distal anastomosis. Before completing the anastomosis, the trachea was examined for any secretions or blood clots in the airways.
Spontaneous breathing usually continued immediately after the completion of the proximal anastomosis. If there were any breathing difficulties, a second look into the trachea was done, and any secretions or clots were removed from the airway. After undisturbed spontaneous breathing occurred, the midline cervicotomy was closed in three layers by using 4–0 Vicryl. The animal was placed in the recovery cage in a lateral decubitus position, and its respiration was noted every 5 min until the animal was fully awake. The overall operative mortality was five animals (19%), and the causes of death were a blood clot near the carina and secretions occluding the epiglottis. Animals were inspected twice a day. If any signs of compromised respiration were noticed, the animals were killed. Heterotopic tracheal allo- and isografts were performed and removed from the omentum as described by Reichenspurner et al. (24
Graft removal.
After lethal dose of pentobarbital (120 mg i.p.), the midline cervicotomy wound was reopened, and the whole trachea including the graft was removed. The orthotopic graft was divided from its midpoint into two segments. One segment, anastomosed to the recipient trachea, was fixed in 10% neutral buffered formalin solution for hematoxylin and eosin (H&E) and Masson’s trichrome staining. The other segment was placed in a tube containing OCT (Miles, Elkhart, IN), and snap frozen in liquid nitrogen, and stored at −80°C for cryosectioning. For histological sections, the formalin-fixed sample was divided into three: one containing the recipient segment, one containing the anastomotic region, and one containing the graft.
Histopathology.
Peritracheal inflammation, infiltration of lymphocytes, monocytes, and spindled cells (myofibroblast, fibroblasts, and smooth muscle cells), and degree of fibrosis were evaluated from the H&E and Masson’s trichrome staining by an experienced pathologist.
Computerized morphometry.
Video images of H&E stained tracheal sections were taken with a Dage MTI-81 high-resolution color camera mounted on a Leica DMRB microscope and analyzed with a C-imaging 1280 morphometric system (Compix Inc., Imaging Systems, Cranberry Township, PA). The quantification of the different analyses of tracheal sections is described in detail by Reichenspurner et al. (24
Immunohistochemistry.
Tracheal sections embedded in OCT were brought to −20°C and 5 μm thin sections were placed onto poly-L-lysine precoated slides (Cat# P-0425, Sigma Diagnostics, St.Louis MO). Sections were air dried at room temperature, and fixed in acetone at −20°C overnight. Sections were rehydrated in buffered phosphate solution for 10 min, then incubated with the one of following primary antibodies (Serotec, Westbury, NY) for 30 min: pan T-cell marker R73 (〈BTCR), W3/25 (CD4), MRC OX-8 (CD8a), ED-1 (macrophage), MHC class II (OX6), or smooth muscle alpha-actin (Sigma). Secondary antibody was applied for 30 min using peroxidase conjugated rabbit anti-mouse immunoglobulin (Dako, Capinteria, CA), and diaminobenzidine tetrahydrochloride was used as the chromogen. The substitution of 1% bovine albumen phosphate buffered saline for the primary antibody served as the negative (reagent) control. Lewis rat lymph nodes were used as positive controls. The sections were semiquantitatively scored by an observer, who was blinded to the experimental groups. For assessment of the different cell types, the number of positive cells per field was counted from 12 fields per tracheal ring. The cells were counted from one section from three different grafts and the results were reported as a median of each of the ranges of the number of positive cells in fields with the lowest and highest density of cells, because the inflammatory cells were not evenly distributed inside the tracheal ring. For the smooth muscle cell α-actin and class II stainings, grading from 0 to 3 was used (0=no intensity, 1=minimal intensity, 2=moderate intensity, 3=strong intensity).
Immunofluorescence staining.
Fluorescently labeled monoclonal antibodies (mAb) specific for BN or Lewis MHC class I antigens (RT1.An or RT1.Al ) were used for detection of the origin of the epithelium. (A kind gift from Dr. Tom Gill and Dr. Heinz Kunz, Department of Pathology, Pittsburgh University School of Medicine). Frozen tracheal tissue sections of orthotopic allografts removed on days 7, 30, and 60 were air-dried and fixed in acetone at −20°C for 10 min and washed for 10 min in phosphate-buffered saline. The sections were then incubated at room temperature for 30 min with a fluorescence-conjugated antibody diluted 1:200 for anti-BN staining, and 1:100 for anti-Lewis staining, then washed again in phosphate-buffered saline and mounted with p-phenyl-diamine medium. For positive and negative controls, sections from Lewis tracheas and BN tracheas were stained with both antibodies. Both negative controls (anti-BN staining of Lewis tissue and anti-Lewis staining of BN tissue) had minimal background staining while positive controls (anti-Lewis staining of Lewis tissue and anti-BN staining of BN tissue) demonstrated a moderate staining of all cell types.
Statistical analysis.
The data obtained by morphometric analysis were analyzed by calculating mean values and the standard errors of mean. The one-way analysis of variance was used for determination of the levels of significance for the differences between the groups when the normality test was passed. Orthotopic allografts were compared with the adjacent recipient tracheas by using a paired t test. For comparisons, which failed the normality test, a nonparametric test (Kruskal Wallis test, Mann-Whitney test) was used. All analyses were performed using software SPSS for Windows (SPSS Inc. Chicago, IL).
RESULTS
Of 22 successfully performed orthotopic tracheal allografts, 13 were removed between days 2 and 10 (one day 2, one day 5, three day 6, six day 7, one day 8, one day 10). Seven recipients were killed due to respiratory distress associated with the retention of tracheal secretions (or an exudate from the oedematous allograft) typically located at the distal anastomosis. In one animal killed on day 8, a plug of granulation tissue at the site of proximal anastomosis obliterated the tracheal lumen by more than 50%. Four allografts were removed on postoperative day 30, and five allografts on postoperative day 60. Two orthotopic isografts were removed on days 30 and 60.
Histology.
Lymphocytic tracheitis and epithelial alterations were noted in orthotopic allografts during the first 10 days indicating initial ischemia and severe alloimmune injury (Fig. 1B ). Half of the luminal circumference was devoid of the epithelium in the day 2 allograft, whereas allografts removed on days 5 and 7 had nearly complete (92%) coverage of the epithelium. Inflammatory cells were abundant in all tracheal layers, including the epithelium. Mild inflammatory cell infiltration was seen at the adjacent recipient trachea. Luminal patency and coverage by cuboidal or attenuated epithelium was noted in day 30 and day 60 allografts (Fig. 1, C and D ). The mean epithelial coverage was 95% on day 30 and 98% on day 60. In most allografts, the cuboidal epithelium was partially covered by cilia (Fig. 1C , inset). The ciliary coverage was, on average, 30% on day 60. Typically, the allografts had a flat oval shape on day 60. All day 30 and day 60 allografts demonstrated mild to moderate intratracheal OAD consisting of spindled cells that were evenly distributed within the circumference of the tracheal wall. No histological evidence of anastomotic rupture was found. The two orthotopic isografts were histologically indistinguishable from the host tracheas.
Figure 1: Histological sections from orthotopic tracheal allografts. A, Normal trachea with intact respiratory epithelium (H&E x25). The epithelial cells were lined by numerous cilia (insert: H&E x200). B, Edematous tracheal wall, lymphocytic infiltration, and damaged epithelium in an orthotopic allograft removed on day 5 (H&E x25). The epithelium has lost its integrity and no cilia were found. Abundant mononuclear cells were present in the epithelial layer and subepithelially (inset: H&E x100). C, An orthotopic allograft removed on day 30 had patent lumen covered by cuboidal epithelium (H&E x25). Cilia lined the luminal surface of the epithelium and the mononuclear cells were less frequent than on day 5 (inset: H&E x100). D, A day 60 orthotopic allograft with a patent lumen covered by attenuated epithelium. (H&E x25). The attenuated epithelial cells did not contain cilia. The number of mononuclear cells in the epithelium and subepithelial layers was less than on day 30 (insert: H&E x100).
Computerized morphometry.
Morphometric assessment was obtained from normal tracheas (n=6), from orthotopic allografts removed on day 2 (n=1), on day 5 (n=1), on day 7 (n=6), on day 8 (n=1), on day 30 (n=4), and on day 60 (n=5), from adjacent host tracheas at the corresponding time points, and from heterotopic tracheal allografts removed on day 30 (n=5).
Obliteration was assessed as the percentage of intratracheal tissue (IT) representing the amount of tissue inside the cartilage ring. No differences were found among day 7, 30, and 60 orthotopic allografts (Fig. 2 ). In orthotopic allografts removed on days 30 and 60, IT was significantly lower than in heterotopic allografts (P <0.001, for both). In orthotopic allografts removed on days 7, 30, and 60, IT was significantly greater than in host tracheas (P =0.008;P =0.17;P =0.045, respectively) or in normal tracheas (P <0.001;P =0.17;P <0.001, respectively).
Figure 2: Tissue inside the cartilage ring±SEM in orthotopic allografts, adjacent recipient tracheas, normal tracheas, and day 30 heterotopic tracheal allografts (het30) as measured by computerized morphometry. Although obliteration was greater in the orthotopic allografts than in the recipient tracheas on days 7 and 60, significantly less obliteration was found in the orthotopic allografts on days 30 and 60 than in the heterotopic allografts (P <0.001). Compared with heterotopic allografts, the orthotopic location of tracheal allografts was associated with less obliterative airway disease (OAD).
Aspect ratio of orthotopic allografts increased from day 7 (mean 1.22±0.08) to day 60 (mean 1.97±0.05), indicating a change of the shape from rounded to oval (P <0.001). Assessment of IT from oval shaped grafts can be biased due to the calculation method, thus, whenever the aspect ratio is more than 1.6, the inner tracheal area might be a more accurate measure of luminal obliteration.
Inner tracheal area was greater in orthotopic allografts than in host tracheas on day 7 (P =0.007), but the difference did not reach significance on days 30 and 60 (P =0.142, P =0.067, respectively) (Fig. 3 ). Inner tracheal area was smaller in orthotopic allografts than in heterotopic allografts removed on day 30 (P =0.015, t test).
Figure 3: Inner tracheal area (±SEM) of orthotopic allografts, adjacent recipient tracheas, normal tracheas, and heterotopic tracheal allografts as measured by computerized morphometry. In the orthotopic allografts, increased tissue area between the cartilage ring and the epithelium was found at all observation points when compared to the recipient tracheas or normal tracheas. Orthotopic allografts had significantly smaller (P =0.015) inner tracheal areas than heterotopic allografts (het30) at the same observation point on day 30, indicating less OAD.
Outer tracheal area was significantly greater in day 7 orthotopic allografts than in day 30 and day 60 allografts, than in normal tracheas (P <0.001, for all), or in host tracheas (P =0.002), indicating swelling and cellular infiltration due to alloimmune response (Fig. 4 ). When the outer tracheal area of normal tracheas was compared with that of orthotopic allografts removed on day 30 and day 60, the differences were not significant (P =0.732, P =0.488, respectively). Outer tracheal area in day 30 orthotopic allografts was significantly greater than in host tracheas (P =0.006), whereas the difference between day 60 orthotopic allografts and host tracheas was not significant (P =0.978).
Figure 4: Outer tracheal area (±SEM) of orthotopic allografts, adjacent recipient tracheas, normal tracheas, and heterotopically transplanted tracheal allografts (het30) as measured by computerized morphometry. As a consequence of tissue swelling and lymphocytic infiltration caused by acute alloimmune response, the outer tracheal area was significantly increased in orthotopic allografts on day 7 when compared to the later time points or nontransplanted tracheas (P <0.001). No significant differences were found at observations after day 7.
Immunohistology.
Immunohistological staining demonstrated ED-1 positive cells (macrophages), pan-T lymphocytes, and CD4+ and CD8+ cells in all layers of the orthotopic tracheal wall on days 30 and 60. The number of positive cells tended to decrease from day 30 to day 60 (Table 1 ). In the epithelium, CD4+ lymphocytes were more abundant than CD8+ lymphocytes. On day 30 and 60, smooth muscle cell α-actin positive cells (myofibroblasts and smooth muscle cells) formed a narrow layer between the epithelium and tracheal cartilage, and in addition, α-actin positive cells were seen outside the cartilage (Fig. 5 ). Class II antibody staining was strongly positive in all soft tissue layers of the tracheal wall including the epithelium.
Table 1: Semiquantitative analysis of immunohistochemical staining for Pan T+ cells, CD4+ lymphocytes, and CD8+ lymphocytes as medians of the highest and lowest numbers of positive cells in each tracheal ring, and intensity of smooth muscle α-actin positive cells from orthotopic tracheal allografts removed on day 30 and on day 60, from heterotopic allografts and isografts removed on day 30, and from normal tracheas (N=3 in each group)
Figure 5: Smooth muscle cell α-actin staining of orthotopic tracheal allografts. A, A day 30 orthotopic allograft exhibited an evenly distributed layer of positive cells between the epithelium and the cartilage (x25). B, Higher magnification of the tracheal wall (x200). C, A day 60 orthotopic allograft had an intense staining of smooth muscle cells and myofibroblasts (x25). D, Higher magnification of the day 60 allograft showing the orientation of the spindled cells (x200).
Phenotype of the epithelium.
Immunofluorescence staining from the orthotopic grafts demonstrated that on day 7 the cuboidal or columnar allograft epithelium was of donor phenotype, whereas the underlying tracheal wall exhibited both recipient and donor phenotype. On day 30, the allografts of BN-origin had a strong anti-Lewis staining in the epithelium, whereas the anti-BN staining was low, and mainly seen in the chondrocytes. On day 60, all allograft cells exhibited anti-Lewis staining and the anti-BN staining was negative, suggesting remodeling of the allografts by cells of the host origin.
DISCUSSION
We have demonstrated that orthotopic tracheal transplantation was technically feasible in rats provided that meticulous surgical technique was applied. When the threat of respiratory distress during acute alloimmune response was avoided, success beyond the first 2 weeks led to long-term tracheal patency that was associated with epithelial regrowth on the allograft. The MHC phenotype on the regenerating orthotopic allograft epithelium was of donor phenotype on day 7, whereas the rest of the tracheal wall exhibited both recipient and donor phenotype. On day 30, the epithelium became completely remodeled by the host epithelium, consistent with our previous finding from heterotopic composite grafts and with a study in dogs where the epithelial phenotype of cryopreserved orthotopic allografts changed from donor type to recipient type between 10 and 20 days after transplantation (29
Because of abundant expression of class II antigens, the airway epithelium is believed to be the primary target of chronic rejection in human lung allografts (7 , 8 , 30 28 22 , 31
Improved revascularization could be one advantage of orthotopic allografts over heterotopic allografts, especially at the anastomotic regions. Nevertheless, the histological picture including tissue odema and epithelial cell necrosis was the same in the orthotopic grafts as described in the heterotopic grafts (3 32 , 33
Respiratory epithelium has a remarkable potential to regenerate and cover tubal structures. In large-animal studies, tubular prosthesis undergo epithelialization and the luminal surface of tracheas reconstructed from cryopreserved allograft cartilage becomes covered by ciliated epithelium within 2 weeks (14 , 34 19 35 19 , 36
The studies of tracheal trauma provide further evidence for the importance of the epithelium in the prevention of obliteration. Epithelial equivalent (fibroblast collagen matrix only) effectively prevents tracheal stenosis after trauma (34 37
Unlike large-animal models where survival of orthotopic tracheal allografts and avoidance of anastomotic rupture or stenosis requires either irradiation, preservation or immunosuppressive medication (38 , 39
In conclusion, orthotopic tracheal allografts in rats are feasible for studies of acute and chronic rejection. We have shown, for the first time, that the development of OAD in orthotopic allografts is significantly inhibited when compared to heterotopic allografts, and that the inhibition of progression of OAD is associated with epithelial regrowth onto the luminal surface of the orthotopic allografts. Further experiments might study the revascularization of the orthotopic allografts and molecular events in the regenerating epithelium. Because OAD is reduced, but not fully prevented, this model might contribute to the studies of chronic airway rejection and testing of new immunosuppressive drugs.
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