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Fast Versus Slow Recruitment Maneuver at Different Degrees of Acute Lung Inflammation Induced by Experimental Sepsis

Santos, Raquel S. PhD; Moraes, Lillian PhD; Samary, Cynthia S. PhD; Santos, Cíntia L. PhD; Ramos, Maíra B. A. RRT; Vasconcellos, Ana P. RRT; Horta, Lucas F. MS; Morales, Marcelo M. MD, PhD; Capelozzi, Vera L. MD, PhD; Garcia, Cristiane S. N. B. PhD; Marini, John J. MD; Gama de Abreu, Marcelo MD, PhD; Pelosi, Paolo MD, FERS; Silva, Pedro L. PhD; Rocco, Patricia R. M. MD, PhD

doi: 10.1213/ANE.0000000000001173
Critical Care, Trauma, and Resuscitation: Research Report

BACKGROUND: Large tidal volume (VT) breaths or “recruitment maneuvers” (RMs) are used commonly to open collapsed lungs, but their effectiveness may depend on how the RM is delivered. We hypothesized that a stepped approach to RM delivery (“slow” RM) compared with a nonstepped (“fast” RM), when followed by decremental positive end-expiratory pressure (PEEP) titration to lowest dynamic elastance, would (1) yield a more homogeneous inflation of the lungs, thus reducing the PEEP obtained during post-RM titration; (2) produce less lung morphofunctional injury, regardless of the severity of sepsis-induced acute lung inflammation; and (3) result in less biological damage in severe, but not in moderate, acute lung inflammation.

METHODS: Sepsis was induced by cecal ligation and puncture surgery in 51 Wistar rats. After 48 hours, animals were anesthetized, mechanically ventilated (VT = 6 mL/kg), and stratified by PO2/fraction of inspired oxygen ratio into moderate (≥300) and severe (<300) acute lung inflammation groups. Each group was then subdivided randomly into 3 subgroups: (1) nonrecruited; (2) RM with continuous positive airway pressure (30 cm H2O for 30 seconds; CPAPRM or fast RM); and (3) RM with stepwise airway pressure increase (5 cm H2O/step, 8.5 seconds/step, 6 steps, 51 seconds; STEPRM or slow RM), with a maximum pressure hold for 10 seconds. All animals underwent decremental PEEP titration to determine the level of PEEP required to optimize dynamic compliance after RM and were then ventilated for 60 minutes with VT = 6 mL/kg, respiratory rate = 80 bpm, fraction of inspired oxygen = 0.4, and the newly adjusted PEEP for each animal. Respiratory mechanics, hemodynamics, and arterial blood gases were measured before and at the end of 60-minute mechanical ventilation. Lung histology and biological markers of inflammation and damage inflicted to endothelial cells were evaluated at the end of the 60-minute mechanical ventilation.

RESULTS: Respiratory system mean airway pressure was lower in STEPRM than that in CPAPRM. The total RM time was greater, and the RM rise angle was lower in STEPRM than that in CPAPRM. In both moderate and severe acute lung inflammation groups, STEPRM reduced total diffuse alveolar damage score compared with the score in nonrecruited rats. In moderate acute lung inflammation, STEPRM rats compared with CPAPRM rats had less endothelial cell damage and angiopoietin (Ang)-2 expression. In severe acute lung inflammation, STEPRM compared with CPAPRM reduced hyperinflation, endothelial cell damage, Ang-2, and intercellular adhesion molecule-1 expressions. RM rise angle correlated with Ang-2 expression.

CONCLUSIONS: Compared with CPAPRM, STEPRM reduced biological markers associated with endothelial cell damage and ultrastructural endothelial cell injury in both moderate and severe sepsis-induced acute lung inflammation.

Supplemental Digital Content is available in the text.Published ahead of print February 1, 2016

From the *Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Laboratory of Experimental Surgery, Faculty of Medicine, and Laboratory of Cellular and Molecular Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; §Department of Pathology, School of Medicine, University of São Paulo, São Paulo, Brazil; Rio de Janeiro Federal Institute of Education, Science and Technology, Rio de Janeiro, Brazil; Department of Medicine, University of Minnesota, Minneapolis/Regions Hospital, Pulmonary and Critical Care Medicine, St Paul, Minnesota; #Pulmonary Engineering Group, Department of Anesthesiology and Intensive Care Therapy, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; and **IRCCS AOU San Martino-IST, Department of Surgical Sciences and Integrated Diagnostics, University of Genoa, Genoa, Italy.

Accepted for publication November 20, 2015.

Published ahead of print February 1, 2016

Funding: This study was supported by the Centers of Excellence Program (PRONEX-FAPERJ), the Brazilian Council for Scientific and Technological Development (CNPq), the Rio de Janeiro State Research Foundation (FAPERJ), the São Paulo State Research Foundation (FAPESP), the Coordination for the Improvement of Higher Level Personnel (CAPES), and the European Community Seventh Framework Programme (TARKINAID, FP7-2007-2013).

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.

Address correspondence and reprint requests to Patricia R. M. Rocco, MD, PhD, Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho, 373, Bloco G-014, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brazil. Address e-mail to prmrocco@biof.ufrj.br.

Sepsis is a leading cause of acute respiratory distress syndrome (ARDS), producing severe impairment of gas exchange and usually requiring invasive mechanical ventilation.1 The heterogeneous distribution of consolidated/atelectatic lung areas during ARDS causes adjoining normal lung areas to become more vulnerable to ventilator-induced lung injury. Strategies to recruit closed lung units and prevent derecruitment might help improve oxygenation and reduce the incidence of ventilator-induced lung injury.2 The use of recruitment maneuvers (RMs), however, is controversial, both in the operating room3,4 and in the intensive care unit.5,6 During the application of RM, the rate and duration of pressure rise are important determinants of both effectiveness and alveolar-capillary damage.7,8 Continuous positive airway pressure at 35 to 45 cm H2O for 30 to 40 seconds (CPAPRM)2 may improve lung morphofunction9 but also can cause injury to the alveolar-capillary membrane because of high inspiratory airflow.10,11 Therefore, RMs that achieve the same maximal pressure by the use of a progressive, stepwise increase in airway pressure (STEPRM or “slow” RM) have been proposed to improve lung mechanics12 and reduce epithelial and endothelial cell damage.8 Most studies that have analyzed the effects of RMs, however, have been performed with a fixed post-RM positive end-expiratory pressure (PEEP) level. Ideally, PEEP should be individually optimized after RMs to preserve any benefit.13–17 PEEP can be optimized on the basis of oxygenation5,18 or respiratory system mechanics,19 but both methods have distinct advantages19,20 and disadvantages.18,21,22

Because it induces a state that closely mimics human sepsis and can produce different degrees of severity, cecal ligation and puncture (CLP) surgery is the gold standard animal model for sepsis research.23 We hypothesized that, when followed by decremental PEEP titration, STEPRM compared with CPAPRM would (1) reduce the PEEP obtained during post-RM titration; (2) produce less lung morphofunctional injury, regardless of the severity of sepsis-induced acute lung inflammation; and (3) result in less biological damage. To test this hypothesis, we compared the effects of STEPRM with CPAPRM followed by decremental PEEP titration on arterial blood gases, lung mechanics, histology, and expression of interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1), angiopoietin (Ang)-1, Ang-2, and Tie-2 receptor tyrosine kinase in rats with moderate and severe acute lung inflammation induced by sepsis.

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METHODS

Detailed methods are described in the Supplemental Digital Content (http://links.lww.com/AA/B359).

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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 (CEUA-019). In 51 adult male Wistar rats (weight 296 ± 43 g), CLP was performed to trigger sepsis-induced acute lung inflammation.6 Forty-eight hours after surgery, animals were anesthetized by an intraperitoneal injection of ketamine (100 mg/kg) and midazolam (5 mg/kg). The tail vein was cannulated (Jelco 24G; Becton Dickinson, Juiz de Fora, MG, Brazil), and anesthesia was maintained with IV ketamine (50 mg/kg/h) and midazolam (2.5 mg/kg/h). Animals were kept in dorsal recumbency throughout the experiment. After midline neck incision, a polyethylene catheter (PE50) was introduced into the right internal carotid artery for blood sampling and measurement of mean arterial blood pressure (MAP). Heart rate, MAP, and rectal temperature were recorded continuously (Networked Multiparameter Veterinary Monitor LifeWindow 6000V; Digicare Animal Health, Boynton Beach, FL). Body temperature was maintained at 37.5 ± 1°C with a heating blanket. Lactated Ringer’s solution (10 mL/kg/h) was administered via the tail vein to keep MAP > 70 mm Hg. If MAP < 70 mm Hg, a Gelafundin® bolus (0.5 mL; B. Braun Medical SA, Melsungen, Germany) was given through the caudal vein. A 14-G cannula was used for tracheostomy, and a fluid-filled tube was inserted in the esophagus for esophageal pressure (Pes) measurements.

After neuromuscular blockade with vecuronium bromide (2 mg/kg IV), animals were ventilated mechanically (Servo-I; MAQUET, Solna, Sweden) in volume-controlled mode with tidal volume (VT) = 6 mL/kg, respiratory rate (RR) = 80 breaths/min, fraction of inspired oxygen (FIO2) = 1.0, and zero end-expiratory pressure for 5 minutes. After animal stabilization, arterial blood (300 μL) was drawn into a heparinized syringe for the measurement of PaO2, PaCO2, and arterial pH (i-STAT; Abbott Laboratories, Chicago, IL) (baseline). Rats were then divided into 2 groups according to the level of oxygenation: severe (PaO2/FIO2 < 300) or moderate (PaO2/FIO2 ≥ 300), which, in previous pilot studies, has correlated with the degree of diffuse alveolar damage (DAD) observed via histological analysis (Supplemental Digital Content, Supplemental Figure 1, http://links.lww.com/AA/B359). Animals from each group were then randomized into 3 subgroups: (1) nonrecruited (non-RM; moderate, n = 8, and severe, n = 9), in which there was no RM, but animals underwent decremental PEEP titration; (2) CPAP of 30 cm H2O for 30 seconds (CPAPRM; moderate, n = 9, and severe, n = 8) followed by decremental PEEP titration; and (3) stepwise increase in airway pressure (Paw; 5 cm H2O/step, 8.5 seconds per step) over 51 seconds (STEPRM; moderate, n = 10, and severe, n = 7), with maximum pressure sustained for 10 seconds followed by decremental PEEP titration (Fig. 1). STEPRM was designed to achieve a pressure-time product comparable with that of CPAPRM (Fig. 2). STEPRM was achieved by the PCV (pressure controlled ventilation) mode, where Paw was increased by 5 cm H2O every 8.5 seconds to a maximum pressure of 30 cm H2O (total of 6 steps) (Fig. 2).

Figure 1

Figure 1

Figure 2

Figure 2

For PEEP titration, VT was kept constant at 6 mL/kg and RR was reduced to 60 bpm to prevent auto-PEEP. PEEP was reduced gradually (in 2-cm H2O decrements) from 11 cm H2O until the lowest level of lung dynamic elastance (E,L). After we detected the lowest level of E,L, PEEP was then reduced followed by an increase in E,L, forming a “U-shaped” curve. The PEEP level was returned to the point with the lowest E,L, which was called “end-PEEP.” No animals were titrated to 0 cm H2O PEEP. Dynamic lung elastance was calculated by the equation of motion.24 No additional RM was performed.

Figure 3

Figure 3

After PEEP titration, animals were ventilated protectively with VT = 6 mL/kg, RR = 80 bpm, FIO2 = 0.4, and the titrated end-PEEP (for each animal) for 60 minutes. Functional data (electrocardiogram, MAP, transpulmonary pressure, and rectal temperature) were collected immediately after RM application and every 15 minutes for 60 minutes. After 1 hour of mechanical ventilation, FIO2 was set at 1.0 and, after 5 minutes, arterial blood gases were analyzed (end-PEEP). Animals were euthanized and their lungs extracted for histologic and molecular biology analysis (Fig. 3). Further information about this step is in the section to follow.

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Lung Mechanics

Airflow, VT, Paw, and Pes were measured before and after RM and PEEP titration.25,26 Transpulmonary pressure (P, L) was calculated at end-inspiration and end-expiration as the difference between Paw and Pes. Respiratory system peak pressure (PRS, peak), respiratory system mean pressure (PRS, mean), and Paw-time product (PRS × T) were calculated. All signals were filtered (200 Hz), amplified by a 4-channel conditioner (SC-24; SCIREQ, Montreal, Quebec, Canada), and sampled at 200 Hz with a 12-bit analog-to-digital converter (National Instruments, Austin, TX). PEEP selection was done online, and all other mechanical data were computed offline using a routine written in MATLAB (version R2007a; The MathWorks Inc., Natick, MA).25

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Histology

Light Microscopy

At the end of the experimental protocol, a laparotomy was performed and heparin (1000 IU) was injected into the tail vein. The trachea was clamped at end-expiration. Lungs were removed en bloc at the PEEP setting determined during PEEP titration. The abdominal aorta and vena cava were sectioned to quickly kill the animal by exsanguination. The right lower lobe was fixed in 4% buffered formaldehyde solution, and 4-μm thick slices were stained with hematoxylin and eosin. Photomicrographs at magnifications of ×100, ×200, and ×400 were obtained from 4 nonoverlapping fields of view per section with the use of a light microscope. DAD was quantified with a weighted scoring system by an expert in lung pathology blinded to the experimental protocol.27 To summarize in brief, scores of 0 to 4 were used to represent the severity of edema, atelectasis, and hyperinflation, with 0 standing for no effect and 4 for maximum severity. In addition, 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. Hyperinflation was characterized by structures with morphology distinct from that of alveoli and wider than 120 μm.28 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 calculated as the sum of each score characteristic and ranged from 0 to 48.

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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 and fixed (2.5% glutaraldehyde and phosphate buffer 0.1 M, pH = 7.4) for electron microscopy (JEOL 1010 Transmission Electron Microscope, Tokyo, Japan). Each electron microscopic image (20 per animal) was analyzed for damage to alveolar-capillary membrane, type I and type II epithelial cells, and endothelial cells at 3 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.8,26

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Messenger RNA Expression of Inflammation and Endothelial Cell Damage Markers

Real-time reverse transcription-polymerase chain reaction (PCR) was performed as described previously.1 Central slices of each right lung were cut, collected in cryotubes, flash-frozen by immersion in liquid nitrogen, and stored at −80°C. Quantitative real-time reverse transcription-PCR was performed to measure biological markers associated with inflammation (IL-6) and damage inflicted to endothelial cells (ICAM-1, Ang-2, Ang-1, and Tie-2 receptor tyrosine kinase). The primers (Invitrogen, Carlsbad, CA) used for gene amplification are listed in the Supplemental Digital Content (http://links.lww.com/AA/B359). Relative mRNA levels were measured with an SYBR green detection system by use of ABI 7500 real-time PCR (Applied Biosystems, Foster City, CA). Samples were measured in triplicate. For each sample, the expression of each gene was normalized to housekeeping gene expression (acidic ribosomal phosphoprotein P0, 36B4) using the 2−ΔΔCt method, where ΔCt = Ct, reference gene − Ct, target gene. Relative gene expression was expressed as fold change relative to non-RM animals.

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Statistical Analysis

The sample size calculation for testing the hypothesis that STEPRM would induce less biological damage compared with CPAPRM in moderate acute lung inflammation was based on pilot studies. The effect size (2.2) was calculated based on the differences in IL-6 mRNA expressions between groups as compared in a previous study done by our group.29 We calculated that a sample size of 8 animals per group would provide the appropriate power (1 − β = 0.8) to identify significant (α = 0.05) differences in biological damage, considering an effect size d = 2.2, 2-sided tests, and multiple comparisons (Bonferroni correction), 3 tests at the α* = 0.05/3 = 0.0167 level (G*Power 3.1.9.2, University of Düsseldorf, Düsseldorf, Germany). Three was chosen as the number of comparisons because there were 3 groups: non-RM versus CPAPRM, CPAPRM versus STEPRM, and non-RM versus STEPRM.

The normality of data was tested by use of the Kolmogorov-Smirnov test with Lilliefors correction. The homogeneity of variances was tested by means of the Levene median test, with all P values ≥ 0.05. Functional variables were tested with 1-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test (Prism, version 5.0a; GraphPad Software, Inc., La Jolla, CA). The Kruskal-Wallis test followed by the Dunn post hoc test was used to compare DAD scores, electron microscopy findings, and molecular biology data. Multiple linear regression analysis was done between independent (PRS, peak; PRS, mean; PRS × T; total RM time; RM rise angle) and dependent (IL-6, Ang-2, ICAM-1) variables among all groups and after stratification into moderate and severe groups. Data are expressed as mean ± SD or as median and interquartile range, as appropriate. All actual post hoc P values according to the respective statistical analysis are provided, and significance was accepted at P < 0.05. Full statistical data, including all P values from Bonferroni or Kruskal-Wallis tests, Lilliefors correction, and the Levene median test, as well as degrees of freedom, sum of squares, and mean squares for all variables, are provided in Supplemental Table 1 of the Supplemental Digital Content (http://links.lww.com/AA/B359).

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RESULTS

Fifty-eight percent of rats survived for 48 hours after sepsis induction to be included in the study protocol. No animals died during the mechanical ventilation strategies. The fluid volume required to keep MAP > 70 mm Hg did not differ between groups, regardless of the severity of acute lung inflammation (Table 1, crystalloids: 1-way ANOVA, P = 0.9018 for moderate group and P = 0.1595 for severe group; colloids: 1-way ANOVA, P = 0.9029 for moderate group and P = 0.1850 for severe group). As reported in Table 2, during RMs: (1) PRS, peak (moderate, P = 0.2128; severe, P = 0.4199) and PRS × T (moderate, P = 0.4142; severe, P = 0.5767) were comparable in STEPRM and CPAPRM; (2) PRS, mean was lower in STEPRM than that in CPAPRM (moderate, P < 0.0001; severe, P < 0.0001); (3) the total RM time was greater in STEPRM than that in CPAPRM (moderate, P < 0.0001; severe, P < 0.0001); and (4) the RM rise angle was lower in STEPRM than that in CPAPRM (moderate, P < 0.0001; severe, P < 0.0001). The time to achievement of lowest dynamic lung elastance during PEEP titration and the end-PEEP level (median = 5 cm H2O) did not differ among groups (1-way ANOVA: moderate, P = 0.2603; severe, P = 0.0778). Furthermore, the transpulmonary pressure at end-PEEP was similar among all groups, regardless of the severity of acute lung inflammation (1-way ANOVA: moderate, P = 0.4038; severe, P = 0.8644). Table 3 shows the results of blood gas analyses at baseline-zero end-expiratory pressure and end-PEEP. In animals with severe acute lung inflammation, PaO2/FIO2 improved regardless of the type of RM applied (non-RM, P = 0.0003; CPAPRM, P = 0.0008; STEPRM, P = 0.0017).

Table 1

Table 1

Table 2

Table 2

Table 3

Table 3

Histologic evaluation revealed greater edema, atelectasis, and alveolar hyperinflation in the severe compared with moderate acute lung inflammation group (Fig. 4). In moderate and severe acute lung inflammation, STEPRM was associated with lower total DAD score than non-RM (Fig. 4, D and H; moderate, P = 0.0281; severe, P = 0.0221). In severe inflammation only, the CPAPRM group also presented a lower total DAD score compared with non-RM animals (Fig. 4H, P = 0.0231). In moderate acute lung inflammation when compared with non-RM and CPAPRM protocols, STEPRM reduced atelectasis (Fig. 4B, STEPRM versus non-RM, P = 0.0201; STEPRM versus CPAPRM, P = 0.0221), but edema and hyperinflation did not differ among non-RM, CPAPRM, and STEPRM (Fig. 4, A and C, Kruskal-Wallis test: P = 0.5301 for edema and P = 0.1169 for hyperinflation). In severe acute lung inflammation, edema (Fig. 4E) and atelectasis (Fig. 4F) were reduced in CPAPRM (edema, P = 0.0234; atelectasis, P = 0.0035) and STEPRM (edema, P = 0.0217; atelectasis, P = 0.0387) compared with non-RM. STEPRM animals exhibited less hyperinflation compared with CPAPRM and non-RM animals (Fig. 4G, STEPRM versus CPAPRM, P = 0.0057; STEPRM versus non-RM, P = 0.0492).

Figure 4

Figure 4

Ultrastructural lung findings are depicted in Figure 5. All animals exhibited cytoplasmic degeneration of alveolar type I and type II epithelial cells, interstitial and alveolar edema, and endothelial cell injury, regardless of the severity of acute lung inflammation. These alterations were more pronounced in severe compared with the moderate acute lung inflammation group. Endothelial cell damage was less pronounced in STEPRM than that in CPAPRM, both in moderate (P = 0.0218) and in severe (P = 0.0257) acute lung inflammation (Table 4).

Table 4

Table 4

Figure 5

Figure 5

The mRNA expression of IL-6 was comparable among non-RM, CPAPRM, and STEPRM in the moderate acute lung inflammation group (1-way ANOVA, P = 0.8540) but greater in CPAPRM compared with STEPRM (P = 0.038) in the severe acute lung inflammation group (Fig. 6).

Figure 6

Figure 6

The mRNA expression of biological markers associated with lung endothelial cell damage (Ang-1, Ang-2, Tie-2, ICAM-1) is shown in Figure 7. In moderate and severe acute lung inflammation, Ang-2 expression was lower in STEPRM compared with non-RM and CPAPRM (Fig. 7, B and F, moderate: STEPRM versus non-RM, P = 0.0372; STEPRM versus CPAPRM, P = 0.0156; severe: STEPRM versus non-RM, P = 0.0040; STEPRM versus CPAPRM, P = 0.0021). In moderate acute lung inflammation, ICAM-1 expression was lower in STEPRM than that in non-RM (Fig. 7D, P = 0.0011), whereas in severe acute lung inflammation, ICAM-1 levels were lower in STEPRM than that in CPAPRM (Fig. 7H, P = 0.0296). In both moderate and severe acute lung inflammation, no differences in Ang-1 and Tie-2 expressions were observed among non-RM, CPAPRM, and STEPRM (Fig. 7, A, C, E, and G; 1-way ANOVA in moderate group: Ang-1, P = 0.4482; Tie-2, P = 0.5178; 1-way ANOVA in severe group: Ang-1, P = 0.6852; Tie-2, P = 0.4822).

Figure 7

Figure 7

In the severe acute lung inflammation group, the RM rise angle correlated with Ang-2 expression (coefficient = 1.955, P = 0.023).

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DISCUSSION

In the present model of sepsis-induced acute lung inflammation, STEPRM followed by individualized PEEP titration resulted in better lung histologic and biochemical parameters compared with CPAPRM. STEPRM reduced endothelial cell damage and Ang-2 expression regardless of the severity of acute lung inflammation. In addition, in severe acute lung inflammation, the increase in Ang-2 expression correlated with the RM rise angle.

Our results are consistent with a “2-hit” model of lung injury. The initial onset of sepsis can be viewed as the “first hit” to the lungs, and subsequent RM ventilation protocols can be considered the “second hit,” with the potential to impose (or attenuate) further lung damage. The overall effects of RMs on lung injury are unknown, although a large RM might be expected to cause some degree of lung injury. Previous studies suggest that RM application may lead to alveolar edema absorption and a decrease in alveolar collapse.29 In our model, RM was applied even at high oxygenation and low levels of atelectasis, which is not common clinically, but may occur during anesthetic induction.30

We measured mRNA expression of IL-6 because it is associated closely with acute-phase mediators and correlates with the severity of sepsis.31 ICAM-1 was chosen because it is increased in endothelial and inflammatory cell tethering.32 Ang-1 and Ang-2 are peptide ligands that bind the Tie-2 receptor tyrosine kinase and are found primarily on endothelial cells. Ang-1 appears to promote vessel stability33 and may also exert anti-inflammatory effects by signaling the downregulation of surface adhesion molecules and E-selectin.34 Ang-2 is a ligand for the Tie-2 receptor and blocks its phosphorylation, thereby promoting vessel destabilization and inflammation24 and correlates positively with the severity of sepsis.35

Unlike in previous studies that evaluated RM,8,10,36 we titrated PEEP individually in each animal because the percentage of recruitable lung areas and the results of PEEP titration vary widely among patients.37 In addition, lung recruitability affects the efficacy of ventilator strategies, such as adjusted PEEP levels.14,38 Nevertheless, respiratory system mean Paw during PEEP titration, the titrated PEEP values, and oxygenation after 1 hour of mechanical ventilation were similar in non-RM, CPAPRM, and STEPRM, regardless of the severity of acute lung inflammation. The absence of difference in titrated PEEP values may result from 2 factors: (1) the end-inspiration Paw (28 cm H2O) reached at the first level of PEEP titration (11 cm H2O) recruited lung units, even with a short respiratory cycle; and (2) in the CLP model, lungs are highly recruitable, despite the severity of inflammation at baseline without PEEP. Once PEEP was titrated, respiratory system driving pressure did not differ between the moderate and severe acute lung inflammation groups.

Our histologic findings indicate that, in both moderate and severe acute lung inflammation, STEPRM was associated with greater recruitment of lung units (less atelectasis) and lower total DAD scores than in the non-RM group. This finding suggests that the greatest level of PEEP used to begin PEEP titration (11 cm H2O) is not enough to fully open lung units. We also found that, in severe acute lung inflammation, STEPRM resulted in less alveolar hyperinflation compared with CPAPRM, suggesting that slow RM may open alveoli more homogeneously.39 The decrease in hyperinflation with STEPRM also may explain reduced endothelial cell damage and Ang-2 expressions in the STEPRM groups. In moderate acute lung inflammation, lung recruitability may be lower,40 explaining why STEPRM, but not CPAPRM, reduced atelectasis and total DAD score.

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Clinical Implications

RMs are low-cost, feasible interventions to optimize mechanical ventilation in critically ill patients. A slow RM allows functional improvement in lung function with less hemodynamic impairment and endothelial cell damage. The present experimental study supports the use of RM using a stepwise increase in Paw. More clinical studies41 are needed to evaluate the role of stepwise RMs in humans.

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Limitations

The present study has several limitations. First, acute lung inflammation was induced by CLP, which may prevent the generalization of our findings to other forms of ARDS; however, we used this model because it is associated with alveolar and interstitial edema, atelectasis, and alveolar hyperinflation.42 Second, animal stratification was based on a PaO2/FIO2 threshold of 300 mm Hg combined with DAD score. Although this threshold was arbitrary, it allocated approximately 50% of animals to each severity group. Although lower cutoff values (e.g., PaO2/FIO2 = 200 mm Hg or 100 mm Hg) may have been closer to those specified in the Berlin definition,43 few animals would have been enrolled in the very low oxygenation group at these thresholds. Thus, our results cannot be extended to other experimental models of sepsis or directly extrapolated to the clinical scenario. In addition, because our main focus was to investigate the effects of different RMs and PEEP titration, we chose not to include a control group without lung injury. Third, we evaluated the effects of different types of RMs after 1 hour of mechanical ventilation and a period of PEEP titration. Hence, we do not know whether the effects we observed were because of RM application or PEEP titration. Finally, blood gases were analyzed only twice, precluding more detailed assessment of changes in oxygenation.

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CONCLUSIONS

In conclusion, STEPRM compared with CPAPRM reduced levels of biological markers associated with endothelial cell damage and ultrastructural endothelial cell injury. Our experimental data require confirmation in clinical studies before clinicians should consider STEPRM superior to the traditional CPAPRM for improvement of lung function and pulmonary protection in moderate and severe sepsis-induced acute lung inflammation.

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DISCLOSURES

Name: Raquel S. Santos, PhD.

Contribution: This author designed the study, conducted the experiments, coordinated data collection and data quality assurance, analyzed the data, and prepared the manuscript.

Attestation: Raquel S. Santos approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript, and is the archival author.

Name: Lillian Moraes, PhD.

Contribution: This author conducted the experiments, analyzed the data, and prepared the manuscript.

Attestation: Lillian Moraes approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Cynthia S. Samary, PhD.

Contribution: This author conducted the experiments, analyzed the data, and prepared the manuscript.

Attestation: Cynthia S. Samary approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Cíntia L. Santos, PhD.

Contribution: This author conducted the experiments, analyzed the data, and prepared the manuscript.

Attestation: Cíntia L. Santos approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Maíra B. A. Ramos, RRT.

Contribution: This author conducted the experiments, analyzed the data, and prepared the manuscript.

Attestation: Maíra B. A. Ramos approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Ana P. Vasconcellos, RRT.

Contribution: This author conducted the experiments, analyzed the data, and prepared the manuscript.

Attestation: Ana P. Vasconcellos approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Lucas F. Horta, MS.

Contribution: This author conducted the experiments, analyzed the data, and prepared the manuscript.

Attestation: Lucas F. Horta approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Marcelo M. Morales, MD, PhD.

Contribution: This author conducted the experiments, analyzed the data, and prepared the manuscript.

Attestation: Marcelo M. Morales approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Vera L. Capelozzi, MD, PhD.

Contribution: This author conducted the experiments, analyzed the data, and prepared the manuscript.

Attestation: Vera L. Capelozzi approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Cristiane S. N. B. Garcia, PhD.

Contribution: This author designed the study, coordinated data collection and data quality assurance, and prepared the manuscript.

Attestation: Cristiane S. N. B. Garcia approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: John J. Marini, MD.

Contribution: This author helped design the study and prepared the manuscript.

Attestation: John J. Marini approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Marcelo G. de Abreu, MD, PhD.

Contribution: This author designed the study and prepared the manuscript.

Attestation: Marcelo G. de Abreu approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Paolo Pelosi, MD, FERS.

Contribution: This author designed the study, coordinated data collection and data quality assurance, and prepared the manuscript.

Attestation: Paolo Pelosi approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Pedro L. Silva, PhD.

Contribution: This author designed the study, conducted the experiments, coordinated data collection and data quality assurance, and prepared the manuscript.

Attestation: Pedro L. Silva approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Patricia R. M. Rocco, MD, PhD.

Contribution: This author designed the study, coordinated data collection and data quality assurance, and prepared the manuscript.

Attestation: Patricia R. M. Rocco approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

This manuscript was handled by: Avery Tung, MD.

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ACKNOWLEDGMENTS

The authors express their gratitude to Mr. Andre Benedito da Silva for animal care, Mrs. Ana Lucia Neves da Silva for her help with microscopy, Mrs. Moira Elizabeth Schottler and Mr. Filippe Vasconcellos for their assistance in editing the manuscript, and MAQUET for providing technical support.

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