Melanie Hasler

In vitro exposure of human fibroblasts to local anaesthetics impairs cell growth

Lidocaine, bupivacaine or ropivacaine are used routinely to manage perioperative pain. Sparse data exist evaluating the effects of local anaesthetics (LA) on fibroblasts, which are involved actively in wound healing. Therefore, we investigated the effects of the three LA to assess the survival, viability and proliferation rate of fibroblasts. Human fibroblasts were exposed to 0·3 mg/ml and 0·6 mg/ml of each LA for 2 days, followed by incubation with normal medium for another 1, 4 or 7 days (group 1). Alternatively, cells were incubated permanently with LA for 3, 6 or 9 days (group 2). Live cell count was assessed using trypan blue staining. Viability was measured by the tetrazolium bromide assay. Proliferation tests were performed with the help of the colorimetric bromodeoxyuridine assay. Production of reactive oxygen species (ROS) was determined, measuring the oxidation of non‐fluorescent‐2,7′‐dichlorofluorescin. Treatment of cells with the three LA showed a concentration‐dependent decrease of live cells, mitochondrial activity and proliferation rate. Group arrangement played a significant role for cell count and proliferation, while exposure time influenced viability. Among the analysed LA, bupivacaine showed the most severe cytotoxic effects. Increased production of ROS correlated with decreased viability of fibroblasts in lidocaine‐ and bupivacaine‐exposed cells, but not upon stimulation with ropivacaine. This study shows a concentration‐dependent cytotoxic effect of lidocaine, bupivacaine and ropivacaine on fibroblasts in vitro, with more pronounced effects after continuous incubation. A possible mechanism of cell impairment could be triggered by production of ROS upon stimulation with lidocaine and bupivacaine.

Effects of blood products on inflammatory response in endothelial cells in vitro.

Background Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. Methods The inflammatory response from pre-activated (endotoxin-stimulated) and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC), platelet concentrates (PC) and fresh frozen plasma (FFP) was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. Results Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. Conclusion Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.

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.

Nanomagnet-based removal of lead and digoxin from living rats

In a number of clinical conditions such as intoxication, bacteraemia or autoimmune diseases the removal of the disease-causing factor from blood would be the most direct cure. However, physicochemical characteristics of the target compounds limit the applicability of classical filtration and diffusion-based processes. In this work, we present a first in vivo magnetic blood purification rodent animal model and demonstrate its ability to rapidly clear toxins from blood circulation using two model toxins with stable plasma levels (lead (Pb(2+)) and digoxin). Ultra-strong functionalized metal nanomagnets are employed to eliminate the toxin from whole blood in an extracorporeal circuit. In the present experimental demonstration over 40% of the toxin (i.e. lead or digoxin) was removed within the first 10 minutes and over 75% within 40 minutes. After capturing the target substance, a magnetic trap prevents the toxin-loaded nanoparticles from entering the blood circulation. Elemental analysis and magnetic hysteresis measurements confirm full particle recovery by simple magnetic separation (residual particle concentration below 1 μg mL(-1) (detection limit)). We demonstrate that magnetic separation-based blood purification offers rapid blood cleaning from noxious agents, germs or other deleterious materials with relevance to a number of clinical conditions. Based on this new approach, current blood purification technologies can be extended to efficiently remove disease-causing factors, e.g. overdosed drugs, bacteria or cancer cells without being limited by filter cut-offs or column surface saturation.

Volatile Anesthetics Improve Survival after Cecal Ligation and Puncture

Background:Sepsis remains a leading cause of death in intensive care units. There is growing evidence that volatile anesthetics have beneficial immunomodulatory effects on complex inflammation-mediated conditions. The authors investigated the effect of volatile anesthetics on the overall survival of mice in a sepsis model of cecal ligation and puncture (CLP). Methods:Mice (N = 12 per treatment group) were exposed to anesthetic concentrations of desflurane, isoflurane, and sevoflurane either during induction of sepsis or when the mice showed pronounced symptoms of inflammation. Overall survival, as well as organ function and inflammation was compared with the CLP group without intervention. Results:With desflurane and sevoflurane conditioning (1.2 minimal alveolar concentration for 2 h immediately after induction of CLP) overall survival was improved to 58% and 83%, respectively, compared with 17% in the untreated CLP group. Isoflurane did not significantly affect outcome. Application of sevoflurane 24 h after sepsis induction significantly improved overall survival to 66%. Conclusions:Administration of the volatile anesthetics desflurane and sevoflurane reduced CLP-induced mortality. Anesthesia may be a critical confounder when comparing study data where different anesthesia protocols were used.

Endotoxin Removal by Magnetic Separation-Based Blood Purification

This work describes a magnetic separation-based approach using polymyxin B-functionalized metal alloy nanomagnets for the rapid elimination of endotoxins from human blood in vitro and functional assays to evaluate the biological relevance of the blood purification process. Playing a central role in gram-negative sepsis, bacteria-derived endotoxins are attractive therapeutic targets. However, both direct endotoxin detection in and removal from protein-rich fluids remains challenging. We present the synthesis and functionalization of ultra-magnetic cobalt/iron alloy nanoparticles and a magnetic separation-based approach using polymyxin B-functionalized nanomagnets to remove endotoxin from human blood in vitro. Conventional chromogenic Limulus Amebocyte Lysate assays confirm decreased endotoxin activity in purified compared to untreated samples. Functional assays assessing key steps in host defense against bacteria show an attenuated inflammatory mediator expression from human primary endothelial cells in response to purified blood samples compared to untreated blood and less chemotactic activity. Exposing Escherichia coli-positive blood samples to polymyxin B-functionalized nanomagnets even impairs the ability of gram-negative bacteria to form colony forming units, thus making magnetic separation based blood purification a promising new approach for future sepsis treatment.

Iron core/shell nanoparticles as magnetic drug carriers: possible interactions with the vascular compartment

AIMS Nanomagnets with metal cores have recently been shown to be promising candidates for magnetic drug delivery due to higher magnetic moments compared with commonly used metal oxides. Successful application strongly relies on a safe implementation that goes along with detailed knowledge of interactions and effects that nanomagnets might impart once entering the body. MATERIALS & METHODS In this work, we put a particular focus on the interactions of ultra-strong metal nanomagnets (≥ three-times higher in magnetization compared with oxide nanoparticles) within the vascular compartment. Individual aspects of possible effects are addressed, including interactions with the coagulation cascade, the complement system, phagocytes and toxic or inflammatory reactions both by blood and endothelial cells in response to nanomagnet exposure. RESULTS We show that carbon-coated metal nanomagnets are well-tolerated by cells of the vascular compartment and have only minor effects on blood coagulation. CONCLUSION These findings provide the fundament to initiate successful first in vivo evaluations opening metal nanomagnets with improved magnetic properties to fascinating applications in nanomedicine.

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.

Intravenous Application of a Primary Sevoflurane Metabolite Improves Outcome in Murine Septic Peritonitis: First Results

Volatile anesthetics are known to have immunomodulatory effects in conditions of organ injury. A recent study in an experimental sepsis model has shown remarkably improved survival when mice were exposed to volatile anesthetics. In the present study, we show that hexafluoroisopropanol – a water-soluble primary sevoflurane metabolite – has beneficial effects on the overall survival in a murine model of cecal ligation and puncture. Seven-day survival as well as tissue damage markers including transaminases and high mobility group box protein-1 were assessed as measures of end organ damage. In animals undergoing cecal ligation and puncture procedure hexafluoroisopropanol conditioning - but not late postconditioning 24 hours after sepsis induction - significantly increased survival rate (17% vs. 77%, p = 0.037) and attenuated secretion of organ damage markers. This study shows survival benefits by administration of the metabolite of a volatile anesthetic. If successfully translated, hexafluoroisopropanol might offer interesting therapeutic opportunities in the future treatment of abdominal sepsis.

Insight into the beneficial immunomodulatory mechanism of the sevoflurane metabolite hexafluoro-2-propanol in a rat model of endotoxaemia

Volatile anaesthetics such as sevoflurane attenuate inflammatory processes, thereby impacting patient outcome significantly. Their inhalative administration is, however, strictly limited to controlled environments such as operating theatres, and thus an intravenously injectable immunomodulatory drug would offer distinct advantages. As protective effects of volatile anaesthetics have been associated with the presence of trifluorinated carbon groups in their basic structure, in this study we investigated the water‐soluble sevoflurane metabolite hexafluoro‐2‐propanol (HFIP) as a potential immunomodulatory drug in a rat model of endotoxic shock. Male Wistar rats were subjected to intravenous lipopolysaccharide (LPS) and thereafter were treated with HFIP. Plasma and tissue inflammatory mediators, neutrophil invasion, tissue damage and haemodynamic stability were the dedicated end‐points. In an endotoxin‐induced endothelial cell injury model, underlying mechanisms were elucidated using gene expression and gene reporter analyses. HFIP reduced the systemic inflammatory response significantly and decreased endotoxin‐induced tissue damage. Additionally, the LPS‐provoked drop in blood pressure of animals was resolved by HFIP treatment. Pathway analysis revealed that the observed attenuation of the inflammatory process was associated with reduced nuclear factor kappa B (NF‐κΒ) activation and suppression of its dependent transcripts. Taken together, intravenous administration of HFIP exerts promising immunomodulatory effects in endotoxaemic rats. The possibility of intravenous administration would overcome limitations of volatile anaesthetics, and thus HFIP might therefore represent an interesting future drug candidate for states of severe inflammation.