G Model

RESPNB 2482 1–8 ARTICLE IN PRESS

Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

journal homepage: www.elsevier.com/locate/resphysiol

1 Effects of lipopolysaccharide-induced inflammation on initial lung

2 fibrosis during open-lung mechanical ventilation in rats
3 Q1 Joerg Krebs a,∗ , Alexander Kolz a , Charalambos Tsagogiorgas a , Paolo Pelosi b ,
4 Patricia R.M. Rocco c , Thomas Luecke a
a
5 Department of Anaesthesiology and Critical Care Medicine, University Medical Centre Mannheim, Medical Faculty Mannheim of the University of
6 Heidelberg, Theodor-Kutzer Ufer 1-3, 68165 Mannheim, Germany
7 Q2 b Department of Surgical Sciences and Integrated Diagnostics, IRCCS AOU San Martino – IST, University of Genoa, Genoa, Italy
c
8 Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho, s/n,
9 Bloco G-014, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brazil
10

11
24 a r t i c l e i n f o a b s t r a c t
12
13 Article history: This study aimed to assess the impact of pulmonary inflammation on early fibrotic response in rats
14 Accepted 2 April 2015 challenged with increasing doses of lipopolysaccharide (LPS). Twenty-four rats were randomized and
15 Available online xxx infused with three different increasing doses of continuous LPS infusion (n = 8/group) while being ven-
16 tilated with low tidal volumes and open-lung positive end-expiratory pressure. Another eight animals
17 Keywords: served as uninjured control group. Hemodynamics, gas exchange, respiratory system mechanics, lung
18 Acute lung injury
histology, ␣-smooth muscle actin, plasma cytokines, and mRNA expression of cytokines and type I and III
19 ␣-Smooth muscle actin
procollagen in lung tissue were assessed. We found impaired hemodynamics and gas exchange as well
20 Inflammation
21 Lipopolysaccharide
as higher histological lung injury scores and ␣-smooth muscle actin expressions in the medium LPS dose
22 Lung fibrosis compared to control and the lower LPS dose. The highest LPS dose did not cause further aggravation of
23 Open lung ventilation these findings. In all LPS groups type I and III procollagen decreased compared to controls and there was
a negative correlation between type III procollagen-RNA expression and proinflammatory mediators.
© 2015 Published by Elsevier B.V.

25

26

1. Introduction 27

The acute respiratory distress syndrome (ARDS) is characterized Q3 28

Abbreviations: ␣-SMA, ␣-smooth muscle actin; ALI, acute lung injury; ARDS,
acute respiratory distress syndrome; BL, baseline; CINC-1, cytokine-induced neu-
by acute onset of bilateral pulmonary infiltrates and hypoxemia 29

trophil chemoattractant 1; Ecw, chest wall static elastance; EL, lung static elastance; caused by different pulmonary and extrapulmonary diseases (Force 30

ELISA, enzyme-linked immunosorbent assay; Ers, respiratory system static elas- et al., 2012). Histomorphological post-mortem examinations reveal 31
tance; FiO2 , fraction of inspired oxygen; g, gram; h, hour; H2 O, water; HR, heart a distinctive chronological pattern of inflammation and fibrosis 32
rate; I:E, inspiratory/expiratory ratio; IL-1␤, interleukin-1␤; IL 6i, nterleukin 6; i.p.,
(Thille et al., 2013) resulting in an accumulation of intraparenchy- 33
intraperitoneal; Kg, kilogram; LPS, lipopolysaccharide; MAP, mean arterial pressure;
mg, milligram; mRNA, messenger RNA; OL-MV, open lung mechanical ventila- matous collagen (Zapol et al., 1979) in lung tissue. 34

tion; PaCO2 , partial pressure of carbon dioxide; PaO2 , partial pressure of oxygen; In the late stages of the disease, type I collagen (PCI) fibers, which 35
PaO2 /FiO2 , ratio of partial pressure arterial oxygen/fraction of inspired oxygen; PC I, are thick and exhibit cross-linked fibrils, are prevalent (Pelosi and 36
type I procollagen; PC III, type III procollagen; PCR, polymerase chain reaction; PEEP, Negrini, 2008). In the initial phase of lung injury, however, there is 37
positive end expiratory pressure; pH, negative logarithm of the molar concentration
of dissolved hydronium ions; Pinsp, ed-inspiratory plateau pressure; Pmean, mean
a predominance of type III collagen (PCIII) fibers, which are more 38

airway pressure; TGF-␤, transforming growth factor ␤; TNF-␣, tumor necrosis factor flexible and susceptible to breakdown. This is an important obser- 39

␣; UI, uninjured control group; VT , tidal volume. vation, as there appears to be a correlation between the initial 40
∗ Corresponding author. Tel.: +49 6321 69538; fax: +49 6321/968074. detection (Madtes et al., 1998) and amount (Marshall et al., 2000) 41
E-mail addresses: Joerg.Krebs@gmx.de (J. Krebs), of PCIII and ARDS-related mortality. 42
Alexander.Kolz@medma.uni-heidelberg.de (A. Kolz),
Charalambos.Tsagogiorgas@umm.de (C. Tsagogiorgas), ppelosi@hotmail.com
The initial synthesis of PCIII can be modulated experimentally 43

(P. Pelosi), prmrocco@biof.ufrj.br (P.R.M. Rocco), Thomas.Luecke@umm.de through various treatment strategies. Silva et al. found an increase 44

(T. Luecke). of PCIII RNA expression in hypervolemic rats with acute lung injury 45

http://dx.doi.org/10.1016/j.resp.2015.04.003
1569-9048/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Krebs, J., et al., Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during
open-lung mechanical ventilation in rats. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.04.003
 G Model
RESPNB 2482 1–8 ARTICLE IN PRESS
2 J. Krebs et al. / Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx

46 induced by cecal ligation and puncture compared to normovolemic a heating blanket, placed in the supine position, tracheotomized, 107

47 animals (Silva et al., 2010). Other authors reported effects on early intubated with a 14G catheter (Kliniject, KLINIKA Medical GmbH, 108

48 PCIII RNA expression caused by different recruitment maneuvers Usingen, Germany), and mechanically ventilated with a neona- 109

49 to open up atelectatic lung regions (Rzezinski et al., 2009; Santiago tal respirator (Babylog 8000, Dräger, Lübeck, Germany). Initially, 110

50 et al., 2010; Silva et al., 2013), different positive end-expiratory a pressure-controlled mode was used with a PEEP of 2 cm H2 O, an 111

51 pressure (PEEP) levels to keep these atelectatic lung regions open inspiratory/expiratory ratio (I:E) of 1:1 and a fraction of inspired 112

52 during end-expiration (Farias et al., 2005; Passaro et al., 2009), as oxygen (FiO2 ) of 0.5. FiO2 was maintained constant throughout 113

53 well as different applied tidal volumes (de Carvalho et al., 2007; the entire experimental period. End-inspiratory plateau pressure 114

54 Krebs et al., 2010). Finally, the application of assisted compared to (Pinsp) was adjusted to maintain a VT of 6 mL/kg. Respiratory rate 115

55 controlled mechanical ventilation (Saddy et al., 2010) reduced PCIII was adjusted to keep PaCO2 within physiological range. MAP was 116

56 RNA expression in experimental ARDS. kept above 60 mmHg during the experiment using additional fluid 117

57 Therefore, knowing the time course of lung fibrosis from the boluses of balanced electrolyte solution (Deltajonin, Deltaselect 118

58 early to late phases of ARDS, as well as the effects of different ven- GmbH, Munich, Germany) and norepinephrine as needed. Fluid vol- 119

59 tilatory strategies that may affect pulmonary fibrosis, might be a ume, weight gain, and norepinephrine requirements were recorded 120

60 valuable tool to reduce ARDS morbidity and mortality. for the 6-h experimental period. 121

61 In recent studies from our group (Krebs et al., 2010, 2011),

62 reduced expression of PCI and PCIII mRNA was found in rats 2.2. Experimental protocol 122

63 ventilated with open-lung mechanical ventilation (OL-MV), con-

64 sisting of an inflating maneuver followed by individualized titrated After a stabilization period of 15 min, the LPS infusion in the 123

65 PEEP and low tidal volumes. These findings were consistent LPS 0.075, LPS 0.75 and LPS 1.5 groups was started. Measurements 124

66 in uninjured animals and animals challenged with a continu- of hemodynamic, respiratory system mechanics and gas exchange 125

67 ous infusion of either lipopolysaccharide (LPS) (extrapulmonary parameters were obtained (baseline, UI BL and LPS BL), after which 126

68 injury model) or a saline washout model (pulmonary injury all groups were ventilated for 6 consecutive hours using OL-MV 127

69 model) and treated with conventional or high-frequency oscillatory (Krebs et al., 2010, 2011, 2014). Briefly, lungs were inflated with 128

70 ventilation. a continuous positive airway pressure of 25 cmH2 O for 40 s, fol- 129

71 The aim of the present study was to evaluate the effects of lowed by a decremental PEEP titration starting at 10 cmH2 O which 130

72 increasing LPS doses on systemic and pulmonary inflammation and was reduced every 10 min in steps of 2 cmH2 O, until the respira- 131

73 on the early fibrotic response. We hypothesized that, when using tory system static elastance (Ers) no longer decreased while VT 132

74 a lung-protective ventilator strategy, the progressive LPS-induced was kept at 6 mL/kg. “Optimal” PEEP (defined as PEEP at min- 133

75 inflammatory response suppresses the early fibrotic response. imal Ers) was applied throughout the 6-h experimental period 134

after re-inflation. Hemodynamics, respiratory mechanics and the 135

results of a blood gas analysis were noted hourly. At the end 136
76 2. Materials and methods
of the experiment (END), PEEP was again reduced to 2 cmH2 O 137

to ensure comparability to BL. Again, a full set of hemodynam- 138

77 The study was approved by the Institutional Review Board
ics, respiratory mechanics and blood gas measurements were 139
78 for the care of animal subjects of the University of Heidelberg,
taken. Body weight was noted as well. Heparin (1000 IU) was 140
79 Mannheim, Germany. All animals received humane care in com-
then injected intravenously and the lungs were excised. During 141
80 pliance with the “Principles of Laboratory Animal Care” formulated
the procedure, the trachea was clamped at 5 cmH2 O. The right 142
81 by the National Society for Medical Research and the Guide for the
lungs were snap-frozen in nitrogen for mRNA extraction and real- 143
82 Care and Use of Laboratory Animals prepared by the U.S. National
time quantitative polymerase chain reaction (PCR) analysis. The left 144
83 Academy of Sciences.
lungs were immersed in 4% formalin and embedded in paraffin for 145

histology. 146

84 2.1. Animal preparation

2.3. Respiratory system, lung and chest wall mechanics 147

85 A total of 32 male Wistar rats (450–500 g) were housed in

86 standard conditions with food and water ad libitum. Twenty-four Respiratory mechanics were calculated as previously described 148

87 rats were randomized and infused with three different increasing (Krebs et al., 2010, 2011, 2014). Respiratory system static elastance 149

88 doses of continuous Escherichia coli LPS (O55:B5, Sigma–Aldrich, (Ers) was computed as the difference between end-inspiratory and 150

89 Hamburg, Germany) infusion [0.075 (LPS 0.075), 0.75 (LPS 0.75) end-expiratory tracheal pressure divided by VT . Chest wall static 151

90 or 1.5 (LPS 1.5) mg/kg/h] (n = 8/group). All animals in the three elastance (Ecw) was calculated as the difference between end- 152

91 LPS groups were primed with 1 mg/kg LPS intraperitoneally (i.p.) inspiratory and end-expiratory esophageal pressure divided by VT , 153

92 24 h prior to the experiment, as previously described (Krebs et al., and lung static elastance (EL) as Ers–Ecw. All measurements were 154

93 2010). Another eight animals received 1 mL saline i.p. 24 h prior to taken during 3–4 s of end-inspiratory and end-expiratory airway 155

94 the experiment, but no LPS during the experimental period, and occlusion, respectively. 156

95 served as uninjured (UI) control group. Anesthesia was induced by

96 i.p. injection of ketamine hydrochloride (50 mg/kg; Ketanest 10%® , 2.4. Histological examination 157

97 Pfizer, Karlsruhe, Germany) and xylazine (2 mg/kg; Rompun® , Bay-

98 erVital, Leverkusen, Germany) and maintained with ketamine as For histological examination, hematoxylin–eosin stains were 158

99 needed. One femoral artery was cannulated for continuous mea- prepared from 4 ␮m-thick slices of the left lung. Two experienced 159

100 surement of mean arterial pressure (MAP) and heart rate (HR) and investigators blinded to group allocation evaluated 10 random 160

101 intermittent collection of blood samples for blood gas analysis. non-coincident fields of view using a conventional light micro- 161

102 Both femoral veins were cannulated with polyethylene catheter scope at 100× magnification. A well-established (Krebs et al., 2010, 162

103 tubing for maintenance of anesthesia and continuous infusion 2011, 2014) five-point (ranging from 0 to 4) scoring system (see 163

104 of LPS. Esophageal pressure was measured using an appropriate online supplement for further details) was used to describe the 164

105 catheter whose correct positioning was confirmed as previously amount of intra- and extra-alveolar hemorrhage, intra-alveolar 165

106 described (Talmor et al., 2006). Animals were then transferred to edema, inflammatory infiltration of the interalveolar septa and 166

Please cite this article in press as: Krebs, J., et al., Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during
open-lung mechanical ventilation in rats. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.04.003
 G Model
RESPNB 2482 1–8 ARTICLE IN PRESS
J. Krebs et al. / Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx 3

167 airspace, atelectasis and overinflation. Furthermore, the scoring 3. Results 224

168 variables were added and a histological total injury score was
169 calculated. ␣-SMA staining was performed with a primary anti- 3.1. Respiratory function and hemodynamics 225

170 body (Santa Cruz Biotechnology, Santa Cruz, USA) using a standard
171 avidin–biotin complex method. The amount of ␣-SMA expressed in All animals survived the entire 6-h experimental period. At END, 226

172 pulmonary tissue was quantified using a specific camera (Olympus oxygenation was decreased and PaCO2 increased in the LPS 0.75 227

173 Color-View2, Olympus Germany, Hamburg, Germany) and soft- and 1.5 groups compared to UI. Progressive hemodynamic deterio- 228

174 ware (Olympus CellF , Olympus Germany, Hamburg, Germany) and ration (increased heart rate and decreased mean arterial pressure) 229

175 computed as percent of total parenchyma. Again, 10 non-coincident was observed with increasing doses of LPS, despite aggressive fluid 230

176 fields of view were evaluated per slide. therapy and norepinephrine infusions (Table 1). 231

177 2.5. Systemic inflammatory response 3.2. Histological examination 232

178 To assess the systemic inflammatory response, the concen- Histological evaluation revealed a progressive increase in hem- 233
179 trations of TNF-␣, IL-1␤, IL-6 and cytokine-induced neutrophil orrhage, inflammation, atelectasis and total lung injury score in LPS 234
180 chemoattractant 1 (CINC-1) were measured in blood plasma after 0.075, LPS 0.75 and LPS 1.5 compared to UI (Table 2). The amount 235
181 the 6-h experimental period by the enzyme-linked immunosorbent of ␣-SMA expressing tissue was reduced with incremental doses of 236
182 assay (ELISA) technique, following the manufacturer’s instructions LPS from UI to LPS 0.075 to LPS 0.75 (Fig. 1). 237
183 (R&D Systems, Abingdon, UK).

3.3. Systemic inflammatory response 238

184 2.6. Real-time quantitative PCR
The incremental doses of LPS from UI to LPS 0.075 and LPS 0.75 239
185 Total mRNA was extracted separately from the right lung induced a significant increase in blood plasma levels of TNF-␣, IL- 240
186 using the TRIzol method (Invitrogen GmbH, Karlsruhe, Germany) 1␤, IL-6 and CINC-1 (Table 3). 241
187 and reverse-transcribed (Superscript II Reverse Transcriptase,
188 Invitrogen GmbH, Karlsruhe, Germany) according to the manu-
189 facturer’s instructions. Real-time PCR was used for quantitative 3.4. Lung tissue inflammatory and fibrotic response 242
190 measurement of TNF-␣, IL-1␤, IL-6, CINC-1, PCI and PCIII mRNA
191 expression using commercially available primers (TaqManTM gene Expression of proinflammatory mediators in lung parenchyma 243
192 expression assay; Applied Biosystems Applera Deutschland GmbH, due to LPS infusion showed a dose-dependent increase up to 244
193 Darmstadt, Germany: Assay ID: ␤-Actin: Rn00667869 m1, TNF␣ LPS 0.75, while there was no significant difference between LPS 245
194 Rn99999017 m1, IL6 Rn99999011 m1, IL1ß Rn00676330 m1, 0.75 and LPS 1.5 (Fig. 2). Continuous infusion of LPS progres- 246
195 CINC-1: Rn00578225 m1 Col1A1 Rn01463848 m1, Col3A1 sively reduced PCI and PCIII RNA expression (Fig. 3). We found a 247
196 Rn01437681 m1). All samples were run in triplicate, normal- strong positive correlation between PC I and PCIII RNA expressions 248
197 ized to the housekeeping gene ␤-actin and expressed as fold (r2 = 0.902, p = 0.000002), and negative correlations between PCIII 249
198 change relative to unventilated control animals. RNA expression and proinflammatory mediators (TNF-␣ vs. PCIII: 250

r2 = −0.5091, p = 0.00145; IL 6 vs. PCIII: r2 = −0.3672, p = 0.0345; 251

CINC vs. PCIII: r2 = −0.432, p = 0.0138). No significant correlations 252

199 2.7. Statistical analysis
were found for PCI. 253

200 The sample size calculation for testing the primary hypothesis
201 (i.e., gene expression of PCIII in lung tissue would be decreased
202 with LPS 1.5 compared to UI) was based on effect estimates
203 obtained from pilot studies, as well as on previous measurements
204 by our group (mean and dispersion, respectively). Accordingly,
205 we expected that a sample size of 8 animals per group would
206 provide the appropriate power (1−ˇ = 0.8) to identify signifi-
207 cant (˛ = 0.05) differences in PCIII gene expression, considering
208 an effect size d = 2.2, a two-sided test, and multiple comparisons
209 (n = 3) (˛* = 0.0167, Bonferroni-adjusted). The normality of the data
210 and the homogeneity of variances were tested by means of the
211 Shapiro–Wilk test and Levene’s median test, respectively. Both con-
212 ditions were satisfied in all instances for physiological data; thus,
213 one-way ANOVA was used followed by Holm–Sidak’s post hoc test
214 as required.
215 Physiological data are expressed as mean ± standard deviation
216 (SD). Data from lung histology, ELISA and PCR are expressed as
217 median (interquartile range) and were tested using Kruskal–Wallis
Fig. 1. Percentage of ␣-SMA-positive lung parenchyma. Data expressed as individ- Q4
218 followed by Tukey’s post hoc test or one-way ANOVA followed
ual measurements and the median. UI, uninjured lung; LPS 0.075, lung injury with
219 by Holm–Sidak’s post hoc test as appropriate. ␣-SMA expression LPS, 6 h of controlled mechanical ventilation, continuous infusion of 0.075 mg/kg/h
220 is expressed as individual data points and medians. For cor- LPS; LPS 0.75, lung injury with LPS, 6 h of controlled mechanical ventilation, contin-
221 relations, Spearman’s rank correlation test was used. Statistical uous infusion of 0.75 mg/kg/h LPS; LPS 1.5, lung injury with LPS, 6 h of controlled
mechanical ventilation, continuous infusion of 1.5 mg/kg/h LPS. (A) p < 0.05 UI vs.
222 analyses were performed using SigmaPlot 11.0 (Systat Software
LPS 0.075; (B) p < 0.05 UI vs. LPS 0.75; (C) p < 0.05 UI vs. LPS 1.5; (D) p < 0.05 UI LPS
223 GmbH, Erkrath, Germany). The level of significance was set at 0.075 vs. LPS 0.75; (E) p < 0.05 LPS 0.075 vs. LPS 1.5; (F) p < 0.05 UI LPS 0.75 vs. LPS
p < 0.05. 1.5.

Please cite this article in press as: Krebs, J., et al., Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during
open-lung mechanical ventilation in rats. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.04.003
 G Model
RESPNB 2482 1–8 ARTICLE IN PRESS
4 J. Krebs et al. / Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx

Table 1
Physiological data at baseline and at the end of the experiment.

UI BL UI LPS BL LPS 0.075 LPS 0.75 LPS 1.5

(n = 8) (n = 8) (n = 24) (n = 8) (n = 8) (n = 8)

RR (bpm) 100.00 ± 0.0 100.00 ± 0.0 100.00 ± 0.0 101.13 ± 3.0 100.25 ± 0.7 100.75 ± 2.1
Pinsp (cmH2 O) 13.13 ± 2.8 13.6 ± 0.9 10.62 ± 1.3 14.38 ± 2.4 14.13 ± 1.2 13.75 ± 1.1
Pmean (cmH2 O) 6.55 ± 1.2 6.40 ± 0.3 5.63 ± 0.8 7.04 ± 1.3 6.60 ± 0.4 6.51 ± 0.5
PEEP (cmH2 O) 1.99 ± 0.0 1.98 ± 0.0 1.97 ± 0.1 2.25 ± 0.7 1.99 ± 0.0 2.25 ± 0.7
Ers (cmH2 O/mL) 3.28 ± 0.7 3.46 ± 0.3 2.87 ± 0.4 4.06 ± 0.8 3.99 ± 0.4 3.95 ± 0.4
Ecw (cmH2 O/mL) 1.08 ± 0.8 1.21 ± 0.6 1.05 ± 0.2 1.16 ± 0.3 1.18 ± 0.3 1.58 ± 0.3
EL (cmH2 O/mL) 2.46 ± 0.6 2.62 ± 0.3 1.83 ± 0.4 3.00 ± 0.7 3.05 ± 0.3 2.84 ± 0.4
PaO2 /FiO2 423.68 ± 39.6 487.25 ± 25.3 493.96 ± 30.1 437.19 ± 67.8 329.19 ± 88.1B,D 318.38 ± 45.1C,E
PaCO2 (mmHg) 43.9 ± 4.4 43.14 ± 3.6 51.34 ± 5.8 50.06 ± 5.2 57.35 ± 5.8B,D 55.33 ± 5.0C
pH 7.35 ± 0.0 7.36 ± 0.0 7.30 ± 0.0 7.34 ± 0.1 7.27 ± 0.0B 7.30 ± 0.1C
HR (bpm) 205.5 ± 35.1 194.25 ± 15.6 354.00 ± 52.6 210.75 ± 15.7 248.25 ± 15.6B 246.38 ± 54.5C
MAP (mmHg) 84.88 ± 13.5 74.69 ± 5.8 99.90 ± 13.6 66.25 ± 5.8 63.75 ± 6.6B 62.19 ± 5.5C
Weight gain (g) 51.88 ± 16.7 94.69 ± 11.0A 133.5 ± 16.4B,D 131.88 ± 18.4C,E
Norepinephrine (␮g/g bodyweight/min) 0.00 ± 0.0 21.56 ± 45.8 75.91 ± 44.4B 61.99 ± 49.2C

UI BL, uninjury lung at baseline; UI End 0.075, uninjury lung, 6 h of controlled mechanical ventilation; LPS BL, lung injury with LPS, baseline; LPS 0.075, lung injury with
LPS, 6 h of controlled mechanical ventilation, continuous infusion of 0.075 mg/kg/h LPS; LPS 0.75, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous
infusion of 0.75 mg/kg/h LPS; LPS 1.5, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous infusion of 1.5 mg/kg/h LPS.
RR, respiratory rate; Pinsp, end-inspiratory plateau pressure; Pmean, mean airway pressure; PEEP, positive end-expiratory pressure; Ers, respiratory system elastance; Ecw,
chest wall elastance; EL, lung elastance; PaO2 /FiO2 , ratio of partial pressure arterial oxygen/fraction of inspired oxygen; PaCO2 , partial pressure of carbon dioxide; pH, negative
logarithm of the molar concentration of dissolved hydronium ions; HR, heart rate; MAP, mean arterial pressure; weight gain at the end of the experimental period in g;
norepinephrine dosage at the end of the experimental period.
Values are expressed as mean ± standard deviation.
A
p < 0.05 UI End vs. LPS 0.075.
B
p < 0.05 UI End vs. LPS 0.75.
C
p < 0.05 UI End vs. LPS 1.5.
D
p < 0.05 LPS 0.075 vs. LPS 0.75.
E
p < 0.05 LPS 0.075 vs. LPS 1.5.
Q5 F p < 0.05 LPS 0.75 vs. LPS 1.5.

Table 2
Histological lung injury score.

UI LPS 0.075 LPS 0.75 LPS 1.5

A B
Hemorrhage 0.0 (0.0/0.3) 0.4 (0.0/0.8) 0.9 (0.3/1.6) 0.3 (0.0/1.0)C
Inflammation 0.3 (0.0/0.8) 2.8 (2.4/3.3)A 3.8 (3.3/4.0)B,D 3.3 (2.5/3.6)C
Atelectasis 1.5 (0.3/2.5) 2.4 (1.8/3.3)A 2.8 (2/3.5)B 2.4 (2.0/3.3)C
Overinflation 0.6 (0.3/1.1) 1.0 (0.4/1.8) 1.0 (0.3/1.5) 1.0 (0.5/1.3)
Edema 0.5 (0.3/0.8) 1.00 (0.8/1.5)A 1.50 (1.00/1.81)B 1.25 (0.9/1.6)C
Total lung injury score 3.0 (1.5/4.6) 7.8 (6.7/8.6)A 9.8 (8.7/11.3)B,D 8.0 (7.2/9.8)C,F

UI, uninjured lung; LPS 0.075, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous infusion of 0.075 mg/kg/h LPS; LPS 0.75, lung injury with LPS, 6 h
of controlled mechanical ventilation, continuous infusion of 0.75 mg/kg/h LPS; LPS 1.5, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous infusion of
1.5 mg/kg/h LPS.
Values are expressed as median and interquartile ranges.
A
p < 0.05 UI vs. LPS 0.075.
B
p < 0.05 UI vs. LPS 0.75.
C
p < 0.05 UI vs. LPS 1.5.
D
p < 0.05 LPS 0.075 vs. LPS 0.75.
Q6 p < 0.05 LPS 0.075 vs. LPS 1.5.
E
F
p < 0.05 LPS 0.75 vs. LPS 1.5.

Table 3
Systemic inflammatory response.

UI LPS 0.075 LPS 0.75 LPS 1.5

TNF-␣ 0.0 (0.0/0.0) 11.4 (8.9/14.6)A 31.7 (21.8/49.6)B 29.4 (23.5/42.1)C

IL-1␤ 0.0 (0.0/0.0) 31.2 (27.7/40.9)A 24.9 (16.5/53.8)B,D 89.6 (46.0/107.9)C,E,F
IL-6 99.9 (78.6/136.6) 2937.0 (1421.0/4111.0)A 15096.0 (6307.0/18831.3)B,D 5202.0 (4357.5/6389.5)C,F
CINC-1 389.0 (356.9/522.4) 5229.5 (4276.8/7941.8)A 26490.5(15526.3/33194.5)B,D 15790.0 (12884.5/22755.5)C

UI, uninjured lung; LPS 0.075, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous infusion of 0.075 mg/kg/h LPS; LPS 0.75, lung injury with LPS, 6 h
of controlled mechanical ventilation, continuous infusion of 0.75 mg/kg/h LPS; LPS 1.5, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous infusion of
1.5 mg/kg/h LPS.
TNF-␣, tumor necrosis factor-␣; IL, interleukin; CINC-1, cytokine-induced neutrophil chemoattractant 1.
Values are expressed as median and interquartile ranges.
A
p < 0.05 UI vs. LPS 0.075.
B
p < 0.05 UI vs. LPS 0.75.
C
p < 0.05 UI vs. LPS 1.5.
D
p < 0.05 LPS 0.075 vs. LPS 0.75.
E
p < 0.05 LPS 0.075 vs. LPS 1.5.
F
p < 0.05 LPS 0.75 vs. LPS 1.5.

Please cite this article in press as: Krebs, J., et al., Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during
open-lung mechanical ventilation in rats. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.04.003
 G Model
RESPNB 2482 1–8 ARTICLE IN PRESS
J. Krebs et al. / Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx 5

Fig. 2. Pro-inflammatory mRNA expression in lung tissue. Values are normalized to the housekeeping gene ␤-actin and expressed as fold change relative to untreated control
animals.
UI, uninjured lung; LPS 0.075, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous infusion of 0.075 mg/kg/h LPS; LPS 0.75, lung injury with LPS, 6 h
of controlled mechanical ventilation, continuous infusion of 0.75 mg/kg/h LPS; LPS 1.5, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous infusion of
1.5 mg/kg/h LPS. TNF ␣, tumor necrosis factor-␣; CINC-1, cytokine-induced neutrophil chemoattractant 1. Values are expressed as median and interquartile ranges. p-values
are indicated above brackets.

254 4. Discussion Lipopolysaccharide, a glycopeptide from the outer membrane 279

of gram-negative bacteria, is a potent activator of the CD14/TLR4 280

255 This study investigated the effects of three incremental receptor dimer mainly located on monocytes and macrophages 281

256 doses of intravenous LPS on respiratory system mechanics, (Wright et al., 1990), and thus triggers the synthesis and excretion 282

257 gas exchange, hemodynamics, lung histology (including ␣-SMA- of various proinflammatory mediators (Matute-Bello et al., 2008). 283

258 staining), cytokine levels in blood plasma, as well as cytokine and Intravenous infusion (Krebs et al., 2010) and i.p. injection (Menezes 284

259 procollagen (PCI and PCIII) mRNA expression in lung tissue in an in et al., 2005) of LPS induces ALI, which is characterized by neu- 285

260 vivo rat model ventilated with low tidal volume and PEEP titrated trophil accumulation in the alveolar and interstitial space, alveolar 286

261 to the minimal static elastance of the respiratory system. The main wall thickening and proteinaceous edema in the alveolar space. In 287

262 findings were: (1) LPS 0.75 was associated with an impairment humans, the administration of incremental LPS doses results in a 288

263 of hemodynamics, gas exchange, total lung injury score and ␣- progressively severe proinflammatory host response (Vedder et al., 289

264 SMA expression compared to UI and LPS 0.075; (2) systemic and 1999). As to be expected, continuous infusion of LPS in our study 290

265 lung tissue proinflammatory markers increased compared to UI resulted in physiological and histological alterations as well as an 291

266 and LPS 0.075; (3) LPS 1.5 did not cause a consistent aggravation of increase of proinflammatory mediators in blood plasma and lung 292

267 these findings; (4) in all LPS groups, PC I and PC III expressions parenchyma. 293

268 decreased compared to controls; and (5) a negative correlation While increasing the LPS dose from 0.075 to 0.75 mg/kg/h 294

269 was observed between PCIII expression and proinflammatory yielded progressively pronounced parenchymal impairment, fur- 295

270 mediators. ther increasing the LPS dose to 1.5 mg/kg/h did not result in 296

271 The current, well-established two-hit model of extrapulmonary additional injury. To the best of our knowledge, this is the first time 297

272 ALI induced by LPS was chosen because it provides hemodynamic that a ceiling effect of intravenous LPS administration is reported. 298

273 stability throughout the 6-h experimental period (Krebs et al., Therefore, we are neither sure whether these findings can be repro- 299

274 2010). Additionally, low tidal volumes of 6 mL/kg (Force et al., 2012) duced using different experimental settings nor are we able to 300

275 and setting PEEP according to the minimal elastance of the respira- provide a biologically sound explanation for this ceiling effect. 301

276 tory system (Gattinoni et al., 2010) sought to minimize additional Interestingly, in the present ALI model using open-lung 302

277 lung injury imposed by the high mechanical stress caused by non- mechanical ventilation, the mRNA expression of PC I and PC 303

278 protective ventilatory strategies (Makiyama et al., 2014). III decreased significantly following LPS infusion, in contrast to 304

Please cite this article in press as: Krebs, J., et al., Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during
open-lung mechanical ventilation in rats. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.04.003
 G Model
RESPNB 2482 1–8 ARTICLE IN PRESS
6 J. Krebs et al. / Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx

2012). Without the cyclic stretch caused by mechanical ventilation, 333

expression of profibrotic markers was markedly reduced. 334

Previous studies (Krebs et al., 2010, 2011) implied that the appli- 335

cation of an open-lung strategy using a low tidal volume, alveolar 336

inflation maneuver and a PEEP level titrated to minimum elastance 337

of the respiratory system does not trigger an uniform increase in 338

PCIII and PCI RNA expression in uninjured animals or in acute lung 339

injury caused by continuous LPS application. Furthermore, whole 340

genome analysis revealed no significant increase in various other 341

profibrotic parameters, such as TGF-␤ and fibronectin, in uninjured 342

animals ventilated with this strategy (Krebs et al., 2014). In line 343

with this decrease in early PCIII, expression we found a significant 344

reduction of ␣-SMA expression on analysis of lung histology in our 345

ALI model. The expression of ␣-SMA indicates transformation of 346

fibroblasts to myofibroblasts (Synenki et al., 2007), which secrete 347

collagen into the extracellular matrix, induce parenchymal fibrosis 348

(Laurent, 1985; Zapol et al., 1979) and acquire contractile proper- 349

ties, which ultimately leads to impaired tissue compliance (Hinz, 350

2012). In this context, several authors investigated animal models 351

of ALI with concomitant parenchymal fibrosis and longer observa- 352

tion periods (Domenici et al., 2004) and found an increased amount 353

of pulmonary ␣-SMA (Cabrera-Benitez et al., 2012), which corre- 354

lated with reduced lung parenchyma compliance and increased 355

lung tissue collagen content (Keshari et al., 2014). An increase 356

in ␣-SMA-positive myofibroblasts has been previously shown in 357

a bleomycin-induced pulmonary fibrosis model in rats after 24 h 358

(Vyalov et al., 1993). 359

In patients with ARDS, myofibroblasts are widely found in 360

Fig. 3. Profibrotic mRNA expression in lung tissue. Values are normalized to the bronchoalveolar lavage fluid and might be indicative of mortality 361
housekeeping gene ␤-actin and expressed as fold change relative to untreated con- (Synenki et al., 2007). Furthermore, Sandbo et al. (Sandbo et al., 362
trol animals.
UI, uninjured lung; LPS 0.075, lung injury with LPS, 6 h of controlled mechanical ven-
2007) demonstrated downregulation of ␣-SMA expression caused 363

tilation, continuous infusion of 0.075 mg/kg/h LPS; LPS 0.75, lung injury with LPS, by reduced mRNA levels and inhibited SMA promoter activity in 364

6 h of controlled mechanical ventilation, continuous infusion of 0.75 mg/kg/h LPS; vascular smooth muscle cells treated with LPS. Dose-dependent 365
LPS 1.5, lung injury with LPS, 6 h of controlled mechanical ventilation, continuous undulation of collagen synthesis and ␣-SMA expression have also 366
infusion of 1.5 mg/kg/h LPS. PCI, type I collagen; PC III, type III collagen. Values are
been noted in skin fibroblasts (Yang et al., 2013). 367
expressed as median and interquartile ranges. p-values are indicated above bracket.
In short, in our model of LPS-induced lung injury, we observed 368

lung histological impairment and increased inflammatory media- 369

tors in blood plasma and lung parenchyma, as well as a significant 370

305 several previously published studies (Domenici et al., 2004; Keshari reduction in PCI, PCIII and ␣-SMA. 371

306 et al., 2014; Silva et al., 2013), which may suggest that fibrogenesis We previously demonstrated that OL-MV is able to standard- 372

307 is affected not only by the route of LPS administration (intratra- ize lung volume history (Nishida et al., 2004) and homogenize 373

308 cheal, intraperitoneal or intravenous) but also by the mechanical lung parenchyma, preventing atelectasis and overinflation during 374

309 ventilation strategy. Intravenous LPS induces neutrophil accu- 12 h of mechanical ventilation in uninjured lungs (Krebs et al., 375

310 mulation, vascular leakage, impairment of the alveolar-capillary 2011) and over 6 h in an LPS-induced lung injury model (Krebs 376

311 membrane and pulmonary edema (Krebs et al., 2010) and therefore et al., 2010). OL-MV might reduce intraparenchymal stress and 377

312 leads to inhomogeneous density distribution in lung parenchyma strain (Cressoni et al., 2014; Makiyama et al., 2014) and thus mini- 378

313 and intraparenchymal stress. Mechanical ventilation with low mize the iatrogenic profibrotic effects of mechanotransduction due 379

314 tidal volumes and adequate PEEP can reduce end-tidal distention to positive-pressure ventilation. It is likely that the native reac- 380

315 (Terragni et al., 2009) and cyclic alveolar closing and reopening tion of lung parenchyma to LPS stimulation might be antifibrotic 381

316 (Duggan et al., 2003; Farias et al., 2005; Gernoth et al., 2009), thus as well, unless tissue structures are exposed to additional, non- 382

317 minimizing detrimental tissue deformation (Cressoni et al., 2014; physiological mechanical stress. Further studies are required to 383

318 Makiyama et al., 2014) and lung parenchyma remodeling (Copland address these issues. 384

319 et al., 2006; Torday et al., 2003). One might speculate that the venti-
320 lator strategy used in the setting of LPS-induced lung inflammation 4.1. Limitations 385

321 can have a profound influence on the early fibrotic response. This
322 hypothesis is supported by previous studies that demonstrated This study has some limitations that must be addressed. First, 386

323 increased PCIII mRNA expression with high-tidal volume ventila- we used a specific model of lung injury with fixed doses of intra- 387

324 tion compared to more protective strategies (Berg et al., 1997; de venously administered LPS, and cannot exclude the possibility that 388

325 Carvalho et al., 2007). Moreover, the application of adequate PEEP different dosages might have resulted in different effects. Based on 389

326 reduces regional stress and strain, thus preventing end-expiratory preliminary studies using this type of LPS and rat strain, we found 390

327 atelectasis-induction and the increase in PCIII mRNA expression that the highest infusion rate of LPS yielded 100% survival rate over 391

328 in rat models of experimental ALI (Passaro et al., 2009). Recent the experimental period of 6 h was 1.5 mg/kg/h. Since we aimed 392

329 investigations using an in vivo double-hit model combining acid to modulate the inflammatory stimulus, different doses of LPS, 393

330 aspiration and high tidal volume-induced cyclic stretch reported namely 0.75 and 0.075 mg/kg/h, were used in order to investigate 394

331 an increase in hydroxyproline (the most important precursor of whether there would be a dose dependent change in profibrotic 395

332 collagen synthesis), TGF-␤, and ␣-SMA (Cabrera-Benitez et al., response. Second, as the observation period lasted only 6 h, the 396

Please cite this article in press as: Krebs, J., et al., Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during
open-lung mechanical ventilation in rats. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.04.003
 G Model
RESPNB 2482 1–8 ARTICLE IN PRESS
J. Krebs et al. / Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx 7

397 present experiment did not evaluate possible long-term effects. References 452

398 In previous studies we reported a negative correlation between

399 inflammation (represented by IL-6-mRNA-expression) and fibrosis Berg, J.T., Fu, Z., Breen, E.C., Tran, H.C., Mathieu-Costello, O., West, J.B., 1997. High 453
lung inflation increases mRNA levels of ECM components and growth factors in 454
400 (represented by PCIII-mRNA-expression) during open lung venti- lung parenchyma. J. Appl. Physiol. 83, 120–128. 455
401 lation with (Krebs et al., 2010) and without (Krebs et al., 2011) Cabrera-Benitez, N.E., Parotto, M., Post, M., Han, B., Spieth, P.M., Cheng, W.E., Val- 456

402 continuous LPS infusion, while using post hoc analysis. Therefore ladares, F., Villar, J., Liu, M., Sato, M., Zhang, H., Slutsky, A.S., 2012. Mechanical 457
stress induces lung fibrosis by epithelial-mesenchymal transition. Crit. Care 458
403 we planned this confirmatory prospective study to investigate the Med. 40, 510–517. 459
404 relationship between inflammation and fibrosis during the early Copland, I.B., Reynaud, D., Pace-Asciak, C., Post, M., 2006. Mechanotransduction of 460
405 phase of acute lung injury and mechanical ventilation with an stretch-induced prostanoid release by fetal lung epithelial cells. Am. J. Physiol. 461
Lung Cell Mol. Physiol. 291, L487–L495. 462
406 open lung approach during an experimental period of 6 h (Krebs Cressoni, M., Cadringher, P., Chiurazzi, C., Amini, M., Gallazzi, E., Marino, A., Brioni, 463
407 et al., 2010, 2011, 2014) Different results due to longer observa- M., Carlesso, E., Chiumello, D., Quintel, M., Bugedo, G., Gattinoni, L., 2014. Lung 464
408 tion periods, different dosages or route of administration of LPS or inhomogeneity in patients with acute respiratory distress syndrome. Am. J. 465
Respir. Crit. Care Med. 189, 149–158. 466
409 different ventilator strategies remain speculative. Third, different
de Carvalho, M.E., Dolhnikoff, M., Meireles, S.I., Reis, L.F., Martins, M.A., Deheinzelin, 467
410 ventilatory strategies and their effects on fibrotic response were D., 2007. Effects of overinflation on procollagen type III expression in experi- 468
411 not evaluated. Fourth, we could not verify whether all initially dere- mental acute lung injury. Crit. Care 11, R23. 469

412 cruited lung parenchyma was inflated with the distending pressure Domenici, L., Pieri, L., Galle, M.B., Romagnoli, P., Adembri, C., 2004. Evolution of 470
endotoxin-induced lung injury in the rat beyond the acute phase. Pathobiology 471
413 of 25 cmH2 O during the inflation maneuver, since no morphologi- 71, 59–69. 472
414 cal analysis with computed tomography was performed. We opted Duggan, M., McCaul, C.L., McNamara, P.J., Engelberts, D., Ackerley, C., Kavanagh, 473

415 to use open lung ventilation with the same ventilator protocol as B.P., 2003. Atelectasis causes vascular leak and lethal right ventricular failure 474
in uninjured rat lungs. Am. J. Respir. Crit. Care Med. 167, 1633–1640. 475
416 described in previous studies (Krebs et al., 2010, 2011, 2014). Farias, L.L., Faffe, D.S., Xisto, D.G., Santana, M.C., Lassance, R., Prota, L.F., Amato, M.B., 476
417 Since we aimed to compare our data with these previously Morales, M.M., Zin, W.A., Rocco, P.R., 2005. Positive end-expiratory pressure 477

418 published investigations, in the present study we adopted a sim- prevents lung mechanical stress caused by recruitment/derecruitment. J. Appl. 478
Physiol. 98, 53–61. 479
419 ilar protocol. Additionally, we showed (Krebs et al., 2010, 2011, Force, A.D.T., Ranieri, V.M., Rubenfeld, G.D., Thompson, B.T., Ferguson, N.D., Caldwell, 480
420 2014) that the amount of atelectasis was not relevant based on his- E., Fan, E., Camporota, L., Slutsky, A.S., 2012. Acute respiratory distress syndrome: 481
421 tological examination. We may only speculate whether a higher the Berlin Definition. JAMA 307, 2526–2533. 482
Gattinoni, L., Carlesso, E., Brazzi, L., Caironi, P., 2010. Positive end-expiratory pres- 483
422 distending pressure results in even less atelectasis. sure. Curr. Opin. Crit. Care 16, 39–44. 484
423 Fifth, any generalization regarding the inverse correlation of Gernoth, C., Wagner, G., Pelosi, P., Luecke, T., 2009. Respiratory and haemodynamic 485
424 inflammation and fibrosis in the initial course of acute lung changes during decremental open lung positive end-expiratory pressure titra- 486
tion in patients with acute respiratory distress syndrome. Crit. Care 13, R59. 487
425 injury would be inappropriate. Finally, we did not investigate
Hinz, B., 2012. Mechanical aspects of lung fibrosis: a spotlight on the myofibroblast. 488
426 the concrete mechanisms resulting in the reduced expression of Proc. Am. Thorac. Soc. 9, 137–147. 489
427 ␣-SMA and did not conduct any selective analysis of ␣-SMA in Keshari, R.S., Silasi-Mansat, R., Zhu, H., Popescu, N.I., Peer, G., Chaaban, H., Lambris, 490

428 fibroblasts/myofibroblasts, but rather in whole lung homogenates. J.D., Polf, H., Lupu, C., Kinasewitz, G., Lupu, F., 2014. Acute lung injury and fibrosis 491
in a baboon model of Escherichia coli sepsis. Am. J. Respir. Cell Mol. Biol. 50, 492
429 Therefore, a distinctive impairment of myofibroblast differentia- 439–450. 493
430 tion cannot be inferred at this point. Krebs, J., Pelosi, P., Tsagogiorgas, C., Zoeller, L., Rocco, P.R., Yard, B., Luecke, T., 2010. 494
Open lung approach associated with high-frequency oscillatory or low tidal vol- 495
ume mechanical ventilation improves respiratory function and minimizes lung 496
431 5. Conclusions injury in healthy and injured rats. Crit. Care 14, R183. 497
Krebs, J., Pelosi, P., Tsagogiorgas, C., Haas, J., Yard, B., Rocco, P.R., Luecke, T., 2011. 498
Time course of lung inflammatory and fibrogenic responses during protective 499
432 In the present model of ALI in rats ventilated with a protective
mechanical ventilation in healthy rats. Respir. Physiol. Neurobiol. 178, 323–328. 500
433 mechanical strategy, continuous infusion of LPS up to 0.75 mg/kg/h Krebs, J., Tsagogiorgas, C., Pelosi, P., Rocco, P.R., Hottenrott, M., Sticht, C., Yard, B., 501
434 induced a dose-dependent increase in inflammatory mediators in Luecke, T., 2014. Open lung approach with low tidal volume mechanical venti- 502
lation attenuates lung injury in rats with massive brain damage. Crit. Care 18, 503
435 blood and lung tissue, with a decrease in fibrogenic response in the
R59. 504
436 lung parenchyma. Laurent, G.J., 1985. Biochemical pathways leading to collagen deposition in pul- 505
monary fibrosis. Ciba Found. Symp. 114, 222–233. 506
Madtes, D.K., Rubenfeld, G., Klima, L.D., Milberg, J.A., Steinberg, K.P., Martin, T.R., 507
437 Author contributions Raghu, G., Hudson, L.D., Clark, J.G., 1998. Elevated transforming growth factor- 508
alpha levels in bronchoalveolar lavage fluid of patients with acute respiratory 509
438 J.K., C.T., P.P., P.R.M.R. and T.L. participated in the study design. distress syndrome. Am. J. Respir. Crit. Care Med. 158, 424–430. 510
Makiyama, A.M., Gibson, L.J., Harris, R.S., Venegas, J.G., 2014. Stress concentration 511
439 J.K., A.K. and T.L. performed the study. J.K., C.T., A.K. and T.L. around an atelectatic region: a finite element model. Respir. Physiol. Neurobiol. 512
440 processed the data and performed the statistical analysis. J.K., 201, 101–110. 513
441 P.P., P.R.M.R. and T.L. wrote the manuscript. All authors read and Marshall, R.P., Bellingan, G., Webb, S., Puddicombe, A., Goldsack, N., McAnulty, R.J., 514
Laurent, G.J., 2000. Fibroproliferation occurs early in the acute respiratory dis- 515
442 approved the final manuscript.
tress syndrome and impacts on outcome. Am. J. Respir. Crit. Care Med. 162, 516
1783–1788. 517

443 Competing interests Matute-Bello, G., Frevert, C.W., Martin, T.R., 2008. Animal models of acute lung 518
injury. Am. J. Physiol. Lung Cell Mol. Physiol. 295, L379–L399. 519
Menezes, S.L., Bozza, P.T., Neto, H.C., Laranjeira, A.P., Negri, E.M., Capelozzi, V.L., 520
444 The authors declare that they have no competing interests. This Zin, W.A., Rocco, P.R., 2005. Pulmonary and extrapulmonary acute lung injury: 521
445 study was supported by departmental funds. inflammatory and ultrastructural analyses. J. Appl. Physiol. 98, 1777–1783. 522
Nishida, T., Suchodolski, K., Schettino, G.P., Sedeek, K., Takeuch, M., Kacmarek, R.M., 523
2004. Peak volume history and peak pressure-volume curve pressures indepen- 524
446 Acknowledgement dently affect the shape of the pressure-volume curve of the respiratory system. 525
Crit. Care Med. 32, 1358–1364. 526
Passaro, C.P., Silva, P.L., Rzezinski, A.F., Abrantes, S., Santiago, V.R., Nardelli, L., Santos, 527
447 The authors thank Mrs. Jutta Schulte for her invaluable help in R.S., Barbosa, C.M., Morales, M.M., Zin, W.A., Amato, M.B., Capelozzi, V.L., Pelosi, 528
448 performing ELISA and TaqMan procedures. P., Rocco, P.R., 2009. Pulmonary lesion induced by low and high positive end- 529
expiratory pressure levels during protective ventilation in experimental acute 530
lung injury. Crit. Care Med. 37, 1011–1017. 531
449 Appendix A. Supplementary data Pelosi, P., Negrini, D., 2008. Extracellular matrix and mechanical ventilation in 532
healthy lungs: back to baro/volotrauma? Curr. Opin. Crit. Care 14, 16–21. 533
Rzezinski, A.F., Oliveira, G.P., Santiago, V.R., Santos, R.S., Ornellas, D.S., Morales, 534
450 Supplementary data associated with this article can be found, in
M.M., Capelozzi, V.L., Amato, M.B., Conde, M.B., Pelosi, P., Rocco, P.R., 535
451 the online version, at http://dx.doi.org/10.1016/j.resp.2015.04.003. 2009. Prolonged recruitment manoeuvre improves lung function with less 536

Please cite this article in press as: Krebs, J., et al., Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during
open-lung mechanical ventilation in rats. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.04.003
 G Model
RESPNB 2482 1–8 ARTICLE IN PRESS
8 J. Krebs et al. / Respiratory Physiology & Neurobiology xxx (2015) xxx–xxx

537 ultrastructural damage in experimental mild acute lung injury. Respir. Physiol. Terragni, P.P., Del Sorbo, L., Mascia, L., Urbino, R., Martin, E.L., Birocco, A., Faggiano, C., 565
538 Neurobiol. 169, 271–281. Quintel, M., Gattinoni, L., Ranieri, V.M., 2009. Tidal volume lower than 6 mL/kg 566
539 Saddy, F., Oliveira, G.P., Garcia, C.S., Nardelli, L.M., Rzezinski, A.F., Ornellas, D.S., enhances lung protection: role of extracorporeal carbon dioxide removal. Anes- 567
540 Morales, M.M., Capelozzi, V.L., Pelosi, P., Rocco, P.R., 2010. Assisted ventilation thesiology 111, 826–835. 568
541 modes reduce the expression of lung inflammatory and fibrogenic mediators in Thille, A.W., Esteban, A., Fernandez-Segoviano, P., Rodriguez, J.M., Aramburu, J.A., 569
542 a model of mild acute lung injury. Intensive Care Med. 36, 1417–1426. Vargas-Errazuriz, P., Martin-Pellicer, A., Lorente, J.A., Frutos-Vivar, F., 2013. 570
543 Sandbo, N., Taurin, S., Yau, D.M., Kregel, S., Mitchell, R., Dulin, N.O., 2007. Downregu- Chronology of histological lesions in acute respiratory distress syndrome with 571
544 lation of smooth muscle alpha-actin expression by bacterial lipopolysaccharide. diffuse alveolar damage: a prospective cohort study of clinical autopsies. Lancet 572
545 Cardiovasc. Res. 74, 262–269. Respir. Med. 1, 395–401. 573
546 Santiago, V.R., Rzezinski, A.F., Nardelli, L.M., Silva, J.D., Garcia, C.S., Maron-Gutierrez, Torday, J.S., Torres, E., Rehan, V.K., 2003. The role of fibroblast transdifferentiation 574
547 T., Ornellas, D.S., Morales, M.M., Capelozzi, V.L., Marini, J., Pelosi, P., Rocco, P.R., in lung epithelial cell proliferation, differentiation, and repair in vitro. Pediatr. 575
548 2010. Recruitment maneuver in experimental acute lung injury: the role of Pathol. Mol. Med. 22, 189–207. 576
549 alveolar collapse and edema. Crit. Care Med. 38, 2207–2214. Vedder, H., Schreiber, W., Yassouridis, A., Gudewill, S., Galanos, C., Pollmacher, T., 577
550 Silva, P.L., Cruz, F.F., Fujisaki, L.C., Oliveira, G.P., Samary, C.S., Ornellas, D.S., Maron- 1999. Dose-dependence of bacterial lipopolysaccharide (LPS) effects on peak 578
551 Gutierrez, T., Rocha, N.N., Goldenberg, R., Garcia, C.S., Morales, M.M., Capelozzi, response and time course of the immune-endocrine host response in humans. 579
552 V.L., Gama de Abreu, M., Pelosi, P., Rocco, P.R., 2010. Hypervolemia induces Inflamm. Res. 48, 67–74. 580
553 and potentiates lung damage after recruitment maneuver in a model of sepsis- Vyalov, S.L., Gabbiani, G., Kapanci, Y., 1993. Rat alveolar myofibroblasts acquire 581
554 induced acute lung injury. Crit. Care 14, R114. alpha-smooth muscle actin expression during bleomycin-induced pulmonary 582
555 Silva, P.L., Moraes, L., Santos, R.S., Samary, C., Ramos, M.B., Santos, C.L., Morales, fibrosis. Am. J. Pathol. 143, 1754–1765. 583
556 M.M., Capelozzi, V.L., Garcia, C.S., de Abreu, M.G., Pelosi, P., Marini, J.J., Rocco, P.R., Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J., Mathison, J.C., 1990. CD14, a 584
557 2013. Recruitment maneuvers modulate epithelial and endothelial cell response receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. 585
558 according to acute lung injury etiology. Crit. Care Med. 41, e256–e265. Science 249, 1431–1433. 586
559 Synenki, L., Chandel, N.S., Budinger, G.R., Donnelly, H.K., Topin, J., Eisenbart, J., Yang, H., Hu, C., Li, F., Liang, L., Liu, L., 2013. Effect of lipopolysaccharide on the 587
560 Jovanovic, B., Jain, M., 2007. Bronchoalveolar lavage fluid from patients with biological characteristics of human skin fibroblasts and hypertrophic scar tissue 588
561 acute lung injury/acute respiratory distress syndrome induces myofibroblast formation. IUBMB Life 65, 526–532. 589
562 differentiation. Crit. Care Med. 35, 842–848. Zapol, W.M., Trelstad, R.L., Coffey, J.W., Tsai, I., Salvador, R.A., 1979. Pul- 590
563 Talmor, D., Sarge, T., O’Donnell, C.R., Ritz, R., Malhotra, A., Lisbon, A., Loring, S.H., monary fibrosis in severe acute respiratory failure. Am. Rev. Respir. Dis. 119, 591
564 2006. Esophageal and transpulmonary pressures in acute respiratory failure. 547–554. 592
Crit. Care Med. 34, 1389–1394.

Please cite this article in press as: Krebs, J., et al., Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during
open-lung mechanical ventilation in rats. Respir. Physiol. Neurobiol. (2015), http://dx.doi.org/10.1016/j.resp.2015.04.003