Ann Thorac Surg 2008;85:1925-1929. doi:10.1016/j.athoracsur.2008.02.084
© 2008 The Society of Thoracic Surgeons
Original Articles: General Thoracic
Effects of Cyclosporine A and Bronchial Transection on Mucociliary Transport in Rats
Rogerio Pazetti, PhDa,
Paulo M. Pêgo-Fernandes, MD, PhDa,*,
Geraldo Lorenzi-Filho, MD, PhDb,
Paulo H.N. Saldiva, MD, PhDc,
Luiz Felipe P. Moreira, MD, PhDd,
Fabio B. Jatene, MD, PhDa
a Laboratory of Thoracic Surgery Research, Department of Cardiopneumology, Faculty of Medicine, University of São Paulo, São Paulo, São Paulo, Brazil
b Pulmonary Division, Department of Cardiopneumology, Faculty of Medicine, University of São Paulo, São Paulo, São Paulo, Brazil
c Laboratory of Experimental Air Pollution, Department of Pathology, Faculty of Medicine, University of São Paulo, São Paulo, São Paulo, Brazil
d Heart Institute of Clinics Hospital, Faculty of Medicine, University of São Paulo, São Paulo, São Paulo, Brazil
Accepted for publication February 11, 2008.
* Address correspondence to Dr Pêgo-Fernandes, Laboratory of Thoracic Surgery Research, Department of Cardiopneumology, Faculty of Medicine, University of São Paulo, Avenida Doutor Enéas de Carvalho Aguiar, 44-2° andar, bloco 2, sala 9, São Paulo-SP, 05403-000, Brazil (Email: paulo.fernandes{at}incor.usp.br).
Presented at the Forty-fourth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 28–30, 2008.
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Abstract
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Background: Posttransplant infection remains the leading cause of morbidity and mortality after lung transplantation. We hypothesized that bronchial transection and immunosuppression by cyclosporine both play a key role in the impairment of airway mucociliary clearance, a basic defense system.
Methods: Sixty-four rats were assigned to four groups of 16 each according to surgical procedure and drug therapy as follows: sham-operated and saline solution; bronchial transection and saline solution; sham-operated and cyclosporine; bronchial transection and cyclosporine (10 mg/kg/day). Eight animals from each group were euthanized on postoperative day 30 or 90. In vitro mucus transportability, in situ mucociliary transport, and ciliary beating frequency were measured.
Results: There was a significant impairment (p < 0.001) on ciliary beating frequency due to either bronchial transection or cyclosporine therapy. In vitro transportability was impaired only in cyclosporine-treated groups (p < 0.001). In situ mucociliary transport was reduced in cyclosporine-treated animals as well as in those that underwent bronchial transection (p < 0.001). This impairment was significantly recovered 90 days after operation. In contrast, the effects of cyclosporine did not change over 90 days of treatment.
Conclusions: These results support our hypothesis that mucociliary clearance is impaired after bronchial transection and cyclosporine therapy. Further studies are necessary to relate this finding with posttransplant infection and also to test some drugs aiming to protect airway mucociliary system.
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Introduction
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Lung transplantation is a therapeutic option for selected patients with end-stage lung disease [1] such as emphysema, fibrosing alveolitis, cystic fibrosis, pulmonary hypertension, and bronchiectasis [2]. Pulmonary infections have remained the most common cause of early and late morbidity and mortality in lung transplant recipients [3].
Chmiel and Davis [4] show that patients with cystic fibrosis become infected due to an impaired mucociliary clearance. The mucociliary system is the pulmonary first line of defense against infections. It continuously removes particles and pathogenic microorganisms that are trapped on the mucus [5]. Several factors involved in the transplantation procedure, such as bronchial transection, denervation, devascularization, anesthesia, and dehydration, can cause transient or permanent injury to the mucociliary system [6].
In this regard, bronchial transection plays an important role in postoperative complications. The consequences of the injury due to bronchial transection can be verified on the ciliated epithelium, either close to the anastomosis area or along the entire distal bronchial stem [7]. After lung transplantation, both mucociliary transport (MCT) and ciliary beating frequency (CBF) are impaired, and the rigidity of the mucus also increases [8]. We have previously shown that MCT is impaired up to 30 days after bronchial transection and reanastomosis [9].
The use of immunosuppression is mandatory after lung transplantation [10], and such agents are a major determinant for posttransplant infectious complications. Immunosuppression may be deleterious to the defense system not only by acting in the immune system but may also have effects at the bronchial level. Actually, in a recent study we found that cyclosporine A (CsA) caused a decrease in mucus production from bronchial respiratory epithelium after a 30-day period of treatment [11].
The present study was designed to answer some important questions concerning the effects of bronchial section and immunosuppression on MCT in rats after lung transplantation: Is there any synergistic effect between them? Is there any recovery on mucociliary system after surgery and therapy? If so, how long does it take?
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Material and Methods
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Experimental Design
Sixty-four male Wistar rats (weight, 300 ± 25 g) were assigned to four groups of 16 rats each according to surgical procedure and drug therapy as follows: sham-operated and saline solution (ShSal); bronchial transection and saline solution (TrSal); sham-operated and cyclosporine A (ShCsA), and bronchial transection and cyclosporine A (TrCsA). The animals were maintained according to the Guide for the Care and Use of Laboratory Animals [12]. Our Institution's Ethical Committee approved the protocol.
Surgical Procedure
Anesthesia was induced with inhaled isoflurane (Isothane, Baxter, Puerto Rico) in a closed chamber, followed by orotracheal intubation with 6.5-cm-long 14-gauge catheter. General anesthesia maintained by inhalation of 2% isoflurane from a nebulizer (Model 1223; Takaoka; São Paulo, Brazil). Ventilation was achieved by a volume-cycled ventilator (Harvard Apparatus, Holliston, MA), with respiratory rate of 70 breaths/min and tidal volume of 10 mL/kg.
A left thoracotomy was performed. The left main stem bronchus was dissected, clamped, and completely transected, followed by an end-to-end anastomosis with continuous running 8-0 polypropylene suture. All sham-operated animals underwent the same surgical procedure, except for the bronchial transection and anastomosis. A stereomicroscope (x8 original magnification) was used for the procedure.
Therapy
All animals were treated subcutaneously either with 50-mg/mL cyclosporine A (SANDIMMUN, Novartis, Switzerland) in a daily dose of 10 mg/kg, or similar volume of saline solution (0.9% sodium chloride, Baxter, São Paulo, Brasil), from operation day to the postoperative day 30 or 90.
Euthanasia
After 30 or 90 days of treatment, 8 animals from each group were anesthetized with intraperitoneal pentobarbital (50 mg/kg) and euthanized by exsanguination, according to the Report of the American Veterinary Medicine Association Panel on Euthanasia [13].
Data Collection and Analysis
The lungs were removed from the thoracic cavity, an incision was performed in the left main stem bronchus, and mucus samples were collected. In vitro mucus transportability was evaluated by using a bullfrog (Rana catesbeiana) palate model, as follows: The transport velocity of the mucus sample placed on an excised frog's palate was determined with the aid of a stereomicroscope equipped with a reticulated eyepiece. The velocity of the rat mucus samples was compared with the transport speed of autologous frog mucus, and the results were expressed as relative speed (rat/frog).
After mucus sample collection, the ventral wall of the left main stem bronchus was opened to expose the ciliated epithelium. The bronchus was placed under a light microscope (Olympus, BX50, Tokyo, Japan) that was connected to a camera (Sony Triniton, 3CCD, Tokyo, Japan). A stroboscope (Machine Vision Strobe, Cedarhurst, NY) was placed in front of the bronchus and CBF was measured by synchronization between cilia movement and stroboscope flashlight. Then, under the same microscope, in situ MCT was timed by direct observation of talc particle movement across the bronchus.
All data were analyzed with Prism software (GraphPad Software Inc, San Diego, CA). Analysis of variance was used to verify the interference and interaction of the factors. Comparisons between groups were performed using the Bonferroni posttest. Results were expressed as mean ± SD and significance was considered for p < 0.05.
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Results
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Our results showed a significant impairment on CBF (Fig 1) at 30 and 90 days in all groups that underwent either bronchial transection (TrSal30, 5.33 ± 0.73; TrSal90, 7.15 ± 0.47) or CsA therapy (ShCsA30, 6.49 ± 1.11; ShCsA90, 6.07 ± 1.20) compared with control groups (ShSal30, 8.35 ± 0.68; ShSal90, 8.98 ± 1.04; p < 0.001). The CBF values were worse when these two factors were present together (TrCsA30, 4.32 ± 0.75; TrCsA90, 5.11 ± 0.63).
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Fig 1. Ciliary beating frequency (mean ± standard deviation) from operated on and unoperated on left main stem bronchi of rats treated with saline or cyclosporine A for 30 or 90 days. There was a significant difference between groups: *vs sham saline (ShSal, white bars); #vs bronchial transection, saline (TrSa, light gray;) and sham cyclosporine A (ShCsA, dark gray); p < 0.001. (TrCsA = bronchial transection treated with cyclosporine A, black bars.)
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There was a significant impairment on in vitro mucus transportability (p < 0.001) only in CsA-treated groups (ShCsA30, 0.65 ± 0.19; TrCsA30, 0.66 ± 0.13; TrCsA90, 0.60 ± 0.11) compared with control groups (ShSal30, 0.95 ± 0.17; ShSal90, 0.86 ± 0.06). Bronchial transection did not affect this variable (Fig 2).
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Fig 2. In vitro mucus transportability (mean ± standard deviation) from operated on and unoperated on left main stem bronchi of rats treated with saline or cyclosporine A for 30 or 90 days. There was a significant difference between groups: *vs sham saline (ShSal, white bars); p < 0.001. (TrSa = bronchial transection, saline light gray; ShCsA = sham operation, cyclosporine A, dark gray; TrCsA = bronchial transection, cyclosporine A, black bars.)
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In situ MCT was significantly slower in all CsA-treated animals (ShCsA30, 0.27 ± 0.06; ShCsA90, 0.26 ± 0.05) as well as in those that underwent bronchial transection (TrSal30, 0.10 ± 0.03; TrSal90, 0.26 ± 0.04) compared with control groups (ShSal30, 0.37 ± 0.05; ShSal90, 0.37 ± 0.07). This in situ MCT impairment of the saline-treated animals on postoperative day 30 was significantly recovered 90 days after bronchial transection (TrSal30 vs TrSal90, p < 0.001). In contrast, CsA-treated animals did not show any significant recovery up to 90 days after the procedure (TrCsA30, 0.07 ± 0.03 vs TrCsA90, 0.12 ± 0.03, p > 0.05). A positive and significant interaction (p < 0.001) was noted between bronchial transection and CsA therapy on in situ MCT after 90 days of treatment (Fig 3).
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Fig 3. In situ mucociliary transport (mean ± standard deviation) from operated on and unoperated on left main stem bronchi of rats treated with saline or cyclosporine A for 30 or 90 days. There was a statistically significant difference between groups: *vs sham saline (ShSal, white bars); #vs bronchial transection, saline (TrSa, light gray;) and sham cyclosporine A (ShCsA, dark gray); p < 0.001. (TrCsA = bronchial transection treated with cyclosporine A, black bars.)
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All collected data as well as comparison between groups concerning CBF, mucus transportability, and MCT are reported in Tables 1, 2, and 3,
respectively.
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Comment
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The present observation of a significant in situ MCT impairment after bronchial transection and CsA therapy is in accordance with our previous results [9, 11]. The present study extends those findings by showing that (1) rats undergoing bronchial transection and CsA therapy showed a further in situ MCT impairment, (2) in situ MCT was recovered after 90 days of bronchial transection only in saline-treated animals, and (3) the effects on in situ MCT were mostly mediated by a decrease on ciliary beating frequency.
The perfect function of MCT depends on an interaction among various factors, such as an intact ciliated epithelium with synchronized ciliary beating, an adequate amount of mucus with ideal viscoelastic properties, and the adequate composition and size of the periciliary fluid layer [14]. All these factors directly depend on good local vascularization and innervation [15]. During lung transplantation, both the arterial and nervous supply of the bronchus are completely interrupted and not reconnected, which can cause many disturbances in mucociliary clearance [16].
In this study we performed a long single anastomosis after bronchial transection, and our results showed an impaired MCT at postoperative day 30, likewise described by Rivero and coworkers [9]. Although there was a significant recovery of the mucociliary function on at postoperative day 90, it did not return to normal values. This early impairment, followed by a late recovery of the mucociliary clearance after 90 days in rats treated with saline solution, points to the regeneration of the ciliated epithelium as a causative factor. Brody and coworkers [17] observed that within postoperative week 1, particle clearance had returned to normal values in transplanted dogs and remained so up to 26 weeks after transplantation. Another study, however, concluded that mucociliary clearance is impaired in most transplanted and denervated lungs for periods of up to several months postoperatively [18]. Our results were similar to those observed by Marelli and coworkers [19], who showed that a recovery of mucociliary function occurred at 12 weeks after operation that could be attributed to revascularization.
A second important factor that increases the risk of pulmonary infection in this immediate postoperative period is the use of immunosuppressant agents to avoid rejection. Cyclosporine A was chosen to be the immunosuppressant agent in our study because of its clinical use at large in posttransplant patients [20]. We administered CsA daily in a subcutaneous dose of 10 mg/kg. Many studies report this is an effective and nontoxic dose that is well tolerated by rats and shows a constant serum concentration [21]. The serum CsA levels we found (1350 ng/mL) were similar to other studies that tested the pharmacokinetics of this drug [22]. In this study, the MCT of the operated on main stem bronchus was markedly impaired after treatment with CsA for 30 days and up to 90 days.
Evidence indicates that CsA blocks the initial stages of lymphocyte activation, inhibiting the production of interleukin-2 and other lymphokines [23, 24]. Theoretically, the CsA mechanism of action includes an influence on cytoplasmic calcium that is an essential component for normal cellular function. Some authors have reported a direct correlation between CsA-induced cytotoxicity and changes in mitochondrial enzyme activity [25]. Cyclosporine A has also the ability to interact with receptors within the nucleus to inhibit genetic transcription of proteins secreted by fibroblasts, endothelial cells, macrophages, and monocytes [26]. Neuringer and coworkers [27] showed that CsA exerts growth inhibitory effect on airway epithelial cells that alters respiratory epithelium integrity. We can infer from these facts that CsA is able to influence the ciliated cells of the respiratory epithelium and impair either the ciliary beating mechanism or even the production and composition of the mucus.
Respiratory mucus must have ideal physical properties (viscosity and elasticity) to allow cilia penetration and to receive their transmitted energy and provide the mucus transport along the ciliated epithelium effectively [28]. To assess the transportability of the mucus samples we used an in vitro model of mucus-depleted frog palate, because the bullfrog's palate is lined with a pseudostratified epithelium similar to that found in human conductive airways [7, 29]. Rubin and coworkers [30] suggested that bullfrog mucus has viscoelastic properties similar to normal mammalian respiratory mucus. Our study showed a significant difference between collected samples from saline-treated groups vs CsA-treated groups, as well as between sham-operated animals vs operated animals. These results support the hypothesis that there is influence of both CsA therapy and bronchial transection on the structure of respiratory mucus, which will ultimately result in an impairment of the transportability by the ciliated epithelium of frog palate.
We concluded that CsA therapy impairs airway mucociliary clearance by decreasing both ciliary beating frequency and in vitro mucus transportability. This effect is more important in transected bronchi, suggesting an additive deleterious effect of CsA administration and bronchial transection on MCT. Such findings are important in the context of pulmonary transplantation, where these two conditions coexist. Because these two factors cannot be avoided in human lung transplantation, we believe that some drugs that are well known for improving mucociliary clearance, such as methylxanthines, sodium cromoglycate, cholinergics, hypertonic saline, and others, must be concomitantly administered with the immunosuppressive therapy. Further studies will be necessary to verify if other immunosuppressant drugs, namely tacrolimus, azathioprine, prednisone, and mycophenolate mofetil, have the same negative effects on this system, and to find a drug able to avoid this undesirable effect on the respiratory epithelium.
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Discussion
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DR MICHAEL S. MULLIGAN (Seattle, WA): I have a question for you. In your manuscript you indicated that the levels of cyclosporine, the serum levels that you were achieving, were approximately 10-fold higher than what we would see clinically, what we would benchmark clinically. And it could be that a significant amount of this relates to cyclosporine toxicity. I can't comment on that because I don't know the mechanism whereby cyclosporine is modulating mucociliary transport anyway. But I would wonder whether or not you had assessed viability, particularly when doing your in situ studies, as to whether or not these airway epithelial cells were still alive or your beating frequency in transport had a net negative effect because a lot of the cells were dead.
DR PEGO-FERNANDES: We know that the metabolism of the rats and the patients are different. And we used the same dose of cyclosporine A reported in the literature as an immunosuppressant and nontoxic dose. And we did a histologic study in another publication in the past year that you see that the cells are okay but produce less mucus than those without the drug.
DR MULLIGAN: But you haven't done a dose response to figure out where that threshold effect is experienced? Because it is tough to translate if your actual levels are 10-fold higher than one would use in a human.
DR PEGO-FERNANDES: Yes. But we used the dose response curve performed by other groups that used rats, and we have another paper showing that this dosage of the cyclosporine A is not toxic.
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Acknowledgments
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This study was performed at the Thoracic and Cardiovascular Surgery Post-Graduation Program of Heart Institute of Clinics Hospital of São Paulo University Medical School, São Paulo, SP, Brazil. It was supported by grants from The State of São Paulo Foundation (FAPESP), São Paulo, SP, Brazil.
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References
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Abstract
Introduction
