Alexander Hoetzel

Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro.

Background:Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. Methods:Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3+ T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti–caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti–cytochrome c Western blotting and enzyme-linked immunosorbent assays. Results:Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3–like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. Conclusion:Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

Heme oxygenase-1 induction by the clinically used anesthetic isoflurane protects rat livers from ischemia/reperfusion injury.

Objective:It was the aim of this study to characterize the influence of isoflurane-induced heme oxygenase-1 (HO-1) expression on hepatocellular integrity after ischemia and reperfusion. Summary Background Data:Abundant experimental data characterize HO-1 as one of the most powerful inducible enzymes that contribute to the protection of the liver and other organs after harmful stimuli. Therapeutic strategies aimed at utilizing the protective effects of HO-1 are hampered by the fact that most pharmacological inducers of this enzyme perturb organ function by themselves and are not available for use in patients because of their toxicity and undesirable or unknown side effects. Methods:Rats were pretreated with isoflurane before induction of partial hepatic ischemia (1 hour) and reperfusion (1 hour). At the end of each experiment, blood and liver tissue were obtained for molecular biologic, histologic, and immunohistochemical analyses. Results:Isoflurane pretreatment increased hepatic HO-1 mRNA, HO-1 protein, HO enzyme activity, and decreased plasma levels of AST, ALT, and α-GST. Histologic analysis of livers obtained from isoflurane-pretreated rats showed a reduction of necrotic areas, particularly in the perivenular region, the predominant site of isoflurane-induced HO-1 expression. In addition, sinusoidal congestion that could otherwise be observed after ischemia/reperfusion was inhibited by the anesthetic. Furthermore, isoflurane augmented hepatic microvascular blood flow and lowered the malondialdehyde content within the liver compared with control animals. Administration of tin protoporphyrin IX inhibited HO activity and abolished the isoflurane-induced protective effects. Conclusions:This study provides first evidence that pretreatment with the nontoxic and clinically approved anesthetic isoflurane induces hepatic HO-1 expression, and thereby protects rat livers from ischemia/reperfusion injury.

Inhaled hydrogen sulfide protects against ventilator-induced lung injury.

Background:Mechanical ventilation still causes an unacceptably high rate of morbidity and mortality because of ventilator-induced lung injury (VILI). Therefore, new therapeutic strategies are needed to treat VILI. Hydrogen sulfide can induce hypothermia and suspended animation-like states in mice. Hydrogen sulfide can also confer antiinflammatory and antiapoptotic effects. This study investigates the organ-protective effects of inhaled hydrogen sulfide during mechanical ventilation. Methods:Mice were ventilated with a tidal volume of 12 ml/kg body weight for 6 h with synthetic air in the absence or presence of hydrogen sulfide (80 parts per million) and, in a second series, at either mild hypothermia or normothermia. Staining of lung sections determined the degree of lung damage by VILI score and apoptotic cells. Bronchoalveolar lavage fluid was analyzed for the cytokines interleukin-1&bgr; and macrophage inflammatory protein-1&bgr; and for neutrophil accumulation. Heme oxygenase-1 and heat shock protein 70 expression were assessed in the lung tissue by Western immunoblot analysis. Results:Mechanical ventilation at both hypothermia and normothermia led to a profound development of VILI, characterized by pulmonary edema, increased apoptosis, cytokine release, neutrophil recruitment, and up-regulation of the stress proteins such as heme oxygenase-1 and heat shock protein 70. In contrast, the application of hydrogen sulfide during ventilation at either mild hypothermia or normothermia prevented edema formation, apoptosis, proinflammatory cytokine production, neutrophil accumulation, and inhibited heme oxygenase-1 expression. Conclusions:Inhalation of hydrogen sulfide during mechanical ventilation protects against VILI by the inhibition of inflammatory and apoptotic responses. Hydrogen sulfide confers lung protection independently of its ability to induce mild hypothermia during ventilation.

Carbon Monoxide Protects against Ventilator-induced Lung Injury via PPAR-γ and Inhibition of Egr-1

RATIONALEnVentilator-induced lung injury (VILI) leads to an unacceptably high mortality. In this regard, the antiinflammatory properties of inhaled carbon monoxide (CO) may provide a therapeutic option.nnnOBJECTIVESnThis study explores the mechanisms of CO-dependent protection in a mouse model of VILI.nnnMETHODSnMice were ventilated (12 ml/kg, 1-8 h) with air in the absence or presence of CO (250 ppm). Airway pressures, blood pressure, and blood gases were monitored. Lung tissue was analyzed for inflammation, injury, and gene expression. Bronchoalveolar lavage fluid was analyzed for protein, cell and neutrophil counts, and cytokines.nnnMEASUREMENTS AND MAIN RESULTSnMechanical ventilation caused significant lung injury reflected by increases in protein concentration, total cell and neutrophil counts in the bronchoalveolar lavage fluid, as well as the induction of heme oxygenase-1 and heat shock protein-70 in lung tissue. In contrast, CO application prevented lung injury during ventilation, inhibited stress-gene up-regulation, and decreased lung neutrophil infiltration. These effects were preceded by the inhibition of ventilation-induced cytokine and chemokine production. Furthermore, CO prevented the early ventilation-dependent up-regulation of early growth response-1 (Egr-1). Egr-1-deficient mice did not sustain lung injury after ventilation, relative to wild-type mice, suggesting that Egr-1 acts as a key proinflammatory regulator in VILI. Moreover, inhibition of peroxysome proliferator-activated receptor (PPAR)-gamma, an antiinflammatory nuclear regulator, by GW9662 abolished the protective effects of CO.nnnCONCLUSIONSnMechanical ventilation causes profound lung injury and inflammatory responses. CO treatment conferred protection in this model dependent on PPAR-gamma and inhibition of Egr-1.

Mitogen-Activated Protein Kinases Regulate Susceptibility to Ventilator-Induced Lung Injury

Background Mechanical ventilation causes ventilator-induced lung injury in animals and humans. Mitogen-activated protein kinases have been implicated in ventilator-induced lung injury though their functional significance remains incomplete. We characterize the role of p38 mitogen-activated protein kinase/mitogen activated protein kinase kinase-3 and c-Jun-NH2-terminal kinase-1 in ventilator-induced lung injury and investigate novel independent mechanisms contributing to lung injury during mechanical ventilation. Methodology and Principle Findings C57/BL6 wild-type mice and mice genetically deleted for mitogen-activated protein kinase kinase-3 (mkk-3 −/−) or c-Jun-NH2-terminal kinase-1 (jnk1 −/−) were ventilated, and lung injury parameters were assessed. We demonstrate that mkk3 −/− or jnk1 −/− mice displayed significantly reduced inflammatory lung injury and apoptosis relative to wild-type mice. Since jnk1−/− mice were highly resistant to ventilator-induced lung injury, we performed comprehensive gene expression profiling of ventilated wild-type or jnk1−/− mice to identify novel candidate genes which may play critical roles in the pathogenesis of ventilator-induced lung injury. Microarray analysis revealed many novel genes differentially expressed by ventilation including matrix metalloproteinase-8 (MMP8) and GADD45α. Functional characterization of MMP8 revealed that mmp8−/− mice were sensitized to ventilator-induced lung injury with increased lung vascular permeability. Conclusions We demonstrate that mitogen-activated protein kinase pathways mediate inflammatory lung injury during ventilator-induced lung injury. C-Jun-NH2-terminal kinase was also involved in alveolo-capillary leakage and edema formation, whereas MMP8 inhibited alveolo-capillary protein leakage.

Thiopental Inhibits the Activation of Nuclear Factor κB

Background Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury. Its long-term administration increases the incidence of nosocomial infections, which contributes to the high mortality rate of these patients. However, the mechanism of its immunosuppressing effect remains unknown. Methods The effect of thiopental (200–1000 &mgr;g/ml) on the activation of the nuclear transcription factor &kgr;B (NF-&kgr;B; electrophoretic mobility shift assays), on NF-&kgr;B–driven reporter gene activity (transient transfection assays), on the expression of NF-&kgr;B target genes (enzyme-linked immunoassays), on T-cell activation (flow cytometric analyses of CD69 expression), and on the content of the NF-&kgr;B inhibitor I&kgr;B-&agr; (Western blotting) was studied in human T lymphocytes in vitro. Results Thiopental inhibited the activation of the transcription factor NF-&kgr;B but did not alter the activity of the cyclic adenosine monophosphate response element binding protein. Other barbiturates (methohexital), anesthetics (etomidate, propofol, ketamine), or opioids (fentanyl, morphine) did not affect NF-&kgr;B activation. Thiopental-mediated suppression of NF-&kgr;B could be observed in Jurkat cells and in primary CD3+ lymphocytes from healthy volunteers, was time- and concentration-dependent, occurred at concentrations that are clinically achieved, and persisted for hours after the incubation. It was associated with an inhibition of NF-&kgr;B–driven reporter gene activity, of the expression of interleukin-2, -6, and -8, and interferon &ggr;, and of the activation of CD3+ lymphocytes. Suppression of NF-&kgr;B appeared to involve reduced degradation of I&kgr;B-&agr;. Conclusion The results demonstrate that thiopental inhibits the activation of NF-&kgr;B and may thus provide a molecular mechanism for some of the immunosuppressing effects associated with thiopental therapy.

Thiopental Inhibits Tumor Necrosis Factor α–induced Activation of Nuclear Factor κB through Suppression of IκB Kinase Activity

Background Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury and is associated with immunosuppressive effects. The authors have recently reported that thiopental inhibits activation of nuclear factor (NF) &kgr;B, a transcription factor implicated in the expression of many inflammatory genes. Thus, it was the aim of the current study to examine the molecular mechanism of this inhibitory effect. Methods The authors tested &ggr;-aminobutyric acid (GABA), the GABAA antagonist bicuculline, and the GABAB antagonist dichlorophenyl-methyl-amino-propyl-diethoxymethyl-phosphinic acid (CGP 52432) in combination with thiopental for their influence on the activation of NF-&kgr;B. In addition, they investigated the direct effect of thiopental on activated NF-&kgr;B DNA binding activity. These experiments were conducted in Jurkat T lymphocytes using electrophoretic mobility shift assays. The presence of the phosphorylated and dephosphorylated NF-&kgr;B inhibitor I&kgr;B&agr; (Western blotting) and I&kgr;B kinase activity were studied in Jurkat T cells and human CD3+ T lymphocytes. In addition, the authors tested the effect of the structural barbiturate analog pairs thiopental–pentobarbital and thiamylal–secobarbital and of thiopental in combination with the thio-group containing chemical dithiothreitol on the activation of NF-&kgr;B. Results GABA did not inhibit NF-&kgr;B activation, and the GABAA and GABAB antagonists bicuculline and CGP did not diminish the thiopental-mediated inhibitory effect on NF-&kgr;B activation. Thiopental did not inhibit activated NF-&kgr;B directly in a cell-free system. The phosphorylation of I&kgr;B&agr; was prevented after incubation with 1,000 &mgr;g/ml thiopental. The same concentration of thiopental also inhibited I&kgr;B kinase activity in tumor necrosis factor–stimulated Jurkat T cells and human CD3+ T lymphocytes (60% suppression, P < 0.05 vs. tumor necrosis factor &agr; alone). Thiobarbiturates (4 × 10−3 m) inhibited NF-&kgr;B activity, whereas equimolar concentrations of the structural oxyanalogs did not. Preincubation of thiopental with dithiothreitol diminished the inhibitory effect. Conclusion Thiopental-mediated inhibition of NF-&kgr;B activation is due to the suppression of I&kgr;B kinase activity and depends at least in part on the thio-group of the barbiturate molecule.

Carbon monoxide prevents ventilator induced lung injury via caveolin-1

Objectives:Carbon monoxide (CO) can confer anti-inflammatory protection in rodent models of ventilator-induced lung injury (VILI). Caveolin-1 exerts a critical role in cellular responses to mechanical stress and has been shown to mediate cytoprotective effects of CO in vitro. We sought to determine the role of caveolin-1 in lung susceptibility to VILI in mice. Furthermore, we assessed the role of caveolin-1 in the tissue-protective effects of CO in the VILI model. Design:Prospective experimental study. Setting:University laboratory. Subjects:Wild type (wt) and caveolin-1 deficient (cav-1−/−) mice. Interventions:Mice were subjected to tracheostomy and arterial cannulation. Wt and cav-1−/− mice were ventilated with a tidal volume of 12 mL/kg body weight and a frequency of 80/minute for 5 minutes as control or for 8 hours with air in the absence or presence of CO (250 parts per million). Bronchoalveolar lavage and histology were used to determine lung injury. Lung sections or homogenates were analyzed for caveolin-1 expression by immunohistochemical staining or Western blotting, respectively. Measurements and Main Results:Ventilation led to an increase in bronchoalveolar lavage protein concentration, cell count, neutrophil recruitment, and edema formation, which was prevented in the presence of CO. Although ventilation alone slightly induced caveolin-1 expression in epithelial cells, the application of CO during the ventilation significantly increased the expression of caveolin-1. In comparison with wt mice, mechanical ventilation of cav-1−/− mice led to a significantly higher degree of lung injury when compared with wt mice. In contrast to its effectiveness in wt mice, CO administration failed to reduce lung-injury markers in cav-1−/− mice. Conclusions:Caveolin-1 null mice are more susceptible to VILI. CO executes lung-protective effects during mechanical ventilation that are dependent, in part, on caveolin-1 expression.

Mechanism of hepatic heme oxygenase-1 induction by isoflurane

Background:The heme oxygenase pathway represents a major cell and organ protective system in the liver. The authors recently showed that isoflurane and sevoflurane up-regulate the inducible isoform heme oxygenase 1 (HO-1). Because the activating cascade remained unclear, it was the aim of this study to identify the underlying mechanism of this effect. Methods:Rats were anesthetized with pentobarbital intravenously or with isoflurane per inhalation (2.3 vol%). Kupffer cell function was inhibited by dexamethasone or gadolinium chloride. Nitric oxide synthases were inhibited by either N&ohgr;-nitro-l-arginine methyl ester or S-methyl thiourea. N-Acetyl-cysteine served as an antioxidant, and diethyldithiocarbamate served as an inhibitor of cytochrome P450 2E1. Protein kinase C and phospholipase A2 were inhibited by chelerythrine or quinacrine, respectively. HO-1 was analyzed in liver tissue by Northern blot, Western blot, immunostaining, and enzymatic activity assay. Results:In contrast to pentobarbital, isoflurane induced HO-1 after 4–6 h in hepatocytes in the pericentral region of the liver. The induction was prevented in the presence of dexamethasone (P < 0.05) and gadolinium chloride (P < 0.05). Inhibition of nitric oxide synthases or reactive oxygen intermediates did not affect isoflurane-mediated HO-1 up-regulation. In contrast, chelerythrine (P < 0.05) and quinacrine (P < 0.05) resulted in a blockade of HO-1 induction. Conclusion:The up-regulation of HO-1 by isoflurane in the liver is restricted to parenchymal cells and depends on Kupffer cell function. The induction is independent of nitric oxide or reactive oxygen species but does involve protein kinase C and phospholipase A2.

The volatile anesthetic isoflurane prevents ventilator-induced lung injury via phosphoinositide 3-kinase/Akt signaling in mice.

BACKGROUND: Mechanical ventilation leads to ventilator-induced lung injury in animals, and can contribute to acute lung injury/acute respiratory distress syndrome in humans. Acute lung injury/acute respiratory distress syndrome currently causes an unacceptably high rate of morbidity and mortality among critically ill patients. Volatile anesthetics have been shown to exert anti-inflammatory and organ-protective effects in vivo. We investigated the effects of the volatile anesthetic isoflurane on lung injury during mechanical ventilation. METHODS: C57BL/6N mice were ventilated with a tidal volume of 12 mL/kg body weight for 6 hours in the absence or presence of isoflurane, and, in a second series, with or without the specific phosphoinositide 3-kinase/Akt inhibitor LY294002. Lung injury was determined by comparative histology, and by the isolation of bronchoalveolar lavage for differential cell counting and analysis of cytokine levels using enzyme-linked immunosorbent assays. Lung homogenates were analyzed for protein expression by Western blotting. RESULTS: Mechanical ventilation caused increases in alveolar wall thickening, cellular infiltration, and an elevated ventilator-induced lung injury score. Neutrophil influx and cytokine (i.e., interleukin-1&bgr;, and macrophage inflammatory protein-2) release were enhanced in the bronchoalveolar lavage of ventilated mice. The expression levels of the stress proteins hemeoxygenase-1 and heat shock protein-70 were elevated in lung tissue homogenates. Isoflurane ventilation significantly reduced lung damage, inflammation, and stress protein expression. In contrast, phosphorylation of Akt protein was substantially increased during mechanical ventilation with isoflurane. Inhibition of phosphoinositide 3-kinase/Akt signaling before mechanical ventilation completely reversed the lung-protective effects of isoflurane treatment in vivo. CONCLUSIONS: Inhalation of isoflurane during mechanical ventilation protects against lung injury by preventing proinflammatory responses. This protection is mediated via phosphoinositide 3-kinase/Akt signaling.