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Impact of Different Ventilation Strategies on Driving Pressure, Mechanical Power, and Biological Markers During Open Abdominal Surgery in Rats

Maia, Lígia de A. PhD*; Samary, Cynthia S. PhD*; Oliveira, Milena V. MSc*; Santos, Cintia L. MD, PhD*; Huhle, Robert PhD; Capelozzi, Vera L. MD, PhD; Morales, Marcelo M. MD, PhD§; Schultz, Marcus J. MD, PhD; Abreu, Marcelo G. MD, PhD; Pelosi, Paolo MD, FERS; Silva, Pedro L. PhD*; Rocco, Patricia Rieken Macedo MD, PhD*

doi: 10.1213/ANE.0000000000002348
Basic Science: Original Laboratory Research Report
Free
SDC

BACKGROUND: Intraoperative mechanical ventilation may yield lung injury. To date, there is no consensus regarding the best ventilator strategy for abdominal surgery. We aimed to investigate the impact of the mechanical ventilation strategies used in 2 recent trials (Intraoperative Protective Ventilation [IMPROVE] trial and Protective Ventilation using High versus Low PEEP [PROVHILO] trial) on driving pressure (ΔPRS), mechanical power, and lung damage in a model of open abdominal surgery.

METHODS: Thirty-five Wistar rats were used, of which 28 were anesthetized, and a laparotomy was performed with standardized bowel manipulation. Postoperatively, animals (n = 7/group) were randomly assigned to 4 hours of ventilation with: (1) tidal volume (VT) = 7 mL/kg and positive end-expiratory pressure (PEEP) = 1 cm H2O without recruitment maneuvers (RMs) (low VT/low PEEP/RM−), mimicking the low-VT/low-PEEP strategy of PROVHILO; (2) VT = 7 mL/kg and PEEP = 3 cm H2O with RMs before laparotomy and hourly thereafter (low VT/moderate PEEP/4 RM+), mimicking the protective ventilation strategy of IMPROVE; (3) VT = 7 mL/kg and PEEP = 6 cm H2O with RMs only before laparotomy (low VT/high PEEP/1 RM+), mimicking the strategy used after intubation and before extubation in PROVHILO; or (4) VT = 14 mL/kg and PEEP = 1 cm H2O without RMs (high VT/low PEEP/RM−), mimicking conventional ventilation used in IMPROVE. Seven rats were not tracheotomized, operated, or mechanically ventilated, and constituted the healthy nonoperated and nonventilated controls.

RESULTS: Low VT/moderate PEEP/4 RM+ and low VT/high PEEP/1 RM+, compared to low VT/low PEEP/RM− and high VT/low PEEP/RM−, resulted in lower ΔPRS (7.1 ± 0.8 and 10.2 ± 2.1 cm H2O vs 13.9 ± 0.9 and 16.9 ± 0.8 cm H2O, respectively; P< .001) and less mechanical power (63 ± 7 and 79 ± 20 J/min vs 110 ± 10 and 120 ± 20 J/min, respectively; P = .007). Low VT/high PEEP/1 RM+ was associated with less alveolar collapse than low VT/low PEEP/RM− (P = .03). E-cadherin expression was higher in low VT/moderate PEEP/4 RM+ than in low VT/low PEEP/RM− (P = .013) or high VT/low PEEP/RM− (P = .014). The extent of alveolar collapse, E-cadherin expression, and tumor necrosis factor-alpha correlated with ΔPRS (r = 0.54 [P = .02], r = −0.48 [P = .05], and r = 0.59 [P = .09], respectively) and mechanical power (r = 0.57 [P = .02], r = −0.54 [P = .02], and r = 0.48 [P = .04], respectively).

CONCLUSIONS: In this model of open abdominal surgery based on the mechanical ventilation strategies used in IMPROVE and PROVHILO trials, lower mechanical power and its surrogate ΔPRS were associated with reduced lung damage.

Published ahead of print July 27, 2017.

From the *Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Centro de Ciências da Saúde, Rio de Janeiro, Brazil; Department of Anesthesiology and Intensive Care Therapy, Pulmonary Engineering Group, University Hospital Dresden, Technische Universität Dresden, Dresden, Germany; Department of Pathology, Faculty of Medicine, University of São Paulo, São Paulo, Brazil; §Laboratory of Cellular and Molecular Physiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Centro de Ciências da Saúde, Rio de Janeiro, Brazil; Department of Intensive Care Medicine and the Laboratory for Experimental Intensive Care and Anesthesiology (L.E.I.C.A), Academic Medical Centre at the University of Amsterdam, Amsterdam, the Netherlands; and Department of Surgical Sciences and Integrated Diagnostics, IRCCS AOU San Martino-IST, University of Genoa, Genoa, Italy.

Accepted for publication June 7, 2017.

Published ahead of print July 27, 2017.

Funding: This study was supported by grants from the Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ), grant number E-26/103.118/2014), Rio de Janeiro, Brazil; and the Brazilian Council for Scientific and Technological Development (CNPq, grant number 469716/2014-2), Brasilia, Brazil.

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website.

This study was presented in part as an abstract at the American Thoracic Society scientific meeting, San Francisco, CA, May 13–18, 2016, and at the European Society of Intensive Care Medicine meeting, Berlin, Germany, October 3–7, 2015.

Reprints will not be available from the authors.

Address correspondence to Patricia Rieken Macedo Rocco, MD, PhD, Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho – CCS, Laboratório de Investigação Pulmonar, Ilha do Fundão, 21941-902, Rio de Janeiro, Brazil. Address e-mail to prmrocco@biof.ufrj.br.

General anesthesia causes a reduction in lung volume, with airway closure and development of atelectasis.1–3 The use of low tidal volumes (VT) during general anesthesia for surgery has been suggested to reduce the occurrence of postoperative pulmonary complications (PPCs), and represents an important element of “protective ventilation.”4–7 On the other hand, the use of low VT may exacerbate the reduction of resting lung volume, increasing airway closure and atelectasis.8 These effects can be prevented if lung volume is restored through application of appropriate positive end-expiratory pressure (PEEP) levels and recruitment maneuvers (RMs).1 Retrospective studies have suggested that low intraoperative VT is associated with increased mortality if accompanied by low9 or high PEEP levels,10 whereas meta-analyses have been able to detect a protective role for VT, but not PEEP, during general anesthesia for surgery.11

Recent randomized controlled trials of intraoperative ventilation for abdominal surgery12,13 compared different ventilation strategies with respect to occurrence of PPCs. In the Intraoperative Protective Ventilation (IMPROVE) trial,12 low VT with moderate PEEP levels and RMs resulted in fewer PPCs compared to high VT and no PEEP. In the Protective Ventilation using High versus Low PEEP (PROVHILO) trial,13 low VT with high PEEP levels and RMs, compared to low VT and low PEEP without RMs, did not protect against PPCs. In addition, a recent meta-analysis showed that higher respiratory system driving pressure was associated with increased risk of PPCs.14 There is no consensus regarding the best intraoperative ventilator strategy for abdominal surgery.5–7 Therefore, the impact of the ventilatory strategies used in IMPROVE and PROVHILO on driving pressure, mechanical power delivered from the ventilator to the respiratory system, and lung damage and inflammation remains unclear. To the best of our knowledge, this is the first standardized, randomized preclinical translational study that clarifies the respiratory and biological effects of mechanical ventilation strategies used in both trials in a rat model of open abdominal surgery. We hypothesized that reduced driving pressure and mechanical power would be associated with less lung damage during abdominal surgery.

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METHODS

Animal Preparation and Experimental Protocol

All procedures on rats were approved by the Ethics Committee of the Carlos Chagas Filho Institute of Biophysics, Health Sciences Center, Federal University of Rio de Janeiro, Brazil (CEUA-019). 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 Research Council Guide for the Care and Use of Laboratory Animals. Experiments conformed with the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” (Council of Europe No. 123, Strasbourg 1985), and the present study followed the Animal Research: Reporting of In Vivo Experiments guidelines for reporting of animal research.15

Thirty-five healthy Wistar rats (weight 394 ± 60 g) were sedated (10 mg/kg of diazepam intraperitoneally) and anesthetized (75 mg/kg of ketamine and 2 mg/kg midazolam intraperitoneally). The tail vein was cannulated (Jelco 24G; Becton Dickinson, Franklin Lakes, NJ) for continuous infusion of 50 mg/kg/h of ketamine, 2 mg/kg/h of midazolam, and 7 mL/kg/h of Ringer’s lactate (B. Braun, Rio de Janeiro, Brazil) during mechanical ventilation. 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), pupil diameter, position of the nictitating membrane, and movement in response to tail stimulation.16 Experiments were started when responses to a noise stimulus (handclap), whisker stimulation, and tail clamping were absent.

Local anesthetic (0.4 mL of 2% lidocaine) was infiltrated, and a tracheostomy was performed via a midline neck incision for a 14-gauge cannula. A catheter was introduced into the right carotid artery for blood sampling and monitoring of mean arterial pressure (MAP). Body temperature was maintained at 37.5 ± 1°C using a heating plate. Gelafundin (B. Braun, São Gonçalo, Rio de Janeiro, Brazil) was administered in 0.5-mL increments to maintain MAP >60 mm Hg. Animals were then paralyzed (0.4 mg of pancuronium intramuscularly, followed by a 0.4-mg intravenous bolus diluted in 1 mL of Ringer’s lactate) and mechanically ventilated (Servo-I; MAQUET, Solna, Sweden) in volume-controlled mode with VT = 7 mL/kg, minute ventilation = 150 mL/min, inspiratory-to-expiratory ratio = 1:2, fraction of inspired oxygen = 0.4, and PEEP = 1 cm H2O for 5 min.

Arterial blood (300 μL) was drawn into a heparinized syringe to determine arterial oxygen partial pressure (Pao2), arterial carbon dioxide partial pressure (Paco2), bicarbonate levels, and arterial pH (pHa; ABL80 FLEX, Radiometer, Copenhagen, Denmark). This was defined as the baseline time point for data collection (baseline). Rats were then randomly assigned, using closed sealed envelopes, to 1 of 4 groups (n = 7/group) to receive the following mechanical ventilation strategies, mimicking the ventilation strategies of IMPROVE or PROVHILO: (1) VT = 7 mL/kg with PEEP = 1 cm H2O and no RMs (low VT/low PEEP/RM−) (PROVHILO); (2) VT = 7 mL/kg with PEEP = 3 cmH2O and RMs, performed through a continuous positive airway pressure of 30 cm H2O for 30 seconds after randomization (before laparotomy) and every 1 hour thereafter (low VT/moderate PEEP/4 RM+) (IMPROVE); (3) VT = 7 mL/kg with PEEP = 6 cm H2O and RMs, performed as described above but only before laparotomy (low VT/high PEEP/1 RM+) (PROVHILO); or (4) VT = 14 mL/kg with PEEP = 1 cm H2O and no RMs (high VT/low PEEP/RM−) (IMPROVE).

After group allocation, laparotomy was performed, and animals were ventilated for 4 hours. The respiratory rate (RR) was adjusted to reach a minute ventilation of 150 mL/min. At the start of mechanical ventilation and 3 hours thereafter, a standardized bowel manipulation was performed as follows: under sterile conditions, lateral retractors were carefully placed, the bowel was gently taken out of the abdominal cavity and reintroduced within 20 seconds. The retractors were left in place, and the abdominal cavity was continuously humidified with warmed saline at 37°C. Respiratory system mechanics, heart rate, MAP, and the amount of fluids infused were measured hourly. At the end of the experiment, animals were killed by injection of sodium thiopental (60 mg/kg), and the lungs were extracted for postmortem analysis (see the timeline of the experiments, Supplemental Digital Content 1, Figure 1, http://links.lww.com/AA/B923). Seven rats were not tracheotomized, operated, or mechanically ventilated, and constituted the healthy nonoperated and nonventilated controls (NO–NV).

Post hoc new experiments were performed to evaluate whether the effects observed in the low VT/moderate PEEP and low VT/high-PEEP groups might be associated with RMs. For these experiments, rats were randomly assigned to 1 of 2 groups (n = 5): VT = 7 mL/kg with PEEP = 3 cm H2O without RMs (low VT/moderate PEEP/RM−) or VT = 7 mL/kg with PEEP = 6 cm H2O without RMs (low VT/high PEEP/RM−). Additional methods related to these groups are provided in Supplemental Digital Content 2, Additional Methods, http://links.lww.com/AA/B924.

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Data Acquisition and Processing

A pneumotachograph (internal diameter = 1.5 mm, length = 4.2 cm, distance between side ports = 2.1 cm) was connected to the tracheal cannula for airflow (V′) measurements. The pressure gradient across the pneumotachograph was determined using a SCIREQ differential pressure transducer (UT-PDP-300, SCIREQ, Montreal, Quebec, Canada). Tidal volume was calculated by digital integration of the flow signal.17 Tracheal pressure (paw) was measured with a SCIREQ differential pressure transducer (UT-PDP-75, SCIREQ).

Airflow and airway pressure were continuously recorded throughout the experiments with a computer running software written in LabVIEW (National Instruments, Austin, TX). All signals were filtered, amplified (SC-24, SCIREQ), and sampled at 200 Hz with a 12-bit analog-to-digital converter (National Instruments). Peak (Ppeak,RS) and mean (Pmean,RS) airway pressures, as well as respiratory system plateau pressure (Pplat,RS), were computed offline by a routine written in MATLAB (Version R2007a; The Mathworks Inc, Natick, MA).18

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Measurement of Mechanical Power, Energy, and Driving Pressure

The mechanical power (Pv) applied by the ventilator to the respiratory system was calculated by multiplication of energy (E) by RR as Pv = E · RR.19,20 Energy applied by the ventilator to the respiratory system was calculated by numerical integration of the inspiratory airway pressure–volume curve versus volume (the area between the inspiratory limb of the pressure–volume curve and the volume axis). Respiratory system driving pressure (ΔPRS) was calculated as the difference between Pplat,RS and minimal pressure per cycle.21,22

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Histology

Heparin (1000 IU) was injected into the tail vein, the trachea was clamped at end-expiration, and the lungs were removed en bloc. The left lung was frozen in liquid nitrogen and submerged in Carnoy’s solution.23,24 Slices (4 μm thick) were stained with hematoxylin and eosin. Lung morphometric analysis was performed using an integrating eyepiece with a coherent system consisting of a grid with 100 points and 50 lines of known length coupled to a conventional light microscope (Olympus BX51; Olympus Latin America, Brazil). The volume fractions of the lung occupied by collapsed alveoli, normal pulmonary areas, or hyperinflated structures were determined by the point-counting technique at a magnification of ×200 across 10 random, noncoincident microscopic fields.25

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Transmission Electron Microscopy of the Lung

Three slices measuring 2 × 2 × 2 mm were cut from 3 different segments of the right lung (apex, middle, and base of the lung). They were then fixed in 2.5% glutaraldehyde and phosphate buffer, 0.1 M (pH = 7.4) for electron microscopy analysis (JEOL 1010 Transmission Electron Microscope; Japan Electron Optics Laboratory Co, Tokyo, Japan). For each electron microscopy image (20 per animal), an injury score was determined. The following parameters were analyzed concerning lung parenchyma: damage to alveolar capillary membrane, type II epithelial cell lesion, and endothelial cell damage.17 Pathological findings were graded on a 5-point, semiquantitative, severity-based scoring system as follows: 0 = normal lung parenchyma; 1 = changes in 1%–25%; 2 = changes in 26%–50%; 3 = changes in 51%–75%; and 4 = changes in 76%–100% of examined tissue.

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Immunohistochemistry

The right lower lung was immersed in immunohistochemistry solution. To evaluate the degree of epithelial cell damage, E-cadherin (the major transmembrane protein of the adherens junction) was analyzed.26 Immunohistochemical procedures were performed on lung sections using a mouse polyclonal antibody against E-cadherin (cat. 610181, BD Transduction Laboratories, 1:300). Visualization and image capture were performed using a light microscope (Eclipse E800, Nikon, Japan) coupled to a digital camera (Evolution, Media Cybernetics Inc, Rockville, MD) and Q-Capture 2.95.0 graphic interface software (version 2.0.5; Quantitative Imaging, Surrey, British Columbia, Canada). Expression of E-cadherin was analyzed using ImagePro Plus software (version 4.5.1, Media Cybernetics).

The pathologist or technician working on lung morphometry, the electron microscopy images, and immunohistochemistry was blinded to the nature of the study.

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Molecular Biology

The right middle lung was flash-frozen by immersion in liquid nitrogen and stored at −80°C for quantification of mRNA expression. Quantitative real-time reverse transcription polymerase chain reaction was performed to measure the expression in lung tissue of type III procollagen (PCIII), a biological marker associated with fibrogenesis; amphiregulin, a marker of pulmonary stretch; receptor for advanced glycation end products (RAGE), a marker of damage inflicted to type I epithelial cells; and vascular cell adhesion molecule 1 (VCAM-1), a marker of endothelial cell damage (for primers, see Supplemental Digital Content 2, Additional Methods, http://links.lww.com/AA/B924). Total RNA was extracted from central slices of frozen lung tissue (RNeasy plus mini kit; Qiagen, Hilden, Germany). RNA concentration was measured by spectrophotometry in a Nanodrop ND-1000 system (Thermo Fisher Scientific, Waltham, MA). First-strand complementary DNA was synthesized from total RNA using the QuantiTect 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, CA). Samples were measured in triplicate. Relative gene expression was calculated as a ratio of the average gene expression levels compared with the reference gene (acidic ribosomal phosphoprotein P0, 36B4)27 and expressed as fold change relative to animals in the NO–NV group.

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Enzyme-Linked Immunosorbent Assay

The right upper lung was immediately frozen in liquid nitrogen and stored at −80°C for enzyme-linked immunosorbent assay. Interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α levels were quantified by enzyme-linked immunosorbent assay in the lung homogenate. All procedures were done according to manufacturer protocol (Peprotech, London, UK) and normalized to total protein as assessed by Bradford’s reagent (Sigma-Aldrich, St Louis, MO).

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Statistical Analysis

Variables were tested for normality using the Kolmogorov–Smirnov test. Parametric data were expressed as means ± SD, and nonparametric data were expressed as median (interquartile range). The main results were presented as treatment effects (difference in means between groups) and their 95% confidence intervals (CIs). To compare respiratory system mechanics and blood gas analysis over time, 2-way repeated-measures ANOVA followed by the Bonferroni post hoc test was used. For interactions between group and time, we adopted a less conservative P value (P< .10) as significant. To compare the NO–NV group with each ventilatory strategy, Student t tests followed by the Bonferroni–Holm procedure were used (P value adjusted for 4 comparisons; P< .0125). The percentage of alveolar collapse, E-cadherin, and the expression of biological markers among mechanical ventilation groups were compared using the Kruskal-Wallis test followed by the Dunn post hoc test. Correlations were assessed using the Spearman test, as data were distributed nonparametrically. All tests were performed in GraphPad Prism version 6.07 (GraphPad Software, La Jolla, CA). The significance level was set at 5%.

The sample size calculation of each group was based on our experimental experience, which allowed detection of significant differences with the smallest possible number of animals, and on the respiratory effects observed in a previous study in rodents using comparable ventilator settings.28 A sample size of 7 animals per group would provide the appropriate power (1 − β = 0.8) to identify significant (α = 0.05) differences in Pplat,RS between ventilatory strategies based on low VT associated with low PEEP without RMs and high PEEP with RMs, taking into account an effect size d = 1.76, a 2-sided test, and a sample size ratio = 1 (G*Power 3.1.9.2, University of Düsseldorf, Düsseldorf, Germany).

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RESULTS

At baseline, respiratory parameters and arterial blood gases were not significantly different across groups (Supplemental Digital Content 3, Table 1, http://links.lww.com/AA/B925). During experiments, heart rate, MAP (Supplemental Digital Content 4, Figure 2, http://links.lww.com/AA/B926), total fluids administered (Supplemental Digital Content 5, Table 2, http://links.lww.com/AA/B927), and arterial blood gases were comparable among groups. Oxygenation, pHa, and bicarbonate levels decreased in all groups during the experiments (Supplemental Digital Content 6, Table 3, http://links.lww.com/AA/B928).

Ppeak,RS, Pmean,RS, Pplat,RS, ΔPRS, energy, and Pv differed among groups and over time (Table). Low VT, regardless of PEEP level, resulted in lower Pplat,RS (P< .0001) and ΔPRS (P< .001) than high VT/low PEEP/RM− (Supplemental Digital Content 7, Figure 3, http://links.lww.com/AA/B929). In low VT/moderate PEEP/4 RM+, Pplat,RS and ΔPRS were lower than in other groups. The low VT/moderate PEEP/4 RM+ and low VT/high PEEP/1 RM+ groups exhibited comparable energy and Pv, which were lower than in low VT/low PEEP/RM− and high VT/low PEEP/RM− (Table).

Table.

Table.

As shown in Figure 1, except for low VT/high PEEP/1 RM+, all ventilation strategies increased the fraction area of alveolar collapse compared to NO–NV. Lung hyperinflation did not differ between groups. Additionally, low VT/high PEEP/1 RM+ was associated with less alveolar collapse (treatment effect: 17.1%; 95% CI, 3.8–30.5; P = .032) than low VT/low PEEP/RM−.

Figure 1.

Figure 1.

Electron microscopy findings of lung parenchyma are depicted in Supplemental Digital Content 8, Figure 4, http://links.lww.com/AA/B930. Damage to type II epithelial cells and alveolar capillary membrane was present in all groups; however, no endothelial cell injury was observed (Supplemental Digital Content 9, Table 4, http://links.lww.com/AA/B931). Low VT/moderate PEEP/4 RM+ was associated with a lower damage to type II epithelial cell compared to high VT/low PEEP/RM− (P = .005).

As shown in Figure 2, E-cadherin expression was lower in mechanically ventilated animals compared to NO–NV. Among the ventilated groups, E-cadherin expression was higher in low VT/moderate PEEP/4 RM+ than in low VT/low PEEP/RM− (treatment effect: −8.8%; 95% CI, −16.0% to −1.5%; P = .013) and high VT/low PEEP/RM− (treatment effect: 9.5%; 95% CI, 11.9%–17.1%; P = .014).

Figure 2.

Figure 2.

Amphiregulin mRNA expression was higher in all mechanically ventilated groups as compared with NO–NV (Figure 3). PCIII mRNA expression was higher (treatment effect: −0.9; 95% CI, −1.7 to −0.2; P = .039) in high VT/low PEEP/RM− than in low VT/high PEEP/1 RM+. RAGE mRNA expression was lower in low VT/high PEEP/1 RM+ than in NO–NV. VCAM-1 mRNA expression did not differ across groups.

Figure 3.

Figure 3.

IL-6 levels were higher in all mechanically ventilated groups than in NO–NV. Low VT/moderate PEEP/4 RM+ was associated with a lower TNF-α level (P = .028) than high VT/low PEEP/RM− (Figure 4).

Figure 4.

Figure 4.

Alveolar collapse, E-cadherin expression, and TNF-α level correlated with ΔPRS (r = 0.54, P = .02; r = −0.48, P = .05; and r = 0.59, P = .09, respectively) and Pv (r = 0.57, P = .02; r = −0.54, P = .02; and r = 0.48, P = .04, respectively; Figure 5), whereas PCIII and VCAM-1 mRNA expressions correlated with ΔPRS (r = 0.52, P = .01 and r = 0.39, P = .006, respectively; Supplemental Digital Content 10, Figure 5, http://links.lww.com/AA/B932). Mechanical power correlated with ΔPRS (r = 0.75, P = .001).

Figure 5.

Figure 5.

To evaluate whether the effects observed in the low VT/moderate PEEP/4 RM+ and low VT/high PEEP/1 RM+ might be associated with RMs, these groups were compared with those without RM: low VT/moderate PEEP/RM− and low VT/high PEEP/RM−. At baseline, respiratory parameters and arterial blood gases were not significantly different across groups (Supplemental Digital Contents 11 and 12, Tables 5 and 6, http://links.lww.com/AA/B933, http://links.lww.com/AA/B934). Low-VT/moderate PEEP/4 RM+ animals exhibited lower pHa and oxygenation when compared to low VT/moderate PEEP/RM− (Supplemental Digital Content 12, Table 6, http://links.lww.com/AA/B934). Low-VT/moderate PEEP/4 RM+ animals also had lower Ppeak,RS, Pmean,RS, Pplat,RS, ΔPRS, energy, and Pv when compared to low-VT/moderate PEEP/RM− animals (Supplemental Digital Content 13, Table 7, http://links.lww.com/AA/B935). Low VT/moderate PEEP/RM− exhibited higher alveolar collapse compared to low VT/moderate PEEP/4 RM+ (Supplemental Digital Content 14, Figure 6, http://links.lww.com/AA/B936). There was no difference in E-cadherin expression between groups (Supplemental Digital Content 15, Figure 7, http://links.lww.com/AA/B937). PCIII mRNA expression was higher in low VT/moderate PEEP/4 RM+ than low VT/moderate PEEP/RM− (Supplemental Digital Content 16, Figure 8, http://links.lww.com/AA/B938). Gene expressions of amphiregulin, RAGE, and VCAM-1 did not differ across groups (Supplemental Digital Content 16, Figure 8, http://links.lww.com/AA/B938). In addition, TNF-α levels were higher in low-VT/high PEEP/RM+ than in low-VT/high PEEP/RM− animals (Supplemental Digital Content 17, Figure 9, http://links.lww.com/AA/B939).

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DISCUSSION

The main findings of the present study conducted in a rat model of open abdominal surgery were: (1) low VT/moderate PEEP/4 RM+ and low VT/high PEEP/1 RM+ were associated with lower ΔPRS and Pv than low VT/low PEEP/RM− and high VT/low PEEP/RM−; (2) low VT/moderate PEEP/4 RM+ led to reduced type II epithelial cell damage compared to high VT/low PEEP/RM−; and (3) the extent of alveolar collapse, E-cadherin expression, and TNF-α expression correlated with ΔPRS and mechanical power. Lower mechanical power and its surrogate ΔPRS were associated with reduced lung damage.

Previous studies have shown that mechanical ventilation is harmful to the mesenteric microcirculation,29 while other authors have reported that injurious mechanical ventilation for 4 hours may elicit an immune response similar to that observed during infections.30 However, there have been no preclinical studies with an experimental design comparable to ours. In our study, ventilation strategies that closely resembled those compared in 2 recent randomized controlled trials were chosen.12,13 To the best of our knowledge, this was the first standardized, randomized preclinical translational study to address the impact of mechanical ventilation strategies on ΔPRS, mechanical power, and biological markers during open abdominal surgery in rats.

The first group (low VT/low PEEP/RM−) mimicked the low VT, low PEEP strategy of the PROVHILO trial.13 The second group (low VT/moderate PEEP/4 RM+) mimicked the protective ventilation strategy of the IMPROVE trial,12 in which low VT, moderate PEEP, and periodic RMs were used. The third group (low VT/high PEEP/1 RM+) mimicked the strategy consisting of low VT, high PEEP, and RMs after intubation and before extubation described in the PROVHILO trial.13 The fourth group (high VT/low PEEP/RM−) mimicked conventional ventilation as used in the IMPROVE trial,12 with high VT, low PEEP, and no RMs. Importantly, all of these mechanical ventilation strategies, including high VT/low PEEP/RM−, are still used in clinical practice.31,32VT levels normalized to body weight were close to those used in clinical trials, as the relationship between lung volume and body mass is approximately constant among mammalian species.33 In contrast, PEEP values in the different groups were adjusted to the respective low, moderate, and high ranges in rats.23,24 Laparotomy followed by standardized manipulation of the bowel and use of retractors were meant to reproduce as closely as possible open abdominal surgery in humans, a setting in which surgical trauma usually induces release of proinflammatory substances into the bloodstream, likely priming the lungs to further insult.34 A 4-hour period was chosen because this corresponded approximately to the average operative time in IMPROVE12 and PROVHILO.13

The fact that low VT/moderate PEEP/4 RM+ substantially reduced ΔPRS, energy, and Pv suggests that this strategy resulted in a more favorable relationship between pressure and volume in the respiratory system as compared to the other protective ventilation strategy. During anesthesia, the resting lung volume (functional residual capacity) is reduced, promoting airway closure and atelectasis.1 In a study using computed tomography, atelectasis was noted to occur in 100% of subjects undergoing general anesthesia.35 Airway closure and atelectasis will be prevented if functional residual capacity is restored by using a suitable PEEP and RMs,1,2 which likely explains the lessened alveolar collapse and prevention of cell–cell contact with maintenance of E-cadherin in the groups in which RMs were applied. On the other hand, if PEEP exceeds the level required to stabilize the lungs, overdistension may result, despite the use of low VT. Thus, moderate levels of PEEP in noninjured lungs may represent a compromise between cyclic overdistension and closing/reopening of lung units.36 In accordance with our findings, reduced ΔPRS is associated with a lower incidence of PPCs regardless of PEEP level and VT.14 Additionally, alveolar collapse was found to correlate with ΔPRS and Pv.

We observed that E-cadherin expression was lower in the mechanically ventilated animals compared to NO–NV. This may be explained by increased mechanical stress at the alveolar epithelial layer, leading to partial loss of cell–cell adhesion. Presence of the soluble fragments of E-cadherin in bronchoalveolar lavage fluid likely reflects epithelial cell injury.37 We evaluated E-cadherin in lung tissue by immunohistochemistry because this technique allows recognition of remaining E-cadherin between epithelial cells both in the airway and in alveolar sites.38 Interestingly, E-cadherin expression was higher in low VT/moderate PEEP/4 RM+ than low VT/low PEEP/RM−, suggesting that a protective ventilation strategy able to reduce ΔPRS, energy, and mechanical power and keep these parameters at relatively low levels might better preserve the alveolar epithelial cell layer. Furthermore, E-cadherin correlated well with ΔPRS and Pv, suggesting that these parameters could be taken into account in lung damage prevention strategies. In contrast to E-cadherin, amphiregulin expression did not show a clear association with ΔPRS and Pv. We hypothesize that amphiregulin more closely reflects overdistension of lung cells, whereas E-cadherin is more closely related to closing and reopening of lung units and shear stress.

Our finding that protective ventilation strategies (ie, those based on the use of low VT) were not associated with relevant differences in IL-6 is worthy of note. The fact that ΔPRS remained in a relatively low range (<15 cm H2O) in most groups might partly explain these findings. Furthermore, noninjured lungs, even if primed by inflammatory mediators released at the surgical site, could theoretically tolerate the stress of mechanical ventilation better. In patients with acute respiratory distress syndrome, ΔPRS >15 cm H2O has been associated with higher mortality rates,39 and in patients undergoing surgery, ΔPRS >13 cm H2O was associated with higher incidence of PPCs, irrespective of VT and PEEP settings.14 In the present study, ΔPRS >15 cm H2O (high VT/low PEEP/RM−) resulted in increased levels of TNF-α and PCIII mRNA expression compared to the ventilation strategy with the lowest ΔPRS. The underlying mechanism is likely related to opening and closing of alveolar units during high VT and low PEEP, leading to greater mechanical stress, which has been associated with PCIII mRNA expression.40 In addition, TNF-α correlated with ΔPRS and mechanical power. In this line, high TNF-α levels in bronchoalveolar lavage have been associated with increased lung inflammation and damage.41

We performed experiments to rule out the contribution of RMs. The level of TNF-α was higher in those animals ventilated with low VT/high PEEP plus RMs than in those with similar ventilator settings but no RMs (Supplemental Digital Content 17, Figure 9, http://links.lww.com/AA/B939). Therefore, the better outcomes observed were due to the combination of moderate PEEP levels and low VT, not to the presence of RMs.

The fact that Pv correlated with ΔPRS is not surprising. Besides airflow, airway pressure, volume, and stored energy in the respiratory system, ΔPRS is one of the major determinants of mechanical power calculations, and especially of the dynamic component.20,21 Thus, in our study, ΔPRS likely reflected dynamic stress on lung structures, serving as a surrogate of Pv.

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Possible Clinical Implications of These Findings

Driving pressure and mechanical power should be considered in the monitoring systems of anesthesia ventilator devices to optimize mechanical ventilation during surgery and thus minimize lung damage.14 Low tidal volume, when combined with lower driving pressure and mechanical power, seems to be one of the most important factors to mitigate lung damage. RMs do not seem to reduce lung injury; this may have important clinical consequences, eg, by limiting their use to rescue purposes or specific clinical situations. Our experimental data do not support the routine use of RM in these conditions. We believe that introducing measurement of driving pressure and mechanical power into clinical practice in the near future should elucidate the safe limits of mechanical ventilation during open abdominal surgery.

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Limitations

This study has several limitations. First, our model of ventilation during open abdominal surgery did not reproduce all aspects of complex clinical scenario in humans, such as the potential influence of patients’ comorbidities (eg, chronic obstructive pulmonary disease), which may influence outcome. Second, the PEEP levels observed were not the same as those reported for intraoperative mechanical ventilation strategies, and cannot be directly extrapolated to the clinical setting. However, a theoretical analysis based on transpulmonary pressures has suggested that PEEP levels in rats correspond to roughly double those observed in humans. This is based on theoretical analysis on the height of the thorax from sternum to vertebra. The in situ transpulmonary pressure of the rat lung is between 1.5 and 2 cm H2O; correspondingly, a PEEP of 6 cm H2O, for example, is about 2 to 3 times higher than this range.42 Third, the rat chest wall has higher compliance compared to that of humans, and accounts for 10% of the airway pressure. Nevertheless, as less airway pressure is dissipated to the chest wall and more to the lungs, we would expect more strain in lung tissue. Therefore, even though this precludes direct comparisons, lung strain—and, consequently, expected lung injury—would be greater. Fourth, the study design precluded identification of the respective contributions of VT, PEEP, and RMs to the biological impact of the mechanical ventilation strategies investigated. When considering these limitations, readers should bear in mind that the study interventions were designed to reproduce the ventilation strategies used in the largest randomized controlled trials in the field,13 with a view to immediate translational aspects. Fifth, although our outcome variables are mutually independent, we did not adjust for multiple outcome variables, which in turn, may have increased the likelihood of false-positive findings. Sixth, since it is methodologically difficult and may induce bias, we did not extract lung tissue from the baseline condition for comparison to that collected at the end of the experiment. Instead, we used the healthy nonoperated and nonventilated group for comparison to baseline. In addition, to evaluate endothelial cell damage, VCAM-1 mRNA expression and electron microscopy findings were evaluated. In healthy animals, VCAM-1 is probably not a sufficient marker of endothelial cell injury43 or might require a longer time course to change. Finally, energy and mechanical power calculations did not differ between energy stored and delivered breath-by-breath to the respiratory system (ie, static and dynamic components).

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CONCLUSIONS

In the model of open abdominal surgery used in this study, based on the mechanical ventilation strategies used in the IMPROVE and PROVHILO trials, lower mechanical power and its surrogate driving pressure were associated with reduced lung damage.

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ACKNOWLEDGMENTS

The authors express their gratitude to Andre Benedito da Silva for animal care, Ana Lucia Neves da Silva for her help with microscopy, and Marcella Rocco for her help with mathematical physics data (Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Rio de Janeiro, Brazil); Ronir Raggio Luiz, PhD (Institute of Public Health Studies, Federal University of Rio de Janeiro), for his help with statistics; and Moira Elizabeth Schottler (Rio de Janeiro) and Filippe Vasconcellos (São Paulo), Brazil, for their assistance in editing the manuscript.

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DISCLOSURES

Name: Lígia de A. Maia, PhD.

Contribution: This author helped in the design and conduction of the study, data collection and analysis, and preparation of the manuscript.

Name: Cynthia S. Samary, PhD.

Contribution: This author helped in the design and conduction of the study and in data collection.

Name: Milena V. Oliveira, MSc.

Contribution: This author helped in the conduction of the study and in data collection.

Name: Cíntia L. Santos, MD, PhD.

Contribution: This author helped in data collection and analysis, and preparation of the manuscript.

Name: Robert Huhle, PhD.

Contribution: This author helped in the conduction of the study and in data collection.

Name: Vera L. Capelozzi, MD, PhD.

Contribution: This author helped in the conduction of the study and in data collection.

Name: Marcelo M. Morales, MD, PhD.

Contribution: This author helped in the conduction of the study, data analysis, and preparation of the manuscript.

Name: Marcus J. Schultz, MD, PhD.

Contribution: This author helped in the design of the study, data analysis, and preparation of the manuscript.

Name: Marcelo G. Abreu, MD, PhD.

Contribution: This author helped in the design and conduction of the study, data analysis, and preparation of the manuscript.

Name: Paolo Pelosi, MD, FERS.

Contribution: This author helped in the design and conduction of the study, data analysis, and preparation of the manuscript.

Name: Pedro L. Silva, PhD.

Contribution: This author helped in the design and conduction of the study, data analysis, and preparation of the manuscript.

Name: Patricia Rieken Macedo Rocco, MD, PhD.

Contribution: This author helped in the design and conduction of the study, data analysis, and preparation of the manuscript.

This manuscript was handled by: Alexander Zarbock, MD.

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