Ammonia exposure by intratracheal instillation causes severe and deteriorating lung injury and vascular effects in mice.

OBJECTIVE: Ammonia (NH3) is a corrosive alkaline gas that can cause life-threatening injuries by inhalation. The aim was to establish a disease model for NH3-induced injuries similar to acute lung injury (ALI) described in exposed humans and investigate the progression of lung damage, respiratory dysfunction and evaluate biomarkers for ALI and inflammation over time. METHODS: Female BALB/c mice were exposed to an NH3 dose of 91.0 mg/kg·bw using intratracheal instillation and the pathological changes were followed for up to 7 days. RESULTS: NH3 instillation resulted in the loss of body weight along with a significant increase in pro-inflammatory mediators in both bronchoalveolar lavage fluid (e.g. IL-1ß, IL-6, KC, MMP-9, SP-D) and blood (e.g. IL-6, Fibrinogen, PAI-1, PF4/CXCL4, SP-D), neutrophilic lung inflammation, alveolar damage, increased peripheral airway resistance and methacholine-induced airway hyperresponsiveness compared to controls at 20 h. On day 7 after exposure, deteriorating pathological changes such as increased macrophage lung infiltration, heart weights, lung hemorrhages and coagulation abnormalities (elevated plasma levels of PAI-1, fibrinogen, endothelin and thrombomodulin) were observed but no increase in lung collagen. Some of the analyzed blood biomarkers (e.g. RAGE, IL-1ß) were unaffected despite severe ALI and may not be significant for NH3-induced damages. CONCLUSIONS: NH3 induces severe acute lung injuries that deteriorate over time and biomarkers in lungs and blood that are similar to those found in humans. Therefore, this model has potential use for developing diagnostic tools for NH3-induced ALI and for finding new therapeutic treatments, since no specific antidote has been identified yet.

Nasal Lavage Fluid as a Biomedical Sample for Verification of Chlorine Exposure.

Chlorine is a toxic chemical that has been used as a chemical warfare agent in recent armed conflicts. There is an urgent need for methods to verify alleged uses of chlorine, and phospholipid chlorohydrins (PL-HOCl) derived from the pulmonary surfactant of exposed victims have previously been proposed as biomarkers of chlorine exposure. Here, we describe an improved protocol for the chemical analysis of these biomarkers and its applicability to biomedical samples from chlorine-exposed animals. By the use of a polymeric solid-phase-supported transesterification of PL-HOCl using ethanolamine, a common biomarker, oleoyl ethanolamide chlorohydrin (OEA-HOCl), was derived from all the diverse oleoyl PL-HOCl that may be formed by chlorine exposure. Compared to native lipid biomarkers, OEA-HOCl represents a larger biomarker pool and is better suited for nano-liquid chromatography--tandem mass spectrometry (nLC-MS-MS analysis), generating 3 amol Limit of Detection (LOD) and a reduced sample carry-over. With the improved protocol, significantly elevated levels of OEA-HOCl were identified in bronchoalveolar lavage fluid (BALF) of chlorine-exposed rats, 2-48 hours after exposure. The difficulty of BALF sampling from humans limits the methods usefulness as a verification tool of chlorine exposure. Conversely, nasal lavage fluid (NLF) is readily collected without advanced equipment. In NLF from chlorine-exposed rats, PL-HOCl were identified and significantly elevated levels of the OEA-HOCl biomarker were detected 2-24 hours after exposure. In order to test the potential of NLF as a biomedical sample for verification of human exposure to chlorine, in-vitro chlorination of human NLF samples was performed. All human in-vitro chlorinated NLF samples exhibited elevated OEA-HOCl biomarker levels, following sample derivatization. These data indicate the potential of human NLF as a biomedical sample for the verification of chlorine exposure, but further work is required to develop and validate the method for the use on real-world samples.

High concentrations of ammonia induced cytotoxicity and bronchoconstriction in a precision-cut lung slices rat model.

Exposure to high concentrations of ammonia (NH3) can cause life-threatening lung damages. The objective of this study was to establish a translational in vitro model for NH3-induced lung injury. Precision-cut lung slices (PCLS) from rats were exposed to NH3 and toxicological responses and cell viability were quantified by analysis of LDH, WST-1, inflammatory mediators (IL-1ß, IL-6, CINC-1, MMP-9, RAGE and IL-18), and by microscopic evaluation of bronchoconstriction induced by electric-field-stimulation (EFS) or methacholine (MCh). Different treatment strategies were assessed to prevent or reverse the damages caused by NH3 using anti-inflammatory, anti-oxidant or neurologically active drugs. Exposure to NH3 caused a concentration-dependent increase in cytotoxicity (LDH/WST-1) and IL-1ß release in PCLS medium. None of the treatments reduced cytotoxicity. Deposition of NH3 (24-59 mM) on untreated PCLS elicited an immediate concentration-dependent bronchoconstriction. Unlike MCh, the EFS method did not constrict the airways in PCLS at 5 h after NH3-exposure (47-59 mM). Atropine and TRP-channel antagonists blocked EFS-induced bronchoconstriction but these inhibitors could not block the immediate NH3-induced bronchoconstriction. In conclusion, NH3 exposure caused cytotoxic effects and lung damages in a concentration-dependent manner and this PCLS method offers a way to identify and test new concepts of medical treatments and biomarkers that may be of prognostic value.

N-acetyl cysteine protects against chlorine-induced tissue damage in an ex vivo model.

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-1ß, IL-6 and CINC-1) in medium or lung tissue homogenate. Exposure to Cl2 induced a concentration-dependent cytotoxicity (LDH/WST-1) and IL-1ß 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.

Comparisons of acute inflammatory responses of nose-only inhalation and intratracheal instillation of ammonia in rats.

Objective: To establish a rat model with respiratory and pulmonary responses caused by inhalation exposure to non-lethal concentrations of ammonia (NH3) that can be used for evaluation of new medical countermeasure strategies for NH3-induced acute lung injury (ALI). This is of great value since no specific antidotes of NH3-induced injuries exist and medical management relies on supportive and symptomatically relieving efforts. Methods: Female Sprague-Dawley rats (8-9 weeks old, 213g ± 2g) were exposed to NH3 using two different exposure regimens; nose-only inhalation or intratracheal instillation. The experiment was terminated 5 h, 24 h, 14 and 28 days post-exposure. Results: Nose-only inhalation of NH3 (9000-15 000 ppm) resulted in increased salivation and labored breathing directly post-exposure. Exposure did not increase inflammatory cells in bronchoalveolar lavage fluid but exposure to 12 000 ppm NH3 during 15 min reduced body weight and induced coagulation abnormalities by increasing serum fibrinogen levels. All animals were relatively recovered by 24 h. Intratracheal instillation of NH3 (1%) caused early symptoms of ALI including airway hyperresponsiveness, neutrophilic lung inflammation and altered levels of coagulation factors (increased fibrinogen and PAI-1) and early biomarkers of ALI (IL-18, MMP-9, TGFß) which was followed by increased deposition of newly produced collagen 14 days later. Histopathology analysis at 5 h revealed epithelial desquamation and that most lesions were healed after 14 days. Conclusions: This study demonstrates that intratracheal instillation can reproduce several early hallmarks of ALI. Our findings therefore support that the intratracheal instillation exposure regimen can be used for new medical countermeasure strategies for NH3-induced ALI.

Anti-inflammatory and anti-fibrotic treatment in a rodent model of acute lung injury induced by sulfur dioxide.

CONTEXT: Inhalation of sulfur dioxide (SO2) affects the lungs and exposure to high concentrations can be lethal. The early pulmonary response after inhaled SO2 involves tissue injury, acute neutrophilic lung inflammation and airway hyperresponsiveness (AHR). In rats, long-term pulmonary fibrosis is evident 14 days post-exposure as indicated by analysis of collagen deposition in lung tissue. Early treatment with a single dose of dexamethasone (DEX,10 mg/kg) significantly attenuates the acute inflammatory response in airways. However, this single DEX-treatment is not sufficient for complete protection against SO2-induced injuries. METHODS: Female Sprague-Dawley rats exposed to SO2 (2200 ppm, nose-only exposure, 10 min) were given treatments (1, 5 and 23 h after SO2-exposure) with the anti-fibrotic and anti-inflammatory substance Pirfenidone (PFD, 200 mg/kg) or DEX (10 mg/kg) to evaluate whether the inflammatory response, AHR and lung fibrosis could be counteracted. RESULTS: Both treatment approaches significantly reduced the total leukocyte response in bronchoalveolar lavage fluid and suppressed pulmonary edema. In contrast to DEX-treatment, PFD-treatment reduced the methacholine-induced AHR to almost control levels and partially suppressed the acute mucosal damage whereas multiple DEX-treatment was the only treatment that reduced collagen formation in lung tissue. CONCLUSIONS: To enable an accurate extrapolation of animal derived data to humans, a detailed understanding of the underlying mechanisms of the injury, and potential treatment options, is needed. The findings of the present study suggest that treatments with the capability to reduce both AHR, the inflammatory response, and fibrosis are needed to achieve a comprehensive mitigation of the acute lung injury caused by SO2.

8-Isoprostane is an early biomarker for oxidative stress in chlorine-induced acute lung injury.

Inhalation of chlorine (Cl2) may cause oxidative acute lung injury (ALI) characterized by pulmonary edema, pneumonitis, and hyperreactive airways. The aim of the study was to identify possible biomarkers for Cl2-induced ALI. Female BALB/c mice were exposed to Cl2 for 15min using two protocols 1) concentration-dependent response (25-200ppm) and 2) time-kinetics (2h-14days post-exposure). Exposure to 50-200ppm Cl2 caused a concentration-dependent inflammatory response with increased expression of IL-1ß, IL-6 and CXCL1/KC in bronchoalveolar lavage fluid 2-6h after exposure which was followed by increased lung permeability and a neutrophilic inflammation 12-24h post-exposure. The early inflammatory cytokine response was associated with a clear but transient increase of 8-isoprostane, a biomarker for oxidative stress, with its maximum at 2h after exposure. An increase of 8-isoprostane could also be detected in serum 2h after exposure to 200ppm Cl2, which was followed by increased levels of IL-6 and CXCL1/KC and signs of increased fibrinogen and PAI-1. Melphalan, a non-oxidizing mustard gas analog, did not increase the 8-isoprostane levels, indicating that 8-isoprostane is induced in airways through direct oxidation by Cl2. We conclude that 8-isoprostane represents an early biomarker for oxidative stress in airways and in the blood circulation following Cl2-exposure.

Differential cellular responses in healthy mice and in mice with established airway inflammation when exposed to hematite nanoparticles.

The aim of this study was to investigate the inflammatory and immunological responses in airways and lung-draining lymph nodes (LDLNs), following lung exposure to iron oxide (hematite) nanoparticles (NPs). The responses to the hematite NPs were evaluated in both healthy non-sensitized mice, and in sensitized mice with an established allergic airway disease. The mice were exposed intratracheally to either hematite NPs or to vehicle (PBS) and the cellular responses were evaluated on days 1, 2, and 7, post-exposure. Exposure to hematite NPs increased the numbers of neutrophils, eosinophils, and lymphocytes in the airways of non-sensitized mice on days 1 and 2 post-exposure; at these time points the number of lymphocytes was also elevated in the LDLNs. In contrast, exposing sensitized mice to hematite NPs induced a rapid and unspecific cellular reduction in the alveolar space on day 1 post-exposure; a similar decrease of lymphocytes was also observed in the LDLN. The results indicate that cells in the airways and in the LDLN of individuals with established airway inflammation undergo cell death when exposed to hematite NPs. A possible explanation for this toxic response is the extensive generation of reactive oxygen species (ROS) in the pro-oxidative environment of inflamed airways. This study demonstrates how sensitized and non-sensitized mice respond differently to hematite NP exposure, and it highlights the importance of including individuals with respiratory disorders when evaluating health effects of inhaled nanomaterials.