Name: mouse model of oleic acid-induced acute respiratory distress syndrome
Uploaded: 2022-06-02T12:50:00
Description: Watch this Scientific Journal Video about Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome at JoVE.com

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<...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome).">The acute respiratory distress syndrome).</a> Journal of Clinical Investigation. 122 (8), 2731-2740 (2012).</li><li>Wang, H. M., Bodenstein, M., Markstaller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+pathology+of+three+widely+used+animal+models+of+acute+lung+injury.">Overview of the pathology of three widely used animal models of acute lung injury.</a> European Surgical Research. 40 (4), 305-316 (2008).</li><li>Moriuchi, H., Zaha, M., Fukumoto, T., Yuizono, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+polymorphonuclear+leukocytes+in+oleic+acid-induced+lung+injury.">Activation of polymorphonuclear leukocytes in oleic acid-induced lung injury.</a> Intensive Care Medicine. 24 (7), 709-715 (1998).</li><li>Julien, M., Hoeffel, J. M., Flick, M. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+edema+in+dog+lung+depleted+of+blood+components.">Permeability edema in dog lung depleted of blood components.</a> Journal of Applied Physiology. 57 (1), 147-153 (1984).</li><li>Vad&#225;sz, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oleic+acid+inhibits+alveolar+fluid+reabsorption:+a+role+in+acute+respiratory+distress+syndrome.">Oleic acid inhibits alveolar fluid reabsorption: a role in acute respiratory distress syndrome.</a> American Journal of Respiratory and Critical Care Medicine. 171 (5), 469-479 (2005).</li><li>Tenghao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+growth+factor-2+reduces+inflammatory+response+to+acute+lung+injury+induced+by+oleic+acid+in+rats+by+regulating+key+proteins+of+the+wnt/&#946;-catenin+signaling+pathway.">Keratinocyte growth factor-2 reduces inflammatory response to acute lung injury induced by oleic acid in rats by regulating key proteins of the wnt/&#946;-catenin signaling pathway.</a> Evidence-Based Complementary and Alternative. 2020, 8350579 (2020).</li><li>Gon&#231;alves-de-Albuquerque, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oleic+acid+induces+lung+injury+in+mice+through+activation+of+the+ERK+pathway.">Oleic acid induces lung injury in mice through activation of the ERK pathway.</a> Mediators of Inflammation. 2012, 956509 (2012).</li><li>Huang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dipyrithione+attenuates+oleic+acid-induced+acute+lung+injury.">Dipyrithione attenuates oleic acid-induced acute lung injury.</a> Pulmonary Pharmacology &amp; Therapeutics. 24 (1), 74-80 (2011).</li><li>Goncalves-de-Albuquerque, C. F., Silva, A. R., Burth, P., Castro-Faria, M. V., Castro-Faria-Neto, H. 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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 in...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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&#39;s balanced Salts</td>
			<td>Sigma-Aldrich</td>
			<td>H4981</td>
			<td></td>
		</tr>
		<tr>
			<td>Heatpad</td>
			<td>tkreprodu&#231;&#227;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&#225;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&#160; PrimoStar</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-1 beta duoSet ELISA</td>
			<td>R&#38;D system</td>
			<td>DY401</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-6 duoSet ELISA</td>
			<td>R&#38;D system</td>
			<td>DY406</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse TNF-alpha duoSet ELISA</td>
			<td>R&#38;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&#252;rk&#39;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</su...

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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 ...

This model induces in mice several key features of the acute respiratory syndrome, such as edema, alveolar permeability, and leukocyte filtration, thus helping researchers better understand ARDS pathophysiology Using oleic to induce RDS better mimics these conditions, while avoiding the risk of altering bloods pH and cause emboli when using pure oleic acid. A well done sedation is very important. Make sure the animal is deeply sedated throughout the procedure.This way, no sudden expand may hinder the of the Doing this procedure step-by-step, allows any associates to perform this procedure correctly. Also, this technique may be applied to study different diseases by changing the injector substance. Start preparing the sodium oleate solution by adding sodium hydroxide tablets or solution in the ultrapure water to elevate the pH to 12 to 13.Then, add oleic acid drop by drop under constant agitation in an ultrasonic bath at 37 degrees Celsius. Next, to perform intratracheal administration of oleic acid, lay the anesthetized mouse in a dorsal decubitus position on a heating pad at 37 degrees Celsius, and make an incision of 0.5 to 1 centimeter in V-shape at the thyroid level. Remove the thyroid to expose the trachea, and inject 50 microliters of the oleate solution.After the injection, close the incision with a synthetic non-absorbable monofilament suture. Then, return the mouse to the home cage and monitor it until complete recovery from the surgery. For intravenous administration, use an ultra fine needle to inject oleic acid into the orbital plexus of the anesthetized mouse.To collect the bronchoalveolar lavage fluid, lay the euthanized mouse in the dorsal decubitus position and make a 1 centimeter incision in the anterior region with surgical scissors. Expose it to the trachea and make a small cut to introduce a 20 gauge intravenous catheter. Connect the catheter to a 1 milliliter sterile syringe to slowly inject 500 microliters of sterile saline into the lungs.Then, aspirate the bronchoalveolar lavage fluid with the same syringe. After repeating the fluid collection 3 to 5 times, transfer the fluid to a sterile micro tube and place the tube on ice. In representative analysis, oleic acid injection increased total leukocytes in intratracheal and intravenous routes compared to the control.Intratracheal administration of oleic acid induces the production of inflammatory mediators, such as tumor necrosis factor alpha, interleukin 6, and interleukin 1 beta, compared to the control. Intratracheal and intravenous administration of oleic acid also increased total protein concentration in the bronchoalveolar lavage fluid. Intratracheal and intravenous injection enhanced Prostaglandin E2 concentration and lipid bodies formation than the control.Further, hematoxylin and eosin staining. Illustrated tissue disruption, hemorrhage and leukocyte infiltration after intravenous and intratracheal administration of oleic acid. After the research performed the technique, the collected samples could be analyzed by to evaluate protein These procedures can help research and understand how can mimic ARDS, and choose and develop new treatments that

Research

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