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The effect of basic fibroblast growth factor and omentopexy on revascularization and epithelial regeneration of heterotopic rat tracheal isografts
The effect of basic fibroblast growth factor and omentopexy on revascularization and epithelial regeneration of heterotopic rat tracheal isografts Donor airway ischemia is a significant problem after clinical lung transplantation despite the use of omentopexy for accelerated local bronchial revascularization. Several growth factors have been shown to induce angiogenesis in vitro and in vivo. In the present study the quantitative effects on tracheal revascularization and epithelial regeneration of omentopexy and continuous local administration of basic fibroblast growth factor were investigated in a heterotopic rat tracheal isograft model. Tracheas were harvested from donor rats and heterotopically implanted into the omentum of syngeneic recipient rats. Animals were randomly assigned to study groups differing only in treatment of the tracheal segments: omental wrap for 2, 7, or 14 days; omental wrap plus continuous local administration of basic fibroblast growth factor for 7 or 14 days; or omental wrap plus local application of saline for 7 or 14 days. Two, 7, or 14 days after the animals were put to death, the vascularity of the tracheal segments and attached omentum and the tracheal epithelial morphology were assessed in a blinded fashion with use of light microscopy and morphometric image analysis. Vascularity in tracheal segments treated with basic fibroblast growth factor was significantly (p < 0.05) greater than in control tracheas after 7 and 14 days. Epithelial regeneration was also improved in the basic fibroblast growth factor-treated groups at days 7 and 14 (p < 0.05). We conclude that continuous local administration of basic fibroblast growth factor enhances early revascularization of tracheal segments induced by omentopexy and accelerates epithelial regeneration in a heterotopic rat tracheal isograft model. (J THoRAc CARDIOVASC SURG 1992;104:180-8)
Eckhard Mayer, MD,* Paulo F. G. Cardoso, MD,a John D. Puskas, MD,a Kleber De Campos, MD,a Tadayuki Oka, MD,a Irving Dardick, MD, FRCPC,b and G. A. Patterson, MD, FRCSC,a Toronto, Ontario, Canada
Acute and chronic ischemic airway complications remain significant problems in clinical lung transplantation despite widespread success of the procedure. I, 2 From the Division of Thoracic Surgery, Department of Surgery" and Department of Pathology," University of Toronto, Toronto General Hospital, Toronto, Ontario, Canada. Supported by The Canadian Cystic Fibrosis Foundation. Received for publication Sept. 26, 1990. Accepted for publication April 29, 1991. Address for reprints: G. A. Patterson, MD, Suite 3108 Queeny Tower, I Barnes Hospital Plaza, St. Louis, MO 63110. *Supported by the Deutsche Forschungsgemeinschaft and the Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University Clinics, Mainz, Germany; present address: Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University Clinics, D-6S00 Mainz, Germany.
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Inadequate airway anastomotic healing has been more problematic in patients undergoing en bloc double lung transplantation with a single tracheal anastomosis than in single lung transplant recipients. I The currently favored bilateral sequential lung transplantation- procedure for patients requiring double lung transplantation may reduce the risk of airway necrosis. However, donor bronchus ischemia remains a significant problem. Although direct restoration of the bronchial artery blood flow has been attempted with some success in canine single lung transplantationv" and clinical lung transplantation, I currently employed techniques in pulmonary transplantation make no attempt to restore the arterial bronchial circulation. Therefore the ischemic donor bronchus is dependent on collateral flow from the pulmonary circulation until local tissue collaterals can develop. Pulmonary to bronchial collateral flow has been
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Effect of bFGF and omentopexy on tracheal isografts
I8I
Tracheal ,._~~~===~~ segment ---:-i/---Omentum
1
Fig. 1. Tracheal segment is stretched on steel wire and connected to miniosmotic pump by a catheter. Trachea is placed on recipient omentum and ready to be wrapped.
Table I. Study groups I
(n
Harvest day Omental wrap
= 7)
o
Saline/pump' bFGF/pump
4B (n = 6) 14
+ +
-t-, Present; -, not present.
extensively studied.i- 6 yet the determinants of retrograde mucosal blood flow are not known. Enhancement of local revascularization appears to be an option to improve donor airway ischemia. Bronchial omentopexy has been demonstrated to enhance canine donor bronchus revascularization within 4 days.i- 8 Other tissue pedicles have been shown to restore bronchial collateral circulation." The omentum has been used routinely, however, in clinical lung transplantation. Omentum contains a rich vascular and lymphatic network. In addition, omental extract has been demonstrated to possess angiogenic properties. 10-12 Several growth factors have been isolated and shown to induce angiogenesis in vitro 13, 14 and in ViVO.15, 16 Basic fibroblast growth factor (bFGF) is considered to have the most potent angiogenic properties.!" The present study was designed to quantify, in a heterotopic rat tracheal isograft model, the effects on tracheal revascularization and epithelial regeneration of omentopexy and continuous local administration of bFGF.
Material and methods Seventy male inbred Lewis rats (250 to 300 gm; Charles RiverCanada Inc., S1.Constant, Quebec, Canada) were used
in this study. Tracheas were resected from 21 donor animals and cut into two segments, each of which was heterotopically implanted into the omentum of 42 recipient animals. Tracheal segments from seven donor rats were harvested and not reimplanted (group 1). Recipient animals were randomly assigned to one of three other study groups: group 2 animals received no treatment except the heterotopic tracheal implantation, and they were killed after 2 days (group 2A, n = 6), 7 days (group 2B, n = 6), or 14days (group 2C, n = 6). Group 3 animals were treated with a local continuous infusion of bFGF at the site of the tracheal omentopexy for 7 days (group3A, n = 6) or l4days (group 3B, n = 6). Group 4 animals were treated with a continuous local application of saline for 7 days (group 4A, n = 6) or 14days (group 4B, n = 6). The experimental conditionsfor each study group are summarized in Table I. The animals were put to death on the last day of treatment (groups 3A and 4A at day 7; groups 3B and 4B at day 14). Donor procedure. Rats were anesthetized with pentobarbital sodium (40 mg/kg intraperitoneally) and heparin was injected (500 IU /kg intraperitoneally). With use of a sterile technique, the upper trachea was exposedthrough a midline incision in the neck. A median sternotomy was performed and the thymus gland was resected. The right anterior vena cava, innominate artery, and aortic arch were dividedbetween ligatures. The distal area of the trachea was dissected free to its bifurcation, and its length was measured. Both main bronchi were divided, and the trachea was bluntly extracted with use of a minimum of sharp dissectionbetween trachea and esophagus. The trachea was divided caudally to the larynx, and the tracheal lumen was
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flushed with 10 ml of sterile saline. With the use of a cork board with a wet sponge, the trachea was stretched to its original length (28 to 32 mm) and maintained in this length by two needles on either end. The trachea was divided into two segments with 10 cartilaginous rings each. The ends of each segment were mounted on a splint consisting of a U-shaped stainless steel wire (No.4 surgical steel, 22 gauge, Ethicon, Inc., Somerville, N.J.) with a length of 14 to 16 mm, according to the original length of the tracheal segment, to avoid shrinking of the extracted segment. The sharp ends of the steel wire were covered with vinyl tubing to avoid injuries to the recipient. The segments were stored in a wet sponge for 10 to 20 minutes during preparation of recipient animals. Drug administration. In groups 3 and 4, bFGF or sterile saline was continuously administered by means of a miniosmotic pump (Alza Corp. Palo Alto, Calif.). Four to 6 hours after implantation, each osmotic pump was weighed and filled with 200 ng of bFGF (isolated from bovine pituitary gland; Collaborative Research Inc., Bedford, Mass.) diluted in 200 JlI of saline (group 3A; pump model m-2ool, nominal pumping rate, 1.0 JlI/hr), 400 ng of bFGF diluted in 200 JlI of saline (group 3B; pump model m-2oo2, nominal pumping rate 0.5 JlI/hr), or 200 JlI of saline (group 4A, model m-2001; group 4B, model m-2002). The pumps were each weighed to ensure correct filling and incubated in sterile saline at 37° C for 4 to 6 hours. A 25 mm vinyl catheter (V-60, Bolab Inc., Lake Havasu City, Ariz.) was closed at one end and perforated with a needle to provide six to eight channels for a distance of 12 mm. The perforated part was attached to the U-formed steel wire with two 6-0 Prolene sutures (Ethicon, Inc., Somerville N.J.). The catheter was filled with the same solution contained in a particular pump and connected to the pump flow regulator. The donor tracheal segment was attached to the steel wire, with a distance of I to 2 mm maintained between the perforated part of the catheter and the trachea (Fig. 1). Recipient procedure. Recipient rats were anesthetized in an induction chamber (halothane, 3%), and anesthesia was maintained with mask ventilation (1.5% to 2.5% halothane; Fio, 1.0). In a sterile fashion the omentum was brought out through a 2 em upper median laparotomy. In group 2 the donor tracheal segment stretched on a steel wire was completely wrapped in omentum by means of two 6-0 Prolene sutures that excluded the wire from the central omental wrap containing the portion of trachea. Tracheal segments in groups 3 and 4 were wrapped in omentum in the same way except that the perforated part of the catheter was included in the central region with the tracheal segment. These, along with the attached pump, were then returned to the abdomen. The abdomen was closed with use of interrupted 4-0 Prolene sutures, and the skin was approximated with staples. After recovery from anesthesia, the animals were placed in cages (one per cage) and fed standard laboratory rat food and water ad libitum. Assessment. Two (group 2A), 7 (groups 2B, 3A, 4A), or 14 (groups 2C, 3B, 4B) days postoperatively, the animals were anesthetized with halothane and the omentum was excised through a right lateral laparotomy. The steel wire and catheter were carefully removed from the tracheal segment wrapped in omentum. In groups 3 and 4 it was confirmed that the pump contained a small residual volume of fluid. The tip of each tracheal specimen was incisedto allow fixation by immersion in 10% buffered formalin. The animals were killed by exsanguination. Tracheas in group I were fixed in 10% buffered formalin immediately after harvest. After embedding in paraffin,
The Jour.nal of Thoracic and Cardiovascular Surgery
4 Jlm thick longitudinal sections of the trachea and adherent
omentum were cut from the midpoint of the cartilaginous trachea, stained with hematoxylin and eosin, and assessed histologically in a blinded fashion without reference to the different experimental conditions. Vascular evaluation. Changes in the vascularity of the tracheal segments and the surrounding omental tissue were assessed with use of morphometric image analysis. To avoid misinterpretations due to artifact at the ends of the tracheal segment and to conform to size constraints of imaging equipment, only the 2 mm segment in the center of the longitudinal section was examined. Image analysis was done with use of a video camera (MTI 65, DAGE-MTI, Michigan City, Ind.) connected to a light microscope (Leitz Colorlux K, Leitz Wetzlar, Germany) to capture the digitized image of each 2 mm segment (X83 magnification) on a video screen (Multi Sync 3D, NEC Information Systems, Boxborough, Mass.) for subsequent analysis. Vesselnumber per square millimeter tracheal (VN /T) and omental (VN /0) area and vessel area per tracheal (VA/ T) and omental (VA/a) area in percent were calculated, with use of the methodology described presently, as the average of three measurements for each trachea. Numerical evaluation of the blood vessels and the area occupied by them within the cartilaginous part of the trachea and the adjacent omentum was performed at x208 magnification. This was done with use of a digitizing pad (Hipad Digitizer, Houston Instruments, Austin, Tex.) and pen linked to an NEC Powermate 386/20 personal computer utilizing niorphometrysoftware (Bioquant IV, R&M Biometrics, Nashville Tenn.). No attempt was made to distinguish between arteries and veins within the tracheal segments and the omentum. Whenever the identification of a blood vessel was equivocal, the X208 magnification picture was saved and the corresponding tracheal area was examined at a x832 magnification. The total number of vessels (VN) and total luminal area occupied by vessels (VA) were measured within an area of trachea (T) determined by an 800 X 800 Jlm' grid centered between two cartilage rings. VN and VA in the adherent area of the omentum (0) were also determined. The measurements were done at three random sites of the center 2 mm part of each trachea. Epithelial assessment. The tracheal epithelium as a "target organ" of revascularization was examined with regard to epithelial regeneration and epithelial thickness. With use of x832 magnification, epithelial regeneration was assessed according to the following scale: 0: No epithelium, single nonconfluent epithelial cells I: Confluent single-layered nonciliated epithelium 2: Confluent multilayered nonciliated epithelium 3: Normal mucociliary epithelium Along the fixed longitudinal section of airway, 10 random areas of epithelium with a length of 200 Jlm each were examined, five sites on cartilage rings and five between rings. The grades of each evaluation were added; therefore the maximal grade representing a morphologically normal epithelium was 30, and the minimum score representing no confluent epithelium was O. Length and area of the mucosa within the three random sites that were evaluated for vascularity were measured, and the epithelial thickness was calculated (thickness = width = areal length). Three thickness values were averaged for each tracheal segment. Statistical analysis. Data analysis was performed with use of SAS software (SAS, Inc., Cary, N.C.). To achieve homoge-
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Effect of bFGF and omentopexy on tracheal isografts
18 3
Fig. 2. Cartilaginous part of trachea after 2 days of omental wrapping (group 2A). Severe submucosal hemorrhage. Epithelial structure is damaged more severely in area underlying cartilage ring than in intercartilaginous area. (Original magnification X417.)
Table II. Tracheal graft vascularity Group
1 (native)
VN/T (jmm 2) VA/T (%)
61.6 ± 8.2 3.07 ± 0.26
2A (OM2D)
2C
2B (OM 7D)
(OM 14D)
3A (bFGF7D)
3B (bFGF 14D)
4A (SAL 7D)
4B (SAL 14D)
24.6* ± 3.2 1.44* ± 0.18
36.5t ± 5.5 1.75* ± 0.17
36.4* ± 2.8 3.43 ± 0.54
45.1 ± 4.1 2.84 ± 0.46
28.8t ± 5.2 2.05 ± 0.43
34.7t ± 4.4 1.99 ± 0.34
Native, No treatment or implantation of tracheal segment; OM, omental wrap; D, day; bFGF, growth factor/pump treatment; SAL, saline/pump treatment; VN/T, vessel number per tracheal area; VA/T, vessel area per tracheal area; - , identification of vessels not possible. All values are mean ± standard error of the mean. 'p < 0.05 vs group I (native tracheas).
tp
< O.Ol vs group
1.
neous variances, all data were transformed to logarithms before one-way or two-way analysis of variance. Contrasts were used for comparing multiple treatments with a common control (native tracheas). Tukey's studentized range test was chosen for multiple comparisons between the groups. The level of significance was defined as 0.05; all values are presented as mean ± standard error of the mean. The animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978).
Results Becauseofmacroscopic(n = 2) or microscopic (n = 1) evidence of infection, three animals were excluded from further histopathologic assessment. Catheter disconnection from the graft site or from the pump flow modulator was not observed in any of the animals.
Tracheal blood vessels. Tracheal vessels in group 2A (omental wrap for 2 days) could not be identified and quantified because of severe hemorrhage (Fig. 2). The VN/T was significantly higher in native tracheas (group 1) than in any other group except the 14-day bFGF group 3B (Table II). VN/T and VA/T were higher after 14 days of omental wrapping (group 2C: VN/T, 36,5% ± 5.5%; VA/T, 1.75% ± 0.17%) in comparison with 7 days (group 2B: VN/T, 24.6% ± 3.2%; VA/T, 1.44% ± 0.18%) without reaching statistical significance. Vascularity was significantly superior in the bFGFtreated tracheas compared with control and saline-treated tracheas. At day 7 the bFGF/pump group (group 3A) showed a significant (p < 0.05) increase in VA/T compared with the nontreatment group 2B and the saline/ pump group 4A (Fig. 3, A). VN/T in the bFGF-treated group (3A) was significantly higher (p < 0.05) than in
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Mayer et al.
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* : p ee.0 6
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GROUP 3A
70 OMENTAL WRAP. n·8
70 SALINE. n'5
70 bFGF. n-8
VN/T (per mm2j
~-----------------=------,
*:
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GROUP 20
NATlYE, n-7
20 OMENTAL
70 OMENTAL
,.0 OMENTAL
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peo.os va G 28
SCORE
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30,----,----,---,--,---------------.------, 30
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--25
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Y. OMENTAL WRAP, SALINE···'
20
10
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70 OMENTAL WRAP. n·8
70 SALINE. n·5
GROUP 3A 7D bFGF,
n-e
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7 DAYS
_
NON-TREATED
14 DAYS _
SALINE
0
bFGF
Fig. 3. Tracheal vascularity at day 7. A, Surface area of blood vessels per tracheal area (VA/T) is significantly higherin basic fibroblast growth factor (bFGF)-treated group than in omental wrap group and saline-treatedgroup. B, Number of vessels per tracheal area (VN/T) is significantly higher in bFGFtreated group than in omental wrap group.
Fig. 4. Epithelial regeneration score. A, Tracheas wrapped in omentum and not treated showed a significant increase in epithelial score over time. B, bFGF-treated groups had a significantly higher epithelial score than omental wrap groups and saline-treated groups at days 7 and 14.
group 2B (omental wrap); the difference between group 3A (bFGF) and the saline-treated group (4A) did not reach statistical significance (Fig. 3, B). At day 14 VA/T was significantly (p < 0.05) increased in the bFGF group (3B) compared with the control group (2C) and salinetreated group (4B) (see Table 11). There was no significant difference in VN/T between the groups after 14 days. Omental blood vessels. After 2 days of omental wrapping, an analysis of omental blood vessels was impossible because there was no firm connection between tracheal segment and omentum. The data of omental vessel numbers (VN/O) and areas (VA/O) are summarized in Table III. After 7 days VN/O was higher in the bFGF group (3A) compared with groups 2B and 4A; however, this difference did not reach statistical significance (p = 0.07). At day 14 there was no difference in VN/O and VA/O between the groups (see Table III).
Epithelial viability. In the nontreated groups there was a significant (p < 0.001) increase of the epithelial scoring grade over time (Fig. 4, A). After 2 days (group 2A) there was no confluent epithelial layer in any of the tracheal segments. Mucosal areas underlying cartilage rings appeared to be damaged more severely than intercartilage areas (see Fig. 2). After 7 days epithelial morphologyvaried from a simple undifferentiated monolayer epithelium to areas with morphologically normal, ciliated epithelium. Most of the tracheal segments harvested after 7 days, however, were characterized by a multilayered epithelium with a high percentage of secretory cells and without ciliated cells (Fig. 5). After 14 days epithelial areas with ciliary differentiation (Fig. 6) alternated with areas characterized by a nonciliated, multilayered epithelium. The thickness of the epithelium in the control groups increased significantly between 2 days and 7 days (group
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Fig. 5. Multilayered, metaplastic, hypersecretory epithelium of a trachea wrapped in omentumfor 7 days. (Group 2B,original magnification X667.) Table III. Omental vascularity Group
1 (native)
VN/O (frnrn2) VA/O (%)
2A (OM 2D)
38
48
28
2C
(OM7D)
(OM 14D)
3A (bFGF7D)
(bFGF 14D)
4A (SAL 7D)
(SAL 14D)
36.3 ± 4.6 2.54 ± 0.24
7\.9 ± 18.1 5.14±1.40
68.6 ± 8.5 4.60 ± 0.89
64.5 ± 9.5 7.22 ± \.00
46.2 ± 4.9 3.46 ± 1.00
56.3 ± 3.6 7.20 ± 1.17
Native, No treatment or implantation of tracheal segment; OM, omental wrap; D, day; bFGF, growth factor/pump treatment; SAL, saline/pump treatment; VN/O, vessel number per omental area; VA/O, vessel area per omental area; - , no omentum or identification of omental vessels not possible. All values are mean ± standard error of the mean.
2A, 7.2 ± 0.69 Jlm; group 2B, 42.6 ± 3.48 um;
p < 0.05). The decrease in thickness of the epithelium between days 7 and 14 was not statistically significant (group 2B, 42.6 ± 3.48 Jlm; group 2C, 31.6 ± 3.43 Jlm). At day 7 the epithelium was significantly thicker than in native tracheas (group 2B, 42.6 ± 3.48 Jlm; group I, 25.4 ± 1.46 Jlm; p < 0.001); there was no difference in thickness of the epithelium between 14-day control tracheas and native tracheas. bFGF-treated tracheas had a significantly (p < 0.05) higher epithelial score grade than nontreated and salinetreated segments after 7 and 14 days (Fig. 4, B). Group 3B (bFGF, 14 days) epithelial score was similar to that of native tracheas (group I, 29 ± 0.3; group 3B, 29.1 ± 0.4; p = 0.96). Discussion Ischemic airway complications have been significant causesof morbidity and mortality after lung transplantation.I, 2 Since the bronchial arterial circulation is generally not surgically reconnected, the donor bronchus is
dependent on retrograde collateral flow from the pulmonary artery circulation and local tissue revascularization. Omentum appears to contain specific angiogenic factors l O- 12 and has proved successful for local revascularization of ischemic tissue. 17-20 Omentum will revascularize ischemic canine donor bronchiv": therefore it has been used routinely to wrap the bronchial anastomoses in clinicallung transplantation. I, 2 With use of a "telescoping" technique of bronchial anastomosis and early postoperative corticosteroids, the San Antonio group-' recently reported satisfactory bronchial healing after single lung transplantation without bronchial omentopexy. In the present study continuous local administration of bFGF significantly increased the vascularity of the initially completely devascularized tracheal segments wrapped in omentum at days 7 and 14. After 2 days of omental wrapping, original tracheal vessels could not be identified because of severe hemorrhage and vascular destruction. The same phenomenon has been observed in canine models after 4 and 5 days.7 8 Although the mechanism of airway revascularization by omentum remains 0
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Fig. 6. Structurally normal, mucociliary epithelium of a trachea wrapped in omentum for 14 days and treated with bFGF. (Group 3B, original magnification X667.)
unclear, our findings suggest that continuous local bFGF administration enhances this process. Because the differences in vascularity between nontreated and bFGF stimulated tracheal segments were less obvious at day 14 compared with day 7, bFGF administration appeared to enhance vessel ingrowth preferentially in the early period after implantation. At day 7 the vascularity of the surrounding omentum also seemed to be increased in the bFGF group compared with the nontreatment group, although the difference did not reach statistical significance. There was no difference in omental vascularity after 14 days. Similar results were reported by Eppley and coworkersl''; they found that the most significant difference in revascularization of mandibular bone grafts between bFGF and control groups occurred at day 10. It is possible that the difference we noted at day 7 might also have been present at day 5 or day 10. We were limited in the number of samples we could process, however, and therefore arbitrarily selected 2, 7, and 14 days. In vivo regeneration of airway epithelium after mechanical and toxic injury has been extensively described in the literature. 22- 27 It has also been shown that differentiation of tracheal epithelium in primary cell culture recapitulates regeneration after injury and normal fetal development of tracheal epithelium.P Although the effects of ischemia on epithelial regeneration are not known, we hypothesized that epithelial regeneration of ischemic tracheal segments might be similar to regeneration after other kinds of injury. The scoring system used in the present study is based on the descriptions of hamster epithelial regeneration. 22-24, 27, 28 After denuding epithelial injury, a flat, monolayered, undifferentiated
epithelium can be observed in the first 2 days. Between days 2 and 6, a multilayered metaplastic epithelium develops by division of secretory cells, and the normal morphology of epithelium with one or two layers of ciliated, secretory, and basal cells is reached between 6 and 8 days. In the present study similar stages of epithelial regeneration were observed; however, the time-course seemed to be delayed compared with regeneration after nonischemic mechanical injury.22-24, 27 After 2 days of omental wrapping, a confluent monolayer of undifferentiated epithelial cells could be found in only a few intercartilaginous areas, whereas there was no confluent epithelium over cartilage rings. The intercartilaginous tissue appears to be the only possible route for revascularization. After 7 days of omental wrapping without treatment, the epithelial score was increased and tracheas were characterized by a multilayered nonciliated epithelium that was similar to the epithelium described 3 to 6 days after mechanical injury and in cell culture. 22-24, 27, 28 At day 14, epithelial areas showing metaplasia alternating with areas of normal epithelium could be found in the nontreated tracheas. Tracheal segments treated with bFGF showed the highest degree of epithelial regeneration at days 7 and 14 and could not be distinguished from native tracheas in terms of epithelial morphology after 14 days. Although a mitogenic effect of bFGF on epithelial cells has been demonstrated.l? we consider the acceleration of epithelial regeneration in our model to be a result of improved revascularization by bFGF. The morphology of submucosal cartilage, difficult to identify and quantitate, was not assessed. We do not know
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whether other bFGF mitogenic effects on fibroblasts or smooth muscle cells 29 or bFGF-induced cartilage repair/" 30 were operative in these devascularized tracheal segments. The heterotopic rat tracheal isograft model used allows the evaluation of local revascularization without the influences of graft rejection, immunosuppression, and retrograde mucosal blood flow from the pulmonary circulation. It could be readily used, for example, to study the effects of rejection or immunosuppression on tracheal revascularization and viability. The use of foreign material such as surgical steel wire, catheters, and minipumps in the peritoneal cavity may induce artifacts in the assessment of revascularization. Implanting the tracheal segments without stretching them, however, would leave in the omentum tracheal segments that are shrunken to almost half of their normal anatomic length, exposing a surface with a high percentage of cartilage. Cartilage has been shown to inhibit the growth of endothelial cells.'! The catheter and minipump were used for continuous local drug administration.V This may be important since in a rabbit model we did not demonstrate any angiogenic effect of bFGF on tracheal revascularization with use of a single topical bFGF application.P A certain degree of foreign body reaction and increased vascularity could be observed, however, after 7 and 14 days in the saline/pump groups compared with the control groups, although differences were not statistically significant. Since the beginning of this study we have adopted clinically the strategy proposed by the San Antonio group" regarding the bronchial anastomosis, and we have been pleased with the preliminary experience in both single and bilateral sequential lung transplantation. Nonetheless, more rapidlocal tissue revascularization of the ischemic donor bronchus would provide even greater security. We conclude that continuous local administration of bFGF enhances the effect of omentopexy on early revascularization and epithelial regeneration of devascularized tracheal segments in a heterotopic rat tracheal isograft model. We would like to acknowledge the excellent technical assistanceofS. Diamant, J. Mates, K. Ekern, and D. Hunter. Assistance with statistical analysis was receivedfrom the Statistical Consulting Service (K. Knight, PhD) of the Department of Statistics, University of Toronto. REFERENCES I. Patterson GA, Todd TR, Cooper JD, et al. Airway complications after double lung transplantation. J THORAC CARDlOVASC SURG 1990;99:14-21. 2. Pasque MK, Cooper JD, Kaiser LR, Haydock DA, Triantafillou A, Trulock EP. Improved technique for bilateral
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lung transplantation-rationale and initial clinical experience. Ann Thorac Surg 1990;49:785-91. 3. Mills NL, Boyd AD, Gherapong C. The significance of bronchial circulation in lung transplantation. J THoRAc CARDIOVASC SURG 1970;60:866-74. 4. Veith FJ, Torres M, Colon J, et al. Transplantation of the left lung in to the right hemithorax to facilitate immediate reconstitution of bronchial artery flow. J THORAC CARDlOVASC SURG 1978;75:141-6. 5. Ladowski JS, Hardesty RL, Griffith BP. The pulmonary artery blood supply to the supracarinal trachea. J Heart Transplant 1984;4:40-2. 6. Barman SA, Ardell JL, Parker JC, Perry ML, Taylor AE. Pulmonary and systemic blood flowcontributions to upper airways in canine lung. Am J PhysioI1988;255:HI130-5. 7. Lima 0, Goldberg M, Peters WJ, Ayabe H, Townsend E, Cooper JD. Bronchial omentopexy in canine lung transplantation. J THORAC CARDIOVASC SURG 1982;83:418-21. 8. Morgan E, Lima 0, 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. 9. Turrentine MW, Kesler KA, Wright CD, et al. Effect of omental, intercostal and internal mammary artery pedicle wraps on bronchial healing. Ann Thorac Surg 1990;49: 574-9. 10. Goldsmith HS, Griffith AL, Catsimpoolas N. Increased vascular perfusion after administration of an omental lipid fraction. Surg Gynecol Obstet 1986;162:579-83. 11. Nottebaert M, Lane JM, Juhn A, et al. Omental angiogenic lipid fraction and bone repair: an experimental study in the rat. J Orthop Res 1989;7:157-69. 12. Cartier R, Brunette I, Hashimoto K, Bourne WM, Schaff HV. Angiogenic factor: a possible mechanism for neovascularization produced by omental pedicles. J THORAC CARDIOVASC SURG 1990;99:264-8. 13. Gospodarowitz D, Baird A, Cheng J, Lui GM, Esch F, Bohlen P. Isolation of fibroblast growth factor from bovine adrenal gland: physiochemicaland biologicalcharacterization. Endocrinology 1986;118:82-90. 14. Folkman J, Klagsbrun M. Angiogenic factors. Science 1987;235:442-7. 15. Thompson JA, Anderson KD, DiPietro JM, et al. Site-directed neovessel formation in vivo.Science 1988;241:134952. 16. Eppley BL, Doucet M, Connolly DT. Feder J. Enhancement of angiogenesis by bFGF in mandibular bone graft healing in the rabbit. J Oral Maxillofac Surg 1988;46: 391-8. 17. Pifarre R, Hufnagel CA. Epicardiectomy and omental graft in acute myocardial infarction. Am J Surg 1968; 115:589-93. 18. Goldsmith JS, Kiely AA, Randal HT. Protection of intrathoracic esophageal anastomoses by omentum. Surgery 1968;63:464-6. 19. Dupont C, Menard Y. Transposition of the greater omentum for reconstruction of the chest wall. Plast Reconstr Surg 1972;49:263-7.
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20. Virkkula L, Eerola S. Use of omental pedicle for treatment of bronchial fistula after lower lobectomy. Scand J Thorac Cardiovasc Surg 1975;9:287-90. 21. Calhoon JH, Grover FL, Gibbons WJ, et al. Single lung transplantation: alternative indications and technique. J THORAC CARDIOVASC SURG 1991;101:816-25. 22. McDowell EM, Becci PJ, Schurch W, Trump BF. The respiratory epithelium. VII. Epidermoid metaplasia of hamster tracheal epithelium during regeneration following mechanical injury. J Nat! Cancer Inst 1979;62:995-1006. 23. Keenan KP, Combs JW, McDowell EM. Regeneration of hamster tracheal epithelium after mechanical injury. I. Focal lesions:quantitative morphologic study of cell proliferation. Virchows Arch [B) 1982;41:193-214. 24. Keenan KP, Wilson TS, McDowell EM. Regeneration of hamster tracheal epithelium after mechanical injury. VI. Histochemical, immunocytochemical and ultrastructural studies. Virchows Arch [B) 1983;43:213-40. 25. Gordon AE, Lane BP. Ciliated cell differentiation in regenerating rat tracheal epithelium. Lung 1984;162:23343. 26. Evans MJ, Shami SG, Cabral-Anderson LJ, Dekker NP. Role of nonciliated cells in renewal of the bronchial epithelium of rats exposed to N0 2. Am J PathoI1986;123:12633. 27. Keenan KP, Saffiotti U, Stinson SF, Riggs CW, McDow-
28.
29.
30.
31.
32.
33.
ell EM. Morphological and cytokinetic responses of hamster airways to intralaryngeal or intratracheal cannulation with instillation of saline or ferric oxide particles in saline. Cancer Res 1989;49:1521-7. McDowell EM, Ben T, Newkirk C, Chang S, DeLuca LM. Differentiation of tracheal mucociliary epithelium in primary cell culture recapitulates normal fetal development and regeneration followinginjury in hamsters. Am J Pathol 1987;129:511-22. Gospodarowicz D. Isolation and characterization of acidic and basic fibroblast growth factor. Methods Enzymo11987; 147:106-19. Cuevas P, Burgos J, Baird A. Basic fibroblast growth factor (FGF) promotes cartilage repair in vivo. Biochem Biophys Res Cornmun 1988;156:611-8. Eisenstein R, Kuettner KE, Neapolitan C, et al. Cartilage extracts inhibit the growth of fibroblasts and endothelial cells in culture. Am J PathoI1975;81:337-43. Urquhart J, Fara JW, Willis KL. Rate-controlled delivery systems in drug and hormone research. Annu Rev Pharmacol ToxicoI1984;24:199-236. Olech VM, Keshavjee SH, Chamberlain DW, Slutsky AS, Patterson GA. The role of basic fibroblast growth factor in the revascularization of rabbit tracheal autografts. Ann Thorac Surg 1991;52:258-64.
Bound volumes available to subscribers Boundvolumesof THEJOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY are availableto subscribers(only)for the 1992 issues from the Publisher, at a cost of $71.00 for domestic,$93.97 for Canadian, and $89.00 for internationalsubscribersfor Vol. 103 (JanuaryJune) and Vol. 104 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertisingis removed.Copiesare shippedwithin 60 days after publicationof the last issueof the volume.The bindingisdurable buckram with the JOURNAL name, volumenumber, and year stamped in goldon the spine. Paymentmust accompany all orders. Contact MosbyYear Book,Inc., SubscriptionServices, 11830 Westline Industrial Drive,St. Louis, Missouri 63146-3318, USA; phone (800) 325-4177, ext. 4351, or (314) 453-4351. Subscriptionsmust be in force to qualify. Boundvolumes are not available in place of a regular JOURNAL subscription.
Eckhard Mayer, MD,* Paulo F. G. Cardoso, MD,a John D. Puskas, MD,a Kleber De Campos, MD,a Tadayuki Oka, MD,a Irving Dardick, MD, FRCPC,b and G. A. Patterson, MD, FRCSC,a Toronto, Ontario, Canada
Acute and chronic ischemic airway complications remain significant problems in clinical lung transplantation despite widespread success of the procedure. I, 2 From the Division of Thoracic Surgery, Department of Surgery" and Department of Pathology," University of Toronto, Toronto General Hospital, Toronto, Ontario, Canada. Supported by The Canadian Cystic Fibrosis Foundation. Received for publication Sept. 26, 1990. Accepted for publication April 29, 1991. Address for reprints: G. A. Patterson, MD, Suite 3108 Queeny Tower, I Barnes Hospital Plaza, St. Louis, MO 63110. *Supported by the Deutsche Forschungsgemeinschaft and the Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University Clinics, Mainz, Germany; present address: Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University Clinics, D-6S00 Mainz, Germany.
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Inadequate airway anastomotic healing has been more problematic in patients undergoing en bloc double lung transplantation with a single tracheal anastomosis than in single lung transplant recipients. I The currently favored bilateral sequential lung transplantation- procedure for patients requiring double lung transplantation may reduce the risk of airway necrosis. However, donor bronchus ischemia remains a significant problem. Although direct restoration of the bronchial artery blood flow has been attempted with some success in canine single lung transplantationv" and clinical lung transplantation, I currently employed techniques in pulmonary transplantation make no attempt to restore the arterial bronchial circulation. Therefore the ischemic donor bronchus is dependent on collateral flow from the pulmonary circulation until local tissue collaterals can develop. Pulmonary to bronchial collateral flow has been
Volume 104 Number 1 July 1992
Effect of bFGF and omentopexy on tracheal isografts
I8I
Tracheal ,._~~~===~~ segment ---:-i/---Omentum
1
Fig. 1. Tracheal segment is stretched on steel wire and connected to miniosmotic pump by a catheter. Trachea is placed on recipient omentum and ready to be wrapped.
Table I. Study groups I
(n
Harvest day Omental wrap
= 7)
o
Saline/pump' bFGF/pump
4B (n = 6) 14
+ +
-t-, Present; -, not present.
extensively studied.i- 6 yet the determinants of retrograde mucosal blood flow are not known. Enhancement of local revascularization appears to be an option to improve donor airway ischemia. Bronchial omentopexy has been demonstrated to enhance canine donor bronchus revascularization within 4 days.i- 8 Other tissue pedicles have been shown to restore bronchial collateral circulation." The omentum has been used routinely, however, in clinical lung transplantation. Omentum contains a rich vascular and lymphatic network. In addition, omental extract has been demonstrated to possess angiogenic properties. 10-12 Several growth factors have been isolated and shown to induce angiogenesis in vitro 13, 14 and in ViVO.15, 16 Basic fibroblast growth factor (bFGF) is considered to have the most potent angiogenic properties.!" The present study was designed to quantify, in a heterotopic rat tracheal isograft model, the effects on tracheal revascularization and epithelial regeneration of omentopexy and continuous local administration of bFGF.
Material and methods Seventy male inbred Lewis rats (250 to 300 gm; Charles RiverCanada Inc., S1.Constant, Quebec, Canada) were used
in this study. Tracheas were resected from 21 donor animals and cut into two segments, each of which was heterotopically implanted into the omentum of 42 recipient animals. Tracheal segments from seven donor rats were harvested and not reimplanted (group 1). Recipient animals were randomly assigned to one of three other study groups: group 2 animals received no treatment except the heterotopic tracheal implantation, and they were killed after 2 days (group 2A, n = 6), 7 days (group 2B, n = 6), or 14days (group 2C, n = 6). Group 3 animals were treated with a local continuous infusion of bFGF at the site of the tracheal omentopexy for 7 days (group3A, n = 6) or l4days (group 3B, n = 6). Group 4 animals were treated with a continuous local application of saline for 7 days (group 4A, n = 6) or 14days (group 4B, n = 6). The experimental conditionsfor each study group are summarized in Table I. The animals were put to death on the last day of treatment (groups 3A and 4A at day 7; groups 3B and 4B at day 14). Donor procedure. Rats were anesthetized with pentobarbital sodium (40 mg/kg intraperitoneally) and heparin was injected (500 IU /kg intraperitoneally). With use of a sterile technique, the upper trachea was exposedthrough a midline incision in the neck. A median sternotomy was performed and the thymus gland was resected. The right anterior vena cava, innominate artery, and aortic arch were dividedbetween ligatures. The distal area of the trachea was dissected free to its bifurcation, and its length was measured. Both main bronchi were divided, and the trachea was bluntly extracted with use of a minimum of sharp dissectionbetween trachea and esophagus. The trachea was divided caudally to the larynx, and the tracheal lumen was
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flushed with 10 ml of sterile saline. With the use of a cork board with a wet sponge, the trachea was stretched to its original length (28 to 32 mm) and maintained in this length by two needles on either end. The trachea was divided into two segments with 10 cartilaginous rings each. The ends of each segment were mounted on a splint consisting of a U-shaped stainless steel wire (No.4 surgical steel, 22 gauge, Ethicon, Inc., Somerville, N.J.) with a length of 14 to 16 mm, according to the original length of the tracheal segment, to avoid shrinking of the extracted segment. The sharp ends of the steel wire were covered with vinyl tubing to avoid injuries to the recipient. The segments were stored in a wet sponge for 10 to 20 minutes during preparation of recipient animals. Drug administration. In groups 3 and 4, bFGF or sterile saline was continuously administered by means of a miniosmotic pump (Alza Corp. Palo Alto, Calif.). Four to 6 hours after implantation, each osmotic pump was weighed and filled with 200 ng of bFGF (isolated from bovine pituitary gland; Collaborative Research Inc., Bedford, Mass.) diluted in 200 JlI of saline (group 3A; pump model m-2ool, nominal pumping rate, 1.0 JlI/hr), 400 ng of bFGF diluted in 200 JlI of saline (group 3B; pump model m-2oo2, nominal pumping rate 0.5 JlI/hr), or 200 JlI of saline (group 4A, model m-2001; group 4B, model m-2002). The pumps were each weighed to ensure correct filling and incubated in sterile saline at 37° C for 4 to 6 hours. A 25 mm vinyl catheter (V-60, Bolab Inc., Lake Havasu City, Ariz.) was closed at one end and perforated with a needle to provide six to eight channels for a distance of 12 mm. The perforated part was attached to the U-formed steel wire with two 6-0 Prolene sutures (Ethicon, Inc., Somerville N.J.). The catheter was filled with the same solution contained in a particular pump and connected to the pump flow regulator. The donor tracheal segment was attached to the steel wire, with a distance of I to 2 mm maintained between the perforated part of the catheter and the trachea (Fig. 1). Recipient procedure. Recipient rats were anesthetized in an induction chamber (halothane, 3%), and anesthesia was maintained with mask ventilation (1.5% to 2.5% halothane; Fio, 1.0). In a sterile fashion the omentum was brought out through a 2 em upper median laparotomy. In group 2 the donor tracheal segment stretched on a steel wire was completely wrapped in omentum by means of two 6-0 Prolene sutures that excluded the wire from the central omental wrap containing the portion of trachea. Tracheal segments in groups 3 and 4 were wrapped in omentum in the same way except that the perforated part of the catheter was included in the central region with the tracheal segment. These, along with the attached pump, were then returned to the abdomen. The abdomen was closed with use of interrupted 4-0 Prolene sutures, and the skin was approximated with staples. After recovery from anesthesia, the animals were placed in cages (one per cage) and fed standard laboratory rat food and water ad libitum. Assessment. Two (group 2A), 7 (groups 2B, 3A, 4A), or 14 (groups 2C, 3B, 4B) days postoperatively, the animals were anesthetized with halothane and the omentum was excised through a right lateral laparotomy. The steel wire and catheter were carefully removed from the tracheal segment wrapped in omentum. In groups 3 and 4 it was confirmed that the pump contained a small residual volume of fluid. The tip of each tracheal specimen was incisedto allow fixation by immersion in 10% buffered formalin. The animals were killed by exsanguination. Tracheas in group I were fixed in 10% buffered formalin immediately after harvest. After embedding in paraffin,
The Jour.nal of Thoracic and Cardiovascular Surgery
4 Jlm thick longitudinal sections of the trachea and adherent
omentum were cut from the midpoint of the cartilaginous trachea, stained with hematoxylin and eosin, and assessed histologically in a blinded fashion without reference to the different experimental conditions. Vascular evaluation. Changes in the vascularity of the tracheal segments and the surrounding omental tissue were assessed with use of morphometric image analysis. To avoid misinterpretations due to artifact at the ends of the tracheal segment and to conform to size constraints of imaging equipment, only the 2 mm segment in the center of the longitudinal section was examined. Image analysis was done with use of a video camera (MTI 65, DAGE-MTI, Michigan City, Ind.) connected to a light microscope (Leitz Colorlux K, Leitz Wetzlar, Germany) to capture the digitized image of each 2 mm segment (X83 magnification) on a video screen (Multi Sync 3D, NEC Information Systems, Boxborough, Mass.) for subsequent analysis. Vesselnumber per square millimeter tracheal (VN /T) and omental (VN /0) area and vessel area per tracheal (VA/ T) and omental (VA/a) area in percent were calculated, with use of the methodology described presently, as the average of three measurements for each trachea. Numerical evaluation of the blood vessels and the area occupied by them within the cartilaginous part of the trachea and the adjacent omentum was performed at x208 magnification. This was done with use of a digitizing pad (Hipad Digitizer, Houston Instruments, Austin, Tex.) and pen linked to an NEC Powermate 386/20 personal computer utilizing niorphometrysoftware (Bioquant IV, R&M Biometrics, Nashville Tenn.). No attempt was made to distinguish between arteries and veins within the tracheal segments and the omentum. Whenever the identification of a blood vessel was equivocal, the X208 magnification picture was saved and the corresponding tracheal area was examined at a x832 magnification. The total number of vessels (VN) and total luminal area occupied by vessels (VA) were measured within an area of trachea (T) determined by an 800 X 800 Jlm' grid centered between two cartilage rings. VN and VA in the adherent area of the omentum (0) were also determined. The measurements were done at three random sites of the center 2 mm part of each trachea. Epithelial assessment. The tracheal epithelium as a "target organ" of revascularization was examined with regard to epithelial regeneration and epithelial thickness. With use of x832 magnification, epithelial regeneration was assessed according to the following scale: 0: No epithelium, single nonconfluent epithelial cells I: Confluent single-layered nonciliated epithelium 2: Confluent multilayered nonciliated epithelium 3: Normal mucociliary epithelium Along the fixed longitudinal section of airway, 10 random areas of epithelium with a length of 200 Jlm each were examined, five sites on cartilage rings and five between rings. The grades of each evaluation were added; therefore the maximal grade representing a morphologically normal epithelium was 30, and the minimum score representing no confluent epithelium was O. Length and area of the mucosa within the three random sites that were evaluated for vascularity were measured, and the epithelial thickness was calculated (thickness = width = areal length). Three thickness values were averaged for each tracheal segment. Statistical analysis. Data analysis was performed with use of SAS software (SAS, Inc., Cary, N.C.). To achieve homoge-
Volume 104 Number 1 July 1992
Effect of bFGF and omentopexy on tracheal isografts
18 3
Fig. 2. Cartilaginous part of trachea after 2 days of omental wrapping (group 2A). Severe submucosal hemorrhage. Epithelial structure is damaged more severely in area underlying cartilage ring than in intercartilaginous area. (Original magnification X417.)
Table II. Tracheal graft vascularity Group
1 (native)
VN/T (jmm 2) VA/T (%)
61.6 ± 8.2 3.07 ± 0.26
2A (OM2D)
2C
2B (OM 7D)
(OM 14D)
3A (bFGF7D)
3B (bFGF 14D)
4A (SAL 7D)
4B (SAL 14D)
24.6* ± 3.2 1.44* ± 0.18
36.5t ± 5.5 1.75* ± 0.17
36.4* ± 2.8 3.43 ± 0.54
45.1 ± 4.1 2.84 ± 0.46
28.8t ± 5.2 2.05 ± 0.43
34.7t ± 4.4 1.99 ± 0.34
Native, No treatment or implantation of tracheal segment; OM, omental wrap; D, day; bFGF, growth factor/pump treatment; SAL, saline/pump treatment; VN/T, vessel number per tracheal area; VA/T, vessel area per tracheal area; - , identification of vessels not possible. All values are mean ± standard error of the mean. 'p < 0.05 vs group I (native tracheas).
tp
< O.Ol vs group
1.
neous variances, all data were transformed to logarithms before one-way or two-way analysis of variance. Contrasts were used for comparing multiple treatments with a common control (native tracheas). Tukey's studentized range test was chosen for multiple comparisons between the groups. The level of significance was defined as 0.05; all values are presented as mean ± standard error of the mean. The animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978).
Results Becauseofmacroscopic(n = 2) or microscopic (n = 1) evidence of infection, three animals were excluded from further histopathologic assessment. Catheter disconnection from the graft site or from the pump flow modulator was not observed in any of the animals.
Tracheal blood vessels. Tracheal vessels in group 2A (omental wrap for 2 days) could not be identified and quantified because of severe hemorrhage (Fig. 2). The VN/T was significantly higher in native tracheas (group 1) than in any other group except the 14-day bFGF group 3B (Table II). VN/T and VA/T were higher after 14 days of omental wrapping (group 2C: VN/T, 36,5% ± 5.5%; VA/T, 1.75% ± 0.17%) in comparison with 7 days (group 2B: VN/T, 24.6% ± 3.2%; VA/T, 1.44% ± 0.18%) without reaching statistical significance. Vascularity was significantly superior in the bFGFtreated tracheas compared with control and saline-treated tracheas. At day 7 the bFGF/pump group (group 3A) showed a significant (p < 0.05) increase in VA/T compared with the nontreatment group 2B and the saline/ pump group 4A (Fig. 3, A). VN/T in the bFGF-treated group (3A) was significantly higher (p < 0.05) than in
1 84
The Journal of Thoracic and Cardiovascular Surgery
Mayer et al.
VAIT ('11.)
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ve G 28, 4A
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GROUP 3A
70 OMENTAL WRAP. n·8
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70 bFGF. n-8
VN/T (per mm2j
~-----------------=------,
*:
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NATlYE, n-7
20 OMENTAL
70 OMENTAL
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*:
--25
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20
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70 SALINE. n·5
GROUP 3A 7D bFGF,
n-e
o B
7 DAYS
_
NON-TREATED
14 DAYS _
SALINE
0
bFGF
Fig. 3. Tracheal vascularity at day 7. A, Surface area of blood vessels per tracheal area (VA/T) is significantly higherin basic fibroblast growth factor (bFGF)-treated group than in omental wrap group and saline-treatedgroup. B, Number of vessels per tracheal area (VN/T) is significantly higher in bFGFtreated group than in omental wrap group.
Fig. 4. Epithelial regeneration score. A, Tracheas wrapped in omentum and not treated showed a significant increase in epithelial score over time. B, bFGF-treated groups had a significantly higher epithelial score than omental wrap groups and saline-treated groups at days 7 and 14.
group 2B (omental wrap); the difference between group 3A (bFGF) and the saline-treated group (4A) did not reach statistical significance (Fig. 3, B). At day 14 VA/T was significantly (p < 0.05) increased in the bFGF group (3B) compared with the control group (2C) and salinetreated group (4B) (see Table 11). There was no significant difference in VN/T between the groups after 14 days. Omental blood vessels. After 2 days of omental wrapping, an analysis of omental blood vessels was impossible because there was no firm connection between tracheal segment and omentum. The data of omental vessel numbers (VN/O) and areas (VA/O) are summarized in Table III. After 7 days VN/O was higher in the bFGF group (3A) compared with groups 2B and 4A; however, this difference did not reach statistical significance (p = 0.07). At day 14 there was no difference in VN/O and VA/O between the groups (see Table III).
Epithelial viability. In the nontreated groups there was a significant (p < 0.001) increase of the epithelial scoring grade over time (Fig. 4, A). After 2 days (group 2A) there was no confluent epithelial layer in any of the tracheal segments. Mucosal areas underlying cartilage rings appeared to be damaged more severely than intercartilage areas (see Fig. 2). After 7 days epithelial morphologyvaried from a simple undifferentiated monolayer epithelium to areas with morphologically normal, ciliated epithelium. Most of the tracheal segments harvested after 7 days, however, were characterized by a multilayered epithelium with a high percentage of secretory cells and without ciliated cells (Fig. 5). After 14 days epithelial areas with ciliary differentiation (Fig. 6) alternated with areas characterized by a nonciliated, multilayered epithelium. The thickness of the epithelium in the control groups increased significantly between 2 days and 7 days (group
Volume 104 Number 1 July 1992
Effect of bFGF and omentopexy on tracheal isografts
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Fig. 5. Multilayered, metaplastic, hypersecretory epithelium of a trachea wrapped in omentumfor 7 days. (Group 2B,original magnification X667.) Table III. Omental vascularity Group
1 (native)
VN/O (frnrn2) VA/O (%)
2A (OM 2D)
38
48
28
2C
(OM7D)
(OM 14D)
3A (bFGF7D)
(bFGF 14D)
4A (SAL 7D)
(SAL 14D)
36.3 ± 4.6 2.54 ± 0.24
7\.9 ± 18.1 5.14±1.40
68.6 ± 8.5 4.60 ± 0.89
64.5 ± 9.5 7.22 ± \.00
46.2 ± 4.9 3.46 ± 1.00
56.3 ± 3.6 7.20 ± 1.17
Native, No treatment or implantation of tracheal segment; OM, omental wrap; D, day; bFGF, growth factor/pump treatment; SAL, saline/pump treatment; VN/O, vessel number per omental area; VA/O, vessel area per omental area; - , no omentum or identification of omental vessels not possible. All values are mean ± standard error of the mean.
2A, 7.2 ± 0.69 Jlm; group 2B, 42.6 ± 3.48 um;
p < 0.05). The decrease in thickness of the epithelium between days 7 and 14 was not statistically significant (group 2B, 42.6 ± 3.48 Jlm; group 2C, 31.6 ± 3.43 Jlm). At day 7 the epithelium was significantly thicker than in native tracheas (group 2B, 42.6 ± 3.48 Jlm; group I, 25.4 ± 1.46 Jlm; p < 0.001); there was no difference in thickness of the epithelium between 14-day control tracheas and native tracheas. bFGF-treated tracheas had a significantly (p < 0.05) higher epithelial score grade than nontreated and salinetreated segments after 7 and 14 days (Fig. 4, B). Group 3B (bFGF, 14 days) epithelial score was similar to that of native tracheas (group I, 29 ± 0.3; group 3B, 29.1 ± 0.4; p = 0.96). Discussion Ischemic airway complications have been significant causesof morbidity and mortality after lung transplantation.I, 2 Since the bronchial arterial circulation is generally not surgically reconnected, the donor bronchus is
dependent on retrograde collateral flow from the pulmonary artery circulation and local tissue revascularization. Omentum appears to contain specific angiogenic factors l O- 12 and has proved successful for local revascularization of ischemic tissue. 17-20 Omentum will revascularize ischemic canine donor bronchiv": therefore it has been used routinely to wrap the bronchial anastomoses in clinicallung transplantation. I, 2 With use of a "telescoping" technique of bronchial anastomosis and early postoperative corticosteroids, the San Antonio group-' recently reported satisfactory bronchial healing after single lung transplantation without bronchial omentopexy. In the present study continuous local administration of bFGF significantly increased the vascularity of the initially completely devascularized tracheal segments wrapped in omentum at days 7 and 14. After 2 days of omental wrapping, original tracheal vessels could not be identified because of severe hemorrhage and vascular destruction. The same phenomenon has been observed in canine models after 4 and 5 days.7 8 Although the mechanism of airway revascularization by omentum remains 0
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Mayer et al.
The Journal of Thoracic and Cardiovascular Surgery
Fig. 6. Structurally normal, mucociliary epithelium of a trachea wrapped in omentum for 14 days and treated with bFGF. (Group 3B, original magnification X667.)
unclear, our findings suggest that continuous local bFGF administration enhances this process. Because the differences in vascularity between nontreated and bFGF stimulated tracheal segments were less obvious at day 14 compared with day 7, bFGF administration appeared to enhance vessel ingrowth preferentially in the early period after implantation. At day 7 the vascularity of the surrounding omentum also seemed to be increased in the bFGF group compared with the nontreatment group, although the difference did not reach statistical significance. There was no difference in omental vascularity after 14 days. Similar results were reported by Eppley and coworkersl''; they found that the most significant difference in revascularization of mandibular bone grafts between bFGF and control groups occurred at day 10. It is possible that the difference we noted at day 7 might also have been present at day 5 or day 10. We were limited in the number of samples we could process, however, and therefore arbitrarily selected 2, 7, and 14 days. In vivo regeneration of airway epithelium after mechanical and toxic injury has been extensively described in the literature. 22- 27 It has also been shown that differentiation of tracheal epithelium in primary cell culture recapitulates regeneration after injury and normal fetal development of tracheal epithelium.P Although the effects of ischemia on epithelial regeneration are not known, we hypothesized that epithelial regeneration of ischemic tracheal segments might be similar to regeneration after other kinds of injury. The scoring system used in the present study is based on the descriptions of hamster epithelial regeneration. 22-24, 27, 28 After denuding epithelial injury, a flat, monolayered, undifferentiated
epithelium can be observed in the first 2 days. Between days 2 and 6, a multilayered metaplastic epithelium develops by division of secretory cells, and the normal morphology of epithelium with one or two layers of ciliated, secretory, and basal cells is reached between 6 and 8 days. In the present study similar stages of epithelial regeneration were observed; however, the time-course seemed to be delayed compared with regeneration after nonischemic mechanical injury.22-24, 27 After 2 days of omental wrapping, a confluent monolayer of undifferentiated epithelial cells could be found in only a few intercartilaginous areas, whereas there was no confluent epithelium over cartilage rings. The intercartilaginous tissue appears to be the only possible route for revascularization. After 7 days of omental wrapping without treatment, the epithelial score was increased and tracheas were characterized by a multilayered nonciliated epithelium that was similar to the epithelium described 3 to 6 days after mechanical injury and in cell culture. 22-24, 27, 28 At day 14, epithelial areas showing metaplasia alternating with areas of normal epithelium could be found in the nontreated tracheas. Tracheal segments treated with bFGF showed the highest degree of epithelial regeneration at days 7 and 14 and could not be distinguished from native tracheas in terms of epithelial morphology after 14 days. Although a mitogenic effect of bFGF on epithelial cells has been demonstrated.l? we consider the acceleration of epithelial regeneration in our model to be a result of improved revascularization by bFGF. The morphology of submucosal cartilage, difficult to identify and quantitate, was not assessed. We do not know
Volume 104 Number 1 July 1992
whether other bFGF mitogenic effects on fibroblasts or smooth muscle cells 29 or bFGF-induced cartilage repair/" 30 were operative in these devascularized tracheal segments. The heterotopic rat tracheal isograft model used allows the evaluation of local revascularization without the influences of graft rejection, immunosuppression, and retrograde mucosal blood flow from the pulmonary circulation. It could be readily used, for example, to study the effects of rejection or immunosuppression on tracheal revascularization and viability. The use of foreign material such as surgical steel wire, catheters, and minipumps in the peritoneal cavity may induce artifacts in the assessment of revascularization. Implanting the tracheal segments without stretching them, however, would leave in the omentum tracheal segments that are shrunken to almost half of their normal anatomic length, exposing a surface with a high percentage of cartilage. Cartilage has been shown to inhibit the growth of endothelial cells.'! The catheter and minipump were used for continuous local drug administration.V This may be important since in a rabbit model we did not demonstrate any angiogenic effect of bFGF on tracheal revascularization with use of a single topical bFGF application.P A certain degree of foreign body reaction and increased vascularity could be observed, however, after 7 and 14 days in the saline/pump groups compared with the control groups, although differences were not statistically significant. Since the beginning of this study we have adopted clinically the strategy proposed by the San Antonio group" regarding the bronchial anastomosis, and we have been pleased with the preliminary experience in both single and bilateral sequential lung transplantation. Nonetheless, more rapidlocal tissue revascularization of the ischemic donor bronchus would provide even greater security. We conclude that continuous local administration of bFGF enhances the effect of omentopexy on early revascularization and epithelial regeneration of devascularized tracheal segments in a heterotopic rat tracheal isograft model. We would like to acknowledge the excellent technical assistanceofS. Diamant, J. Mates, K. Ekern, and D. Hunter. Assistance with statistical analysis was receivedfrom the Statistical Consulting Service (K. Knight, PhD) of the Department of Statistics, University of Toronto. REFERENCES I. Patterson GA, Todd TR, Cooper JD, et al. Airway complications after double lung transplantation. J THORAC CARDlOVASC SURG 1990;99:14-21. 2. Pasque MK, Cooper JD, Kaiser LR, Haydock DA, Triantafillou A, Trulock EP. Improved technique for bilateral
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