Sevoflurane posttreatment prevents oxidative and inflammatory injury in ventilator-induced lung injury.

Mechanical ventilation is a life-saving clinical treatment but it can induce or aggravate lung injury. New therapeutic strategies, aimed at reducing the negative effects of mechanical ventilation such as excessive production of reactive oxygen species, release of pro-inflammatory cytokines, and transmigration as well as activation of neutrophil cells, are needed to improve the clinical outcome of ventilated patients. Though the inhaled anesthetic sevoflurane is known to exert organ-protective effects, little is known about the potential of sevoflurane therapy in ventilator-induced lung injury. This study focused on the effects of delayed sevoflurane application in mechanically ventilated C57BL/6N mice. Lung function, lung injury, oxidative stress, and inflammatory parameters were analyzed and compared between non-ventilated and ventilated groups with or without sevoflurane anesthesia. Mechanical ventilation led to a substantial induction of lung injury, reactive oxygen species production, pro-inflammatory cytokine release, and neutrophil influx. In contrast, sevoflurane posttreatment time dependently reduced histological signs of lung injury. Most interestingly, increased production of reactive oxygen species was clearly inhibited in all sevoflurane posttreatment groups. Likewise, the release of the pro-inflammatory cytokines interleukin-1β and MIP-1β and neutrophil transmigration were completely prevented by sevoflurane independent of the onset of sevoflurane administration. In conclusion, sevoflurane posttreatment time dependently limits lung injury, and oxidative and pro-inflammatory responses are clearly prevented by sevoflurane irrespective of the onset of posttreatment. These findings underline the therapeutic potential of sevoflurane treatment in ventilator-induced lung injury

Thiopental inhibits global protein synthesis by repression of eukaryotic elongation factor 2 and protects from hypoxic neuronal cell death.

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited

Static compliance before and after sevoflurane application.

<p>Static compliance before and after sevoflurane application.</p

Effect of sevoflurane posttreatment on ventilator-induced lung injury.

<p>Mice were either non-ventilated (control), or they were mechanically ventilated with 12 ml/kg for 6 h either under ketamine + acepromazine (6h KET) or sevoflurane (6h SEV) anesthesia or a combination of both: KET for 5, 3, or 1 h, followed by SEV for another 1, 3, or 5 h as indicated (5h KET + 1h SEV, 3h KET + 3h SEV, 1h KET + 5h SEV). Lung tissue sections were stained with hematoxylin and eosin. Representative pictures are shown for each experimental group as indicated (200x, A). High power fields were randomly assigned to measure alveolar wall thickness (B), and to calculate a ventilator-induced lung injury (VILI) score (C). Data represent means ± SD 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 KET group; ⁺<i>P</i><0.05 vs. 5h KET + 1h SEV group; °<i>P</i><0.05 vs. 3h KET + 3h SEV group; ^&<i>P</i><0.05 vs. 1h KET + 5h SEV group.</p

Effect of sevoflurane posttreatment on oxidative stress.

<p>Mice were either non-ventilated (control), or they were mechanically ventilated with 12 ml/kg for 6 h either under ketamine + acepromazine (6h KET) or sevoflurane (6h SEV) anesthesia or a combination of both: KET for 5, 3, or 1 h, followed by SEV for another 1, 3, or 5 h as indicated (5h KET + 1h SEV, 3h KET + 3h SEV, 1h KET + 5h SEV). Lung tissue sections were stained by DHE (A). Representative pictures are shown for each experimental group as indicated (200x, A). ROS fluorescence intensity per cell was measured and expressed as fold induction compared to control group (B). Data represent means ± SD for n = 4 (5h KET + 1h SEV) or n = 7/group. ANOVA (Tukey`s post hoc test), *<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 6h KET group.</p

Inhibitors of protein synthesis reduce hypoxic neuronal damage.

<p>Cellular damage in human neuronal SK-N-SH cells was induced by oxygen deprivation (closed symbols) in an atmosphere containing 5% CO₂, 95% N₂ for 0–72 h and determined by an LDH release assay. Control cells were cultured in 5% CO₂, 21% O₂, and 74% N₂ (open symbols). In (A), cellular damage was measured in the presence (squares) or absence (rhombi) of fetal calf serum. In (B), 5 µg/ml cycloheximide (closed triangles) or 2 µg/ml actinomycin D (closed rhombi) were added to the cells in serum containing growth medium (squares). In (C), 0.1 mM thiopental (closed circles) or 0.5 mM thiopental (closed triangles) were added to the cells in serum containing growth medium (squares). Values represent the mean ± standard deviations of four separate experiments. Experimental groups were statistically analyzed by performing two-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. Statistically significant differences within groups shown for (A) are: serum treated oxygen deprived SK-N-SH cells versus serum treated normoxic control cells (***, p<0.001). Statistically significant differences within serum treated groups shown for (B) are: normoxic control cells versus oxygen deprived SK-N-SH cells (***, p<0.001); and oxygen deprived SK-N-SH cells versus oxygen-deprived cells treated with 2 µg/ml actinomycin D (^§§§, p<0.001) or versus oxygen-deprived cells treated with 5 µg/ml cycloheximide (^###, p<0.001). Statistically significant differences within serum treated groups shown for (C) are: normoxic control cells versus oxygen deprived SK-N-SH cells (***, p<0.001), versus oxygen-deprived cells treated with 0.1 mM thiopental (^{<>\raster(60%)="rg1"<>}, p<0.05; ^{<>\raster(60%)="rg1"<><>\raster(60%)="rg1"<><>\raster(60%)="rg1"<>}, p<0.001) or versus oxygen-deprived cells treated with 0.5 mM thiopental (^{<>\raster(60%)="rg2"<><>\raster(60%)="rg2"<><>\raster(60%)="rg2"<>}, p<0.001); and oxygen deprived SK-N-SH cells versus oxygen-deprived cells treated with 0.1 mM thiopental (^{<>\raster(60%)="rg3"<><>\raster(60%)="rg3"<><>\raster(60%)="rg3"<>}, p<0.001) or versus oxygen-deprived cells treated with 0.5 mM thiopental (^¥¥¥, p<0.001).</p

Thiopental induces eEF2 phosphorylation.

<p>SK-N-SH cells were treated with 10 µM –2 mM thiopental for 6 h (A) or with 0.5 mM thiopental for 10 min to 12 h (B) and analyzed by immunoblotting with an anti-human phospho-eEF2 threonine 56 antibody (upper blots) or an eEF2 antibody that detects endogenous levels of eEF2 independently of phosphorylation (lower blots). Data are representative of four independent experiments.</p

Inhibitors of protein synthesis preserve intracellular ATP-content during oxygen deprivation.

<p>Human neuronal SK-N-SH cells were cultured in an oxygen-free atmosphere for 12–72 h in the presence of 5 µg/ml cycloheximide (closed triangles), 0.1 mM thiopental (closed rhombi), 0.5 mM thiopental (asterisks), or left untreated (closed squares). Cells, cultured in a normoxic atmosphere (open squares) served as a control. ATP content of cells was measured in lysates by an ATP-driven luciferase assay. Determined relative light units (RLU) were normalized to protein content and represent the means ± standard deviations of three independent experiments. Experimental groups were statistically evaluated by performing two-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. Statistical differences of oxygen deprived SK-N-SH cells (closed squares) compared to oxygen-deprived cells treated with 5 µg/ml cycloheximide (^###, p<0.001), 0.5 mM thiopental (^{<>\raster(60%)="rg3"<>}, p<0.05; ^{<>\raster(60%)="rg3"<><>\raster(60%)="rg3"<>}, p<0.01), 0.1 mM thiopental (^¥¥, p<0.01), or untreated control cells (^§§, p<0.01; ^§§§, p<0.001) are shown.</p

Thiopental inhibits protein synthesis, ameliorates hypoxic damage, and maintains energy balance during oxygen deprivation in primary cortical neurons.

<p>In (A), cortical neurons were treated with 10 µM −2 mM thiopental for 30 min and analyzed for phosphorylation of eEF2 and AMPK by immunoblotting. In (B), cortical neurons were left untreated or were exposed to 0.1–2 mM thiopental for 6 h and then pulse labeled with 200 µCi of [³⁵S]methionine for an additional 2 h. Cellular lysates were separated by 10% SDS-PAGE and the amounts of newly synthesized proteins were detected by autoradiography on dried electrophoresis gels. In (C/D), cortical neurons were exposed to hypoxia for 48 h in the presence or absence of 0.5 mM thiopental. Cellular damage was determined by an LDH-release assay (C). The relative intracellular ATP-content was measured by an ATP-driven luciferase assay (D). Values represent the mean ± standard deviations of three independent experiments. Statistical evaluation of experimental groups was performed by one-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. The statistically significant difference of oxygen-deprived cortical neurons in the presence or absence of thiopental is shown (***, p<0.001).</p