Simvastatin attenuates ventilator-induced lung injury in mice

Abstract

Introduction: Mechanical ventilation (MV) is a life saving intervention in acute respiratory failure without alternative. However, particularly in pre-injured lungs, even protective ventilation strategies may evoke ventilator-induced lung injury (VILI), which is characterized by pulmonary inflammation and vascular leakage. Adjuvant pharmacologic strategies in addition to lung protective ventilation to attenuate VILI are lacking. Simvastatin exhibited anti-inflammatory and endothelial barrier stabilizing properties in vitro and in vivo.

Methods: Mice were ventilated (12 ml/kg; six hours) and subjected to simvastatin (20 mg/kg) or sham treatment. Pulmonary microvascular leakage, oxygenation, pulmonary and systemic neutrophil and monocyte counts and cytokine release in lung and blood plasma were assessed. Further, lung tissue was analyzed by electron microscopy.

Results: Mechanical ventilation induced VILI, displayed by increased pulmonary microvascular leakage and endothelial injury, pulmonary recruitment of neutrophils and Gr-1high monocytes, and by liberation of inflammatory cytokines in the lungs. Further, VILI associated systemic inflammation characterized by blood leukocytosis and elevated plasma cytokines was observed. Simvastatin treatment limited pulmonary endothelial injury, attenuated pulmonary hyperpermeability, prevented the recruitment of leukocytes to the lung, reduced pulmonary cytokine levels and improved oxygenation in mechanically ventilated mice.

Conclusions: High-dose simvastatin attenuated VILI in mice by reducing MV-induced pulmonary inflammation and hyperpermeability.

Figure 1

Simvastatin improved oxygenation in VILI. (a) Peripheral Oxygen Saturation (SpO₂) was monitored continuously and (b) P/F ratio was assessed at the end of the 6 h ventilation period in simvastatin (6 h Vent. + Simva) or sham (6 h Vent.) treated mice. Simvastatin treatment prevented the decline of SpO₂ and improved oxygenation in VILI. (a: 6 h Vent. N = 8; 6 h Vent. + Simva n = 10; b: 6 h Vent. N = 7; 6 h Vent. + Simva n = 9; *P < 0.05).

Figure 2

Simvastatin reduced VILI-associated lung hyperpermeability. Human serum albumin (HSA; 1 mg) was injected 90 minutes prior to termination of the experiment. In non-ventilated simvastatin (NV + Simva) or sham (NV) treated mice, and in ventilated and simvastatin (6 h Vent. + Simva) or sham (6 h Vent.) treated mice, HSA levels in plasma and BAL were determined. Simvastatin treatment reduced VILI associated lung hyperpermeability. (NV n = 6; NV + Simva n = 7; 6 h Vent. N = 7; 6 h Vent. + Simva n = 6; *P < 0.05, ***P < 0.001).

Figure 3

Simvastatin reduced VILI-associated endothelial injury. In lung sections of non ventilated, sham treated mice (NV) (a, b) and non-ventilated, simvastatin treated mice (NV + Simva) (c, d), structurally intact capillaries containing numerous caveolae and vesicles in endothelial cells were seen. In lung sections of ventilated, sham treated mice (6 h Vent.) (e, f), capillaries showed pronounced endothelial cell swelling as well as loss of intracellular vesicles and caveolae. Lungs of ventilated, simvastatin treated mice (6 h Vent. + Simva) (g, h) had intact capillaries, and neither signs of endothelial cell swelling, nor reduction of intracellular vesicles was observed. (Representative images out of n = 8 each group are shown. Bar 2 μm).

Figure 4

Simvastatin treatment limited VILI-associated pulmonary leukocyte infiltration. After 6 h mechanical ventilation (MV) of simvastatin (6 h Vent + Simva) or sham treated mice (6 h Vent.) and in non-ventilated sham (NV) or simvastatin (NV + Simva) treated mice, leukocytes isolated from whole left lung tissue and from blood were differentiated by flow cytometry. MV increased pulmonary PMN (a) and GR-1^high monocytes (b). Simvastatin reduced PMN and monocyte counts in the lungs of ventilated mice. MV also increased circulating blood neutrophils (c) and Gr-1^high monocytes (d), whereas leukocyte (e) and lymphocyte (f) counts were not significantly altered by MV (F). PMN and Gr-1^high monocyte counts were higher in Simvastatin treated, ventilated mice (6 h Vent. + Simva), as compared to sham treated, ventilated mice (6 h Vent.). (a-b: NV n = 6; NV + Simva n = 7; 6 h Vent. N = 7; 6 h Vent. + Simva n = 6. c-d: NV n = 9; NV + Simva n = 9; 6 h Vent. N = 8; 6 h Vent. + Simva n = 8; *P < 0.05; **P < 0.01, ***P < 0.001).

Figure 5

Simvastatin attenuated VILI-associated pulmonary cytokine production. Simvastatin (6 h Vent. + Simva) or sham treated mice (6 h Vent.) subjected to 6 h of mechanical ventilation, and non-ventilated sham (NV) or simvastatin (NV + Simva) treated mice were sacrificed. Cytokine levels in homogenized lung tissue were quantified. MV evoked an increase of pulmonary cytokines. Simvastatin treatment limited VILI associated production of IL-1β, MIP-1α and IL-12p40. (n = 8 each group; *P < 0.05, **P < 0.01, ***P < 0.001). (b.t., below threshold).