The procedures used in this study were approved by the Ethics Committee on the Use of Animals of the Oswaldo Cruz Foundation (CEUA licenses n°002-08, 36/10 and 054/2015). Male Swiss Webster mice weighing between 20-30 g, provided by the Institute of Science and Technology in Biomodels (ICTB) of the Oswaldo Cruz Foundation (FIOCRUZ), were used for the experiments. The animals were kept in ventilated isolators in the Pavilhão Ozório de Almeida's vivarium, and water and food were available ad libitum. They were exposed to a 12 h/12 h light and dark cycle.
1. Preparation of sodium oleate solution
2. Induction of lung injury by oleic acid
3. Bronchoalveolar lavage fluid collection (BALF)
4. Total and differential cell analysis in BALF
5. Determination of total protein in BALF
6. Enzyme immunosorbent assays
7. Lipid body staining and counting
8. Statistical analysis
In an uninjured lung, alveolar fluid clearance occurs by the transport of ions through the intact alveolar epithelial layer. The osmotic gradient carries fluid from the alveoli into the pulmonary interstitium, where it is drained by lymphatic vessels or reabsorbed. Na/K-ATPase drives this transport11. OA is an inhibitor of Na/K-ATPase27 and sodium channel21, which may contribute to edema formation, as we have already suggested23. The exacerbated inflammatory response in ARDS leads to alveolar damage, increased endothelial and epithelial permeability, and accumulation of alveolar fluid rich in protein and inflammatory cells, causing edema. The edema causes the lungs to increase breathing rate due to the accumulation of interstitial fluid and gas exchange impairment, resulting in hypoxemia and respiratory failure28. Cytokines such as TNF-α and vascular endothelial growth factor (VEGF) destabilize VE-cadherin bonds, contributing to increased endothelial permeability and alveolar fluid accumulation7.
OA injection increased total leukocytes in intratracheal and intravenous routes (Figure 3). It was necessary to induce OA for lung injury by the intravenous route rather than the intratracheal route. The present work showed an increase in neutrophil counts in BALF at 6 h, with the peak at 24 h and decreasing at 48 h and 72 h. A higher concentration of IL-6, IL-1β, and TNF-α in the BALF was observed after 24 h of OA intratracheal instillation23 (Figure 4). OA prevents edema clearance and can trigger the formation of edema rich in protein by both intravenous and intratracheal routes15,23. The lung edema was assessed by total protein assay in BALF, showing that i.v. and i.t. administration increased total protein concentration (Figure 5). Lipid bodies are intracellular organelles containing substrate and enzymes to eicosanoids production8,29. The formation of lipid bodies enhances the production of lipid mediators, and it can be used to access cell activation. Intratracheal and intravenous OA injection enhanced lipid bodies formation and PGE2 concentration23 after 24 h (Figure 6). OA injection also induced tissue disruption, hemorrhage, and leukocyte infiltration in intratracheal and intravenous routes, as shown in the hematoxylin and eosin (H&E) staining histology (Figure 7). Also, OA causes alteration in lung function19. Thus, oleic acid-induced lung injury presents numerous ARDS features.
Figure 1: The individual steps in the intratracheal administration protocol. (A) A mouse is anesthetized using 5% isofluorane and 2 L/min of O2. (B) A tracheal incision with a surgical scissor in mice in dorsal decubitus position. (C) Intratracheal instillation using an insulin syringe. Created with BioRender.com. Please click here to view a larger version of this figure.
Figure 2: The individual steps in the intravenous administration protocol. (A) A mouse is anesthetized with 5% isofluorane and 2L/min of O2. (B) Intravenous injection using an insulin syringe by the medial canthus. Created with BioRender.com. Please click here to view a larger version of this figure.
Figure 3: Administration of OA induces leukocyte activation in the BALF of mice. Total leukocytes in intravenous (i.v) and intratracheal administration (i.t) (A) and an illustrative photomicrograph (1000x magnification) in intratracheal administration (i.t.) stained with May-Grünwald-Giemsa (B) were performed 24 h after the OA challenge. Scale bar = 10 μm. The same volume of sterile saline was administered to the control group. Each bar represents the mean ± SEM of at least seven animals. *P < 0.05, compared to controls. Please click here to view a larger version of this figure.
Figure 4: Intratracheal administration (i.t) of OA induces the production of inflammatory mediators in the lung of mice. TNF-α (A), IL-6 (B), IL-1β (C) were measured 24 h after the challenge. Sterile saline was administered to the control group. Each bar represents the mean ± SEM for at least six animals. *P < 0.05, compared to controls. Please click here to view a larger version of this figure.
Figure 5: Total protein content in BALF 24 h after OA injection. Intratracheal (i.t.) and intravenous (i.v.) administration of OA increases total protein in the BALF of mice. The Control group received the same volume of sterile saline. The results are means ± SEM from at least six different animals. *P < 0.0001, compared to controls. Please click here to view a larger version of this figure.
Figure 6: Lipid body formation in leukocytes and PGE2 production in BALF of OA treated mice. Intratracheal (i.t) and intravenous (i.v) administration of OA induces inflammatory mediators and lipid bodies accumulation in the BALF of mice (A) and (B), respectively. (C) Illustrative photomicrograph of lipid bodies (1000x magnification) stained in the lungs of the animals with osmium tetroxide (OsO4) 24 h after OA challenge. The arrows point to the lipid bodies. Scale bar = 10 μm. Controls received the same volume of saline. The results are means ± SEM from seven animals. *P < 0.05, compared to controls. Please click here to view a larger version of this figure.
Figure 7: Illustrative pulmonary histology in mice. (A) Control mice treated with saline and no sign of hemorrhage. (B) Intravenous administration of OA (i.v). (C) Intratracheal administration (i.t) with tissue alterations. H&E staining was performed. Magnification, 1000x. Scale bar = 50 μm. Please click here to view a larger version of this figure.
Anesthetic vaporizer | SurgiVet | model 100 | |
Braided slik thread with needle number 5 | Shalon medical | N/A | |
Cabinet vivarium | Insight | Model EB273 | |
Centrifuge | Eppendorf | 5430/5430R | |
Cytofunnel | ThermoFisher | 11-025-48 | |
Drontal puppy | Bayer | N/A | |
Hank's balanced Salts | Sigma-Aldrich | H4981 | |
Heatpad | tkreprodução | TK-500 | |
Hydrocloric Acid | Sigma-Aldrich | 30721 | |
Insulin syringe Ultrafine | BD | 328322 | |
Isoforine 1mL/mL | Cristália | N/A | |
Ketamine | Syntec | N/A | |
May-Grunwald-Giemsa | Sigma-Aldrich | 205435 | |
Micro BCA Protein Assay Kit | ThermoFisher | 23235 | |
Microscope PrimoStar | Carl Zeiss | ||
Mouse IL-1 beta duoSet ELISA | R&D system | DY401 | |
Mouse IL-6 duoSet ELISA | R&D system | DY406 | |
Mouse TNF-alpha duoSet ELISA | R&D system | DY410 | |
Neubauer chamber improved bright-line | Global optics | ||
Oleic Acid (99%) | Sigma-Aldrich | O1008 | |
Osmium tetroxide solution (4%) | Sigma-Aldrich | 75632 | |
Peripheral Intravenous Catherter 20 G | BD Angiocath | 388333 | |
Prism 8 (graphic and statistic software) | Graphpad | N/A | |
Prostaglandin E2 ELISA Kit -Monoclonal | Cayman Chemical | 514010 | |
Shandon Cytospin 3 | ThermoFisher | N/A | |
Sodium hydroxide | Merck | 1,06,49,81,000 | |
Spectrophotometer | Molecular Devices | SpectraMax ABS plus | |
Swiss webster mice | ICTB/FIOCRUZ | N/A | |
Syringe 1 mL | BD | 990189 | |
Tris-base | Bio Rad | 161-0719 | Electrophoresis purity reagent |
Türk's solution | Sigma-Aldrich | 93770 | |
Xilazine | Syntec | N/A |
Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.
Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.
Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.