Ropivacaine Decreases Inflammation in Experimental Endotoxin-induced Lung Injury
Blumenthal, Stephan M.D.*; Borgeat, Alain M.D.†; Pasch, Thomas M.D.‡; Reyes, Livia§; Booy, Christa§; Lambert, Maud M.D.∥; Schimmer, Ralph C. M.D., M.B.A.#; Beck-Schimmer, Beatrice M.D.**
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 μm 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.
INTRATRACHEAL application of bacterial endotoxin leads to an inflammatory reaction with pathologic characteristics, resembling an acute respiratory distress syndrome. This acute lung injury (ALI) represents a suitable experimental system to investigate the immunopathologic mechanisms of acute respiratory distress syndrome. Studies using lipopolysaccharide to induce lung injury have mainly focused on interactions involving the vascular compartment; however, interest in the epithelial compartment has increased since its contribution to the inflammatory reaction by interaction of effector cells (macrophages, neutrophils) with target cells (airway epithelial cells) has been shown.1–3
A local intervention through the airways is a possible diagnostic and therapeutic option.
Both rat pulmonary artery endothelial cells (RPAECs) and alveolar epithelial cells (AECs) express and secrete various inflammatory mediators, such as adhesion molecules (intercellular adhesion molecule 1 [ICAM-1]), cytokines (tumor necrosis factor α [TNF-α]) and chemokines (cytokine-induced neutrophil chemoattractant 1 [CINC-1], monocyte chemoattractant protein 1 [MCP-1]).3–7
The airway epithelial is thought to play an important role in the initiation and exacerbation of inflammatory response within the airways, recruiting effector cells through the expression and production of these inflammatory mediators.
Local anesthetics have been shown to have antiinflammatory properties, influencing a broad spectrum of effector cell functions such as phagocytosis and superoxide production, which are relevant to inflammatory diseases.8,9
Ropivacaine, a newer local anesthetic, shows many of the properties of other highly liposoluble local anesthetics but benefits from a larger safety margin.10–12
Few studies exist regarding the possible protective effects of this local anesthetic in inflammatory situations.13
We hypothesized decreased inflammatory reactions and attenuation of ALI in the presence of ropivacaine. To explore this research questions, the immune modulating effect of ropivacaine was evaluated in a model of lipopolysaccharide-induced lung injury in rat AECs and RPAECs (in vitro) and in rat lung (in vivo).
Materials and Methods
All animal experiments and animal care were approved by the Swiss Veterinary Health Authorities, Zurich, Switzerland.
Alveolar Epithelial Cells
L2 cells (CCL 149; ATCC, Rockville, MD), a clonally isolated cell line from the adult rat lung of alveolar epithelial cell type II origin, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% HEPES buffer.14
Experiments were performed with confluent cells from passages 10–20. Before cell stimulation, the medium was changed to DMEM–1% FBS.
Rat Pulmonary Artery Endothelial Cells
Rat pulmonary artery endothelial cells, kindly provided by Roscoe Warner, Ph.D. (Research Assistant Professor, Department of Pathology, University of Michigan, Ann Arbor, Michigan), were cultured in DMEM, supplemented with 10% FBS, 1% penicillin, and 1% HEPES. RPAECs from passages 25–35 were grown until confluence.
Stimulation with Lipopolysaccharide
Confluent monolayers of AECs and RPAECs were exposed to Escherichia coli serotype 055:B5 lipopolysaccharide (20 μg/ml; Sigma, Buchs, Switzerland) for 4 h, diluted in DMEM–1% FBS, or to phosphate-buffered saline (PBS) in DMEM-1% FBS as a control.
In a first experimental setup, different blocking concentrations (100, 10, and 1 μm) of ropivacaine (AstraZeneca, Zug, Switzerland) were tested for the effect of ICAM-1 expression in AECs and RPAECs. Lipopolysaccharide and ropivacaine were added at the same time to the cells. One micromolar proved to be the most effective concentration. For the final experiments, four different groups were designed: PBS–PBS, PBS–1 μm ropivacaine, lipopolysaccharide–PBS, and lipopolysaccharide–1 μm ropivacaine. Cells were collected after 4 h of stimulation. Ropivacaine-induced cytotoxicity was excluded performing lactate dehydrogenase assays (Promega, Madison, WI).
RNA Extraction and Reverse-transcription Polymerase Chain Reaction
Total RNA from AECs and RPAECs was extracted using TRIZOL® (Invitrogen, Life Technologies, Carlsbad, CA) as described previously, and messenger RNA (mRNA) for ICAM-1 was determined.15
For analysis, reverse-transcription polymerase chain reaction was performed using a polymerase chain reaction core kit (Applied Biosystems, Rotkreuz, Switzerland). Random hexanucleotide primers and murine leukemia virus reverse transcriptase were used for complementary DNA synthesis. Reverse transcription was performed with 0.8 μg RNA at 20°C for 5 min, at 42°C for 30 min, and at 99°C for 5 min. Primers located on separate exons were designed to assess expression ICAM-1 (table 1
). For the primer pair, optimal cycling conditions were tested to detect target genes within the logarithmic amplification phase. Reverse-transcription polymerase chain reaction products were resolved on 1.5% agarose gels (Ultra Pure; Invitrogen, Basel, Switzerland) and stained with ethidium bromide.
Isolation of Neutrophils
Venous blood was obtained from healthy adult volunteers and anticoagulated with citrate–dextrose solution (Sigma, Buchs, Switzerland) as described previously.16
Neutrophils were isolated by gradient centrifugation over Ficoll-Paque (Amersham Pharmacia, Biotech, Dubendorf, Switzerland) followed by 1% dextran (Amersham Pharmacia, Biotech, Dubendorf, Switzerland) sedimentation for 1 h to separate neutrophils from erythrocytes. After centrifugation of the supernatant, the contaminating erythrocytes were lysed. The remaining neutrophils were resuspended in Hank’s balanced salt solution without calcium and magnesium (Life Technologies, Basel, Switzerland). The entire procedure was performed at 15°C.
Assay of Neutrophil Adherence
Alveolar epithelial cells or RPAECs were plated in DMEM–10% FBS on tissue culture–treated 96-well microtiter plates (NUNC Products, Life Technologies, Basel, Switzerland) and grown to confluence at 37°C with 5% CO2. After stimulation with E. coli serotype 0.55:B5 lipopolysaccharide (20 μg/ml) in DMEM–1% FBS or a mixture of lipopolysaccharide and 1 μm ropivacaine for 4 h, 1 × 106 neutrophils were added to each well, and the microtiter plates were incubated for 15 min at 37°C with 5% CO2. This incubation step allowed neutrophils to settle onto the epithelial cell monolayer before being stimulated with phorbol 12-myristate 13-acetate (final concentration of 100 ng/well). Thirty minutes later, nonadherent cells were removed by washing twice with DMEM. Finally, the number of adherent neutrophils was determined by counting them in a defined area using a Neubauer chamber.
Equation (Uncited)Image Tools
Cytotoxicity was measured using a nonradioactive standard lactate dehydrogenase (LDH) assay (Promega). AECs or RPAECs were seeded into the wells of a 96-well culture dish. Cells were stimulated with lipopolysaccharide (20 μg/ml) for 4 h. Neutrophils (1 × 106
) in PBS–0.02% bovine serum albumin were added to each well, followed by a stimulation with phorbol 12-myristate 13-acetate 15 min later as described. After incubation for 5 h, the supernatant was removed from each well and centrifuged at 700g
for 5 min. Some target cells were incubated with lysing solution for 45 min (target cell maximum LDH release). Supernatant was also collected from wells of AECs or RPAECs, which had not been incubated with neutrophils (spontaneous LDH release). Supernatant from wells, incubated with neutrophils, were defined as experimental release. LDH content was determined by measuring red formazan resulting from the conversion of tetrazolium salt into this substrate by LDH. Cytotoxicity was calculated according to the following formula:
Animal Model of Acute Lung Injury
Pathogen-free male Wistar rats (250–300 g) were housed in individual isolator cages within the Animal Care Facilities at the University of Zurich. Rats were anesthetized with subcutaneously administered Hypnorm® (fentanyl–fluanisone; Janssen, Beerse, Belgium), 0.25 ml/kg body weight; Domitor® (medetomidine; Pfizer, Inc., Westchester, PA), 0.25 ml/kg body weight; and 0.1% atropine, 0.05 ml/kg body weight.
The lowest concentration of ropivacaine with best effect was determined in pilot experiments (data not shown). In a first series of experiments, lung injury was induced by intratracheal instillation of 150 μg lipopolysaccharide in 300 μl PBS either with or without 1 mm ropivacaine. For intravenous blocking experiments, which were performed in a second series, 300 μl of 1 mm ropivacaine or 300 μl PBS for control animals were applied intravenously immediately after intratracheal lipopolysaccharide instillation. In both series, four groups were tested (PBS–PBS, PBS–ropivacaine, lipopolysaccharide–PBS, and lipopolysaccharide–ropivacaine), and every group consisted of five animals. Five hours after onset of lung injury, animals were killed, and bronchoalveolar lavage was performed, followed by excision of the lung.
For bronchoalveolar lavage, 10 ml PBS was gently instilled into the lungs, withdrawn, and reinstilled three times and collected. Bronchoalveolar lavage fluid (BALF) was centrifuged at 700g. Supernatant was aliquoted and frozen at −20°C. Cell pellets from centrifuged BALF were assessed for differential cell counts using cytospins and Diff-Quick (Dade Behring, Duedingen, Switzerland).
Determination of Inflammatory Mediators in BALF
To study inflammatory changes in the respiratory compartment, the following mediators were determined: TNF-α, CINC-1, and MCP-1. Enzyme-linked immunosorbent assays (ELISAs) were performed according to the manufacturer’s recommendations, using OptEIA rat TNF-α and MCP-1 ELISA (Pharmingen, San Diego, CA) and rat CINC-1 ELISA (R&D Systems, Abingdon, United Kingdom), respectively. Optical density was determined at 492 nm.
Extravasation of albumin was assessed by a direct ELISA according to a previous protocol.17
Briefly, coating carbonate buffer (0.1 m carbonate, pH 9.5) was used to dilute samples (BALF from injured lungs, 1:1,000), and a standard curve was created with recombinant rat albumin (RDI, Flanders, NJ). Ninety-six–well plates were coated with samples and recombinant albumin and incubated overnight at 4°C. A first polyclonal rabbit anti-rat albumin antibody (RDI; 10 μg/ml) was incubated for 1 h at 4°C (rabbit immunoglobulin G used as a control), followed by a 1-h incubation at 4°C with a secondary horseradish peroxidase–labeled goat anti-rabbit antibody (Sigma, Buchs, Switzerland). Color reaction was developed with o
-phenylenediamine dihydrochloride (Sigma), the reaction was stopped with 3 m H2
, and optical density was determined at 492 nm (ELISA reader; Bioconcept, Allschwil, Switzerland).
Results are expressed as mean ± SEM. Increases were expressed in percentage or absolute values. Ropivacaine-induced reduction in the presence of lipopolysaccharide was calculated as the relative change compared with lipopolysaccharide without ropivacaine results (= 100%). All experiments were conducted at least five times. Analysis of variance with post–analysis of variance comparison was performed to assess the statistical significance of differences at a 5% level. Densitometry data are expressed as percentage values of controls (control = 100%).
Ropivacaine-induced Change of ICAM-1 mRNA Expression in AECs and RPAECs
Alveolar epithelial cells were stimulated with lipopolysaccharide for 4 h with or without the presence of ropivacaine. Control cells were exposed to PBS. Several concentrations of this local anesthetic were tested in a first experimental setup to assess the blocking effect of ropivacaine: 100, 10, and 1 μm final concentration of ropivacaine. RNA was extracted, and reverse-transcription polymerase chain reaction was performed. Upon lipopolysaccharide stimulation, an up-regulation of mRNA for ICAM-1 of 263% could be demonstrated compared with control cells (P
< 0.005; fig. 1A
). Coincubation of the cells with the local anesthetic attenuated increased ICAM-1 expression according to the used ropivacaine concentration. Whereas 100 μm ropivacaine did not show any blocking effect, 10 μm ropivacaine down-regulated ICAM-1 expression by 27% (P
< 0.05). One micromolar ropivacaine as the most effective concentration had an inhibitory effect showing a decrease of ICAM-1 expression by 41% (P
Changes in mRNA for ICAM-1 showed a similar pattern in RPAECs as demonstrated in AECs (fig. 1B
). Lipopolysaccharide enhanced ICAM-1 expression with an increase of 107% (P
< 0.05). Ropivacaine at 100 and 10 μm did not significantly alter mRNA for ICAM-1, but 1 μm ropivacaine decreased up-regulated ICAM-1 expression in endothelial cells by 24% (P
Adherence of Neutrophils to Alveolar Epithelial and Pulmonary Endothelial Cells after Lipopolysaccharide Exposure: Role of Ropivacaine
Adherence assays were performed using neutrophils to define the biologic role of ropivacaine in regard to up-regulated ICAM-1 (fig. 2A
). AECs were exposed to lipopolysaccharide for 4 h, followed by an incubation with phorbol 12-myristate 13-acetate–stimulated neutrophils. Adherence of these effector cells to endotoxin-exposed cells increased substantially by 1,115% compared with control cells (P
< 0.00001). Ropivacaine administration blocked this enhanced adherence by 58% (P
In the case of RPAECs, increased adhesion of neutrophils (692% increase, P
< 0.0001) was attenuated by 44% with ropivacaine (P
< 0.005; fig. 2B
Damage to Alveolar Epithelial and Pulmonary Endothelial Cells Caused by Neutrophils
We also studied the ability of ropivacaine to influence neutrophil-induced damage to AECs and RPAECs. Phorbol 12-myristate 13-acetate–stimulated neutrophils were incubated with lipopolysaccharide-stimulated AECs or with control cells, and cytotoxicity was determined. Figure 3
shows LDH release as an indirect measurement of cell death. Neutrophils as a source of possible LDH release could be excluded by trypan blue staining. Control AECs exposed to stimulated neutrophils showed a cytotoxicity of 32%, whereas cytotoxicity of lipopolysaccharide-stimulated AECs increased to 58% (P
< 0.01; fig. 3A
). With ropivacaine administration, cytotoxicity was observed to decrease by 28% (P
Similar data were obtained with RPAECs (fig. 3B
). Lipopolysaccharide induced an increase of cytotoxicity by 111% (P
< 0.05). This increase was attenuated in the presence of ropivacaine by 33% (P
Effect of Ropivacaine on Neutrophil Accumulation in Lipopolysaccharide-injured Lungs
To evaluate cellular elements quantitatively and qualitatively in the respiratory compartment, BALF cells were analyzed, with focus on neutrophils. As seen in figure 4A
, neutrophil content increased 5 h after lipopolysaccharide stimulation from 0.2 × 106
to 43 × 106
< 0.00001). Intratracheal application of ropivacaine in lipopolysaccharide-injured lungs attenuated neutrophil accumulation by 56% (P
Intervention with intravenously applied ropivacaine reduced neutrophil recruitment by 81% (fig. 4B
) compared with lipopolysaccharide–PBS animals (P
Ropivacaine and Inflammatory Mediators in the Respiratory Epithelial Compartment
To determine possible neutrophil chemoattractants, responsible for the ropivacaine-induced attenuation of neutrophil recruitment, protein expression of the important inflammatory mediators TNF-α, CINC-1, and MCP-1 in BALF was assessed.
Tumor necrosis factor α concentration increased from 0.24 ng/ml in PBS animals to 8.54 ng/ml in endotoxin-injured animals (P
< 0.00001; fig. 5A
). In the presence of intratracheally applied ropivacaine, TNF-α expression was attenuated by 48% (P
< 0.005). With intravenous ropivacaine, TNF-α concentration was decreased by 78% (P
< 0.0005; fig. 5B
In PBS animals, a baseline CINC-1 concentration of 0.15 ng/ml was measured in the respiratory compartment (fig. 6A
). Upon lipopolysaccharide stimulation, it increased to 1.07 ng/ml (P
< 0.0005) and was attenuated by intratracheally applied ropivacaine by 59% (P
< 0.001). Intravenous ropivacaine reduced CINC-1 by 69% (P
< 0.005; fig. 6B
As previously shown, MCP-1 plays a crucial role in neutrophil recruitment in the early endotoxin-induced lung injury and was therefore assessed in this study as well.18
In control lungs, a MCP-1 concentration of 0.58 ng/ml was measured, which increased to 34.08 ng/ml in lipopolysaccharide-injured lungs (P
< 0.0005; fig. 7A
). In the presence of intratracheal ropivacaine, this increase was reduced to 17.02 ng/ml (51% down-regulation, P
< 0.01). For intravenous intervention of ropivacaine, an 81% reduction was found (P
< 0.001; fig. 7B
Albumin in the respiratory compartment is a reliable diagnostic parameter for the change of alveolocapillary permeability. To evaluate the effect of ropivacaine on vascular permeability, albumin content was determined in BALF. Intratracheally applied lipopolysaccharide caused an increase of albumin from 81 to 1,093 ng/ml (P
< 0.00005; fig. 8A
). In animals treated with ropivacaine intratracheally, a 46% attenuation of the albumin content was observed (P
< 0.0005), whereas intravenous ropivacaine application decreased the albumin content by 38% (P
< 0.05; fig. 8B
In the current study, we have shown that ropivacaine is an effective antiinflammatory substance in a model of endotoxin-induced lung injury in vitro and in vivo. The use of in vitro ropivacaine significantly decreased ICAM-1–mediated neutrophil adherence and effector cell–induced killing in pulmonary endothelial and epithelial cells. In a model of ALI in vivo, ropivacaine showed effect on two important parameters of inflammation: It attenuated accumulation of effector cells, possibly by down-regulation of key chemotaxins, and in addition reduced alveolocapillary permeability. This diminished inflammatory response to lipopolysaccharide was observed not only for intravenous but also for intratracheal application of ropivacaine.
Lidocaine and other local anesthetics were recently shown to have various inhibitory effects in inflammatory processes. Most of these studies were performed in neutrophils, with focus on the expression of CD11b/CD18 or superoxide anion release.19–21
All of these experiments aimed at the function of effector cells such as neutrophils and macrophages. Our goal was to determine the impact of ropivacaine, not on effector, but on target cells, which were exposed to noxious substances.
In a first approach, we tested the effect of ropivacaine on lipopolysaccharide-injured endothelial and epithelial lung cells in vitro
. Because several studies have highlighted the importance of the respiratory epithelial compartment in the process of lung inflammation, we studied not only the vascular but also the respiratory compartment.2,3,18,22
Ropivacaine suppressed both endothelial and epithelial ICAM-1 expression, with ICAM-1 being a key adhesion molecule at the beginning of the inflammatory reaction. Importantly, possible biologic consequences of decreased ICAM-1 expression, such as adhesion of effector cells and target cell killing, were diminished as well in our study. The antiinflammatory effect of local anesthetics on epithelial and endothelial cells described herein represents new information.
The effect of ropivacaine regarding mRNA expression for ICAM-1 represented a reverse concentration–response relation (fig. 1
). Ropivacaine might interact with a membrane receptor, inducing an inhibitory cell-signaling pathway and thereby negatively interfering with the lipopolysaccharide-induced pathways such as toll-like receptors, CD14, or lipopolysaccharide-binding protein. With higher concentrations, ropivacaine molecules might change configuration, therefore not being able to interact anymore with the according membrane receptor. Regarding clinical applicability of ropivacaine, the phenomenon of its specific concentration–response curve would imply a certain positive aspect with regard to the margin of safety.
Another interesting phenomenon was the missing blocking effect of ropivacaine on cultured cells in the absence of lipopolysaccharide (fig. 1
). This might give rise to the hypothesis that ropivacaine exclusively interferes with lipopolysaccharide-depending intracellular pathways.
Lidocaine has already been studied in vivo
to determine a potential inhibitory effect of this local anesthetic substance in acute lung inflammation. Lidocaine significantly attenuated lung injury in a model of endotoxemia.23–25
A similar protective effect with lidocaine was achieved in acid aspiration, reperfusion injury, and hyperoxia-induced lung injury.26–28
However, none of these studies evaluated ropivacaine or an intratracheal application.
There are several reasons to consider and further investigate the use of the new local anesthetic ropivacaine in inflammatory lung processes because it might be safer. Although lidocaine has shown beneficial effects regarding the inflammation process in ALI, it was declared as a potentially harmful drug in patients with ALI.29
In addition, some local anesthetics have been implicated as a cause of ALI.30,31
It is known from lidocaine that inhibition of neutrophils also suppresses microbial clearance functions during infections. Other neutrophil-directed antiinflammatory agents, which have also been shown to inhibit ALI related to noninfectious causes, ultimately worsened the outcome in infection-related ALI.32,33
Cardiotoxicity, as one of the most severe complications of local anesthetics, is another issue that raises concern when these drugs are used.34,35
Although lidocaine has the longest history of being given systemically, there are also concerns, which have been defined. Lidocaine, when used to suppress ventricular fibrillation in acute myocardial infarction, has increased the incidence of asystolic arrest.35
In addition, lidocaine might worsen congestive heart failure in patients with ALI.34,36
Finally, ropivacaine has been shown to have a wider margin of safety as compared with lidocaine in experimental studies. Lidocaine, 0.6%, but not 0.1% ropivacaine, can induce apoptosis on a T-cell line, and higher neurotoxicity of lidocaine compared with ropivacaine was observed when tested on dorsal root ganglion neurons from chick embryos.37,38
Another interesting aspect of our study is the attenuation of ALI after intratracheal instillation of ropivacaine. The intratracheal approach essentially represents a local rather than a systemic administration, which would allow the potential effect to be targeted to the lungs with a possible reduction of undesired systemic effects. A study performed with lidocaine has shown that intravenous and intratracheal applications of lidocaine have the same effect regarding treatment of bronchial hyperactivity/constriction.39
However, plasma concentration of lidocaine after intratracheally applied lidocaine treatment was only one third compared with systemic application. As direct instillation of endotoxin into lungs produces a characteristic pattern of inflammation similar to that found in acute respiratory distress syndrome, therapy directed toward limiting the local inflammatory response would be an option for improving outcomes in humans with acute respiratory distress syndrome.22
In summary, we have shown that ropivacaine has strong antiinflammatory effects on neutrophils, but also on endothelial and epithelial lung cells in vitro and in vivo. Beside the topical effect of this local anesthetic, when intratracheally instilled, inflammatory actions were inhibited by low and clinically relevant doses of ropivacaine. It will be of further interest to elucidate the protective effects of ropivacaine in vivo to answer critical questions regarding the efficacy and safety of this new local anesthetic in the context of ALI.
The authors thank Irene Odermatt (Art Designer, Institute of Anesthesiology, University of Zurich, Zurich, Switzerland) and Christian Gasser (Art Designer, Institute of Physiology, University of Zurich) for development of illustrations.
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