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JoVE Journal Immunology and Infection

Immunology and Infection

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

Published: June 2, 2022 doi: 10.3791/63566
Automatic Translation
*1,2,5, *1,2,6, 1,3,4, 1,3, 1,2,4,5,6
1Laboratório de Imunofarmacologia, Fundação Oswaldo Cruz, FIOCRUZ, 2Laboratório de Imunofarmacologia, Departamento de Bioquímica, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro, 3Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto Oswaldo Cruz, FIOCRUZ, 4Programa de Pós-Graduação em Biologia Molecular e Celular, Universidade Federal do Estado do Rio de Janeiro, 5Programa de Pós-Graduação em Ciências e Biotecnologia, Universidade Federal Fluminense, 6Programa de Pós-Graduação em Neurociências, Universidade Federal Fluminense
* These authors contributed equally

Summary

The present protocol describes a lung injury model in mice using oleic acid to mimic acute respiratory distress syndrome (ARDS). This model increases the inflammatory mediators on edema and decreases lung compliance. Oleic acid is used in the salt form (oleate) since this physiological form avoids the risk of embolism.

Abstract

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.

Introduction

Ashbaugh et al.1, in 1967, first described the acute respiratory distress syndrome (ARDS) and since then has been through multiple revisions. According to the Berlin definition, ARDS is a pulmonary inflammation that leads to an acute respiratory failure and hypoxemia (PaO2/FiO2 > 300 mm Hg) due to imbalance in the ventilation to perfusion ratio, diffuse bilateral alveolar damage (DAD) and infiltrate, increased lung weight, and edema2,3. The pulmonary parenchyma is a complex cellular environment compounded by epithelial, endothelial, and other cells. These cells form barriers and structures responsible for gas exchange and homeostasis in the alveoli3. The most abundant cells within the epithelial barrier are alveolar type I cells (AT1) with a larger surface area for gas exchange and fluid management through Na/K-ATPase. Also, the alveolar type II cells (AT2) produce surfactant, reducing surface tension in the alveoli4. Underneath, endothelial cells form a semipermeable barrier separating the pulmonary circulation from the interstitium. Its functions include detecting stimuli, coordinating inflammatory responses, and cellular transmigration5. The endothelial cells also regulate gas exchange, vascular tonus, and coagulation5. Therefore, endothelial and epithelial function disturbances may exacerbate a proinflammatory phenotype, causing lung damage leading to ARDS5.

ARDS development is risk-associated with bacterial and viral pneumonia or indirect factors such as non-pulmonary sepsis, trauma, blood transfusions, and pancreatitis6. These conditions cause the release of pathogens-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), inducing proinflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, and IL-85. TNF-α is linked to vascular-endothelial cadherin (VE-cadherin) degradation in endothelial barrier disruption and leucocyte infiltration into the lung parenchyma. Neutrophils are the first cells to migrate, attracted by IL-8 and LTB45,7,8. Neutrophils further increase proinflammatory cytokines, reactive oxygen species (ROS)9, and neutrophil extracellular traps (NETs) formation generating extra endothelial and epithelial damage10. Epithelial damage prompts inflammation and activation of Toll-like receptors in AT2 cells and resident macrophages, inducing the release of chemokines attracting inflammatory cells to the lungs4. Also, the production of cytokines like interferon-β (INFβ) causes TNF-related apoptosis-inducing receptors (TRAIL), leading ATII cells to apoptosis, impairing fluid and ion clarence4. The disruption of endothelial and epithelial barrier structure allows the influx of fluid, proteins, red blood cells, and leukocytes into the alveolar space, causing edema. With edema established, the pulmonary effort to maintain breathing and gas exchange is altered11. Hypercapnia and hypoxemia induce cell death and sodium transport disturbance, aggravating alveolar edema due to poor clearance capacity10. ARDS also has elevated levels of IL-17A, associated with organ dysfunction, increased percentage of alveolar neutrophils, and alveolar permeability9.

There have been ongoing advances in research on the pathophysiology, epidemiology, and treatment of ARDS in recent years12,13. However, ARDS is a heterogeneous syndrome despite the progress in therapeutic research resulting in mechanical ventilation and fluid therapy optimization. Thus, a more effective direct pharmacological treatment is still needed10, and animal studies may help unveil ARDS mechanisms and targets for intervention.

Current ARDS models are not able to fully replicate the pathology. Thus, researchers often choose the model that could better fit their interests. For instance, the lipopolysaccharide (LPS) induction model induces ARDS by endotoxic shock triggered mainly by TLR414. HCl induction mimics acid aspiration, and the damage is neutrophilic-dependent14. On the other hand, the current sodium oleate model induces endothelial damage that increases vascular permeability and edema. Furthermore, using sodium oleate instead of oleic acid in liquid form avoids embolism risks and alteration in the blood pH15.

Animals models for ARDS
Preclinical studies in animal models help understand the pathology and are essential for new ARDS treatments research. The ideal animal model needs to have characteristics resembling the clinical situation and good reproducibility of disease mechanisms with relevant pathophysiological features of each disease stage, evolution, and repair14. Several animal models are used to assess acute lung injury in ARDS pre-clinically. However, as all models have limitations, they do not fully reproduce the human pathology6,14,16. The oleic acid-induced ARDS is used in different animal species17. Pigs18, sheeps19, and dogs20 submitted to OA injection present numerous clinical features of the disease with alveolar-capillary membrane dysfunction and increased permeability with protein and cell infiltration.

For instance, OA at 1.25 µM intravenously injected blocked transepithelial transport leading to alveolar edema15. Alternatively, in the in vitro model using A549 cells, OA at a concentration of 10 µM did not change the epithelial sodium channel (eNAC) or the expression of Na/K-ATPase. However, OA seems to associate with both channels, directly inhibiting their activity21. OA intravenous injection at 0.1 mL/kg caused lung tissue congestion and swelling, reduced alveolar spaces with thickened alveolar septa, and increased inflammatory and red blood cell counts22. Also, OA induced apoptosis and necrosis in endothelial and epithelial cells in the lung15. The injection of a tris-oleate solution, intratracheally in mice, enhanced neutrophil infiltration and edema as early as 6 h after stimulation23. OA injection at 24 h increased proinflammatory cytokine levels (i.e., TNF-α, IL-6, and IL-1β)23. In addition, intravenous (orbital plexus) injection of 10 µM of a tris-oleate inhibits pulmonary Na/K-ATPase activity, similar to ouabain at 10-3 µM, a selective enzyme inhibitor. Also, OA induces inflammation with cell infiltration, formation of lipid bodies, and production of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2)22,24. Therefore, oleic acid-induced ARDS generates edema, hemorrhage, neutrophil infiltration, increased myeloperoxidase (MPO) activity, and ROS24. Hence, OA administration is a well-established model for lung injury22,25. All the results presented in this article that has OA represents the salt form, sodium oleate.

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Protocol

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

  1. Use oleic acid to prepare a 100 mmol/L of sodium oleate stock solution in any sterile tube or glass flask.
    NOTE: A 50 mL (final volume) solution was prepared for the present work, but the volume must be adjusted as per the experimental need. The solution must be always prepared in sterile tubes or glass containers.
    1. First, add NaOH tablets or solution in ultrapure water to elevate the pH. A pH value of 12-13 is recommended for a 25 mL volume.
      NOTE: Alternatively, Tris base can be used to prepare the Tris-oleate solution.
    2. Add the oleic acid (see Table of Materials) very slowly, drop by drop, under constant agitation in an ultrasonic bath at 37 °C.
      NOTE: If oleic acid precipitation occurs, start all over from the beginning.
    3. Once oleic acid is completely dissolved, carefully adjust the pH to 7.4, drop by drop under stirring, with ultra-pure diluted HCl and then adjust to the final volume of 50 mL.
      ​NOTE: Freshly prepare the working oleate solutions. Alternatively, the solution may be aliquoted, stocked, and maintained at -20 °C in a nitrogen-enriched environment to avoid oxidation for no longer than a month. Avoid frozen-refrozen cycles.

2. Induction of lung injury by oleic acid

  1. Perform the intratracheal administration of oleic acid.
    1. Anesthetize the mice using 5% isoflurane with 2 L/min of O2 employing a veterinary anesthetic vaporizer (Figure 1A). Remove the fur at the incision area with depilatory cream and disinfect the area with three alternating rounds of betadine scrub and alcohol using sterile gauze. Confirm the depth of anesthesia by toe pinch.
      NOTE: Use sterile gloves and instruments during the procedure. Use a drape to cover the animal and expose only the incision site. Perform the experiment in a biological safety cabinet to avoid isofluorane escape to the environment. Analgesics are not administered as they may inhibit the inflammatory response.
    2. After anesthesia, lay the animal in a dorsal decubitus position and make an incision (0.5-1 cm) in V-shape at the thyroid level. Gently displace the thyroid to expose the trachea (Figure 1B) and inject 50 µL of the prepared oleate solution (step 1).
      NOTE: The mice were divided into two groups, with eight animals in each group. The lung injury group receives sodium-oleate solution at 25 mM (1.25 µmol), and the control group receives 50 µL of sterile saline by instillation into the trachea of each mouse with an insulin syringe (volume 300 µL, 30 G) (Figure 1C).
    3. Suture the mice's incision site with a synthetic non-absorbable monofilament suture, return it to their cage, and monitor it until complete recovery from the surgery. During all procedures, maintain the animals on a heating pad at 37 °C.
      NOTE: Mice usually take up to 15 min to recover from surgery.
  2. Perform intravenous administration of oleic acid.
    1. After anesthesia (step 2.1.1, Figure 2A), inject intravenously into the orbital plexus by inserting the ultrafine needle (see Table of Materials) in the medial canthus of the eye socket (Figure 2B).
      ​NOTE: The mice were divided into two groups, with eight animals in each group. Each group receives 100 µL of the sodium-oleate solution at 10 µmol of OA per animal, while the control group receives 100 µL of sterile saline.
  3. After the surgery, monitor the animals daily for adverse reactions. Humane endpoints for euthanasia include adverse reactions, convulsions, and coma.

3. Bronchoalveolar lavage fluid collection (BALF)

  1. Euthanize the mice with an intraperitoneal lethal dose of ketamine (300 mg/Kg) and xylazine (30 mg/Kg) (see Table of Materials).
  2. Lay the animal in the dorsal decubitus position, make an incision of approximately 1 cm with surgical scissors in the animals' anterior region, expose the trachea and make a small cut to introduce an intravenous catheter (20 G).
  3. Connect the catheter to a 1 mL sterile syringe, slowly and gradually inject 0.5 mL of sterile saline into the lungs, and then aspirate the fluid from the BALF with the same syringe. Repeat it 3-5 times, and transfer it to a sterile microtube, placing them in ice.
    ​NOTE: The samples can be stored at -20 °C for up to 6 months.

4. Total and differential cell analysis in BALF

  1. For total cell count, dilute 20 µL of BALF in 180 µL (10x dilution) of Turk's solution (see Table of Materials). Perform the counting using a Neubauer chamber under an optical microscope with a 40x objective.
  2. For differential count, put 100 µL of BALF in the cellular funnel containing slides and centrifuge it at 22.86 x g for 5 min at 4 °C in a cytocentrifuge, and stain with May-Grunwald (15%, pH 7.2)-Giemsa (1:10) (see Table of Materials). Proceed with cell count in a light microscope with immersion objective.

5. Determination of total protein in BALF

  1. Determine the total BALF supernatant protein by a commercial protein quantification kit and read the absorbance at 562 nm using a spectrophotometer following the manufacturer's instructions (see Table of Materials).

6. Enzyme immunosorbent assays

  1. Centrifuge BALF at 1,200 x g for 10 min at 4 °C. Then collect the supernatant with a pipette and store it at -80 °C for assays of TNF-α, IL-1β, IL-6, and PGE215,23,25.
    NOTE: The centrifugation in step 6.1 makes the BALF cell-free.
    1. Perform the cytokines assays on cell-free BALF using a commercial ELISA kit according to the manufacturer's instructions. Carry out the PGE2 assay using an enzyme immunoassay (EIA) kit following the manufacturer's instructions (see Table of Materials).

7. Lipid body staining and counting

  1. Fix the leukocytes on cytospin slides using 3.7% formaldehyde in Ca2+, Mg2+ free Hank's buffered salt solution (HBSS, pH 7.4) and stain with 1.5% OsO4 while still moist3 (see Table of Materials). Then, count the lipid bodies per cell in 50 consecutive leukocytes from each slide using the oil-immersion objective lens of the microscope.

8. Statistical analysis

  1. Perform statistical analysis using graphing and statistics software (see Table of Materials). Express the results as mean ± SEM and analyze by one-Way Anova followed by a post-test Newman-Keuls-Student26. Consider the differences significant when P < 0.05.

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Representative Results

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
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
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
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
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
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
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
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.

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Discussion

Selecting the correct ARDS model is essential to carry out the preclinical studies, and the evaluator must consider all the possible variables, such as age, sex, administration methods, and others6. The chosen model must reproduce the disease based on risk factors such as sepsis, lipid embolism, ischemia-reperfusion of the pulmonary vasculature, and other clinical risks14. However, no animal model used for ARDS can recreate all the human syndrome's features. Multiple injuring agent models include LPS, OA, hydrochloric acid, bacteria, and viruses6. Also, different administration methods are used, more commonly, intratracheal, intranasal, or intravenous. The pulmonary ischemia-reperfusion model causes capillary rupture and accumulation of intra-alveolar protein6. LPS-induced lung injury is widely used and leads to acute injury of epithelial and endothelial barriers. LPS binds to the Toll-like receptor 4 (TLR-4) in the airway epithelium triggering NF-κB activation enhancing the production of cytokines and chemokines, attracting inflammatory cells30, which leads to a robust neutrophilic alveolitis6. However, the model shows variations between strains and species of animals, decreasing the reproducibility of the results in animals for human patients with ARDS30.

The HCl-injury model mimics ARDS by acidic content aspiration. The low pH in the lungs induces an acute inflammatory response followed by a late fibrotic injury. The damage is neutrophil-dependent, causing alveolar hemorrhage, edema, and impaired fluid clearance. However, humans do not aspirate only HCl but a complex gastric content with a pH often higher than 1.531. These and other ARDS models have been extensively reviewed elsewhere31. Among all the models used, the oleic acid-induced ARDS model is the most ideal14.

The sodium oleate model causes lung damage, induces apoptosis and necrosis of alveolar cells, and enhances cytokines production such as TNFα, IL-8, IL-6, IL-1β, and MIP-1α19. OA also induces proteases and elastase expression with hemorrhage causing severe lung injury25. OA reproduces the disease due to lipid embolism, increased pulmonary vascular permeability, and extravascular fluid with radiographic infiltrates32. Also, patients with ARDS have higher plasmatic OA concentration15,22,24.

OA intratracheal administration induces inflammatory mediators' production similar to clinical ARDS and decreases lung compliance and gas exchange25,32. OA intravenous injection promotes the histomorphological and physiological aspects of the disease32. Serum albumin is a potent OA ligand which can explain why this route requires a higher OA (10 µmol)15 amount than intratracheal (1.25 µmol)23 to induce lung damage. Indeed, our group showed a correlation between OA/albumin disbalance and higher death risk in patients with leptospirosis33.

As with every other model, there are some disadvantages presented in the model. When not administered in salt form, oleic acid may cause toxic effects and variations due to blood emulsification. As demonstrated in this article, using its salt form decreases the toxic effects and avoids two problems: emboli formation and pH fluctuation in blood and the lungs. Also, it ensures that the pulmonary injury is caused by the oleate and not by a secondary effect15. In addition, the preparation of oleic acid in salt form in this model does not require conjugation with albumin. Researches show albumin's beneficial effects in reducing inflammation and vascular permeability. Furthermore, albumin restores hemodynamic and respiration in lung injury patients. Thus, conjugating albumin with OA could impair its impact on the animal, reducing the models' viability33,34.

At the molecular level, sodium oleate inhibits sodium-potassium ATPase (NKA) and sodium channel (eNac), which impair ions transportation, increasing vascular permeability and edema formation15. Also, OA can bind to free fatty acid receptor 1 (FFAR1), increasing intracellular Ca2+ concentration, which triggers kinases signaling proteins such as PI3K and MAPK, leading to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and enhancing the inflammatory response25.

In summary, although no model can fully reproduce ARDS features, they are valuable tools to study the disease. Preclinical research is crucial in understanding the pathophysiology of ARDS and the development of new treatments. The intratracheal and intravenous administration of OA, in salt form, generates reliable and reproducible ARDS models, making it a golden model to study ARDS.

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Disclosures

The authors declare no conflict of interest.

Acknowledgments

This research was funded by the Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Grant 001, Programa de Biotecnologia da Universidade Federal Fluminense (UFF), Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Figure 1 and Figure 2 are created with BioRender.com.

Materials

Name Company Catalog Number Comments
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

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Mouse Model Oleic Acid-induced Acute Respiratory Distress Syndrome ARDS Pollutant Exposure Cigarette Smoke Infectious Agents Fatty Acids Animal Models Pathomechanism Limitations Oleic Acid (OA) Harmful Effects On The Lung Lung Injury Emboli Disrupting Tissue Altering PH Impairing Edema Clearance Endothelial Injury Alveolar Permeability Inflammation Membrane Hyaline Formation Cell Death Injection Of OA (in Salt Form) Physiological Form Of OA At PH 7
Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome
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de Oliveira Rodrigues, S., Patricio… More

de Oliveira Rodrigues, S., Patricio de Almeida, M. A., Castro-Faria-Neto, H. C., Silva, A. R., Felippe Gonçalves-de-Albuquerque, C. Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome. J. Vis. Exp. (184), e63566, doi:10.3791/63566 (2022).

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