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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 2  |  Issue : 4  |  Page : 126-131

Lung recruitment maneuver with and without nebulized albuterol for acute lung injury: a randomized, controlled study


1 Department of Anesthesia, Qena Faculty of Medicine, South Valley University, Qena, Egypt
2 Department of Chest Diseases, Qena Faculty of Medicine, South Valley University, Qena, Egypt

Date of Submission12-May-2015
Date of Acceptance22-Nov-2015
Date of Web Publication17-Mar-2016

Correspondence Address:
Salah M Asida
Department of Anesthesia, Qena University Hospital, Qena Faculty of Medicine, South Valley University, Qena 83523
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2356-9115.178908

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  Abstract 

Background
β2-Adrenoceptor agonists accelerate the resolution of pulmonary edema in experimental and clinical studies. We tested the hypothesis that combining nebulized albuterol with lung recruitment maneuver (RM) during mechanical ventilation would improve oxygenation and accelerate the resolution of pulmonary edema more than would RM alone in patients with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS).
Materials and methods
In this randomized, controlled, single-blinded study, 60 patients admitted to the ICU of Qena University Hospitals and suffering from ALI and/or ARDS were recruited. All patients were subjected to the lung RM. They were assigned to two equal groups: the albuterol group (30 patients), in which patients were medicated with nebulized albuterol 1 mg diluted in 10 ml saline; and the control group (30 patients), in which patients were given 10 ml nebulized saline instead of albuterol. This regimen was repeated every 8 h for 3 days. Alveolar fluid samples were withdrawn before and 1 h after the lung recruitment for protein concentration measurement in the alveolar fluid.
Results
We found a significant difference between the two groups regarding PaO2 (P = 0.003) and PaO2/FiO2 (P = 0.023) before and after RM ± albuterol. No significant difference between the two groups was found regarding alveolar fluid clearance and alveolar protein concentration.
Conclusion
RM with nebulized albuterol improved oxygenation but the addition of albuterol was not beneficial regarding alveolar fluid clearance in terms of lowering alveolar protein concentration in ALI/ARDS patients.
Trial registration
The study was registered in the Australian Newzealand clinical trial registry (ANZCTR), with the number, ACTRN12613000836730, and website, http://www.ANZCTR.org.au/ACTRN12613000836730.aspx.

Keywords: acute lung injury, albuterol, recruitment maneuver


How to cite this article:
Asida SM, Badawy MS. Lung recruitment maneuver with and without nebulized albuterol for acute lung injury: a randomized, controlled study. Res Opin Anesth Intensive Care 2015;2:126-31

How to cite this URL:
Asida SM, Badawy MS. Lung recruitment maneuver with and without nebulized albuterol for acute lung injury: a randomized, controlled study. Res Opin Anesth Intensive Care [serial online] 2015 [cited 2020 Jun 4];2:126-31. Available from: http://www.roaic.eg.net/text.asp?2015/2/4/126/178908


  Introduction Top


The patients of acute lung injury (ALI) suffer from inflammation of the pulmonary blood vessels, which leads to protein-rich fluid leak [when it proceeds to acute respiratory distress syndrome (ARDS)] from the pulmonary circulation into the pulmonary interstitium and alveolar lumen. This impairs gas exchange and gas diffusion across the alveolocapillary membrane, which worsens oxygenation [1]. To improve oxygenation by removing this protein-rich fluid accumulated in the alveoli, we searched for a method to stimulate resolution of this pulmonary edema. It is well known that recruitment maneuver (RM) improves arterial oxygenation and respiratory mechanics by increasing the number of opened alveoli in nonaerated lung areas, thus increasing the amount of oxygen available for gas exchange [2]. In addition, alveolar recruitment reduces lung stretch and hence the inflammatory reaction caused by mechanical ventilation and, therefore, increase alveolar fluid clearance [3].

Bronchodilators are used frequently for patients with acute respiratory failure without a known airway disease. Beneficial effects of this therapy include improved mucociliary clearance [4], more improved lung mechanics, and observed decrease in the work of breathing [5]. On the basis of preclinical observations, it was found that treatment with β2-agonists could reduce pulmonary edema and accelerate alveolar fluid clearance [6]. The primary hypothesis of this study was that the treatment of patients with ALI/ARDS with albuterol combined with lung RM would accelerate the resolution of alveolar edema and improve patients' outcome than would the treatment with RM alone. The aim of this study was to assess the efficacy of the addition of albuterol in improving the response to RM in the form of acceleration of alveolar fluid clearance and patient's oxygenation.


  Materials and methods Top


This randomized, single-blinded, controlled study was carried out in Qena University Hospital's ICU from 2013 to mid 2014. Written informed consent was taken from patients' relatives or caregivers.

Ethical approval for this study was provided by the Ethical Committee of Qena Faculty of Medicine, Qena, Egypt (Chairperson Professor Ahmad Abolyosr) in January 2013.

The study is registered in the Australian Newzealand clinical trial registry (ANZCTR) at the number, ACTRN12613000836730, and website, http://www.ANZCTR.org.au/ACTRN12613000836730.aspx.

Assigning patients and ventilator settings

We enrolled 60 patients, who were either admitted directly to the ICU or referred to the ICU from the Chest Diseases Department. The study interventions began within 12 h of ALI/ARDS confirmation of diagnosis. Patients were randomly assigned to one of two groups according to a computer-generated randomization table. In group A patients received nebulized albuterol just after RM, and in group P, patients received nebulized saline solution as a placebo just after RM. Enrollment and assigning patients to each group were done by a physician (Dr Asida) different from the one who did the nebulization (Dr Shahat, who was blinded to the nature of the nebulized drug). Patients had to fulfill the criteria of the ALI/ARDS according to the 2012 Berlin definition of ALI/ARDS [7] to be included in the study [Table 1]. Exclusion criteria were refusal of consent (by relatives), age younger than 18 years or older than 65 years, chronic respiratory insufficiency (chronic obstructive pulmonary disease), restrictive respiratory insufficiency, increased intracranial pressure, bronchopleural fistula, the persistence of unstable postsurgical hemodynamics despite appropriate supportive therapy, acute myocardial infarction, and neuromuscular disease.
Table 1: Patients' characteristics

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On admission, plain chest radiographic films of the patients were taken before and after RM for confirmation of diagnosis and follow-up. Arterial blood samples were taken for blood gas analysis and for calculating the PO 2 /FiO 2 to assess the degree of severity of ALI/ARDS. Patients were put to sleep (if necessary) by using propofol 1% 1-2 mg/kg intravenously or midazolam 2-5 mg intravenously and intubated after relaxation with atracurium 0.4-0.6 mg/kg intravenously, followed by midazolam 2.5-3.5 μg/kg/min infused intravenously in 50 ml crystalloid solution by a syringe pump. Atracurium was continued as 3.5 μg/kg/min for relaxation and proper measurement of lung mechanics.

Patients were ventilated with Evita 2 ventilator (Drager, Germany). Ventilator was set on volume-controlled mechanical ventilation mode, with a tidal volume of 6 ml/kg and respiratory rate that kept normocapnia. The fraction of inspired oxygen was set at 0.6. Inspiration : expiration time ratio was set as 1:2. Positive end-expiratory pressure (PEEP) was set either 5 cmH 2 O above the lower inflection point or 10 cmH 2 O in the absence of the lower inflection point (this was before and after RM).

Radial or femoral artery catheter was inserted (Arrow Inc., Erding, Germany) for invasive blood pressure monitoring and withdrawal of arterial blood samples for the measurement of arterial blood gases. Patients were also monitored for noninvasive blood pressure, pulse-oximetry, and ECG. A urethral catheter was used for urine output measurement. SpO 2 , heart rate, and mean blood pressure were recorded every 30 min; serial blood gas analysis was carried out every hour; the recorded data for statistical analysis were only those obtained just before, during, and after the RM and nebulization. Medical management of patients was carried out according to the protocols of the ICU of Qena University Hospital for every case according to the cause of ALI/ARDS.

Bronchoalveolar sampling

Bronchoalveolar samples were collected before and 1 h after RM using a 8-Fr 51 cm catheter, which was passed through the suction port of a fiberoptic laryngoscope, which in turn was passed through the endotracheal tube through swivel connection in order not to interrupt ventilation and wedged into the distal airway of a diseased lung area (guided by radiographic film) with contentious suction applied to it (-400 cmH 2 O) to obtain edema fluid at least 1 ml, which was collected in a sterile Eppendorf tube for immediate analysis (Eppendorf SA, Hamburg, Germany) in the clinical pathology lab. After centrifugation for 5 min at 10 000 rpm, the supernatant was recovered for quantifying protein concentration according to the biuret method in a modular analyzer (Roche Hitachi, Mannheim, Germany). Percent alveolar fluid clearance was calculated as = 100×{1-(protein concentration pre-RM/protein concentration post-RM)}, and expressed as %/hour by dividing the number by the time separating the two alveolar samples (just before and 1 h after RM). Pressure-volume (P-V) curves were measured on an Evita 2 ventilator using the low constant flow method described by Lim et al. [8]. During the maneuver, the peak airway pressure was limited to 40 cmH 2 O. P-V curves were measured in zero end expiratory pressure (ZEEP) and at PEEP for each patient, and the corresponding lung volume (exhaled tidal volume) was plotted to define the lower inflection point. The difference in lung volume at PEEP and ZEEP was considered as the amount of lung recruitment.

Recruitment maneuver

The RM was carried out by increasing peak airway pressure so as to get a plateau pressure of 25 cmH 2 O. PEEP was set at 10 cmH 2 O above the lower inflection point for 30 s and repeated for 10 min or 10 cmH 2 O above the prerecruitment PEEP level. If necessary, tidal volume was decreased to maintain peak airway pressure below the upper inflection point or below 40 cmH 2 O if the upper inflection point could not be identified on the ZEEP P-V curve. The whole duration of this RM and nebulization was about 10 min. It was repeated every 8 h for 3 days for every patient. If hypotension (below 60 mmHg mean arterial blood pressure) occurred, the RM was immediately stopped. A positive response to RM was defined as an increase in arterial oxygen tension by 20% of the baseline value.

Albuterol

We used nebulized albuterol (farcoline ampoule, 0.5 mg/ml; Pharco, Egypt) in a dose of 1 mg of albuterol solution (two ampoules farcoline) dissolved into 10 ml of normal saline. The solution was sterile and isotonic. Using Jet Nebulizer (with assumed inhaled particle size of 1-5 μm) connected to the ventilator through the inspiratory limb, albuterol was nebulized through the endotracheal tube of patients after the end of each lung RM, which was every 8 h for 3 days. The physician who performed nebulization was blinded to the nature of the nebulized drug. Nebulization was stopped if tachycardia occurred (heart rate 15% more than that recorded before nebulization).

Measurements

Protein concentration in the alveolar fluid sample withdrawn before and 1 h after lung recruitment (RM) with or without albuterol was measured and recorded. PO 2 and PO 2 /FiO 2 were also calculated before and 1 h after RM. Heart rate and SpO 2 were continuously recorded from the pulse oximeter; invasive blood pressure was measured and recorded every 30 min.

Statistical analysis

On the basis of previously published data we assumed a confidence level of 95% as appropriate. To detect 15% difference in protein concentration and PO 2 /FiO 2 ratio, the sample size of 30 cases per group (60 cases total) was calculated (http://www.surveysystem.com).

Statistical analysis was carried out using the SPSS program version 17 (USA). All data were expressed as mean ± SD. Patient's baseline clinical characteristics in the two groups were compared using the χ2 -test. Changes in cardiorespiratory parameters, as well as protein concentration in the bronchoalveolar lavage, were analyzed by using the unpaired t-test. P value (for all post RM values) less than 0.05 was considered statistically significant.


  Results Top


This study was conducted on 60 patients admitted to the ICU of Qena University Hospital. The cause of admission is shown in [Table 1]. The patients' characteristics were comparable in the two groups; no significant difference was found between the two groups [Table 1].

We found no significant difference in mean heart rate and mean blood pressure between the two groups, and when compared with prerecruitment data, although heart rate was slightly higher in the albuterol group, it was not statistically nor clinically significant [Table 2], [Table 3] and [Table 4].
Table 2: Cardiorespiratory parameters of the fi rst day of the study

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Table 3: Cardiorespiratory parameters of the second day of the study.

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Table 4: Cardiorespiratory parameters of the third day of the study

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Ventilator setting parameters were comparable for the two groups; no significant difference was recorded [Table 2], [Table 3] and [Table 4].

We found that the PaO 2 values for the 3 study days were higher in the albuterol group than in the placebo group; the difference was statistically significant. The PaO 2 /FiO 2 ratio was higher in the albuterol group than in the placebo group, as we found statistically significant difference between the two groups [Table 2], [Table 3] and [Table 4].

No significant difference was found between the two groups regarding protein concentration in the alveolar fluid samples after the RM with albuterol (group A) or without albuterol (control group). In addition, we found no significant difference between the two groups regarding alveolar fluid clearance, throughout the duration (3 days) of the study [Table 5].
Table 5: Protein concentration (g/dl) in bronchoalveolar samples

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  Discussion Top


This study was conducted in the ICU of Qena University Hospital on 60 patients admitted for the management of ALI (and/or ARDS) due to various causes. To improve oxygenation and decrease alveolar fluid accumulation, which impairs gas exchange in the lung and worsens the patient's general condition, we considered the lung RM as a part of the treatment protocol.

This study showed improved oxygenation in the form of increased PaO 2 1 h after the RM with albuterol nebulization and also the PaO 2 /FiO 2 ratio throughout the duration (3 days) of the study [Table 2], [Table 3] and [Table 4]. We also found improved alveolar fluid clearance after RM but with no significant difference between the albuterol group and the placebo group [Table 5]. However, there was a difference in protein concentration between pre-RM with post-RM for each group.

The question is, could this improved oxygenation and alveolar fluid clearance be attributed to RM alone or RM and nebulized albuterol? In other words, does albuterol administration have any role in improving oxygenation and alveolar fluid clearance?

The beneficial effects of RM on oxygenation and alveolar fluid clearance have been established and reported in many studies [2],[9],[10]. There are many techniques for lung recruitment. All of these methods focus on temporarily increasing the airway pressure above the set PEEP to open the closed alveoli in the distal lung areas and filling it with oxygen to allow more oxygen for gas exchange and optimize ventilation/perfusion ratio [2],[3]. The RM carried out in our study was similar to that carried out in the National Heart, Lung and Blood Institute ARDS clinical trials network [11].

Constantin and colleagues in 2007, assessing the effect of RM on oxygenation and alveolar fluid clearance, compared the patients' responses to RM as regards improved oxygenation with those who did not respond to RM; they found that responders maintained net alveolar fluid clearance as the protein concentration in the alveolar fluid samples increased after 1 and 4 h after RM. Thus, they demonstrated that RM not only improved oxygenation but also alveolar fluid clearance at the same time [12]. In our study, we did not measure protein concentration or oxygenation parameters after 4 h of RM (±albuterol), as was done in a study by Constantin, as it was satisfactory to us to demonstrate improved oxygenation and alveolar fluid clearance after the administration of albuterol following RM. The results of our study were similar to the results obtained in the study by Constantine regarding the effect of RM alone on oxygenation and alveolar fluid clearance.

Grasso and colleagues in 2002 demonstrated (as was in our study) the beneficial effect of RM on patient's oxygenation and net alveolar fluid clearance. A total of 22 patients with ARDS were studied during the ARDSnet lung-protective ventilatory strategy: tidal volume was set at 6 ml/kg, PEEP and FiO 2 were set at SpO 2 of 90-95%, and/or a PaO 2 of 60-80 mmHg (baseline) was used. They reported an increase PaO 2 /FiO 2 by 20 ± 3% in nonresponders (n = 11) and by 175 ± 23% (n = 11) in responders [9].

Similar findings were reported in a study by Valente Barbas in 2003, who selected published medical literature from 1972 to 2002 and his personal observations. He reported that in the clinical setting, RMs that use a continuous positive airway pressure of 40 cmH 2 O for 40 s improve oxygenation in patients with early ARDS, who do not have an impairment in the chest wall. High intermittent PEEP, intermittent sighs, or high-pressure-controlled ventilation improves short-term oxygenation in ARDS patients [10].

Several studies were conducted to assess the role of albuterol in accelerating the resolution of pulmonary edema and in protecting alveolar epithelium from injury due to mechanical ventilation in ALI/ARDS patients [12],[13],[14],[15].

Perkins et al. in 2006 [15] conducted a study on the effect of intravenous infusion of albuterol (15 μg/kg/h) or placebo for 7 days on 40 patients on mechanical ventilation during the treatment of ALI. They concluded that further research was required to confirm the efficacy and safety of intravenous salbutamol (albuterol) in patients with ALI/ARDS, although their trial provided the first proof that in humans with ALI/ARDS, sustained treatment with intravenous β-agonists reduces extravascular lung water. Perkins used salbutamol (albuterol) intravenously, but in the present study we used albuterol by inhalational route on the basis of previous reports of the efficiency of nebulized albuterol in reducing alveolar edema [11] and believing that the nebulization can ensure maximum bioavailability of this drug at the target site of action, namely the alveoli and bronchial tree. The intravenous route distributes the drug all over the body, which may decrease its concentration in the lungs and decrease its desired effect, which may require the use of higher dose with its possible cardiovascular side effects.

Has the use of nebulized albuterol to our treatment protocol added any benefit regarding oxygenation, alveolar edema clearance, and to patient's outcome? Indeed, the results of this study did not support the use of albuterol. We found no statistically significant difference between the group that received nebulized albuterol and the group that received placebo apart from the improved oxygenation, which can be attributed to RM only [Table 2], [Table 3] and [Table 4]. We found many studies that agree with the results of this study. Matthay and colleagues in 2011 [11] published a multicenter, clinical trial conducted on 282 patients with ALI who were put on mechanical ventilation. Patients were randomized to receive aerosolized albuterol (5 mg) or saline placebo every 4 h for up to 10 days. The ventilator-free days were their primary outcome variable. The researchers found no significant difference between the albuterol and placebo groups. They concluded that aerosolized albuterol does not improve clinical outcomes in patients with ALI, and recommended against the routine use of β2-agonist therapy in mechanically ventilated patients with ALI. In our study, we used a smaller dose of albuterol and tested the short-term effects of RM ± albuterol, and found results similar to those of a study by Matthay in 2011. We administered 1 mg of albuterol three times instead of four times daily, as was done in Matthay's study, to decrease the possibility of side effects of albuterol on our patient's hemodynamics, namely tachycardia, cardiac arrhythmias, and hypokalemia. In this study, heart rate values were elevated in the albuterol group, but we found them to be statistically significant. In addition, we found no serious adverse effects, including ventricular dysrhythmias or atrial fibrillation.

Lastly, why could not the patient benefit from albuterol nebulization? Many explanations can be given for this: the alveolar epithelium is usually not intact in ALI/ARDS patients, thus rendering it unresponsive to albuterol [16]; in addition, the alveolar edema makes a barrier for albuterol to reach alveolar capillaries as it dilutes the delivered drug. Another possibility is the occurrence of down-regulation of β2 receptors during albuterol therapy over the study period. However, most experimental studies [14],[17] suggested that this is a minor factor, especially if we consider the short period of the study during which the patients receive albuterol.

It is to be noted that our study did not test whether there was a difference in the response of patients to RM ± nebulized albuterol regarding the primary cause of ALI/ARDS: pulmonary and extrapulmonary.


  Conclusion Top


This study showed no clinical benefit from using nebulized albuterol in a combination with RM during the management of ALI/ARDS patients. We believe that the improved oxygenation parameters and alveolar fluid clearance could be mainly attributed to RM. The improved alveolar fluid clearance and reduction of alveolar protein concentration was obvious when comparing protein concentration before RM ± albuterol with that after RM [Table 5], as we found improved alveolar fluid clearance but with the same degree in the two groups, showing that the use of albuterol was not effective regarding this point.


  Acknowledgements Top


The authors thank Professor Abdelrahman Abdelhamid, Professor of Clinical Pathology, Qena University Hospital for his help in measuring alveolar protein concentration.

Financial support was obtained from South Valley University and Qena University Hospital.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Folkesson HG, Matthay MA. Alveolar epithelial ion and fluid transport: recent progress. Am J Respir Cell Mol Biol 2006; 35:10-19.  Back to cited text no. 1
    
2.
Moran I, Zavala E, Fernandez R, Blanch L, Mancebo J. Recruitment maneuvers in acute lung injury/acute respiratory distress syndrome. Eur Resp J Suppl 2003; 42:37S-42S.  Back to cited text no. 2
    
3.
Frank JA, McAuley DF, Gutierrez JA, Daniel BM, Dobbs I, Matthay MA. Different effects of sustained inflation recruitment maneuvers on alveolar epithelial and lung endothelial injury. Crit Care Med 2005; 33:181-188.  Back to cited text no. 3
    
4.
Bennett WD. Effect of b-adrenergic agonists on mucociliary clearance. J Allergy Clin Immunol 2002; 110:S291-S297.  Back to cited text no. 4
    
5.
Mancebo J, Amaro P, Lorino H, Lemaire F, Harf A, Brochard L. Effects of albuterol inhalation on the work of breathing during weaning from mechanical ventilation. Am Rev Respir Dis 1991; 144:95-100.  Back to cited text no. 5
    
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Chang LH, Honiden S, Haithcock JA, Das AM, Short KA, Nierman DM, Carson SS. Utilization of bronchodilators in ventilated patients without obstructive airways disease. Respir Care 2007; 52:154-158.  Back to cited text no. 6
    
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Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, et al. Acute respiratory distress syndrome: the Berlin Definition. JAMA 2012; 307:2526-2533.  Back to cited text no. 7
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8.
Lim CM, Koh Y, Park W, Chin JY, Shim TS, Lee SD, et al. Mechanistic scheme and effect of 'extended sigh' as a recruitment maneuver in patients with acute respiratory distress syndrome: a preliminary study. Crit Care Med 2001; 29:1255-1260.  Back to cited text no. 8
    
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Grasso S, Mascia L, Del Turco M, Malacarne P, Giunta F, Brochard L, et al. Effects of recruiting maneuvers in patients with acute respiratory distress syndrome ventilated with protective ventilatory strategy. Anesthesiology 2002; 96:795-802.  Back to cited text no. 9
    
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Valente Barbas CS. Lung recruitment maneuvers in acute respiratory distress syndrome and facilitating resolution. Crit Care Med 2003; 31:S265-S271.  Back to cited text no. 10
    
11.
Matthay MA, Brower RG, Carson S, Douglas IS, Eisner M, Hite D, et al., National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome (ARDS) Clinical Trials Network. Randomized, placebo-controlled clinical trial of an aerosolized β2 -agonist for treatment of acute lung injury. Am J Respir Crit Care Med 2011; 184:561-568.  Back to cited text no. 11
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12.
Constantin JM, Cayot-Constantin S, Roszyk L, Futier E, Sapin V, Dastugue B, et al. Response to recruitment maneuver influences net alveolar fluid clearance in acute respiratory distress syndrome. Anesthesiology 2007; 106:944-951.  Back to cited text no. 12
    
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Sakuma T, Gu X, Wang Z, Maeda S, Sugita M, Sagawa M, et al. Stimulation of alveolar epithelial fluid clearance in human lungs by exogenous epinephrine. Crit Care Med 2006; 34:676-681.  Back to cited text no. 13
    
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Sartori C, Fang X, McGraw DW, Koch P, Snider ME, Folkesson HG, Matthay MA. Selected contribution: long-term effects of b2-adrenergic receptor stimulation on alveolar fluid clearance in mice. J Appl Physiol 2002; 93:1875-1880.  Back to cited text no. 14
    
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Perkins GD, McAuley DF, Thickett DR, Gao F. The beta-agonist lung injury trial (BALTI): a randomized placebo-controlled clinical trial. Am J Respir Crit Care Med 2006; 173:281-287.  Back to cited text no. 15
    
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Atabai K, Ware LB, Snider ME, Koch P, Daniel B, Nuckton TJ, Matthay MA. Aerosolized b2-adrenergic agonists achieve therapeutic levels in the pulmonary edema fluid of ventilated patients with acute respiratory failure. Intensive Care Med 2002;28:705-711.  Back to cited text no. 16
    
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Maron MB, Folkesson HG, Stader SM, Hodnichak CM. Impaired alveolar liquid clearance after 48-h isoproterenol infusion spontaneously recovers by 96 h of continuous infusion. Am J Physiol Lung Cell Mol Physiol 2006; 291:L252-L256.  Back to cited text no. 17
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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