Physiological Effects of Different Recruitment Maneuvers for Improved Lung Inhomogeneity in a Pig Model of ARDS

Background: In acute respiratory distress syndrome (ARDS), lung recruitment maneuvers can recruit collapsed alveoli in gravity-dependent lung regions, improving the homogeneity of ventilation distribution. This study used electrical impedance tomography (EIT) to investigate the physiological effects of different recruitment maneuvers for alveolar recruitment in a pig model of ARDS. Methods: ARDS was induced in ten healthy male pigs with repeated bronchoalveolar lavage until the arterial partial pressure of oxygen (PaO 2 )/fraction of inspired oxygen (FiO 2 ) (P/F ratio) was < 100 mmHg and remained stable for 30 minutes (T ARDS ). ARDS pigs underwent three sequential recruitment maneuvers, including sustained inflation (SI), increments of positive end-expiratory pressure (PEEP) (IP), and pressure-controlled ventilation (PCV) applied in random order, with 30 mins at a PEEP of 5 cmH 2 O between maneuvers. Respiratory mechanics, hemodynamics, arterial blood gas, and EIT were recorded at baseline, T ARDS , and before and after each recruitment maneuver. Results: In all ten pigs, ARDS was successfully induced with a mean 2.8±1.03L (2800±1032.80ml) bronchoalveolar lavages. PaO 2 , SO 2 , P/F, and compliance were significantly improved after recruitment with SI, IP or PCV (all p<0.05), and there were no significant differences between maneuvers. Global inhomogeneity (GI) was significantly decreased after recruitment with SI, IP, or PCV. There were no significant differences in GI before or after recruitment with the different maneuvers. The decrease in GI (ΔGI) was significantly greater after recruitment with IP compared to SI (p=0.023), but there was no significant difference in ΔGI between IP and PCV. Conclusion: SI, IP, and PCV increased oxygenation, and regional and global compliance of the respiratory system, and decreased inhomogeneous gas distribution in ARDS pigs. IP significantly improved inhomogeneity of the lung compared to SI.


Background
Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by a decrease in functional lung size 1 . The pathophysiology of ARDS includes diffuse alveolar collapse 3 and acute exudative lesions distributed in a gravitationally dependent gradient 4 . Although this disease was first 3 defined almost 50 years ago, the hospital mortality rate for patients with severe ARDS remains high, estimated at 46% 2 .
Lung recruitment maneuvers, including sustained inflation (SI), increments of positive end-expiratory pressure (PEEP) (IP), and pressure-controlled ventilation (PCV), can improve oxygenation and increase respiratory system compliance in patients with ARDS. Recruitment maneuvers can recruit collapsed alveoli in gravity-dependent lung regions and improve the homogeneity of ventilation distribution, but may cause alveolar overdistention and lead to ventilator-associated lung injury in non-dependent regions. 5 A randomized controlled trial showed that SI and PCV improved the arterial partial pressure of oxygen (PaO 2 )/fraction of inspired oxygen (FiO 2 ) (P/F ratio) in 40 patients with ARDS, and the P/F was significantly increased after PCV compared to SI 6 . However, dynamic regional information on changes in lung ventilation after recruitment maneuvers has not been reported.
Recruitment and overdistention during lung recruitment have been evaluated by chest X-ray, computed tomography, and lung ultrasound. Electrical impedance tomography (EIT) is a non-invasive, radiation-free technique that can be used for bedside monitoring of lung tissue aeration during breathing. EIT allows semi-continuous, real-time measurement of changes in electrical resistivity within lung tissue and provides information on regional ventilation distributions 7,8 . Domenighetti 9 reported that EIT can be used to measure impedance changes and assess regional ventilation distribution during tidal breathing. The EIT-based global inhomogeneity (GI) index has been developed as a tool to quantify tidal volume distribution within the lung 10 .
Previous research has focused on the effect of recruitment maneuvers on gas exchange and hemodynamics. Literature describing the influence of recruitment maneuvers on global inhomogeneity and regional ventilation distribution is scarce. This study used EIT to investigate the physiological effects of different recruitment maneuvers that achieve the same maximum pressure for alveolar recruitment in a porcine model of ARDS. Findings will inform clinical decision-making around recruitment maneuvers while minimizing the risk of barotrauma in individuals with ARDS.

Methods 4
The protocol for this study was approved by the Science and Technological Committee and the Animal Use and Care Committee of the University School of Medicine, Nanjing, China. Domestic pigs (Sus scrofa domesticus) were purchased from a local farmer (Qinglongshan animal breeding farm, JiangShu, China). Animal experiments were performed in accordance with the Guidance for the Care and Use of Laboratory Animals 11 .

Animal Preparation
Pigs were housed on straw in a cage and fed with a standard diet 12 . Prior to the study, the animals were fasted overnight. Ten healthy male pigs (body weight 50.3±1.5 kg) were anesthetized with an intramuscular injection of ketamine hydrochloride (3 mg/kg), atropine (2 mg/kg) and fentanyl citrate (2 mg/kg) and an intravenous infusion of propofol (1-2 mg/kg·h), fentanyl citrate (0.5-1 μg/kg·h), midazolam (0.1 mg/kg·h), and atracurium (0.4 mg/kg·h) and placed in the supine position on a thermo-regulated operating table. During surgery, pigs received balanced electrolyte solution (5 ml/kg/h), pigs' body temperature was maintained at 37.5°C, and pigs' mean arterial pressure (MAP) was maintained > 60 mmHg with rapid infusions of 0.9% saline (20 ml/kg), as needed.

Experiment Protocol
Baseline measurements (T Baseline ) were made after pigs had stabilized for 30 minutes. Subsequently, a pig model of ARDS was established using bilateral lung lavage with isotonic saline (30 ml/kg; 38°C) 5 infused through a funnel. Negative pressure was applied to the proximal portion of an endotracheal tube to remove excessive fluid. Alveolar lavage was repeated every 10 min until the P/F ratio decreased to less than 100 mmHg and remained stable for 30 min (T ARDS ); then, FiO 2 was set at 0.4. ARDS pigs underwent three sequential recruitment maneuvers, including SI, IP and PCV applied in random order according to a random number table, with 30 mins at a PEEP of 5 cmH 2 O between maneuvers ( Figure 1). SI was performed using continuous positive airway pressure (CPAP) held at 40 cmH 2 O for 40 secs 13 . For IP, PEEP was increased from 5 cmH 2 O to a maximum of 40 cmH 2 O in 5 cmH 2 O increments, with each increment lasting 30 secs, and retuned to 5 cmH 2 O in the reverse process. For PCV, peak pressure was 40 cmH 2 O, inspiratory to expiratory ratio was 1:2, and PEEP was 20 cmH 2 O for 2 min. For IP and PCV, respiratory rate was set to 0. Respiratory mechanics, hemodynamic parameters, arterial blood gas, and EIT were recorded at T Baseline , T ARDS , and before and after each recruitment maneuver. MAP, CVP, and PAWP were monitored using calibrated pressure transducers. Blood gases were evaluated with an automated blood gas analyser (Nova M; Nova Biomedical, Waltham, MA, USA).

EIT Measurements and Analysis
EIT measurements (PulmoVista 500; Dräger Medical GmbH, Lübeck, Germany) were performed for 3 minutes each at T Baseline , T ARDS , and before and after each recruitment maneuver as previously described 14 . EIT data were generated by applying small alternate electrical currents through 16 electrodes located equidistant apart on a belt positioned around the pigs' thorax, 5cm above the xyphoid process. A reference electrocardiogram (ECG) electrode was positioned on the abdomen. Current applications and voltage measurements were automatically selected to be compatible with the image reconstruction algorithm. The images were continuously recorded and reconstructed at 40 Hz (Draeger EIT Data Analysis Tool 61).
Four regions of interests (ROI) of the same size and shape consisting of contiguous pixels were identified within EIT images obtained during tidal breathing. A cross section of the lung (ventral to 6 dorsal) was divided into four equal parts, namely ROI1, ROI2, ROI3 and ROI4. 15 Tidal volume distribution within the lung was quantified using the GI, as previously described. 16 For each breathing cycle, the median value of a tidal image, in which each pixel represented the difference in impedance between end-inspiration and end-expiration, was calculated. The absolute difference between the median value and every pixel value was summed to indicate the variation in the tidal volume distribution. The GI index was adjusted by normalization to the sum of the impedance values. A smaller GI index represented a more homogeneous distribution, and a larger GI index indicated a more inhomogeneous ventilation. The decrease in GI (ΔGI) with each recruitment maneuver was calculated as the difference in GI before and after recruitment.
General anesthesia was maintained throughout the study. After completion of the experiments, the animals were euthanized while in deep anesthesia by an intravenous injection of thiopental.

Statistical Analyses
Statistical analyses were performed using SPSS v20 (Chicago, IL, USA). Differences in global inhomogeneity and changes in global and regional end-expiratory lung impedance among different recruitment maneuvers were investigated. Comparisons were made between values obtained before and after each recruitment maneuver. For non-normally distributed data, results are expressed as median and interquartile range, and comparisons were made with the Wilcoxon rank test. For data that was normally distributed, results are expressed as mean and standard deviation, and comparisons were made with paired samples t tests and Bonferroni correction. p < 0.05 was considered statistically significant.
The recruitment maneuvers did not cause hemodynamic instability, and there were no significant differences in hemodynamic parameters after recruitment with the different maneuvers (Table 1). No animals died during the experiments. PaO 2 , SO 2 , and P/F were significantly improved after recruitment with SI, IP or PCV (all p<0.05), and there were no significant differences between maneuvers. The recruitment maneuvers had no effect on PaCO 2 or pH (Table 1).
Overall respiratory system compliance was significantly increased after recruitment with SI, IP, or PCV (p< 0.05) ( Table 1). The recruitment maneuvers had no effect on compliance in non-gravitydependent lung regions. Compliance was significantly increased in gravity-dependent lung regions after lung recruitment with IP or PCV, and there were no significant differences between maneuvers  (Figure 3). There were no significant differences in ΔGI with the different maneuvers. The ΔGI was significantly greater after recruitment with IP compared to SI (p=0.023), but there was no significant difference in ΔGI between IP and PCV ( Figure 4).

Discussion
This study used EIT to investigate the physiological effects of different recruitment maneuvers that achieve the same maximum pressure when held for different time spans, including SI, IP and PCV, for alveolar recruitment in a pig model of ARDS. Findings showed that these recruitment maneuvers increased oxygenation and compliance in overall and gravity-dependent lung regions, and decreased inhomogeneous gas distribution in the ARDS lung, with no adverse effects on hemodynamics immediately after the maneuver. In a previous study 17 , hemodynamic parameters were monitored during recruitment maneuvers in models of ventilator-induced and oleic acid lung injury; findings showed no differences in hemodynamics during the various recruitment maneuvers.
Patients with ARDS can suffer from inhomogeneous gas distribution, which leads to ventilationperfusion mismatching, a high dead-space fraction, and the potential for ventilator-induced lung injury (VILI). Recruitment maneuvers aim to open collapsed alveoli and improve oxygenation and respiratory system compliance. However, recruitment maneuvers can over-distend aerated alveoli, and ventilation at high inflation pressures can lead to VILI.
Heterogeneous lung structure (i.e, collapsed and overexpanded contiguous lung regions) is increasingly recognized as a key risk factor for inhomogeneous gas distribution, VILI, and mortality in mechanically ventilated patients 18 . Recent studies showed that the extent of lung inhomogeneities increase with the severity of ARDS 19 , and a protective ventilatory strategy may not be sufficient to minimize VILI in patients with ARDS whose disease process is characterized by an inhomogeneous distribution of pulmonary lesions that includes a small, nondependent, normally aerated compartment and a large, dependent, nonaerated compartment 20,21 .
In the present study, the inhomogeneous distribution of lung alterations in the pig model of ARDS was directly assessed using EIT. EIT has several advantages compared to established imaging techniques such as CT as it is radiation free and applicable at the bedside. In previous studies, Zhao 16 16 . In the present study, we used the GI index as a direct representation of global inhomogeneity in tidal ventilation in ARDS pigs. As the GI index is 0.40 ± 0.05 in patients under anesthesia without pulmonary disease, the GI index was expected to be > 0.45 in our experimental animals. We assessed the change in inhomogeneity with various recruitment maneuvers. Our results showed that recruitment maneuvers were able to decrease the inhomogeneity of the lung, possibly because of their unique ability to couple regional recruitment with preserved diaphragm activity, both of which are able to increase homogeneity of ventilation 15,22,23 . Previous studies have shown different recruitment maneuvers are associated with differences in oxygenation, respiratory system compliance, hyperinflation, and hemodynamics 24,25,26,27 . However, a ventilation strategy with aggressive lung recruitment may increase mortality in patients with ARDS 28 .
The present study showed that IP significantly improved inhomogeneity of the lung compared to SI and PCV in ARDS pigs. These data suggest that evaluating the effect of recruitment maneuvers with 9 EIT could play a role in minimizing VILI. Results of this study should be extrapolated to the clinical setting with caution, considering the differences in the shape of the thorax between pigs and humans.
Clinical trials are required to evaluate the efficacy and safety of recruitment maneuvers in patients with ARDS, and no recommendations about specific recruitment maneuvers can be made at this time.
Our study was associated with several limitations. First, maximal recruitment of the lung was not achieved with any maneuver. A peek pressure of 40 cmH 2 O may not have been sufficient for opening certain alveoli in ARDS pigs. Borges 28  Maximal recruitment would further improve the heterogeneity of the lung. Failure to achieve maximal recruitment of the lung would affect monitoring of end-expiratory lung impedance. Second, the relative impedance changes monitored by EIT may have been affected by cardiac movement and errors in the reconstruction algorithm. Last, all parameters were measured after recruitment maneuvers and resorption atelectasis could not be measured as EIT was used for monitoring dynamic ventilation distribution.

Conclusions
This study used EIT to show that different recruitment maneuvers that achieve the same maximum pressure, including SI, IP, and PCV, increased oxygenation and overall and regional compliance, and decreased inhomogeneous gas distribution with no adverse effects on hemodynamics in ARDS pigs. IP significantly improved inhomogeneity of the lung compared to SI and PCV. Further studies are needed to confirm the clinical significance of these findings.

Availability of data and materials
The datasets used during the current study are available from the corresponding author on reasonable request.

Authors' contributions
XFP was responsible for conception and design of the study; acquisition, analysis and interpretation of data; and drafting and revising the article for final approval before publication. PC and WLH was responsible for design of the study; acquisition and analysis of data; and revising the article. LL and LSQ participated in data analysis and interpretation of the results. GFM participated in interpretation of the results and writing the article. YY participated in data analysis; interpretation of the results; and writing the article. HYZ was responsible for the conception and design of the study; analysis and interpretation of data; drafting and revising the article, providing important intellectual content; and final approval before publication. All authors read and approved the manuscript.

Ethics approval
The study was approved by the Science and Technological Committee and the Animal Use and Care