Hypoxia sensing by hepatic stellate cells leads to VEGF-dependent angiogenesis and may contribute to accelerated liver regeneration.

Portal vein ligation (PVL) induces liver growth prior to resection. Associating liver partition and portal vein ligation (PVL plus transection=ALPPS) or the addition of the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG) to PVL both accelerate growth via stabilization of HIF-α subunits. This study aims at clarifying the crosstalk of hepatocytes (HC), hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) in accelerated liver growth. In vivo, liver volume, HC proliferation, vascular density and HSC activation were assessed in PVL, ALPPS, PVL+DMOG and DMOG alone. Proliferation of HC, HSC and LSEC was determined under DMOG in vitro. Conditioned media experiments of DMOG-exposed cells were performed. ALPPS and PVL+DMOG accelerated liver growth and HC proliferation in comparison to PVL. DMOG alone did not induce HC proliferation, but led to increased vascular density, which was also observed in ALPPS and PVL+DMOG. Activated HSC were detected in ALPPS, PVL+DMOG and DMOG, again not in PVL. In vitro, DMOG had no proliferative effect on HC, but conditioned supernatant of DMOG-treated HSC induced VEGF-dependent proliferation of LSEC. Transcriptome analysis confirmed activation of proangiogenic factors in hypoxic HSC. Hypoxia signaling in HSC induces VEGF-dependent angiogenesis. HSC play a crucial role in the cellular crosstalk of rapid liver regeneration.

Sevoflurane Protects Hepatocytes From Ischemic Injury by Reducing Reactive Oxygen Species Signaling of Hepatic Stellate Cells: Translational Findings Based on a Clinical Trial.

BACKGROUND: Randomized controlled trials (RCTs) data demonstrate that sevoflurane postconditioning improves clinical outcomes of liver resection with inflow occlusion, presumably due to hepatocyte protection from ischemic injury. However, mechanisms remain unclear. This study examines liver biopsy samples obtained in an RCT of sevoflurane postconditioning to test the hypothesis that sevoflurane attenuates hepatocyte apoptosis. METHODS: Messenger ribonucleic acid (mRNA) of pro- and antiapoptotic regulators Bax and B-cell lymphoma 2 (Bcl2) was examined in hepatic biopsies obtained during the RCT. Hepatic stellate cells (HSCs) and hepatocytes were exposed to hypoxia/reoxygenation (H/R) in vitro to evaluate the effect of sevoflurane postconditioning on apoptosis. The role of HSC as a potential apoptosis trigger in hepatocytes through the production of reactive oxygen species induced by H/R was explored by transferring supernatants from H/R-exposed HSC to hepatocytes as target cells. RESULTS: In patients of the RCT, the Bax/Bcl2 mRNA ratio in liver tissue was markedly decreased in the sevoflurane arm (25% ± 21% reduction; P = .001). In vitro, H/R increased reactive oxygen species production in HSC by 33% ± 16% (P = .025), while it was abolished in the presence of sevoflurane (P < .001). In hepatocytes, caspase was minimally activated by H/R. However, incubation of hepatocytes with supernatants of HSC, previously exposed to H/R, increased caspase activity by 28% ± 13% (P < .001). When exposed to supernatants from HSC undergoing sevoflurane postconditioning, caspase activation in hepatocytes was reduced by 20% ± 9% (P < .001), similarly to the sevoflurane effect on the BAX/Bcl2 mRNA ratio in the liver samples. CONCLUSIONS: The study shows that sevoflurane postconditioning affects apoptosis of hepatocytes after ischemia-reperfusion injury in patients. It also demonstrates that HSC may be the effector cells of sevoflurane protection.

Sevoflurane protects rat brain endothelial barrier structure and function after hypoxia-reoxygenation injury.

BACKGROUND: After cerebral injury blood-brain barrier disruption significantly impairs brain homeostasis. Volatile anesthetics have been shown to be protective in ischemia-reperfusion injury scenarios. Their impact on brain endothelial cells after hypoxia-reoxygenation (H/R) has not yet been studied in detail. METHODS: Rat brain endothelial cells (RBE4) were exposed to severe hypoxia and reoxygenated in air in the presence or absence of sevoflurane. Changes in dextran permeability and architecture of the cellular junctional proteins ZO-1 and ß-catenin were measured. To determine necrosis and apoptosis rate DNA content, LDH release and caspase activity were quantified. The role of vascular endothelial growth factor (VEGF) as an inflammatory mediator increasing vascular permeability was assessed. At the same time, it was evaluated if sevoflurane effects are mediated through VEGF. Results were analyzed by unpaired t-tests or one way-analysis of variance followed by Bonferroni's correction. RESULTS: H/R led to a 172% increase in permeability (p<0.001), cell swelling and qualitatively but not quantitatively modified expression of ZO-1, ß-catenin and F-actin. In the presence of sevoflurane during reoxygenation, barrier function improved by 96% (p = 0.042) in parallel to a decrease of the cell size and less re-arranged junction proteins and F-actin. Sevoflurane-induced improvement of the barrier function could not be explained on the level of necrosis or apoptosis as they remained unchanged independent of the presence or absence of the volatile anesthetic. Increased expression of VEGF after H/R was attenuated by sevoflurane by 34% (p = 0.004). Barrier protection provided by sevoflurane was similar to the application of a blocking VEGF-antibody. Furthermore, the protective effect of sevoflurane was abolished in the presence of recombinant VEGF. CONCLUSIONS: In H/R-induced rat brain endothelial cell injury sevoflurane maintains endothelial barrier function through downregulation of VEGF, which is a key player not only in mediating injury, but also with regard to the protective effect of sevoflurane.

Sevoflurane attenuates systemic inflammation compared with propofol, but does not modulate neuro-inflammation: A laboratory rat study.

BACKGROUND: Septic encephalopathy is believed to be a result of neuro-inflammation possibly triggered by endotoxins, such as lipopolysaccharides (LPS). Modulation of the immune system is a property of volatile anaesthetics. OBJECTIVE: We aimed to investigate the systemic and cerebral inflammatory response in a LPS-induced sepsis model in rats. We compared two different sedation strategies, intravenous propofol and the volatile anaesthetic sevoflurane, with the hypothesis that the latter may attenuate neuro-inflammatory processes. DESIGN: Laboratory rat study. SETTING: Basic research laboratories at the University Hospital Zurich and University Zurich Irchel between August 2014 and June 2016. PATIENTS: A total of 32 adult male Wistar rats. INTERVENTIONS: After tracheotomy and mechanical ventilation, the anaesthetised rats were monitored before sepsis was induced by using intravenous LPS or phosphate-buffered saline as control. Rats were sedated with propofol (10 mg kg h) or sevoflurane (2 vol%) continuously for 12 h. MAIN OUTCOME MEASURES: Systemic inflammatory markers such as cytokine-induced neutrophil chemo-attractant protein 1, monocyte chemo-tactic protein-1 and IL-6 were determined. The same cytokines were measured in brain tissue. Cellular response in the brain was assessed by defining neutrophil accumulation with myeloperoxidase and also activation of microglia with ionised calcium-binding adaptor molecule-1 and astrocytes with glial fibrillary acidic protein. Finally, brain injury was determined. RESULTS: Animals were haemodynamically stable in both sedation groups treated with LPS. Blood cytokine peak values were lower in the sevoflurane-LPS compared with propofol-LPS animals. In brain tissue of LPS animals, chemoattractant protein-1 was the only significantly increased cytokine (P = 0.003), however with no significance between propofol and sevoflurane. After LPS challenge, cerebral accumulation of neutrophils was observed. Microglia activation was pronounced in the hippocampus of animals treated with LPS (P = 0.006). LPS induced prominent astrogliosis (P < 0.001). There was no significant difference in microglia or astrocyte activation or apoptosis in the brain between sevoflurane and propofol. CONCLUSION: We have shown that systemic attenuation of inflammation by the volatile anaesthetic sevoflurane did not translate into attenuated neuro-inflammation in this LPS-induced inflammation model. TRIAL REGISTRATION: Animal approval No. 134/2014, Veterinäramt Zürich.

Sevoflurane Abolishes Oxygenation Impairment in a Long-Term Rat Model of Acute Lung Injury.

BACKGROUND: Patients experiencing acute lung injury (ALI) often need mechanical ventilation for which sedation may be required. In such patients, usually the first choice an intravenously administered drug. However, growing evidence suggests that volatile anesthetics such as sevoflurane are a valuable alternative. In this study, we evaluate pulmonary and systemic effects of long-term (24-hour) sedation with sevoflurane compared with propofol in an in vivo animal model of ALI. METHODS: Adult male Wistar rats were subjected to ALI by intratracheal lipopolysaccharide (LPS) application, mechanically ventilated and sedated for varying intervals up to 24 hours with either sevoflurane or propofol. Vital parameters were monitored, and arterial blood gases were analyzed. Inflammation was assessed by the analysis of bronchoalveolar lavage fluid (BALF), cytokines (monocyte chemoattractant protein-1 [MCP-1], cytokine-induced neutrophil chemoattractant protein-1 [CINC-1], interleukin [IL-6], IL-12/12a, transforming growth factor-ß, and IL-10) in blood and lung tissue and inflammatory cells. The alveolocapillary barrier was indirectly assessed by wet-to-dry ratio, albumin, and total protein content in BALF. Results are presented as mean ± standard deviation. RESULTS: After 9 hours of ventilation and sedation, oxygenation index was higher in the LPS/sevoflurane (LPS-S) than in the LPS/propofol group (LPS-P) and reached 400 ± 67 versus 262 ± 57 mm Hg after 24 hours (P < .001). Cell count in BALF in sevoflurane-treated animals was lower after 18 hours (P = .001) and 24 hours (P < .001) than in propofol controls. Peak values of CINC-1 and IL-6 in BALF were lower in LPS-S versus LPS-P animals (CINC-1: 2.7 ± 0.7 vs 4.0 ± 0.9 ng/mL; IL-6: 9.2 ± 2.3 vs 18.9 ± 7.1 pg/mL, both P < .001), whereas IL-10 and MCP-1 did not differ. Also messenger RNAs of CINC-1, IL-6, IL-12a, and IL-10 were significantly higher in LPS-P compared with LPS-S. MCP-1 and transforming growth factor-ß showed no differences. Wet-to-dry ratio was lower in LPS-S (5.4 ± 0.2 vs 5.7 ± 0.2, P = .016). Total protein in BALF did not differ between P-LPS and S-LPS groups. CONCLUSIONS: Long-term sedation with sevoflurane compared with propofol improves oxygenation and attenuates the inflammatory response in LPS-induced ALI. Our findings suggest that sevoflurane may improve lung function when used for sedation in patients with ALI.

Hypoxia of the growing liver accelerates regeneration.

BACKGROUND: After portal vein ligation of 1 side of the liver, the other side regenerates at a slow rate. This slow growth may be accelerated to rapid growth by adding a transection between the 2 sides, i.e., performing portal vein ligation and parenchymal transection. We found that in patients undergoing portal vein ligation and parenchymal transection, portal vein hyperflow in the regenerating liver causes a significant reduction of arterial flow due to the hepatic arterial buffer response. We postulated that the reduction of arterial flow induces hypoxia in the regenerating liver and used a rat model to assess hypoxia and its impact on kinetic growth. METHODS: A rat model of rapid (portal vein ligation and parenchymal transection) and slow regeneration (portal vein ligation) was established. Portal vein flow and pressure data were collected. Liver regeneration was assessed in rats using computed tomography, proliferation with Ki-67, and hypoxia with pimonidazole and HIF-1α staining. RESULTS: The rat model confirmed acceleration of regeneration in portal vein ligation and parenchymal transection as well as the portal vein hyperflow seen in patients. Additionally, tissue hypoxia was observed after portal vein ligation and parenchymal transection, while little hypoxia staining was detected after portal vein ligation. To determine if hypoxia is a consequence or an inciting stimulus of rapid liver regeneration, we used a prolyl-hydroxylase blocker to activate hypoxia signaling pathways in the slow model. This clearly accelerated slow to rapid liver regeneration. Inversely, abrogation of hypoxia led to a blunting of rapid growth to slow growth. The topical application of prolyl-hydroxylase inhibitors on livers in rats induced spontaneous areas of regeneration. CONCLUSION: This study shows that pharmacologically induced hypoxic signaling accelerates liver regeneration similar to portal vein ligation and parenchymal transection. Hypoxia is likely an accelerator of liver regeneration. Also, prolyl-hydroxylase inhibitors may be used to enhance liver regeneration pharmaceutically.

Inflammatory Kidney and Liver Tissue Response to Different Hydroxyethylstarch (HES) Preparations in a Rat Model of Early Sepsis.

BACKGROUND: Tissue hypoperfusion and inflammation in sepsis can lead to organ failure including kidney and liver. In sepsis, mortality of acute kidney injury increases by more than 50%. Which type of volume replacement should be used is still an ongoing debate. We investigated the effect of different volume strategies on inflammatory mediators in kidney and liver in an early sepsis model. MATERIAL AND METHODS: Adult male Wistar rats were subjected to sepsis by cecal ligation and puncture (CLP) and assigned to three fluid replenishment groups. Animals received 30mL/kg of Ringer's lactate (RL) for 2h, thereafter RL (75mL/kg), hydroxyethyl starch (HES) balanced (25mL/kg), containing malate and acetate, or HES saline (25mL/kg) for another 2h. Kidney and liver tissue was assessed for inflammation. In vitro rat endothelial cells were exposed to RL, HES balanced or HES saline for 2h, followed by stimulation with tumor necrosis factor-α (TNF-α) for another 4h. Alternatively, cells were exposed to malate, acetate or a mixture of malate and acetate, reflecting the according concentration of these substances in HES balanced. Pro-inflammatory cytokines were determined in cell supernatants. RESULTS: Cytokine mRNA in kidney and liver was increased in CLP animals treated with HES balanced compared to RL, but not after application of HES saline. MCP-1 was 3.5fold (95% CI: 1.3, 5.6) (p<0.01) and TNF-α 2.3fold (95% CI: 1.2, 3.3) (p<0.001) upregulated in the kidney. Corresponding results were seen in liver tissue. TNF-α-stimulated endothelial cells co-exposed to RL expressed 3529±1040pg/mL MCP-1 and 59±23pg/mL CINC-1 protein. These cytokines increased by 2358pg/mL (95% CI: 1511, 3204) (p<0.001) and 29pg/ml (95% CI: 14, 45) (p<0.01) respectively when exposed to HES balanced instead. However, no further upregulation was observed with HES saline. PBS supplemented with acetate increased MCP-1 by 1325pg/mL (95% CI: 741, 1909) (p<0.001) and CINC-1 by 24pg/mL (95% CI: 9, 38) (p<0.01) compared to RL. Malate as well as HES saline did not affect cytokine expression. CONCLUSION: We identified HES balanced and specifically its component acetate as pro-inflammatory factor. How important this additional inflammatory burden on kidney and liver function is contributing to the sepsis-associated inflammatory burden in early sepsis needs further evaluation.

Inflammatory response of lung macrophages and epithelial cells after exposure to redox active nanoparticles: effect of solubility and antioxidant treatment.

The effects of an exposure to three mass-produced metal oxide nanoparticles-similar in size and specific surface area but different in redox activity and solubility-were studied in rat alveolar macrophages (MAC) and epithelial cells (AEC). We hypothesized that the cell response depends on the particle redox activity and solubility determining the amount of reactive oxygen species formation (ROS) and subsequent inflammatory response. MAC and AEC were exposed to different amounts of Mn3O4 (soluble, redox-active), CeO2 (insoluble, redox-active), and TiO2 (insoluble, redox-inert) up to 24 h. Viability and inflammatory response were monitored with and without coincubation of a free-radical scavenger (trolox). In MAC elevated ROS levels, decreased metabolic activity and attenuated inflammatory mediator secretion were observed in response to Mn3O4. Addition of trolox partially resolved these changes. In AEC, decreased metabolic activity and an attenuated inflammatory mediator secretion were found in response to CeO2 exposure without increased production of ROS, thus not sensitive to trolox administration. Interestingly, highly redox-active soluble particles did not provoke an inflammatory response. The data reveal that target and effector cells of the lung react in different ways to particle exposure making a prediction of the response depending on redox activity and intracellular solubility difficult.

Modulation of early inflammatory response by different balanced and non-balanced colloids and crystalloids in a rodent model of endotoxemia.

The use of hydroxyethyl starch (HES) in sepsis has been shown to increase mortality and acute kidney injury. However, the knowledge of the exact mechanism by which several fluids, especially starch preparations may impair end-organ function particularly in the kidney, is still missing. The aim of this study was to measure the influence of different crystalloid and colloid fluid compositions on the inflammatory response in the kidney, the liver and the lung using a rodent model of acute endotoxemia. Rats were anesthetized and mechanically ventilated. Lipopolysaccharide (5 mg/kg) was administered intravenously. After one hour crystalloids [lactate-buffered (RLac) or acetate-buffered (RAc)] were infused i.v. (30 ml/kg) in all groups. At 2 hours rats either received different crystalloids (75 ml/kg of RLac or RAc) or colloids (25 ml/kg of HES in saline or HES in RAc or gelatin in saline). Expression of messenger RNA for cytokine-induced neutrophil chemoattractant-1 (CINC-1), monocyte chemotactic protein-1 (MCP-1), necrosis factor α (TNFα) and intercellular adhesion molecule 1 (ICAM-1) was assessed in kidney, liver and lung tissue by real-time PCR after 4 hours. The use of acetate-buffered solutions was associated with a significantly higher expression of CINC-1 and TNFα mRNA in the liver, in the kidney and in the lung. Only marginal effects of gelatin and hydroxyethyl starch on mRNA expression of inflammatory mediators were observed. The study provides evidence that the type of buffering agent of different colloidal and crystalloid solutions might be a crucial factor determining the extent of early end-organ inflammatory response in sepsis.

Fluorinated groups mediate the immunomodulatory effects of volatile anesthetics in acute cell injury.

Volatile anesthetics are known to attenuate inflammatory response and tissue damage markers in acute organ injury. It is unclear whether these beneficial effects of volatile anesthetics are mediated by the ether basic structure or by characteristics of their halogenations. We describe in an in vitro model of acute inflammation in pulmonary cells that halogenation (fluorinated carbon groups) is responsible for the immunomodulatory effects. The inflammatory response after coexposure to endotoxin and sevoflurane, diethyl-ether, or various water-soluble molecules carrying trifluorinated carbon (CF(3)) groups was evaluated in pulmonary epithelial and endothelial cells and in neutrophils. In epithelial and endothelial cells, expression of inflammatory mediators to LPS stimulation was dose-dependently decreased upon exposure to sevoflurane and other molecules with CF(3) groups. This was not observed for diethyl-ether or structure-similar nonfluorinated molecules. In neutrophils, chemotactic activity, as well as expression of surface CD11b and CD62L, was positively modified by molecules carrying CF(3) groups. Cytotoxicity could be excluded. These findings for the first time reveal in an in vitro model of acute inflammation that the immunomodulatory effects are not limited to volatile anesthetics but are associated with a much broader class of CF(3) group-containing molecules. The immunomodulatory effects could now be provided in a hydrophilic, injectable formulation for the treatment of patients suffering from acute organ injury, such as acute lung injury, in environments not suitable for volatile anesthetics.

The effect of hydroxyethyl starches (HES 130/0.42 and HES 200/0.5) on activated renal tubular epithelial cells.

BACKGROUND: Acute renal failure is a frequent complication of sepsis. Hydroxyethyl starch (HES) is widely used in the treatment of such patients. However, the effect of HES on renal function during sepsis remains controversial. We established an in vitro model of tumor necrosis factor-alpha (TNF-alpha)-stimulated human proximal tubular epithelial (HK-2) cells to assess the possible effects of HES 130/0.42 and HES 200/0.5 on these activated cells. METHODS: HK-2 cells were stimulated with TNF-alpha in the presence or absence of HES 130/0.42 or 200/0.5. After 4, 10, and 18 h of incubation, monocyte chemoattractant protein-1 (MCP-1), a key chemoattractant for neutrophils and macrophages, was measured. In addition, viability and cytotoxicity assays were performed. RESULTS: MCP-1 expression was doubled upon TNF-alpha exposure. In the presence of 2% and 4% HES 200/0.5 in 98% (96%) medium over a stimulation time period of 10 h and 18 h, the MCP-1 concentration was decreased between 26% and 56% (P < 0.05). TNF-alpha stimulation resulted in a significant decrease of viability by 53%-63%, whereas viability decreased by only 32%-40% in coincubation with HES 130/0.42 (P < 0.005) and remained even less affected by TNF-alpha in the presence of HES 200/0.5 (P < 0.001). The TNF-alpha-induced cell death rate was attenuated in the presence of HES 200/0.5 (P < 0.05). CONCLUSIONS: This in vitro study shows that both HES products modulate cell injury upon inflammatory stimulation. The effect was more pronounced in the HES 200/0.5 group than for HES 130/0.42, suggesting a possible biological difference between the HES types.

Sevoflurane ameliorates gas exchange and attenuates lung damage in experimental lipopolysaccharide-induced lung injury.

BACKGROUND: Acute lung injury is a common complication in critically ill patients. Several studies suggest that volatile anesthetics have immunomodulating effects. The aim of the current study was to assess possible postconditioning with sevoflurane in an in vivo model of endotoxin-induced lung injury. METHODS: Rats were anesthetized, tracheotomized, and mechanically ventilated. Lipopolysaccharide (saline as control) was administered intratracheally. Upon injury after 2 h of propofol anesthesia, general anesthesia was continued with either sevoflurane or propofol for 4 h. Arterial blood gases were measured every 2 h. After 6 h of injury, bronchoalveolar lavage was performed and lungs were collected. Total cell count, albumin content, concentrations of the cytokines cytokine-induced neutrophil chemoattractant-1 and monocyte chemoattractant protein-1, and phospholipids were analyzed in bronchoalveolar lavage fluid. Expression of messenger RNA for the two cytokines and for surfactant protein B was determined in lung tissue. Histopathologic examination of the lung was performed. RESULTS: Significant improvement of the ratio of oxygen tension to inspired oxygen fraction was shown with sevoflurane (mean + or - SD: 243 + or - 94 mmHg [32.4 kPa]) compared with propofol (88 + or - 19 mmHg [11.7 kPa]). Total cell count representing effector cell recruitment as well as albumin content as a measure of lung permeability were significantly decreased in the sevoflurane-lipopolysaccharide group compared with the propofol-lipopolysaccharide group in bronchoalveolar lavage fluid. Expression of the cytokines protein in bronchoalveolar lavage fluid as well as messenger RNA in lung tissue was significantly lower in the sevoflurane-lipopolysaccharide group compared with the propofol-lipopolysaccharide group. CONCLUSIONS: Postconditioning with sevoflurane attenuates lung damage and preserves lung function in an in vivo model of acute lung injury.

The immunomodulatory effect of sevoflurane in endotoxin-injured alveolar epithelial cells.

BACKGROUND: Endotoxin-induced lung injury is a useful experimental system for the characterization of immunopathologic mechanisms in acute lung injury. Although alveolar epithelial cells (AEC) are directly exposed to volatile anesthetics, there is limited information about the effect of anesthetics on these cells. In this study we investigated the effect of pretreatment with the inhaled anesthetic sevoflurane on lipopolysaccharide (LPS)-injured AEC. METHODS: AEC were incubated with 1.1 vol % sevoflurane for 0.5 h, followed by LPS stimulation for 5 h. Expression of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1beta (MIP-1beta), macrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophil chemoattractant-1 (CINC-1), and intercellular adhesion molecule-1 (ICAM-1) was analyzed. In addition, functional tests were performed through chemotaxis and adherence assays to underline the biological relevance of the findings. RESULTS: Exposure of AEC to sevoflurane resulted in a 50% downregulation of MCP-1 protein in the sevoflurane-LPS group when compared with non-sevoflurane- LPS cells (P < 0.05). MIP-1beta concentration in LPS-stimulated cells decreased by 32% with sevoflurane (P < 0.05), MIP-2 by 29% (P < 0.05), and CINC-1 by 20% (P < 0.05). ICAM-1 protein expression was attenuated by 36% (P < 0.05). This inhibition caused substantial changes in the inflammatory response of neutrophils. 33% less chemotactic activity was seen in sevoflurane-treated LPS cells (P < 0.001) as well as 47% decreased adhesion of neutrophils to AEC (P < 0.001). CONCLUSIONS: This study shows that sevoflurane alters the LPS-induced inflammatory response, not only with respect to the expression pattern of inflammatory mediators, but also regarding the biological consequences with less accumulation of effector cells such as neutrophils.

Ropivacaine decreases inflammation in experimental endotoxin-induced lung injury.

BACKGROUND: Endotoxin causes acute lung injury, which can lead to acute respiratory distress syndrome. Because local anesthetics are known to attenuate inflammatory reactions, ropivacaine was tested for its possible antiinflammatory effect in lipopolysaccharide-induced lung injury in rat alveolar epithelial cells (AECs) and rat pulmonary artery endothelial cells (RPAECs) in vitro and in vivo. METHODS: AECs and RPAECs were stimulated for 4 h with lipopolysaccharide or lipopolysaccharide and 1 mum ropivacaine. Messenger RNA (mRNA) for intercellular adhesion molecule 1 was assessed. Isolated neutrophils were incubated with stimulated target cells to quantify adhesion and neutrophil-induced cytotoxicity in AECs and RPAECs. In vivo, lipopolysaccharide was instilled intratracheally with or without 1 mm intratracheally or intravenously administered ropivacaine. Bronchoalveolar lavage was performed 5 h later to determine neutrophil and albumin content, as well as concentrations of inflammatory mediators. RESULTS: In AECs and RPAECs, ropivacaine attenuated lipopolysaccharide-induced up-regulation of mRNA for intercellular adhesion molecule 1 by 41% and 24%, respectively (P < 0.05). In the presence of ropivacaine, increased neutrophil adhesion was down-regulated by 58% and 44% (P < 0.005), whereas cytotoxicity in AECs and RPAECs was diminished by 28% and 33%, respectively (P < 0.05). Enhanced neutrophil count in lipopolysaccharide lungs was reduced by 56% in the presence of intratracheally instilled ropivacaine (81% with intravenous ropivacaine; P < 0.005). Albumin was decreased by 46% with intratracheal ropivacaine (38% with intravenous ropivacaine; P < 0.05), and inflammatory mediators were decreased by 48-59% (69-81% with intravenous ropivacaine; P < 0.01). CONCLUSIONS: Ropivacaine intervention substantially attenuated the inflammatory response in acute lung injury and thus may carry an interesting potential for antiinflammatory treatment.

Inflammatory response of tracheobronchial epithelial cells to endotoxin.

Respiratory epithelial cells play a crucial role in the inflammatory response in endotoxin-induced lung injury, an experimental model for acute lung injury. To determine the role of epithelial cells in the upper respiratory compartment in the inflammatory response to endotoxin, we exposed tracheobronchial epithelial cells (TBEC) to lipopolysaccharide (LPS). Expression of inflammatory mediators was analyzed, and the biological implications were assessed using chemotaxis and adherence assays. Epithelial cell necrosis and apoptosis were determined to identify LPS-induced cell damage. Treatment of TBEC with LPS induced enhanced protein expression of cytokines and chemokines (increases of 235-654%, P < 0.05), with increased chemotactic activity regarding neutrophil recruitment. Expression of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was enhanced by 52-101% (P < 0.0001). This upregulation led to increased adhesion of neutrophils, with >95% adherence to TBEC after LPS stimulation, which could be blocked by either ICAM-1 (69%) or VCAM-1 antibodies (55%) (P < 0.05). Enhanced neutrophil-induced necrosis of TBEC was observed when TBEC were exposed to LPS. Reduced neutrophil adherence by ICAM-1 or VCAM-1 antibodies resulted in significantly lower TBEC death (52 and 34%, respectively, P < 0.05). Therefore, tight adherence of neutrophils to TBEC appears to promote epithelial cell killing. In addition to indirect effector cell-induced TBEC death, direct LPS-induced cell damage was seen with increased apoptosis rate in LPS-stimulated TBEC (36% increase of caspase-3, P < 0.01). These data provide evidence that LPS induces TBEC killing in a necrosis- and apoptosis-dependent manner.

Pulmonary aspiration: new therapeutic approaches in the experimental model.

BACKGROUND: Acute lung injury caused by gastric aspiration is a frequent occurrence in unconscious patients. Acute respiratory distress syndrome in association with gastric aspiration carries a mortality of up to 30% and accounts for up to 20% of deaths associated with anesthesia. Although the clinical condition is well known, knowledge about the exact inflammatory mechanisms is still incomplete. This study was performed to define the role of alveolar macrophages in this inflammatory response. In addition, potentially modifying effects of intratracheally applied nuclear factor kappaB inhibitor pyrrolidine dithiocarbamate were investigated. METHODS: Rat alveolar macrophages were depleted by intratracheal administration of clodronate liposomes, and lung injury was evaluated 6 h after instillation of 0.1N hydrochloric acid. In a second set of experiments, pyrrolidine dithiocarbamate was intratracheally instilled 3 h after hydrochloric acid application, and injury parameters were determined. RESULTS: Depletion of alveolar macrophages resulted in decreased production of inflammatory mediators in acid aspiration (23-80% reduction of messenger RNA or protein of inflammatory mediators; P < 0.05) and consequently also in diminished neutrophil recruitment (36% fewer neutrophils; P < 0.01). Treatment with pyrrolidine dithiocarbamate was highly effective in decreasing neutrophil recruitment (66%; P < 0.01) and vascular permeability (80%; P < 0.001). CONCLUSIONS: These data suggest that alveolar macrophages play an essential role in the inflammatory response of acid-induced lung injury. For the first time, attenuation of acid-induced lung injury with an inhibitor, applied after the onset of injury, is shown.

Alveolar macrophages regulate neutrophil recruitment in endotoxin-induced lung injury.

BACKGROUND: Alveolar macrophages play an important role during the development of acute inflammatory lung injury. In the present study, in vivo alveolar macrophage depletion was performed by intratracheal application of dichloromethylene diphosphonate-liposomes in order to study the role of these effector cells in the early endotoxin-induced lung injury. METHODS: Lipopolysaccharide was applied intratracheally and the inflammatory reaction was assessed 4 hours later. Neutrophil accumulation and expression of inflammatory mediators were determined. To further analyze in vivo observations, in vitro experiments with alveolar epithelial cells and alveolar macrophages were performed. RESULTS: A 320% increase of polymorphonuclear leukocytes in bronchoalveolar lavage fluid was observed in macrophage-depleted compared to macrophage-competent lipopolysaccharide-animals. This neutrophil recruitment was also confirmed in the interstitial space. Monocyte chemoattractant protein-1 concentration in bronchoalveolar lavage fluid was significantly increased in the absence of alveolar macrophages. This phenomenon was underlined by in vitro experiments with alveolar epithelial cells and alveolar macrophages. Neutralizing monocyte chemoattractant protein-1 in the airways diminished neutrophil accumulation. CONCLUSION: These data suggest that alveolar macrophages play an important role in early endotoxin-induced lung injury. They prevent neutrophil influx by controlling monocyte chemoattractant protein-1 production through alveolar epithelial cells. Alveolar macrophages might therefore possess robust anti-inflammatory effects.

Acid-induced lung injury: role of nuclear factor-kappaB.

BACKGROUND: Aspiration of acidic gastric contents leads to acute lung injury and is still one of the most common clinical events associated with acute lung injury. This study was performed to assess acid-induced lung inflammation in vitro and in vivo with respect to the time pattern of activated transcription factor nuclear factor-kappaB (NF-kappaB) and proinflammatory molecules. METHODS: L2 cells (alveolar epithelial cells) were exposed for various periods to a medium with a pH of 6. In the in vivo model, 1 ml/kg of 0.1 n acidic solution was instilled into the lungs of rats. NF-kappaB binding activity and expression pattern of inflammatory mediators were determined. Blocking studies were performed with the NF-kappaB inhibitor pyrrolidine dithiocarbamate. RESULTS: In vitro NF-kappaB binding activity showed a biphasic expression pattern with a first peak at 1 h and a second one at 6-8 h. In acid-injured rat lungs, NF-kappaB binding activity was confirmed in a biphasic manner with a first increase at 0.5-2 h (608 +/- 93% and 500 +/- 15%, respectively, P < 0.05) and a second peak at 8 h (697 +/- 35% increase, P < 0.005). Whole lung mRNA for macrophage inflammatory protein-1beta and macrophage inflammatory protein-2 showed a similar expression pattern, which could explain the biphasic neutrophil recruitment. Intratracheal pyrrolidine dithiocarbamate attenuated lung injury as evidenced by a reduction of neutrophil accumulation and expression of inflammatory mediators. CONCLUSIONS: These data suggest that NF-kappaB binding activity plays a key role in molecular and cellular events in acid-induced lung injury.