Free
Critical Care Medicine  |   February 2010
Redox Balance and Cellular Inflammation in the Diaphragm, Limb Muscles, and Lungs of Mechanically Ventilated Rats
Author Affiliations & Notes
  • Judith Marín-Corral, M.D.
    *
  • Leticia Martínez-Caro, B.Sc., Ph.D.
  • José A. Lorente, M.D., Ph.D.
  • Marta de Paula, Ph.D.
  • Lara Pijuan, M.D., Ph.D.
    §
  • Nicolas Nin, M.D., Ph.D.
  • Joaquim Gea, M.D., Ph.D.
  • Andrés Esteban, M.D., Ph.D.
  • Esther Barreiro, M.D., Ph.D.
    #
  • * Ph.D. Student, Pulmonology Department-Muscle and Respiratory System Research Unit, Institut Municipal d'Investigació Mèdica (IMIM)-Hospital del Mar, Parc de Recerca Biomèdica de Barcelona (PRBB); Department of Medicine, Medical School, Universitat Autònoma de Barcelona, Barcelona, Spain. † Post-doctoral Investigator, ‡ Investigator, Servicio de Medicina Intensiva, Hospital Universitario de Getafe, Getafe, Madrid, Spain; Centro de Investigación en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Bunyola, Majorca, Balearic Islands, Spain. § Assistant Professor, Department of Medicine, Medical School, Universitat Autònoma de Barcelona; Department of Pathology, IMIM-Hospital del Mar, Barcelona, Spain. ∥ Professor, # Associate Professor, Pulmonology Department-Muscle and Respiratory System Research Unit, IMIM-Hospital del Mar, PRBB; CIBERES, ISCIII; Health and Experimental Sciences Department, Universitat Pompeu Fabra, PRBB, Barcelona, Spain.
Article Information
Critical Care Medicine / Critical Care / Respiratory System
Critical Care Medicine   |   February 2010
Redox Balance and Cellular Inflammation in the Diaphragm, Limb Muscles, and Lungs of Mechanically Ventilated Rats
Anesthesiology 2 2010, Vol.112, 384-394. doi:10.1097/ALN.0b013e3181c38bed
Anesthesiology 2 2010, Vol.112, 384-394. doi:10.1097/ALN.0b013e3181c38bed
What We Already Know about This Topic
  • ❖ High tidal volume ventilation produces oxidative stress and inflammation in the lung, but whether it produces similar effects in other organs is less clear
What This Article Tells Us That Is New
  • ❖ In normal rats, high tidal volume ventilation produced minimal or no oxidative stress and inflammation in the skeletal muscle and the diaphragm
  • ❖ High tidal volume ventilation may not induce diaphragmatic dysfunction via oxidative stress and inflammation
ACUTE lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common conditions in intensive care units. Regardless of the severity of respiratory failure, the most common cause of death in patients with ARDS is shock and multiorgan failure.1 Despite the lifesaving potential ofmechanical ventilation in patients with ALI and ARDS, complications arising out of this sort of therapy in critically ill patients should also be considered. In this regard, previous studies have shown that mechanical ventilation strategies using high tidal volume (VT) and low positive end-expiratory pressure (PEEP) cause alveolar disruption and pulmonary edema, thus activating inflammatory pathways.2,3 On this basis, it became clear that in patients with ARDS and ALI, certain strategies of mechanical ventilation might enhance lung injury, a condition known as ventilator-induced lung injury (VILI).4 The pathophysiology of VILI, however, has not yet been fully elucidated.
Oxidative stress, defined as the imbalance between oxidants and antioxidants in favor of the former, has been suggested to be involved, among other factors, in the development and perpetuation of VILI.5 For instance, a reduction in antioxidant activity along with an increase in malondialdehyde was observed in the fluid lining of mechanically ventilated lungs.6,7 In addition, protein tyrosine nitration, a marker of nitrosative stress, was also shown to be increased in the lungs of patients with ALI.8 Interestingly, evidence from animal studies demonstrated that inflammatory cells and proinflammatory cytokines also contributed to the pathogenesis of VILI.9,10 
Recently, the line has also been put forward that high VTmechanical ventilation induced organ injury other than lung injury and systemic inflammation in several animal models of VILI.11–17 It remains unknown, however, whether high VTmechanical ventilation exerts deleterious effects on skeletal muscles, including the diaphragm, thus potentially influencing the weaning of patients from the ventilator. Moreover, studies investigating early molecular events induced by VILI in different organs and tissues are also lacking. On this basis, our study explored whether high VTmechanical ventilation per se  may induce early molecular changes such as increased oxidative stress levels and inflammation in the diaphragm, limb muscles, and lungs in an in vivo  experimental model of VILI applied to healthy rats for 1 h. In fact, the VILI model used in the current investigation has already been used for exclusively exploring pulmonary and systemic effects of high VTin healthy animals.10–19 Accordingly, our objective was to specifically explore early molecular events involving oxidative stress, antioxidant mechanisms, and inflammation in the diaphragms, limb muscles (fast-and slow-twitch types), and lungs of healthy rats exposed to high VTmechanical ventilation for 1 h.
Materials and Methods
Animals and Study Protocol
All animal experiments were conducted at the Hospital Universitario de Getafe (Madrid, Spain). This is a controlled study designed in accordance with both the ethical standards on animal experimentation (EU 609/86 CEE, Real Decreto 1201/05 BOE 252, Spain) at the Hospital Universitario de Getafe and the Helsinki convention for the use and care of animals. All experiments were approved by the Animal Research Committee at the Hospital Universitario de Getafe.
Three independent groups of pathogen-free healthy male Sprague-Dawley rats (Harlan Iberica, Spain, weight 325-375 g, n = 8/group) were studied using well-validated methodologies, which have been previously published by our group and others.10–19 Two different ventilatory strategies were used in healthy rats to evaluate the effects of lung overdistension in the presence or absence of PEEP10–19 : moderate VTmechanical ventilation (VT9 ml/kg, PEEP = 5 cm H2O), Group 1, and high VTventilation (VT35 ml/kg, PEEP = 0 cm H2O), Group 2. In both groups of rats, respiratory parameters were set as follows: respiratory rate, 70 bpm; inspiratory time, 0.3 s; expiratory time, 0.56 s; and inspired fraction of oxygen, 0.45. All animals were initially ventilated for an equilibration period of 10 min using moderate VTventilation parameters before they were assigned to either Group 1 or Group 2 (moderate and high VTmechanical ventilation, respectively). Both groups of animals were mechanically ventilated for 1 h, after being assigned to the corresponding group of the mechanical ventilation strategy. A third group of nonventilated rats (intact animals) was also studied (Group 3, control). Importantly, in ventilated animals, respiratory drive was abolished by the high respiratory rate set in the ventilator, because no spontaneous breaths were observed. Lung mechanics, hemodynamics, and circulatory conditions were measured both at time 0 and after the 1-h period of mechanical ventilation in each group of ventilated rats.
At the end of the experimental period (1 h), the following muscles and organs were removed in all animals during anesthesia: diaphragm, gastrocnemius, soleus, tibialis anterior, and lungs. The muscle and organ specimens were immediately frozen in liquid nitrogen and subsequently stored at −80°C. Furthermore, tissue specimens corresponding to the diaphragm, gastrocnemius, and lungs were also immersed in an alcohol-formol bath for 2 h and were embedded in paraffin. Frozen tissues were used for the redox marker analyses (immunoblotting), whereas paraffin-embedded tissues were used for the assessment of inflammation (immunohistochemical analyses). (See also Animals and Study Protocol in Supplemental Digital Content 1, in which full details on the animal experimentation and protocol is provided, .)
Biologic Studies
All biologic analyses were conducted in the same laboratory at the IMIM-Hospital del Mar (Barcelona, Spain).
Oxidative Stress Markers.
The effects of reactive oxygen species (ROS) and reactive nitrogen species on muscle proteins were evaluated using immunoblotting according to the methods published previously.20–26 (See also Oxidative Stress Markers in Supplemental Digital Content 1, in which full details on the redox balance analyses are provided, .)
Inflammation in the Diaphragm and Gastrocnemius Muscles.
To evaluate the presence of inflammatory cells in these muscles, immunohistochemical analyses were conducted in all the diaphragms and gastrocnemius of the three groups of animals by using similar methods published else where.27,28 (See also Inflammation in Diaphragm and Gastrocnemius Muscles in Supplemental Digital Content 1, in which full details on the muscle inflammation assessment are described, .)
Inflammation in the Lungs.
Inflammatory infiltrates were determined in the lungs of the three groups of rats using hematoxylin-eosin staining according to the methodologies previously published.29,30 See also Inflammation in the Lungs in Supplemental Digital Content 1, in which full details on the evaluation of lung inflammation levels are provided, .
Statistical Analysis
This study is designed on the basis of a two-tailed hypothesis. Clinical and physiologic data are presented as mean (SD). Biologic data are presented as median and interquartile range in the tables, whereas they are presented as box and whisker plots in the figures. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, 12.0 version for windows, SPSS Inc., Chicago, IL). Mechanical ventilation-induced changes in clinical and physiologic variables were explored using paired Student-t  test compared with baseline in both moderate- and high-VTrat groups. Kruskal-Wallis test was used to examine significant differences among the three study groups, for each biologic variable and tissue. Furthermore, nonparametric Mann-Whitney U test was also used to specifically explore significant differences in biologic variables of the study between each group of mechanically ventilated rats and control animals. The sample size was based on previous studies10–19 and on assumptions of 80% power to detect an improvement of more than 20% in measured outcomes at a level of significance of P  less than or equal to 0.05. In all the biologic variables involving the study of redox balance (total reactive carbonyls, malondialdehyde-protein adducts, protein tyrosine nitration, Mn-superoxide dismutase (Mn-SOD), and catalase), mean difference between groups was estimated at a minimum of 25%, and SD was approximately 25-30% of the mean value for each of the variables.
Results
Effects of Mechanical Ventilation
Compared with time 0, peak airway pressure was significantly increased in rats exposed to high VTfor 1 h, while exhibiting a significant decrease in the dynamic compliance of the respiratory system (table 1). Furthermore, high-VTrats showed hypotension, hypoxemia, acidosis, and hyperlactatemia after the 1-h ventilation period compared with levels at time 0 (table 1).
Table 1.  Changes in Blood Gases, Hemodynamic, and Mechanical Ventilatory Parameters in Rats Subjected to Mechanical Ventilation with Moderate-tidal (9 ml/kg) and High-tidal Volume (35 ml/kg)
Image not available
Table 1.  Changes in Blood Gases, Hemodynamic, and Mechanical Ventilatory Parameters in Rats Subjected to Mechanical Ventilation with Moderate-tidal (9 ml/kg) and High-tidal Volume (35 ml/kg)
×
Oxidative Stress Markers
Total Reactive Carbonyls.
Representative immunoblots corresponding to protein carbonylation in the diaphragm and gastrocnemius muscles of the rats are illustrated in figures 1A and B, respectively. In the diaphragms, total protein carbonylation was significantly lower in high-VTanimals than in either moderate-VTrats or control animals (fig. 1C). In the gastrocnemius, protein carbonylation levels were significantly reduced in high-VTrats compared with control animals (fig. 1D). High-VTand moderate-VTanimals showed significantly lower levels of muscle protein carbonylation in the soleus and tibialis anterior compared with control rats (figs. 1E and F, respectively). In the rat lungs, reactive carbonyl levels did not significantly differ among the three study groups (table 2).
Fig. 1. Representative examples of protein oxidation (total carbonyl groups) immunoblots in the diaphragms (A  ) of control (n = 8), moderate tidal volume (VT, n = 8), and high VT(n = 8) mechanically ventilated rats. Several oxidized proteins of different molecular weights (MW, in kilo Daltons) were observed in all muscles. Representative immunoblots of oxidized proteins of different MW, in kilodaltons, in the gastrocnemius (B  ) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total reactive carbonyl groups to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Reactive carbonyl levels were significantly reduced in the diaphragms (C  ) of the high-VTrats compared with either control or moderate-VTanimals. In the gastrocnemius (D  ), reactive carbonyls were significantly decreased in high-VTrats compared with controls and almost significantly reduced compared with moderate-VTrodents. In the soleus (E  ) and tibialis anterior (F  ), reactive carbonyl levels were significantly decreased in high- and moderate-VTrats compared with controls.
Fig. 1. Representative examples of protein oxidation (total carbonyl groups) immunoblots in the diaphragms (A 
	) of control (n = 8), moderate tidal volume (VT, n = 8), and high VT(n = 8) mechanically ventilated rats. Several oxidized proteins of different molecular weights (MW, in kilo Daltons) were observed in all muscles. Representative immunoblots of oxidized proteins of different MW, in kilodaltons, in the gastrocnemius (B 
	) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total reactive carbonyl groups to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Reactive carbonyl levels were significantly reduced in the diaphragms (C 
	) of the high-VTrats compared with either control or moderate-VTanimals. In the gastrocnemius (D 
	), reactive carbonyls were significantly decreased in high-VTrats compared with controls and almost significantly reduced compared with moderate-VTrodents. In the soleus (E 
	) and tibialis anterior (F 
	), reactive carbonyl levels were significantly decreased in high- and moderate-VTrats compared with controls.
Fig. 1. Representative examples of protein oxidation (total carbonyl groups) immunoblots in the diaphragms (A  ) of control (n = 8), moderate tidal volume (VT, n = 8), and high VT(n = 8) mechanically ventilated rats. Several oxidized proteins of different molecular weights (MW, in kilo Daltons) were observed in all muscles. Representative immunoblots of oxidized proteins of different MW, in kilodaltons, in the gastrocnemius (B  ) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total reactive carbonyl groups to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Reactive carbonyl levels were significantly reduced in the diaphragms (C  ) of the high-VTrats compared with either control or moderate-VTanimals. In the gastrocnemius (D  ), reactive carbonyls were significantly decreased in high-VTrats compared with controls and almost significantly reduced compared with moderate-VTrodents. In the soleus (E  ) and tibialis anterior (F  ), reactive carbonyl levels were significantly decreased in high- and moderate-VTrats compared with controls.
×
Table 2.  Oxidative Stress Markers in the Skeletal Muscles and Lungs of Mechanically Ventilated and Control Rats
Image not available
Table 2.  Oxidative Stress Markers in the Skeletal Muscles and Lungs of Mechanically Ventilated and Control Rats
×
Malondialdehyde-protein Adducts.
In the diaphragm and gastrocnemius of the rats (figs. 2A and B, respectively), total malondialdehyde-protein adducts levels did not significantly differ among the three groups. In the soleus and tibialis anterior (figs. 2C and D, respectively), high-VTand moderate-VTanimals showed significantly lower levels of muscle malondialdehyde-protein adducts compared with control rats. In the rat lungs, however, high-VTanimals showed significantly greater levels of total malondialdehyde-protein adducts compared with either moderate-VTrats or control animals (figs. 2E and F).
Fig. 2. Optical densities in the box plots are expressed as the ratio of the optical densities of total malondialdehyde (MDA)-protein adducts to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. There was an almost significant decrease in MDA-protein adducts in the diaphragms (A  ) and gastrocnemius (B  ) muscles of the high-tidal volume (VT, n = 8) rats compared with the controls (n = 8). In the soleus (C  ) and tibialis anterior (D  ), MDA-protein adduct levels were significantly decreased in high- and moderate-VTrats (n = 8) compared with the controls. Representative immunoblots of MDA-protein adducts of different molecular weights (MW), in kilo Daltons, in the lungs (E  ) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total MDA-protein adducts to those of GAPDH in the lungs (F  ). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted (F  ). Lungs of high-VTrats exhibited a significant increase in MDA-protein adducts compared with either controls or moderate-VT animals. The lungs of moderate-VTrodents showed an almost significant increase in MDA-protein adducts compared with control animals (F  ).
Fig. 2. Optical densities in the box plots are expressed as the ratio of the optical densities of total malondialdehyde (MDA)-protein adducts to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. There was an almost significant decrease in MDA-protein adducts in the diaphragms (A 
	) and gastrocnemius (B 
	) muscles of the high-tidal volume (VT, n = 8) rats compared with the controls (n = 8). In the soleus (C 
	) and tibialis anterior (D 
	), MDA-protein adduct levels were significantly decreased in high- and moderate-VTrats (n = 8) compared with the controls. Representative immunoblots of MDA-protein adducts of different molecular weights (MW), in kilo Daltons, in the lungs (E 
	) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total MDA-protein adducts to those of GAPDH in the lungs (F 
	). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted (F 
	). Lungs of high-VTrats exhibited a significant increase in MDA-protein adducts compared with either controls or moderate-VT animals. The lungs of moderate-VTrodents showed an almost significant increase in MDA-protein adducts compared with control animals (F 
	).
Fig. 2. Optical densities in the box plots are expressed as the ratio of the optical densities of total malondialdehyde (MDA)-protein adducts to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. There was an almost significant decrease in MDA-protein adducts in the diaphragms (A  ) and gastrocnemius (B  ) muscles of the high-tidal volume (VT, n = 8) rats compared with the controls (n = 8). In the soleus (C  ) and tibialis anterior (D  ), MDA-protein adduct levels were significantly decreased in high- and moderate-VTrats (n = 8) compared with the controls. Representative immunoblots of MDA-protein adducts of different molecular weights (MW), in kilo Daltons, in the lungs (E  ) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total MDA-protein adducts to those of GAPDH in the lungs (F  ). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted (F  ). Lungs of high-VTrats exhibited a significant increase in MDA-protein adducts compared with either controls or moderate-VT animals. The lungs of moderate-VTrodents showed an almost significant increase in MDA-protein adducts compared with control animals (F  ).
×
Protein Tyrosine Nitra tion.
In the diaphragm and gastrocnemius of the rats (figs. 3A and B, respectively), high-VTrats showed significantly lower levels of muscle protein tyrosine nitration compared with moderate-VTanimals, but not controls. In the soleus and tibialis anterior of the rats (figs. 3C and D, respectively), high-VTand moderate-VTrats showed lower levels of muscle protein nitration compared with control animals. In the rat lungs, protein tyrosine nitration levels did not significantly differ among the three study groups (table 2).
Fig. 3. Optical densities in the box plots are expressed as the ratio of the optical densities of total protein tyrosine nitration to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. In the diaphragms (A  ) and gastrocnemius (B  ) muscles of the high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats, protein nitration levels were significantly reduced compared with moderate-VTanimals (n = 8) but not controls (n = 8). In the soleus (C  ) and tibialis anterior (D  ), protein tyrosine nitration levels were significantly decreased in high- and moderate-VTrats compared with the controls.
Fig. 3. Optical densities in the box plots are expressed as the ratio of the optical densities of total protein tyrosine nitration to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. In the diaphragms (A 
	) and gastrocnemius (B 
	) muscles of the high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats, protein nitration levels were significantly reduced compared with moderate-VTanimals (n = 8) but not controls (n = 8). In the soleus (C 
	) and tibialis anterior (D 
	), protein tyrosine nitration levels were significantly decreased in high- and moderate-VTrats compared with the controls.
Fig. 3. Optical densities in the box plots are expressed as the ratio of the optical densities of total protein tyrosine nitration to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. In the diaphragms (A  ) and gastrocnemius (B  ) muscles of the high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats, protein nitration levels were significantly reduced compared with moderate-VTanimals (n = 8) but not controls (n = 8). In the soleus (C  ) and tibialis anterior (D  ), protein tyrosine nitration levels were significantly decreased in high- and moderate-VTrats compared with the controls.
×
Antioxidant Mechanisms
Mn-SOD.
In the diaphragm, gastrocnemius, soleus, and tibialis anterior of the rats, Mn-SOD protein levels did not significantly differ among the three groups of animals (table 2). In the rat lungs, however, Mn-SOD protein levels were significantly reduced in both high-VTand moderate-VTanimals compared with the controls (fig. 4A). Moreover, lung Mn-SOD levels were significantly lower in the high-VT rats compared with moderate-VTanimals (fig. 4A).
Fig. 4. Optical densities in the box plots are expressed as the ratio of the optical densities of Mn-SOD (A  ) and catalase (B  ) protein contents to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lungs of the rat. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Levels of Mn-SOD protein content (A  ) were lower in the lungs of high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats compared with either controls (n = 8) or moderate-VTanimals (n = 8). Levels of Mn-SOD content in the lungs of moderate-VTrats were also significantly reduced compared with the controls. Levels of catalase protein (B  ) were lower in the lungs of high-VTrats compared with controls, but not with those in moderate-VTanimals. Levels of catalase content in the lungs of moderate-VTrats were also significantly reduced compared with the controls (B  ).
Fig. 4. Optical densities in the box plots are expressed as the ratio of the optical densities of Mn-SOD (A 
	) and catalase (B 
	) protein contents to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lungs of the rat. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Levels of Mn-SOD protein content (A 
	) were lower in the lungs of high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats compared with either controls (n = 8) or moderate-VTanimals (n = 8). Levels of Mn-SOD content in the lungs of moderate-VTrats were also significantly reduced compared with the controls. Levels of catalase protein (B 
	) were lower in the lungs of high-VTrats compared with controls, but not with those in moderate-VTanimals. Levels of catalase content in the lungs of moderate-VTrats were also significantly reduced compared with the controls (B 
	).
Fig. 4. Optical densities in the box plots are expressed as the ratio of the optical densities of Mn-SOD (A  ) and catalase (B  ) protein contents to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lungs of the rat. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Levels of Mn-SOD protein content (A  ) were lower in the lungs of high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats compared with either controls (n = 8) or moderate-VTanimals (n = 8). Levels of Mn-SOD content in the lungs of moderate-VTrats were also significantly reduced compared with the controls. Levels of catalase protein (B  ) were lower in the lungs of high-VTrats compared with controls, but not with those in moderate-VTanimals. Levels of catalase content in the lungs of moderate-VTrats were also significantly reduced compared with the controls (B  ).
×
Catalase.
In the diaphragm, gastrocnemius, soleus, and tibialis anterior of the rats, catalase protein levels did not significantly differ among the three groups of rats (table 2). Nevertheless, in the rat lungs, catalase protein levels were significantly reduced in both high-VTand moderate-VTanimals compared with controls (fig. 4B).
Inflammatory Cells
Diaphragms and Gastrocnemius Muscles.
Human tonsils were used as positive controls for both leukocyte and macrophage immunohistochemical identification (figs. 5A and B, respectively). Figures 5C and Dillustrate inflammatory cell infiltration within the diaphragm fibers of a high-VTrat and a control animal, respectively. Although intramuscular inflammatory cell infiltration was extremely low in the respiratory and limb muscles of the three study groups of rats, there was a statistically significant increase in inflammatory cell counts in the diaphragm and gastrocnemius (figs. 5E and F, respectively) of high-VTrats compared with either controls or moderate-VTanimals.
Fig. 5. Immunohistochemical localization of both leukocytes (A  ) and macrophages (B  ) in human tonsils (positive controls, ×400). Although intramuscular inflammatory cell levels were low in all groups of rats, diaphragm fibers of high-tidal volume (VT) rats exhibited more prominent infiltration (C  ) than the control diaphragms (D  ). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Total inflammatory cell counts (inflammatory cells/muscle section area in square millimeters [mm2]) were significantly greater in the diaphragms (E  ) of high-VTmechanically ventilated (MV) rats (n = 8) compared with either moderate-VTanimals (n = 8) or the controls (n = 8). Diaphragm inflammatory cell counts were also greater in moderate-VTrats than in the controls (E  ). In the gastrocnemius (F  ), inflammatory cell counts (inflammatory cells/muscle section area in mm2) of high-VTrats were significantly increased compared with either controls or moderate-VTrats. Gastrocnemius inflammatory cell counts did not significantly differ between moderate-VTrats and controls (F  ).
Fig. 5. Immunohistochemical localization of both leukocytes (A 
	) and macrophages (B 
	) in human tonsils (positive controls, ×400). Although intramuscular inflammatory cell levels were low in all groups of rats, diaphragm fibers of high-tidal volume (VT) rats exhibited more prominent infiltration (C 
	) than the control diaphragms (D 
	). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Total inflammatory cell counts (inflammatory cells/muscle section area in square millimeters [mm2]) were significantly greater in the diaphragms (E 
	) of high-VTmechanically ventilated (MV) rats (n = 8) compared with either moderate-VTanimals (n = 8) or the controls (n = 8). Diaphragm inflammatory cell counts were also greater in moderate-VTrats than in the controls (E 
	). In the gastrocnemius (F 
	), inflammatory cell counts (inflammatory cells/muscle section area in mm2) of high-VTrats were significantly increased compared with either controls or moderate-VTrats. Gastrocnemius inflammatory cell counts did not significantly differ between moderate-VTrats and controls (F 
	).
Fig. 5. Immunohistochemical localization of both leukocytes (A  ) and macrophages (B  ) in human tonsils (positive controls, ×400). Although intramuscular inflammatory cell levels were low in all groups of rats, diaphragm fibers of high-tidal volume (VT) rats exhibited more prominent infiltration (C  ) than the control diaphragms (D  ). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Total inflammatory cell counts (inflammatory cells/muscle section area in square millimeters [mm2]) were significantly greater in the diaphragms (E  ) of high-VTmechanically ventilated (MV) rats (n = 8) compared with either moderate-VTanimals (n = 8) or the controls (n = 8). Diaphragm inflammatory cell counts were also greater in moderate-VTrats than in the controls (E  ). In the gastrocnemius (F  ), inflammatory cell counts (inflammatory cells/muscle section area in mm2) of high-VTrats were significantly increased compared with either controls or moderate-VTrats. Gastrocnemius inflammatory cell counts did not significantly differ between moderate-VTrats and controls (F  ).
×
Lungs.
Hematoxylin-eosin staining of a lung section from both control and moderate-VTrats are shown in figures 6A and B, respectively. Abundant inflammatory cells were observed within the lung inflammatory cell infiltrates in moderate-VTrats (fig. 6C). Interestingly, lung inflammatory cell infiltrates were more abundant, but were of a smaller size, in high-VTrats (figs. 6D and E) compared with moderate-VTanimals (fig. 6C). Abundant inflammatory cells were observed within the corresponding infiltrates (fig. 6F). The number of inflammatory infiltrates was significantly greater in the lungs of high-VTrats compared with either control or moderate-VTrodents (fig. 6G). However, total lung inflammatory infiltrated area was significantly increased in both high- and moderate-VTrats compared with control animals (fig. 6H).
Fig. 6. Representative hematoxylin-eosin staining of a lung section from a control rat (A  , ×40). Representative hematoxylin-eosin infiltrates corresponding to a lung section from a moderate tidal volume (VT) rat (B  , ×40). The presence of the abundant inflammatory cells in the same infiltrate can be seen at ×400 (C  ). High-VTinduced an increase in the number of lung inflammatory cell infiltrates (D  and E  , ×40). Abundant inflammatory cells were also seen in these infiltrates (F  , ×400). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. The number of infiltrates (total number of infiltrates/total lung section area in square millimeters [mm2]) was greater in the lungs of high-VTmechanically ventilated (MV) rats (n = 8) compared with either control (n = 8) or moderate-VT(n = 8) animals (G  ). The number of lung infiltrates did not differ between moderate-VTrats and controls (G  ). Total lung inflammatory infiltrated area (total inflammatory infiltrated area in mm2/total lung section area in mm2) was significantly greater in high-VTrats compared with controls, but not moderate-VTrats (H  ). Total lung inflammatory infiltrated area was also significantly larger in the moderate-VTrats compared with controls (H  ).
Fig. 6. Representative hematoxylin-eosin staining of a lung section from a control rat (A 
	, ×40). Representative hematoxylin-eosin infiltrates corresponding to a lung section from a moderate tidal volume (VT) rat (B 
	, ×40). The presence of the abundant inflammatory cells in the same infiltrate can be seen at ×400 (C 
	). High-VTinduced an increase in the number of lung inflammatory cell infiltrates (D 
	and E 
	, ×40). Abundant inflammatory cells were also seen in these infiltrates (F 
	, ×400). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. The number of infiltrates (total number of infiltrates/total lung section area in square millimeters [mm2]) was greater in the lungs of high-VTmechanically ventilated (MV) rats (n = 8) compared with either control (n = 8) or moderate-VT(n = 8) animals (G 
	). The number of lung infiltrates did not differ between moderate-VTrats and controls (G 
	). Total lung inflammatory infiltrated area (total inflammatory infiltrated area in mm2/total lung section area in mm2) was significantly greater in high-VTrats compared with controls, but not moderate-VTrats (H 
	). Total lung inflammatory infiltrated area was also significantly larger in the moderate-VTrats compared with controls (H 
	).
Fig. 6. Representative hematoxylin-eosin staining of a lung section from a control rat (A  , ×40). Representative hematoxylin-eosin infiltrates corresponding to a lung section from a moderate tidal volume (VT) rat (B  , ×40). The presence of the abundant inflammatory cells in the same infiltrate can be seen at ×400 (C  ). High-VTinduced an increase in the number of lung inflammatory cell infiltrates (D  and E  , ×40). Abundant inflammatory cells were also seen in these infiltrates (F  , ×400). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. The number of infiltrates (total number of infiltrates/total lung section area in square millimeters [mm2]) was greater in the lungs of high-VTmechanically ventilated (MV) rats (n = 8) compared with either control (n = 8) or moderate-VT(n = 8) animals (G  ). The number of lung infiltrates did not differ between moderate-VTrats and controls (G  ). Total lung inflammatory infiltrated area (total inflammatory infiltrated area in mm2/total lung section area in mm2) was significantly greater in high-VTrats compared with controls, but not moderate-VTrats (H  ). Total lung inflammatory infiltrated area was also significantly larger in the moderate-VTrats compared with controls (H  ).
×
Discussion
In this study, in contrast to our initial hypothesis, early molecular events after administration of high VTmechanical ventilation to healthy rats were characterized by a significant reduction in both protein carbonylation and nitration in the diaphragm and limb muscles of the animals, while inducing a significant increase in protein oxidation in their lungs. Interestingly, in the muscle specimens, modifications in oxidative stress markers were not accompanied by any significant variation in the content of the antioxidant mechanisms Mn-SOD or catalase, whereas significant reductions in the levels of these antioxidant enzymes were observed in the lungs. Furthermore, although there was a statistically significant increase in inflammatory cell counts in the diaphragm and gastrocnemius muscles of high-VTrats compared with control animals, such an increase was quite likely to be of small biologic relevance in terms of contribution to inflammatory and oxidative stress events in either respiratory or limb muscles of the mechanically ventilated animals. Finally, inflammatory cell infiltrates were greater in the lungs of mechanically ventilated rats compared with the nonventilated controls.
Muscle Redox Balance and Inflammation
In the current investigation, against our original hypothesis, high VTmechanical ventilation induced a significant decrease in the levels of oxidative stress, as measured by protein carbonylation and nitration, in the diaphragms, gastrocnemius, soleus, and tibialis anterior muscles compared with nonventilated animals. Carbonyl formation (ketones and aldehydes) is an important detectable marker of protein oxidation. Carbonyl groups can be formed by the direct reaction of proteins with ROS, leading to the formation of protein derivatives containing highly reactive carbonyl groups,31,32 and by Michael-addition reactions of lysine, cysteine, or histidine residues with unsaturated aldehydes (hydroxynonenal and malondialdehyde) formed during the peroxidation of polyunsaturated fatty acids.31,32 Conversely, protein tyrosine nitration is usually considered a marker of excessive reactive nitrogen species production in the tissues. Peroxynitrite, which is formed from the near-diffusion limited reaction between nitric oxide and superoxide anions, is the most widely accepted mechanism of in vivo  tyrosine nitration in the skeletal muscles.33,34 
In the current investigation, modifications observed in protein carbonylation and nitration in both the respiratory and limb muscles of healthy animals ventilated at high VTmay also be part of the systemic effects of mechanical ventilation in the organs and systems other than the lungs. Indeed, high VTmechanical ventilation of healthy rats was already shown to induce cardiovascular, liver, and gut injury in addition to systemic inflammation.11–14,17,19 In this study, high-VTventilated rats exhibited hypotension, hypoxemia, acidosis, and hyperlactatemia—findings that are in keeping with previous reports.17–19 Although the organ and skeletal muscle blood flow was not measured in this study, it would be possible to conclude that perfusion of organs and muscles in high-VTrats was quite likely to be reduced as a result of the hypotension exhibited by these animals after the 1-h period of mechanical ventilation. Decreased blood flow and hypoxemia may well account for the reduced oxidative stress levels detected in both the respiratory and limb muscles of high-VTrodents after mechanical ventilation compared with control animals. Interestingly, protein carbonylation (both reactive carbonyls and malondialdehyde-protein adducts) and nitration (3-nitrotyrosine immunoreactivity) levels were also significantly decreased in the soleus and tibialis anterior muscles of mechanically ventilated rats, using moderate VT(9 ml/kg). Although not tested in the current investigation, it could be hypothesized that mechanical ventilation per se  could alter vascular function and blood flow15,19 in healthy animals, especially that of more distal and smaller muscles, such as the tibialis anterior and the soleus. Clearly, future studies will be designed to further explore this hypothesis.
It is worth noting that in none of the muscles examined, protein levels of the potent antioxidants Mn-SOD or catalase were modified by any of the mechanical ventilation strategies used in this study. These findings reinforce the concept that reductions in protein carbonylation and nitration levels detected in the diaphragm and limb muscles of mechanically ventilated healthy rats were most likely due to decreased ROS production within the skeletal muscle fibers. On the grounds that certain amounts of ROS and reactive nitrogen species are required for normal muscle force production, it could also be anticipated that decreased oxidative stress levels in the diaphragm and the limb muscles of mechanically ventilated animals may depress the muscle performance. Although not examined in the present investigation, Reid et al.  35 already demonstrated that depletion of ROS by incubation of diaphragm fiber bundles with the antioxidant catalase or by enzymatic removal of superoxide anions substantially depressed muscle contractile function. The results of their investigations led to the conclusion that in unfatigued muscles, ROS are mandatory for optimal contractile performance by facilitating, for instance, excitation-contraction coupling.35 In conclusion, mechanical ventilation-induced oxidative stress cannot be assumed in all organs or tissues, at least in early stages. Administration of antioxidants36–38 might not all be justified in these specific models of VILI.
Despite the statistically significant increase in inflammatory cells detected in the diaphragms of ventilated rats (both high and moderate VT) and in the gastrocnemius of high-VTanimals, such results are likely to be of small biologic relevance, because total levels of inflammatory cells were, in fact, extremely low in both respiratory and limb muscles of the three groups of rodents. These findings are in keeping with recent data obtained in our laboratory (Esther Barreiro, M.D., Ph.D., unpublished observations, 2009), in which intramuscular inflammatory cell counts were also shown to be extremely low in the diaphragm, external intercostals, and vastus lateralis muscles of chronic obstructive pulmonary disease patients and control subjects.
Lung Redox Balance and Inflammation
A redox imbalance in the lungs has already been demonstrated in several models of VILI.5–8 In our study, protein carbonylation levels, as measured by malondialdehyde-protein adducts, but not protein tyrosine nitration, were significantly greater in the lungs of high-VTrats than in controls. Lung content of the antioxidants Mn-SOD and catalase, however, were significantly reduced in both groups of mechanically ventilated rodents compared with controls. Previous reports have also shown a decrease in antioxidant capacity in healthy lungs exposed to high VTventilation6,39 and an increase in malondialdehyde concentrations in the alveolar lining fluid.7 The underlying mechanisms accounting for such a redox imbalance are probably based on the cyclic stretch of the lungs induced by mechanical ventilation. In this regard, exposure of the lung epithelial and endothelial cells to cyclic stretch increased ROS production as early as 30 min.40,41 Besides, intense cyclic stretch also reduces glutathione and increases glutathione oxidation,40,42 suggesting that redox imbalance as a result of cyclic stretch is a major contributor to the onset and perpetuation of VILI.5 
On the basis of the previous documented immunohistochemical tyrosine nitration of lung proteins in patients with ARDS,8 nitrosative stress through protein nitration by reactive nitrogen species was also a proposed contributor to the pathogenesis of ALI. In this study, however, lung protein tyrosine nitration levels did not significantly differ among the three groups of animals. Moreover, we do not believe that tyrosine nitration of lung proteins through the action of leukocyte myeloperoxidases has taken place in mechanically ventilated rats, because the rise in inflammatory cell infiltrates was not accompanied by increased protein tyrosine nitration in their lungs. It is quite likely that mechanical ventilation for longer periods of time would have been associated with enhanced protein tyrosine nitration levels through either myeloperoxidase- or peroxynitrite-mediated mechanisms.
In the lungs of high-VTventilated rodents, the number and size of the inflammatory infiltrates was significantly greater than in the control group. Furthermore, the size of the infiltrated area, but not the number of infiltrates, was also significantly greater in moderate-VTrats compared with nonventilated animals. This led us to the observation that inflammatory infiltrates, although less abundant, were slightly larger in moderate-VTrats compared with high-VTanimals. Moreover, it should also be mentioned that the increase in inflammatory cell infiltrates observed in the lungs of mechanically ventilated animals is consistent with previous reports, in which the levels of several inflammatory cytokines, such as interleukin-1 β, interleukin-6, tumor necrosis factor-α, interleukin-8, and CXC chemokines,10,43,44 were also shown to be increased in the lungs of healthy animals exposed to high VTmechanical ventilation.
Study Critique
In the current investigation, the first limitation is related to the clinical relevance of the findings encountered by using this specific model of VILI, in which lung injury was induced by the administration of high VTmechanical ventilation to healthy rats for a short period of time. In humans, 12 ml/kg has been shown to induce harmful effects on lungs,45 whereas this VTwould be harmless in healthy rats. Furthermore, standard mechanical ventilation in humans has been recently shown to induce marked atrophy of the diaphragm fibers and increased proteolysis as a result of inactivity.46 It is worth noting that the ventilatory strategies used in the current investigation were within the scope of those used in previously published reports from our group and other investigators,10–19,47–49 in which key physiologic alterations defining the pulmonary and nonpulmonary effects of high VTmechanical ventilation were demonstrated. In this regard, it is common practice to apply high-extreme conditions (clinical and/or physiologic) in completely healthy animals to generate contrast and to ensure that biologic differences among groups cannot be the subject of methodological concerns. On this basis, it is possible to conclude that compared with humans, administration of much larger VTis required in healthy rats to elicit lung injury in addition to pathophysiologic and biologic responses. Furthermore, these different ventilatory strategies were used in this study with the aim of exploring lung overdistension in the presence or absence of PEEP. Importantly, in previously published investigations,10–19 these ventilatory strategies were shown to yield similar end-inspiratory lung volumes. Hence, we believe that administration of a high VTcan be perfectly justified in this model.
The second limitation is related to the short period of mechanical ventilation administered to the animals. However, our main goal in this experimental study was to explore the early molecular events involving redox balance of slow- and fast-twitch muscles and lungs in response to high VTmechanical ventilation in an animal model of VILI. Future studies will be definitely designed to assess medium- and long-term effects of VILI in organs and muscles by using a wider range of ventilatory strategies including lower VT. Importantly, in the present investigation, inflammation and posttranslational oxidative modifications of both muscle and lung proteins have been evaluated in healthy rodents exposed to high VT, thus showing significant variations in both the respiratory and limb muscles and the lungs compared with control animals.
Despite these recognized limitations, we believe that the study of events taking place very early after the pulmonary insult (high VTstretch) is of clinical relevance, because early occurrence of oxidative stress during high VTmechanical ventilation cannot be assumed in all organs or tissues. In fact, a reduction, rather than an increase, in oxidative stress was observed in both the respiratory and limb muscles of healthy rats exposed to large VTand VILI for a relatively short period of time.
Conclusions
Our study provides evidence that even with the use of extremely large VTand the creation of VILI in healthy rodents, there was no increase in oxidative stress in the diaphragm or limb muscles. In fact, at a particular time point, high VTmechanical ventilation did not induce the same oxidative and inflammatory events in all the rat tissues, whereas oxidative stress and inflammation increased in the lungs and 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.
The authors thank Francesc Sánchez, Sandra Mas, and Maitane Pérez (Laboratory Technicians, Pulmonology Department-Muscle and Respiratory System Research Unit, Institut Municipal d'Investigació Mèdica-Hospital del Mar, Parc de Recerca Biomèdica de Barcelona, Barcelona, Catalonia, Spain) for their technical assistance in the laboratory and Dr. Rafael Marcos, M.D., Ph.D. (Associate Professor, Unitat d'Epidemiologia i Registre de Càncer de Girona, Pla Director d'Oncologia, Departament de Salut, Institut d'Investigació Biomèdica de Girona, Girona, Catalonia, Spain), for his generous help with the statistical analyses.
References
Esteban A, Anzueto A, Frutos F, Alía I, Brochard L, Stewart TE, Benito S, Epstein SK, Apezteguía C, Nightingale P, Arroliga AC, Tobin MJ; Mechanical Ventilation International Study Group: Characteristics and outcomes in adult patients receiving mechanical ventilation: A 28-day international study. JAMA 2002; 287:345–55Esteban, A Anzueto, A Frutos, F Alía, I Brochard, L Stewart, TE Benito, S Epstein, SK Apezteguía, C Nightingale, P Arroliga, AC Tobin, MJ Mechanical Ventilation International Study Group,
Webb HH, Tierney DF: Experimental pulmonary edema due to intermittent positive pressure ventilation with high inflation pressures: Protection by positive end-expiratory pressure. Am Rev Respir Dis 1974; 110:556–65Webb, HH Tierney, DF
Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network. N Engl J Med 2000; 342:1301–8
Tremblay LN, Slutsky AS: Ventilator-induced lung injury: From the bench to the bedside. Intensive Care Med 2006; 32:24–33Tremblay, LN Slutsky, AS
Reddy SP, Hassoun PM, Brower R: Redox imbalance and ventilator-induced lung injury. Antioxid Redox Signal 2007; 9:2003–12Reddy, SP Hassoun, PM Brower, R
Collard KJ, Godeck S, Holley JE, Quinn MW: Pulmonary antioxidant concentrations and oxidative damage in ventilated premature babies. Arch Dis Child Fetal Neonatal Ed 2004; 89:F412–6Collard, KJ Godeck, S Holley, JE Quinn, MW
Cheng YJ, Chan KC, Chien CT, Sun WZ, Lin CJ: Oxidative stress during 1-lung ventilation. J Thorac Cardiovasc Surg 2006; 132:513–8Cheng, YJ Chan, KC Chien, CT Sun, WZ Lin, CJ
Haddad IY, Pataki G, Hu P, Galliani C, Beckman JS, Matalon S: Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J Clin Invest 1994; 94:2407–13Haddad, IY Pataki, G Hu, P Galliani, C Beckman, JS Matalon, S
Kawano T, Mori S, Cybulsky M, Burger R, Ballin A, Cutz E, Bryan AC: Effect of granulocyte depletion in a ventilated surfactant-depleted lung. J Appl Physiol 1987; 62:27–33Kawano, T Mori, S Cybulsky, M Burger, R Ballin, A Cutz, E Bryan, AC
Tremblay L, Valenza F, Ribeiro SP, Li J, Slutsky AS: Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest 1997; 99:944–52Tremblay, L Valenza, F Ribeiro, SP Li, J Slutsky, AS
Nin N, Peñuelas O, de Paula M, Lorente JA, Fernández-Segoviano P, Esteban A: Ventilation-induced lung injury in rats is associated with organ injury and systemic inflammation that is attenuated by dexamethasone. Crit Care Med 2006; 34:1093–8Nin, N Peñuelas, O de Paula, M Lorente, JA Fernández-Segoviano, P Esteban, A
Chiumello D, Pristine G, Slutsky AS: Mechanical ventilation affects local and systemic cytokines in an animal model of acute respiratory distress syndrome. Am J Respir Crit Care Med 1999; 160:109–16Chiumello, D Pristine, G Slutsky, AS
Choi WI, Quinn DA, Park KM, Moufarrej RK, Jafari B, Syrkina O, Bonventre JV, Hales CA: Systemic microvascular leak in an in vivo  rat model of ventilator-induced lung injury. Am J Respir Crit Care Med 2003; 167:1627–32Choi, WI Quinn, DA Park, KM Moufarrej, RK Jafari, B Syrkina, O Bonventre, JV Hales, CA
Guery BP, Welsh DA, Viget NB, Robriquet L, Fialdes P, Mason CM, Beaucaire G, Bagby GJ, Neviere R: Ventilation-induced lung injury is associated with an increase in gut permeability. Shock 2003; 19:559–63Guery, BP Welsh, DA Viget, NB Robriquet, L Fialdes, P Mason, CM Beaucaire, G Bagby, GJ Neviere, R
Nin N, Valero JA, Lorente JA, de Paula M, Fernández-Segoviano P, Sánchez-Ferrer A, Esteban A: Large tidal volume mechanical ventilation induces vascular dysfunction in rats. J Trauma 2005; 59:711–6Nin, N Valero, JA Lorente, JA de Paula, M Fernández-Segoviano, P Sánchez-Ferrer, A Esteban, A
Jerng JS, Hsu YC, Wu HD, Pan HZ, Wang HC, Shun CT, Yu CJ, Yang PC: Role of the renin-angiotensin system in ventilator-induced lung injury: An in vivo  study in a rat model. Thorax 2007; 62:527–35Jerng, JS Hsu, YC Wu, HD Pan, HZ Wang, HC Shun, CT Yu, CJ Yang, PC
Nin N, Lorente JA, de Paula M, El Assar M, Vallejo S, Peñuelas O, Fernández-Segoviano P, Ferruelo A, Sánchez-Ferrer A, Esteban A: Rats surviving injurious mechanical ventilation show reversible pulmonary, vascular and inflammatory changes. Intensive Care Med 2008; 34:948–56Nin, N Lorente, JA de Paula, M El Assar, M Vallejo, S Peñuelas, O Fernández-Segoviano, P Ferruelo, A Sánchez-Ferrer, A Esteban, A
Nin N, Lorente JA, De Paula M, Fernández-Segoviano P, Peñuelas O, Sánchez-Ferrer A, Martínez-Caro L, Esteban A: Aging increases the susceptibility to injurious mechanical ventilation. Intensive Care Med 2008; 34:923–31Nin, N Lorente, JA De Paula, M Fernández-Segoviano, P Peñuelas, O Sánchez-Ferrer, A Martínez-Caro, L Esteban, A
Martínez-Caro L, Lorente JA, Marín-Corral J, Sánchez-Rodríguez C, Sánchez-Ferrer A, Nin N, Ferruelo A, de Paula M, Fernández-Segoviano P, Barreiro E, Esteban A: Role of free radicals in vascular dysfunction induced by high tidal volume ventilation. Intensive Care Med 2009; 35:1110–9Martínez-Caro, L Lorente, JA Marín-Corral, J Sánchez-Rodríguez, C Sánchez-Ferrer, A Nin, N Ferruelo, A de Paula, M Fernández-Segoviano, P Barreiro, E Esteban, A
Levine RL, Williams JA, Stadtman ER, Shacter E: Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 1994; 233:346–57Levine, RL Williams, JA Stadtman, ER Shacter, E
Requena JR, Fu MX, Ahmed MU, Jenkins AJ, Lyons TJ, Thorpe SR: Lipoxidation products as biomarkers of oxidative damage to proteins during lipid peroxidation reactions. Nephrol Dial Transplant 1996; 11:48–53Requena, JR Fu, MX Ahmed, MU Jenkins, AJ Lyons, TJ Thorpe, SR
Barreiro E, de la Puente B, Minguella J, Corominas JM, Serrano S, Hussain SN, Gea J: Oxidative stress and respiratory muscle dysfunction in severe chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2005; 171:1116–24Barreiro, E de la Puente, B Minguella, J Corominas, JM Serrano, S Hussain, SN Gea, J
Barreiro E, Nowinski A, Gea J, Sliwinski P: Oxidative stress in the external intercostal muscles of patients with obstructive sleep apnoea. Thorax 2007; 62:1095–101Barreiro, E Nowinski, A Gea, J Sliwinski, P
Bustamante V, Casanova J, López de Santamaría E, Mas S, Sellarés J, Gea J, Gáldiz JB, Barreiro E: Redox balance following magnetic stimulation training in the quadriceps of patients with severe COPD. Free Radic Res 2008; 42:939–48Bustamante, V Casanova, J López de Santamaría, E Mas, S Sellarés, J Gea, J Gáldiz, JB Barreiro, E
Barreiro E, Rabinovich R, Marin-Corral J, Barberà JA, Gea J, Roca J: Chronic endurance exercise induces quadriceps nitrosative stress in patients with severe COPD. Thorax 2009; 64:13–9Barreiro, E Rabinovich, R Marin-Corral, J Barberà, JA Gea, J Roca, J
Barreiro E, Garcia-Martínez C, Mas S, Ametller E, Gea J, Argilés JM, Busquets S, López-Soriano FJ: UCP3 overexpression neutralizes oxidative stress rather than nitrosative stress in mouse myotubes. FEBS Lett 2009; 583:350–6Barreiro, E Garcia-Martínez, C Mas, S Ametller, E Gea, J Argilés, JM Busquets, S López-Soriano, FJ
Gosker HR, Kubat B, Schaart G, van der Vusse GJ, Wouters EF, Schols AM: Myopathological features in skeletal muscle of patients with chronic obstructive pulmonary disease. Eur Respir J 2003; 22:280–5Gosker, HR Kubat, B Schaart, G van der Vusse, GJ Wouters, EF Schols, AM
Montes de Oca M, Torres SH, De Sanctis J, Mata A, Hernández N, Tálamo C: Skeletal muscle inflammation and nitric oxide in patients with COPD. Eur Respir J 2005; 26:390–7Montes de Oca, M Torres, SH De Sanctis, J Mata, A Hernández, N Tálamo, C
Stapleton CM, Jaradat M, Dixon D, Kang HS, Kim SC, Liao G, Carey MA, Cristiano J, Moorman MP, Jetten AM: Enhanced susceptibility of staggerer (RORalphasg/sg) mice to lipopolysaccharide-induced lung inflammation. Am J Physiol Lung Cell Mol Physiol 2005; 289:L144–52Stapleton, CM Jaradat, M Dixon, D Kang, HS Kim, SC Liao, G Carey, MA Cristiano, J Moorman, MP Jetten, AM
Li YT, Yao CS, Bai JY, Lin M, Cheng GF: Anti-inflammatory effect of amurensin H on asthma-like reaction induced by allergen in sensitized mice. Acta Pharmacol Sin 2006; 27:735–40Li, YT Yao, CS Bai, JY Lin, M Cheng, GF
Stadtman ER, Levine RL: Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 2003; 25:207–18Stadtman, ER Levine, RL
Requena JR, Levine RL, Statdman ER: Recent advances in the analysis of oxidized proteins. Amino Acids 2003; 25:221–6Requena, JR Levine, RL Statdman, ER
Beckman JS, Koppenol WH: Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and ugly. Am J Physiol 1996; 271:C1424–37Beckman, JS Koppenol, WH
Barreiro E, Comtois AS, Gea J, Laubach VE, Hussain SN: Protein tyrosine nitration in the ventilatory muscles: Role of nitric oxide synthases. Am J Respir Cell Mol Biol 2002; 26:438–46Barreiro, E Comtois, AS Gea, J Laubach, VE Hussain, SN
Reid MB, Khawli FA, Moody MR: Reactive oxygen in skeletal muscle: III. Contractility of unfatigued muscle. J Appl Physiol 1993; 75:1081–7Reid, MB Khawli, FA Moody, MR
Jepsen S, Herlevsen P, Knudsen P, Bud MI, Klausen NO: Antioxidant treatment with N-acetylcysteine during adult respiratory distress syndrome: A prospective, randomized, placebo-controlled study. Crit Care Med 1992; 20:918–23Jepsen, S Herlevsen, P Knudsen, P Bud, MI Klausen, NO
Suter PM, Domenighetti G, Schaller MD, Laverrière MC, Ritz R, Perret C: N-acetylcysteine enhances recovery from acute lung injury in man. A randomized, double-blind, placebo-controlled clinical study. Chest 1994; 105:190–4Suter, PM Domenighetti, G Schaller, MD Laverrière, MC Ritz, R Perret, C
Bernard GR, Wheeler AP, Arons MM, Morris PE, Paz HL, Russell JA, Wright PE: A trial of antioxidants N-acetylcysteine and procysteine in ARDS. The Antioxidant in ARDS Study Group. Chest 1997; 112:164–72Bernard, GR Wheeler, AP Arons, MM Morris, PE Paz, HL Russell, JA Wright, PE
Jafari B, Ouyang B, Li LF, Hales CA, Quinn DA: Intracellular glutathione in stretch-induced cytokine release from alveolar type-2 like cells. Respirology 2004; 9:43–53Jafari, B Ouyang, B Li, LF Hales, CA Quinn, DA
Chapman KE, Sinclair SE, Zhuang D, Hassid A, Desai LP, Waters CM: Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 2005; 289:L834–41Chapman, KE Sinclair, SE Zhuang, D Hassid, A Desai, LP Waters, CM
Chess PR, O'Reilly MA, Sachs F, Finkelstein JN: Reactive oxidant and p42/44 MAP kinase signaling is necessary for mechanical strain-induced proliferation in pulmonary epithelial cells. J Appl Physiol 2005; 99:1226–32Chess, PR O'Reilly, MA Sachs, F Finkelstein, JN
Papaiahgari S, Yerrapureddy A, Hassoun PM, Garcia JG, Birukov KG, Reddy SP: EGFR-activated signaling and actin remodeling regulate cyclic stretch-induced NRF2-ARE activation. Am J Respir Cell Mol Biol 2007; 36:304–12Papaiahgari, S Yerrapureddy, A Hassoun, PM Garcia, JG Birukov, KG Reddy, SP
Frank JA, Pittet JF, Wray C, Matthay MA: Protection from experimental ventilator-induced acute lung injury by IL-1 receptor blockade. Thorax 2008; 63:147–53Frank, JA Pittet, JF Wray, C Matthay, MA
Halbertsma FJ, Vaneker M, Scheffer GJ, van der Hoeven JG: Cytokines and biotrauma in ventilator-induced lung injury: A critical review of the literature. Neth J Med 2005; 63:382–92Halbertsma, FJ Vaneker, M Scheffer, GJ van der Hoeven, JG
Gajic O, Frutos-Vivar F, Esteban A, Hubmayr RD, Anzueto A: Ventilator settings as a risk factor for acute respiratory distress syndrome in mechanically ventilated patients. Intensive Care Med 2005; 31:922–6Gajic, O Frutos-Vivar, F Esteban, A Hubmayr, RD Anzueto, A
Levine S, Nguyen T, Taylor N, Friscia ME, Budak MT, Rothenberg P, Zhu J, Sachdeva R, Sonnad S, Kaiser LR, Rubinstein NA, Powers SK, Shrager JB: Rapid disuse atrophy of diaphragm fibers in mechanically ventilated humans. N Engl J Med 2008; 358:1327–35Levine, S Nguyen, T Taylor, N Friscia, ME Budak, MT Rothenberg, P Zhu, J Sachdeva, R Sonnad, S Kaiser, LR Rubinstein, NA Powers, SK Shrager, JB
Imai Y, Parodo J, Kajikawa O, de Perrot M, Fischer S, Edwards V, Cutz E, Liu M, Keshavjee S, Martin TR, Marshall JC, Ranieri VM, Slutsky AS: Injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome. JAMA 2003; 289:2104–12Imai, Y Parodo, J Kajikawa, O de Perrot, M Fischer, S Edwards, V Cutz, E Liu, M Keshavjee, S Martin, TR Marshall, JC Ranieri, VM Slutsky, AS
Peng X, Abdulnour RE, Sammani S, Ma SF, Han EJ, Hasan EJ, Tuder R, Garcia JG, Hassoun PM: Inducible nitric oxide synthase contributes to ventilator-induced lung injury. Am J Respir Crit Care Med 2005; 172:470–9Peng, X Abdulnour, RE Sammani, S Ma, SF Han, EJ Hasan, EJ Tuder, R Garcia, JG Hassoun, PM
Takenaka K, Nishimura Y, Nishiuma T, Sakashita A, Yamashita T, Kobayashi K, Satouchi M, Ishida T, Kawashima S, Yokoyama M: Ventilator-induced lung injury is reduced in transgenic mice that overexpress endothelial nitric oxide synthase. Am J Physiol Lung Cell Mol Physiol 2006; 290:L1078–86Takenaka, K Nishimura, Y Nishiuma, T Sakashita, A Yamashita, T Kobayashi, K Satouchi, M Ishida, T Kawashima, S Yokoyama, M
Fig. 1. Representative examples of protein oxidation (total carbonyl groups) immunoblots in the diaphragms (A  ) of control (n = 8), moderate tidal volume (VT, n = 8), and high VT(n = 8) mechanically ventilated rats. Several oxidized proteins of different molecular weights (MW, in kilo Daltons) were observed in all muscles. Representative immunoblots of oxidized proteins of different MW, in kilodaltons, in the gastrocnemius (B  ) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total reactive carbonyl groups to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Reactive carbonyl levels were significantly reduced in the diaphragms (C  ) of the high-VTrats compared with either control or moderate-VTanimals. In the gastrocnemius (D  ), reactive carbonyls were significantly decreased in high-VTrats compared with controls and almost significantly reduced compared with moderate-VTrodents. In the soleus (E  ) and tibialis anterior (F  ), reactive carbonyl levels were significantly decreased in high- and moderate-VTrats compared with controls.
Fig. 1. Representative examples of protein oxidation (total carbonyl groups) immunoblots in the diaphragms (A 
	) of control (n = 8), moderate tidal volume (VT, n = 8), and high VT(n = 8) mechanically ventilated rats. Several oxidized proteins of different molecular weights (MW, in kilo Daltons) were observed in all muscles. Representative immunoblots of oxidized proteins of different MW, in kilodaltons, in the gastrocnemius (B 
	) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total reactive carbonyl groups to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Reactive carbonyl levels were significantly reduced in the diaphragms (C 
	) of the high-VTrats compared with either control or moderate-VTanimals. In the gastrocnemius (D 
	), reactive carbonyls were significantly decreased in high-VTrats compared with controls and almost significantly reduced compared with moderate-VTrodents. In the soleus (E 
	) and tibialis anterior (F 
	), reactive carbonyl levels were significantly decreased in high- and moderate-VTrats compared with controls.
Fig. 1. Representative examples of protein oxidation (total carbonyl groups) immunoblots in the diaphragms (A  ) of control (n = 8), moderate tidal volume (VT, n = 8), and high VT(n = 8) mechanically ventilated rats. Several oxidized proteins of different molecular weights (MW, in kilo Daltons) were observed in all muscles. Representative immunoblots of oxidized proteins of different MW, in kilodaltons, in the gastrocnemius (B  ) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total reactive carbonyl groups to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Reactive carbonyl levels were significantly reduced in the diaphragms (C  ) of the high-VTrats compared with either control or moderate-VTanimals. In the gastrocnemius (D  ), reactive carbonyls were significantly decreased in high-VTrats compared with controls and almost significantly reduced compared with moderate-VTrodents. In the soleus (E  ) and tibialis anterior (F  ), reactive carbonyl levels were significantly decreased in high- and moderate-VTrats compared with controls.
×
Fig. 2. Optical densities in the box plots are expressed as the ratio of the optical densities of total malondialdehyde (MDA)-protein adducts to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. There was an almost significant decrease in MDA-protein adducts in the diaphragms (A  ) and gastrocnemius (B  ) muscles of the high-tidal volume (VT, n = 8) rats compared with the controls (n = 8). In the soleus (C  ) and tibialis anterior (D  ), MDA-protein adduct levels were significantly decreased in high- and moderate-VTrats (n = 8) compared with the controls. Representative immunoblots of MDA-protein adducts of different molecular weights (MW), in kilo Daltons, in the lungs (E  ) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total MDA-protein adducts to those of GAPDH in the lungs (F  ). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted (F  ). Lungs of high-VTrats exhibited a significant increase in MDA-protein adducts compared with either controls or moderate-VT animals. The lungs of moderate-VTrodents showed an almost significant increase in MDA-protein adducts compared with control animals (F  ).
Fig. 2. Optical densities in the box plots are expressed as the ratio of the optical densities of total malondialdehyde (MDA)-protein adducts to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. There was an almost significant decrease in MDA-protein adducts in the diaphragms (A 
	) and gastrocnemius (B 
	) muscles of the high-tidal volume (VT, n = 8) rats compared with the controls (n = 8). In the soleus (C 
	) and tibialis anterior (D 
	), MDA-protein adduct levels were significantly decreased in high- and moderate-VTrats (n = 8) compared with the controls. Representative immunoblots of MDA-protein adducts of different molecular weights (MW), in kilo Daltons, in the lungs (E 
	) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total MDA-protein adducts to those of GAPDH in the lungs (F 
	). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted (F 
	). Lungs of high-VTrats exhibited a significant increase in MDA-protein adducts compared with either controls or moderate-VT animals. The lungs of moderate-VTrodents showed an almost significant increase in MDA-protein adducts compared with control animals (F 
	).
Fig. 2. Optical densities in the box plots are expressed as the ratio of the optical densities of total malondialdehyde (MDA)-protein adducts to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. There was an almost significant decrease in MDA-protein adducts in the diaphragms (A  ) and gastrocnemius (B  ) muscles of the high-tidal volume (VT, n = 8) rats compared with the controls (n = 8). In the soleus (C  ) and tibialis anterior (D  ), MDA-protein adduct levels were significantly decreased in high- and moderate-VTrats (n = 8) compared with the controls. Representative immunoblots of MDA-protein adducts of different molecular weights (MW), in kilo Daltons, in the lungs (E  ) of control, moderate-VT, and high-VTmechanically ventilated rats. Optical densities in the box plots are expressed as the ratio of the optical densities of total MDA-protein adducts to those of GAPDH in the lungs (F  ). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted (F  ). Lungs of high-VTrats exhibited a significant increase in MDA-protein adducts compared with either controls or moderate-VT animals. The lungs of moderate-VTrodents showed an almost significant increase in MDA-protein adducts compared with control animals (F  ).
×
Fig. 3. Optical densities in the box plots are expressed as the ratio of the optical densities of total protein tyrosine nitration to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. In the diaphragms (A  ) and gastrocnemius (B  ) muscles of the high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats, protein nitration levels were significantly reduced compared with moderate-VTanimals (n = 8) but not controls (n = 8). In the soleus (C  ) and tibialis anterior (D  ), protein tyrosine nitration levels were significantly decreased in high- and moderate-VTrats compared with the controls.
Fig. 3. Optical densities in the box plots are expressed as the ratio of the optical densities of total protein tyrosine nitration to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. In the diaphragms (A 
	) and gastrocnemius (B 
	) muscles of the high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats, protein nitration levels were significantly reduced compared with moderate-VTanimals (n = 8) but not controls (n = 8). In the soleus (C 
	) and tibialis anterior (D 
	), protein tyrosine nitration levels were significantly decreased in high- and moderate-VTrats compared with the controls.
Fig. 3. Optical densities in the box plots are expressed as the ratio of the optical densities of total protein tyrosine nitration to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each muscle. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. In the diaphragms (A  ) and gastrocnemius (B  ) muscles of the high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats, protein nitration levels were significantly reduced compared with moderate-VTanimals (n = 8) but not controls (n = 8). In the soleus (C  ) and tibialis anterior (D  ), protein tyrosine nitration levels were significantly decreased in high- and moderate-VTrats compared with the controls.
×
Fig. 4. Optical densities in the box plots are expressed as the ratio of the optical densities of Mn-SOD (A  ) and catalase (B  ) protein contents to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lungs of the rat. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Levels of Mn-SOD protein content (A  ) were lower in the lungs of high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats compared with either controls (n = 8) or moderate-VTanimals (n = 8). Levels of Mn-SOD content in the lungs of moderate-VTrats were also significantly reduced compared with the controls. Levels of catalase protein (B  ) were lower in the lungs of high-VTrats compared with controls, but not with those in moderate-VTanimals. Levels of catalase content in the lungs of moderate-VTrats were also significantly reduced compared with the controls (B  ).
Fig. 4. Optical densities in the box plots are expressed as the ratio of the optical densities of Mn-SOD (A 
	) and catalase (B 
	) protein contents to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lungs of the rat. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Levels of Mn-SOD protein content (A 
	) were lower in the lungs of high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats compared with either controls (n = 8) or moderate-VTanimals (n = 8). Levels of Mn-SOD content in the lungs of moderate-VTrats were also significantly reduced compared with the controls. Levels of catalase protein (B 
	) were lower in the lungs of high-VTrats compared with controls, but not with those in moderate-VTanimals. Levels of catalase content in the lungs of moderate-VTrats were also significantly reduced compared with the controls (B 
	).
Fig. 4. Optical densities in the box plots are expressed as the ratio of the optical densities of Mn-SOD (A  ) and catalase (B  ) protein contents to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lungs of the rat. Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Levels of Mn-SOD protein content (A  ) were lower in the lungs of high-tidal volume (VT, n = 8) mechanically ventilated (MV) rats compared with either controls (n = 8) or moderate-VTanimals (n = 8). Levels of Mn-SOD content in the lungs of moderate-VTrats were also significantly reduced compared with the controls. Levels of catalase protein (B  ) were lower in the lungs of high-VTrats compared with controls, but not with those in moderate-VTanimals. Levels of catalase content in the lungs of moderate-VTrats were also significantly reduced compared with the controls (B  ).
×
Fig. 5. Immunohistochemical localization of both leukocytes (A  ) and macrophages (B  ) in human tonsils (positive controls, ×400). Although intramuscular inflammatory cell levels were low in all groups of rats, diaphragm fibers of high-tidal volume (VT) rats exhibited more prominent infiltration (C  ) than the control diaphragms (D  ). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Total inflammatory cell counts (inflammatory cells/muscle section area in square millimeters [mm2]) were significantly greater in the diaphragms (E  ) of high-VTmechanically ventilated (MV) rats (n = 8) compared with either moderate-VTanimals (n = 8) or the controls (n = 8). Diaphragm inflammatory cell counts were also greater in moderate-VTrats than in the controls (E  ). In the gastrocnemius (F  ), inflammatory cell counts (inflammatory cells/muscle section area in mm2) of high-VTrats were significantly increased compared with either controls or moderate-VTrats. Gastrocnemius inflammatory cell counts did not significantly differ between moderate-VTrats and controls (F  ).
Fig. 5. Immunohistochemical localization of both leukocytes (A 
	) and macrophages (B 
	) in human tonsils (positive controls, ×400). Although intramuscular inflammatory cell levels were low in all groups of rats, diaphragm fibers of high-tidal volume (VT) rats exhibited more prominent infiltration (C 
	) than the control diaphragms (D 
	). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Total inflammatory cell counts (inflammatory cells/muscle section area in square millimeters [mm2]) were significantly greater in the diaphragms (E 
	) of high-VTmechanically ventilated (MV) rats (n = 8) compared with either moderate-VTanimals (n = 8) or the controls (n = 8). Diaphragm inflammatory cell counts were also greater in moderate-VTrats than in the controls (E 
	). In the gastrocnemius (F 
	), inflammatory cell counts (inflammatory cells/muscle section area in mm2) of high-VTrats were significantly increased compared with either controls or moderate-VTrats. Gastrocnemius inflammatory cell counts did not significantly differ between moderate-VTrats and controls (F 
	).
Fig. 5. Immunohistochemical localization of both leukocytes (A  ) and macrophages (B  ) in human tonsils (positive controls, ×400). Although intramuscular inflammatory cell levels were low in all groups of rats, diaphragm fibers of high-tidal volume (VT) rats exhibited more prominent infiltration (C  ) than the control diaphragms (D  ). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. Total inflammatory cell counts (inflammatory cells/muscle section area in square millimeters [mm2]) were significantly greater in the diaphragms (E  ) of high-VTmechanically ventilated (MV) rats (n = 8) compared with either moderate-VTanimals (n = 8) or the controls (n = 8). Diaphragm inflammatory cell counts were also greater in moderate-VTrats than in the controls (E  ). In the gastrocnemius (F  ), inflammatory cell counts (inflammatory cells/muscle section area in mm2) of high-VTrats were significantly increased compared with either controls or moderate-VTrats. Gastrocnemius inflammatory cell counts did not significantly differ between moderate-VTrats and controls (F  ).
×
Fig. 6. Representative hematoxylin-eosin staining of a lung section from a control rat (A  , ×40). Representative hematoxylin-eosin infiltrates corresponding to a lung section from a moderate tidal volume (VT) rat (B  , ×40). The presence of the abundant inflammatory cells in the same infiltrate can be seen at ×400 (C  ). High-VTinduced an increase in the number of lung inflammatory cell infiltrates (D  and E  , ×40). Abundant inflammatory cells were also seen in these infiltrates (F  , ×400). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. The number of infiltrates (total number of infiltrates/total lung section area in square millimeters [mm2]) was greater in the lungs of high-VTmechanically ventilated (MV) rats (n = 8) compared with either control (n = 8) or moderate-VT(n = 8) animals (G  ). The number of lung infiltrates did not differ between moderate-VTrats and controls (G  ). Total lung inflammatory infiltrated area (total inflammatory infiltrated area in mm2/total lung section area in mm2) was significantly greater in high-VTrats compared with controls, but not moderate-VTrats (H  ). Total lung inflammatory infiltrated area was also significantly larger in the moderate-VTrats compared with controls (H  ).
Fig. 6. Representative hematoxylin-eosin staining of a lung section from a control rat (A 
	, ×40). Representative hematoxylin-eosin infiltrates corresponding to a lung section from a moderate tidal volume (VT) rat (B 
	, ×40). The presence of the abundant inflammatory cells in the same infiltrate can be seen at ×400 (C 
	). High-VTinduced an increase in the number of lung inflammatory cell infiltrates (D 
	and E 
	, ×40). Abundant inflammatory cells were also seen in these infiltrates (F 
	, ×400). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. The number of infiltrates (total number of infiltrates/total lung section area in square millimeters [mm2]) was greater in the lungs of high-VTmechanically ventilated (MV) rats (n = 8) compared with either control (n = 8) or moderate-VT(n = 8) animals (G 
	). The number of lung infiltrates did not differ between moderate-VTrats and controls (G 
	). Total lung inflammatory infiltrated area (total inflammatory infiltrated area in mm2/total lung section area in mm2) was significantly greater in high-VTrats compared with controls, but not moderate-VTrats (H 
	). Total lung inflammatory infiltrated area was also significantly larger in the moderate-VTrats compared with controls (H 
	).
Fig. 6. Representative hematoxylin-eosin staining of a lung section from a control rat (A  , ×40). Representative hematoxylin-eosin infiltrates corresponding to a lung section from a moderate tidal volume (VT) rat (B  , ×40). The presence of the abundant inflammatory cells in the same infiltrate can be seen at ×400 (C  ). High-VTinduced an increase in the number of lung inflammatory cell infiltrates (D  and E  , ×40). Abundant inflammatory cells were also seen in these infiltrates (F  , ×400). Standard box plots with median (twenty-fifth and seventy-fifth percentiles) and whiskers (at minimum and maximum values) are depicted. The number of infiltrates (total number of infiltrates/total lung section area in square millimeters [mm2]) was greater in the lungs of high-VTmechanically ventilated (MV) rats (n = 8) compared with either control (n = 8) or moderate-VT(n = 8) animals (G  ). The number of lung infiltrates did not differ between moderate-VTrats and controls (G  ). Total lung inflammatory infiltrated area (total inflammatory infiltrated area in mm2/total lung section area in mm2) was significantly greater in high-VTrats compared with controls, but not moderate-VTrats (H  ). Total lung inflammatory infiltrated area was also significantly larger in the moderate-VTrats compared with controls (H  ).
×
Table 1.  Changes in Blood Gases, Hemodynamic, and Mechanical Ventilatory Parameters in Rats Subjected to Mechanical Ventilation with Moderate-tidal (9 ml/kg) and High-tidal Volume (35 ml/kg)
Image not available
Table 1.  Changes in Blood Gases, Hemodynamic, and Mechanical Ventilatory Parameters in Rats Subjected to Mechanical Ventilation with Moderate-tidal (9 ml/kg) and High-tidal Volume (35 ml/kg)
×
Table 2.  Oxidative Stress Markers in the Skeletal Muscles and Lungs of Mechanically Ventilated and Control Rats
Image not available
Table 2.  Oxidative Stress Markers in the Skeletal Muscles and Lungs of Mechanically Ventilated and Control Rats
×