Pre- and posttreatment with hydrogen sulfide prevents ventilator-induced lung injury by limiting inflammation and oxidation

<div><p>Although essential in critical care medicine, mechanical ventilation often results in ventilator-induced lung injury. Low concentrations of hydrogen sulfide have been proven to have anti-inflammatory and anti-oxidative effects in the lung. The aim of this study was to analyze the kinetic effects of pre- and posttreatment with hydrogen sulfide in order to prevent lung injury as well as inflammatory and oxidative stress upon mechanical ventilation. Mice were either non-ventilated or mechanically ventilated with a tidal volume of 12 ml/kg for 6 h. Pretreated mice inhaled hydrogen sulfide in low dose for 1, 3, or 5 h prior to mechanical ventilation. Posttreated mice were ventilated with air followed by ventilation with hydrogen sulfide in various combinations. In addition, mice were ventilated with air for 10 h, or with air for 5 h and subsequently with hydrogen sulfide for 5 h. Histology, interleukin-1β, neutrophil counts, and reactive oxygen species formation were examined in the lungs. Both pre-and posttreatment with hydrogen sulfide time-dependently reduced or even prevented edema formation, gross histological damage, neutrophil influx and reactive oxygen species production in the lung. These results were also observed in posttreatment, when the experimental time was extended and hydrogen sulfide administration started as late as after 5 h air ventilation. In conclusion, hydrogen sulfide exerts lung protection even when its application is limited to a short or delayed period. The observed lung protection is mediated by inhibition of inflammatory and oxidative signaling.</p></div

Effect of expanded H₂S posttreatment on ventilator-induced lung damage.

<p>Mice spontaneously breathed air (control) or for 6 h, or they were mechanically ventilated with 12 ml/kg either with air alone (6 h air, 10 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). Another group of mice was first mechanically ventilated with air alone for 5 h, followed by ventilation with 80 ppm H₂S for another 5 h. Lung sections were stained with H&E. Representative pictures are shown for each experimental group as indicated (A). Alveolar wall thickness was measured (B) and ventilator-induced lung injury (VILI) score was calculated (C). Data represent means ± SEM for n = 6/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h air vent group; ^&<i>P</i><0.05 vs. 10h air vent group.</p

Effect of H₂S posttreatment on lung inflammation and oxidative stress.

<p>Mice spontaneously breathed air (control) or for 6 h, or they were mechanically ventilated with 12 ml/kg for 6 h either with air alone (6 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). All other mice were first mechanically ventilated with air alone for 5, 3, or 1 h, followed by ventilation with 80 ppm H₂S for another 1, 3, or 5 h as indicated. Bronchoalveolar lavage (BAL) IL-1β cytokine content was determined by ELISA (A). The fraction of neutrophil cells was measured in BAL fluid by cytospin analysis (B). Lung tissue sections were stained with dihydroethidium (C). Representative pictures are shown for each experimental group as indicated in C. ROS fluorescence intensity was measured, calculated, and expressed as fold induction compared to control group (D). Data represent means ± SEM for n = 7/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h air vent group; ^&<i>P</i><0.05 vs. 5h air vent + 1h H₂S vent group; ⁺<i>P</i><0.05 vs. 3h air vent + 3h H₂S vent group.</p

Effect of H₂S pretreatment on ventilator-induced lung injury.

<p>Mice spontaneously breathed air (control) or for 6 h, or they were mechanically ventilated with 12 ml/kg for 6 h either with air alone (6 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). All other mice spontaneously breathed air supplemented with 80 ppm H₂S 1, 3, or 5 h prior to mechanical ventilation with air for another 6 h as indicated. Lung sections were stained with H&E. Representative pictures are shown for each experimental group as indicated (A). Alveolar wall thickness was measured (B) and ventilator-induced lung injury (VILI) score was calculated (C). Data represent means ± SEM for n = 7/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h air vent group; ^&<i>P</i><0.05 vs. 1h H₂S pre + 6h air vent group.</p

Effect of expanded H₂S posttreatment on lung inflammation and oxidative stress.

<p>Mice spontaneously breathed air (control) or for 6 h, or they were mechanically ventilated with 12 ml/kg either with air alone (6 h air, 10 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). Another group of mice was first mechanically ventilated with air alone for 5 h, followed by ventilation with 80 ppm H₂S for another 5 h. Bronchoalveolar lavage (BAL) IL-1β cytokine content was determined by ELISA (A). The fraction of neutrophil cells was measured in BAL fluid by cytospin analysis (B). Lung tissue sections were stained with dihydroethidium (C). Representative pictures are shown for each experimental group as indicated in C. ROS fluorescence intensity was measured, calculated, and expressed as fold induction compared to control group (D). Data represent means ± SEM for n = 6/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h air vent group; ^&<i>P</i><0.05 vs. 10h air vent group; ⁺<i>P</i><0.05 vs. 5h air vent + 5h H₂S vent group.</p

Study design and timeline.

<p>(A) <b>Pretreatment.</b> Mice spontaneously breathed air (control) for 6 h or were mechanically ventilated with 12 ml/kg for 6 h with either air alone (6 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). All other mice spontaneously breathed air supplemented with 80 ppm H₂S for 1, 3, or 5 h prior to mechanical ventilation with air for another 6 h as indicated. (B) <b>Posttreatment.</b> Mice spontaneously breathed air (control) for 6 h or were mechanically ventilated with 12 ml/kg for 6 h with either air alone (6 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). All other mice were first mechanically ventilated with air alone for 5, 3, or 1 h, followed by ventilation with 80 ppm H₂S for another 1, 3, or 5 h as indicated. (C) <b>Expanded Posttreatment</b>. Mice spontaneously breathed air (control) for 6 h or were mechanically ventilated with 12 ml/kg with either air in the absence or presence of 80 ppm H₂S (6 h air, 6 h H₂S) or were ventilated for 10 h with air alone (10 h air) or for 5 h with air alone followed by ventilation with H₂S for another 5 h (5 h air + 5 h H₂S).</p

Effect of H₂S posttreatment on ventilator-induced lung injury.

<p>Mice spontaneously breathed air (control) or for 6 h, or they were mechanically ventilated with 12 ml/kg for 6 h either with air alone (6 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). All other mice were first mechanically ventilated with air alone for 5, 3, or 1 h, followed by ventilation with 80 ppm H₂S for another 1, 3, or 5 h as indicated. Lung sections were stained with H&E. Representative pictures are shown for each experimental group as indicated (A). Alveolar wall thickness was measured (B) and ventilator-induced lung injury (VILI) score was calculated (C). Data represent means ± SEM for n = 7/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h air vent group; ^&<i>P</i><0.05 vs. 5h air vent + 1h H₂S vent group; ⁺<i>P</i><0.05 vs. 3h air vent + 3h H₂S vent group; °<i>P</i><0.05 vs. 1h air vent + 5h H₂S vent group.</p

Effect of H₂S pretreatment on lung inflammation and oxidative stress.

<p>Mice spontaneously breathed air (control) or for 6 h, or they were mechanically ventilated with 12 ml/kg for 6 h either with air alone (6 h air) or air supplemented with 80 ppm H₂S (6 h H₂S). All other mice spontaneously breathed air supplemented with 80 ppm H₂S 1, 3, or 5 h prior to mechanical ventilation with air for another 6 h as indicated. Bronchoalveolar lavage (BAL) IL-1β cytokine content was determined by ELISA (A). The fraction of neutrophil cells was measured in BAL fluid by cytospin analysis (B). Lung tissue sections were stained with dihydroethidium (C). Representative pictures are shown for each experimental group as indicated in C. ROS fluorescence intensity was measured, calculated, and expressed as fold induction compared to control group (D). Data represent means ± SEM for n = 6/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h air vent group.</p

Genetic Targets of Hydrogen Sulfide in Ventilator-Induced Lung Injury – A Microarray Study

<div><p>Recently, we have shown that inhalation of hydrogen sulfide (H₂S) protects against ventilator-induced lung injury (VILI). In the present study, we aimed to determine the underlying molecular mechanisms of H₂S-dependent lung protection by analyzing gene expression profiles in mice. C57BL/6 mice were subjected to spontaneous breathing or mechanical ventilation in the absence or presence of H₂S (80 parts per million). Gene expression profiles were determined by microarray, sqRT-PCR and Western Blot analyses. The association of Atf3 in protection against VILI was confirmed with a Vivo-Morpholino knockout model. Mechanical ventilation caused a significant lung inflammation and damage that was prevented in the presence of H₂S. Mechanical ventilation favoured the expression of genes involved in inflammation, leukocyte activation and chemotaxis. In contrast, ventilation with H₂S activated genes involved in extracellular matrix remodelling, angiogenesis, inhibition of apoptosis, and inflammation. Amongst others, H₂S administration induced Atf3, an anti-inflammatory and anti-apoptotic regulator. Morpholino mediated reduction of Atf3 resulted in elevated lung injury despite the presence of H₂S. In conclusion, lung protection by H₂S during mechanical ventilation is associated with down-regulation of genes related to oxidative stress and inflammation and up-regulation of anti-apoptotic and anti-inflammatory genes. Here we show that Atf3 is clearly involved in H₂S mediated protection.</p></div

Effect of reduced Atf3 protein synthesis on VILI.

<p>(A) Representative pictures of H&E stained lung tissue from control animals and animals mechanically ventilated with synthetic air in the presence or absence of H₂S. Control-Morpholino or Atf3-Morpholino treated mice were ventilated with supplemented H₂S. (B) Ventilator-induced lung injury (VILI) score was measured from the histology samples and (C) the relative amount of neutrophils was determined by cytospin analysis of BALF. Data represent means ±SEM for n = 4/group. Analysis of variance (Student–Newman–Keuls post hoc test), *P<0.05 vs. control; ^#P<0.05 vs. H₂S control-Morpfolino; ^§P<0.05 vs. air-ventilated group.</p