Influence of mechanical ventilation and sepsis on redox balance in diaphragm, myocardium, limb muscles, and lungs.

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ORIGINAL ARTICLE Influence of mechanical ventilation and sepsis on redox balance in diaphragm, myocardium, limb muscles, and lungs Q16

ALBA CHACON-CABRERA, YENY ROJAS, LETICIA MARTINEZ-CARO, MONICA VILA-UBACH, ESTEBAN, JOSE A. LORENTE1, and NICOLAS NIN, ANTONIO FERRUELO, ANDRES 1 ESTHER BARREIRO BARCELONA AND MADRID, SPAIN

Mechanical ventilation (MV), using high tidal volumes (VT), causes lung (ventilatorinduced lung injury [VILI]) and distant organ injury. Additionally, sepsis is characterized by increased oxidative stress. We tested whether MV is associated with enhanced oxidative stress in sepsis, the commonest underlying condition in clinical acute lung injury. Protein carbonylation and nitration, antioxidants, and inflammation (immunoblotting) were evaluated in diaphragm, gastrocnemius, soleus, myocardium, and lungs of nonseptic and septic (cecal ligation and puncture 24 hours before MV) rats undergoing MV (n 5 7 per group) for 150 minutes using 3 different strategies (low VT [VT 5 9 mL/kg], moderate VT [VT 5 15 mL/kg], and high VT [VT 5 25 mL/kg]) and in nonventilated control animals. Compared with nonventilated control animals, in septic and nonseptic rodents (1) diaphragms, limb muscles, and myocardium of high-VT rats exhibited a decrease in protein oxidation and nitration levels, (2) antioxidant levels followed a specific fiber-type distribution in slowand fast-twitch muscles, (3) tumor necrosis factor a (TNF-a) levels were higher in respiratory and limb muscles, whereas no differences were observed in myocardium, and (4) in lungs, protein oxidation was increased, antioxidants were rather decreased, and TNF-a remained unmodified. In this model of VILI, oxidative stress does not occur in distant organs or skeletal muscles of rodents after several hours of MV with moderate-to-high VT, whereas protein oxidation levels were increased in the lungs of the animals. Inflammatory events were moderately expressed in skeletal muscles and lungs of the MV rats. Concomitant sepsis did not strongly affect the MV-induced effects on muscles, myocardium, or lungs in the rodents. (Translational Research 2014;-:1–19) Abbreviations: --- ¼ ---

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Jose A. Lorente and Esther Barreiro share the senior position.

From the Pulmonology Department-Muscle and Respiratory System Research Unit, Institut Municipal d’Investigacio Medica (IMIM)-Hospital del Mar, Parc de Recerca Biomedica de Barcelona (PRBB), Barcelona, Spain; Health and Experimental Sciences Department (CEXS), Universitat Pompeu Fabra, PRBB, Barcelona, Spain; Centro de Investigacion en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Madrid, Spain; Servicio de Medicina Intensiva, Fundacion para la Investigacion Biomedica, Hospital Universitario de Getafe, Getafe, Madrid, Spain; Servicio de Medicina Intensiva, Hospital de Torrejon, Torrejon de Ardoz, Madrid, Spain; Universidad Europea de Madrid, Madrid, Spain.

Submitted for publication January 14, 2014; revision submitted July 8, 2014; accepted for publication July 9, 2014. Reprint requests: Esther Barreiro, Pulmonology Department-Muscle and Respiratory System Research Unit, IMIM-Hospital del Mar, PRBB, C/Dr Aiguader, 88, E-08003 Barcelona, Spain; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.07.003

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Translational Research - 2014

Chacon-Cabrera et al

AT A GLANCE COMMENTARY Chacon-Cabrera A, et al. Background

Mechanical ventilation (MV), using high tidal volumes, causes lung (ventilator-induced lung injury) and distant organ injury. Additionally, sepsis is characterized by increased oxidative stress. MV may be associated with enhanced oxidative stress in sepsis, the commonest underlying condition in clinical acute lung injury. Translational Significance

Medium-term changes in redox balance induced by high-tidal volume MV were characterized by increased oxidative stress in the lungs, whereas causing a decline of these phenomena in respiratory and limb muscles, and myocardium. Inflammatory events were moderately expressed in skeletal muscles and lungs of the MV rats. Concomitant sepsis did not strongly affect the MVinduced effects on muscles, myocardium, or lungs in the rodents.

INTRODUCTION

In intensive care units (ICU), acute lung injury (ALI) and acute respiratory distress syndrome are common conditions that cause death in patients as a result of shock and multiorgan failure.1 In these patients, mechanical ventilation (MV) is a lifesaving strategy that may entail serious complications in lungs such as alveolar disruption and pulmonary edema,2 a process known as ventilator-induced lung injury (VILI).3 Several cellular and molecular mechanisms have been involved in the development and perpetuation of VILI.4 Inflammatory cells and proinflammatory cytokines were shown to participate in the pathophysiology of VILI.5,6 In the lining fluid of mechanically ventilated lungs, antioxidant activity was decreased,7 whereas malondialdehyde levels were increased.8 Levels of nitrosative stress were also shown to be increased in the lungs of patients with ALI.9 Moreover, in another study,10 it was also reported that increased levels of protein oxidation and inflammation took place in lungs from rats exposed to high-tidal volume (VT) MV. Recently, caveolin-1 was shown to play a key role in the inflammatory events and endothelial hyperpermeability taking place in the lungs during MV.11 In several animal models of VILI,10,12-18 high-VT MV of short duration was also shown to induce injury in distant organs other than the lungs. In a recent

investigation from our group,10 the diaphragm and both slow- and fast-twitch limb muscles exhibited a significant decline in oxidative stress markers and low levels of intramuscular inflammatory cell infiltration in response to high-VT MV of very short duration (1 hour). We concluded from that study that perfusion of organs and muscles in the high-VT rats was likely to be reduced as a result of the hypotension exhibited by the rodents after the 1-hour period of MV.10 However, in that study,10 we used a rather high VT (35 mL/kg) for a very short period of time (1 hour). Whether MV using lower VT (more clinically relevant) for longer periods of time and in a more clinically realistic context (ie, sepsis, as the most common risk factor for ALI) induces similar molecular events in muscles and organs other than the lungs remains to be answered. Patients with respiratory failure often require MV in the context of impaired gas exchange and muscle dysfunction.19,20 On the other hand, ICU-acquired weakness has been shown to be a serious complication leading to prolonged MV and a long stay in the ICU.19-23 On the basis of this, the study of the effects of MV on muscle status is of great clinical relevance. Several factors such as hypoxemia, increased vascular resistance, acidosis, enhanced energy requirements, inflammation, and oxidative stress are proposed contributors to peripheral and respiratory muscle contractile dysfunction in septic patients,24 and in animal models of sepsis and endotoxemia.25-28 Whether MV per se may influence the underlying cellular and molecular events of poor muscle condition in models of sepsis has not been identified so far. In the present study, we hypothesized that mediumterm molecular events such as oxidative stress and inflammation may develop in the lungs and distant organs including respiratory and limb muscles of rats with and without sepsis exposed to several levels of MV. The model of VILI used in the present investigation has already been used in previous studies.6,10,12-18,29,30 Accordingly, our objectives were to specifically explore the medium-term molecular events involving oxidative stress, antioxidant mechanisms, and inflammation in the diaphragms, limb muscles (fast- and slow-twitch types), myocardium, and lungs of healthy rats exposed to several degrees of MV for 150 minutes. The influence of 24 hours of sepsis on the same muscles and organs was also evaluated in rodents exposed to identical MV strategies. MATERIALS AND METHODS

See the online data supplement for additional methodological details regarding animal experiments, molecular biology and statistical analyses.

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The studies on animal models were conducted at the Hospital Universitario de Getafe (Madrid, Spain). This study has been designed in accordance with the ethical regulations on animal experimentation (EU 2010/63 CEE, Real Decreto 53/2013 BOE 34, Spain) at the Hospital Universitario de Getafe and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (1986). The study conforms to the current international ethical guidelines for animal research. All animal experiments were approved by the Animal Research Committee at the Hospital Universitario de Getafe. Animal models of MV with and without sepsis. Pathogen-free healthy male Sprague-Dawley rats (Harlan Iberica, Spain, weight 370–380 g, n 5 7 per group) were used for the purpose of the investigation. Sepsis was induced by cecal ligation and puncture as previously described.31 Briefly, animals were anesthetized before surgery with intraperitoneal boluses of diazepam (5 mg/kg) and ketamine (90 mg/kg) as needed. The surgical procedure was performed under sterile conditions. A midline laparotomy incision was made and the cecum was exposed and ligated at 1.0–1.5 cm distal to the ileocecal valve. The ileocecal valve was also ligated. The cecum was then punctured once (16 ga) distal and 3 times (14 ga) proximal to the ligature site. Gentle pressure was applied to the cecum until the intraluminal content was exteriorized. Then, the bowel was returned to the peritoneal cavity and levofloxacin (7 mg/kg intraperitoneally) was administered. Finally, the midline laparotomy was closed in 2 layers. Sham-operated animals underwent laparotomy and bowel manipulation, but no puncture was done. Right after closure of the laparotomy, 0.9% saline solution (50 mL/kg subcutaneously ) was administered. Animals were left in their cages with water ad libitum for 24 hours before MV. After an equilibration period of 30 minutes using MV (VT 5 9 mL/kg), rats were exposed to the following MV protocols for 150 minutes in both sepsis and nonsepsis models: (1) nonventilated control group; (2) low-VT group (VT 5 9 mL/kg 1 PEEP 5 5 cm H2O); (3) moderate-VT group (VT 5 15 mL/kg 1 PEEP 5 5 cm H2O); and (4) high-VT group (VT 5 25 mL/kg 1 PEEP 5 0 cm H2O). Therefore, a total number of 8 groups of rats were used for the purpose of the present study. In both experimental models, respiratory parameters were set as follows: respiratory rate, 70 bpm; inspiratory time, 0.3 seconds; expiratory time, 0.56 seconds; and inspired fraction of oxygen, 0.45. The model of VILI used in the present investigation has been previously demonstrated to be an appropriate experimental model for this purpose.1-10 Animals.

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Unlike previous investigations10,16-18 in which a more aggressive model of lung injury was used (ie, VT 35 mL/kg for 60 minutes), in the present study, we chose to apply VT 25 mL/kg for 150 minutes to the rodents to make the model more clinically relevant (eg, inducing injury by the use of a VT more similar to that used in patients). This model features an inflammatory pulmonary response induced by MV. In rats undergoing VILI using this ventilatory strategy, different metabolomic patterns and signs of lung injury (increased peak inspiratory pressure, hypoxemia, hypotension, signs of diffuse alveolar damage at histologic examination) were demonstrated.32 In addition, this model also induces changes in several cytokines measured in the circulation or in lung tissue (unpublished data). The ven- Q8 tilatory pattern used in the different groups aimed to represent extremes of lung injury, from no or minimal injury using low VT with a low level of PEEP (VT 9 mL/kg, PEEP 5 15 cm H2O) to severe injury using large VT with no PEEP (VT 25 mL/kg, PEEP 5 0 cm H2O). We arbitrarily chose to use some level of PEEP in the intermediate group (VT 15 mL/kg, PEEP 5 15 cm H2O). As PaCO2 is expected to differ in the different groups, dead-space ventilation (the air volume of the airway and the ventilatory system not participating in gas exchange) was increased in animals ventilated with high VT by increasing the length of the ventilatory circuit to attain comparable values of PaCO2 during the ventilation period. The objective to keep PaCO2 constant was thought to be preferable to changing the respiratory rate, as respiratory rate per se may be a determinant of lung injury. Monitoring of animals. In each group of ventilated rats, lung mechanics (dynamic respiratory system compliance and peak airway pressure), hemodynamics (blood pressure), and circulatory conditions (blood gases, pH, and lactate) were measured both at baseline t 5 0 immediately after the equilibration period at 30 minutes and at t 5 150 minutes right after 150 minutes of MV. In ventilated animals, respiratory drive was abolished by the high respiratory rate set in the ventilator as no spontaneous breaths were observed. Body temperature was maintained during the entire duration of the experiment by placing the animals over a thermopad (Challoner Marketing Ltd, Buckinghamshire England, UK). Tissue sampling. At the end of the experimental period (150 minutes), animals were sacrificed by exsanguination and the following muscles and organs were removed in all animals: diaphragm, gastrocnemius (fast-twitch muscle), soleus (slow-twitch muscle), myocardium, and lungs. Muscle and organ specimens were immediately frozen in liquid nitrogen and subsequently stored at 280 C. Frozen tissues were further used for


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the quantification of redox markers and inflammation using immunoblotting techniques. Biological studies. All biological experiments were performed in the same laboratory at IMIM-Hospital del Mar (Barcelona, Spain). The effects of reactive oxygen and nitrogen species on muscle proteins were evaluated according to methodologies published elsewhere.24,26-28,33-37 Statistical analysis. This is a study designed on the basis of a 2-tail hypothesis. Clinical and physiological data are presented as the mean (standard deviation). Biological data are presented as box and whisker plots in the figures. Statistical analyses were performed using the Statistical Package for the Social Sciences (Portable SPSS, PASW Statistics 18.0 version for windows; SPSS Inc, Chicago, IL). MV-induced changes in clinical and physiological variables were explored using the paired Student t test compared with baseline (t 5 0 and 30 minutes) in the 3 groups of rats mechanically ventilated in both experimental models (sepsis and nonsepsis). In each experimental model, the Kruskal-Wallis test was used to examine potential significant differences among the 4 study groups of rats (control group, 9, 15, and 25 mL) for each biological variable and tissue. Furthermore, nonparametric Mann-Whitney test was also used to specifically explore significant differences in the study of biological variables between each group of mechanically ventilated rats and the control animals or rats ventilated with the lowest VT (9 mL). The sample size was based on previous studies3,6,10,11,16-18,27,29,30,38 and on assumptions of 80% power to detect differences of more than 20% in measured outcomes at a level of significance of P , 0.05. In all the biological variables involving the study of redox balance (total reactive carbonyls, malondialdehyde-protein adducts, protein tyrosine nitration, Mn-superoxide dismutase [SOD], CuZnSOD, and catalase), mean difference between groups was estimated at a minimum of 25% and standard deviation was approximately 25%–30% of the mean value for each of the variables. RESULTS Physiological effects of MV in rats with and without sepsis. In both models (sepsis and nonsepsis), mechan-

ically ventilated rats exposed to 9, 15, and 25 mL/kg for 2.5 hours compared with baseline exhibited a significant increase in peak airway pressure, whereas the dynamic compliance of the respiratory system decreased in those animals (Table I). After MV, arterial pressure was significantly reduced in all groups of rats in both experimental models (Table I). Levels of pH were not significantly modified by any of the MV strategies in either model (Table I). Compared with baseline, blood levels of lactate significantly decreased in rodents

exposed to VT 5 9 mL/kg and VT 5 15 mL/kg, but not to VT 5 25 mL/kg, after MV (Table I). Levels of arterial partial pressure of oxygen and carbon dioxide were not significantly modified by any of the MV strategies in either model (Table I). Biological markers in skeletal muscles, myocardium, and lungs. Diaphragm. As shown in Fig 1A–D and

Supplementary Fig E2A and B and Table E1, in general levels of protein oxidation and nitration and those of SODs were decreased or not modified in the respiratory muscles of the mechanically ventilated rodents (highest VT) compared with the controls and 9 mL rats in both septic and nonseptic experimental groups. In the same muscles, protein content of the antioxidant catalase did not differ between mechanically ventilated rats and controls in any of the models. In nonseptic rodents, protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemoxygenase (HO)-1 were significantly increased in both 15 and 25 mL groups compared with controls or 9 mL animals, whereas the opposite effect was shown in the muscles of the septic rats. Compared with controls and 9 mL rats, protein levels of the cytokine tumor necrosis factor (TNF)-a were significantly increased in the diaphragms of the mechanically ventilated animals without sepsis, whereas the opposite effect was observed in the muscles of the septic rodents. Gastrocnemius. As illustrated in Fig 2A–D and Supplementary Fig E3A and B and Table E2, levels of protein oxidation and nitration did not differ or were reduced in the gastrocnemius of mechanically ventilated rodents (highest VT) compared with controls or 9 mL groups in both septic and nonseptic models. In the latter animals, levels of CuZn-SOD were increased in the limb muscle of the mechanically ventilated rodents compared with controls and 9 mL groups, whereas no differences in this parameter were detected among the groups of septic rats. In the same muscles, protein levels of both Nrf2 and HO-1 markers did not differ among the study groups of animals in any of the experimental models. Protein levels of TNF-a were only increased in the gastrocnemius of nonseptic animals mechanically ventilated using 15 and 25 mL compared with the controls. Soleus. As shown in Fig 3A–D and Supplementary Fig E4A and B and Table E3, in both experimental models, protein oxidation and nitration levels were in general reduced in the soleus of the mechanically ventilated rats (highest VT) compared with controls and 9 mL groups. Protein content of SODs and catalase, especially the latter was significantly increased in the soleus of mechanically ventilated rats (highest VT) compared with controls and 9 mL animals. In nonseptic animals, the protein content of Nrf2 did not differ among any


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Table I. Functional, mechanical, and hemodynamic characteristics in rats exposed to several degrees of mechanical ventilation with and without sepsis FLA 5.2.0 DTD TRSL809_proof 23 August 2014 2:10 pm ce

VT 5 9 mL/kg (n 5 7)

Paw (cm H2O) Nonsepsis Sepsis CRS (mL/cm H2O) Nonsepsis Sepsis MAP (mm Hg) Nonsepsis Sepsis pH Nonsepsis Sepsis Lactate (mmol/L) Nonsepsis Sepsis PaO2 (mm Hg) Nonsepsis Sepsis PaCO2 (mm Hg) Nonsepsis Sepsis

VT 5 15 mL/kg (n 5 7)

Baseline

t 5 150 min

Baseline

16.9 6 1.57 16.0 6 1.48

18.8 6 0.90, P 5 0.001 17.2 6 1.66, P 5 0.003

17.3 6 1.7 18.4 6 3.51

18.7 6 1.5, P 5 0.047 19.4 6 2.3, P 5 0.018

28.3 6 2.14 27.4 6 4.11

39.3 6 4.89, P 5 0.000 39.3 6 11.66, P 5 0.016

0.33 6 0.05 0.32 6 0.03

0.28 6 0.03, P 5 0.005 0.28 6 0.03, P 5 0.002

0.35 6 0.03 0.34 6 0.07

0.33 6 0.03, P 5 0.032 0.32 6 0.06, P 5 0.011

0.41 6 0.03 0.38 6 0.05

0.26 6 0.03, P 5 0.000 0.37 6 0.09, P 5 0.016

121.9 6 17.53 107.4 6 22.9

92.9 6 12.39, P 5 0.006 86.3 6 17.20, P 5 0.022

132.8 6 12.7 103.6 6 18.1

111.0 6 8.2, P 5 0.043 91.3 6 15.88, P 5 0.033

141.9 6 18.7 110.3 6 25

87.6 6 20.07, P 5 0.001 64.3 6 15.77, P 5 0.005

7.40 6 0.04 7.36 6 0.06

7.33 6 0.04, P . 0.05 7.35 6 0.06, P . 0.05

7.39 6 0.04 7.4 6 0.06

7.34 6 0.07, P . 0.05 7.31 6 0.11, P . 0.05

1.8 6 1.04 4.4 6 1.8

1.6 6 0.7, P . 0.05 3.6 6 1.97, P . 0.05

7.4 6 0.02 7.45 6 0.06 1.5 6 0.41 3.6 6 1.9

7.38 6 0.65, P . 0.05 7.44 6 0.07, P . 0.05 1.1 6 0.22, P 5 0.03 1.8 6 0.73, P 5 0.031

183.3 6 8.48 182.3 6 12.65

192.0 6 15.34, P . 0.05 186.0 6 18.51, P . 0.05

34.9 6 5.4 25.9 6 3.02

35.6 6 6.53, P . 0.05 26.4 6 2.23, P . 0.05

1.7 6 0.29 3.9 6 1.6 177.6 6 20.48 175.3 6 35.29 36.3 6 7.9 26.3.5 6 1.2

t 5 150 min

VT 5 25 mL/kg (n 5 7)

0.9 6 0.36, P 5 0.001 1.8 6 1.13, P 5 0.006

Baseline

t 5 150 min

165.0 6 21.33, P . 0.05 197.5 6 35.43, P . 0.05

158.0 6 32.61 195 6 12.5

119.2 6 64.6, P . 0.05 207.8 6 32.2, P . 0.05

42.9 6 4.6, P . 0.05 29.0 6 1.7, P . 0.05

35.2 6 6.59 28.7 6 5.15

38.3 6 5.82, P . 0.05 30.6 6 5.56, P . 0.05


Abbreviations: CRS, dynamic respiratory system compliance; MAP, mean arterial pressure; n, number of samples; Paw, peak airway pressure; PaO2, arterial partial pressure of oxygen; PaCO2, arterial partial pressure of carbon dioxide; t, time; VT, tidal volume. Data are presented as the mean (standard deviation). Variables are measured at baseline (t 5 0, 30 minutes after the equilibration period) and right after 150 minutes of mechanical ventilation (t 5 150 minutes). Statistics: Statistical significance is represented by the actual P values. In all the groups, comparisons were made between t 5 150 minutes and t 5 0 minutes.

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Fig 1. (A) Representative immunoblots of oxidative stress and inflammatory markers in the diaphragm of nonseptic rats in nonventilated controls (N 5 2) and mechanically ventilated animals with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (B) Representative immunoblots of oxidative stress and inflammatory markers in the diaphragm of nonventilated controls with sepsis (N 5 2) and septic rodents mechanically ventilated with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (C) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the diaphragm. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of mechanically ventilated (MV) rats with and without sepsis (left and right panels, respectively): total reactive carbonyls (top left panels), MDA-protein adducts (bottom left panels), and protein tyrosine nitration (top right panels). (D) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the diaphragm. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of MV rats with and without sepsis (left and right panels, respectively): Mn-SOD (top left panels), CuZn-SOD (medium left panels), catalase (bottom left panels), Nrf2 (top right panels), HO-1 (medium right panels), and TNF-a (bottom right panels). VT, tidal volume; MDA, malondialdehyde; MnSOD, manganese superoxide dismutase; CuZnSOD, copper zinc superoxide dismutase; Nrf, nuclear factor erythroid 2-related factor; HO, hemoxygenase; TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTL, control; OD, optical density.

of the study groups, whereas in septic rodents, soleus Nrf2 levels were significantly greater in all groups of mechanically ventilated animals than in the controls. In the soleus of nonseptic rats, HO-1 levels did not differ among any of the study groups, whereas in septic rodents HO-1 protein content was greater in the soleus of mechanically ventilated animals compared with controls. In addition, soleus of 15 and 25 mL groups of septic rats showed an increase in HO-1 levels compared with 9 mL group. Levels of TNF-a were significantly greater in the limb muscle of the mechanically ventilated rats with sepsis than in the controls. Myocardium. As illustrated in Fig 4A–D and Supplementary Fig E5A and B and Table E4, in nonseptic rodents, protein oxidation and nitration levels were

significantly decreased in the heart of mechanically ventilated rodents (highest VT) compared with controls and 9 mL rats, whereas in the septic animals, protein carbonylation levels were increased. Interestingly, protein content of Mn-SOD was increased in the myocardium of the heart of mechanically ventilated rodents (highest VT) compared with controls and 9 mL nonseptic rats, whereas levels of CuZn-SOD were increased in the same organ of the mechanically ventilated animals in the model of sepsis. In general, catalase protein content was reduced in the myocardium of nonseptic rats, whereas no differences were detected between groups in the septic rodents. In nonseptic rats, myocardium Nrf2 levels were significantly reduced in both 9 and 15 mL animals compared with controls and 25 mL


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Fig 1. (continued).


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Fig 2. (A) Representative immunoblots of oxidative stress and inflammatory markers in the gastrocnemius of nonseptic rats in nonventilated controls (N 5 2) and mechanically ventilated animals with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (B) Representative immunoblots of oxidative stress and inflammatory markers in the gastrocnemius of nonventilated controls with sepsis (N 5 2) and septic rodents mechanically ventilated with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (C) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the gastrocnemius. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of mechanically ventilated (MV) rats with and without sepsis (left and right panels, respectively): total reactive carbonyls (top left panels), MDA-protein adducts (bottom left panels), and protein tyrosine nitration (top right panels). (D) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the gastrocnemius. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of MV rats with and without sepsis (left and right panels, respectively): Mn-SOD (top left panels), CuZn-SOD (medium left panels), catalase (bottom left panels), Nrf2 (top right panels), HO-1 (medium right panels), and TNF-a (bottom right panels). VT, tidal volume; MDA, malondialdehyde; MnSOD, manganese superoxide dismutase; CuZnSOD, copper zinc superoxide dismutase; Nrf, nuclear factor erythroid 2-related factor 2; HO, hemoxygenase; TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTL, control; OD, optical density.

rats, whereas in the myocardium of the septic animals, Nrf2 levels did no differ among any of the study groups. Myocardium HO-1 protein content was significantly lower in nonseptic mechanically ventilated animals than in the controls, whereas in septic animals, myocardium HO-1 levels did not differ among any of the study groups. TNF-a protein levels did not differ between groups in the heart of the animals in any experimental model. Lungs. As shown in Fig 5A–D and Supplementary Fig E6A and B and Table E5 in both experimental models, the lungs exhibited a significant rise in protein oxidation levels only in the mechanically ventilated rodents exposed to the highest VT (25 mL) compared with both controls and 9 mL groups of animals. Lung protein tyrosine nitration levels did not differ between

groups in any of the experimental models. In the lungs of nonseptic rats, Mn-SOD levels were significantly increased in the mechanically ventilated rats compared with the controls, whereas levels of this enzyme were reduced in the 25 mL rodents compared with controls and 9 mL groups in septic rats. Nevertheless, protein levels of CuZn-SOD were decreased in the lungs of the mechanically ventilated rats (highest VT) compared with controls and 9 mL nonseptic rodents in both experimental models. Lung catalase protein levels were increased in the 15 mL rodents compared with both controls and 9 mL groups of nonseptic animals, whereas its levels were reduced in the 15 and 25 mL groups compared with the controls in septic rodents. In nonseptic rats, lung Nrf2 levels were significantly reduced in the 15 mL animals compared with controls and 9 mL


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Fig 2. (continued).


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Fig 3. (A) Representative immunoblots of oxidative stress and inflammatory markers in the soleus of nonseptic rats in nonventilated controls (N 5 2) and mechanically ventilated animals with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (B) Representative immunoblots of oxidative stress and inflammatory markers in the soleus of nonventilated controls with sepsis (N 5 2) and septic rodents mechanically ventilated with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (C) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the soleus. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of mechanically ventilated (MV) rats with and without sepsis (left and right panels, respectively): total reactive carbonyls (top left panels), MDA-protein adducts (bottom left panels), and protein tyrosine nitration (top right panels). (D) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the soleus. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of MV rats with and without sepsis (left and right panels, respectively): Mn-SOD (top left panels), CuZn-SOD (medium left panels), catalase (bottom left panels), Nrf2 (top right panels), HO-1 (medium right panels), and TNF-a (bottom right panels). VT, tidal volume; MDA, malondialdehyde; MnSOD, manganese superoxide dismutase; CuZnSOD, copper zinc superoxide dismutase; Nrf, nuclear factor erythroid 2-related factor 2; HO, hemoxygenase; TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTL, control; OD, optical density.

groups. In the lungs of septic animals, Nrf2 protein content was lower in the 25 mL animals than in controls and 9 mL groups, and Nrf2 levels were also reduced in the 15 mL group compared with 9 mL rodents. In nonseptic animals, protein content of HO-1 was reduced in the lungs of the mechanically ventilated rats compared with controls, and the lungs of 25 mL animals showed a decrease in HO-1 levels compared with 9 mL rats. Lung HO-1 levels of septic animals were decreased in the 15 and 25 mL rats compared with both controls and 9 mL groups. In the lungs of nonseptic animals, protein TNF-a levels were increased in the 9 mL rats, whereas they were reduced in the 25 mL rats compared with the controls. Interestingly, lung TNF-a protein

levels did not differ significantly between groups in septic animals. DISCUSSION Redox balance and inflammation in skeletal muscles and myocardium. In the mechanically ventilated

rats, protein oxidation and nitration levels did not increase in general in any of the muscles or organs analyzed in the present study. Interestingly, 24 hours of sepsis only slightly modified or even attenuated the biological effects induced by MV per se in the different muscles and organs. Nevertheless, the findings encountered in the investigation are in line with a previous study from our group10 in which a decline in several


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Fig 4. (A) Representative immunoblots of oxidative stress and inflammatory markers in the myocardium of nonseptic rats in nonventilated controls (N 5 2) and mechanically ventilated animals with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (B) Representative immunoblots of oxidative stress and inflammatory markers in the myocardium of nonventilated controls with sepsis (N 5 2) and septic rodents mechanically ventilated with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (C) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the myocardium. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of mechanically ventilated (MV) rats with and without sepsis (left and right panels, respectively): total reactive carbonyls (top left panels), MDA-protein adducts (bottom left panels), and protein tyrosine nitration (top right panels). (D) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the myocardium. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of MV rats with and without sepsis (left and right panels, respectively): Mn-SOD (top left panels), CuZn-SOD (medium left panels), catalase (bottom left panels), Nrf2 (top right panels), HO-1 (medium right panels), and TNF-a (bottom right panels). VT, tidal volume; MDA, malondialdehyde; MnSOD, manganese superoxide dismutase; CuZnSOD, copper zinc superoxide dismutase; Nrf2, nuclear factor erythroid 2-related factor 2; HO, hemoxygenase; TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTL, control; OD, optical density.

oxidative stress markers was observed in respiratory and limb muscles (both fast- and slow-twitch fiber types) of rats exposed to MV for a shorter duration (60 minutes) and higher VT (35 mL/kg). The novelty in the present study is that we used a wider range of VT than in our previous investigation10 and for a longer period of time to test whether levels of VT more closely related to those used in clinical practice also induce lung and distant injury. In addition, the myocardium was also analyzed to elucidate whether skeletal muscle inactivity imposed by sedation and passive MV is responsible for the previously reported effects of MV on redox balance.10 Finally, to make the model of VILI more clinically relevant, we also

analyzed MV-induced changes in redox balance in septic rats as sepsis is the most common condition leading to respiratory failure and the requirement for MV in the ICU. Importantly, the reduction in oxidative stress markers detected in the heart shared similarities to a great extent to those examined in the respiratory and limb muscles of the same animals, especially in the absence of sepsis. These findings suggest that the activity of the muscle does not seem to play a major role in the effects induced by MV in the rodents, because levels of protein oxidation were similarly decreased in active muscles (myocardium) and inactive limb muscles. Furthermore, it could also be concluded that the decrease in protein


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Fig 5. (A) Representative immunoblots of oxidative stress and inflammatory markers in the lungs of nonseptic rats in nonventilated controls (N 5 2) and mechanically ventilated animals with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (B) Representative immunoblots of oxidative stress and inflammatory markers in the lungs of nonventilated controls with sepsis (N 5 2) and septic rodents mechanically ventilated with 9 mL/kg (N 5 2), 15 mL/kg (N 5 2), and 25 mL/kg (N 5 2) groups, respectively. GAPDH is shown as loading control. (C) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the lungs. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of mechanically ventilated (MV) rats with and without sepsis (left and right panels, respectively): total reactive carbonyls (top left panels), MDA-protein adducts (bottom left panels), and protein tyrosine nitration (top right panels). (D) Optical densities in the box plots are expressed as the ratio of the optical densities of each studied marker to those of GAPDH in the lungs. Standard box plots with median (25th and 75th percentiles) and whiskers (at minimum and maximum values) are depicted in the diaphragm of MV rats with and without sepsis (left and right panels, respectively): Mn-SOD (top left panels), CuZn-SOD (medium left panels), catalase (bottom left panels), Nrf2 (top right panels), HO-1 (medium right panels), and TNF-a (bottom right panels). VT, tidal volume; MDA, malondialdehyde; MnSOD, manganese superoxide dismutase; CuZnSOD, copper zinc superoxide dismutase; Nrf, nuclear factor erythroid 2-related factor 2; HO, hemoxygenase; TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTL, control; OD, optical density.

oxidation and nitration observed in skeletal and heart muscles of the exposed rodents is part of the systemic effects of MV in organs other than the lungs. Indeed, previous investigations3,6,10,11,16-18,29,30,38 have already demonstrated that MV induces systemic effects such as inflammation and injury of several organs other than the lungs. In the present study, mechanically ventilated rats exhibited hypotension, especially in the septic animals receiving the highest VT. Additionally, blood lactate levels decreased in the VT 5 9 mL/kg and VT 5 15 mL/kg groups, but not in the VT 5 25 mL/kg group. These findings are in agreement with previous reports in which hypotension was also observed in models of MV in rodents.10,16-18,29 Also, similarly to previous

studies,10,16,17,29 relevant findings in the investigation were the significant reduction in the dynamic compliance of the respiratory system and the increase in peak airway pressure detected in all study groups in both experimental models, especially in animals receiving the highest VT. Taken together, the pathophysiological alterations induced by MV in the respiratory and cardiovascular systems suggest that perfusion of muscles and organs could be severely altered in the animals, especially in septic rodents exposed to the highest VT. A reduction in blood flow in tissues and vital organs (heart) could lead to decreased levels of oxidant production after the MV period compared with baseline. Protein oxidation and nitration levels were, indeed, decreased in the diaphragms, limb muscles,


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Fig 5. (continued).


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and myocardium of the mechanically ventilated rats especially in the high-VT group, irrespective of sepsis. Levels of the cytosolic enzyme CuZn-SOD seemed to follow a specific pattern of expression in the different muscles or models. For instance, although in soleus and myocardium, CuZn-SOD protein levels were increased in the septic mechanically ventilated rats compared with their nonventilated controls, a significant decrease or no differences were observed in the diaphragms and gastrocnemius of the septic rodents, respectively. Interestingly, in nonseptic animals, MV induced an increase in CuZn-SOD levels in gastrocnemius, whereas a decrease was observed in soleus and myocardium muscles. Taken together, these findings suggest the presence of a differential fiber-type pattern of expression of this potent cellular antioxidant enzyme system in slow- and fasttwitch myofibers in response to MV partly influenced by sepsis. MV alone induced a decrease in catalase protein levels only in gastrocnemius and myocardium muscles of nonseptic rodents. Interestingly, the soleus exhibited a significant rise in catalase content in both models of mechanically ventilated animals. However, sepsis did not modify catalase protein levels in diaphragm, gastrocnemius, or the heart. In line with this, it was demonstrated that in external intercostals and vastus lateralis of patients with sepsis,24 levels of antioxidants (Mn-SOD and catalase) did not differ from those detected in the controls. It is likely that sepsis induces alterations per se in enzyme content and activity27 in skeletal muscles that may prevent their upregulation even if oxidative stress develops. Future investigations may be designed to specifically explore these mechanisms. The cytoprotective signaling pathway Nrf2 is constitutively expressed in all tissues and seems to be a master regulator of age-related disorders, cancer, and degenerative diseases. Nrf2 mediates multiple cellular responses against oxidative stress, inflammation, cancer, and aging through the activation of numerous genes.39 In the present study, Nrf2 levels were increased in the diaphragm of the nonseptic mechanically ventilated rats, whereas the opposite effect was seen in the same muscle in septic rats. Interestingly, in the soleus of septic mechanically ventilated rats a significant rise in Nrf2 was observed compared with the nonventilated controls. In the gastrocnemius and myocardium muscles no significant differences or a decrease was rather seen in the mechanically ventilated rodents compared with the controls in both experimental models. Importantly, a similar pattern of expression of the inducible isoform HO-1 was observed in the same muscles in the mechanically ventilated animals in both models. These findings are not surprising, because Nrf2 is known to regulate

HO-1 expression as a general cytoprotective response to oxidative stress. Taken together, it would be possible to conclude that the activity of the muscle and the fibertype composition may account for the differences in expression of the antioxidant pathways Nrf2 and HO-1, which seem to be more abundantly expressed in slow-twitch fibers. In the diaphragm and gastrocnemius of nonseptic mechanically ventilated rats, levels of TNF-a were increased, whereas no significant differences were observed in the soleus or myocardium of the same rodents. Moreover, in the respiratory muscle of the mechanically ventilated rodents, sepsis induced a decrease in TNF-a protein expression, whereas the soleus exhibited a significant rise in levels of the cytokine in the same animals. The myocardium or gastrocnemius did not experience any significant difference with respect to the nonseptic animals. In the present investigation, inflammatory cell counts have not been measured in muscles. In a previous study from our group,10 although inflammatory cell numbers increased in response to high-VT MV in skeletal muscles, their levels were extremely low to exert any biological effects on those muscles. Taken together, it would be possible to conclude from the present findings that the muscle fibers per se are likely to synthesize enhanced levels of TNF-a in response to MV in fast-twitch muscles such as the diaphragm and gastrocnemius. Surprisingly, sepsis induced a significant increase in TNF-a only in the slow-twitch muscle of the mechanically ventilated rodents. These findings are not in agreement with previous reports in which levels of inflammatory cytokines were increased in respiratory and limb muscles of patients with sepsis24 and animal models of sepsis.40,41 Indeed, they could be explained by the particular time course of release of the pleiotropic cytokine after the insult in the different fiber types. In the present study, levels of protein oxidation as measured by both MDA-protein adducts and reactive carbonyls were significantly increased in the lungs of the mechanically ventilated rats, especially in those of the highest VT in both experimental models. These findings are in total agreement with previous investigations in which oxidative stress levels were enhanced in the lungs in several models of VILI.4,7-10 Protein tyrosine nitration levels, however, did not differ in mechanically ventilated rats from those detected in nonventilated control animals in either experimental model. These findings are also in line with a previous study,10 in which protein nitration levels remained unmodified after 1 hour of high-VT MV in rodents. Nevertheless, a previous report reported that patients with ALI exhibited higher levels of immunohistochemical protein tyrosine nitration expression in their


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lungs.9 It would be possible to conclude that MV for longer periods of time administered to actual patients, as opposed to rodents, may induce higher levels of protein nitration in the diseased lungs through either peroxynitrite- or myeloperoxidase-mediated mechanisms.26,38 Importantly, protein content of the antioxidant Mn-SOD was increased in the lungs of the nonseptic mechanically ventilated rats, whereas concomitant sepsis induced a significant decrease. A similar pattern of expression was observed for the antioxidant catalase in response to either MV or sepsis. Nonetheless, protein levels of CuZn-SOD, Nrf2, and HO-1 were rather decreased in the lungs of mechanically ventilated rats in both experimental models. Results encountered in the present study are somehow in contrast with a previous report in which Mn-SOD and catalase protein contents were shown to be reduced in lungs in a model of VILI in which nonseptic animals were mechanically ventilated using high VT for 1 hour.10 Differences in the experimental models and the degree of cyclic stretch in the lungs induced by MV, especially in the highest VT groups, may account for the discrepancies observed among studies.7,8,10,42 In the present study, in both models, levels of the cytokine TNF-a did not significantly differ in the lungs of the mechanically ventilated rats from those detected in the nonventilated control rodents. These findings are in contrast to previously published results in which inflammatory cell infiltrates10 and levels of proinflammatory cytokines and chemokines6,43,44 were shown to be increased in the lungs in several models of VILI. Differences in the study design and experimental models, and the fact that actual protein levels, but not mRNA, were measured in the present study may account for the discrepancies encountered across investigations.6,10,43,44 Study critique. It is worth mentioning that the ventilatory strategies used in the present investigation were within the scope of those used in previously published reports from our group and other investigators,6,10,1218,29,30,45-47 in which key physiological alterations defining the pulmonary and nonpulmonary effects of high-VT MV were demonstrated. In this regard, it is common practice to apply high-extreme conditions (clinical and/or physiological) in animals, including those with 24 hours of sepsis, to generate contrast and to ensure biological differences among groups that cannot be the subject of methodological concerns. On the basis of this, one may conclude that compared with humans, administration of much larger VT is required to induce the desired damaging effects such as lung injury and the pathophysiological and biological responses in the other

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organs. Furthermore, different ventilatory strategies were used in the present study with the aim to explore lung overdistension in the presence or absence of PEEP, and its potential systemic effects on tissues other than the lungs. We acknowledge, however, that it would have also been of interest to analyze the effects of these ventilatory strategies on lung permeability and cellular inflammation in fluids such as bronchoalveolar lavage. Nonetheless, as the focus of the present investigation was to study the effects of several ventilatory strategies on the skeletal muscles of the exposed animals, bronchoalveolar lavage fluid was not collected from any of the experimental groups of rodents. Future studies will certainly address this specific question. Moreover, a previous study has already reported the presence of abundant inflammatory cell infiltrates in the lungs of rats exposed to several strategies of MV.10 A limitation is related to the clinical relevance of the findings encountered using this specific model of VILI, in which lung injury was induced by the administration of relatively high-VT MV to healthy and septic rats for a relatively short period of time. In humans, 12 mL/kg have been shown to induce harmful effects on lungs,48 whereas this VT would result harmless in healthy rats. Furthermore, standard MV in humans was shown to induce marked atrophy of the diaphragm fibers and increased proteolysis as a result of inactivity.49 Despite the recognized limitations, we believe that the study of medium-term events taking place after the pulmonary insult (high VT-induced stretch) is of clinical relevance, because early occurrence of oxidative stress during high-VT MV cannot be assumed in all organs or tissues. In fact, a reduction, rather than an increase, in oxidative stress was observed in skeletal muscles and even myocardium of rats exposed to large VT and VILI for a relatively short period of time (2.5 hours) in the presence or absence of sepsis. Speculations. The study demonstrates that in this model of VILI, oxidative stress does not take place in distant organs or skeletal muscles of rodents after several hours of MV with moderate-to-high VT, whereas protein oxidation levels were indeed increased in the lungs of the animals. Inflammatory events were moderately expressed in the skeletal muscles and absent in the lungs of the mechanically ventilated rats. Concomitant sepsis did not strongly affect, or even attenuated, the MVinduced effects on muscles, myocardium, or lungs in the rodents. The present results have potential clinical implications, because oxidative stress and inflammatory events cannot be assumed in all organs or tissues even in the presence of sepsis. Antioxidant therapies, therefore, should not be administered to these patients.


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ACKNOWLEDGMENTS

Q10



Conflicts of Interest: All authors have read the Journal’s policy on disclosure of potential conflicts of interest and have none to declare. All authors have also read the journal’s authorship agreement. Editorial Support: None to declare. This study has been supported by CIBERES, FIS 12/ 02534, FIS 11/02029, FIS 11/02791, and FIS 12/02898; 2009-SGR-393; SEPAR 2010; SEPAR 2013; FUCAP 2011; FUCAP 2012; and Marat o TV3 (MTV3-071010) (Spain). Dr Esther Barreiro was a recipient of the ERS COPD Research Award 2008. Supplementary Data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.trsl.2014.07.003. REFERENCES

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