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Ann Thorac Surg 2006;82:465-471
© 2006 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

Protective Effect of a Nebulized ß₂-Adrenoreceptor Agonist in Warm Ischemic–Reperfused Rat Lungs

^a Department of Thoracic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
^b Department of Experimental Therapeutic, Translational Research Center, Kyoto University Hospital, Kyoto, Japan

Accepted for publication January 3, 2006.

^_* Address correspondence to Dr Wada, Department of Thoracic Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin, Kyoto 606-8507, Japan. (Email: wadah{at}kuhp.kyoto-u.ac.jp).

	Abstract

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
BACKGROUND: It seems inevitable that non–beating-heart donors will be utilized to resolve the shortage of donors for clinical lung transplantation. The control of warm ischemia-reperfusion injury is crucial in manipulating non–beating-heart donors. We hypothesized that nebulization of a ß₂-adrenoreceptor agonist, salmeterol xinafoate (SLM), during warm ischemia would increase lung tissue cyclic adenosine monophosphate (cAMP) levels, resulting in lung protection.

METHODS: Two studies were conducted. The first investigated the effect of SLM nebulization during ischemia on pulmonary ischemia-reperfusion injury, using an isolated rat lung-perfusion model. The heart-lung block was excised with cannulation of the pulmonary artery and vein, exposed to 55 minutes of ischemia at 37°C, and subsequently reperfused for 60 minutes. Several parameters were measured during reperfusion. In the second study, to measure changes in lung tissue cAMP levels during warm ischemia with or without SLM nebulization, rat lungs were harvested and exposed to 60 minutes of warm ischemia with ventilation.

RESULTS: Salmeterol xinafoate nebulization significantly decreased the pulmonary shunt fraction, airway resistance, and pulmonary vascular resistance. It also inhibited pulmonary edema throughout the reperfusion period. Lung tissue cAMP was effectively maintained by SLM nebulization at the end of reperfusion. Myeloperoxidase activity in the lungs was decreased significantly by SLM nebulization. Lung tissue cAMP levels decreased during the 60 minutes of warm ischemia, but increased with SLM nebulization (p < 0.01).

CONCLUSIONS: Our results confirmed that SLM nebulization during warm ischemia maintained lung tissue cAMP levels, resulting in the alleviation of pulmonary warm ischemia-reperfusion injury.

	Introduction

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The shortage of brain-dead donors remains a critical problem for clinical lung transplantation. To resolve this problem, so-called marginal donors have begun to be utilized, but their numbers are also limited. An alternative method could be the use of lungs from non–beating-heart donors (NBHDs). Several previous studies have considered the potential for lung transplantation from NBHDs [1, 2]. We also previously investigated and reported on the relationship between energy levels and mitochondrial dysfunction, and warm ischemia after circulatory arrest [3, 4].

In 2000, Steen and associates [5] successfully performed lung transplantation from uncontrolled NBHDs. Warm ischemia inevitably occurs in NBHDs during circulatory arrest. The acceptable period of warm ischemia is short, which limits the frequency of lung transplantation from NBHDs. The inhibition of warm ischemia-reperfusion (I-R) injury is, therefore, crucial to facilitate lung transplantation from NBHDs. Several attempts have been made to alleviate warm I-R injury, but have failed to sufficiently improve lung function in organs transplanted from NBHDs [1, 2, 6].

It is difficult to ensure that drugs that are administered intravenously after cardiac arrest are stably distributed. We therefore focused on transalveolar administration as a drug delivery route after cardiac arrest, because transalveolar access is simple and feasible without surgical intervention. Salmeterol xinafoate (SLM), which is a ß₂-adrenoreceptor agonist with a remarkably long duration of action, has been widely used as a long-acting bronchodilator [7]. Salmeterol xinafoate elevates intracellular 3',5-cyclic adenosine monophosphate (cAMP) through its activation of ß₂ adrenoreceptors. Previous reports indicate that ß₂-adrenoreceptor agonists protect the lungs against I-R injury [8–11]. However, to date, no studies have confirmed the effects of nebulized ß₂-adrenoreceptor agonists on lung protection immediately after warm ischemia.

In the current study, we hypothesized that nebulized SLM would elevate lung tissue cAMP levels, which decrease during warm ischemia, and that the increased levels would restore pulmonary function against I-R injury.

	Material and Methods

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Animal Treatment and Material
Male Lewis rats weighing 280 g to 320 g (Japan SLC, Hamamatsu, Japan) were used in this study. All animals received humane care in compliance with the Principles of Laboratory Animal Care, formulated by the US National Society for Medical Research, and the "Guide for the Care and Use of Laboratory Animals," prepared by the US Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). The study protocol was approved by the Ethical Committee of the Faculty of Medicine at Kyoto University, Japan.

Aerosol Delivery
Salmeterol xinafoate (0.12 mM) was dissolved in N,N-dimethylformamide (N,N-DMF; 0.5%) and diluted with saline (0.9%) to achieve the final concentrations shown in parentheses. Hereafter, N,N-DMF and saline are referred to as "the vehicle." The vehicle with or without SLM was aerosolized by a jet nebulizer (PARI Jet-Nebulizer 73-1963; Hugo-Sachs Elektronik Harvard Apparatus, March-Hugstetten, Germany), which was inserted into the inspiratory loop of the ventilator. The operating pressure of the compressed air was 0.15 MPa. In this system, 100% of the particles were maintained below 10 µm, and 60% were maintained below 2.5 µm [12]. In each experiment, 2 mL fluid was nebulized for 10 minutes at the beginning of ischemia.

Investigation of I-R Injury
Isolated rat lung-perfusion model
Isolated rat lung perfusion (Model 829; Hugo-Sachs Elektronik Harvard Apparatus) was performed with the perfusate of diluted homologous blood, as previously reported [13, 14]. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg), intubated after tracheotomy, and ventilated during surgery. After a medial abdominal incision, the animals were heparinized (500 IU/body) through the inferior vena cava. Heparin was administered to enable ex vivo perfusion of the lungs. A median sternotomy was performed, and the pulmonary artery and vein were cannulated directly and connected to the perfusion circuit. The perfusate flow was increased gradually to 10 mL/min, and the heart and lung block was harvested to avoid lung ischemia. The test lung was then placed in the artificial thorax and ventilated with ambient air at negative pressure under the following conditions: respiratory rate = 60 cycles/min; peak inspiratory and expiratory chamber pressures = –8 and –4 cmH₂O, respectively; ratio of inspiratory duration = 50%. The perfusate consisted of heparinized whole blood obtained from 2 additional rats, and saline containing 4% albumin. The pH of the perfusate was adjusted to between 7.3 and 7.4 with sodium bicarbonate. The perfusate was driven by a roller pump at a constant flow of 10 mL/min. The artificial thorax, the perfusate circuit and the airway were water-jacketed, and the temperature was maintained at 37°C throughout the experiment. The perfusate, which was deoxygenated in a glass-made deoxygenator filled with a gas mixture of nitrogen (92%) and carbon dioxide (8%), was pumped into the pulmonary artery of the test lung. The inner surface of the deoxygenator was spherical, so as to maximize its surface area; the perfusate could therefore be deoxygenated while dribbling along the inner surface. The lowered partial oxygen tension created by the deoxygenator mimicked the mixed venous blood in vivo. The perfusate from the pulmonary vein was drained and recirculated to the deoxygenator. Using a pressure-equilibration vessel, the pulmonary venous pressure was kept constant at +2 cmH₂O against the hilum.

Experimental protocol
The animals were randomly allocated to three groups: SLM, control, and sham. In the SLM and control groups, 55 minutes of warm ischemia was started by interrupting the perfusion after 15 minutes for stabilization. Ventilation was continued during the ischemic period. The lungs were then reperfused for 60 minutes with an initial five-step increase in flow rates as follows: 1 mL/min (0 to 2 minutes), 3 mL/min (2 to 4 minutes), 5 mL/min (4 to 6 minutes), 7 mL/min (6 to 8 minutes), and 10 mL/min (8 to 60 minutes). In the SLM group (n = 8), nebulization (10 minutes) of SLM was started immediately after the beginning of ischemia. In the control group (n = 8), nebulization (10 minutes) of the vehicle was started immediately after the beginning of ischemia. In the sham group (n = 5), the lungs were continuously perfused, and the vehicle was nebulized during the last 10 minutes of stabilization. At the end of the stabilization, the time was set at 0, and 60 minutes of perfusion was started (Fig 1).

View larger version (11K): [in this window] [in a new window]   Fig 1. Protocols for the rat lung ischemia–reperfusion experiments. In the salmeterol xinafoate (SLM) and control groups, 55 minutes of warm ischemia was started by interrupting the perfusion after 15 minutes for stabilization. Ventilation was continued during the ischemic period. The lungs were then reperfused for 60 minutes. In the SLM group, nebulization (10 minutes) of SLM was started immediately after the beginning of ischemia. In the control group, nebulization (10 minutes) of the vehicle was started immediately after the beginning of ischemia. In the sham group, the lungs were continuously perfused, with the vehicle being nebulized during the last 10 minutes of stabilization. At the end of stabilization, the time was set at 0, and 60 minutes of perfusion was started.

Determination of cAMP levels after reperfusion
The vasculature of the right lower lobe of each studied lung was flushed with warmed saline (37°C) at the end of reperfusion to measure the level of cAMP in the lung tissue alone. At harvest, the lung tissue was quickly flattened with brass tongs (precooled with liquid nitrogen), immediately soaked in liquid nitrogen, and then lyophilized overnight. After the dry weights were measured, the lung tissues were homogenized in ice-cold 0.1 N HCl. Once the homogenates had been centrifuged at 3,000g for 15 minutes at 4°C, the levels of cAMP in the supernatants were measured with a radioimmunoassay kit (cAMP assay kit; Yamasa, Chiba, Japan). At the same time, protein levels in the lung tissues were measured according to the method described by Lowry and coworkers [15].

Myeloperoxidase activity
The level of myeloperoxidase (MPO) was measured in the left lobe of each of the studied lungs, which were resected at 60 minutes after reperfusion and snap frozen in liquid nitrogen until the assays were performed (MPO Assay Kit; CytoStore, Calgary, Canada). Briefly, tissue was homogenized in potassium phosphate buffer containing hexadecyl trimethyl ammonium bromide. After the homogenate had been centrifuged, the supernatant was decanted. The MPO activity in each sample was measured using a standard chromogenic spectrophotometric technique.

Determination of adenosine nucleotide levels
The right middle lobe of each studied lung was collected immediately after flushing the pulmonary vascular bed with warmed saline (37°C) at the end of reperfusion. At harvest, the lung tissue was quickly flattened with brass tongs (precooled with liquid nitrogen), immediately soaked in liquid nitrogen, and then lyophilized overnight. After the dry weight had been measured, the lung tissue was homogenized in ice-cold 0.5 N perchloric acid. Once the homogenate had been centrifuged at 3,000g for 10 minutes at 4°C, the supernatant was neutralized using 5 N KOH. After the extract had been centrifuged at 3,000g for 10 minutes at 4°C, the supernatant was used to measure the levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) by high-performance liquid chromatography using a column (Shim-pack CLC-ODS [15 cm x 6.0 mm]; Shimadzu, Japan) and 100 mM sodium phosphate buffer (pH 6.0) at a wavelength of 260 nm.

Changes in cAMP Levels During Warm Ischemia
Isolation and ischemia of rat lungs
Rats were anesthetized, intubated after tracheotomy, and ventilated during surgery as described above. The animals were also heparinized (500 IU/rat) using the same protocol. A median sternotomy was performed, and the pulmonary artery was cannulated directly. The abdominal aorta, vena cava, and right and left ventricles were incised to facilitate free flow of pulmonary venous effluent. The pulmonary vascular bed was flushed with 50 mL saline (37°C) at a pressure of 20 cmH₂O to measure the level of cAMP in the lung tissue alone. After flushing the pulmonary vasculature, the right middle lobe was resected to determine the baseline data. The heart and lung block was harvested, and then placed in a glass chamber (artificial thorax) and ventilated with ambient air at negative pressure under the conditions described above. After 60 minutes, the right lower lobe was removed from the same test lung. The temperature was maintained at 37°C throughout the experiment.

Experimental protocol
The animals were randomly allocated to two groups: SLM and control. In the SLM group (n = 5), the lungs were exposed to 60 minutes of warm ischemia, and nebulization (10 minutes) of SLM was started immediately after the beginning of ischemia. In the control group (n = 5), the lungs were exposed to 60 minutes of warm ischemia, and nebulization (10 minutes) of the vehicle was started immediately after the beginning of ischemia.

Determination of cAMP levels during warm ischemia
At harvest, the lung tissues were flattened with brass tongs, soaked in liquid nitrogen, and lyophilized overnight. The cAMP assay of the lung tissue was prepared as described above.

Statistical Analysis
All statistical analyses were performed using StatView 5.0 software (Abacus Concepts, Berkeley, California) on an AT-compatible computer. All values are presented as the mean ± SEM. Data were evaluated using one-way analysis of variance (ANOVA), Scheffe's post hoc multiple comparison test, and Student's paired t test. A probability (p) value less than 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Investigation of I-R Injury
The body weights of the rats were similar in all three groups (300 ± 6 g, 302 ± 5 g, and 297 ± 7 g in the SLM, control, and sham groups, respectively). Hemoglobin levels, WBC counts, and platelet counts of the perfusate from the deoxygenator, both before ischemia and throughout the entire reperfusion period, did not differ among the three groups. The blood gas data for the perfusate from the deoxygenator, both before ischemia and throughout the entire reperfusion period, did not differ among the three groups. All of the lungs in the three groups were reperfused successfully during 60 minutes of postischemic reperfusion. Thus, consistent with previous reports [13, 14], our isolated rat lung-perfusion model remained stable throughout the observation period.

Pulmonary Function Tests
Throughout the entire reperfusion period, the pulmonary shunt fraction in the SLM group was significantly lower than that in the control group (p < 0.01), and was similar to that in the sham group (Fig 2). Thus, SLM nebulization improved the lung gas exchange. Dynamic airway resistance in the control group was significantly higher at both 10 and 60 minutes of reperfusion than in the sham group (p < 0.05). By contrast, at 10, 50, and 60 minutes of reperfusion, the dynamic airway resistance in the SLM group was significantly lower than in the control group (p < 0.05; Fig 3). Warm ischemia for 55 minutes significantly elevated PVR in the control group at 10, 40, 50, and 60 minutes of reperfusion compared with the sham group (p < 0.01). Throughout the entire reperfusion period, SLM nebulization significantly decreased PVR compared with the control group (p < 0.01; Fig 4). The weight of the test lung in the control group at 20, 30, 40, 50, and 60 minutes of reperfusion was significantly higher than in the sham group (p < 0.01). The weight gain of the test lung during reperfusion indicated pulmonary edema in this model. Salmeterol xinafoate nebulization during warm ischemia significantly inhibited pulmonary edema throughout the entire reperfusion period (p < 0.05; Fig 5).

View larger version (8K): [in this window] [in a new window]   Fig 2. Pulmonary shunt fraction. Values are expressed as the mean ± SEM. **p < 0.01 between the salmeterol xinafoate group (solid circles) and the control group (boxes). p < 0.01 between the sham group (open circles) and the control group. (BL = baseline.)

View larger version (11K): [in this window] [in a new window]   Fig 3. Dynamic airway resistance. Values are expressed as the mean ± SEM. *p < 0.05 or **p < 0.01 between the salmeterol xinafoate group (solid circles) and the control group (boxes). p < 0.05 between the sham group (open circles) and the control group. (BL = baseline.)

View larger version (11K): [in this window] [in a new window]   Fig 4. Pulmonary vascular resistance. Values are expressed as the mean ± SEM. **p < 0.01 between the salmeterol xinafoate group (solid circles) and the control group (boxes). p < 0.01 between the sham group (open circles) and the control group. (BL = baseline.)

View larger version (10K): [in this window] [in a new window]   Fig 5. Lung weight gain. Values are expressed as the mean ± SEM. *p < 0.05 or **p < 0.01 between the salmeterol xinafoate group (solid circles) and the control group (boxes). p < 0.01 between the sham group (open circles) and the control group. (BL = baseline.)

View larger version (32K): [in this window] [in a new window]   Fig 6. Lung tissue cyclic adenosine monophosphate (cAMP) levels measured (A) after 60 minutes of warm ischemia and (B) after 60 minutes of reperfusion. Values are expressed as the mean ± SEM. *p < 0.05 and **p < 0.01. (SLM = salmeterol xinafoate.)

View larger version (12K): [in this window] [in a new window]   Fig 7. Myeloperoxidase activity in the studied lungs at 60 minutes after reperfusion. Values are expressed as the mean ± SEM. *p < 0.05 versus the control group. (SLM = salmeterol xinafoate.)

	Comment

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This study clearly showed that SLM nebulization during warm ischemia maintained lung tissue cAMP levels, thereby alleviating pulmonary I-R injury. In this study, transalveolar administration (that is, nebulization) corresponded to a drug delivery route specific to the lung alone. Nebulization has several advantages. First, as no surgical intervention is necessary, it should be more easily accepted with fewer associated ethical problems. Second, as almost all NBHDs have been intubated and ventilated, it is only necessary to connect the nebulizer to the endotracheal tube, so the warm ischemic time might be shortened. Third, physicians other than chest surgeons could easily manipulate the inhalation system. Nebulization is therefore a promising option for lung preservation. Moreover, a combination of nebulization and topical cooling of NBHD lungs (that is, nebulization before the insertion of pleural catheters), as reported by Steen and coworkers [5], could have added effectiveness.

Elevated intracellular cAMP levels are known to protect the lungs against various injuries [10, 11, 16–18]. Intracellular cAMP levels in the lungs are reportedly decreased by lung injury resulting from hypoxia [19]. The current study demonstrated that intracellular cAMP levels in the lungs decreased during warm ischemia; moreover, nebulization after ischemia alone, which simulates the situation after cardiac arrest, maintained intracellular cAMP levels in the lungs. These results are consistent with previous reports showing that pulmonary parenchymal cells persist even after cardiac arrest [20, 21], and confirm that the pathways remain at least partly active. In a lung warm-ischemia model, when substances that elevated intracellular cAMP levels (such as the ß₂-adrenoreceptor agonist isoproterenol [22] and the phosphodiesterase inhibitor rolipram [18]) were administered intravenously at reperfusion, I-R injury in the lungs was reportedly alleviated; however, intravenous administration at reperfusion might decrease systemic pressure. Schutte and colleagues [16] reported that aerosolized prostanoids administered immediately after warm ischemia attenuated pulmonary I-R injury. However, our current study is the first to show that a clinically used ß₂-adrenoreceptor agonist can inhibit pulmonary warm I-R injury. Salmeterol xinafoate is already in widespread clinical use. The clinical application of SLM to control pulmonary I-R injury would therefore be easy and safe in terms of drug toxicity.

Infiltrating leukocytes are a major factor in I-R injury [23]. Diminished intracellular cAMP in the endothelial cells during hypoxia was previously reported to increase leukocyte adhesion [24]. In the current study, MPO activity in the SLM group was significantly lower than in the control group. This finding indicated that elevating intracellular cAMP levels through SLM administration inhibited leukocyte infiltration, resulting in lung protection. It is possible that a reperfusion period of 60 minutes might be too short to detect changes in MPO levels in lung tissue. However, a previous report showed that inhaled nitric oxide decreased MPO levels in an isolated rat lung-perfusion model with 60 minutes of warm ischemia followed by 60 minutes of reperfusion [25]. In addition, ICAM-1 protein levels were reported to increase significantly after just 30 minutes of ischemia in an isolated rat lung [26]. These reports support our finding that SLM administration affected MPO levels after 60 minutes of reperfusion. In the current study, we found no significant elevation of lung tissue ATP levels at the end of reperfusion in the SLM group. Lung protection by SLM might be associated with an improved energy state in the lung tissue. Cyclic adenosine monophosphate is produced from intracellular ATP by adenylyl cyclase. In the SLM group, surplus ATP was consumed for the production of cAMP, so it is possible that lung tissue ATP levels decreased in proportion to the elevation of cAMP levels.

In the current study, the periods of warm ischemia (55 minutes) and reperfusion (60 minutes) were relatively brief. As a next step in the investigation of lung transplantation from NBHDs, it will be necessary to confirm the effect of SLM nebulization over a longer period of reperfusion using large animal models. Non–beating-heart donor lungs have a necessary period of warm ischemia (as addressed by this model); however, they are also usually subjected to a period of cold ischemia. In future experiments with large animal models, we intend to include a period of cold ischemia to mimic the clinical conditions of NBHDs. Our model, in which the lungs are ventilated with negative pressure, is better suited to measuring pulmonary physiology under spontaneous respiration than models with positive pressure ventilation [27]. However, positive pressure ventilation would have been more suitable in the current study, because there is no spontaneous respiration after cardiac arrest. In future experiments using large animal models, we intend to ventilate the lungs with positive pressure to mimic the clinical conditions of NBHDs. In addition, a stand-off period is required after the declaration of death before further actions can be carried out with human NBHDs. We therefore intend to apply a stand-off period of around 5 minutes before SLM nebulization in future studies using large animal models.

In conclusion, we used an isolated rat lung-perfusion model to confirm that SLM nebulization during warm ischemia maintains lung tissue cAMP levels, resulting in the alleviation of pulmonary I-R injury. Our data show that SLM nebulization could be a simple and effective method for use in lung transplantation from NBHDs after cardiac arrest.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We would like to thank Dr Hajime Nakamura from the Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Japan, for scientific and technical support.

	References

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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