This research was funded by the Instituto Oswaldo Cruz, Funda&#231;&#227;o Oswaldo Cruz (FIOCRUZ), Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) Grant 001, Programa de Biotecnologia da Universidade Federal Fluminense (UFF), Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Funda&#231;&#227;o Carlos Chagas Filho de Amparo &#224; Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq). Figure 1 and Figure 2 are created with BioRender.com.

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&#176;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 Pavilh&#227;o Oz&#243;rio de Almeida&#39;s vivarium, and water and food were available ad libitum. They were exposed to a 12 h/12 h light and dark cycle.&#160;
1. Preparation of sodium oleate solution
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Add the oleic acid (see Table of Materials) very slowly, drop by drop, under constant agitation in an ultrasonic bath at 37 &#176;C. 
		NOTE: If oleic acid precipitation occurs, start all over from the beginning.</li>
		<li>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. 
		&#8203;NOTE: Freshly prepare the working oleate solutions. Alternatively, the solution may be aliquoted, stocked, and maintained at -20 &#176;C in a nitrogen-enriched environment to avoid oxidation for no longer than a month. Avoid frozen-refrozen cycles.</li>
	</ol>
	</li>
</ol>
2. Induction of lung injury by oleic acid
<ol>
	<li>Perform the intratracheal administration of oleic acid.
	<ol>
		<li>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.</li>
		<li>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 &#181;L 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 &#181;mol), and the control group receives 50 &#181;L of sterile saline by instillation into the trachea of each mouse with an insulin syringe (volume 300 &#181;L, 30 G) (Figure 1C).</li>
		<li>Suture the mice&#39;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 &#176;C. 
		NOTE: Mice usually take up to 15 min to recover from surgery.</li>
	</ol>
	</li>
	<li>Perform intravenous administration of oleic acid.
	<ol>
		<li>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). 
		&#8203;NOTE: The mice were divided into two groups, with eight animals in each group. Each group receives 100 &#181;L of the sodium-oleate solution at 10 &#181;mol of OA per animal, while the control group receives 100 &#181;L of sterile saline.</li>
	</ol>
	</li>
	<li>After the surgery, monitor the animals daily for adverse reactions. Humane endpoints for euthanasia include adverse reactions, convulsions, and coma.</li>
</ol>
3. Bronchoalveolar lavage fluid collection (BALF)
<ol>
	<li>Euthanize the mice with an intraperitoneal lethal dose of ketamine (300 mg/Kg) and xylazine (30 mg/Kg) (see Table of Materials).</li>
	<li>Lay the animal in the dorsal decubitus position, make an incision of approximately 1 cm with surgical scissors in the animals&#39; anterior region, expose the trachea and make a small cut to introduce an intravenous catheter (20 G).</li>
	<li>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. 
	&#8203;NOTE: The samples can be stored at -20 &#176;C for up to 6 months.</li>
</ol>
4. Total and differential cell analysis in BALF
<ol>
	<li>For total cell count, dilute 20 &#181;L of BALF in 180 &#181;L (10x dilution) of Turk&#39;s solution (see Table of Materials). Perform the counting using a Neubauer chamber under an optical microscope with a 40x objective.</li>
	<li>For differential count, put 100 &#181;L of BALF in the cellular funnel containing slides and centrifuge it at 22.86 x g for 5 min at 4 &#176;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.</li>
</ol>
5. Determination of total protein in BALF
<ol>
	<li>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&#39;s instructions (see Table of Materials).</li>
</ol>
6. Enzyme immunosorbent assays
<ol>
	<li>Centrifuge BALF at 1,200 x g for 10 min at 4 &#176;C. Then collect the supernatant with a pipette and store it at -80 &#176;C for assays of TNF-&#945;, IL-1&#946;, IL-6, and PGE215,23,25. 
	NOTE: The centrifugation in step 6.1 makes the BALF cell-free.
	<ol>
		<li>Perform the cytokines assays on cell-free BALF using a commercial ELISA kit according to the manufacturer&#39;s instructions. Carry out the PGE2 assay using an enzyme immunoassay (EIA) kit following the manufacturer&#39;s instructions (see Table of Materials).</li>
	</ol>
	</li>
</ol>
7. Lipid body staining and counting
<ol>
	<li>Fix the leukocytes on cytospin slides using 3.7% formaldehyde in Ca2+, Mg2+ free Hank&#39;s buffered salt solution (HBSS, pH 7.4) and stain with 1.5% OsO4&#160;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.</li>
</ol>
8. Statistical analysis
<ol>
	<li>Perform statistical analysis using graphing and statistics software (see Table of Materials). Express the results as mean &#177; SEM and analyze by one-Way Anova followed by a post-test Newman-Keuls-Student26. Consider the differences significant when P &#60; 0.05.</li>
</ol>

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The authors declare no conflict of interest.

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&#39;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-&#954;B 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 TNF&#945;, IL-8, IL-6, IL-1&#946;, and MIP-1&#945;19. 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&#39; 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 &#181;mol)15 amount than intratracheal (1.25 &#181;mol)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.&#160;In addition, the preparation of oleic acid in salt form in this model does not require conjugation with albumin. Researches show albumin&#39;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&#39; 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-&#954;B) 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.

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 &#62; 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-&#945;, IL-1&#946;, IL-6, and IL-85. TNF-&#945; 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-&#946; (INF&#946;) 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 &#181;M intravenously injected blocked transepithelial transport leading to alveolar edema15. Alternatively, in the in vitro model using A549 cells, OA at a concentration of 10 &#181;M 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-&#945;, IL-6, and IL-1&#946;)23. In addition, intravenous (orbital plexus) injection of 10 &#181;M of a tris-oleate inhibits pulmonary Na/K-ATPase activity, similar to ouabain at 10-3 &#181;M, 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.

<table><tbody><tr><td>Anesthetic vaporizer</td><td>SurgiVet</td><td>model 100</td><td></td></tr><tr><td>Braided slik thread with needle number 5</td><td>Shalon medical</td><td>N/A</td><td></td></tr><tr><td>Cabinet vivarium</td><td>Insight</td><td>&amp;nbsp;Model EB273</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5430/5430R</td><td></td></tr><tr><td>Cytofunnel</td><td>ThermoFisher</td><td>11-025-48</td><td></td></tr><tr><td>Drontal puppy</td><td>Bayer</td><td>N/A</td><td></td></tr><tr><td>Hank&#039;s balanced Salts</td><td>Sigma-Aldrich</td><td>H4981</td><td></td></tr><tr><td>Heatpad</td><td>tkreprodu&amp;ccedil;&amp;atilde;o</td><td>TK-500</td><td></td></tr><tr><td>Hydrocloric Acid</td><td>Sigma-Aldrich</td><td>30721</td><td></td></tr><tr><td>Insulin syringe Ultrafine</td><td>BD</td><td>328322</td><td></td></tr><tr><td>Isoforine 1mL/mL</td><td>Crist&amp;aacute;lia</td><td>N/A</td><td></td></tr><tr><td>Ketamine</td><td>Syntec</td><td>N/A</td><td></td></tr><tr><td>May-Grunwald-Giemsa</td><td>Sigma-Aldrich</td><td>205435</td><td></td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>ThermoFisher</td><td>23235</td><td></td></tr><tr><td>Microscope&amp;nbsp; PrimoStar</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Mouse IL-1 beta duoSet ELISA</td><td>R&amp;amp;D system</td><td>DY401</td><td></td></tr><tr><td>Mouse IL-6 duoSet ELISA</td><td>R&amp;amp;D system</td><td>DY406</td><td></td></tr><tr><td>Mouse TNF-alpha duoSet ELISA</td><td>R&amp;amp;D system</td><td>DY410</td><td></td></tr><tr><td>Neubauer chamber improved bright-line</td><td>Global optics</td><td></td><td></td></tr><tr><td>Oleic Acid (99%)</td><td>Sigma-Aldrich</td><td>O1008</td><td></td></tr><tr><td>Osmium tetroxide solution (4%)</td><td>Sigma-Aldrich</td><td>75632</td><td></td></tr><tr><td>Peripheral Intravenous Catherter 20 G</td><td>BD Angiocath</td><td>388333</td><td></td></tr><tr><td>Prism 8 (graphic and statistic software)</td><td>Graphpad</td><td>N/A</td><td></td></tr><tr><td>Prostaglandin E2 ELISA Kit -Monoclonal</td><td>Cayman Chemical</td><td>514010</td><td></td></tr><tr><td>Shandon Cytospin 3</td><td>ThermoFisher</td><td>N/A</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>1,06,49,81,000</td><td></td></tr><tr><td>Spectrophotometer</td><td>Molecular Devices</td><td>SpectraMax ABS plus</td><td></td></tr><tr><td>Swiss webster mice</td><td>ICTB/FIOCRUZ</td><td>N/A</td><td></td></tr><tr><td>Syringe 1 mL</td><td>BD</td><td>990189</td><td></td></tr><tr><td>Tris-base</td><td>Bio Rad</td><td>161-0719</td><td>Electrophoresis purity reagent</td></tr><tr><td>T&amp;uuml;rk&#039;s solution</td><td>Sigma-Aldrich</td><td>93770</td><td></td></tr><tr><td>Xilazine</td><td>Syntec</td><td>N/A</td><td></td></tr></tbody></table>

mouse model of oleic acid-induced acute respiratory distress syndrome

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.

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-&#945; 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-1&#946;, and TNF-&#945; 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&#38;E) staining histology (Figure 7). Also, OA causes alteration in lung function19. Thus, oleic acid-induced lung injury presents numerous ARDS features.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63566/63566fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63566/63566fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63566/63566fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63566/63566fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63566/63566fig03.jpg" /> 
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-Gr&#252;nwald-Giemsa (B) were performed 24 h after the OA challenge. Scale bar = 10 &#956;m. The same volume of sterile saline was administered to the control group. Each bar represents the mean &#177; SEM of at least seven animals. *P &#60; 0.05, compared to controls. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63566/63566fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63566/63566fig04.jpg" /> 
Figure 4: Intratracheal administration (i.t) of OA induces the production of inflammatory mediators in the lung of mice. TNF-&#945; (A), IL-6 (B), IL-1&#946; (C) were measured 24 h after the challenge. Sterile saline was administered to the control group. Each bar represents the mean &#177; SEM for at least six animals. *P &#60; 0.05, compared to controls. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63566/63566fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63566/63566fig05.jpg" /> 
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 &#177; SEM from at least six different animals. *P &#60; 0.0001, compared to controls. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63566/63566fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63566/63566fig06.jpg" /> 
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 &#956;m. Controls received the same volume of saline. The results are means &#177; SEM from seven animals. *P &#60; 0.05, compared to controls. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63566/63566fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63566/63566fig07.jpg" /> 
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&#38;E staining was performed. Magnification, 1000x. Scale bar = 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63566/63566fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome at JoVE.com

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

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.

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette ...

mouse-model-of-oleic-acid-induced-acute-respiratory-distress-syndrome

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.1K Views.  Fundação Oswaldo Cruz, FIOCRUZ. Dit model induceert bij muizen verschillende belangrijke kenmerken van het acute respiratoire syndroom, zoals oedeem, alveolaire permeabiliteit en leukocytenfiltratie, waardoor onderzoekers de pathofysiologie van ARDS beter kunnen begrijpen Het gebruik van oliezuur om RDS te induceren bootst deze aandoeningen beter na, terwijl het risico op het veranderen van de pH van het bloed en het veroorzaken van embolieën bij gebruik van puur oliezuur wordt vermeden. Een goed uitgevoerde sedatie is erg...

Video: Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Medicine

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome (ARDS)

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% in severe cases. Further research efforts are needed to better understand the complex pathophysiology of this disease. There are different well-established animal models to induce acute lung injury but none has been able to adequately mimic the complex pathomechanisms of ARDS. The most crucial factor for the development of this condition is the damage to the alveolar capillary unit. The combination of two well-established lung injury models allow us to mimic in more detail the underlying pathomechanism. Bronchoalveolar lavage (BAL) leads to surfactant depletion as well as alveolar collapse. The repeated instillation of fluid volumes causes subsequent hypoxemia. Surfactant depletion is a key factor of ARDS in humans. BAL is often combined with other lung injury approaches, but not with a second hit followed by oleic acid injection (OAI) yet. Oleic acid injection leads to severely impaired gas exchange, a deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The OAI mimics most of the expected effects of ARDS consisting of extended inflammation of lung tissue with an increase of alveolar leakage and gas exchange impairment. A disadvantage of the combination of different models is the difficulty to determine the influence to the lung injury caused by BAL alone, OAI alone or both together. The model presented in this report represents the combination of BAL and OAI as a new double-hit lung injury model. This new model is easy to implement and an alternative to study different therapeutic approaches in ARDS in the future.

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome ARDS

Different, complex animal models exist to study the pathophysiology of acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage and oleic acid injection induced lung injury is suitable as a new double-hit animal model for studying the acute respiratory distress syndrome.

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% ...

Oleic Acid&#45;Induced Acute Respiratory Decompensation in Pigs

A Porcine Model of Acute Respiratory Failure with a Continuous Infusion of Oleic Acid

This protocol outlines an acute respiratory distress model utilizing centrally administered oleic acid infusion in Yorkshire pigs. Prior to experimentation, each pig underwent general anesthesia, endotracheal intubation, and mechanical ventilation, and was equipped with bilateral&#160;jugular vein central vascular access catheters. Oleic acid was administered through a dedicated pulmonary artery catheter at a rate of 0.2 mL/kg/h. The infusion lasted for 60-120 min, inducing respiratory distress. Throughout the experiment, various parameters including heart rate, respiratory rate, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, end-tidal carbon dioxide, peak airway pressures, and plateau pressures were monitored. Around the 60 min mark, decreases in partial arterial oxygen pressure (PaO2) and fraction of oxygen-saturated hemoglobin (SpO2) were observed. Periodic hemodynamic instability, accompanied by acute increases in pulmonary artery pressures, occurred during the infusion. Post-infusion, histological analysis of the lung parenchyma revealed changes indicative of parenchymal damage and acute disease processes, confirming the effectiveness of the model in simulating acute respiratory decompensation.

Author Spotlight: Exploring Venous Waveforms for Non&#45;Invasive Respiratory Monitoring in Pigs

Infusing oleic acid continuously into the pulmonary artery of an anesthetized adult pig induces acute respiratory failure, enabling controlled experimentation during acute respiratory decompensation.

This protocol outlines an acute respiratory distress model utilizing centrally administered oleic acid infusion in Yorkshire pigs. Prior to ...

Citric Acid-Induced Mouse Model for Cough Hypersensitivity

Establishment of a Mouse Model with Cough Hypersensitivity via Inhalation of Citric Acid

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable economic burden. Increased cough sensitivity is a pathophysiological hallmark of chronic cough. It has been observed that cough hypersensitivity is related to airway inflammation, remodeling of airway sensory nerves, and alterations in the central nervous system. However, the precise molecular mechanisms remain unclear and require further elucidation using suitable animal models. Previous studies have utilized guinea pigs as models for studying cough, but these models present several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. In addition, guinea pigs typically exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. In this study, we established a mouse model with cough hypersensitivity&#160;via&#160;continuous inhalation of citric acid (CA). This model is straightforward to operate and yields reproducible results, making it a valuable tool for further studies on the mechanisms and potential novel treatments for chronic cough.

This protocol describes the development of a mouse model with cough hypersensitivity, which can serve as an ideal model for studying the mechanisms of chronic cough.

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable ...

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of acute lung injury or acute inflammation. The current most commonly used methods in acute lung injury mouse models are an intraperitoneal injection of LPS and tracheostomy for the tracheal infusion of LPS. However, the former method lacks lung targeting and damages other organs, and the latter method induces operative trauma, infection risk, and a low survival rate. Here, we recommend a noninvasive oropharyngeal endotracheal intubation method for LPS instillation in mice. In this method, LPS is noninvasively introduced into the trachea through the oropharyngeal cavity to be instilled into the lung with the help of an apparatus for endotracheal intubation. This method not only assures lung targeting but also avoids damage and the risk of death in the animals. We expect that this approach will become widely used in the field of acute lung injury.

Here, we propose a protocol for intratracheal lipopolysaccharide (LPS) delivery via noninvasive oropharyngeal endotracheal intubation. This method minimizes the trauma of the surgical procedure for the animal and accurately delivers LPS to the trachea and then to the lungs.

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of ...

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

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