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N-acetyl cysteine protects against chlorine-induced tissue damage in an ex vivo model
Toxicology Letters 322 (2020) 58–65
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
N-acetyl cysteine protects against chlorine-induced tissue damage in an ex vivo model
T
Lina Ågren, Linda Elfsmark, Christine Akfur, Lars Hägglund, Barbro Ekstrand-Hammarström, Sofia Jonasson* Swedish Defence Research Agency, CBRN Defence and Security, Umeå, Sweden
A R T I C LE I N FO
A B S T R A C T
Keywords: Precision-cut lung slices (PCLS) Chlorine Lung-injury N-acetyl cysteine Treatment Rat
High-level concentrations of chlorine (Cl2) can cause life-threatening lung injuries and the objective in this study was to understand the pathogenesis of short-term sequelae of Cl2-induced lung injury and to evaluate whether pre-treatment with the antioxidant N-acetyl cysteine (NAC) could counteract these injuries using Cl2-exposed precision-cut lung slices (PCLS). The lungs of Sprague-Dawley rats were filled with agarose solution and cut into 250 μm-thick slices that were exposed to Cl2 (20−600 ppm) and incubated for 30 min. The tissue slices were pre-treated with NAC (5−25 mM) before exposure to Cl2. Toxicological responses were analyzed after 5 h by measurement of LDH, WST-1 and inflammatory mediators (IL-1β, IL-6 and CINC-1) in medium or lung tissue homogenate. Exposure to Cl2 induced a concentration-dependent cytotoxicity (LDH/WST-1) and IL-1β release in medium. Similar cytokine response was detected in tissue homogenate. Contraction of larger airways was measured using electric-field-stimulation method, 200 ppm and control slices had similar contraction level (39 ± 5%) but in the 400 ppm Cl2 group, the evoked contraction was smaller (7 ± 3%) possibly due to tissue damage. NAC-treatment improved cell viability and reduced tissue damage and the contraction was similar to control levels (50 ± 11%) in the NAC treated Cl2-exposed slices. In conclusion, Cl2 induced a concentration-dependent lung tissue damage that was effectively prevented with pre-treatment with NAC. There is a great need to improve the medical treatment of acute lung injury and this PCLS method offers a way to identify and to test new concepts of treatment of Cl2-induced lung injuries.
1. Introduction Chlorine (Cl2) is a reactive oxidizing industrial gas that is highly toxic. Due to its high toxicity, Cl2 has historically been used as a chemical weapon and is still considered a terrorist threat and used in armed conflicts globally (Winder, 2001; Szinicz, 2005; Jones et al., 2010). In contact with water, Cl2 produces hydrochloric and hypochlorous acids, which can lead to a variety of respiratory injuries both in upper and lower airways if Cl2 is accidentally inhaled and high-level concentrations of Cl2 can cause life-threatening lung injuries (White and Martin, 2010). The applicability of precision-cut lung slices (PCLS) to study the activation of early toxic responses was tested with the aim to understand the pathogenesis of short-term sequelae of Cl2-induced lung-injury. The hallmarks of acute lung injury after Cl2 exposure, such as edema, influx of inflammatory cells and airway hyperresponsiveness (AHR), are more difficult to investigate using PCLS (White and Martin, 2010). However, by using PCLS, the number of animals can be reduced
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and the model has been used to evaluate different types of drugs prior to in vivo testing. The PCLS technique has been used for decades and is widely used to examine airway responses in different species. Major advantages of the PCLS model are the close resemblance in morphology and functionality of the respiratory tract, the numerous number of slices obtained from one animal, and the diversity of intact living pulmonary cells present in the slices (Henjakovic et al., 2008; Davidovich et al., 2013; Lyons-Cohen et al., 2017). The aim of the present study was to investigate whether PCLS have the potential to serve as a generally applicable and effective tool in mimicking chemical-induced lung injury observed in vivo. First, tissue slices were exposed to Cl2 using increasing concentrations of Cl2 and the cytotoxicity was determined as necrotic cell death measured by leakage of the cytosolic enzyme lactate dehydrogenase (LDH) into culture medium (Maes et al., 2015). Loss of metabolic enzyme activity assessed with water-soluble tetrazolium salt (WST-1) was also analyzed in Cl2exposed PCLS. For analyzing the inflammatory response, three
Corresponding author at: Swedish Defence Research Agency, CBRN Defence and Security, SE-901 82, Umeå, Sweden. E-mail address: sofi[email protected] (S. Jonasson).
https://doi.org/10.1016/j.toxlet.2020.01.006 Received 11 September 2019; Received in revised form 7 January 2020; Accepted 9 January 2020 Available online 18 January 2020 0378-4274/ © 2020 Published by Elsevier B.V.
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Fig. 1. Chlorine. The effects of Cl2-exposure analyzed in PCLS medium, A) controls exposed to the balance gas N2, release of B) LDH at 5 h, transformation of C) WST-1 at 2 h (200 and 400 ppm) and expression of the cytokines D) IL-1β, E) CINC-1 and F) IL-6. Measurement of bronchoconstriction induced by EFS-stimulation of PCLS G) PCLS exposed to 200 or 400 ppm Cl2 and at different time-points H) 1−2 h or 5 h post-exposure. Induction of airway constriction by I) methacholine (MCh) in PCLS exposed to Cl2. Data (A-F) show fold-change from control and all values indicate mean ± SEM and significant differences compared to non-exposed control slices are indicated (*p < 0.05, **p < 0.01, and ***p < 0.001). Data (G-I) show the change in % from the baseline airway area (defined as 100 % area size) and all values indicate mean ± SEM and significant differences compared to non-exposed control slices are indicated *control vs 200 ppm, # control (1−2 h) vs 400 ppm (1−2 h) and ¤ control (4−5 h) vs 400 ppm (4−5 h) (*#p < 0.05 and ***,###,¤¤¤ p < 0.001). The number of experiments in each group are presented in fig. A–I.
melting agarose solution (0.75% in incubation medium) after tracheotomy and cooled on ice. The hardened lung was removed from the animal and lobes were separated. Extraction of lung tissue was performed directly postmortem to conserve vitality of the tissue. Tissue cores with a penetrating airway in its center were prepared by using an 8 mm biopsy punch (Histolab, Sweden). Cores were placed in a CryoPure 1.8 mL tube (Sarstedt, Germany) and surrounded by agarose (3% in 150 mM NaCl, Sigma-Aldrich, Sweden). Tissue slices (250 μm) were cut from the cores vertical to the airway by the aid of a Krumdieck tissue slicer (Alabama Research and Development, Munford, AL). Lung slices (n = 88–128 slices/rat) were collected in a petri dish containing 25 mL PCLS-incubation medium and kept in a CO2 incubator at 37 °C. The medium was changed every 30 min during the first 2 h and then every hour for the next 4 h which serves as a stabilization period after slicing to reduce subsequent stress caused from slicing (Liberati et al., 2010). Before overnight incubation, gentamicin (125 μL, 10 mg/ml, Sigma-Aldrich) was added to the PCLS incubation medium and 12 h later the PCLS incubation medium was changed again before the experiment began.
endpoints were used, ELISA assay for CINC-1, IL-6 and IL-1β release both in PCLS culture medium and in tissue homogenate. All these three pro-inflammatory cytokines have been shown to increase in vivo shortly after Cl2-exposure (Jonasson et al., 2013a; Wigenstam et al., 2016b) and have a role in the initial response in the lung repair mechanism and in the development of fibrosis (Papiris et al., 2018). Furthermore, these cytokines are also found in the bronchoalveolar lavage fluids of patients with acute lung injury or acute respiratory distress syndrome (Kiehl et al., 1998; Nys et al., 2003). The aim of this study was also to evaluate whether pre-treatment with a well-characterized substance N-acetyl cysteine (NAC), due to its powerful antioxidant properties (Wigenstam et al., 2015), could counteract the Cl2-induced damages in PCLS. 2. Material and methods 2.1. Preparation of precision-cut lung slices Twenty-two animals were entered into this study. Lungs were prepared from healthy female Sprague-Dawley rats (220–260 g) obtained from Envigo RMS B.V, Netherlands. The animals were kept under controlled conditions in the animal facility providing food and water ad libitum. Animal experiments were approved by the regional ethics committee on animal experiments in Umeå, Sweden according to Directive 2010/64/EU. Animals were anaesthetized with sodium pentobarbital (i.p.) and exsanguinated by cutting the aorta. The lungs were filled with low-
2.2. Preparation of cutting and incubation PCLS medium For use in the Krumdieck slicer, an EBSS cutting medium was prepared by dissolving 0.265 g/L CaCl2 ×H2O, 0.2 g/L MgSO4 ×7H2O, 0.4 g/L KCl, 6.8 g/L NaCl, 0.14 g/L NaH2PO4 ×H2O, 3.0 g/L Glucose, 2.2 g/L NaHCO3, and 6.0 g/L HEPES in MQ water and pH was set to 7.2 59
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Fig. 2. Cell viability. Cell viability (calcein staining intensity) at 5 h in PCLS sections of A) non-exposed controls and in PCLS sections exposed to increasing concentrations of Cl2; B) 200 ppm, C) 400 ppm, D) 600 ppm, or E) Triton X as a negative control. F) Quantification of calcein staining intensity in representative photos of tissue sections from each group using ImageJ (the number of experiments in each group are presented below bars). Values in F represents mean ± SEM and significant differences compared to control are indicated (*** p < 0.001). Photos were taken at 2.5x magnification and analyzed using a fluorescence microscope (495 nm/515 nm).
reduced pH in the medium to 7.1 (5 mM), 6.6 (15 mM) and 6.1 (25 mM). Tissue slices were pre-treated during 30 min and then washed once in PCLS medium prior exposure to Cl2. Control slices were exposed to increasing doses of NAC to evaluate if NAC itself induced toxic effects due to e.g. pH-changes or cell toxicity. Slices were also exposed to NAC 25 mM where the pH of the medium was adjusted to 7.2 as a negative control for the pH changes.
(Sigma-Aldrich). For tissue culturing; a PCLS incubation medium (referred from now on as PCLS medium) containing identical ingredients but with added solutions of Na-Pyruvate (10 mL 10 mM, SigmaAldrich), MEM-Non-essential Amino acid solution 100X (20 mL, SigmaAldrich), MEM-vitamin solution 100X (10 mL, Sigma-Aldrich) and LGlutamine solution (10 mL 200 mM, Sigma-Aldrich) were added and the pH was set to 7.2. The solution was sterile filtered and stored in 4 °C.
2.5. Sample preparation of tissue homogenate and PCLS medium
2.3. Cl2-exposure of PCLS
After removing the PCLS medium (1 ml/well), slices were weighed. The PCLS medium was centrifuged (5 min, 4 °C, 2000 rpm) to remove particulate matter. Slices (n = 4 from each well) was stored at −70 °C until preparation of tissue homogenate. Slices were homogenized in 200 μl PCLS medium using an electric homogenizer (Ika T8 Ultra Turrax Homogenizer) while on ice and then centrifuged (15 min, 4 °C, 2000 rpm) to remove debris. All samples were stored in -70 °C until ELISA-analysis.
The lung slices were placed (n = 4/well) in 0.5 mL PCLS medium in each well using a 24-well plate. A Cl2 gas cylinder (Air Liquide, Germany, 1 mol% Cl2, 99 mol% nitrogen (N2)) was connected to a plastic tube with a catheter (Microlance 21 G, Becton Dickinson (BD) Sweden) directly inserted into the 0.5 mL PCLS medium. A valve (Swagelok, Sweden) was connected to the tube between the manometer on the gas cylinder and the exposure catheter locking the gas flow to 0.4 mL/min which gives 1.2 mg/min (Soap film flowmeter, HewlettPackard 1-10−100 mL). Constant Cl2 gas flow and different exposuredurations was applied to establish a concentration-response (20−600 ppm generated during 2−36 s exposures). The 24-well plate was stored at room temperature (RT) for 30 min after exposure until the PCLS medium (1 mL/well) was changed. The Cl2-exposed plate was stored at RT to avoid chlorine-contamination of non-exposed slices kept in the CO2 incubator. Control slices were sham-treated with 100 mol% N2 to examine if exposure to a non-toxic gas flow could induce cell stress or toxic effects. During incubation, the plates were kept in a CO2 incubator at 37 °C until termination of experiment, at 2 or 5 h post exposure. The pH in the PCLS medium was reduced 15 min after exposure to 200 ppm or 400 ppm Cl2 to 7.13 and 7.0 respectively.
2.6. Analysis of cytotoxicity and live cell staining in PCLS Cytotoxicity of substances was determined using the cell proliferation kit WST-1 (Cellpro-Roche, Sigma-Aldrich) and LDH cytotoxicity detection kit (Cytodet-Roche, Sigma-Aldrich) according to the manufacturer’s instructions (Roche Applied Science) and analyzed using an ELISA reader (Thermo Scientific Mutilskan FC, Thermo Fischer Scientific Oy, Vantaa, Finland). Analysis of ELISA data was performed using the software program for the ELISA reader (SkanIt for Multiskan FC 3.1. Inc. Thermo Fischer Scientific Oy, Vantaa, Finland). Both LDH and WST-1 content was normalized to the amount of tissue (mg) in each well. Viability of PCLS was determined after 5 h by using Calcein AM solution (4 mM in DMSO, Sigma-Aldrich) according to the manufacturer’s instructions and analyzed using a fluorescence microscope (Leica DMR, Leica microsystems). The excitation wavelength was 495 nm and the emission was detected at 515 nm. All photos were taken by using the same settings for all samples.
2.4. NAC pre-treatments of PCLS The antioxidant N-acetyl cysteine (NAC, Sigma-Aldrich) was dissolved in PCLS medium at three different concentrations (5 mM, 15 mM and 25 mM). The pH of each solution was measured and adding NAC 60
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Fig. 3. NAC toxicity and bronchoconstriction. Treatment effects of 5−25 mM N-acetylcysteine (NAC) on LDH release into PCLS medium at 5 h post-exposure to A) 200 ppm or B) 400 ppm Cl2 and NAC-induced effects on WST-1 (compared to control 100 %) at 2 h post exposure to either C) 200 ppm or, D) 400 ppm Cl2. Effects of NAC-treatment (25 mM) on EFS-provoked bronchoconstriction in PCLS at E) 1−2 h or F) 5 h post-exposure to 400 Cl2. Data (A-D) show fold-change from control and all values indicate mean ± SEM. Significant differences compared to control slices are indicated (*p < 0.05, and ***p < 0.001). Data (E-F) show the change in % from the baseline airway area (defined as 100 % area size) and all values indicate mean ± SEM and significant differences are indicated *400 vs 400 ppm +25 mM NAC and #control vs 400 ppm (***,### p < 0.001) at both 1−2 h and 4−5 h after exposure to Cl2. Data (E-F) showing airways before EFS-impulse (set to 100% airway area), maximal contraction directly after impulse (Max) and at relaxation (After). The number of experiments in each group are presented in fig. A–F.
Schlepütz et al. (Schleputz et al., 2011). Photographs were taken before impulse (baseline), directly after impulse (maximum contraction) and 50 s after impulse (relaxation) and the airway area was measured in each photo using an image analysis software (ImageJ 1.52a, National Institutes of Health, USA). The baseline airway area was defined as 100% area size. Photographs were taken 1−2 h or 4−5 h after Cl2exposure and the groups were randomized.
2.7. Analysis of pro-inflammatory cytokines in PCLS medium and tissue The presence of the pro-inflammatory mediators, IL-1β, IL-6 and CINC-1, in PCLS medium or lung tissue homogenate were analyzed individually using specific enzyme linked assay (ELISA) kits (R&D Systems, Inc.) according to the manufacturer’s instructions. The optical density was measured using an ELISA reader and the data was analyzed using the instrument’s software program, referring to the individual standards added to each plate. The total cytokine content of PCLS medium and lung tissue homogenate was normalized to the amount of tissue (mg) in each well.
2.8.2. Methacholine Slices were placed individually in 12 well plates with 2 mL PCLSmedium. The larger airways were studied by microscopy (Leica DM IRB, 10X) and the airway constriction was evoked by adding 25 μL of 8 nM methacholine (MCh, Sigma-Aldrich). Photographs of the baseline airway area were taken before MCh exposure and then every 15 s during 3 min to assess the airway contraction. The airway area was measured in each photo with image analysis software (ImageJ 1.52a). Airway area baseline was defined as 100% area size. Photographs were taken 1−2 h or 4−5 h after Cl2-exposure and the groups were randomized.
2.8. Microscopy analysis of airway contractions in PCLS 2.8.1. Electrical field stimulation Slices were placed individually in 12 well plates with 2 mL PCLSmedium. The larger airways were studied by microscopy (Leica DM IRB, 10X) and airway constriction was electrically evoked by placing the PCLS between two platinum electrodes applying an electrical impulse (Frequency 50 Hz, train width 1 ms, and an output current of 200 mA for 2.5 s). The method used has previously been described by 61
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Fig. 4. NAC bronchoconstriction. Representative photos of PCLS sections from unexposed controls (upper panel) and chlorine (400 ppm Cl2)-exposed PCLS (middle panel) at 1−2 h with and without 25 mM NAC (lower panel) using EFS-stimulation. Photos were taken at 10x magnification using light microscopy, (left column) showing airways before EFS-impulse (set to 100% airway area), maximal contraction directly after impulse (middle column) and at relaxation (right column).
Fig. 5. NAC immune response. Effects of NAC-treatment (5−25 mM) on immune responses induced by 400 ppm Cl2-exposure, measured as release of the cytokines A) IL-1β, B) IL-6, and C) CINC-1 into PCLS medium or in lung tissue homogenates at 5 h. Data show fold-change from control and all values indicate mean ± SEM. Significant differences indicated # all groups vs. control group and *all groups vs. 400-ppm Cl2 group (*,#p < 0.05, **,##p < 0.01, and ***,###p < 0.001). The number of experiments in each group are presented in fig. A–C.
considered significant. The statistical analyses were carried out and graphs were prepared using GraphPad Prism program (version 6.0 GraphPad Software Inc., San Diego, CA, USA).
2.9. Statistics Data are shown as mean ± standard error of mean (SEM) in the graphs. Statistical significance was assessed by parametric methods using one-way analysis of variance (ANOVA) or two-way analysis of variance (ANOVA) to determine differences between groups, followed by Bonferroni post hoc test. A statistical result of p < 0.05 was 62
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cytokine-responses was also shown in lung tissue homogenates after 400 ppm Cl2 exposures (Fig. 5) except for the IL-1β levels that were reduced in the homogenate. The functionality of airways was measured and the larger central airways showed stronger bronchoconstriction than smaller peripheral airways using EFS. The airways of 200 ppm and control lung slices contracted to similar extent (39 ± 5%, Fig. 1G) at 1−2 h after exposure but exposure to 400 ppm Cl2 reduced the ability of the airway to contract, approximately to 7 ± 3% compared to airway area before applying EFS impulse. As displayed in Fig. 1H, the larger airways in control lung slices showed strong bronchoconstriction also after 5 h and the lack of ability to contract was still evident in the 400-ppm Cl2 group. Airway smooth muscle contractions were also induced by exposing the PCLS to MCh (Fig. 1I) at 1−2 h post-exposure to Cl2. Bronchoconstriction was rapidly induced in control lung tissue slices, reducing the airway area by 53 ± 6%, compared to initial values. Slices exposed to 200 and 400 ppm Cl2 had a reduced airway area by approximately 36 ± 6%, a response that was not significantly stronger than in the controls. 3.2. Antioxidant pre-treatment of PCLS reduces Cl2-induced toxicity After 30 min pre-treatment with NAC (5, 15 and 25 mM) the PCLS were exposed to 200 or 400 ppm Cl2, reducing LDH levels to similar levels as in the non-exposed control group (Fig. 3A-B) which indicates that NAC can protect against loss of cell membrane integrity and cell necrosis. The higher doses of NAC also preserved the mitochondrial activity (WST-1) after both 200 and 400 ppm Cl2 exposures (Fig. 3C-D) and re-established bronchoconstriction (Fig. 3E-F and Fig. 4) in lung slices at 1−2 h post-exposure to 400 ppm Cl2 Fig. 4. In 400 ppm-exposed PCLS, NAC prevented release of IL-1β into the PCLS-medium and increased IL-1β levels in lung tissue homogenate significantly (Fig. 5A). After pre-treatment with 5 mM NAC the decrease of IL-6 in PCLS medium was restored to control levels but higher doses of NAC (15 and 25 mM) further increased the cytokine expression to levels significantly higher than in the corresponding control (Fig. 5B). As displayed in Fig. 5C, NAC had similar effect on the levels of CINC-1 in PCLS medium, increasing the levels significantly higher than in controls at the highest concentration 25 mM. Both IL-6 and CINC-1 was significantly increased in NAC-treated lung tissue slices after Cl2 exposure but not to control levels (Fig. 5B and C). Control experiments revealed that pre-treatment with NAC (5−25 mM) alone also caused an increase in IL-6 and IL-1β release from otherwise non-exposed control slices at 5 h compared to healthy non-treated control slices (Fig. 6A). Non-exposed control slices pre-treated with NAC alone had, however, normal levels of IL-6 and CINC-1 in lung homogenate at 5 h (Fig. 6B). Adjusting the pH of the highest concentration of NAC (25 mM) to the same pH as in the PCLS medium did not remediate the observed release of pro-inflammatory cytokines from untreated controls. LDH in PCLS medium (absorbance at 492 nm) was also slightly increased after treatment with 25 mM NAC (from 0.03 ± 0.003 to 0.06 ± 0.003) and using pH-adjusted 25 mM NAC (pH 7.2) further augmented the leakage of LDH to 0.07 ± 0.02 (Fig. 6A). After pre-treatment with 25 mM NAC, the functionality of the larger airways was evaluated both at 1−2 h and at 4−5 h post 400 ppm Cl2 exposure. The bronchoconstriction was approximately 7 ± 3% in Cl2exposed slices, and by pre-treating tissue sections with 25 mM NAC, the airways could contract to similar magnitude as in control PCLS, approximately 50 ± 11% in comparison to initial airway area before the EFS impulse was applied (Fig. 3E-F and Fig. 4).
Fig. 6. Control experiments NAC. Effects of 5−25 mM NAC-treatment on the expression of IL-6, IL-1β and CINC-1 in healthy non-exposed PCLS measured as A) released into medium or B) in lung tissue homogenates at 5 h. * indicate treatment with 25 mM NAC with adjusted pH similar to control medium. Data show fold-change from control and all values indicate mean ± SEM. Significant differences compared to control slices are indicated (#p < 0.05 and ###p < 0.001). The number of experiments in each group are presented in fig. A–B.
3. Results 3.1. Toxic effects induced by Cl2 exposure in PCLS Before using the PCLS to investigate Cl2-induced lung damage, the method of preparing PCLS cultures itself was evaluated for effects on inflammatory cytokine response and cell toxicity. The aim of this experiment was to make sure that the tissue damage caused by PCLS methodology would not interfere with the study of lung damage caused by Cl2. Exposing the slices to the balance gas N2 alone did not cause any significant cell toxicity in the lung tissue but a small statistically significant stress response was observed as IL-1β release in PCLS medium (Fig. 1A). After 30 min exposure to Cl2 and 5 h of incubation, LDH was present in the PCLS medium indicating loss of cell membrane integrity as a consequence of necrotic cell death after Cl2-exposure. Previous studies have shown that LDH release can be regarded either as a direct measurement of a cytotoxic response or as an indirect measure of cell viability. As displayed in Fig. 1B, the LDH release increased with increasing concentration (200−500 ppm) of Cl2. The metabolic activity of the cells was also constrained, measured as the amount of cleaved WST-1 in the tissue (Fig. 1C), showing that higher concentrations of Cl2 significantly reduced the mitochondrial enzyme activity. In addition to LDH and WST-1, viable cells in Cl2-exposed PCLS were stained with calcein at 5 h after exposure (Fig. 2), showing a concomitant decrease in cell viability as the Cl2 concentrations were increased. A concentration-dependent decrease of the pro-inflammatory cytokines CINC-1 and IL-6 was also observed in PCLS medium after exposures to 300−600 ppm Cl2. In contrary, the release of IL-1β increased concentration dependently after exposures to 200−500 ppm. Similar
4. Discussion The main aim of this study was to evaluate PCLS as a suitable ex vivo alternative approach to animal experiments to study the pathogenesis of short-term sequelae of Cl2-induced lung injury. Another objective 63
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MCh-stimulation in the 400-ppm Cl2 group induced more bronchoconstriction than after EFS-stimulation suggesting that Cl2 damages the neuronally mediated airway constriction in airways. Increased reactivity after MCh than EFS in Cl2 exposed slices may also be explained by extensive shedding of epithelial cells, which would result in a more comprehensive triggering of afferent nerve endings potentially leading to increased release of physiological active neuropeptides (Barnes, 1995). This may in turn also lead to increased sensitivity to exogenous provocations (such as MCh) and AHR. Even though the Cl2 exposed slices did not respond as effective as controls, MCh causes contraction of airway smooth muscles by stimulating the muscarinic cholinergic receptors that can be found in both airways and lung parenchyma. A previous study by Schlepütz et al. (Schleputz et al., 2012) has shown that EFS acts on neurons and not directly on airway smooth muscles and that may also be a possible explanation to the different responses in the 400 ppm-exposed slices between the two methods for inducing bronchoconstriction. Treatment with NAC effectively prevented cellular damage and cell death in lung tissue exposed to Cl2 and the contractility after EFS-stimulation of larger airways was preserved after high-dose NAC-treatment. NAC has been shown to protect the alveolar epithelium from reactive oxygen species production both in vitro and in vivo and pre-treatment of lung cells to NAC also decreased the susceptibility to oxidant damage by strengthening intracellular antioxidant defence systems (Simon and Suttorp, 1985; Wagner et al., 1989; Hoffer et al., 1996; Felton et al., 2009). NAC has been shown to interact with numerous biochemical pathways, as an antioxidant drug both due to direct and indirect antioxidant mechanisms (Rushworth and Megson, 2014). NAC breaks thiolated proteins thus releasing free thiols, which are more efficient as antioxidants than NAC and boost the synthesis of GSH and other reduced proteins, which in some cases have an important direct antioxidant activity (Samuni et al., 2013; Aldini et al., 2018). NAC, which has a high content of cysteine, acts as an intracellular GSH precursor that replenishes GSH in GSH-depleted cells, thus minimizing oxidative stress in both endothelial cells and smooth muscle cells (Samuni et al., 2013; Santus et al., 2014; Aldini et al., 2018). Other mechanisms of NAC that have been described are based on its anti-inflammatory properties, endogenous neuromodulator and neurotransmitter signaling functions, mucolytic properties and disulphide breaking activity (Samuni et al., 2013; Aldini et al., 2018). Although several in vivo studies have found that NAC significantly prevents or inhibits oxidative stress under certain conditions after Cl2exposure (Akdur et al., 2008; Leustik et al., 2008; Yadav et al., 2010), a clear molecular mechanism through which NAC exerts this activity is still not known (Samuni et al., 2013; Aldini et al., 2018). In this study, the aim was not to investigate the NAC mechanism more deeply but to investigate if NAC could reverse the symptoms induced after Cl2 exposure ex vivo. Whilst the protective mechanisms of action of NAC remain to be fully clarified, this study provides evidence to support further investigation of NAC as treatment of chemical-induced lung injury. There is a great need to improve medical treatment of acute lung injury and this method offers a way to identify new concepts of treatment of Cl2induced lung injuries. One advantage with this method is the possibility to use human lung tissue e.g. from cancer surgery, for evaluation which enables a more accurate extrapolation of derived ex vivo data to human conditions. This may also give important directions about relevant treatments, aiming to improve the therapeutic effect in humans exposed to high concentrations of toxic chemical gases.
was also to investigate if this PCLS model could be appropriate for drug discovery. With the PCLS model it is possible to test various drugs on the same slice preparation, which also avoids the inter-individual variations and comparable levels of information can be obtained from fewer animals. For the direct analysis of dynamic changes regarding pharmacological responses, a microscopic observation using live lung slices could be obtained where bronchoconstriction can be followed by observing single airways. It is well known that high-level concentrations of Cl2 can cause life threatening lung injuries with onset of symptoms within minutes to hours but that effective treatment of these respiratory injuries and symptoms are still lacking (Williams, 1997; Kales and Christiani, 2004; Evans, 2005; White and Martin, 2010). It has previously been shown in vivo that mice and rats exposed to 200–300 ppm Cl2 develop a neutrophilic pulmonary injury manifested by e.g. pulmonary edema, cardiovascular effects, pulmonary fibrosis and AHR in close accordance with clinical findings in humans exposed to Cl2 (White and Martin, 2010; Jonasson et al., 2013b; Wigenstam et al., 2016b). In this study, NAC was used as pre-treatment for Cl2 exposure due to its powerful antioxidant properties and its potency as free-radical scavenger, and this study evaluates whether pre-treatment with NAC could protect against acute injuries in Cl2-exposed PCLS. Antioxidants have been reported previously to be effective as rescue treatment for Cl2-induced lung injury involving e.g. airway epithelial cell regeneration and NAC has been shown to minimize the oxidative stress and its downstream effects (Akdur et al., 2008; McGovern et al., 2010, 2011; Zarogiannis et al., 2011; Wigenstam et al., 2015). NAC has also been used as treatment using ex vivo rat lung slices exposed to sulphur mustard or perfluoroisobutene and were non-toxic to all cultured rat lung slices at a dose of 5 mM (Hobbs et al., 1993; Wilde and Upshall, 1994). In this present study, all tested NAC doses were non-toxic to the rat PCLS. The contraction of airways in rat PCLS are located to larger bronchi and only minor changes are observed peripherally in small airways (bronchioles) after either MCh or EFS stimulation. The bronchoconstriction of larger airways (bronchi) was most evident in healthy control slices which is not directly comparable to our findings in vivo, using the same animal strain, where tracheostomized rats connected to a small animal ventilator (flexiVent™, Scireq®), used for recording bronchoconstriction, only showed minor constriction (Wigenstam et al., 2016a, b). Ex vivo Cl2 exposure differs from in vivo exposure, when primarily upper and lower airways are being exposed by inhalation, since the entire lung tissue section is directly being exposed ex vivo, including both airways and tissue interstitium. Following chemical exposure, lung repair mechanisms are often initiated immediately, involving cytokine release that change progressively with increased lung damage and reduced airway function (Jonasson et al., 2013a; Elfsmark et al., 2018). Cautious interpretation of experimental results from both in vivo and PCLS ex vivo studies are needed but since the models also show similarities in responses such as cytotoxicity and oxidative stress, PCLS could still be proposed as a method for drug discovery to screen for new interventions. Another challenge is how to treat the PCLS to display characteristics of early fibrosis that can mimic some of the long-term effects observed after Cl2 exposure in vivo (Jonasson et al., 2013a; Alsafadi et al., 2017). Increasing concentrations of Cl2 formed a concentration-dependent pro-inflammatory tissue injury and a decreased metabolic enzyme activity. Disregarding different time-points and exposure set-up systems, the ex vivo model shows a lack of contractility response when using EFSstimulation at higher concentrations of Cl2 (400 ppm) in comparison to in vivo studies, but on the other hand the AHR has not to our awareness been tested in vivo within a few hours after Cl2-exposure. Most in vivo studies have performed AHR measurements at 24 h post-exposure (Samal et al., 2012; Jonasson et al., 2013a; Wigenstam et al., 2016b). When using MCh-stimulation, the bronchoconstriction was comparable between the 200- ppm and 400-ppm Cl2 group respectively and neither of the concentrations differed significantly from control slices. The
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgement
Maes, M., Vanhaecke, T., Cogliati, B., Yanguas, S.C., Willebrords, J., Rogiers, V., Vinken, M., 2015. Measurement of Apoptotic and necrotic cell death in primary hepatocyte cultures. Methods Mol. Biol. 1250, 349–361. McGovern, T.K., Powell, W.S., Day, B.J., White, C.W., Govindaraju, K., KarmoutyQuintana, H., Lavoie, N., Tan, J.J., Martin, J.G., 2010. Dimethylthiourea protects against chlorine induced changes in airway function in a murine model of irritant induced asthma. Respir. Res. 11, 138. McGovern, T., Day, B.J., White, C.W., Powell, W.S., Martin, J.G., 2011. AEOL10150: a novel therapeutic for rescue treatment after toxic gas lung injury. Free Radic. Biol. Med. 50, 602–608. Nys, M., Deby-Dupont, G., Habraken, Y., Legrand-Poels, S., Kohnen, S., Ledoux, D., Canivet, J.L., Damas, P., Lamy, M., 2003. Bronchoalveolar lavage fluids of ventilated patients with acute lung injury activate NF-kappaB in alveolar epithelial cell line: role of reactive oxygen/nitrogen species and cytokines. Nitric Oxide 9, 33–43. Papiris, S.A., Tomos, I.P., Karakatsani, A., Spathis, A., Korbila, I., Analitis, A., Kolilekas, L., Kagouridis, K., Loukides, S., Karakitsos, P., Manali, E.D., 2018. High levels of IL-6 and IL-8 characterize early-on idiopathic pulmonary fibrosis acute exacerbations. Cytokine 102, 168–172. Rushworth, G.F., Megson, I.L., 2014. Existing and potential therapeutic uses for N-acetylcysteine: the need for conversion to intracellular glutathione for antioxidant benefits. Pharmacol. Ther. 141, 150–159. Samal, A.A., Honavar, J., Brandon, A., Bradley, K.M., Doran, S., Liu, Y., Dunaway, C., Steele, C., Postlethwait, E.M., Squadrito, G.L., Fanucchi, M.V., Matalon, S., Patel, R.P., 2012. Administration of nitrite after chlorine gas exposure prevents lung injury: effect of administration modality. Free Radic. Biol. Med. 53, 1431–1439. Samuni, Y., Goldstein, S., Dean, O.M., Berk, M., 2013. The chemistry and biological activities of N-acetylcysteine. Biochim. Biophys. Acta 1830, 4117–4129. Santus, P., Corsico, A., Solidoro, P., Braido, F., Di Marco, F., Scichilone, N., 2014. Oxidative stress and respiratory system: pharmacological and clinical reappraisal of N-acetylcysteine. COPD 11, 705–717. Schleputz, M., Uhlig, S., Martin, C., 2011. Electric field stimulation of precision-cut lung slices. J. Appl. Physiol. 110 (1985), 545–554. Schleputz, M., Rieg, A.D., Seehase, S., Spillner, J., Perez-Bouza, A., Braunschweig, T., Schroeder, T., Bernau, M., Lambermont, V., Schlumbohm, C., Sewald, K., Autschbach, R., Braun, A., Kramer, B.W., Uhlig, S., Martin, C., 2012. Neurally mediated airway constriction in human and other species: a comparative study using precision-cut lung slices (PCLS). PLoS One 7, e47344. Simon, L.M., Suttorp, N., 1985. Lung cell oxidant injury: decrease in oxidant mediated cytotoxicity by N-acetylcysteine. Eur. J. Respir. Dis. Suppl. 139, 132–135. Szinicz, L., 2005. History of chemical and biological warfare agents. Toxicology 214, 167–181. Wagner, P.D., Mathieu-Costello, O., Bebout, D.E., Gray, A.T., Natterson, P.D., Glennow, C., 1989. Protection against pulmonary O2 toxicity by N-acetylcysteine. Eur. Respir. J. 2, 116–126. White, C.W., Martin, J.G., 2010. Chlorine gas inhalation: human clinical evidence of toxicity and experience in animal models. Proc. Am. Thorac. Soc. 7, 257–263. Wigenstam, E., Koch, B., Bucht, A., Jonasson, S., 2015. N-acetyl cysteine improves the effects of corticosteroids in a mouse model of chlorine-induced acute lung injury. Toxicology 328, 40–47. Wigenstam, E., Elfsmark, L., Bucht, A., Jonasson, S., 2016a. Inhaled sulfur dioxide causes pulmonary and systemic inflammation leading to fibrotic respiratory disease in a rat model of chemical-induced lung injury. Toxicology 368-369, 28–36. Wigenstam, E., Elfsmark, L., Koch, B., Bucht, A., Jonasson, S., 2016b. Acute respiratory changes and pulmonary inflammation involving a pathway of TGF-beta1 induction in a rat model of chlorine-induced lung injury. Toxicol. Appl. Pharmacol. 309, 44–54. Wilde, P.E., Upshall, D.G., 1994. Cysteine esters protect cultured rodent lung slices from sulphur mustard. Hum. Exp. Toxicol. 13, 743–748. Williams, J.G., 1997. Inhalation of chlorine gas. Postgrad. Med. J. 73, 697–700. Winder, C., 2001. The toxicology of chlorine. Environ. Res. 85, 105–114. Yadav, A.K., Bracher, A., Doran, S.F., Leustik, M., Squadrito, G.L., Postlethwait, E.M., Matalon, S., 2010. Mechanisms and modification of chlorine-induced lung injury in animals. Proc. Am. Thorac. Soc. 7, 278–283. Zarogiannis, S.G., Jurkuvenaite, A., Fernandez, S., Doran, S.F., Yadav, A.K., Squadrito, G.L., Postlethwait, E.M., Bowen, L., Matalon, S., 2011. Ascorbate and deferoxamine administration after chlorine exposure decrease mortality and lung injury in mice. Am. J. Respir. Cell Mol. Biol. 45, 386–392.
Per-Åke Gradmark and Andreas Larsson are gratefully acknowledged for excellent technical assistance with the EFS set-up and the Cl2exposure. This study has been financially supported by the Swedish Ministry of Defence and the National Board of Health and Welfare through the Swedish Center for Disaster Toxicology. References Akdur, O., Sozuer, E.M., Ikizceli, I., Avsarogullari, L., Ozturk, F., Muhtaroglu, S., Ozkan, S., Durukan, P., 2008. Experimental inhalation of chlorine gas produced with a different method; effects of N-acetyl cysteine on acute pulmonary damage. Toxicol. Mech. Methods 18, 739–743. Aldini, G., Altomare, A., Baron, G., Vistoli, G., Carini, M., Borsani, L., Sergio, F., 2018. NAcetylcysteine as an antioxidant and disulphide breaking agent: the reasons why. Free Radic. Res. 52, 751–762. Alsafadi, H.N., Staab-Weijnitz, C.A., Lehmann, M., Lindner, M., Peschel, B., Konigshoff, M., Wagner, D.E., 2017. An ex vivo model to induce early fibrosis-like changes in human precision-cut lung slices. Am. J. Physiol. Lung Cell Mol. Physiol. 312, L896–L902. Barnes, P.J., 1995. Overview of neural mechanisms in asthma. Pulm. Pharmacol. 8, 151–159. Davidovich, N., Huang, J., Margulies, S.S., 2013. Reproducible uniform equibiaxial stretch of precision-cut lung slices. Am. J. Physiol. Lung Cell Mol. Physiol. 304, L210–220. Elfsmark, L., Agren, L., Akfur, C., Bucht, A., Jonasson, S., 2018. 8-Isoprostane is an early biomarker for oxidative stress in chlorine-induced acute lung injury. Toxicol. Lett. 282, 1–7. Evans, R.B., 2005. Chlorine: state of the art. Lung 183, 151–167. Felton, V.M., Borok, Z., Willis, B.C., 2009. N-acetylcysteine inhibits alveolar epithelialmesenchymal transition. Am. J. Physiol. Lung Cell Mol. Physiol. 297, L805–812. Henjakovic, M., Sewald, K., Switalla, S., Kaiser, D., Muller, M., Veres, T.Z., Martin, C., Uhlig, S., Krug, N., Braun, A., 2008. Ex vivo testing of immune responses in precisioncut lung slices. Toxicol. Appl. Pharmacol. 231, 68–76. Hobbs, M.J., Butterworth, M., Cohen, G.M., Upshall, D.G., 1993. Structure-activity relationships of cysteine esters and their effects on thiol levels in rat lung in vitro. Biochem. Pharmacol. 45, 1605–1612. Hoffer, E., Baum, Y., Tabak, A., Taitelman, U., 1996. N-acetylcysteine increases the glutathione content and protects rat alveolar type II cells against paraquat-induced cytotoxicity. Toxicol. Lett. 84, 7–12. Jonasson, S., Koch, B., Bucht, A., 2013a. Inhalation of chlorine causes long-standing lung inflammation and airway hyperresponsiveness in a murine model of chemical-induced lung injury. Toxicology 303, 34–42. Jonasson, S., Wigenstam, E., Koch, B., Bucht, A., 2013b. Early treatment of chlorineinduced airway hyperresponsiveness and inflammation with corticosteroids. Toxicol. Appl. Pharmacol. 271, 168–174. Jones, R., Wills, B., Kang, C., 2010. Chlorine gas: an evolving hazardous material threat and unconventional weapon. West. J. Emerg. Med. 11, 151–156. Kales, S.N., Christiani, D.C., 2004. Acute chemical emergencies. N. Engl. J. Med. 350, 800–808. Kiehl, M.G., Ostermann, H., Thomas, M., Muller, C., Cassens, U., Kienast, J., 1998. Inflammatory mediators in bronchoalveolar lavage fluid and plasma in leukocytopenic patients with septic shock-induced acute respiratory distress syndrome. Crit. Care Med. 26, 1194–1199. Leustik, M., Doran, S., Bracher, A., Williams, S., Squadrito, G.L., Schoeb, T.R., Postlethwait, E., Matalon, S., 2008. Mitigation of chlorine-induced lung injury by low-molecular-weight antioxidants. Am. J. Physiol. Lung Cell Mol. Physiol. 295, L733–743. Liberati, T.A., Randle, M.R., Toth, L.A., 2010. In vitro lung slices: a powerful approach for assessment of lung pathophysiology. Expert Rev. Mol. Diagn. 10, 501–508. Lyons-Cohen, M.R., Thomas, S.Y., Cook, D.N., Nakano, H., 2017. Precision-cut mouse lung slices to visualize live pulmonary dendritic cells. J. Vis. Exp.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
N-acetyl cysteine protects against chlorine-induced tissue damage in an ex vivo model
T
Lina Ågren, Linda Elfsmark, Christine Akfur, Lars Hägglund, Barbro Ekstrand-Hammarström, Sofia Jonasson* Swedish Defence Research Agency, CBRN Defence and Security, Umeå, Sweden
A R T I C LE I N FO
A B S T R A C T
Keywords: Precision-cut lung slices (PCLS) Chlorine Lung-injury N-acetyl cysteine Treatment Rat
High-level concentrations of chlorine (Cl2) can cause life-threatening lung injuries and the objective in this study was to understand the pathogenesis of short-term sequelae of Cl2-induced lung injury and to evaluate whether pre-treatment with the antioxidant N-acetyl cysteine (NAC) could counteract these injuries using Cl2-exposed precision-cut lung slices (PCLS). The lungs of Sprague-Dawley rats were filled with agarose solution and cut into 250 μm-thick slices that were exposed to Cl2 (20−600 ppm) and incubated for 30 min. The tissue slices were pre-treated with NAC (5−25 mM) before exposure to Cl2. Toxicological responses were analyzed after 5 h by measurement of LDH, WST-1 and inflammatory mediators (IL-1β, IL-6 and CINC-1) in medium or lung tissue homogenate. Exposure to Cl2 induced a concentration-dependent cytotoxicity (LDH/WST-1) and IL-1β release in medium. Similar cytokine response was detected in tissue homogenate. Contraction of larger airways was measured using electric-field-stimulation method, 200 ppm and control slices had similar contraction level (39 ± 5%) but in the 400 ppm Cl2 group, the evoked contraction was smaller (7 ± 3%) possibly due to tissue damage. NAC-treatment improved cell viability and reduced tissue damage and the contraction was similar to control levels (50 ± 11%) in the NAC treated Cl2-exposed slices. In conclusion, Cl2 induced a concentration-dependent lung tissue damage that was effectively prevented with pre-treatment with NAC. There is a great need to improve the medical treatment of acute lung injury and this PCLS method offers a way to identify and to test new concepts of treatment of Cl2-induced lung injuries.
1. Introduction Chlorine (Cl2) is a reactive oxidizing industrial gas that is highly toxic. Due to its high toxicity, Cl2 has historically been used as a chemical weapon and is still considered a terrorist threat and used in armed conflicts globally (Winder, 2001; Szinicz, 2005; Jones et al., 2010). In contact with water, Cl2 produces hydrochloric and hypochlorous acids, which can lead to a variety of respiratory injuries both in upper and lower airways if Cl2 is accidentally inhaled and high-level concentrations of Cl2 can cause life-threatening lung injuries (White and Martin, 2010). The applicability of precision-cut lung slices (PCLS) to study the activation of early toxic responses was tested with the aim to understand the pathogenesis of short-term sequelae of Cl2-induced lung-injury. The hallmarks of acute lung injury after Cl2 exposure, such as edema, influx of inflammatory cells and airway hyperresponsiveness (AHR), are more difficult to investigate using PCLS (White and Martin, 2010). However, by using PCLS, the number of animals can be reduced
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and the model has been used to evaluate different types of drugs prior to in vivo testing. The PCLS technique has been used for decades and is widely used to examine airway responses in different species. Major advantages of the PCLS model are the close resemblance in morphology and functionality of the respiratory tract, the numerous number of slices obtained from one animal, and the diversity of intact living pulmonary cells present in the slices (Henjakovic et al., 2008; Davidovich et al., 2013; Lyons-Cohen et al., 2017). The aim of the present study was to investigate whether PCLS have the potential to serve as a generally applicable and effective tool in mimicking chemical-induced lung injury observed in vivo. First, tissue slices were exposed to Cl2 using increasing concentrations of Cl2 and the cytotoxicity was determined as necrotic cell death measured by leakage of the cytosolic enzyme lactate dehydrogenase (LDH) into culture medium (Maes et al., 2015). Loss of metabolic enzyme activity assessed with water-soluble tetrazolium salt (WST-1) was also analyzed in Cl2exposed PCLS. For analyzing the inflammatory response, three
Corresponding author at: Swedish Defence Research Agency, CBRN Defence and Security, SE-901 82, Umeå, Sweden. E-mail address: sofi[email protected] (S. Jonasson).
https://doi.org/10.1016/j.toxlet.2020.01.006 Received 11 September 2019; Received in revised form 7 January 2020; Accepted 9 January 2020 Available online 18 January 2020 0378-4274/ © 2020 Published by Elsevier B.V.
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Fig. 1. Chlorine. The effects of Cl2-exposure analyzed in PCLS medium, A) controls exposed to the balance gas N2, release of B) LDH at 5 h, transformation of C) WST-1 at 2 h (200 and 400 ppm) and expression of the cytokines D) IL-1β, E) CINC-1 and F) IL-6. Measurement of bronchoconstriction induced by EFS-stimulation of PCLS G) PCLS exposed to 200 or 400 ppm Cl2 and at different time-points H) 1−2 h or 5 h post-exposure. Induction of airway constriction by I) methacholine (MCh) in PCLS exposed to Cl2. Data (A-F) show fold-change from control and all values indicate mean ± SEM and significant differences compared to non-exposed control slices are indicated (*p < 0.05, **p < 0.01, and ***p < 0.001). Data (G-I) show the change in % from the baseline airway area (defined as 100 % area size) and all values indicate mean ± SEM and significant differences compared to non-exposed control slices are indicated *control vs 200 ppm, # control (1−2 h) vs 400 ppm (1−2 h) and ¤ control (4−5 h) vs 400 ppm (4−5 h) (*#p < 0.05 and ***,###,¤¤¤ p < 0.001). The number of experiments in each group are presented in fig. A–I.
melting agarose solution (0.75% in incubation medium) after tracheotomy and cooled on ice. The hardened lung was removed from the animal and lobes were separated. Extraction of lung tissue was performed directly postmortem to conserve vitality of the tissue. Tissue cores with a penetrating airway in its center were prepared by using an 8 mm biopsy punch (Histolab, Sweden). Cores were placed in a CryoPure 1.8 mL tube (Sarstedt, Germany) and surrounded by agarose (3% in 150 mM NaCl, Sigma-Aldrich, Sweden). Tissue slices (250 μm) were cut from the cores vertical to the airway by the aid of a Krumdieck tissue slicer (Alabama Research and Development, Munford, AL). Lung slices (n = 88–128 slices/rat) were collected in a petri dish containing 25 mL PCLS-incubation medium and kept in a CO2 incubator at 37 °C. The medium was changed every 30 min during the first 2 h and then every hour for the next 4 h which serves as a stabilization period after slicing to reduce subsequent stress caused from slicing (Liberati et al., 2010). Before overnight incubation, gentamicin (125 μL, 10 mg/ml, Sigma-Aldrich) was added to the PCLS incubation medium and 12 h later the PCLS incubation medium was changed again before the experiment began.
endpoints were used, ELISA assay for CINC-1, IL-6 and IL-1β release both in PCLS culture medium and in tissue homogenate. All these three pro-inflammatory cytokines have been shown to increase in vivo shortly after Cl2-exposure (Jonasson et al., 2013a; Wigenstam et al., 2016b) and have a role in the initial response in the lung repair mechanism and in the development of fibrosis (Papiris et al., 2018). Furthermore, these cytokines are also found in the bronchoalveolar lavage fluids of patients with acute lung injury or acute respiratory distress syndrome (Kiehl et al., 1998; Nys et al., 2003). The aim of this study was also to evaluate whether pre-treatment with a well-characterized substance N-acetyl cysteine (NAC), due to its powerful antioxidant properties (Wigenstam et al., 2015), could counteract the Cl2-induced damages in PCLS. 2. Material and methods 2.1. Preparation of precision-cut lung slices Twenty-two animals were entered into this study. Lungs were prepared from healthy female Sprague-Dawley rats (220–260 g) obtained from Envigo RMS B.V, Netherlands. The animals were kept under controlled conditions in the animal facility providing food and water ad libitum. Animal experiments were approved by the regional ethics committee on animal experiments in Umeå, Sweden according to Directive 2010/64/EU. Animals were anaesthetized with sodium pentobarbital (i.p.) and exsanguinated by cutting the aorta. The lungs were filled with low-
2.2. Preparation of cutting and incubation PCLS medium For use in the Krumdieck slicer, an EBSS cutting medium was prepared by dissolving 0.265 g/L CaCl2 ×H2O, 0.2 g/L MgSO4 ×7H2O, 0.4 g/L KCl, 6.8 g/L NaCl, 0.14 g/L NaH2PO4 ×H2O, 3.0 g/L Glucose, 2.2 g/L NaHCO3, and 6.0 g/L HEPES in MQ water and pH was set to 7.2 59
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Fig. 2. Cell viability. Cell viability (calcein staining intensity) at 5 h in PCLS sections of A) non-exposed controls and in PCLS sections exposed to increasing concentrations of Cl2; B) 200 ppm, C) 400 ppm, D) 600 ppm, or E) Triton X as a negative control. F) Quantification of calcein staining intensity in representative photos of tissue sections from each group using ImageJ (the number of experiments in each group are presented below bars). Values in F represents mean ± SEM and significant differences compared to control are indicated (*** p < 0.001). Photos were taken at 2.5x magnification and analyzed using a fluorescence microscope (495 nm/515 nm).
reduced pH in the medium to 7.1 (5 mM), 6.6 (15 mM) and 6.1 (25 mM). Tissue slices were pre-treated during 30 min and then washed once in PCLS medium prior exposure to Cl2. Control slices were exposed to increasing doses of NAC to evaluate if NAC itself induced toxic effects due to e.g. pH-changes or cell toxicity. Slices were also exposed to NAC 25 mM where the pH of the medium was adjusted to 7.2 as a negative control for the pH changes.
(Sigma-Aldrich). For tissue culturing; a PCLS incubation medium (referred from now on as PCLS medium) containing identical ingredients but with added solutions of Na-Pyruvate (10 mL 10 mM, SigmaAldrich), MEM-Non-essential Amino acid solution 100X (20 mL, SigmaAldrich), MEM-vitamin solution 100X (10 mL, Sigma-Aldrich) and LGlutamine solution (10 mL 200 mM, Sigma-Aldrich) were added and the pH was set to 7.2. The solution was sterile filtered and stored in 4 °C.
2.5. Sample preparation of tissue homogenate and PCLS medium
2.3. Cl2-exposure of PCLS
After removing the PCLS medium (1 ml/well), slices were weighed. The PCLS medium was centrifuged (5 min, 4 °C, 2000 rpm) to remove particulate matter. Slices (n = 4 from each well) was stored at −70 °C until preparation of tissue homogenate. Slices were homogenized in 200 μl PCLS medium using an electric homogenizer (Ika T8 Ultra Turrax Homogenizer) while on ice and then centrifuged (15 min, 4 °C, 2000 rpm) to remove debris. All samples were stored in -70 °C until ELISA-analysis.
The lung slices were placed (n = 4/well) in 0.5 mL PCLS medium in each well using a 24-well plate. A Cl2 gas cylinder (Air Liquide, Germany, 1 mol% Cl2, 99 mol% nitrogen (N2)) was connected to a plastic tube with a catheter (Microlance 21 G, Becton Dickinson (BD) Sweden) directly inserted into the 0.5 mL PCLS medium. A valve (Swagelok, Sweden) was connected to the tube between the manometer on the gas cylinder and the exposure catheter locking the gas flow to 0.4 mL/min which gives 1.2 mg/min (Soap film flowmeter, HewlettPackard 1-10−100 mL). Constant Cl2 gas flow and different exposuredurations was applied to establish a concentration-response (20−600 ppm generated during 2−36 s exposures). The 24-well plate was stored at room temperature (RT) for 30 min after exposure until the PCLS medium (1 mL/well) was changed. The Cl2-exposed plate was stored at RT to avoid chlorine-contamination of non-exposed slices kept in the CO2 incubator. Control slices were sham-treated with 100 mol% N2 to examine if exposure to a non-toxic gas flow could induce cell stress or toxic effects. During incubation, the plates were kept in a CO2 incubator at 37 °C until termination of experiment, at 2 or 5 h post exposure. The pH in the PCLS medium was reduced 15 min after exposure to 200 ppm or 400 ppm Cl2 to 7.13 and 7.0 respectively.
2.6. Analysis of cytotoxicity and live cell staining in PCLS Cytotoxicity of substances was determined using the cell proliferation kit WST-1 (Cellpro-Roche, Sigma-Aldrich) and LDH cytotoxicity detection kit (Cytodet-Roche, Sigma-Aldrich) according to the manufacturer’s instructions (Roche Applied Science) and analyzed using an ELISA reader (Thermo Scientific Mutilskan FC, Thermo Fischer Scientific Oy, Vantaa, Finland). Analysis of ELISA data was performed using the software program for the ELISA reader (SkanIt for Multiskan FC 3.1. Inc. Thermo Fischer Scientific Oy, Vantaa, Finland). Both LDH and WST-1 content was normalized to the amount of tissue (mg) in each well. Viability of PCLS was determined after 5 h by using Calcein AM solution (4 mM in DMSO, Sigma-Aldrich) according to the manufacturer’s instructions and analyzed using a fluorescence microscope (Leica DMR, Leica microsystems). The excitation wavelength was 495 nm and the emission was detected at 515 nm. All photos were taken by using the same settings for all samples.
2.4. NAC pre-treatments of PCLS The antioxidant N-acetyl cysteine (NAC, Sigma-Aldrich) was dissolved in PCLS medium at three different concentrations (5 mM, 15 mM and 25 mM). The pH of each solution was measured and adding NAC 60
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Fig. 3. NAC toxicity and bronchoconstriction. Treatment effects of 5−25 mM N-acetylcysteine (NAC) on LDH release into PCLS medium at 5 h post-exposure to A) 200 ppm or B) 400 ppm Cl2 and NAC-induced effects on WST-1 (compared to control 100 %) at 2 h post exposure to either C) 200 ppm or, D) 400 ppm Cl2. Effects of NAC-treatment (25 mM) on EFS-provoked bronchoconstriction in PCLS at E) 1−2 h or F) 5 h post-exposure to 400 Cl2. Data (A-D) show fold-change from control and all values indicate mean ± SEM. Significant differences compared to control slices are indicated (*p < 0.05, and ***p < 0.001). Data (E-F) show the change in % from the baseline airway area (defined as 100 % area size) and all values indicate mean ± SEM and significant differences are indicated *400 vs 400 ppm +25 mM NAC and #control vs 400 ppm (***,### p < 0.001) at both 1−2 h and 4−5 h after exposure to Cl2. Data (E-F) showing airways before EFS-impulse (set to 100% airway area), maximal contraction directly after impulse (Max) and at relaxation (After). The number of experiments in each group are presented in fig. A–F.
Schlepütz et al. (Schleputz et al., 2011). Photographs were taken before impulse (baseline), directly after impulse (maximum contraction) and 50 s after impulse (relaxation) and the airway area was measured in each photo using an image analysis software (ImageJ 1.52a, National Institutes of Health, USA). The baseline airway area was defined as 100% area size. Photographs were taken 1−2 h or 4−5 h after Cl2exposure and the groups were randomized.
2.7. Analysis of pro-inflammatory cytokines in PCLS medium and tissue The presence of the pro-inflammatory mediators, IL-1β, IL-6 and CINC-1, in PCLS medium or lung tissue homogenate were analyzed individually using specific enzyme linked assay (ELISA) kits (R&D Systems, Inc.) according to the manufacturer’s instructions. The optical density was measured using an ELISA reader and the data was analyzed using the instrument’s software program, referring to the individual standards added to each plate. The total cytokine content of PCLS medium and lung tissue homogenate was normalized to the amount of tissue (mg) in each well.
2.8.2. Methacholine Slices were placed individually in 12 well plates with 2 mL PCLSmedium. The larger airways were studied by microscopy (Leica DM IRB, 10X) and the airway constriction was evoked by adding 25 μL of 8 nM methacholine (MCh, Sigma-Aldrich). Photographs of the baseline airway area were taken before MCh exposure and then every 15 s during 3 min to assess the airway contraction. The airway area was measured in each photo with image analysis software (ImageJ 1.52a). Airway area baseline was defined as 100% area size. Photographs were taken 1−2 h or 4−5 h after Cl2-exposure and the groups were randomized.
2.8. Microscopy analysis of airway contractions in PCLS 2.8.1. Electrical field stimulation Slices were placed individually in 12 well plates with 2 mL PCLSmedium. The larger airways were studied by microscopy (Leica DM IRB, 10X) and airway constriction was electrically evoked by placing the PCLS between two platinum electrodes applying an electrical impulse (Frequency 50 Hz, train width 1 ms, and an output current of 200 mA for 2.5 s). The method used has previously been described by 61
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Fig. 4. NAC bronchoconstriction. Representative photos of PCLS sections from unexposed controls (upper panel) and chlorine (400 ppm Cl2)-exposed PCLS (middle panel) at 1−2 h with and without 25 mM NAC (lower panel) using EFS-stimulation. Photos were taken at 10x magnification using light microscopy, (left column) showing airways before EFS-impulse (set to 100% airway area), maximal contraction directly after impulse (middle column) and at relaxation (right column).
Fig. 5. NAC immune response. Effects of NAC-treatment (5−25 mM) on immune responses induced by 400 ppm Cl2-exposure, measured as release of the cytokines A) IL-1β, B) IL-6, and C) CINC-1 into PCLS medium or in lung tissue homogenates at 5 h. Data show fold-change from control and all values indicate mean ± SEM. Significant differences indicated # all groups vs. control group and *all groups vs. 400-ppm Cl2 group (*,#p < 0.05, **,##p < 0.01, and ***,###p < 0.001). The number of experiments in each group are presented in fig. A–C.
considered significant. The statistical analyses were carried out and graphs were prepared using GraphPad Prism program (version 6.0 GraphPad Software Inc., San Diego, CA, USA).
2.9. Statistics Data are shown as mean ± standard error of mean (SEM) in the graphs. Statistical significance was assessed by parametric methods using one-way analysis of variance (ANOVA) or two-way analysis of variance (ANOVA) to determine differences between groups, followed by Bonferroni post hoc test. A statistical result of p < 0.05 was 62
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cytokine-responses was also shown in lung tissue homogenates after 400 ppm Cl2 exposures (Fig. 5) except for the IL-1β levels that were reduced in the homogenate. The functionality of airways was measured and the larger central airways showed stronger bronchoconstriction than smaller peripheral airways using EFS. The airways of 200 ppm and control lung slices contracted to similar extent (39 ± 5%, Fig. 1G) at 1−2 h after exposure but exposure to 400 ppm Cl2 reduced the ability of the airway to contract, approximately to 7 ± 3% compared to airway area before applying EFS impulse. As displayed in Fig. 1H, the larger airways in control lung slices showed strong bronchoconstriction also after 5 h and the lack of ability to contract was still evident in the 400-ppm Cl2 group. Airway smooth muscle contractions were also induced by exposing the PCLS to MCh (Fig. 1I) at 1−2 h post-exposure to Cl2. Bronchoconstriction was rapidly induced in control lung tissue slices, reducing the airway area by 53 ± 6%, compared to initial values. Slices exposed to 200 and 400 ppm Cl2 had a reduced airway area by approximately 36 ± 6%, a response that was not significantly stronger than in the controls. 3.2. Antioxidant pre-treatment of PCLS reduces Cl2-induced toxicity After 30 min pre-treatment with NAC (5, 15 and 25 mM) the PCLS were exposed to 200 or 400 ppm Cl2, reducing LDH levels to similar levels as in the non-exposed control group (Fig. 3A-B) which indicates that NAC can protect against loss of cell membrane integrity and cell necrosis. The higher doses of NAC also preserved the mitochondrial activity (WST-1) after both 200 and 400 ppm Cl2 exposures (Fig. 3C-D) and re-established bronchoconstriction (Fig. 3E-F and Fig. 4) in lung slices at 1−2 h post-exposure to 400 ppm Cl2 Fig. 4. In 400 ppm-exposed PCLS, NAC prevented release of IL-1β into the PCLS-medium and increased IL-1β levels in lung tissue homogenate significantly (Fig. 5A). After pre-treatment with 5 mM NAC the decrease of IL-6 in PCLS medium was restored to control levels but higher doses of NAC (15 and 25 mM) further increased the cytokine expression to levels significantly higher than in the corresponding control (Fig. 5B). As displayed in Fig. 5C, NAC had similar effect on the levels of CINC-1 in PCLS medium, increasing the levels significantly higher than in controls at the highest concentration 25 mM. Both IL-6 and CINC-1 was significantly increased in NAC-treated lung tissue slices after Cl2 exposure but not to control levels (Fig. 5B and C). Control experiments revealed that pre-treatment with NAC (5−25 mM) alone also caused an increase in IL-6 and IL-1β release from otherwise non-exposed control slices at 5 h compared to healthy non-treated control slices (Fig. 6A). Non-exposed control slices pre-treated with NAC alone had, however, normal levels of IL-6 and CINC-1 in lung homogenate at 5 h (Fig. 6B). Adjusting the pH of the highest concentration of NAC (25 mM) to the same pH as in the PCLS medium did not remediate the observed release of pro-inflammatory cytokines from untreated controls. LDH in PCLS medium (absorbance at 492 nm) was also slightly increased after treatment with 25 mM NAC (from 0.03 ± 0.003 to 0.06 ± 0.003) and using pH-adjusted 25 mM NAC (pH 7.2) further augmented the leakage of LDH to 0.07 ± 0.02 (Fig. 6A). After pre-treatment with 25 mM NAC, the functionality of the larger airways was evaluated both at 1−2 h and at 4−5 h post 400 ppm Cl2 exposure. The bronchoconstriction was approximately 7 ± 3% in Cl2exposed slices, and by pre-treating tissue sections with 25 mM NAC, the airways could contract to similar magnitude as in control PCLS, approximately 50 ± 11% in comparison to initial airway area before the EFS impulse was applied (Fig. 3E-F and Fig. 4).
Fig. 6. Control experiments NAC. Effects of 5−25 mM NAC-treatment on the expression of IL-6, IL-1β and CINC-1 in healthy non-exposed PCLS measured as A) released into medium or B) in lung tissue homogenates at 5 h. * indicate treatment with 25 mM NAC with adjusted pH similar to control medium. Data show fold-change from control and all values indicate mean ± SEM. Significant differences compared to control slices are indicated (#p < 0.05 and ###p < 0.001). The number of experiments in each group are presented in fig. A–B.
3. Results 3.1. Toxic effects induced by Cl2 exposure in PCLS Before using the PCLS to investigate Cl2-induced lung damage, the method of preparing PCLS cultures itself was evaluated for effects on inflammatory cytokine response and cell toxicity. The aim of this experiment was to make sure that the tissue damage caused by PCLS methodology would not interfere with the study of lung damage caused by Cl2. Exposing the slices to the balance gas N2 alone did not cause any significant cell toxicity in the lung tissue but a small statistically significant stress response was observed as IL-1β release in PCLS medium (Fig. 1A). After 30 min exposure to Cl2 and 5 h of incubation, LDH was present in the PCLS medium indicating loss of cell membrane integrity as a consequence of necrotic cell death after Cl2-exposure. Previous studies have shown that LDH release can be regarded either as a direct measurement of a cytotoxic response or as an indirect measure of cell viability. As displayed in Fig. 1B, the LDH release increased with increasing concentration (200−500 ppm) of Cl2. The metabolic activity of the cells was also constrained, measured as the amount of cleaved WST-1 in the tissue (Fig. 1C), showing that higher concentrations of Cl2 significantly reduced the mitochondrial enzyme activity. In addition to LDH and WST-1, viable cells in Cl2-exposed PCLS were stained with calcein at 5 h after exposure (Fig. 2), showing a concomitant decrease in cell viability as the Cl2 concentrations were increased. A concentration-dependent decrease of the pro-inflammatory cytokines CINC-1 and IL-6 was also observed in PCLS medium after exposures to 300−600 ppm Cl2. In contrary, the release of IL-1β increased concentration dependently after exposures to 200−500 ppm. Similar
4. Discussion The main aim of this study was to evaluate PCLS as a suitable ex vivo alternative approach to animal experiments to study the pathogenesis of short-term sequelae of Cl2-induced lung injury. Another objective 63
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MCh-stimulation in the 400-ppm Cl2 group induced more bronchoconstriction than after EFS-stimulation suggesting that Cl2 damages the neuronally mediated airway constriction in airways. Increased reactivity after MCh than EFS in Cl2 exposed slices may also be explained by extensive shedding of epithelial cells, which would result in a more comprehensive triggering of afferent nerve endings potentially leading to increased release of physiological active neuropeptides (Barnes, 1995). This may in turn also lead to increased sensitivity to exogenous provocations (such as MCh) and AHR. Even though the Cl2 exposed slices did not respond as effective as controls, MCh causes contraction of airway smooth muscles by stimulating the muscarinic cholinergic receptors that can be found in both airways and lung parenchyma. A previous study by Schlepütz et al. (Schleputz et al., 2012) has shown that EFS acts on neurons and not directly on airway smooth muscles and that may also be a possible explanation to the different responses in the 400 ppm-exposed slices between the two methods for inducing bronchoconstriction. Treatment with NAC effectively prevented cellular damage and cell death in lung tissue exposed to Cl2 and the contractility after EFS-stimulation of larger airways was preserved after high-dose NAC-treatment. NAC has been shown to protect the alveolar epithelium from reactive oxygen species production both in vitro and in vivo and pre-treatment of lung cells to NAC also decreased the susceptibility to oxidant damage by strengthening intracellular antioxidant defence systems (Simon and Suttorp, 1985; Wagner et al., 1989; Hoffer et al., 1996; Felton et al., 2009). NAC has been shown to interact with numerous biochemical pathways, as an antioxidant drug both due to direct and indirect antioxidant mechanisms (Rushworth and Megson, 2014). NAC breaks thiolated proteins thus releasing free thiols, which are more efficient as antioxidants than NAC and boost the synthesis of GSH and other reduced proteins, which in some cases have an important direct antioxidant activity (Samuni et al., 2013; Aldini et al., 2018). NAC, which has a high content of cysteine, acts as an intracellular GSH precursor that replenishes GSH in GSH-depleted cells, thus minimizing oxidative stress in both endothelial cells and smooth muscle cells (Samuni et al., 2013; Santus et al., 2014; Aldini et al., 2018). Other mechanisms of NAC that have been described are based on its anti-inflammatory properties, endogenous neuromodulator and neurotransmitter signaling functions, mucolytic properties and disulphide breaking activity (Samuni et al., 2013; Aldini et al., 2018). Although several in vivo studies have found that NAC significantly prevents or inhibits oxidative stress under certain conditions after Cl2exposure (Akdur et al., 2008; Leustik et al., 2008; Yadav et al., 2010), a clear molecular mechanism through which NAC exerts this activity is still not known (Samuni et al., 2013; Aldini et al., 2018). In this study, the aim was not to investigate the NAC mechanism more deeply but to investigate if NAC could reverse the symptoms induced after Cl2 exposure ex vivo. Whilst the protective mechanisms of action of NAC remain to be fully clarified, this study provides evidence to support further investigation of NAC as treatment of chemical-induced lung injury. There is a great need to improve medical treatment of acute lung injury and this method offers a way to identify new concepts of treatment of Cl2induced lung injuries. One advantage with this method is the possibility to use human lung tissue e.g. from cancer surgery, for evaluation which enables a more accurate extrapolation of derived ex vivo data to human conditions. This may also give important directions about relevant treatments, aiming to improve the therapeutic effect in humans exposed to high concentrations of toxic chemical gases.
was also to investigate if this PCLS model could be appropriate for drug discovery. With the PCLS model it is possible to test various drugs on the same slice preparation, which also avoids the inter-individual variations and comparable levels of information can be obtained from fewer animals. For the direct analysis of dynamic changes regarding pharmacological responses, a microscopic observation using live lung slices could be obtained where bronchoconstriction can be followed by observing single airways. It is well known that high-level concentrations of Cl2 can cause life threatening lung injuries with onset of symptoms within minutes to hours but that effective treatment of these respiratory injuries and symptoms are still lacking (Williams, 1997; Kales and Christiani, 2004; Evans, 2005; White and Martin, 2010). It has previously been shown in vivo that mice and rats exposed to 200–300 ppm Cl2 develop a neutrophilic pulmonary injury manifested by e.g. pulmonary edema, cardiovascular effects, pulmonary fibrosis and AHR in close accordance with clinical findings in humans exposed to Cl2 (White and Martin, 2010; Jonasson et al., 2013b; Wigenstam et al., 2016b). In this study, NAC was used as pre-treatment for Cl2 exposure due to its powerful antioxidant properties and its potency as free-radical scavenger, and this study evaluates whether pre-treatment with NAC could protect against acute injuries in Cl2-exposed PCLS. Antioxidants have been reported previously to be effective as rescue treatment for Cl2-induced lung injury involving e.g. airway epithelial cell regeneration and NAC has been shown to minimize the oxidative stress and its downstream effects (Akdur et al., 2008; McGovern et al., 2010, 2011; Zarogiannis et al., 2011; Wigenstam et al., 2015). NAC has also been used as treatment using ex vivo rat lung slices exposed to sulphur mustard or perfluoroisobutene and were non-toxic to all cultured rat lung slices at a dose of 5 mM (Hobbs et al., 1993; Wilde and Upshall, 1994). In this present study, all tested NAC doses were non-toxic to the rat PCLS. The contraction of airways in rat PCLS are located to larger bronchi and only minor changes are observed peripherally in small airways (bronchioles) after either MCh or EFS stimulation. The bronchoconstriction of larger airways (bronchi) was most evident in healthy control slices which is not directly comparable to our findings in vivo, using the same animal strain, where tracheostomized rats connected to a small animal ventilator (flexiVent™, Scireq®), used for recording bronchoconstriction, only showed minor constriction (Wigenstam et al., 2016a, b). Ex vivo Cl2 exposure differs from in vivo exposure, when primarily upper and lower airways are being exposed by inhalation, since the entire lung tissue section is directly being exposed ex vivo, including both airways and tissue interstitium. Following chemical exposure, lung repair mechanisms are often initiated immediately, involving cytokine release that change progressively with increased lung damage and reduced airway function (Jonasson et al., 2013a; Elfsmark et al., 2018). Cautious interpretation of experimental results from both in vivo and PCLS ex vivo studies are needed but since the models also show similarities in responses such as cytotoxicity and oxidative stress, PCLS could still be proposed as a method for drug discovery to screen for new interventions. Another challenge is how to treat the PCLS to display characteristics of early fibrosis that can mimic some of the long-term effects observed after Cl2 exposure in vivo (Jonasson et al., 2013a; Alsafadi et al., 2017). Increasing concentrations of Cl2 formed a concentration-dependent pro-inflammatory tissue injury and a decreased metabolic enzyme activity. Disregarding different time-points and exposure set-up systems, the ex vivo model shows a lack of contractility response when using EFSstimulation at higher concentrations of Cl2 (400 ppm) in comparison to in vivo studies, but on the other hand the AHR has not to our awareness been tested in vivo within a few hours after Cl2-exposure. Most in vivo studies have performed AHR measurements at 24 h post-exposure (Samal et al., 2012; Jonasson et al., 2013a; Wigenstam et al., 2016b). When using MCh-stimulation, the bronchoconstriction was comparable between the 200- ppm and 400-ppm Cl2 group respectively and neither of the concentrations differed significantly from control slices. The
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgement
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