Pressure support ventilation (PSV) is one of the most common modes of assisted spontaneous breathing in patients with mild acute respiratory distress syndrome (ARDS).1 The main advantages of PSV over fully controlled ventilation are improvement in gas exchange,2 maintenance of muscle activity3 and overall haemodynamic stability due to a reduced need for sedation,2,4 However, PSV may result in ventilator-induced lung injury (VILI),5,6 as lungs with preexisting damage are more susceptible to increased stress, and regional changes in transpulmonary pressure may be associated with pendelluft (movement of air from more recruited regions to less recruited regions during early inspiration without a gain in tidal volume). In addition, negative pleural pressures have been shown to yield negative alveolar pressures and increased vascular pressure, thus worsening alveolar edema.7 A recent study in animals with severe ARDS reported that spontaneous respiratory effort at low positive end-expiratory pressures (PEEPs) improved oxygenation but promoted pendelluft, whereas an optimised PEEP setting after recruitment mitigated the deleterious effects of spontaneous breathing, thus decreasing inspiratory effort and pendelluft.8 In the clinical setting, use of assisted spontaneous breathing has been indicated only in mild ARDS.1 To date, no study has evaluated the effects of spontaneous assisted breathing with different levels of PEEP on VILI in ARDS.
We hypothesised that in experimental mild ARDS the beneficial effects of PSV (improving lung function and mitigating VILI) would depend on the level of PEEP. We thus evaluated the effects of different PEEP levels, in both PSV and protective pressure-controlled ventilation (PCV), in which the driving pressure was adjusted to achieve a VT of approximately 6 ml kg−1, on lung mechanics and histology, arterial blood gases, biological markers associated with inflammation and types I and II epithelial cell damage in a rat model of mild ARDS.
The current study was approved by the Animal Care and Use Committee (CEUA: 059-15) of the Health Sciences Center, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil (Chairperson Prof M. Frajblat) on 16 June 2015. All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the US National Academy of Sciences Guide for the Care and Use of Laboratory Animals. The current study followed the Animal Research: Reporting In Vivo Experiments guidelines for reporting of animal research.9 The study started on June 2015.
Animal preparation and experimental protocol
Thirty-five Wistar rats [weight 310 ± 19 g] received Escherichia coli lipopolysaccharide (LPS, serotype O55:B5, LPS-B5 Ultrapure: TLR4 agonist; Invivogen, San Diego, California, USA) intratracheally (200 μg), suspended in 0.9% saline solution (total volume 200 μl), to induce mild ARDS.10,11 Twenty-four hours later animals were premedicated with diazepam (10 mg kg−1, Compaz; Cristália, Itapira, São Paulo, Brazil), ketamine (100 mg kg−1, Ketamin-S+; Cristália), and midazolam (2 mg kg−1, Dormicum; UniãoQuímica, São Paulo, São Paulo, Brazil) intraperitoneally. Following local anaesthesia with 2% lidocaine (0.4 ml), a midline neck incision and tracheostomy were performed. An intravenous catheter (Jelco 24G; Becton Dickinson and Company, Franklin Lakes, New Jersey, USA) was inserted into the tail vein for continuous infusion of midazolam (2 mg kg−1 h−1), ketamine (50 mg kg−1 h−1) and Ringer's lactate (7 ml kg−1 h−1, B. Braun, Crissier, Switzerland). Seven of the 35 rats were used for lung histology and molecular biology analysis and were not mechanically ventilated (nonventilated).Twenty-six hours after the LPS challenge, these animals were paralysed, and killed with an intravenous injection of thiopentone and their lungs removed without clamping the trachea.
For the remaining rats, the right internal carotid artery was cannulated with an 18G catheter (Arrow International, Cleveland, Ohio, USA) for blood sampling, gas analysis (Radiometer ABL80 FLEX, Copenhagen NV, Denmark), and mean arterial blood pressure (MAP) monitoring (Networked Multiparameter Veterinary Monitor LifeWindow 6000 V; Digicare Animal Health, Boynton Beach, Florida, USA). Changes in oesophageal pressure (Poes), which reflect chest wall pressure, were measured with a 30-cm-long water-filled catheter (PE205, Becton Dickinson and Company) with side holes at the tip, connected to a differential pressure transducer (UT-PL-400; SCIREQ, Montreal, Quebec, Canada). The catheter was passed into the stomach and then slowly pulled back into the oesophagus; proper positioning was assessed using the ‘occlusion test’.12 Heart rate, MAP, and rectal temperature were continuously monitored (Networked Multiparameter Veterinary Monitor LifeWindow 6000 V). Body temperature was maintained at 37.5 ± 1 °C using a heating bed. Gelafundin (B. Braun, São Gonçalo, Rio de Janeiro, Brazil) was administered intravenously in 0.5-ml increments as needed to maintain MAP more than 60 mmHg.
Animals were mechanically ventilated (Servo-i; MAQUET, Solna, Sweden) in PSV mode using the following settings: driving pressure adjusted to achieve a tidal volume (VT) of 6 ml kg−1, FIO2 = 0.4 and PEEP = 0 cmH2O. BASELINE data were obtained at this time point. Rats were then randomised by the sealed-envelope method to receive PCV or PSV (Supplemental Digital Content, SDC; Fig. 1, http://links.lww.com/EJA/A136). During PCV, animals were paralysed by intravenous administration of pancuronium bromide (2 mg kg−1, Cristália), and mechanically ventilated with FIO2 = 0.4 and PEEP = 0 cmH2O. After 5 min, one further randomisation was done according to PEEP level (2 or 5 cmH2O). Lung mechanics and arterial blood gases were measured after randomisation (INITIAL) and after 2 h of mechanical ventilation (FINAL). In both PCV and PSV, the driving pressure was adjusted to achieve a VT of approximately 6 ml kg−1. At FINAL, animals were killed by intravenous injection of sodium thiopentone 60 mg kg−1 (Cristália), the trachea was clamped at PEEP 2 cmH2O or 5 cmH2O depending on group allocation, and the lungs were removed en bloc for histology and molecular biology analysis. All groups were analysed at the same time point, that is, at 26 h (24 h after intratracheal administration of E. coli LPS and 2 h, even for nonventilated animals).
Data acquisition and lung mechanics
Airflow, as well as airway (Paw) and oesophageal (Poes) pressures, were continuously recorded throughout the experiments by a computer running customer-made software written in LabVIEW (National Instruments; Austin, Texas, USA).11VT was calculated by digital integration of the airflow signal. All signals were amplified in a four-channel signal conditioner (SC-24; SCIREQ) and sampled at 200 Hz with a 12-bit analogue-to-digital converter (National Instruments). Transpulmonary pressure (PL) was calculated during inspiration and expiration as the difference between Paw and Poes. Peak [peak transpulmonary pressure (PpeakL)] transpulmonary pressure (PL = Paw − Poes) and the oesophageal pressure generated 100 ms after onset of inspiratory effort (P0.1) were calculated.13 The respiratory rate was calculated from Poes swings as the frequency per minute of each type of breathing cycle. The pressure–time product (PTP) per breath was calculated as the integral of ΔPoes over time (PTPminute).14 The ratio between inspiratory and total time (Ti : Ttot) in each ventilator strategy was also calculated. During PCV, the mechanical ventilator provides external inspiratory power at each breath cycle (WOBL). WOBL is calculated as the integral of the inspiratory PL versus the inspired volume curve by numerical integration (trapezoidal rule), as depicted in SDC Fig. 2, http://links.lww.com/EJA/A136.15 During PSV (pressure support spontaneous breathing), mechanical power is provided by the mechanical ventilator in association with the respiratory muscles.16 In the current study, total mechanical power (POBL) was measured as POBL = WOBL × RR.17 All mechanical data were computed offline by a routine written in MATLAB (Version R2007a; The Mathworks Inc, Natick, Massachusetts, USA).18
Diffuse alveolar damage
The left lung was fixed in 4% buffered formalin and embedded in paraffin. Sections (3-μm thick) were cut longitudinally from the central zone with a microtome and stained with haematoxylin-eosin for histological analysis. Photomicrographs at magnifications of ×25, ×100 and ×400 were obtained from eight nonoverlapping fields of view per section using a light microscope (Olympus BX51; Olympus Latin America Inc., Sao Paolo, Brazil). Diffuse alveolar damage (DAD) was quantified by an expert in lung pathology (V.L.C.) blinded to group assignment, using a weighted scoring system.19 The following lung histological features were analysed: interstitial oedema, haemorrhage, atelectasis and over distension. A scale of 0 to 4 was used to represent the severity of each of these feature, with 0 standing for no effect and 4 for maximum severity. In addition, the extent of involvement in each field of view was scored on a scale of 0 to 4, with 0 standing for no appearance and 4 for complete involvement. Scores were calculated as the product of severity and extent of each feature, ranging from 0 to 16, and the total DAD score was the sum of these four features and thus ranged from 0 to 64.
To analyse the adherent junction protein E-cadherin, immunohistochemical procedures were performed on 4-μm-thick, paraffin-embedded kidney sections using a mouse polyclonal antibody against E-cadherin (cat. #610181, BD Transduction Laboratories, 1 : 250). After dewaxing and rehydrating, endogenous peroxidase was blocked with 3% H2O2 in methanol for 15 min. Heat-mediated antigen retrieval and enzymatic techniques were performed according to the specific antibody. After blocking the nonspecific binding of immunoglobulins to the tissue, primary antibodies were then incubated overnight at 4 °C in a humidified chamber for approximately 16 h. The sections were then washed in 0.25% Tween/phosphate-buffered saline solution for 5 min and the secondary antibodies incubated (Nichirei-Histofine Simple Stain Rat MAX-PO-Mouse). The chromogen substrate was diaminobenzidine (Liquid DAB, Dako, cat. #K3468). Negative control slides were incubated with mouse isotype immunoglobulins or with antibody diluent solution. Visualisation and image capture was performed using a light microscope (Eclipse E800; Nikon, Tokyo, Japan) coupled to a digital camera (Evolution; Media Cybernetics Inc., Bethesda, Maryland, USA) with the Q-Capture 2.95.0 graphic interface software (version 2.0.5; Quantitative Imaging, Surrey, British Columbia, Canada). High-resolution images (2048 × 1536 pixelbuffer) were captured away from airways. After calibration of program settings, images were analysed in the ImagePro Plus software environment (version 4.5.1; Media Cybernetics, Rockville, Maryland, USA).
Biological markers of inflammation, type II alveolar cell mechanotransduction and type I epithelial cell damage
Quantitative real-time reverse transcription PCR was performed to measure biological markers associated with inflammation [IL-6, cytokine-induced neutrophil chemoattractant (CINC)-1], type II alveolar cell mechanotransduction [surfactant protein-B (SP-B)] and type I alveolar cell damage [receptor for advanced glycation end products (RAGE)]. The primer sequences are listed in supplementary digital content Table 1. Central slices of right lung were cut, collected in cryotubes, quick-frozen by immersion in liquid nitrogen and stored at −80 °C. Total RNA was extracted from frozen tissues using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's recommendations. RNA concentration was measured by spectrophotometry in a Nanodrop ND-1000 system (NanoDrop Products, Wilmington DE USA). First-strand cDNA was synthesised from total RNA using a Quantitec reverse transcription kit (Qiagen). Relative mRNA levels were measured with a SYBR green detection system using ABI 7500 real-time PCR (Applied Biosystems, Foster City, California, USA). Samples were measured in triplicate. For each sample, the expression of each gene was normalised to housekeeping gene expression (acidic ribosomal phosphoprotein P0, 36B4)20 and expressed as fold change relative to nonventilated, using the 2−ΔΔCt method, where ΔCt = Ct (reference gene) − Ct (target gene). All analyses were performed by one of the authors (C.L.S.), who was blinded to group assignment.
A sample size of seven animals per group would provide the appropriate power (1 − β = 0.8) to identify significant (α = 0.05) differences in peak transpulmonary pressure between PCV and PSV,21 taking into account an effect size d = 1.73, a two-sided test, and a sample size ratio = 1 (G*Power 188.8.131.52; University of Düsseldorf, Düsseldorf, Germany).
The Kolmogorov–Smirnov test with Lilliefors’ correction was used to assess normality of data, whereas the Levene median test was used to evaluate the homogeneity of variances. To compare functional parameters over time, two-way repeated-measures ANOVA followed by the Holm–Šidák post-hoc test was used, with a significance level of 5% (P < 0.05). For DAD, immunohistochemistry and molecular biology, nonventilated animals were included and the Kruskal–Wallis test followed by Dunn's multiple comparisons test was used. Parametric data are expressed as mean ± SD, and nonparametric data, as median (interquartile range). Correlation analysis was performed with Spearman's rank correlation method (Spearman's ρ). All tests were performed using GraphPad Prism v6.01 (GraphPad Software, La Jolla, California, USA). Significance was established at P less than 0.05.
No animals died during the protocol. At BASELINE, no differences in VT, Ti/Ttot, respiratory rate, PpeakL, PTPminute, P0.1, PaO2/FiO2, arterial pH (pHa), PaCO2, HCO3− and MAP were observed among groups (SDC Table 2, http://links.lww.com/EJA/A136). Concerning blood gas analysis, a significant time effect (P < 0.0001), regardless of group, was observed in PaO2 : FiO2 ratio, with no major changes in other variables (pHa, PaCO2, HCO3−). MAP did not differ among groups (Table 1).
Differential effects of positive end-expiratory pressure 5 vs. 2 cmH2O in pressure-controlled ventilation
PpeakL was lower at PEEP = 5 cmH2O than PEEP = 2 cmH2O (P < 0.0001) (Table 1). At PEEP = 5 cmH2O, compared with PEEP = 2 cmH2O, DAD score was decreased (P = 0.03), mainly due to reduced atelectasis (P = 0.01, Table 2), whereas E-cadherin tissue expression was increased (P = 0.02, Fig. 1 and SDC Fig. 3, http://links.lww.com/EJA/A136), thus suggesting epithelial cell integrity. Only at PEEP = 2 cmH2O (Fig. 2) were IL-6 and CINC-1 mRNA expressions increased compared with nonventilated animals (P = 0.008 and 0.04, respectively).
Differential effects of positive end-expiratory pressure 5 vs. 2 cmH2O in pressure support ventilation
Again, PpeakL was lower at PEEP = 5 cmH2O than PEEP = 2 cmH2O (P < 0.001). At PEEP = 5 cmH2O, compared with PEEP = 2 cmH2O, DAD score was decreased (P = 0.005), mainly due to interstitial oedema (P < 0.001, Table 2), and E-cadherin tissue expression was increased (P = 0.02, Fig. 1 and SDC Fig. 3, http://links.lww.com/EJA/A136). CINC-1 and IL-6 expressions were higher at PEEP = 2 cmH2O compared with PEEP = 5 cmH2O (P = 0.02 for both), while SP-B expression was lower at PEEP = 2 cmH2O (P = 0.03). Only at PEEP = 2 cmH2O were IL-6 and CINC-1 mRNA expressions increased compared with nonventilated animals (P = 0.007 and 0.04, respectively, Fig. 2). In PSV, no significant changes were observed in RAGE mRNA expression between PEEP 2 and 5 cmH2O.
Pressure-controlled ventilation compared with pressure support ventilation at similar positive end-expiratory pressure levels
Considering each PEEP level (2 or 5 cmH2O), VT, Ti/Ttot and respiratory rate did not differ between PCV and PSV. At both PEEP levels (2 and 5 cmH2O), PpeakL was higher in PSV compared with PCV (P = 0.0003 for both) (Table 1). No differences were observed in DAD score, biological markers of inflammation, or type II and type I epithelial cell damage comparing PCV with PSV at the same PEEP level. POBL was higher in PCV than PSV at similar PEEP levels (SDC Table 3, http://links.lww.com/EJA/A136).
Correlation between mechanical parameters and inflammatory markers considering both ventilator strategies (pressure-controlled ventilation and pressure support ventilation) and positive end-expiratory pressure levels
PpeakL correlated positively with IL-6 (ρ = 0.62, P = 0.0007) and CINC-1 (ρ = 0.50, P = 0.01), and negatively with E-cadherin expression (ρ = −0.67, P = 0.0002) (Fig. 3). POBL correlated positively with CINC-1 in PCV (ρ = 0.87, P = 0.0002) and PSV (ρ = 0.63, P = 0.02) (SDC Fig. 4, http://links.lww.com/EJA/A136).
In the model of mild ARDS used in this study, we found that in PSV, PEEP = 5 cmH2O compared with PEEP = 2 cmH2O was associated with reduced PpeakL, DAD score and IL-6 and CINC-1 expressions, whereas it increased SP-B mRNA expression and maintained alveolar epithelial cell integrity. Therefore, in mild ARDS, PEEP level should be carefully adjusted to mitigate VILI in PSV.
Mild acute respiratory distress syndrome model
Mild ARDS was induced by LPS, resulting in DAD, inflammation and physiologic alterations, such as oxygenation impairment and reduced compliance.22 PSV was compared with a protective mechanical ventilation strategy because both have been applied in mild ARDS in the clinical setting.23 Pressure support settings in PSV are usually adjusted according to expiratory tidal volume.24 However, less attention is given to adjustment of PEEP levels, which can lead to low end-expiratory volume and, possibly, harmful outcomes.25 Low (2 cmH2O) and moderate (5 cmH2O) PEEP levels, as usually set for small animals under controlled mechanical ventilation, were used in the current study.26 In addition, PEEP = 5 cmH2O was chosen on the basis of a previous study by our group which showed no major changes in lung mechanics overtime in rats with endotoxin-induced mild ARDS under PCV,11 thus suggesting maintenance of alveolar stability. In PSV, pressure was adjusted to keep VT within a range known to be protective (6 ml kg−1) in mild ARDS.27
Differential effects of positive end-expiratory pressure 5 vs. 2 cmH2O in pressure-controlled ventilation
PpeakL was lower at PEEP = 5 cmH2O than at 2 cmH2O. The increment in PEEP level yielded better alveolar stability associated with reduced alveolar collapse28and preservation of E-cadherin tissue expression.29 E-cadherin is present between the alveolar epithelial cells and, by maintaining their cohesion, maintains the alveolar-capillary compartmentalisation.30 Previous studies have shown that reduced E-cadherin expression in lung tissue suggests alveolar epithelial cell damage.31–33 In addition, mRNA expression of biomarkers associated with lung inflammation (IL-6 and CINC-1) was higher in animals ventilated with PEEP = 2 cmH2O than in PEEP = 5 cmH2O, which is consistent with the E-cadherin levels.
Differential effects of positive end-expiratory pressure 5 vs. 2 cmH2O in pressure support ventilation
PpeakL was lower at PEEP = 5 cmH2O than at 2 cmH2O. This may be associated with reduced interstitial oedemas a result of the higher PEEP and increased ventilated lung area: these effects may reduce the transpulmonary pressure throughout the lung.34 We observed that animals ventilated in PSV mode with PEEP = 2 cmH2O had a higher interstitial oedema score (Table 2), which may be attributed to elevated transvascular pressure and lung perfusion35 and atelectrauma and pendelluft.6,36,37 The repetitive opening and closing of small airways and alveoli (resulting in atelectrauma) and the presence of pendelluft can provide a driving force for fluid displacement from small vessels to airspaces, especially in the setting of increased alveolar capillary permeability.38 Moreover, E-cadherin expression reduction is one of the main molecular events responsible for dysfunction in cell–cell adhesion, which acts towards alveolar–capillary decompartmentalisation, thus facilitating the shifting of cytokines from the alveolar space to the vascular side.39 In addition, SP-B mRNA expression was higher in PSV with PEEP = 5 cmH2O than at 2 cmH2O. The alveolar stabilisation associated with reduced epithelial cell damage triggers dynamic mechanotransduction in alveolar type II epithelial cells, which upregulates both release and production of SP-B.29
Comparison between pressure-controlled ventilation and pressure support ventilation at the same positive end-expiratory pressure level
At both PEEP levels (2 and 5 cmH2O), PpeakL was higher in PSV compared with PCV. As we measured the delta transpulmonary pressure by subtracting the PEEP level, this increase is explained by the oesophageal pressure decay observed in PSV, but not in PCV, in which the oesophageal swing is upward. The difference in delta transpulmonary pressure between PSV and PCV has been demonstrated elsewhere8 at comparable PEEP levels. Although PpeakL was higher in PCV than PSV, no major differences were observed in DAD score, E-cadherin tissue expression or inflammatory markers at comparable PEEP levels, which denotes the importance of setting a proper PEEP level. On the other hand, the mechanical power of breathing (POBL) (SDC Table 3, http://links.lww.com/EJA/A136) was higher in PCV compared with PSV (SDC Fig. 2, http://links.lww.com/EJA/A136). This is explained by the definition of mechanical power of breathing: during PCV (passive ventilation) pressure is transmitted from an external source (mechanical ventilator) to the lungs, whereas during PSV (spontaneous breathing) mechanical power is provided by the mechanical ventilator in association with the respiratory muscles. In this study, the portion of WOBL and POBL calculated represents only the mechanical ventilator source. Therefore, the WOBL and POBL values were reduced in PSV compared with PCV, which may have an impact on VILI.
Correlations between mechanical parameters and inflammatory markers considering both ventilator strategies (pressure-controlled ventilation and pressure support ventilation) and positive end-expiratory pressure levels
Significant correlations of PpeakL with IL-6, CINC-1, and E-cadherin expression were observed. In both PCV and PSV, at PEEP = 2 cmH2O, PpeakL was associated with higher levels of IL-6 and CINC-1 expressions but lower E-cadherin tissue expression, suggesting greater inflammation and damage to alveolar epithelium. In contrast, at PEEP = 5 cmH2O, with the decrease in PpeakL, IL-6 and CINC-1 mRNA expressions were lower, whereas epithelial integrity was maintained, as detected by E-cadherin tissue expression.
As mechanical power of breathing represents energy transfer from the mechanical ventilator to the lungs and, during PSV, there is an added effort of the respiratory muscles, the correlations of POBL with inflammatory markers were calculated separately (SDC Fig. 4, http://links.lww.com/EJA/A136). Nevertheless, POBL was significantly and positively correlated with CINC-1 mRNA expression in both PCV and PSV, suggesting that mechanical power of breathing plays a role in VILI regardless of ventilator strategy.16
The current study has several limitations that should be considered. First, as we used LPS to induce mild pulmonary ARDS in rats, our results cannot be directly extrapolated to other models or to human ARDS. Second, our target was the induction of mild ARDS in rats, but the PaO2 : FiO2 ratio at INITIAL (PEEP = 5 cmH2O) was 175 to 199, which is classified clinically as moderate injury.40 However, lung histology showed atelectasis associated with interstitial oedema but without alveolar oedema, thus suggesting mild lung damage.22,41 Third, as the observation time was relatively short (2 h), we did not assess protein levels of markers of inflammation, fibrosis or endothelial cell damage; nevertheless, we were able to conduct an immunohistochemical assessment of E-cadherin, which is constitutively expressed between epithelial cells, and evaluate protection of the alveolar-capillary membrane. Finally, gene expression was evaluated by relative expression, not by actual concentrations. However, previous studies42,43 have shown that 2−ΔΔCt is a suitable method to analyse relative changes in gene expression from real-time quantitative PCR experiments,44 and when assessing PCR efficiency, it is as reliable as other methods.45
In our model of mild ARDS during PSV, a PEEP of 5 cmH2O but not of 2 cmH2O reduced lung damage and inflammatory markers while maintaining epithelial integrity. Therefore, during PSV in mild ARDS, the level of PEEP should be carefully adjusted to mitigate VILI.
Acknowledgements relating to this article
Assistance with the study: we express our gratitude to Mr Andre Benedito da Silva for animal care, Mrs Ana Lucia Neves da Silva for her help with microscopy, Ms Marcella Rocco for her help with mathematical physics data, Mrs Moira Elizabeth Schottler and Mr Filippe Vasconcellos for their assistance in editing the manuscript and Prof RonirRaggio Luiz for his help with statistics.
Financial support and sponsorship: the current work was supported by grants from the Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ) (grant number E-26/103.118/2), Rio de Janeiro, Brazil; and the Brazilian Council for Scientific and Technological Development (CNPq) (grant number 469716/2014-2), Brasilia, Brazil.
Conflicts of interest: none.
Presentation: preliminary data for this study were presented as a poster at the American Thoracic Society (ATS) Conference, 13 to 18 May 2016, San Francisco, United States.
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