The extent of ventilator-induced lung injury in mice partly depends on duration of mechanical ventilation

Abstract

Background. Mechanical ventilation (MV) has the potential to initiate ventilator-induced lung injury (VILI). The pathogenesis of VILI has been primarily studied in animal models using more or less injurious ventilator settings. However, we speculate that duration of MV also influences severity and character of VILI. Methods. Sixty-four healthy C57Bl/6 mice were mechanically ventilated for 5 or 12 hours, using lower tidal volumes with positive end-expiratory pressure (PEEP) or higher tidal volumes without PEEP. Fifteen nonventilated mice served as controls. Results. All animals remained hemodynamically stable and survived MV protocols. In both MV groups, PaO2 to FiO2 ratios were lower and alveolar cell counts were higher after 12 hours of MV compared to 5 hours. Alveolar-capillary permeability was increased after 12 hours compared to 5 hours, although differences did not reach statistical significance. Lung levels of inflammatory mediators did not further increase over time. Only in mice ventilated with increased strain, lung compliance declined and wet to dry ratio increased after 12 hours of MV compared to 5 hours. Conclusions. Deleterious effects of MV are partly dependent on its duration. Even lower tidal volumes with PEEP may initiate aspects of VILI after 12 hours of MV.

Figures

Figure 1

Hemodynamic parameters. Systolic blood pressures, heart rates, and peripheral oxygen saturation (SpO2) remained stable throughout 5 hours ((a)–(c)) and 12 hours ((d)–(f)) of mechanical ventilation (MV). Data are presented as median (IQR) of 12–15 ((a)-(b), (d)-(e)), or 3–6 ((c), (f)) mice per group (circle = LVT; square = HVT). LVT, HVT = LVT/PEEP or HVT/ZEEP ventilator settings.

Figure 2

Respiratory parameters. Lung function is represented by the PaO2 to FiO2 ratio (PaO2/FiO2) (a) and static compliance of the respiratory system (b). The reference (REF) represents the median PaO2/FiO2 from 7 mice ventilated with LVT/PEEP for 30 minutes, that is, 442.4 mmHg (396.2 to 501.0) (a) or the combined static compliance of LVT/PEEP and HVT/ZEEP-ventilated mice at t = 0 h (b). The static compliance did not differ between the two groups at t = 0 h. Data are presented as scatter plot (median) of 11–15 (a) or 4–8 (b) mice per group (triangle = REF; circle = LVT; square = HVT). *Illustrates primary statistical analysis (*P < 0.05, **P < 0.01). NVC = nonventilated controls; LVT, HVT = LVT/PEEP or HVT/ZEEP ventilator settings; 5 h, 12 h = 5 or 12 hours of ventilation.

Figure 3

Edema formation. (a): Edema formation is represented by wet to dry ratios of lung tissue (Wet/Dry). Data are presented as scatter plot (median) of 7-8 mice per group (triangle = NVC; circle = LVT; square = HVT). *Illustrates primary statistical analysis (*P < 0.05, **P < 0.01); #illustrates secondary statistical analysis (#P < 0.05). (b) In LVT/PEEP and HVT/ZEEP-ventilated mice, correlation analyses were performed between static compliance of respiratory system and wet/dry of pulmonary tissue. Linear correlations, Pearson correlation coefficients (r), and P values are depicted. NVC = nonventilated controls; LVT, HVT = LVT/PEEP or HVT/ZEEP ventilator settings; 5 h, 12 h = 5 or 12 hours of ventilation.

Figure 4

Alveolar-capillary permeability. ((a)-(b)) Alveolar-capillary permeability is represented by total protein and immunoglobulin (Ig)M levels in bronchoalveolar lavage fluid (BALF). (c) Alveolar epithelial type 1 cell injury is represented by receptor for advanced glycation end-products (RAGE) levels in BALF [18]. Data are presented as scatter plot (median) of 7-8 mice per group (triangle = NVC; circle = LVT; square = HVT). *Illustrates primary statistical analysis (*P < 0.05, **P < 0.01); #Illustrates secondary statistical analysis (#P < 0.05, ##P < 0.01). NVC = nonventilated controls; LVT, HVT = LVT/PEEP or HVT/ZEEP ventilator settings; 5 h, 12 h = 5 or 12 hours of ventilation.

Figure 5

Cell infiltration. (a) Total cell counts were measured in bronchoalveolar lavage fluid (BALF). ((b)-(c)) Differential cell counts were performed on BALF cytospin preparations to determine neutrophil and macrophage infiltration. Data are presented as scatter plot (median) of 6–8 mice per group (triangle = NVC; circle = LVT; square = HVT). *Illustrates primary statistical analysis (*P < 0.05, **P < 0.01); #illustrates secondary statistical analysis (##P < 0.01). NVC = nonventilated controls; LVT, HVT = LVT/PEEP or HVT/ZEEP ventilator settings; 5 h, 12 h = 5 or 12 hours of ventilation.

Figure 6

Inflammatory mediators. ((a)-(b)) Protein levels of the proinflammatory cytokines interleukin (IL)-1β and IL-6 were determined in supernatant of total lung homogenates (HMG). ((c)–(d)) In addition, protein levels of the chemotactic cytokines keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 were determined. Data are presented as scatter plot (median) of 7-8 mice per group (triangle = NVC; circle = LVT; square = HVT). *Illustrates primary statistical analysis (*P < 0.05, **P < 0.01); #illustrates secondary statistical analysis (#P < 0.05, ##P < 0.01). NVC = nonventilated controls; LVT, HVT = LVT/PEEP or HVT/ZEEP ventilator settings; 5 h, 12 h = 5 or 12 hours of ventilation.

Figure 7

Histopathology. Lung sections were stained with hematoxylin eosin (H&E) to analyze lung histopathology (magnification 100x). Total histopathology score was determined by the sum of the score for 4 pathologic parameters: (a) edema, (b) hemorrhage, (c) interstitial cell infiltration, and (d) hyaline membranes. NVC = nonventilated controls; LVT, HVT = LVT/PEEP or HVT/ZEEP ventilator settings; 5 h, 12 h = 5 or 12 hours of ventilation.