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>
	<li>Ashbaugh, D. G., Bigelow, D. B., Petty, T. L., Levine, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+in+adults.">Acute respiratory distress in adults.</a> Lancet. 2 (7511), 319-323 (1967).</li><li>The ARDS Definition Task Force. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome:+The+Berlin+definition.">Acute respiratory distress syndrome: The Berlin definition.</a> JAMA. 307 (23), 2526-2533 (2012).</li><li>Hewitt, R. J., Lloyd, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+immune+responses+by+the+airway+epithelial+cell+landscape.">Regulation of immune responses by the airway epithelial cell landscape.</a> Nature Reviews Immunology. 21 (6), 347-362 (2021).</li><li>Zepp, J. A., Morrisey, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+crosstalk+in+the+development+and+regeneration+of+the+respiratory+system.">Cellular crosstalk in the development and regeneration of the respiratory system.</a> Nature Reviews Molecular Cell Biology. 20 (9), 551-566 (2019).</li><li>Millar, F. R., Summers, C., Griffiths, M. J., Toshner, M. R., Proudfoot, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pulmonary+endothelium+in+acute+respiratory+distress+syndrome:+insights+and+therapeutic+opportunities.">The pulmonary endothelium in acute respiratory distress syndrome: insights and therapeutic opportunities.</a> Thorax. 71 (5), 462 (2016).</li><li>D'Alessio, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+acute+lung+injury+and+ARDS.">Mouse models of acute lung injury and ARDS.</a> Methods in Molecular Biology. 1809, 341-350 (2018).</li><li>Corada, M., 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=Vascular+endothelial-cadherin+is+an+important+determinant+of+microvascular+integrity+in+vivo.">Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (17), 9815-9820 (1999).</li><li>Bozza, P. T., 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=Leukocyte+lipid+body+formation+and+eicosanoid+generation:+cyclooxygenase-independent+inhibition+by+aspirin.">Leukocyte lipid body formation and eicosanoid generation: cyclooxygenase-independent inhibition by aspirin.</a> PNAS. 93 (20), 11091-11096 (1996).</li><li>Mikacenic, C., 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=Interleukin-17A+is+associated+with+alveolar+inflammation+and+poor+outcomes+in+acute+respiratory+distress+syndrome.">Interleukin-17A is associated with alveolar inflammation and poor outcomes in acute respiratory distress syndrome.</a> Critical Care Medicine. 44 (3), 496-502 (2016).</li><li>Matthay, M. A., 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=Acute+respiratory+distress+syndrome.">Acute respiratory distress syndrome.</a> Nature Reviews Disease Primers. 5 (1), 18 (2019).</li><li>Huppert, L. A., Matthay, M. A., Ware, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+acute+respiratory+distress+syndrome.">Pathogenesis of acute respiratory distress syndrome.</a> Seminars in Respiratory and Critical Care Medicine. 40 (1), 31-39 (2019).</li><li>Matthay, M. A., McAuley, D. F., Ware, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+trials+in+acute+respiratory+distress+syndrome:+challenges+and+opportunities.">Clinical trials in acute respiratory distress syndrome: challenges and opportunities.</a> The Lancet Respiratory Medicine. 5 (6), 524-534 (2017).</li><li>Fan, E., Brodie, D., Slutsky, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome:+advances+in+diagnosis+and+treatment.">Acute respiratory distress syndrome: advances in diagnosis and treatment.</a> JAMA. 319 (7), 698-710 (2018).</li><li>Matute-Bello, G., Frevert, C. W., Martin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+acute+lung+injury.">Animal models of acute lung injury.</a> The American Journal of Physiology-Lung Cellular and Molecular Physiology. 295 (3), 379-399 (2008).</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+inhibits+lung+Na/K-ATPase+in+mice+and+induces+injury+with+lipid+body+formation+in+leukocytes+and+eicosanoid+production.">Oleic acid inhibits lung Na/K-ATPase in mice and induces injury with lipid body formation in leukocytes and eicosanoid production.</a> Journal of Inflammation. 10 (1), 34 (2013).</li><li>Matthay, M. A., Ware, L. B., Zimmerman, G. 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. R. <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+lung+injury+in+sheep.">Oleic acid lung injury in sheep.</a> Journal of Applied Physiology. 60 (2), 433-440 (1986).</li><li>Hofman, W. F., Ehrhart, I. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=acute+respiratory+distress+syndrome:+role+of+oleic+acid-triggered+lung+injury+and+inflammation.">acute respiratory distress syndrome: role of oleic acid-triggered lung injury and inflammation.</a> Mediators of Inflammation. 2015, 260465 (2015).</li><li>McHugh, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+comparison+analysis+testing+in+ANOVA.">Multiple comparison analysis testing in ANOVA.</a> Biochemia Medica (Zagreb). 21 (3), 203-209 (2011).</li><li>Swarts, H. G. P., Schuurmans Stekhoven, F. M. A. H., De Pont, J. J. H. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+unsaturated+fatty+acids+to+Na+,K+-ATPase+leading+to+inhibition+and+inactivation.">Binding of unsaturated fatty acids to Na+,K+-ATPase leading to inhibition and inactivation.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1024 (1), 32-40 (1990).</li><li>Swenson, K. E., Swenson, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+acute+respiratory+distress+syndrome+and+COVID-19+lung+injury.">Pathophysiology of acute respiratory distress syndrome and COVID-19 lung injury.</a> Critical Care Clinics. 37 (4), 749-776 (2021).</li><li>Bozza, P. T., Magalh&#227;es, K. G., Weller, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+lipid+bodies+-+Biogenesis+and+functions+in+inflammation.">Leukocyte lipid bodies - Biogenesis and functions in inflammation.</a> Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1791 (6), 540-551 (2009).</li><li>Chen, H., Bai, C., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+the+lipopolysaccharide-induced+acute+lung+injury+model+in+respiratory+medicine.">The value of the lipopolysaccharide-induced acute lung injury model in respiratory medicine.</a> Expert Review of Respiratory Medicine. 4 (6), 773-783 (2010).</li><li>Martin, T. R., Matute-Bello, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+and+emerging+hypotheses+for+acute+lung+injury.">Experimental models and emerging hypotheses for acute lung injury.</a> Critical Care Clinics. 27 (3), 735-752 (2011).</li><li>Schuster, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARDS:+clinical+lessons+from+the+oleic+acid+model+of+acute+lung+injury.">ARDS: clinical lessons from the oleic acid model of acute lung injury.</a> American Journal of Respiratory and Critical Care Medicine. 149 (1), 245-260 (1994).</li><li>Martins, C. A., 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=The+relationship+of+oleic+acid/albumin+molar+ratio+and+clinical+outcomes+in+leptospirosis.">The relationship of oleic acid/albumin molar ratio and clinical outcomes in leptospirosis.</a> Heliyon. 7 (3), 06420 (2021).</li><li>Yu, M. -. y. a. l., 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=Hypoalbuminemia+at+admission+predicts+the+development+of+acute+kidney+injury+in+hospitalized+patients:+A+retrospective+cohort+study.">Hypoalbuminemia at admission predicts the development of acute kidney injury in hospitalized patients: A retrospective cohort study.</a> PLOS ONE. 12 (7), 0180750 (2017).</li></ol>

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...

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 ...

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

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

Research

JoVE Journal

Immunology and Infection

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 ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...