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Ann Thorac Surg 2001;71:332-336
© 2001 The Society of Thoracic Surgeons

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Original article: general thoracic

Bronchial transsection and reanastomosis in pigs with and without bronchial arterial circulation

^a Department of Cardiothoracic Surgery, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
^b Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
^c Department of Medical Anatomy, Section B, The Panum Institute, University of Copenhagen, Copenhagen, Denmark

Accepted for publication May 26, 2000.

Address reprint requests to Dr Gade, Department of Thoracic Surgery, RT 2152, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
e-mail: johngade{at}dadlnet.dk

	Abstract

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Background. The bronchial artery may be vital to the bronchi and lung parenchyma, but results of lung transplantation have raised doubts. This study was performed to examine the effect of bronchial arterial devascularization on bronchial morphology after bronchial transsection and reanastomosis.

Methods. In 6 pigs (study group), the left main bronchus was transsected, reanastomosed, and devascularized. Six control pigs had the same operation without devascularization. After 1 week, bronchial arterial angiography was performed, and specimens were examined with conventional histology and scanning electron microscopy.

Results. Histology showed significant changes (inflammation, edema, and fibrosis) in bronchi and lung parenchyma of the study group compared with the unoperated side (p = 0.028) and with the control group (p = 0.050). Scanning electron microscopy showed significant ciliary denudation in the study group’s left bronchus compared with the unoperated side (p = 0.043) and with the control group (p = 0.0071).

Conclusions. The loss of cilia of the bronchial epithelium and the occurrence of inflammation, edema, and fibrosis in bronchi and lung parenchyma 1 week postoperatively were significantly related to the absence of the bronchial arterial circulation.

	Introduction

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The lungs have a dual blood supply: the pulmonary and bronchial circulations. It was demonstrated that, because of the bronchial arterial circulation, the pulmonary circulation could be interrupted without necrosis of the bronchi or lung parenchyma [1]. This was apparently confirmed by a study in which injection of vinyl chloride into the bronchial arteries was lethal to dogs within 1 week [2], and by other studies showing atelectasis, edema, and ulceration of the bronchial mucosa and lungs in the absence of a bronchial arterial circulation [3–5]. With the appearance of human lung transplantation, however, doubt was thrown on this knowledge. In human lung transplantation, the lung parenchyma is deprived of its normal bronchial arterial blood supply, but apart from initial problems with anastomotic dehiscence, the changes mentioned above have been infrequent. When bronchial arterial revascularization appeared, it was originally performed to avoid bronchial dehiscence in lung transplantation [6–8], and was expected to have other beneficial effects on the lungs [9–11]. However, a beneficial effect of bronchial arterial revascularization remains to be documented clinically, because no randomized investigations have been performed. Accordingly, further controlled experimental studies are needed to clarify the importance of the bronchial artery to the lungs.

The purpose of the present study was to compare the morphological changes in the lung parenchyma and bronchi of pigs undergoing bronchial transsection and reanastomosis with and without the bronchial arterial blood supply.

	Material and methods

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Twelve female pigs from a production herd (Danish Domestic/Yorkshire, crossbreed DDY), weighing between 36 and 44 kg, were used. The pigs were weighed again before section. Pre- and postoperatively, they were kept in the laboratory yard with artificial daylight and fed twice daily.

Surgical and radiographic procedure
The pigs underwent surgery under general anesthesia induced by thiopental 50 mg/kg (Pentothal; Abbott, Gentofte, Denmark), and maintained with NO₂, with pentobarbital 50 mg/mL (Mebumal; Nycomed DAK, Uppsala, Sweden) and fentanyl 50 µg/mL (Haldid; Janssen, Beerse, Belgium) infusion, and with pancuron 2 mg/mL (Pavulon; Organon Teknika, Boxtel, The Netherlands) bolus injections. A left lateral thoracotomy was performed in all the pigs, and the left main bronchus was transsected and reanastomosed with a continuous, nonabsorbable suture (Prolene 4-0). In 6 pigs, the left bronchial arterial branches and all possible collaterals to the left main bronchus were clamped or cut (study group). In the other 6 pigs, division and reanastomosis of the bronchus was carried out attempting not to damage bronchial arterial branches (control group). The unoperated right side served as control for both groups. The pigs received cefuroxim (Zinacef; Glaxo Wellcome, Brondby, Denmark) 1.5 g IV preoperatively and buprenorphin (Anorphin; A/S GEA) 0.6 mg IM postoperatively twice daily for 2 days. Antibiotics were continued only if the pigs had fever. There was no tube drainage, but expansion of the lung was documented with a plain radiograph after operation. Bronchoscopy was carried out preoperatively and postoperatively. The pigs were allowed to survive 1 week, and bronchoscopy was repeated before section. The sectioning procedure has been described earlier [12, 13]. In short, the heart-lung block was perfused with saline, removed with the entire mediastinum from the thyroid cartilage to the diaphragm, and prepared with cannulation for contrast injection into the esophageal, broncho-esophageal, and coronary arteries. A plain radiograph was made before angiography. The patency of the bronchial arterial branches was examined angiographically.

The porcine bronchial blood supply originates from the bronchoesophageal artery, which divides into several bronchial branches and a branch for the upper thoracic esophagus. These porcine bronchial branches are equivalent to the human bronchial arteries. The principal porcine bronchi are each followed by two major bronchial arterial branches that divide into minor branches. In the following, the major branches are named the left lateral and medial, and the right lateral and medial bronchial branches [12]. There are frequent anastomoses with the esophageal artery (which supplies the lower esophagus directly from the aorta) and the coronary arteries [13].

Microscopy procedures
After angiography, a specimen was taken 3 to 4 cm distal to the anastomosis, including 3 to 4 cm of the principal left bronchus and the surrounding lung parenchyma, and similarly from the right bronchus. Specimens were fixed by immersion in 2% glutaraldehyde in 0.05 mol/L phosphate buffer (pH 7.4). After fixation, each specimen was divided in two: one half for conventional histologic examination, the other for scanning electron microscopy.

Histology
Specimens for light microscopy were routinely processed for paraffin blocking, cut in 3- to 5-µm-thick sections, and stained with hematoxylin-eosin, van Gieson-Hansen/Alcian, and PAS. A pathologist (C.B.A.), experienced in the field, examined the sections together with the first author. Sections were blinded to the investigators during the examination. The following details were recorded: presence or absence of cilia; epithelial metaplasia; and signs of inflammation, fibrosis, and/or edema of the mucosa, submucosa, and cartilage, of peribronchial connective tissue, and of the intersegmentary lung septa. The findings in each category were scored as absent (1), minor (2), moderate (3), and severe (4) changes. Scoring was based on agreement between the investigators. The sum of scores was used to compare the differences between the left principal bronchi of the two groups (Mann-Whitney U test). The left/right scores within each group were also compared (Wilcoxon matched pairs test). The median number of goblet cells was counted in four microscopy fields (x100).

Scanning electron microscopy
Annular bronchial segments (approximately 5 mm long) were prepared for examination by the osmium-thiocarbohydrazide (OTOTO) method [14]. The samples were rinsed in 0.15 mol/L phosphate buffer (pH 7.4) and postfixed in 1% OsO₄ in 0.12 mol/L phosphate buffer (pH 7.4) overnight. After rinsing and preparation procedures, the specimens were divided in quarters and mounted on stubs, with colloidal carbon as an adhesive, and sputter-coated with chromium (Edvard’s XE200 Xenosput). Examination and photography were carried out in a Philips FEG 30 scanning electron microscope operated at 1 to 10 kV. The scanning images were evaluated with respect to the presence of cilia and scored as normal (1), or as devoid in 10% to 25% of the examined area (2), in 25% to 50% (3), or in 50% or more (4). The scores of the left side were compared between groups (Mann-Whitney U test), and the left/right scores were compared within groups (Wilcoxon matched pairs test).

Statistics
Nonparametric statistics were used for categorical data and for skewed continuous data. The Mann-Whitney U test was used for comparison between the study group and the control group. The Wilcoxon matched pairs test was used for left/right comparison within each group. Quartiles were used for population description. Parametric statistics were used for continuous data (Student’s t test and standard deviation). Calculations were based on the intention to treat principle. A computer program was used to perform calculations (Statistica 5.0; Statsoft). A level of significance of p less than or equal to 0.05 was chosen.

Ethics
The pigs received humane care in accordance with the national Danish regulations on experimental animal welfare. The investigation was approved by the Danish Inspection on Animal Welfare.

	Results

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Clinical course and angiographic findings
All the pigs survived for the scheduled time. The control group had an average weight increase (±SD) of 1 (±1.4) kg, and the study group had an average weight loss of 0.2 (±1.1) kg (p = 0.170). A bronchoscopy before section revealed no signs of bronchial dehiscence or necrosis in either group. A clinically important bronchial stenosis causing retention of secretion was present in 2 study pigs and in 1 control pig. The lung parenchyma was clinically and radiographically normal in all the control pigs, but edematous congestion was clinically present in 3 study pigs, as well as on a plain radiograph of removed heart-lung block. Angiography on the study group showed complete interruption of the left medial and lateral bronchial branches in 5 pigs and partial interruption in 1 pig. There was partial interruption in 2 control pigs.

Histology
In the study group, severe morphologic changes in the left bronchi and intersegmentary lung septa were found in 5 pigs, and moderate changes were found in 1 pig. Inflammation and edema were the most frequent findings, but fibrosis, absence of cilia, and epithelium metaplasia were also found. Comparison between the left and right sides showed severe changes in the left bronchus and lung as mentioned above, and minor or no changes in the right (p = 0.028). Semiquantitative scorings are shown in Table 1. In the control group, 5 pigs had no or minor changes on the operated left side, and 1 pig had major changes. In this pig, angiography showed that the left lateral bronchial arterial branch had been unintentionally interrupted. Compared with the right side, these changes were not significant (p = 0.280) in the control group as a whole.

View larger version (95K): [in this window] [in a new window]   Fig 1. Section of left (operated) side of study pig. (A) Shows submucosal edema and peribronchial inflammation, fibrosis, and edema. No lung parenchyma is seen adjacent to the cartilage as in normal pigs. (Arrow) Mucosal ulceration (Hematoxylin and eosin [H&E], x40). (B) Shows inflammation, ciliary loss, edema, and slight fibrosis (H&E, x100). (C) Section with broadened interstitial septum showing edema, fibrosis, and inflammation (H&E, x100).

View larger version (157K): [in this window] [in a new window]   Fig 2. Section of left (operated) side of control pig showing normal bronchus with surrounding normal lung tissue. (Arrow) normal intersegmental septum (Hematoxylin and eosin, x40).

View larger version (189K): [in this window] [in a new window]   Fig 3. (A) Scanning electron microscopy of mucosa from the operated side of study group shows severe ciliary loss (x1,000). (Inset) Overview showing that more than 50% of the surface has suffered ciliary loss (x100). (B) Scanning electron microscopy of mucosa from the operated side of control group. The whole surface is covered with evenly distributed cilia as in normal pigs (x1,000). (Inset) Overview (x100).

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Other investigators have examined interruption of the bronchial circulation using different methods. A classic canine study demonstrated severe bronchial necrosis leading to pneumonia and death within 1 week after the injection of vinyl chloride into the bronchial artery [2]. However, vinyl chloride may interrupt the whole vascular bed of the bronchi [15], including the contribution from the pulmonary artery, and Ellis and associates [2] may have shown the consequences of no blood circulation at all. Another canine study demonstrated alveolar edema and disruption after ligation of the bronchial artery, but several operations were performed within 72 hours, and the findings may be attributed to the number of operations [5]. We used a relatively short observation time, which does not exclude reversibility of the morphologic changes. Thus, the bronchial mucosal blood flow has been found to return to its baseline level 2 weeks after interruption of the bronchial arterial circulation [16], and complete, spontaneous revascularization after lower lobe autotransplantation was demonstrated after 90 days [4]. Our finding of ciliary denudation of the epithelium could explain the findings of another study [17], which demonstrated significantly lowered clearance of mucous from the airways 3 weeks after canine autotransplantation. A later study found partial recovery after 12 weeks [18].

It is known that ciliary beat frequency depends on the oxygen tension in the airways, but little is known about factors that influence ciliary and airway epithelium maintenance and turnover. Epithelium turnover was rather slow in normal murine distal airways [19], and in rats after isograft lung transplantation [20], but with an ability to accelerate after toxic damage and allograft lung transplantation, respectively. Turnover in humans was higher in inflamed cystic fibrosis patients than in controls [21]. We found no reports that related the bronchial artery to mucosal cell turnover. It is not known whether the bronchial mucosal oxygen demand can be supplied also by absorption from the bronchial lumen, or whether it depends on blood supply alone.

Together with other experimental studies [3, 4, 18], the present study indicated that the preservation of normal bronchial mucosal morphology depends on a bronchial arterial circulation. These findings are contrary to the experience in lung transplantation, after which the lung and bronchi normally survive without acute, ischemic changes, even though the bronchial arterial circulation has been completely interrupted. The clinical importance of the bronchial artery to the lung is therefore less clear. One explanation of this apparent discrepancy is that biopsies from lung transplantation patients, both mucosal and transbronchial biopsies, are relatively small and changes may be overlooked. Another explanation may be that changes are temporary and have recovered when biopsies are made 1 to 2 weeks after transplantation. Experimental long-term studies are needed to clarify this aspect. However, the finding of fibrosis in our study would normally be expected to be irreversible. If this is true, it raises the question as to whether obliterative bronchiolitis is initiated in this way.

It has been suggested that reestablishment of systemic blood supply in lung transplantation would reduce the frequency of infections and rejections [9, 10, 22], dehiscence [6, 9], ischemic stenosis [8], and perhaps even the bronchiolitis obliterans syndrome [9, 10, 22]. However, these presumptions, all mentioned in the discussion of the studies, could not be substantiated, because a control group was lacking. However, most of these suggestions, if true, would also be relevant to carinal resections and sleeve resections of the bronchi, and indeed preservation of peribronchial tissue has been recommended for carinal resections [23].

In conclusion, the present investigation has shown that severe morphologic changes in airways and lung parenchyma 1 week after bronchial transsection and reanastomosis are significantly related to the absence of a bronchial arterial circulation.

	Acknowledgments

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This work was supported by grants from the following institutions and foundations: The Danish Medical Association Research Foundation, The Danish National Association against Lung Diseases, The Danish Hospital Foundation for Medical Research, The Beckett Fund, The Leo Research Foundation, Felo Aps, The Ib Henriksen Fund, and The Dagmar Marshall Fund. We thank Mary-Ann Gleie, The Panum Institute, and Karin Jensen, Rigshospitalet laboratory technicians, for valuable assistance.

	References

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