Impact of different frequencies of controlled breath and pressure-support levels during biphasic positive airway pressure ventilation on the lung and diaphragm in experimental mild acute respiratory distress syndrome.

BACKGROUND: We hypothesized that a decrease in frequency of controlled breaths during biphasic positive airway pressure (BIVENT), associated with an increase in spontaneous breaths, whether pressure support (PSV)-assisted or not, would mitigate lung and diaphragm damage in mild experimental acute respiratory distress syndrome (ARDS).
MATERIALS AND METHODS: Wistar rats received Escherichia coli lipopolysaccharide intratracheally. After 24 hours, animals were randomly assigned to: 1) BIVENT-100+PSV0%: airway pressure (Phigh) adjusted to VT = 6 mL/kg and frequency of controlled breaths (f) = 100 bpm; 2) BIVENT-50+PSV0%: Phigh adjusted to VT = 6 mL/kg and f = 50 bpm; 3) BIVENT-50+PSV50% (PSV set to half the Phigh reference value, i.e., PSV50%); or 4) BIVENT-50+PSV100% (PSV equal to Phigh reference value, i.e., PSV100%). Positive end-expiratory pressure (Plow) was equal to 5 cmH2O. Nonventilated animals were used for lung and diaphragm histology and molecular biology analysis.
RESULTS: BIVENT-50+PSV0%, compared to BIVENT-100+PSV0%, reduced the diffuse alveolar damage (DAD) score, the expression of amphiregulin (marker of alveolar stretch) and muscle atrophy F-box (marker of diaphragm atrophy). In BIVENT-50 groups, the increase in PSV (BIVENT-50+PSV50% versus BIVENT-50+PSV100%) yielded better lung mechanics and less alveolar collapse, interstitial edema, cumulative DAD score, as well as gene expressions associated with lung inflammation, epithelial and endothelial cell damage in lung tissue, and muscle ring finger protein 1 (marker of muscle proteolysis) in diaphragm. Transpulmonary peak pressure (Ppeak,L) and pressure-time product per minute (PTPmin) at Phigh were associated with lung damage, while increased spontaneous breathing at Plow did not promote lung injury.
CONCLUSION: In the ARDS model used herein, during BIVENT, the level of PSV and the phase of the respiratory cycle in which the inspiratory effort occurs affected lung and diaphragm damage. Partitioning of inspiratory effort and transpulmonary pressure in spontaneous breaths at Plow and Phigh is required to minimize VILI.

Introduction

Inappropriate mechanical ventilation settings in patients with the acute respiratory distress syndrome (ARDS) may result in ventilation-induced lung injury (VILI). VILI is believed to involve a proinflammatory response, leading to lung structural and peripheral organ damage [1]. The use of protective low tidal volume under controlled mechanical ventilation is the only ventilator strategy known to reduce mortality in ARDS [2]. However, controlled mechanical ventilation may lead to diaphragmatic weakness [3,4], thus delaying the weaning process [3]. Partial ventilatory support can be implemented in mild-to-moderate forms of ARDS [5-7]. Since it requires less sedation and no neuromuscular blockade, it prevents muscle atrophy [8] and is associated with better cardiovascular performance [9,10], shorter time on mechanical ventilation, and shorter intensive care unit (ICU) stay [10,11]. On the other hand, spontaneous breathing during assisted mechanical ventilation may aggravate lung injury, since it can increase patient-ventilator asynchrony and work of breathing, leading to so-called patient self-inflicted lung injury (P-SILI) [12-14]. In recent decades, different partial ventilatory support modes have been proposed [15]. During biphasic positive airway pressure ventilation (BIVENT), a combination of time-cycled controlled breaths at two levels of continuous positive airway pressure and spontaneous breathing is allowed at both low and high airway pressure phases [16]. In experimental ARDS, Saddy et al. reported reduced lung and diaphragm damage with lower frequency of controlled breaths during BIVENT [17]. The combination of BIVENT with pressure support ventilation (PSV), when compared with pressure controlled ventilation, has been found to reduce lung damage [18]. We hypothesized that a decrease in frequency of control breaths during BIVENT, associated with an increase in spontaneous breaths, whether pressure support (PSV)-assisted or not, would mitigate lung and diaphragm damage in ARDS. The present study evaluated respiratory variables, histology, biological markers associated with VILI, and markers of diaphragmatic injury under different frequencies of control breaths and PSV in a rat model of experimental mild ARDS.

Materials and methods

Study approval

This study was approved by the Ethics Committee of the Healthy Science Center (CEUA no. 103/16), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the U.S. National Academy of Sciences. The present study followed the ARRIVE guidelines for reporting of animal research [19]. Animals came from the Breeding Facility of Healthy Science Center of Federal University of Rio de Janeiro. Conventional animals were housed at a controlled temperature (23°C) and controlled light–dark cycle (12–12 h), with free access to water and food. No acclimation was done.

Animal preparation and experimental protocol

Forty male Wistar rats (mean weight 292±20g) were anesthetized under spontaneous breathing with 1.5–2.0% isoflurane (Isoforine®; Cristália, Itapira, SP, Brazil) and subjected to intratracheal instillation of 9.6×106 EU/mL Escherichia coli lipopolysaccharide (Merck Millipore, Burlington, Massachusetts, USA), diluted in 200 μL of 0.9% saline solution.

After 24 h, animals were premedicated intraperitoneally (i.p.) with midazolam (1–2 mg/kg) and anesthetized with ketamine (100 mg/kg, i.p.). An intravenous (i.v.) catheter (Jelco 24G, Becton, Dickinson and Company, New Jersey, NJ, USA) was inserted into the tail vein, and anesthesia induced and maintained with midazolam (2 mg/kg/h) and ketamine (50 mg/kg/h). During spontaneous breathing, anesthetic depth was evaluated by the response to light touch with a fingertip on the rat’s whiskers (0 = awake, fully responsive to surroundings; 1 = not responsive to surroundings, rapid response to whisker stimulation; 2 = slow response; 3 = unresponsive to whisker stimulation) [20],.pupil diameter, position of the nictitating membrane, and movement in response to tail stimulation [21,22]. Experiments were started when responses to a noise stimulus (handclap), whisker stimulation, and tail clamping were absent. The depth of the anesthesia was monitored via mean arterial pressure, heart rate and respiratory rate throughout the experiment.

Body temperature was maintained at 37.5 ± 1°C with a heating bed (EFF 421, INSIGHT®, Brazil). After local infiltration of 0.4 mL lidocaine (1%), a tracheostomy was performed and a polyethylene cannula (PE 240, Intramedic®, Clay-Adams Inc, New York, USA; internal diameter 1.8 mm, length 7.5 cm) was introduced into the trachea. A second catheter (18G; Arrow International, USA) was then placed in the right internal carotid artery for blood sampling and gas analysis (Radiometer ABL80 FLEX, Copenhagen NV, Denmark), as well as monitoring of mean arterial pressure (MAP) (Networked Multiparameter Veterinary Monitor LifeWindow 6000 V; Digicare Animal Health, Boynton Beach, FL, USA). Animals were adapted to an airway pressure transducer (UT-PDP-70; SCIREQ, Canada) and a two-sidearm pneumotachograph (internal diameter 2.7 mm, length 25.7 mm, internal volume 0.147 ml, airflow resistance 0.0057 cm H2O·ml-1·s-1) [23] connected to a differential pressure transducer (UT-PDP-02, SCIREQ, Montreal, QC, Canada), for airflow (V’) measurement. A 30-cm-long water-filled catheter (PE-205; Becton, Dickinson and Company) with side holes at the tip, connected to a differential pressure transducer (UT-PL-400; SCIREQ, Canada), was used to measure the esophageal pressure. Briefly, the esophageal catheter was passed into the stomach and then slowly returned into the esophagus; its proper positioning was assessed using the “occlusion test” [24].

Animals were mechanically ventilated (SERVO-i; MAQUET, Solna, Sweden) in assisted pressure-controlled ventilation (A-PCV) with ΔP set to achieve a tidal volume (VT) of 6 mL/kg, positive end-expiratory pressure (PEEP) of 0 cmH2O, I:E (inspiratory: expiratory ratio) of 1:2, respiratory rate (RR) of 100 breaths per minute (bpm), and FiO2 (inspired oxygen fraction) of 0.4 at BASELINE-ZEEP, to evaluate whether the degree of lung damage was similar between ARDS groups. Flow trigger sensitivity was adjusted at BASELINE-PEEP (INITIAL) for adequate inspiratory effort, according to esophageal pressure variation (ΔPes). No additional changes to flow trigger sensitivity were made at any point during the experiment [25]. Shortly thereafter (defined as the INITIAL time point), animals were randomly assigned to one of four groups of BIVENT:

BIVENT-100+PSV0% (n = 8) with Phigh to achieve VT = 6 mL/kg, time at high and low pressures (Thigh, and Tlow, respectively) of 0.3 s, RR 100 bpm;

BIVENT-50+PSV0% (n = 8), with Phigh to achieve VT = 6 mL/kg, Thigh and Tlow of 0.3 and 0.9 s, respectively, RR 50 bpm;

BIVENT-50+PSV50% (n = 8) with Phigh to achieve VT = 6 mL/kg, pressure support ventilation of half the Phigh value (PSV50%), Thigh and Tlow of 0.3 and 0.9 s, respectively, RR 50 bpm;

BIVENT-50+PSV100% (n = 8), with Phigh to achieve VT = 6 mL/kg, pressure support ventilation equal to Phigh (PSV100%), Thigh and Tlow of 0.3 and 0.9 s, respectively, RR 50 bpm (.

A. Experimental design. BIVENT-100+PSV0% (n = 8) with Phigh to achieve VT = 6 mL/kg, Time at high and low pressures (Thigh and Tlow, respectively) = 0.3 s, RR = 100 bpm; BIVENT-50+PSV0% (n = 8), with Phigh to achieve VT = 6 mL/kg, Thigh and Tlow = 0.3 and = 0.9 s, respectively, RR = 50 bpm; BIVENT-50+PSV50% (n = 8) with Phigh to achieve VT = 6 mL/kg, pressure support ventilation of half value of Phigh (PSV50%), Thigh and Tlow = 0.3 and 0.9 s, respectively, RR = 50 bpm; and BIVENT-50+PSV100% (n = 8) with Phigh to achieve VT = 6 mL/kg, pressure support ventilation equal Phigh (PSV100%), Thigh and Tlow = 0.3 and = 0.9 s, respectively, RR = 50 bpm. ARDS: Acute respiratory distress syndrome; BIVENT: Biphasic positive airway pressure; NV: Nonventilated; LPS: Lipopolysaccharide; Phigh: High level of continuous positive airway pressure; Plow: Low level of continuous positive airway pressure. Thigh: Time spent in Phigh; Tlow: Time spent in Plow; RR: Respiratory rate; PSV: Pressure support ventilation. B. Timeline of the experiments. i.t.: Intratracheal; VT: Tidal volume; I:E: Inspiratory-to-expiratory ratio; PEEP: Positive end-expiratory pressure; FiO2: Fraction of inspired oxygen.

Phigh was adjusted across all groups to achieve VT = 6 mL/kg, while PSV adjustments were 50% or 100% of the Phigh level adjusted for each animal. Spontaneous breathing activity was allowed during all ventilatory strategies, including BIVENT-100+PSV0%. Sedation and anesthesia were adjusted to keep adequacy of inspiratory efforts during mechanical ventilation. The Plow level, which reflects PEEP, was set at 5 cmH2O, based on previous observations from our group showing that higher PEEP levels would lead to deterioration in respiratory mechanics in a similar rat model of ARDS [26]. We did not discriminate whether PSV occurred at Plow vs. Phigh, since the SERVO-i ventilator enables PSV only during Tlow. In all groups, FiO2 = 0.4 was maintained for 1 hour, at which time blood gas analysis (Radiometer, Copenhagen, Denmark) and mechanical data were obtained (timepoint FINAL) (. At timepoint FINAL, heparin was injected (1,000 IU i.v.), and animals were euthanized by overdose of sodium thiopental (60 mg/kg i.v.; Cristália, Brazil). The trachea was clamped at Plow = 5 cmH2O, lungs were removed en bloc for histology and molecular biology analysis, and a surgical line was placed in the left bronchus to maintain lung volume at Plow = 5 cmH2O. The right lung was immediately frozen in liquid nitrogen for molecular biology analyses. The diaphragm was also removed at the end of the experiments. Eight of 40 rats were instilled with E. coli LPS, but not ventilated (NV); these animals were used for molecular biology analysis.

Data acquisition and respiratory system mechanics

Airflow, airway pressure (Paw), and Pes were recorded continuously throughout the experiments by a computer running custom-made software written in LabVIEW (National Instruments, USA). All signals were amplified in a three-channel signal conditioner (TAM-DHSE Plugsys Transducers Amplifiers, Module Type 705/2, Harvard Apparatus, Holliston, Massachusetts, USA) and sampled at 200 Hz with a 12-bit analog-to-digital converter (National Instruments; Austin, Texas, USA). All mechanical data were computed offline by a routine written in MATLAB (Version R2007a; The Mathworks Inc., USA) (Please see in the supplement custom-made software written in LabVIEW and routine written in MATLAB for data analysis).

VT was calculated by digital integration of the flow signal. Coefficient of variation (CV) of VT was determined among 600 sampled cycles by the ratio of standard deviation divided by mean values of VT. The total respiratory rate (RR) was calculated from the Pes swings as the frequency per minute of each type of breathing cycle. Mean transpulmonary pressure (Pmean,L) and peak transpulmonary pressure (Ppeak,L) were calculated as the difference between tracheal and esophageal pressure. Inspiratory time divided by total respiratory cycle time (Ti/Ttot) was calculated. The pressure–time product per minute (PTPmin) was calculated as the integral of ΔPes over one minute. The ΔPes reflects the total variation of esophageal pressure during the inspiratory effort. All mechanical parameters were extracted from four different types of breathing cycles as follows: 1) mixed respiratory cycles (M), i.e., negative Pes swings with simultaneous ventilator inspiratory cycling; 2) spontaneous breath cycles, without PSV, at high airway pressure (Phigh), i.e., negative Pes swings at Phigh not followed by ventilator cycling; 3) spontaneous breath cycles, without PSV, at low airway pressure (Plow), i.e., negative Pes swings at Plow not followed by ventilator cycling; and 4) spontaneous breath at pressure support (PSV), only present in groups BIVENT-50+PSV50% and BIVENT-50+PSV100% during the Tlow phase (

Histology

Diffuse alveolar damage

The left lung was fixed in 4% formaldehyde solution and embedded in paraffin. Sections (4 μm thick) were cut longitudinally from the central zone with a microtome and stained with hematoxylin–eosin for histologic analysis. Photomicrographs at magnifications of ×25, ×100, and ×400 were obtained from eight non-overlapping fields of view per section under a light microscope (Olympus BX51; Olympus Latin America Inc., Brazil). Diffuse alveolar damage (DAD) score was quantified by an expert in lung pathology (V.L.C.) blinded to group assignment [27]. Briefly, scores of 0 to 4 were used to represent overdistension, interstitial edema, and alveolar collapse, with 0 standing for no effect and 4 for maximum severity. Additionally, the extent of each scored characteristic per field of view was determined on a scale of 0 to 4, with 0 standing for no visible evidence and 4 for complete involvement. Scores were calculated as the product of severity and extent of each feature, on a range of 0 to 16. The cumulative DAD score was the sum of these three features and thus ranged from 0 to 48 [28].

Electron microscopy

Three slices measuring 2×2×2 mm were cut from three different segments of the right lung and from the right diaphragm and fixed in 2.5% glutaraldehyde and 0.1 M phosphate buffer (pH = 7.4) for transmission electron microscopy (TEM) (JEOL 1010 Transmission Electron Microscope, Tokyo, Japan). Each TEM image (20 per animal) was analyzed for damage to epithelial and endothelial cells, basement membrane, and extracellular matrix at three different magnifications. Pathologic findings were graded on a 5-point semiquantitative severity-based scoring system as follows: 0 = normal lung parenchyma, 1 = changes in 1% to 25% of examined tissue, 2 = changes in 26% to 50% of examined tissue, 3 = changes in 51% to 75% of examined tissue, and 4 = changes in 76% to 100% of examined tissue [29]. For diaphragm analysis, the following aspects were assessed on TEM: 1) myofibril abnormalities, defined as disruption of myofibril bundles or disorganized myofibrillar pattern with edema of the Z-disc and 2) mitochondrial injury with abnormal, swollen mitochondria and abnormal cristae. The pathologic findings were again graded on a 5-point semiquantitative severity-based scoring system, as follows: 0 = normal diaphragm, 1 = changes in 1% to 25%, 2 = changes in 26% to 50%, 3 = changes in 51% to 75%, and 4 = changes in 76% to 100% of examined tissue. The pathologist working on light microscopy and TEM images (V.L.C.) was blinded to group assignment.

Molecular biology analysis of lung and diaphragm tissue

Quantitative real-time reverse transcription polymerase chain reaction was performed to measure biological markers associated with inflammation (tumor necrosis factor [TNF]-α), alveolar stretch (amphiregulin), epithelial cell damage (club cell protein 16), endothelial cell damage [vascular cell adhesion molecule (VCAM)-1], and extracellular matrix damage (decorin) in lung tissue, as well as markers of muscle proteolysis [muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx/atrogin-1)] in the right diaphragm. The primer sequences are listed in . Central slices of right lung and right diaphragm were cut, collected in cryotubes, flash-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. The RNA concentration was measured by spectrophotometry in a Nanodrop ND-2000 system. First-strand cDNA was synthesized from total RNA using a Quantitec reverse transcription kit (Qiagen, Hilden, Germany). Relative mRNA concentrations were measured with a SYBR green detection system using ABI 7500 real-time polymerase chain reaction (Applied Biosystems, Foster City, CA, USA). Samples were measured in triplicate. For each sample, the expression of each gene was normalized to that of the housekeeping gene 36B4 (acidic ribosomal phosphoprotein P0) and expressed as fold change relative to NV, using the 2-ΔΔCt method, where ΔCt = Ct (reference gene)–Ct (target gene). All analyses were performed by two authors (M.A.A., C.L.S.), who were blinded to group assignment.

Statistical analysis

The sample size was judiciously calculated to minimize the use of animals. A sample of 8 animals per group would provide the appropriate power (1-β = 0.8) to identify significant (α = 0.05) differences in alveolar collapse between BIVENT-100+PSV0% and BIVENT50+PSV0% [17], taking into account an effect size d = 1.72, a two-sided test, and a sample size ratio of 1 (G*Power 3.1.9.2, University of Düsseldorf, Germany). The Kolmogorov–Smirnov test with Lilliefors’ correction was used to assess normality of data, while the Levene median test was used to evaluate the homogeneity of variances. For comparisons between BIVENT-100 and BIVENT-50 groups, either Student’s t-test or the Mann–Whitney U test was used as appropriate. For comparisons within BIVENT-50 groups, one-way ANOVA with Holm-Šídák’s post-hoc test (<0.05) or the Kruskal-Wallis test followed by Dunn’s test were used. All tests were performed in GraphPad Prism v8.4.0 (GraphPad Software, La Jolla, CA, USA). Significance was established at P < 0.05 (two-sided).

Results

There were no missing data at any time point in the study. Two animals died due to hemodynamic compromise during the pilot phase. At BASELINE ZEEP, PaO2/FiO2 was lower than 300 mmHg in all groups (S2 Table). MAP was higher than 70 mmHg throughout the experiments. At timepoint FINAL, PaO2/FiO2, pHa, PaCO2, and HCO3- did not significantly differ between BIVENT-100+PSV0% and BIVENT-50+PSV0%, nor among the BIVENT-50+PSV0%, BIVENT-50+PSV50%, and BIVENT-50+PSV100% groups (). The amount of fluid infused did not differ between groups (. Adjusted Phigh and PSV levels are shown in .

BIVENT-100+PSV0% vs BIVENT-50+PSV0% group

The CV of VT was lower in BIVENT-100+PSV0% than BIVENT-50+PSV0% (). Among the mixed cycles, BIVENT-100+PSV0% presented higher Ti/Ttot and RR compared to BIVENT-50+PSV0%. No significant changes were observed in PTPmin and ΔPes between groups.

NV animals showed higher alveolar collapse and cumulative DAD score compared to BIVENT-100+PSV0% and BIVENT-50+PSV0%. The score of overdistension and interstitial edema was higher in NV than BIVENT-50+PSV0%. BIVENT-100+PSV0% cumulative DAD score compared to BIVENT-50+PSV0% ().

Representative photomicrographs of lung parenchyma stained with hematoxylin–eosin.

NV (nonventilated), BIVENT-100+PSV0% and BIVENT-50+PSV0%: The frequency of control breaths is 100 and 50 breaths/min, respectively. BIVENT-50+PSV50%: PSV set to half the value of Phigh (PSV50%). BIVENT-50+PSV100%: PSV equal to the value at Phigh (PSV100%). Note the preserved microscopic architecture of the lung parenchyma in BIVENT-50+PSV50% animals. AD: Alveolar duct. Asterisk: Interstitial edema. Arrows: Areas of alveolar collapse. Scale bar = 50 μm.

Endothelial cell damage was greater in NV compared to than BIVENT-50+PSV0% ().

Photomicrographs of electron microscopy of the lung.

The ultrastructure of the alveolar–capillary barrier shows varying degrees of injury to epithelial/endothelial cells and the basement membrane, as well as collagen fiber deposition in the septal interstitium. NV: Nonventilated. Note that BIVENT-50+PSV50% induced more epithelial (Ep) and endothelial cell (Ed) apoptosis, irregularity and thickness of the basement membrane (arrows), and collagen fiber deposition (*) in the alveolar-capillary barrier than BIVENT-50+PSV0%. In contrast, less epithelial (Ep) and endothelial cell (Ed) apoptosis, greater basement membrane integrity (arrows), and less collagen fiber deposition (*) can be seen in BIVENT-50+PSV100% compared to BIVENT-50+PSV50%.

BIVENT-100 + PSV0% group showed increased amphiregulin gene expression in comparison to BIVENT-50+PSV0% ().

Real-time polymerase chain reaction analysis of biological markers for inflammation (tumor necrosis factor [TNF]-α), epithelial cell damage (club cell secretory protein [CC-16]), endothelial cell damage (vascular cell adhesion molecule [VCAM]-1), alveolar stretch (amphiregulin), and extracellular matrix damage (decorin).

Box plots represent the median and interquartile range of 8 animals. Relative gene expression was calculated as a ratio of the average gene expression levels compared with the reference gene (36B4) and expressed as fold change relative to respective NV (nonventilated). Comparisons between BIVENT-100+PSV0% and BIVENT-50+PSV0% groups were done by the Mann–Whitney U test (p<0.05). For comparisons within BIVENT-50 groups, the Kruskal-Wallis test with Dunn’s post-hoc test was used (p<0.05).

Myofibril abnormality score was higher in BIVENT-100+PSV0% than NV and BIVENT-50+PSV0%, mitochondrial injury did not differ among NV, BIVENT-100+PSV0% and BIVENT-100+PSV50% ().

Electron microscopy of the diaphragm.

Photomicrographs are representative of data obtained from diaphragm sections of eight animals per group. Myofibril damage with Z-disc edema and mitochondrial injury (Mt) was greater in BIVENT-100+PSV0% compared to BIVENT-50+PSV0%. Diaphragmatic mitochondrial damage was more intense in BIVENT-50+PSV50% than BIVENT-50+PSV100%. Sarcomere disarrangement (double arrows) and Z-disc edema were more pronounced during BIVENT-50+PSV50% compared to BIVENT-50+PSV100%. NV: Nonventilated.

MAFbx gene expression was higher in BIVENT-100+PSV0% than BIVENT-50+PSV0% ().

Real-time polymerase chain reaction analysis of biological markers for proteolysis [muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx/atrogin-1)].

Box plots represent the median and interquartile range of 8 animals. Relative gene expression was calculated as a ratio of the average gene expression levels compared with the reference gene (36B4) and expressed as fold change relative to respective NV (nonventilated). Comparisons between BIVENT-100+PSV0% and BIVENT-50+PSV0% groups were done by the Mann–Whitney U test (p<0.05). For comparisons within BIVENT-50 groups, the Kruskal-Wallis test with Dunn’s post-hoc test was used (p<0.05).

Comparisons among the BIVENT-50 + PSV0%, BIVENT-50 + PSV50%, and BIVENT-50 + PSV100% groups

Among total cycles, the CV of VT was lower in BIVENT-50+PSV50% than BIVENT-50+PSV0% and BIVENT-50+PSV100% groups. In addition, Pmean,L, PTPmin, and ΔPes were lower in BIVENT-50+PSV100% than BIVENT-50 + PSV50% animals (). Among Plow cycles, VT, airflow, RR, and PTPmin were lower in BIVENT-50+PSV100% than BIVENT-50+PSV0%. Among Phigh cycles, RR, Ppeak,L, and PTPmin were higher in BIVENT-50+PSV50% than BIVENT-50+PSV0%. Among mixed (M) cycles, Ti/Ttot was higher, while Pmean,L was lower in BIVENT-50+ PSV100% compared to BIVENT-50+PSV50%. Among PSV cycles, ΔPes was lower in BIVENT-50+PSV100% than BIVENT-50+PSV50% animals. Overdistension, alveolar collapse, and cumulative DAD score were higher in BIVENT-50+PSV50% than BIVENT-50+PSV0%, while BIVENT-50+PSV100% showed less interstitial edema and alveolar collapse, as well as a lower cumulative DAD score, compared to BIVENT-50+PSV50% (). BIVENT-50+PSV50% showed more damage to epithelial and endothelial cells, basement membrane, and extracellular matrix compared to BIVENT-50 + PSV0%. The BIVENT-50 + PSV100% group exhibited less basement membrane damage compared to BIVENT-50+PSV50% ().

TNF-α, VCAM-1, amphiregulin, and decorin gene expressions were higher in BIVENT-50+ PSV50% than BIVENT-50+PSV0%. On the other hand, BIVENT-50+PSV100% showed reduced TNF-α, CC-16, and VCAM-1 gene expression compared to BIVENT-50+PSV50% (). The BIVENT-50+PSV100% group exhibited higher amphiregulin expression than BIVENT-50+PSV0% animals.

BIVENT-50+PSV50% showed more myofibril abnormalities than BIVENT-50+PSV0% and BIVENT-50+PSV100% (). The mitochondrial injury score was higher in BIVENT-50+PSV50% than BIVENT-50+PSV0% and BIVENT-50+PSV100%. MURF-1 gene expression was higher in BIVENT-50+PSV50% than in BIVENT-50+PSV0% and BIVENT-50+PSV100% (). No significant changes were observed in MAFbx expression among BIVENT-50 groups ().

Discussion

In the rat model of mild ARDS used herein, at a low protective VT (6 mL/kg), we found that the decrease in the frequency of controlled breaths (BIVENT-100+PSV0% versus BIVENT-50+PSV0%) reduced DAD score, amphiregulin expression in lung tissue and MAFbx expression in diaphragm. In BIVENT-50 groups, the increase in PSV (BIVENT-50+PSV50% versus BIVENT-50+PSV100%) yielded better lung mechanics and less alveolar collapse, interstitial edema, cumulative DAD score, basement membrane damage, as well as gene expressions of TNF-α, CC-16, and VCAM-1 in lung tissue, and MURF-1 expression in diaphragm. Transpulmonary peak pressure (Ppeak,L) and pressure–time product per minute (PTPmin), both at Phigh, were associated with lung damage, while increased rate of spontaneous breaths at Plow was not. In short, total values of PTPmin (inspiratory effort) and Ppeak,L (transpulmonary pressure) did not contribute towards reduction of VILI; however, partitioning of these parameters between spontaneous breaths at Plow and at Phigh is required during BIVENT to optimize ventilator settings.

We used a model of mild lung injury induced by intratracheal instillation of E. coli lipopolysaccharide (E. coli LPS) because it reproduces several characteristics of mild human ARDS [30]. We observed mean PaO2/FiO2 < 300 mmHg at BASELINE-ZEEP; nevertheless, in small animals, changes in lung function and histology (alveolar collapse, neutrophil infiltration, and edema) are more closely related to the degree of lung damage than oxygenation levels are [30]. The model used herein is a two-hit model: endotoxin (first hit) induced alveolar and interstitial edema, alveolar–capillary barrier changes, and elevated markers of inflammation within the first hour after tracheal instillation, increasing progressively until the 24-h timepoint, when mechanical ventilation strategies (second hit) were analyzed [30,31]. After the first hit, both the lung [32,33] and diaphragm [34] are more prone to injury. BIVENT is characterized by two levels (Phigh and Plow) of continuous positive airway pressure with unrestricted spontaneous breathing [17,18,35]. Additionally, BIVENT can be combined with PSV, as classically done in previous studies [18,36,37]. Adding PSV is expected to achieve a reduction in work of breathing [38] and increased alveolar recruitment. By gradually increasing the pressure support according to Phigh (0, 50%, and 100%) within BIVENT-50 groups, a “U-shaped” response was observed according to histological and molecular biology parameters: lung and diaphragm protection was observed in BIVENT-50+PSV0% and BIVENT-50+PSV100% groups, whereas BIVENT-50+PSV50% impaired both lungs and the diaphragm.

BIVENT at different controlled breaths without PSV (BIVENT-100+PSV0% vs BIVENT-50+PSV0%)

BIVENT-100+PSV0% was associated with a lower CV of VT than BIVENT-50+PSV0%. This can be explained by the higher number of mandatory cycles. The CV of VT achieved at BIVENT-50+PSV0% was 32%, which may reduce lung damage [39,40]. Accordingly, the increased rate of spontaneous breathing at Plow during BIVENT-50+PSV0% was associated with reduced cumulative DAD score, mainly due to less alveolar collapse, less damage to epithelial/endothelial cells and basement membrane, and lower amphiregulin gene expression, which denotes less alveolar stretch [41]. Oxygenation did not differ between groups. This is consistent with the fact that oxygenation was associated with a balance between alveolar collapse and overdistension. Moreover, during assisted breathing, not only lung morphology but also regional perfusion distribution may play a relevant role in oxygenation [36].

By reducing the number of controlled cycles from BIVENT-100+PSV0% to BIVENT-50+PSV0%, spontaneous breathing cycles may occur, mainly at Plow within a protective VT range. Both the appropriate degree of variability of respiratory pattern and better maintenance of respiratory muscle tone may improve recruitment and maintenance of airway patency through the modulation of different airway pressures and inspiratory times, ultimately maximizing lung recruitment and stabilization [40,42,43], without causing diaphragm injury. In this line, we may further infer that the maintenance of respiratory muscle tone during BIVENT-50+PSV0%, but not in BIVENT-100+PSV0%, may have contributed to low diaphragm score and decreased expression of proteolysis markers. Accordingly, spontaneous breathing, compared to controlled mechanical ventilation, did not result in a significant decline in diaphragm protein synthesis [44], which corroborates our hypothesis.

BIVENT-50 at different PSV (BIVENT-50+PSV0%, BIVENT-50+PSV50%, and BIVENT-50+PSV100%)

In BIVENT-50+PSV50%, compared to BIVENT-50+PSV0%, VT did not change; however, Ppeak,L was higher at Phigh, reflecting vigorous efforts, which may have contributed to increase the level of PTPmin and ΔPes. The PTPmin was calculated as the integral of ΔPes over one minute and may better reflect inspiratory effort than esophageal pressure swing per se. Total respiratory rate did not differ among groups. Total ΔPes (variation of esophageal pressure during the inspiratory effort) was higher in BIVENT-50+PSV50% compared to BIVENT-50+PSV100%, mainly due to the increase during PSV (assisted and spontaneous breaths). In BIVENT-50+PSV50%, for the same airway pressure set on the ventilator, the higher the ΔPes at Phigh (7.3 ± 3.6 cmH2O), the higher the Ppeak,L (22.1 ± 3.0 cmH2O). On the other hand, when no pressure support was given (BIVENT-50+PSV0%), the lower the ΔPes at Phigh (4.4 ± 0.3 cmH2O), the lower the Ppeak,L (15.3 ± 1.2 cmH2O). We hypothesized that the increased expression of genes implicated in lung inflammation, extracellular matrix damage, and alveolar stretch in BIVENT-50+PSV50% animals may be attributed to increased Ppeak,L and PTPmin at Phigh. Moreover, since BIVENT-50+PSV50% animals exhibited greater atelectasis and overdistension, the increase in Ppeak,L might also reflect a reduction in lung compliance. Increased inspiratory effort may lead an imbalanced diaphragm length-tension relationship [45] and thus culminate in diaphragmatic injury. In this line, Ppeak,L is an important driver of lung damage also when PSV (without BIVENT) is gradually reduced [38,46].

On the other hand, during BIVENT-50+PSV0%, animals showed inspiratory effort at spontaneous breathing mainly in the Plow phase, which may protect the lungs against overdistension and triggering of biological markers. We may infer that the presence of spontaneous breathing activity at Plow may mitigate VILI.

Animals tended to breathe spontaneously more at Plow than at Phigh when the level of pressure support increased from 50 to 100%. In this line, the SERVO-i ventilator allows PSV breaths only at Plow and not at Phigh. Therefore, in BIVENT-50+PSV50%, 8 ± 1 (mean ± SD) spontaneous breaths occurred at Phigh, whereas in BIVENT-50+PSV100%, 9 ± 2 spontaneous breaths occurred at Plow. In this context, if the level of pressure support is low, spontaneous breaths (assisted or not) are favored at higher lung volumes. On the other hand, if the level of pressure support is high (BIVENT-50+PSV100%), spontaneous breaths tend to be favored at lower lung volumes, resulting in less lung stretch and diaphragm injury, which is consistent with the literature [18,38]. BIVENT-50+PSV100% seems to be the most promising ventilation mode.

Possible clinical implications of study findings

The findings of the present study expand the knowledge based on assisted mechanical ventilation strategies by showing that, during BIVENT, both the frequency of controlled breaths and the levels of pressure support (0%, 50% and 100%) affect lung and diaphragm damage differently. In addition, lung injury was worse if the ventilator was set to promote spontaneous efforts at Phigh level, such as observed at BIVENT-50+PSV50%. When BIVENT is set with different mandatory (controlled) and spontaneous breaths (PSV-assisted or not), PTPmin (as a surrogate of inspiratory effort) and Ppeak,L need to be measured during spontaneous breaths at Phigh and Plow. In this line, animals ventilated at BIVENT-50+PSV0% and BIVENT-50+PSV100% tended to breathe at lower pressures (Plow), whereas during BIVENT-50+PSV50%, they adapted at higher pressures (Phigh), resulting in VILI and diaphragmatic damage. This reinforces the concept of the utility of esophageal pressure measurement at the bedside to optimize assisted breathing when targeted to minimize lung and diaphragm injury.

Limitations

Some limitations of this study must be noted. First, an experimental model of mild pulmonary ARDS induced by intratracheal E. coli LPS instillation was used, which does not reproduce all features of human ARDS, does not apply to other degrees of ARDS severity, and is not representative of extrapulmonary ARDS. Second, the extent of alveolar permeability (measured by the protein content in bronchoalveolar lavage fluid) was not evaluated. Third, we chose not to ventilate healthy animals in order to avoid an overly large number of groups, and then increased the number of animals per group to maintain the power of the study. Finally, the ventilation period was limited to 1 hour, since longer periods of ventilation would have required infusion of additional fluids or even vasopressors to maintain MAP, which might have confounded the readouts. Therefore, we cannot guarantee that similar alterations would be maintained for longer periods. Nevertheless, 1 hour of mechanical ventilation was enough to observe molecular changes in key biological markers related to VILI and diaphragmatic proteolysis.

Conclusions

In the ARDS model used herein, during BIVENT, the level of PSV and the phase of the respiratory cycle in which the inspiratory effort occurs affected lung and diaphragm damage. Lung injury was not influenced by the total values of inspiratory effort or transpulmonary pressure. Partitioning of these parameters in spontaneous breaths at Plow and Phigh is required to minimize VILI.

Forward and reverse oligonucleotide sequences of target gene primers.

(DOCX)

Click here for additional data file.

Mean arterial pressure, amount of fluids infused, and arterial blood gases at timepoint BASELINE-ZEEP.

(DOCX)

Click here for additional data file.

Mean arterial pressure, amount of fluids infused, and arterial blood gases at timepoint FINAL.

(DOCX)

Click here for additional data file.

Respiratory parameters adjusted at the ventilator at BASELINE-ZEEP, INITIAL and FINAL.

(DOCX)

Click here for additional data file.

Custom-made software written in LabVIEW and routine written in MATLAB for data analysis.

(DOCX)

Click here for additional data file.

4 Mar 2021

PONE-D-20-40795

Impact of different frequencies of controlled breath and pressure-support levels during biphasic positive airway pressure ventilation on the lung and diaphragm in experimental mild acute respiratory distress syndrome

PLOS ONE

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Regarding the presented results, the strongest signal appears related to the bipap50-psv50 group. Looking at the difference in table 1, 8 breaths were at pHigh in the bipap50-psv50 group (no breaths at plow: why?), while 9 breaths at pLow for the bipap50-psv100 group (no breaths at phigh: why?). A different respiratory pattern could be the reason leading to sili? Why the psv 50 rats breathe at pHigh and the psv100 at pLow?

Another point that needs to be clarified is the peak transpulmonary pressure: the bipap50-psv50 group showed 22 at pHigh vs.15 in the bipap50-psv0 group; how do you explain? I expect that it should be the same. Is it possible that 22 is due to PSV over the pHigh (although, based on the methods section, that should not be the case: pHigh sholud refer to the spontaneous non PSV breaths at cpapHigh). If not, did you discriminate if PSV breaths occurred at pLow vs. pHigh? A triggered PSV cycle above pHigh might lead to overdistention and stretch, possibly explaining some of your results.

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Reviewer #1: COMMENTS TO AUTHORS

The authors hypothesized that lung and diaphragm injury may be altered by levels of pressure support and frequency of controlled-mechanical breath during Bilevel ventilation. By using mild lung injury model, the authors found that 1) lung injury was less when spontaneous effort was facilitated by decreasing mandatory mechanical breath during Bilevel ventilation; 2) but lung injury was deteriorated when spontaneous effort assisted by pressure support occurred on a top of Phigh level. The reviewer found that the current study was intriguing and covered a hot topics regarding how physicians facilitated spontaneous breathing in lung injury. But on the other hand, it was difficult to grasp what the main message was from the current version of manuscript due to numbers of groups.

# message

The reviewer thinks the current study has two messages; first, lung injury was decreased when spontaneous effort was facilitated during Plow by decreasing mandatory mechanical breath during Bilevel ventilation (BIVENT100+PS0 vs. BIVENT50+PS0), second, lung injury was deteriorated if ventilatory settings were manipulated to promote spontaneous effort assisted by pressure support on a top of Phigh level (BIVENT50+PS50). Especially 2nd message is important and should be stressed in a text. Thus, the reviewer suggests the authors to clarify this in conclusion and discussion, in order to let the message more straightforward. Supplemental figure-1 showing sample waveforms indeed helps readers to grasp the message so that this should be implemented in main text.

# potential mechanism

What was surprising to me is that PS-assisted spontaneous effort was observed only 8/mins on a top of Phigh (BIVENT50+PS50), but injury was significantly different from others. How was that possible by such a low respiratory rate occurring during Phigh?

Reviewer #2: In this study, Thompson et al evaluated the lung and diaphragm injury in an experimental model of E.coli induced ARDS, followed by different BIVENT. The authors stated that the frequency of controlled breaths and the PSV level during BIVENT can affect lung and diaphragm damage.

In my opinion this is a very interesting study, that could help to test different mechanical ventilation strategies in ARDS patients. The present study is well planned and well written. For these reasons I have only minor comments

- In the results, there is no mention about the mortality of the animals, during the 24 hours after E.coli administration. Is it possible to add this aspect?

- Among the experimental groups the authors did not consider a group of rat only mechanically ventilated without LPS insult. Please explain the reason of this choice.

- I have only a doubt regarding the time of ventilation. I think (looking to available literature) that only 1 hour of mechanical ventilation is not sufficient to induce a diaphragm injury and a structural disorganization. Please hypothesize a possible explanation about this aspect.

- No differences in terms of oxygenation were found among the experimental groups. Was this result expected?

- In the text, the BIVENT-50-PSV100% was not deeply analyzed. In the Discussion, in “Comparisons across the BIVENT-50 groups” section, the focus was about the comparison between PSV0 versus PSV50. The authors could give more attention on PSV100 results and comparisons versus the other group, since the BIVENT-50-PSV100 seems to be the most promising ventilation method.

Reviewer #3: Thompson AF. et al. reported the effect of Bilevel at 2 set up of RR and with or without PS (i.e. BILEVEL 50) on macroscopic and microscopic variables of lung and diaphragm injury in a preclinical investigation using a rodent model of lung injury by IT instillation of E. Coli LPS.

The primary aim of the study is based on a sample size justified to evaluate the difference in alveolar collapse (i.e. 1 of the 3 items used to estimate DAD) among Bilevel settings using a higher or a lower fixed RR (i.e. 100 versus 50, respectively) and with no PS.

The authors further evaluate the presence of differences on the respiratory parameters only in the lower RR group of BILEVEL (i.e. 50 bpm) according to different levels of PS.

Although the work is of potential clinical interest at bedside, I think that some bias exists in both the animal model and in the interpretation of the study results that does not clearly stick to the study findings.

1. At first, the authors refer to an animal model of mild ARDS. I am not sure that this is correct. According to the criteria of Berlin, PEEP must be included to characterize the severity of ARDS – at the “initial time point” the authors state that PF was lower of 300 in all groups – however, as reported in the methods section – PEEP was 0 at baseline before randomization – this does not guarantee that levels of PF are below 300 in the presence of some level of PEEP at baseline. Furthermore, 1 h of ventilation with PEEP=5 cmH2O brings the PF ratio up to an average value way above 300 (FINAL timepoint) in all groups – and this further confirms that the definition of ARDS is not accurate. Data of gas exchange at baseline should be reported.

2. Quantification of TV of 6 mL/Kg – I have a similar thought as previously observed -considering that the quantification of a TV = 6 ml/kg would report a different driving pressure at ZEEP compared to a DP estimated at a level of PEEP=5 cmH2O – this suggests that the levels of PSV used after randomization to achieve 6 mL/kg was based on an estimation of DP performed at ZEEP and not at PEEP=5 – I expect a different DP for the same TV at ZEEP versus PEEP 5 because of a different position on the PV curve. The authors should report average levels of PSV used in the different groups and the average level of Phigh of the BILEVEL in all groups.

3. It is not clear what the authors mean about the following sentence: “Flow trigger sensitivity was adjusted for adequate inspiratory effort, according to esophageal pressure (Pes) decay”? Did the authors use a fixed flow trigger in all the PSV experiments, didn’t they? The use of different thresholds of flow trigger may make unreliable the study findings as it means that this variable was not kept the same among different PSV experiments.

4. Furthermore, the different PSV groups showed a total RR – despite not significantly different I assume because of the low sample size among the groups - ranging from an average of 81 (PSV 50%) to 113 bpm (PSV 100%) with an increasing level of PSV – which is kind of counterintuitive…I can’t buy for what I see this U-shaped concept.. As first, I would ask whether the level of sedation was kept constant among the different PSV steps or not as in a study previously published by the same investigators (doi.org/10.1371/journal.pone.0246891). Actually, I would expect a lower RR in the PSV100% group in the absence of brain injury.. Furthermore, this makes me uncomfortable about the calculation of the pressure–time product per minute (PTPmin) that was calculated as the integral of ΔPes over one minute – this is, certainly, affected by a different RR among the study groups – so I am not sure if the difference among groups in PTP is because of the animal respiratory effort or because of the different RR. Looking at the PTP of the 2 groups on BILEVEL 100 versus 50 and no PSV – it seems unlikely that the PTP in the BILEVEL 50 and no PSV does not differ compared to the BILEVEL 100 and no PSV in the presence of a 50% decrease of fixed breaths. Levels of Pes should be reported among the study groups.

5. Variability of Vt is quite harsh to interpret as at PSV0% versus PSV 100% CV of TV is basically the same

6. About the DAD score, I am not sure to understand such lower levels of edema and collapse (i.e. media of 2) and a median of 5 for overdistension in the presence of quite low levels of mean lung pressure (i.e. mean value of 4.1) in the PSV100% BILEVEL50

7. Significance was established at p<0.05 – was a two-sided p-value, is it correct? Please add this information.

8. Table 1: Airlow > typo, change it into airflow

9. Discussion: “Therefore, there is a certain threshold of PSV in BIVENT-50 that may yield a continuous excessive stress.” It is a speculation please remove it – the study wasn’t powered to assess a difference in stress among the groups - stress was not the primary study aim – furthermore the data in BILEVEL 50 PSV 50% shows a higher Pmean,L - although not significant compared to other groups - a lower variation of TV and a lower RR. However, in figure 2, despite a pretty low RR in BILEVEL 50 PSV 50%, the swings of Pes were quite limited compared to PSV0% and even PSV100% - this is quite a surprise to me looking at the PTP - again any difference in the assisted flow trigger or sedation?

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18 May 2021

Response to Editor´s Comments

Regarding the presented results, the strongest signal appears related to the bipap50-psv50 group. Looking at the difference in table 1, 8 breaths were at pHigh in the bipap50-psv50 group (no breaths at plow: why?), while 9 breaths at pLow for the bipap50-psv100 group (no breaths at phigh: why?). A different respiratory pattern could be the reason leading to sili? Why the psv 50 rats breathe at pHigh and the psv100 at pLow?

Response: We thank the Editor for this important observation. Indeed, by increasing the level of pressure support from 50% to 100%, animals tend to breathe spontaneously more at Plow than at Phigh. The SERVO-i ventilator allows PSV breaths only at Plow and not at Phigh. Therefore, in BIVENT-50+PSV50%, 8 ± 1 (mean ± SD) spontaneous breaths occurred at Phigh, whereas 9 ± 2 spontaneous breaths occurred at Plow in BIVENT-50+PSV100%. In this context, if the level of pressure support is low (BIVENT-50+PSV50%), spontaneous breaths (PSV assisted or not) are favored at higher lung volumes. On the other hand, if the level of pressure support is high (BIVENT-50+PSV100%), breaths tend to be favored at lower lung volumes, resulting in less lung stretch and diaphragm injury, as described in the literature. We have better specified and discussed this observation in the revised manuscript.

We also agree with the Editor that different respiratory patterns as well as spontaneous breaths at Plow and Phigh may affect VILI. In this line, the expression of genes associated with lung inflammation, epithelial and endothelial cell damage, extracellular matrix damage, and diaphragmatic injury were greater at BIVENT-50+PSV50%, in which spontaneous breaths occurred at Phigh, resulting in increased peak transpulmonary pressure (Ppeak,L) and pressure–time product per minute (PTPmin), which may have favored greater atelectasis and overdistension, thus worsening VILI. In contrast, an increased rate of spontaneous breaths at Plow did not promote lung injury. In short, total values of PTPmin (inspiratory effort) and Ppeak,L (transpulmonary pressure) did not contribute towards reduction of VILI; however, partitioning of these parameters between spontaneous breaths at Plow and Phigh is required during BIVENT to optimize ventilator settings.

In BIVENT-50+PSV50%, compared to BIVENT-50+PSV0%, VT did not change; however, Ppeak,L was higher at Phigh, reflecting vigorous efforts, which may have contributed to increase the level of PTPmin and �Pes. The PTPmin was calculated as the integral of ΔPes over one minute and may better reflect inspiratory effort than esophageal pressure swing per se. The total respiratory rate did not differ among groups. Total �Pes (variation of esophageal pressure during the inspiratory effort) was higher in BIVENT-50+PSV50% compared to BIVENT-50+PSV100%, mainly due to the increase during PSV (assisted and spontaneous breaths). In BIVENT-50+PSV50%, for the same airway pressure set on the ventilator, the higher the �Pes at Phigh (7.3 ± 3.6 cmH2O), the higher the Ppeak,L (22.1 ± 3.0 cmH2O). On the other hand, when no pressure support was given (BIVENT-50+PSV0%), the lower the �Pes at Phigh (4.4 ± 0.3 cmH2O), the lower the Ppeak,L (15.3 ± 1.2 cmH2O). We hypothesized that the increased expression of genes implicated in lung inflammation, extracellular matrix damage, and alveolar stretch in BIVENT-50+PSV50% animals may be attributed to increased Ppeak,L and PTPmin at Phigh. We have modified the Discussion to clarify this issue.

Another point that needs to be clarified is the peak transpulmonary pressure (Ppeak,L): the bipap50-psv50 group showed 22 at pHigh vs.15 in the bipap50-psv0 group; how do you explain? I expect that it should be the same. Is it possible that 22 is due to PSV over the pHigh (although, based on the methods section, that should not be the case: pHigh sholud refer to the spontaneous non PSV breaths at cpapHigh). If not, did you discriminate if PSV breaths occurred at pLow vs. pHigh? A triggered PSV cycle above pHigh might lead to overdistention and stretch, possibly explaining some of your results.

Response: We agree with the Editor’s comments. As described in the Method section, Phigh (high airway pressure) reflects spontaneous breath cycles without PSV. Even though there were negative Pes variations at Phigh, this was not followed by ventilator cycling, since SERVO-I ventilator (MAQUET, Solna, Sweden) enables PSV only during Plow. We have now better explained this point in the Method section of the revised manuscript.

We have now added the �Pes in the Table 1, as requested by one Reviewer. At BIVENT-50+PSV50%, Ppeak,L increased as well as PTPmin at Phigh, which may reflect excessive inspiratory effort. The PTPmin was calculated as the integral of ΔPes over one minute and may better reflect inspiratory effort than esophageal pressure decay per se. Nevertheless, we have now presented the maximum ΔPes decay in order to better reflect the lung stretch caused by spontaneous effort. Moreover, both components of PTPmin, i.e., RR and ΔPes, are now shown separately. Total RR did not differ among groups. On the other hand, total �Pes, which reflects the variation of esophageal pressure during the inspiratory effort, was higher in BIVENT-50+PSV50% than BIVENT-50+PSV100%. In BIVENT-50+PSV50%, total �Pes was highly influenced by �Pes observed during PSV cycles. As a consequence, the higher the �Pes at Phigh (7.3 ± 3.6 cmH2O) for the same airway pressure set on the ventilator, the higher Ppeak,L will be, as observed in BIVENT-50+PSV50% (22.1 ± 3.0 cmH2O). On the other hand, when no pressure support was given, the lower the �Pes at Phigh (4.4 ± 0.3 cmH2O) for the same airway pressure set on the ventilator, the lower Ppeak,L will be (15.3 ± 1.2 cmH2O).

These findings have important clinical implications. Lung injury was worse if the ventilator was set to promote spontaneous efforts at Phigh, such as observed at BIVENT-50+PSV50%. When BIVENT is set with different mandatory (controlled) and spontaneous breaths (PSV assisted or not), PTPmin (as a surrogate of inspiratory effort) and Ppeak,L at Phigh and Plow during spontaneous breath should be measured by esophageal pressure in order to optimize and individualize these ventilatory settings, thus reducing the risk of lung and diaphragm injury. In addition, BIVENT-50+PSV50% was associated with higher atelectasis and overdistension; thus, the increase in Ppeak,L and �Pes might also reflect reduction in lung compliance. We have better explained these important and critical issues in the revised version of the manuscript.

Response to Reviewers´ Comments

Reviewer#1

The authors hypothesized that lung and diaphragm injury may be altered by levels of pressure support and frequency of controlled-mechanical breath during Bilevel ventilation. By using mild lung injury model, the authors found that 1) lung injury was less when spontaneous effort was facilitated by decreasing mandatory mechanical breath during Bilevel ventilation; 2) but lung injury was deteriorated when spontaneous effort assisted by pressure support occurred on a top of Phigh level. The reviewer found that the current study was intriguing and covered a hot topics regarding how physicians facilitated spontaneous breathing in lung injury. But on the other hand, it was difficult to grasp what the main message was from the current version of manuscript due to numbers of groups.

Response: We thank the Reviewer for these positive comments. In the revised version of the manuscript, several modifications were done to clarify the main message of the study.

# message

The reviewer thinks the current study has two messages; first, lung injury was decreased when spontaneous effort was facilitated during Plow by decreasing mandatory mechanical breath during Bilevel ventilation (BIVENT100+PS0 vs. BIVENT50+PS0), second, lung injury was deteriorated if ventilatory settings were manipulated to promote spontaneous effort assisted by pressure support on a top of Phigh level (BIVENT50+PS50). Especially 2nd message is important and should be stressed in a text. Thus, the reviewer suggests the authors to clarify this in conclusion and discussion, in order to let the message more straightforward. Supplemental figure-1 showing sample waveforms indeed helps readers to grasp the message so that this should be implemented in main text.

Response: We thank the Reviewer for these important suggestions, which have now been incorporated to the main text. Figure 1 has been moved to the main manuscript.

Additionally, we would like to clarify that at Phigh there was no PSV, only spontaneous breath cycles. Even though there were negative Pes variations at Phigh, this was not followed by ventilator cycling since SERVO-I ventilator (MAQUET, Solna, Sweden) enables PSV only during Plow phase. We have now better explained this point in the Method section.

As requested by the Reviewer, we have now added a sentence in the “possible clinical implications of study findings” section, as follows: “Lung injury was worse if the ventilator was set to promote spontaneous efforts at Phigh level, such as observed at BIVENT-50+PSV50%. When BIVENT is set with different mandatory (controlled) and spontaneous breaths (PSV assisted or not), PTPmin (as a surrogate of inspiratory effort) and Ppeak,L need to be measured during spontaneous breaths at Phigh and Plow. In this line, animals ventilated at BIVENT-50+PSV0% and BIVENT-50+PSV100% tended to breathe at lower pressures (Plow), whereas during BIVENT-50+PSV50%, they adapted at higher pressures (Phigh), resulting in VILI and diaphragmatic damage. This reinforces the concept of the utility of esophageal pressure measurement at the bedside to optimize assisted breathing when targeted to minimize lung and diaphragm injury.

Furthermore, we better clarified our conclusion: “In the ARDS model used herein, during BIVENT, the level of PSV and the phase of the respiratory cycle in which the inspiratory effort occurs affected lung and diaphragm damage. Lung injury was not influenced by the total values of inspiratory effort or transpulmonary pressure. Partitioning of these parameters in spontaneous breaths at Plow and Phigh is required to minimize VILI”.

# potential mechanism

What was surprising to me is that PS-assisted spontaneous effort was observed only 8/mins on a top of Phigh (BIVENT50+PS50), but injury was significantly different from others. How was that possible by such a low respiratory rate occurring during Phigh?

Response: We thank the Reviewer for this important question. Indeed, by increasing the level of pressure support from 50% to 100%, animals tend to breathe spontaneously more at Plow than at Phigh. The SERVO-i ventilator allows PSV breaths only at Plow and not at Phigh. Therefore, in BIVENT-50+PSV50%, 8 ± 1 (mean ± SD) spontaneous breaths occurred at Phigh, whereas 9 ± 2 spontaneous breaths occurred at Plow in BIVENT-50+PSV100%. In this context, if the level of pressure support is low (BIVENT-50+PSV50%), spontaneous breaths (PSV assisted or not) are favored at higher lung volumes and pressures. On the other hand, if the level of pressure support is high (BIVENT-50+PSV100%), breaths tend to be favored at lower lung volumes and pressures, resulting in less lung stretch and diaphragm injury, as described in the literature.

These findings have important clinical implications. Lung injury was worse if the ventilator was set to promote spontaneous efforts at Phigh, such as observed at BIVENT-50+PSV50%. When BIVENT is set with different mandatory (controlled) and spontaneous breaths (PSV assisted or not), PTPmin (as a surrogate of inspiratory effort) and Ppeak,L at Phigh and Plow during spontaneous breath should be measured by esophageal pressure in order to optimize and individualize these ventilatory settings, thus reducing the risk of lung and diaphragm injury. In addition, BIVENT-50+PSV50% was associated with higher atelectasis and overdistension; thus, the increase in Ppeak,L and �Pes might also reflect reduction in lung compliance. We have better explained these important and critical issues in the revised version of the manuscript.

Reviewer #2

In this study, Thompson et al evaluated the lung and diaphragm injury in an experimental model of E.coli induced ARDS, followed by different BIVENT. The authors stated that the frequency of controlled breaths and the PSV level during BIVENT can affect lung and diaphragm damage.

In my opinion this is a very interesting study, that could help to test different mechanical ventilation strategies in ARDS patients. The present study is well planned and well written. For these reasons I have only minor comments

Response: We thank the Reviewer for these positive comments.

- In the results, there is no mention about the mortality of the animals, during the 24 hours after E.coli administration. Is it possible to add this aspect?

Response: We thank the Reviewer for this important question. Two animals died due to hemodynamic compromise during the pilot phase. We have now included this information in the revised manuscript.

- Among the experimental groups the authors did not consider a group of rat only mechanically ventilated without LPS insult. Please explain the reason of this choice.

Response: We chose not to ventilate healthy animals in order to avoid an overly large number of groups and then increased the number of animals per group to maintain the power of the study as well as to focus on the main hypothesis. The Limitation section has been modified to better clarify this issue.

- I have only a doubt regarding the time of ventilation. I think (looking to available literature) that only 1 hour of mechanical ventilation is not sufficient to induce a diaphragm injury and a structural disorganization. Please hypothesize a possible explanation about this aspect.

Response: We agree with the Reviewer that 1 hour of mechanical ventilation is not sufficient to observe structural alterations in the diaphragm using light microscopy. However, such a short period in small animals does enable us to evaluate early changes in diaphragm using molecular biology (RT-PCR) and electron microscopy. Therefore, we measured the gene expression (mRNA levels) of markers associated with proteolysis: muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx/atrogin-1). According to previous studies from our group (Cruz, PlosOne 2021), 1 hour of mechanical ventilation was sufficient to detect diaphragm changes in TNF-alpha levels associated with different partial ventilatory support modes. It should be pointed out that the biological stimulus occurred 24 hours before, with LPS instillation. Thus, LPS animals are more prone to injury compared to healthy animals being subjected solely to mechanical ventilation. In relation to electron microscopy, we mainly focused on diaphragmatic mitochondrial damage, sarcomere disarrangement, and Z-disc edema. According to previous studies from our group (Saddy, Critical Care 2013), diaphragm injury was observed as well as the presence of vacuoles. In short, in small animals, the first hit is the administration of LPS 24 h before mechanical ventilation, whereas the second hit is the mechanical ventilation strategy itself. After the first hit, both the lung (Felix et al., Anesthesiology, 2019; Rocco & Marini, ICM 2021) and diaphragm (Shimada et al., Immunity, 2012) are primed and thus more prone to injury. The manuscript has been modified accordingly.

- No differences in terms of oxygenation were found among the experimental groups. Was this result expected?

Response: In our first paper (Saddy et al., Intensive Care Med, 2010) using this ventilatory mode, changes in oxygenation were observed when assisted ventilation modes were compared with PCV. Oxygenation improvement was associated with reduced atelectasis. However, when comparisons were done between assisted ventilation modes, no significant differences were observed in oxygenation (Saddy et al., Crit Care, 2013, Cruz et al. PlosOne 2021); this is consistent with the fact that oxygenation was associated with a balance between alveolar collapse and overdistension. Moreover, during assisted breathing, not only lung morphology but also regional perfusion distribution may play a relevant role in oxygenation (Carvalho et al., JAP 2011). The Discussion section has been modified to clarify this issue.

- In the text, the BIVENT-50-PSV100% was not deeply analyzed. In the Discussion, in “Comparisons across the BIVENT-50 groups” section, the focus was about the comparison between PSV0 versus PSV50. The authors could give more attention on PSV100 results and comparisons versus the other group, since the BIVENT-50-PSV100 seems to be the most promising ventilation method.

Response: We thank the Reviewer for this comment. The manuscript has been modified accordingly: “if the level of pressure support is high (BIVENT-50+PSV100%), spontaneous breaths tend to be favored at lower lung volumes, resulting in less lung stretch and diaphragm injury, which is consistent with the literature. BIVENT-50+PSV100% seems to be the most promising ventilation mode.”

These results have important clinical implications. “Lung injury was worse if the ventilator was set to promote spontaneous efforts at Phigh level, such as observed at BIVENT-50+PSV50%. When BIVENT is set with different mandatory (controlled) and spontaneous breaths (PSV assisted or not), PTPmin (as a surrogate of inspiratory effort) and Ppeak,L need to be measured during spontaneous breaths at Phigh and Plow. In this line, animals ventilated at BIVENT-50+PSV0% and BIVENT-50+PSV100% tended to breathe at lower pressures (Plow), whereas during BIVENT-50+PSV50%, they adapted at higher pressures (Phigh), resulting in VILI and diaphragmatic damage. This reinforces the concept of the utility of esophageal pressure measurement at the bedside to optimize assisted breathing when targeted to minimize lung and diaphragm injury.”

We have also clarified the conclusion: “During BIVENT, the level of PSV and the phase of the respiratory cycle in which the inspiratory effort occurs affected lung and diaphragm damage. Lung injury was not influenced by the total values of inspiratory effort or transpulmonary pressure. Partitioning of these parameters in spontaneous breaths at Plow and Phigh is required to minimize VILI.

Reviewer #3

Thompson AF. et al. reported the effect of Bilevel at 2 set up of RR and with or without PS (i.e. BILEVEL 50) on macroscopic and microscopic variables of lung and diaphragm injury in a preclinical investigation using a rodent model of lung injury by IT instillation of E. Coli LPS.

The primary aim of the study is based on a sample size justified to evaluate the difference in alveolar collapse (i.e. 1 of the 3 items used to estimate DAD) among Bilevel settings using a higher or a lower fixed RR (i.e. 100 versus 50, respectively) and with no PS.

The authors further evaluate the presence of differences on the respiratory parameters only in the lower RR group of BILEVEL (i.e. 50 bpm) according to different levels of PS.

Although the work is of potential clinical interest at bedside, I think that some bias exists in both the animal model and in the interpretation of the study results that does not clearly stick to the study findings.

Response: We thank the Reviewer for these comments, and modifications will be incorporated to clarify the main message.

1. At first, the authors refer to an animal model of mild ARDS. I am not sure that this is correct. According to the criteria of Berlin, PEEP must be included to characterize the severity of ARDS – at the “initial time point” the authors state that PF was lower of 300 in all groups – however, as reported in the methods section – PEEP was 0 at baseline before randomization – this does not guarantee that levels of PF are below 300 in the presence of some level of PEEP at baseline. Furthermore, 1 h of ventilation with PEEP=5 cmH2O brings the PF ratio up to an average value way above 300 (FINAL timepoint) in all groups – and this further confirms that the definition of ARDS is not accurate. Data of gas exchange at baseline should be reported.

Response: We thank the Reviewer for this important question. We have now included the gas-exchange at BASELINE ZEEP as requested in a new supplemental table. It can be noted that all groups presented a mean PaO2/FiO2 <300 mmHg.

We understand the Reviewer’s concern regarding experimental ARDS vs. clinical ARDS criteria. Nevertheless, experimental mild ARDS induced by intratracheal instillation of E. coli lipopolysaccharide reproduces several features of mild human ARDS (Matute-Bello G, 2011). According to the ATS Acute Lung Injury in Animals Study Group (Matute-Bello G, 2011), in small animals, changes in lung histology associated with increased neutrophil infiltration are more closely associated with lung damage than evaluation of oxygenation. Thus, in experimental rather than clinical settings, oxygenation is not as useful a parameter to evaluate the degree of lung injury. In the revised manuscript, the Discussion section has been modified accordingly.

The levels of PSV used after randomization to achieve 6 mL/kg was based on an estimation of DP performed at ZEEP and not at PEEP=5 – I expect a different DP for the same TV at ZEEP versus PEEP 5 because of a different position on the PV curve. The authors should report average levels of PSV used in the different groups and the average level of Phigh of the BILEVEL in all groups.

Response: We thank the Reviewer for this question. The levels of PSV used after randomization to achieve 6 mL/kg were based on the estimation of �P performed at PEEP=5 cmH2O. Indeed, immediately after PEEP was added (INITIAL) no significant changes in �P was observed, however, a progressive increase in �P was found at the end of the experiment in BIVENT-50+PSV50%. We have now reported the range of adjusted PSV as well as the range of adjusted Phigh (at BASELINE ZEEP, INITIAL and FINAL) of the BIVENT in a new supplemental table.

3. It is not clear what the authors mean about the following sentence: “Flow trigger sensitivity was adjusted for adequate inspiratory effort, according to esophageal pressure (Pes) decay”? Did the authors use a fixed flow trigger in all the PSV experiments, didn’t they? The use of different thresholds of flow trigger may make unreliable the study findings as it means that this variable was not kept the same among different PSV experiments.

Response: We thank the Reviewer for this question. We have now better described how the flow trigger sensitivity was adjusted. Flow trigger sensitivity was adjusted at BASELINE-PEEP (INITIAL) for adequate inspiratory effort, according to esophageal pressure variation (�Pes). No additional changes to flow trigger sensitivity were made at any point during the experiment.

4. Furthermore, the different PSV groups showed a total RR – despite not significantly different I assume because of the low sample size among the groups - ranging from an average of 81 (PSV 50%) to 113 bpm (PSV 100%) with an increasing level of PSV – which is kind of counterintuitive…I can’t buy for what I see this U-shaped concept.. As first, I would ask whether the level of sedation was kept constant among the different PSV steps or not as in a study previously published by the same investigators (doi.org/10.1371/journal.pone.0246891). Actually, I would expect a lower RR in the PSV100% group in the absence of brain injury.

Response: Indeed, we did not find difference in total RR among groups. Moreover, no significant changes were observed between BIVENT-50+PSV50% and BIVENT-50+PSV100%. During spontaneous breathing, the level of anesthesia was assessed by evaluating pupil size, position, and response to light, position of the nictating membrane, and movement in response to tail stimulation. Sedation and anesthesia were kept constant throughout the experiment. Method section has been modified to better clarify this issue. BIVENT-50+PSV50% compared to BIVENT-50+PSV100% resulted in spontaneous breaths at Phigh, leading to increased lung and diaphragm damage.

Furthermore, this makes me uncomfortable about the calculation of the pressure–time product per minute (PTPmin) that was calculated as the integral of ΔPes over one minute – this is, certainly, affected by a different RR among the study groups – so I am not sure if the difference among groups in PTP is because of the animal respiratory effort or because of the different RR. Looking at the PTP of the 2 groups on BILEVEL 100 versus 50 and no PSV – it seems unlikely that the PTP in the BILEVEL 50 and no PSV does not differ compared to the BILEVEL 100 and no PSV in the presence of a 50% decrease of fixed breaths. Levels of Pes should be reported among the study groups.

Response: We thank the Reviewer for this comment.

We did not detect differences in total RR; however, we found differences concerning compensatory increase in RR in different phases of BIVENT, whether at Thigh or Tlow. If total RR would be a determinant of total PTPmin, they would show similar behavior among different groups. We can observe that total PTPmin was lower in BIVENT-50+PSV100% compared to 0% and 50% of PSV even at similar total RR. This infers lower overall inspiratory effort in the BIVENT-50+PSV100% group. The PTPmin was calculated as the integral of ΔPes over one minute and may better reflect inspiratory effort than esophageal pressure decay per se. Nevertheless, we have now shown the maximum ΔPes decay in order to better reflect the lung stretch caused by spontaneous effort. Both components of PTPmin, i.e., RR and ΔPes, are now shown separately. Total RR did not differ among groups. On the other hand, total �Pes, which reflects the variation of esophageal pressure during the inspiratory effort, was higher in BIVENT-50+PSV50% than BIVENT-50+PSV100%. In BIVENT-50+PSV50%, total �Pes was highly influenced by �Pes observed during PSV cycles since they were adjusted to half the pressure support compared to full support in BIVENT-50+PSV100%. We have explained this issue more clearly in the revised manuscript.

5. Variability of Vt is quite harsh to interpret as at PSV0% versus PSV 100% CV of TV is basically the same

Response: We computed the variability of VT as a possible explanation for adequacy of natural (or most) neurological drive. We observed that, at 0% or 100% of PSV, the animals maintained physiological CV of VT, which is associated with positive outcomes, according to previous literature (Thammanomai et al. J. Appl Physiol, 2008).

6. About the DAD score, I am not sure to understand such lower levels of edema and collapse (i.e. media of 2) and a median of 5 for overdistension in the presence of quite low levels of mean lung pressure (i.e. mean value of 4.1) in the PSV100% BILEVEL50

Response: We thank the Reviewer for this question. These levels of interstitial edema, alveolar collapse, and overdistension are in accordance with previous studies from our group using mild lung damage induced by endotoxin (Felix et al., Anesthesiology, 2019). Moreover, these morphological changes may explain the low levels of mean transpulmonary pressure.

7. Significance was established at p<0.05 – was a two-sided p-value, is it correct? Please add this information.

Response: Yes, a two-sided p-value. We have now added this information to the manuscript.

8. Table 1: Airlow > typo, change it into airflow

Response: We apologize for this mistake. We have now corrected it.

9. Discussion: “Therefore, there is a certain threshold of PSV in BIVENT-50 that may yield a continuous excessive stress.” It is a speculation please remove it – the study wasn’t powered to assess a difference in stress among the groups - stress was not the primary study aim – furthermore the data in BILEVEL 50 PSV 50% shows a higher Pmean,L - although not significant compared to other groups - a lower variation of TV and a lower RR. However, in figure 2, despite a pretty low RR in BILEVEL 50 PSV 50%, the swings of Pes were quite limited compared to PSV0% and even PSV100% - this is quite a surprise to me looking at the PTP - again any difference in the assisted flow trigger or sedation?

Response: This sentence has been removed for clarity. As previously discussed, we did not observe difference in total RR in the BILEVEL-50-PSV50% group. However, we observed a different distribution of RR according to distinct phases of BIVENT.

As previously mentioned, flow trigger sensitivity was adjusted at BASELINE-PEEP (INITIAL) for adequate inspiratory effort, according to esophageal pressure (Pes) decay. No additional changes to flow trigger sensitivity were done at any point in the experiment. The depth of the anesthesia was monitored via mean arterial pressure, heart rate and respiratory rate throughout the experiment.

29 Jul 2021

Impact of different frequencies of controlled breath and pressure-support levels during biphasic positive airway pressure ventilation on the lung and diaphragm in experimental mild acute respiratory distress syndrome

PONE-D-20-40795R1

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Additional Editor Comments:

PLEASE CORRECT A TYPO IN THE ABSTRACT, FIRST LINE OF THE RESULTS, should read : bivent 50+psv0, compared to bivent 100 psv0, reduced...

Reviewers' comments:

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Reviewer #2: All comments have been addressed

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Reviewer #3: Yes

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Reviewer #2: I think that this study is very interesting. The authors answered completely all my questions. For these reason my suggestion is to accept this article.

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12 Aug 2021

PONE-D-20-40795R1

Impact of different frequencies of controlled breath and pressure-support levels during biphasic positive airway pressure ventilation on the lung and diaphragm in experimental mild acute respiratory distress syndrome

Dear Dr. Rocco:

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Table 1

Respiratory mechanical parameters at timepoint FINAL.

Parameter	Cycle	BIVENT-100 + PSV0%	BIVENT-50
			BIVENT-50+PSV0%	BIVENT- 50+PSV50%	BIVENT-50+PSV100%
VT (mL/kg)	M	5.8 ± 0.9	5.9 ± 1.0	6.2 ± 0.8	5.5 ± 0.9
	PSV	-	-	4.8 ± 1.0	5.0 ± 0.7
	P low	2.5 ± 1.4	3.9 ± 1.8	-	1.7 ± 0.6#
	P high	-	5.8 ± 0.9	7.2 ± 2.0	-
	Total	5.8 ± 1.3	5.2 ± 1.2	5.8 ± 0.7	5.0 ± 0.6
CV of VT (%)	M	14 ± 11	15 ± 13	12 ± 8	13 ± 3
	PSV	-	-	12 ± 5	16 ± 5
	P low	18 ± 20	23 ± 13	-	39 ± 18
	P high	-	51 ± 9	36 ± 19	-
	Total	4 ± 4	32 ± 11**	12 ± 6#	28 ± 9†
Airflow (mL/s)	M	12.1 ± 3.6	11.9 ± 3.5	11.8 ± 1.9	11.5 ± 2.3
	PSV	-	-	8.5 ± 1.9	10.2 ± 1
	P low	6.4 ± 3.1	8.0 ± 2.7	-	3.7 ± 2.1#
	P high	-	10.2 ± 1.9	6.8 ± 1.8
	Total	11.6 ± 1.8	10.5 ± 2.5	10.8 ± 1.7	10.2 ± 1.4
RR (bpm)	M	96 ± 4	51 ± 4**	51 ± 2	49 ± 2
	PSV	-	-	48 ± 10	51 ± 9
	P low	18 ± 9	45 ± 16	-	9 ± 2#
	P high	-	5 ± 1	8 ± 1#	-
	Total	110 ± 6	93 ± 25	81 ± 28	113 ± 19
Ppeak, L (cmH2O)	M	15.6 ± 3.8	13.7 ± 1.6	13.9 ± 2.9	14.6 ± 3.1
	PSV	-	-	12.1 ± 1.7	13.9 ± 3.2
	P low	11.8 ± 3.8	10.5 ± 2.3	-	10.6 ± 1.5
	P high	-	15.3 ± 1.2	22.1 ± 3.0#	-
	Total	15.5 ± 3.4	12.3 ± 1.7	13.1 ± 2.1	13.8 ± 3.0
Pmean, L (cmH2O)	M	5.9 ± 0.9	6.8 ± 1.4	7.4 ± 1.7	5.4 ± 0.6†
	PSV	-	-	3.1 ± 0.5	3.4 ± 0.5
	P low	2.4 ± 1.3	3.5 ± 2.0	-	1.9 ± 0.6
	P high	-	2.8 ± 0.1	3.1 ± 0.4	-
	Total	5.6 ± 1.1	4.9 ± 1.3	6.1 ± 2.6	4.1 ± 0.3†
Ti/Ttot (s)	M	0.6 ± 0.1	0.5 ± 0.1**	0.4 ± 0.1	0.6 ± 0.1#†
	PSV	-	-	0.5 ± 0.1	0.5 ± 0.1
	P low	0.6 ± 0.1	0.6 ± 0.1	-	0.6 ± 0.1
	P high	-	0.6 ± 0.1	0.6 ± 0.1	-
	Total	0.6 ± 0.1	0.5 ± 0.1	0.4 ± 0.1	0.6 ± 0.1†
PTPmin (cmH2O*sec/min)	M	53 + 37	44 ± 20	42 ± 15	27 ± 16
	PSV	-	-	46 ± 16	35 ± 8
	P low	19 ± 19	43 ± 22	-	10 ± 2#
	P high	-	2 ± 1	9 ± 4#	-
	Total	67 ± 30	62 ± 26	50 ± 18	26 ± 18#†
ΔPes (cmH2O)	M	1.7 ± 1.4	1.4 ± 1.1	1.3 ± 1.2	1.7 ± 1.4
	PSV	-	-	3.3 ± 1.0	0.8 ± 0.5†
	P low	4.2 ± 2.9	3.7 ± 1.5	-	3.8 ± 1.5
	P high	-	4.4 ± 0.3	7.3 ± 3.6	-
	Total	2.1 ± 1.5	1.9 ± 1.1	2.7 ± 0.9	1.2 ± 0.6†

Values are given as mean ± standard deviation (SD) of 8 animals in each group. Comparisons between BIVENT-100+PSV0% and BIVENT-50+PSV0% groups were done using Student t-test (p<0.05).

**vs. BIVENT-100+PSV0%. Comparisons among BIVENT-50 groups were done using One-Way ANOVA followed by Holm-Šídák post hoc test (p<0.05)

# vs BIVENT-50 + PSV0%

† vs BIVENT-50+PSV50%.

BIVENT: Biphasic positive airway pressure at different rates of time-cycled controlled breaths: 100 and 50 breaths/min; PSV0%:no pressure support ventilation; PSV50%: Pressure support ventilation 50% Phigh; PSV100%: Pressure support ventilation 100% Phigh; M = mixed, assisted breaths; Phigh = spontaneous breaths at high continuous positive airway pressure; Plow: Spontaneous breaths at low continuous positive airway pressure; PSV: Pressure support ventilation; Total: Mean data for mixed, PSV, Plow, and Phigh; VT: Tidal volume; CV of VT: Coefficient of variation of tidal volume; RR: Respiratory rate; Ppeak, L: Transpulmonary peak pressure; Pmean, L: Transpulmonary mean pressure; Ti/Ttot: Inspiratory time divided by total respiratory cycle time; PTPmin: Pressure–time product per minute; ΔPes: Esophageal pressure swing.

Table 2

Diffuse alveolar damage score.

Features of diffuse alveolar damage score	NV	BIVENT-100+PSV0%	BIVENT-50
			BIVENT-50+PSV0%	BIVENT-50+PSV50%	BIVENT-50+PSV100%
Overdistension (0–16)	4 (4–6)	4 (2–4)	2.5 (2–4)*	6 (4.5–8.25)#	5 (3.25–7.5)
Interstitial Edema (0–16)	6 (6–8.75)	4 (3–8.25)	4 (3.25–4)*	5 (4–6)	2 (2–4)†
Alveolar Collapse (0–16)	9 (6.5–11.25)	5 (4–6)*	3 (2–4)* **	6 (4–8.75)#	2 (2–3.5)†
Cumulative DAD score (0–48)	20 (19–22)	12.5 (11–18.5)*	9 (8.25–12)* **	18 (14.5–21)#	10.5 (7.75–12)†

Cumulative diffuse alveolar damage score (scores arithmetically averaged from two independent investigators) representing injury from variables: Overdistension, interstitial edema, and alveolar collapse. Values are given as median (interquartile range) of 8 animals in each group. Comparisons among NV, BIVENT-100+PSV0%, and BIVENT-50+PSV0% groups as well as among BIVENT-50 groups were done by Kruskal-Wallis followed by Dunn’s test. (p<0.05) *vs NV

**vs BIVENT-100+PSV0%

#vs BIVENT-50+PSV0%, †vs BIVENT-50+PSV50%. DAD: Diffuse alveolar damage. NV: Nonventilated. BIVENT: Biphasic positive airway pressure at different rates of time-cycled controlled breaths (100 and 50 breaths/min); PSV0%: No pressure support ventilation; PSV50%: Pressure support ventilation 50% Phigh; PSV100%: Pressure support ventilation 100% Phigh; Phigh = spontaneous breaths at high continuous positive airway pressure.

Table 3

Semiquantitative analysis of lung electron microscopy.

Features of lung electron microscopy	NV	BIVENT-100+PSV0%	BIVENT-50
			BIVENT-50+PSV0%	BIVENT-50+PSV50%	BIVENT-50+PSV100%
Endothelial cell damage	3 (2–3.25)	2.5 (2–3)	2 (1.75–2)* **	3 (2.75–3.25)#	2.5 (2–3)
Epithelial cell damage	3 (2–3.25)	3.5 (2–4)	2 (1.75–2.25)**	3.5 (3–4)#	3 (2.75–4)
Basement membrane damage	3 (2–4)	3 (2.75–3.25)	2 (1.75–2.25)**	3.5 (2.75–4)#	2 (1.75–2.25)†
ECM damage	2.5 (2–3)	3 (2–3)	2 (1–3)	3.5 (3–4)#	2.5 (1.75–3)

Ultrastructure features of electron microscopy of the lung (scores arithmetically averaged from two independent investigators) representing injury from variables: Endothelial apoptosis, epithelial apoptosis, basement membrane damage and cumulative score. Values are given as median (interquartile range) of 8 animals in each group. Comparisons among NV, BIVENT-100+PSV0%, and BIVENT-50+PSV0% groups as well as among BIVENT-50 groups were done by Kruskal-Wallis followed by Dunn’s test. (p<0.05) *vs NV

**vs BIVENT-100+PSV0%.

#vs BIVENT-50+PSV0%

†vs BIVENT-50+PSV50%. ECM: Extracellular matrix. NV: Nonventilated. BIVENT: Biphasic positive airway pressure at different rates of time-cycled controlled breaths (100 and 50 breaths/min); PSV0%: No pressure support ventilation; PSV50%: Pressure support ventilation 50% Phigh; PSV100%: Pressure support ventilation 100% Phigh; Phigh = spontaneous breaths at high continuous positive airway pressure.

Table 4

Semiquantitative analysis of diaphragm electron microscopy.

Features of diaphragm electron microscopy	NV	BIVENT-100+PSV0%	BIVENT-50
			BIVENT-50+PSV0%	BIVENT-50+PSV50%	BIVENT-50+PSV100%
Myofibril abnormality	2 (1–2)	2.5 (2–3)*	2 (1.25–2)**	3 (2–3)#	1.5 (1–2)†
Mitochondrial injury	2 (2–2)	2.5 (2–3)	2 (2–2)	3 (3–4)#	2 (1–2)†

Ultrastructure features of electron microscopy of the diaphragm (scores arithmetically averaged from two independent investigators) representing injury from these two variables: (1) myofibril abnormalities, defined as disruption of myofibril bundles or disorganized myofibrillar pattern with Z-disk edema, and (2) mitochondrial injury with abnormal swollen mitochondria and abnormal cristae. Values are given as median (interquartile range) of 8 animals in each group. Comparisons among NV, BIVENT-100+PSV0%, and BIVENT-50+PSV0% groups as well as among BIVENT-50 groups were done by Kruskal-Wallis followed by Dunn’s test. (p<0.05) *vs NV

**vs BIVENT-100+PSV0%.

#vs BIVENT-50+PSV0%

†vs BIVENT-50+PSV50%. NV: Nonventilated. BIVENT: Biphasic positive airway pressure at different rates of time-cycled controlled breaths (100 and 50 breaths/min); PSV0%: No pressure support ventilation; PSV50%: Pressure support ventilation 50% Phigh; PSV100%: Pressure support ventilation 100% Phigh; Phigh = spontaneous breaths at high continuous positive airway pressure.