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Eur J Cardiothorac Surg 1999;15:37-44
© 1999 Elsevier Science NL
a Department of Cardiothoracic Surgery, The National University Hospital (Rigshospitalet), Copenhagen, Denmark
b Department of Pathology, The National University Hospital (Rigshospitalet), Copenhagen, Denmark
Received 22 June 1998; received in revised form 27 October 1998; accepted 25 November 1998.
Corresponding author. Department of Cardiothoracic Surgery, RT 2152, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark. Tel.: +45-3-545-2627; fax: +45-3-545-2548; e-mail: bar@rh.dk
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Abstract |
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 Top Abstract Introduction
2. Materials and methods...Statistics
ResultsDiscussion
Conclusions
References |
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Key Words: Lung transplantation • Direct bronchial artery revascularization • Ciliary beat frequency
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Introduction |
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TopAbstractIntroduction
2. Materials and methods...Statistics
ResultsDiscussion
Conclusions
References |
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LTx with success was not possible until in the 1980s when the Toronto lung transplant group began developing their transplantation program [1]. Single lung transplantations (SLTx) were performed with better results and fewer bronchial problems than double lung transplantation (DLTx). In Toronto DLTx was performed en-bloc with a tracheal anastomosis without any attempt to directly re-establish the bronchial artery circulation. The clinical results with this DLTx method were poor due to frequent ischemic dehiscence of the airway anastomosis [2]. The en-bloc method was abandoned and instead two-lung transplantations were performed as bilateral single lung transplantations with distal main bronchus anastomoses (bilateral lung transplantation). Three European centers have, however, developed and applied methods for direct bronchial artery revascularization (BAR) [3] [4] [5]. BAR has proved to be possible and associated with good results after en bloc DLTx as well as after SLTx [6] [7].
Even if single and bilateral lung transplantations can be performed with few airway healing problems they still leave the airways without normal arterial blood supply, perfused only by venous blood from the pulmonary circulation and neovascularization. The resulting ischemia could be expected to have negative consequences beyond airway healing, consequences which can possibly be prevented by successful BAR with systemic supply of oxygenated blood.
Since the start of the lung transplant program in Copenhagen BAR has been routinely performed in all lung transplantations whenever possible. We do agree, however, that it remains to be proven that BAR really improves the results after lung transplantation.
One presumed benefit of BAR is reduced frequency and severity of infections in the transplanted lungs. Preserved/improved resistance against infections could be an effect of preserved mucociliary clearance in lungs transplanted with BAR compared to without BAR. The ciliary beat frequency (CBF) has been used in previous studies as an indicator of mucociliary clearance [8].
Two groups have previously investigated CBF in lung transplant recipients, with contradictory results [9] [10]. One group studied five patients and found that the CBF proximal and distal to the airway anastomosis was not significantly different, nor did the CBF of the transplanted bronchus differ from that of untransplanted controls [9]. However, another group studied six patients and found that the CBF of the transplanted lung bronchus was significantly lower than the CBF of the contralateral (native lung) bronchus [10].
The original purpose of the present study was to investigate whether the re-establishment of normal bronchial artery flow in transplanted lungs would contribute to preserved or even improved mucociliary clearance indirectly measured by the CBF of epithelium cells in bronchial mucosa samples.
From January 1992 until July 1997 BAR has been used in 64 en-bloc DLTx, ten heart lung transplantations (HLTx) and 25 SLTx in Copenhagen. Six SLTx were performed without any attempt to perform BAR. The present study only includes patients after SLTx. SLTx has been performed with and without BAR, the choice made by the donor bronchial artery anatomy allowing revascularization of only one lung or the surgeons willingness/ability to perform successful BAR. In addition, the SLTx patient has one native lung for comparison.
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2. Materials and methods |
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TopAbstractIntroduction
2. Materials and methods...Statistics
ResultsDiscussion
Conclusions
References |
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The operative result of BAR was examined 1 month post-transplant by internal mammary-bronchial arteriography. In eight patients bronchial arteriography showed complete revascularization according to our previously published classification system (i.e. all transplanted lung lobes were supplied by at least one bronchial artery ramus) [11].
In ten patients BAR had failed. One patient could not be classified according to this system due to poor quality of the arteriography.
From all 25 patients airway epithelium samples were obtained during bronchoscopic examinations scheduled as part of the routine follow up or acute examinations due to suspected infection or rejection. No premedication was given. Anesthesia was induced with propofol, alfentanil, pancuronium and suxamethonium and the patients were intubated and ventilated with 100% O2.
Bronchoscopy was performed using a flexible bronchoscope. Presence of erythema or increased secretion in the airways was registered.
All samples were obtained within the first 10 min after induction of anesthesia using a small bronchoscope biopsy forceps. Samples were always taken in the sequence: 1, trachea; 2, right bronchus and 3, left bronchus. All biopsies were taken superficially from the cartilaginous part of the airway circumference. Tracheal biopsies were taken approximately 2 cm proximal to the primary carina. The bronchial biopsies were taken just proximal to the ostium of the 6th segment.
According to a randomization code-letter the assisting nurse placed the biopsies in two sets of test tubes numbered 1, 2 and 3. The placement of the cells was `blinded' to the investigator (Nørgaard) who performed all the bronchoscopic samplings and further microscopic investigations.
The test tubes contained Krebs–Ringer solution (NaCl 122 mmol/l, KCl 3 mmol/l, MgSO4 1.2 mmol/l, KH2PO4 0.4 mmol/l, NaHCO3 25 mmol/l, CaCl 1.3 mmol/l (pH 7.4), sterile-filtered).
During the same bronchoscopy transbronchial biopsies were always taken from the transplanted lung parenchyma and histologically examined for the presence of rejection.
Measurement of ciliary beat frequency (CBF)
Immediately after sampling the test tubes containing the biopsies were placed at 0°C (i.e. submerged in a container with ice and water) until measurements were made. The delay from the first until the last biopsy was measured was approximately 1 h. One set of biopsies was used for histology, while the others were used for CBF measurement (
Fig. 1
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Histological analysis
The biopsies were sent for histology immersed in cold Krebs–Ringer. Within 3 h after sampling the biopsies were pipetted onto two sets of microscopy glasses and dried. One set was stained using hematoxylin and eosin and the other using May–Giemsa–Grünwald.
First, the cells were studied at 400x magnification and the epithelial cells were identified as cylinder epithelium, metaplastic epithelium or squamous epithelium. If these epithelium cell types constituted more than approximately 1% of all epithelium cells, 100 cells were counted from random microscope vision-fields to estimate the proportional representation of each epithelium type.
Secondly in each microscopy slide, using 1000x magnification (oil immersion) cylinder epithelial cells containing cilia basal bodies were identified and the proportion of ciliated versus deciliated cells was established by counting the first 100 cells seen in the microscope. The slides on which fewer than 100 cells with basal bodies could be identified were excluded from statistical analysis.
When all recordings and calculations had been made the randomization code letters were opened and the statistical analyses performed.
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Statistics |
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TopAbstractIntroduction
2. Materials and methods...Statistics
ResultsDiscussion
Conclusions
References |
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Results |
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TopAbstractIntroduction
2. Materials and methods...Statistics
ResultsDiscussion
Conclusions
References |
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Median CBF of the transplanted lungs with no BAR (n=13) was 4.4 (range: 2.3–7.1 beats/s, while median CBF of the corresponding native lungs was 4.6 (range: 2.5–7.9 beats/s). This difference was not statistically significant ( Fig. 2 ). Correlation analysis (Spearman's rank correlation) revealed no significant correlation between CBF and the interval from transplantation to sampling (P=0.43).
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In the native bronchus samples all 13 patients had less than 1% metaplastic cells or squamous epithelium cells although five had traces of these epithelium types.
In the samples taken from the transplanted bronchus two patients had traces of metaplastic epithelium while one patient had traces of squamous epithelium, one patient had 33% cylinder epithelium and 67% metaplastic cells and one patient had 69% cylinder epithelium and 31% metaplastic cells in the examined sample.
Nine patients had a sufficiently high number of cells with basal bodies in both the native bronchus and transplanted bronchus samples to be included in the analysis of occurrence of ciliated cells. The mean percentage of cells with basal bodies which were ciliated was 78 (range 40–97%) in transplanted bronchi versus 82 (range 69–100%) in native bronchi (not significant).
Single lung transplantation with complete BAR
Eight of the investigated patients (five women, three men) had complete BAR. The samples were taken 161±205 (SD) days post-transplant (12–553 days).
Median CBF of the transplanted lungs with complete BAR was 5.0 (range: 2.9–7.9 beats/s, while median CBF of the corresponding native lungs was 5.2 (range: 2.3–10.9 beats/s). This difference was not statistically significant ( Fig. 2). Correlation analysis (Spearman's rank correlation) revealed no significant correlation between CBF and the interval from transplantation to sampling.
One set of samples for histology was lost. All seven patients included in the histological analysis had only cylinder epithelium in both transplanted and native bronchus samples.
Only five patients had a sufficiently high number of cells with basal bodies in both the native bronchus and transplanted bronchus samples to be included in the analysis of occurrence of ciliated cells. The mean percentage of cells with basal bodies which were ciliated was 80 (range: 54–90%) in transplanted bronchi versus 84 (range 76–94%) in native bronchi (not significant).
Comparisons between no BAR versus complete BAR
When CBFs of transplanted lungs with complete BAR (mean 5.0 beats/s) were compared to CBFs of transplanted lungs with no BAR (mean 4.4 beats/s) no statistical difference could be found (
Fig. 2). CBFs of the native lung from patients with complete BAR (mean 4.6 beats/s) were not significantly different from CBFs of native lungs from patients with no BAR.
Comparing the percentage of ciliated cells in the transplanted bronchus with BAR (n=5) 80 (range 54–90%) and without BAR (n=9) 78 (range 40–97%) no significant difference could be demonstrated.
Five of 13 samples (38%) from transplanted bronchi with no BAR had abundance of metaplastic and/or squamous epithelium cells, while none of the samples from transplanted bronchi with complete BAR contained any of these cell types (Fishers exact test (two-tailed) P=0.11). Also five of 13 samples from native lungs contained metaplastic/squamous epithelium cells. In three patients metaplastic/squamous epithelium cells were found in both the transplanted and the native lung.
Comparisons of CBF of native lungs versus transplanted lungs (irrespective of BAR)
CBF of transplanted lungs (mean 4.6 beats/s (n=22)) versus CBF of native lungs (mean 4.9 beats/s (n=22)) were not significantly different.
Rejection, bronchitis, airway erythema and increased secretion
TBB samples taken at the same bronchoscopy as the epithelium samples showed that nine patients had no histological signs of rejection (A0), five had minimal (A1) rejection, four had mild rejection (A2), and two had moderate rejection (A3). Two sets of samples contained to little material to be evaluated. Statistical analysis revealed no significant difference between CBFs of the transplanted airways with rejection (A1, A2 or A3) versus no rejection (A0).
In the same TBB samples 12 patients (of which nine had CBF measurements performed) had histological signs of infection/inflammation (bronchitis) while four had no signs of infection/inflammation. Nine samples could not be evaluated for bronchitis. Statistical analysis revealed no correlation between the CBF of transplanted airways and the histological finding of bronchitis versus no bronchitis.
In all 21 patients where CBF had been successfully measured notes had been taken on the presence of airway erythema and/or excessive secretion. No correlation could be demonstrated between CBF and the presence of airway erythema in native and/or transplanted lungs (Mann–Whitney U-test P=0.15). However, excessive secretion in native and/or transplanted lungs, was correlated to increased CBF (Mann–Whitney U-test P=0.02) ( Fig. 3 ).
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Discussion |
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TopAbstractIntroduction
2. Materials and methods...Statistics
ResultsDiscussion
Conclusions
References |
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There exists no widely accepted methods or guidelines on how to study CBF. In this study the simplest possible methods, both regarding handling of the specimens and performing the CBF measurements were applied. We tried to minimize the problems of previous studies: the sampling of specimens, the CBF recording method, and the observer bias in relation to cell selection. Some of the previous investigators have used nylon brushes to collect cell samples for CBF measurements. With this method cells may be damaged from brushing, cells sticking to the brush may not be representative, and cells from consecutive samples may be mixed with cells from previous samples remaining in the working channel of the bronchoscope. In this study superficial bronchus biopsies were taken using a flexible bronchoscope biopsy forceps, providing epithelium strips of multiple connected cells with preserved intercellular junctions. Although taken superficially, the epithelium biopsies usually consisted of all layers of the mucosa, not exclusively columnar epithelium cells. With this method the epithelium samples were transported through the biopsy channel of the bronchoscope enclosed in the biopsy forceps, preventing mixing of cells from consecutive samples.
In (unpublished) pilot experiments we used a photoelectric method for measuring CBF (microphoto-occilography [13]). Very often ciliated cells could be beautifully visualized in the microscope but yet, due to the orientation of the cells in the specimen, it was not possible to obtain a sufficient photoelectric signal for the CBF to be measured. Using the photoelectric method measuring CBF of 5 cells often took a very long time, ranging from 5 min up to 1 h during which period the cells were kept at 37°C and consuming energy. The photoelectric method also carries a significant risk of technical errors. In the search for a better method to measure the CBF of every ciliated cell which could be visualized in an expedite, precise, cheap, technically uncomplicated and foremost unbiased manner, we found the method of videorecording allowing recording of an unlimited number of randomly selected cells from each sample.
The previous investigators who found CBFs of transplanted lungs to be lower than CBFs of native lungs selected for comparison the cells having the highest or the lowest CBF in the specimens in an unblinded manner [10]. To avoid/minimize bias our selection of cells for measurement was done strictly according to the protocol (see Section 2).
Also in relation to specimen preservation there are factors to discuss. Previous investigators have kept sampled cells in tissue culture Medium 199 (Flow Laboratories). Tissue culture Medium 199 contains a wide variety of amino acids, salts, glucose and phosphates including ATP and AMP. It is well known that increasing ATP or AMP concentration will increase CBF [14]. Other substances contained in Medium 199 may also influence CBF. Since the processes controlling CBF are far from fully understood we chose to use a simple medium for the specimens until CBF measurement not to resuscitate cells actively and thereby possibly increase CBF. The choice of Krebs–Ringer solution carries a risk of depriving the cells of substances necessary for maintaining or regulating CBF. The observation of cells spontaneously increasing or reducing their CBF during ongoing recording was taken as an indicator of good cell integrity.
Normally the beating of cilia will stop as intracellular energy is consumed. Cooling of the cells to 0°C was introduced in order to reduce (stop) energy consumption in the cells during the transport period from sampling until re-warming and videotaping. The cold period until videotaping for the `1' sample was approximately 30 min and for the `3' samples approximately 90 min. The delay of up to 90 min from sampling to videotaping was shown not to affect the results ( Fig. 4).
The patients were not randomized to receive BAR or not, but anatomical and perioperative factors selected and divided the patients into the two compared groups, `no BAR' versus `complete BAR'.
There are several possible explanations for the low (lower than in our DLTx material) BAR success rate in the present study. When dividing the bloc anatomical realities often results in less than ideal conditions for revascularization of more than one lung. In SLTx BAR was performed as the last anastomosis, after the pulmonary circulation had been re-established and the lungs reperfused. Heparin was not given or given only in a low dose and clotting of the bronchial circulation is a possibility. Further more this material represents the learning experience of several surgeons, both when it comes to dividing the bloc and to performing the BAR.
Histologically metaplasia was observed in samples from lungs without BAR but no changed ratio between ciliated and non-ciliated columnar epithelium cells was demonstrated. We were not able to confirm the finding in one previous study of reduced CBF in transplanted lungs [10].
In the present study CBF in the airways of transplanted lungs did not differ from CBF of the corresponding native lungs. As CBF of the transplanted lungs without BAR was found to be the same as CBF of the native lungs in this study, it was of no surprise that no effect of BAR could be demonstrated.
If our findings of identical CBF in native and transplanted lungs are correct it could be that CBF is a low energy function which is controlled by humoral substances [15] in the blood or mucus, as these are identical for the two lungs, and not by direct innervation of the epithelium as suggested on theoretical grounds [16]. Normal innervation persists in the native lungs while the transplanted lungs are denervated.
A correlation between increased CBF and excessive secretion in the airways was found and indicates a direct local induction of increased CBF by the presence of excessive secretion and/or perhaps by increased mucus viscosity or changed mucus composition. We have, however, not further accessed the probably important aspects of the production and characteristics of mucus. Mucus production is normally very scanty and quantification and sampling are difficult tasks. As previously mentioned, direct studies of mucociliary clearance are also associated with certain difficulties and have so far been few in lung transplanted patients [12].
Our study showed the same CBF in all lungs, native and transplanted irrespective of BAR or interval from transplantation to sampling. Spontaneous neovascularization of the airways seems to be sufficient to normalize the CBF of the epithelium early after transplantation. If CBF is insensitive to airway ischemia it is a bad indicator to use in studies of the importance of BAR in lung transplantation. In support of this hypothesis, previous investigators have found that the airway cilia of laboratory animals (canines) are oxygenated directly from the surface [17]. Whether this holds true in humans is unknown, but seems reasonable since the oxygen partial pressure in the airways lumen is approximately 160 mmHg compared with 95 mmHg in the arterial blood. Even if vascular oxygen supply would not be a limiting factor for CBF the nutritional supply and washout of catabolic waste products should still mainly depend on a satisfactory blood flow.
It can be argued that the native lungs of SLTx patients are still very sick and therefore do not necessarily have normal CBF. A study with normal control lungs, although academically interesting, would be impossible to set up.
Before closing the issue of epithelium function and mucociliary clearance after lung transplantation there may be a need for another direct study of mucociliary clearance (ideally including simultaneous CBF measurements), provided the technical difficulties can be overcome.
Although not statistically significant there seemed to be one positive effect of BAR observed in this study. In some lungs with no BAR metaplastic and/or squamous epithelium cells were present in the airway samples, while none of the samples from lungs with complete BAR had traces of these epithelium types. An explanation may be that some areas of the epithelium growth layer (near the basal membrane) is affected by inadequate perfusion. To better quantify the metaplastic/squamous epithelium changes further studies of more and larger samples are needed.
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Conclusions |
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TopAbstractIntroduction
2. Materials and methods...Statistics
ResultsDiscussion
Conclusions
References |
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Excessive secretion in the airways was associated with increased CBF.
Histological findings indicated that BAR did not influence presence of ciliated cells or presence of cilia on columnar epithelium cells. Successful BAR may prevent epithelium metaplasia.
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Acknowledgments |
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References |
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TopAbstractIntroduction
2. Materials and methods...Statistics
ResultsDiscussion
Conclusions
References |
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