Name: A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide (Video) | JoVE Journal
Uploaded: 2014-12-15T13:00:00.000+00:00
Duration: 8 min 33 s
Description: Learn to generate pulmonary neutrophilia using aerosolized lipopolysaccharide in mice. This method enables reproducible assessment of neutrophil recruitment and lung inflammation through bronchoalveolar lavage analysis.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We would like to thank Kerstin Thim (AstraZeneca, Lund, Sweden), Benita Dahlberg and Dr. Anders Eklund (Karolinska Institutet, Stockholm, Sweden) as well as Dr. Martin Stampfli (McMaster University, Hamilton, ON, Canada) for skillful assistance and expert advice.

The animal studies were approved by the Northern Stockholm animal welfare ethics committee. The experimental procedures were performed in compliance with Swedish law.
1. Generating an LPS Aerosol
<ol>
	<li>Dissolve 0.5 g purified P. aeruginosa LPS in 50 ml sterile saline with gentle agitation and verify dissolution. Dilute 1 ml dissolved LPS in 9 ml sterile saline, to a final concentration of 1 mg/ml. Protect from light with aluminum foil and store at -20 °C.</li>
	<li>Thaw solubilized LPS in the dark at room temperature and mix well immediately before use.</li>
	<li>In a ventilated level II biohazard hood, insert a red inlet into a nebulizer, and connect the nebulizer to pressurized room air via the tubing provided by the manufacturer (review scheme presenting the experimental devices in Figure 1). 
	CAUTION: appropriate personal protective equipment, including a half face piece reusable respirator with particulate filters, goggles, gloves and protective garments should be used during the course of exposure.</li>
</ol>
<img alt="Nebulizer system diagram with mouse in plexiglass box for aerosol exposure study; tubing and filter." src="/files/ftp_upload/51470/51470fig1highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1: Schematic presentation of the experimental devices used for generating an aerosol. The inlet of the nebulizer is connected to an air supply. The outlet of the nebulizer is first connected to a flow meter via a 15.9 mm tube and an air filter, and air supply is adjusted to 5.0 L/m at 2 kbar pressure. The outlet is next connected to a Plexiglas box fitted with removable lids and 5 mm holes to prevent pressure build-up.
<ol start="4">
	<li>Connect the outlet of the nebulizer to a mass flowmeter via an air filter. Connect the mass flowmeter to an electrical supply.</li>
	<li>Adjust the air supply to 5 L/min, with pressure remaining at 1.0-2.0 bar.</li>
	<li>Remove the mass flowmeter and disconnect the air supply.</li>
	<li>Connect the outlet of the nebulizer to a 15.9 mm tube, which bifurcates and connects to two Plexiglas boxes with the dimensions: 150 x 163 x 205 mm, fitted with removable lids. Each box should have a 5 mm hole in the side opposing the inlet, to prevent pressure build-up.</li>
	<li>Place up to 5 mice in each Plexiglas box and close the lids.</li>
	<li>Open the nebulizer and fill the insert with at least 4 ml LPS dissolved in sterile saline or vehicle alone (sterile saline; the volume should not exceed 8 ml). Re-connect the inlet to the air supply.</li>
	<li>Allow the aerosol to flow into the closed Plexiglas boxes for 10 min. Monitor the animals continuously. Make sure the air supply remains tightly secured to the inlet of the nebulizer.</li>
	<li>Disconnect the air supply. With the lids closed, let the animals remain in the Plexiglas boxes for 2 min.</li>
	<li>Open the lids and allow for the aerosol to disperse, and return the animals to the cages. If the animals appear wet, place the cages on a heating pad set to low heat, to prevent hypothermia.</li>
	<li>Monitor the animals continuously for the first 30 min, and thereafter every 2 hr for the first 6 hr. The animals should exhibit normal respiratory pattern and activity during and after the nebulization procedure.</li>
</ol>
2. Bronchoalveolar Lavage (BAL)
<ol>
	<li>At the experimental end point, deeply anesthetize the animals with isoflurane to effect as recommended by your institution’s veterinary staff.  Pinch the hind paw to check for a withdrawal reflex to ensure sufficient depth of anesthesia to conduct major surgery. Spray down the fur of the animals with 70% ethanol.</li>
	<li>Open the abdomen using scissors, and sever the aorta to exsanguinate the animal. Place a piece of tissue over the abdomen to soak up the blood. </li>
	<li>Follwing euthanasia, use a single anterior-posterior cut of the scissors to expose the thorax. Lift up the rib cage by the anterior tip of the sternum and use the scissors to puncture the diaphragm at the most ventral point, without cutting into any lung lobe. Open the ribcage by making two cuts in the anterior-posterior direction (meeting below the jaw).</li>
	<li>Gently pull apart the rib cage using forceps, and cut the trachea below the larynx.</li>
	<li>Lift up the trachea with the forceps and remove the lungs by cutting the ligaments connecting the lobes to the thoracic cavity, and gently pulling by the adipose and cardiac tissue.</li>
	<li>Insert a polyethylene tube (inner diameter: 0.58 mm; outer diameter: 0.965 mm) into the trachea and secure the tube with a string of silk thread.</li>
	<li>Tie off the multilobe (the four lobes of the right lung) with silk thread.</li>
	<li>Insert a 23 gauge needle into the polyethylene tube and slowly inject 250 µl of ice-cold sterile PBS into the single lobe with a 1 ml syringe.</li>
	<li>Carefully tap the lungs 30 times and collect the liquid through the syringe. Repeat the procedure with 200 µl PBS (approximately 300 µl PBS should be recovered from the single lobe from the total injected volume of 450 µl).</li>
	<li>Keep the bronchoalveolar lavage fluid (BALF) on ice, or enumerate the BAL cells immediately. Count the cells with a heamocytometer, using Turk solution to stain the cells11. Calculate the total cell number by multiplying the number of cells with the dilution factor of the staining solution and the volume within the counted field of the heamocytometer.</li>
	<li>Prepare differential cell counts from cytocentrifuged cells as described elsewhere11.</li>
</ol>
3. Formalin Fixation of Lung Tissue for Histological Assessment
<ol>
	<li>Remove the multilobe and snap-freeze on dry ice. Store at -80 °C.</li>
	<li>Mount a 60 ml syringe with the plunger removed on a metal support stand. Fill the syringe with 10% formalin to the height of 20 cm above the laboratory bench, representing 20 cm of constant pressure.</li>
	<li>Connect the 23 gauge needle secured to the trachea to the 60 ml syringe via a plastic tube with a valve to control the flow of formalin.</li>
	<li>Insufflate the lung lobe with formalin for 5 min. Disconnect the needle and remove it together with the polyethylene tube from the trachea while pulling on the silk thread to close the trachea and retain the pressure in the lung.</li>
	<li>Submerge the lung lobe in formalin and fix for 24 hr at 4 °C.</li>
	<li>Wash the fixed tissue three times for at least 20 min with 70% ethanol, and dehydrate to xylene through the following regimen (1 hr each):
	<ul>
		<li>3x 70% ethanol</li>
		<li>3x 95% ethanol</li>
		<li>3x 100% ethanol</li>
		<li>3x xylene</li>
		<li>1x (liquid) paraffin</li>
	</ul>
	</li>
	<li>Embed dehydrated tissue in paraffin, cut in 4-5 µm sections, and stain with hematoxylin and eosin to allow for histological assessment.</li>
</ol>

<ol><li>King, J. D., Kocincova, D., Westman, E. L., Lam, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review%3A+Lipopolysaccharide+biosynthesis+in+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Innate+Immun%22%5BJournal%5D)">Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.</a> Innate Immun. 15, 261-312 (2009).</li>
<li>Grommes, J., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contribution+of+neutrophils+to+acute+lung+injury%5BTitle%5D)+AND+%22Mol+Med%22%5BJournal%5D)">Contribution of neutrophils to acute lung injury.</a> Mol Med. 17, 293-307 (2011).</li>
<li>Pesci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+cells+and+mediators+in+bronchial+lavage+of+patients+with+chronic+obstructive+pulmonary+disease%5BTitle%5D)+AND+%22Eur+Respir+J%22%5BJournal%5D)">Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease.</a> Eur Respir J. 12, 380-386 (1998).</li>
<li>Hoogerwerf, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+Inflammation+Induced+by+Lipoteichoic+Acid+or+Lipopolysaccharide+in+Humans%5BTitle%5D)+AND+%22Am.+J.+Respir.+Crit.+Care+Med%22%5BJournal%5D)">Lung Inflammation Induced by Lipoteichoic Acid or Lipopolysaccharide in Humans.</a> Am. J. Respir. Crit. Care Med. 178, 34-41 (2008).</li>
<li>Scheuchenzuber, W. J., Eskew, M. L., Zarkower, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+humoral+responses+to+Escherichia+coli+and+sheep+red+blood+cell+antigens+introduced+via+the+respiratory+tract%5BTitle%5D)+AND+%22Exp+Lung+Res%22%5BJournal%5D)">Comparative humoral responses to Escherichia coli and sheep red blood cell antigens introduced via the respiratory tract.</a> Exp Lung Res. 13, 97-112 (1987).</li>
<li>Asti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipopolysaccharide-induced+lung+injury+in+mice.+I.+Concomitant+evaluation+of+inflammatory+cells+and+haemorrhagic+lung+damage%5BTitle%5D)+AND+%22Pulm+Pharmacol+Ther%22%5BJournal%5D)">Lipopolysaccharide-induced lung injury in mice. I. Concomitant evaluation of inflammatory cells and haemorrhagic lung damage.</a> Pulm Pharmacol Ther. 13, 61-69 (2000).</li>
<li>Brand, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Total+deposition+of+therapeutic+particles+during+spontaneous+and+controlled+inhalations%5BTitle%5D)+AND+%22Journal+of+pharmaceutical+sciences%22%5BJournal%5D)">Total deposition of therapeutic particles during spontaneous and controlled inhalations.</a> Journal of pharmaceutical sciences. 89, 724-731 (2000).</li>
<li>Liu, F., Li, W., Pauluhn, J., Trubel, H., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipopolysaccharide-induced+acute+lung+injury+in+rats%3A+comparative+assessment+of+intratracheal+instillation+and+aerosol+inhalation%5BTitle%5D)+AND+%22Toxicology%22%5BJournal%5D)">Lipopolysaccharide-induced acute lung injury in rats: comparative assessment of intratracheal instillation and aerosol inhalation.</a> Toxicology. 304, 158-166 (2013).</li>
<li>Skerrett, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+type+1+TNF+receptor+in+lung+inflammation+after+inhalation+of+endotoxin+or+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">Role of the type 1 TNF receptor in lung inflammation after inhalation of endotoxin or Pseudomonas aeruginosa.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 276, L715-L727 (1999).</li>
<li>Roos, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+epithelial-C%2FEBPbeta+contributes+to+LPS-induced+inflammation+and+its+suppression+by+formoterol%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Lung epithelial-C/EBPbeta contributes to LPS-induced inflammation and its suppression by formoterol.</a> Biochem Biophys Res Commun. 423, 134-139 (2012).</li>
<li>Didon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+epithelial+CCAAT%2Fenhancer-binding+protein-beta+is+necessary+for+the+integrity+of+inflammatory+responses+to+cigarette+smoke%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Lung epithelial CCAAT/enhancer-binding protein-beta is necessary for the integrity of inflammatory responses to cigarette smoke.</a> Am J Respir Crit Care Med. 184, 233-242 (2011).</li>
<li>Silverpil, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Negative+feedback+on+IL-23+exerted+by+IL-17A+during+pulmonary+inflammation%5BTitle%5D)+AND+%22Innate+Immunity%22%5BJournal%5D)">Negative feedback on IL-23 exerted by IL-17A during pulmonary inflammation.</a> Innate Immunity. 19, 479-492 (2013).</li>
<li>Mercer, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteinase-Activated+Receptor-1%2C+CCL2+and+CCL7+Regulate+Acute+Neutrophilic+Lung+Inflammation%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+Cell+and+Molecular+Biology%22%5BJournal%5D)">Proteinase-Activated Receptor-1, CCL2 and CCL7 Regulate Acute Neutrophilic Lung Inflammation.</a> American Journal of Respiratory Cell and Molecular Biology. , (2013).</li>
<li>Korkmaz, B., Horwitz, M. S., Jenne, D. E., Gauthier, F. Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases. Pharmacological Reviews. 62, 726-759 (2010).</li>
<li>Bafadhel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+Exacerbations+of+COPD%3A+Identification+of+Biological+Clusters+and+Their+Biomarkers%5BTitle%5D)+AND+%22Am.+J.+Respir.+Crit.+Care+Med%22%5BJournal%5D)">Acute Exacerbations of COPD: Identification of Biological Clusters and Their Biomarkers.</a> Am. J. Respir. Crit. Care Med. , 201104-200597 (2011).</li>
<li>Hurst, J. R., Perera, W. R., Wilkinson, T. M. A., Donaldson, G. C., Donaldson, G. C., Wedzicha, G. C. Systemic and Upper and Lower Airway Inflammation at Exacerbation of Chronic Obstructive Pulmonary. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHurst%2C+JR+%22Am.+J.+Respir.+Crit.+Care+Med%22">Am. J. Respir. Crit. Care Med</a>. 173, 71-78 (2006).</li>
<li>Rabe, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+Strategy+for+the+Diagnosis%2C+Management%2C+and+Prevention+of+Chronic+Obstructive+Pulmonary+Disease%3A+GOLD+Executive+Summary%5BTitle%5D)+AND+%22Am.+J.+Respir.+Crit.+Care+Med%22%5BJournal%5D)">Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary.</a> Am. J. Respir. Crit. Care Med. 176, 532-555 (2007).</li>
</ol>

Abraham Roos has received a lecturer fee from Boehringer Ingelheim, travel grants from AstraZeneca R&amp;D and a research grant from AstraZeneca R&amp;D. Magnus Nord is a full time employee of AstraZeneca R&amp;D and has received research grants from AstraZeneca R&amp;D. Johan Grunewald and Tove Berg are co-investigators on an unrelated research project funded by AstraZeneca R&amp;D.

Aerosolized LPS generates an inflammatory response in the airways, characterized by neutrophils in the epithelial submucosa, spaces surrounding the conducting airways, as well as the alveolar spaces. This is, together with the increased total protein content in BALF, indicative of plasma leakage, representative of the pathology of acute lung injury. As LPS induces a sterile inflammation, the reaction is independent of the adaptive immune response, and there are limitations to the relevance to bacterial infections. The te...

Lipopolysaccharide (LPS) is a cell wall component of gram negative bacteria1. Challenge to LPS is a well-documented model of acute lung injury, a syndrome characterized by acute neutrophilic inflammation and edema2. In addition, pulmonary neutrophilia is also a hallmark of chronic obstructive pulmonary disease (COPD)3, and LPS challenge in humans has been used to model COPD exacerbations4. Thus, experimental models of LPS exposure are clinically relevant and valuable tools to understand human pathology.
The objective of the pulmonary delivery of aerosolized LPS described here is to generate a neutrophilic inflammatory response in the conducting and respiratory airways, without systemic involvement. Several techniques of LPS challenge have been described previously. Intra-venous injection of LPS is the most commonly used route of administration. Although this technique is easily accessible, the primary damage is to the endothelium, with secondary destruction of the pulmonary epithelium following neutrophil migration to the lung. Intra-venous administration also induces systemic inflammation2, which may complicate the clinical picture in animal models. Systemic inflammation is in contrast not observed with intra-tracheal administration. This technique, however, is labor intensive and requires anesthetics as well as considerable training5,6 . Furthermore, pulmonary deposition by this route of administration is dependent on breathing7. Thus, pulmonary deposition is affected by the depth of anesthesia needed for the intra tracheal administration and variable deposition in the airways may be observed. In contrast, pulmonary delivery with aerosolized LPS requires minimal training, and can easily be accomplished on a large number of animals with little or no variation between individuals5,8 . A recent study confirms that aerosol delivery is superior to the intra-tracheal route with regard to deposition, and that more relevant doses of LPS induce neutrophilic inflammation with this model8.
Previous studies have demonstrated that challenge to aerosolized Psuedomonas aeruginosa LPS generates a marked inflammatory response in the airway lumen and lung parenchyma, including the alveolar spaces9,10. The inflammation is characterized by a predominance of neutrophils and presence of pulmonary edema, and can thus be used to address pathogenesis of acute lung injury and gain further knowledge of the mechanisms contributing to disease pathology.

<table><tbody><tr><td>Purified &lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt; LPS</td><td>Sigma-Aldrich</td><td></td><td>Harmful. Recomended purification. LPS purified from other bactria may be used.</td></tr><tr><td>Pari LC sprint star nebulizer</td><td>PARI Respiratory Equipment Inc.</td><td>023G1250</td><td></td></tr><tr><td>TSI mass flowmeter 4040</td><td>TSI</td><td>4040</td><td>Alternative product from supplier may be used.</td></tr><tr><td>Saint-Gobain 15.9 mm Tygon tube</td><td>Sigma-Aldrich</td><td>Z685704</td><td>Recomended brand.</td></tr><tr><td>Plexiglas boxes with removable lids</td><td>Custom built</td><td>N/A</td><td>150 x 163 x 205 mm (a 2 mm hole on the side).&amp;nbsp;</td></tr><tr><td>3M Half Facepiece Reusable Respirator</td><td>3M</td><td>7503</td><td>Recomended brand.</td></tr><tr><td>3M Advanced Particulate Filters (P100)&amp;nbsp;</td><td>3M</td><td>2291</td><td>Recomended brand.</td></tr><tr><td>Scissors</td><td>VWR</td><td>233-1104</td><td>Preferred scissors may be used.</td></tr><tr><td>Forceps</td><td>VWR</td><td>232-1313</td><td>Preferred forceps may be used.</td></tr><tr><td>Intramedic PE50 polyethylene tube</td><td>BD</td><td>427411</td><td>Recomended brand.</td></tr><tr><td>Ethicon 2-0 Perma-hand silk tread</td><td>VWR</td><td>95056-992</td><td>Recomended brand.</td></tr><tr><td>26 &amp;frac12; gage needle</td><td></td><td></td><td>Alternative suppliers exist.</td></tr><tr><td>1 ml BD slip-tip syringe, non-sterile</td><td><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025" target="_blank"><img src="/img/new_window.png"> BD</a></td><td>301205</td><td>Alternative suppliers exist.</td></tr><tr><td>60 ml BD Luer-Lok syringe, non-sterile, polypropolene</td><td>BD</td><td>301035</td><td>Alternative suppliers exist.</td></tr><tr><td>Fluka Hematoxylin-Eosin</td><td>Sigma-Aldrich</td><td>3972</td><td>Alternative suppliers exist.</td></tr><tr><td>T&amp;uuml;rk's solution</td><td>Merck Millipore</td><td>109277</td><td></td></tr><tr><td>Table top centrifuge</td><td></td><td></td><td>Alternative manufacturers exist.</td></tr><tr><td>Cytospin 4 cytocentrifuge</td><td>Thermo Scientific</td><td>A78300003</td><td>Alternative centrifuge can be used.</td></tr><tr><td>HEMA-3 stat pack</td><td>Fisher Scientific</td><td>23-123-869</td><td>Alternative staining kits exists.</td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td>Alternative suppliers exist.</td></tr></tbody></table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

Challenge to aerosolized P. aeruginosa LPS usually yields a marked inflammatory response in the airway lumen and alveolar space, characterized by a predominance of neutrophils at both early and late time points.
Aerosolized LPS induces pulmonary neutrophilia
C57BL/6by and BALB/c mice were exposed to aerosolized P. aeruginosa LPS or vehicle alone and neutrophils were enumerated in BALF. The total cell number in BALF of C57BL/6by mice exposed to an aero...

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

The overall goal of this procedure is to generate consistent and reproducible airway neutrophilia by exposure to aerosolized LPS. This is accomplished by first loading LPS suspended in saline into a nebulizer connected to an exposure chamber. Next mice are exposed to the aerosolized LPS and then bronchoalveolar lavage is collected and inflammatory cells in the lavage are enumerated.Finally, the lung tissue is fixed in, formally in for embedding and sectioning and immunohistochemical analysis. Ultimately, the total and differential cell counts of the bronchoalveolar lavage can be used to assess neutrophil recruitment to the lungs. The main advantages of this technique over existing methods like intratracheal LPS administration are the even distribution of aerosolized LPS throughout the lungs, the ease of performance and the minimal training required for successful application To generate an LPS aerosol using proper PPE and within a ventilated level two biohazard hood begin by inserting a red inlet into a nebulizer and then connecting the nebulizer to pressurized room air by the tubing provided by the manufacturer.Next, use an air filter to connect the outlet of the nebulizer to a mass flow meter, and then connect the mass flow meter to an electrical supply. Adjust the air flow to five liters per minute, keeping the pressure at one to two bar, and then remove the mass flow meter and disconnect the airflow. Next, connect the outlet of the nebulizer to a 15.9 millimeter bifurcating tube connected to 2 1 50 by 1 63 by 2 0 5 millimeter plexiglass boxes fitted with removable lids.Each box should have a five millimeter hole in the side opposing the inlet to prevent pressure buildup. Now place up to five mice into each box and close the lids. Then open the nebulizer.Fill the insert with four to eight milliliters of LPS dissolved in saline or saline vehicle alone, and reconnect the inlet to the air supply. Allow the aerosol to flow into the closed plexiglass boxes with continuous monitoring of the animals and making sure the air supply remains tightly secured to the inlet of the nebulizer during the aerosolization. After 10 minutes, disconnect the air supply and leave the animals in the plexiglass boxes for another two minutes.Then open the lids. Allow the aerosol to disperse and return the mice to their cages at the experimental endpoint For each animal in turn, first use scissors to make a single anterior posterior cut to expose the thorax. Next, lift the ribcage by the anterior tip of the sternum and puncture the diaphragm at the most ventral point without cutting into any lung tissue.Then open the ribcage with two cuts that meet below the jaw in the anterior posterior direction. Once the ribcage has been opened, use forceps to gently pull it apart and cut the trachea below the larynx. Now lift the trachea and cut the ligaments connecting the lung lobes to the thoracic cavity.Then gently pull the lungs and trachea up and away from the adipose and cardiac tissue and place them on a piece of syringe package paper. Next, insert a 0.965 millimeter polyethylene tube into the trachea and secure it with a string of silk thread. Tie off the multi lobe with silk thread as well, and then insert a 23 gauge needle into the polyethylene tube.Then to collect the bronchoalveolar lavage, use a one milliliter syringe to slowly inject 250 microliters of ice called sterile PBS into the single lobe, and to carefully tap the lungs and against the surface of the work bench 30 times slowly draw the liquid back into the syringe and collect it in a tube after repeating the procedure with 200 microliters of fresh PBS, place the bronchoalveolar lavage on ice for later analysis. To fix the lung tissue for histological analysis, remove the multi lobe and snap. Freeze it on dry ice.Store the lobe at minus 80 degrees Celsius. To fix the tissue for histological analysis, mount a 60 milliliter syringe without a plunger onto a metal support stand insufflate the single lobe with formalin for five minutes. Then disconnect the needle and pull on the silk thread to remove it together with the polyethylene tube from the trachea.Simultaneously closing the trachea and retaining the pressure in the lung. Next, submerge the lobe in Formin for 24 hours at four degrees Celsius. The next day, wash the fixed tissue three times for at least 20 minutes each time in 70%ethanol.After embedding the dehydrated tissue in para film, cut the lobe into four to five micron sections and stain the sections with hematin and eoin for histological assessment. The total cell number in the bronchoalveolar lavage fluid of mice exposed to an aerosol generated with vehicle only is typically around or below 200, 000 cells. The cells consist of 95 to 100%mononuclear cells with only a few lymphocytes and no neutrophils.Mice challenged with one milligram per milliliter of aerosolized.LPS. However, exhibit an increased total cell number in the bronchoalveolar lavage fluid, typically greater than 500, 000 cells after six hours with cell infiltrates remaining high after 24 hours, and with a cell profile shifted towards a predominance of neutrophils. A similar inflammatory cell profile and pulmonary neutrophilia is observed with nebulization of five milligrams per milliliter LPS as well.Neutrophils were observed in the epithelial submucosa as well as in the spaces surrounding the conducting airways and blood vessels after six and 24 hours of LPS challenge, but not in control. Saline treated mice. The total protein content in the bronchoalveolar lavage fluid of LPS challenge mice is increased compared to mice exposed to saline and the expression of the neutrophil chemo attractant chemokines c, XCL one and C XCL two are increased in LPS challenged mice as well.Though this method can provide insights into LPs induced lung inflammation. It can also be applied to other disease models such as bacterial or viral infections, as the aerosolized vapor can be adapted to deliver many different challenges.

a-method-for-generating-pulmonary-neutrophilia-using-aerosolized

Research

JoVE Journal

Immunology and Infection

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

Source: Yuan, Z., et al. Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury. J. Vis. Exp. (2018)This video demonstrates an in vivo mouse model for inducing and analyzing acute lung injury (ALI) using intraperitoneal administration of bacterial lipopolysaccharide. The inflammatory cascade triggered by alveolar macrophages and neutrophils leads to epithelial and endothelial barrier disruption, allowing fluid and immune cells to accumulate in the alveoli. Alveolar...

Lipopolysaccharide-Induced Acute Lung Injury in Mice for Exosome and Immune Analysis

This video describes the procedure for generating an air pouch in the subcutaneous tissue of mice and collecting inflammatory exudates in response to lipopolysaccharide administration in vivo. This video also covers the analysis of the neutrophil infiltration in the air pouch.

An Air Pouch Mouse Model for Lipopolysaccharide-Induced Inflammatory Exudate Collection

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Mice exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid that are packaged with miRNAs. Using a co-culture system, we show that exosomes released in the BAL fluid disrupt expression of tight junction proteins in bronchial epithelial cells and increase expression of pro-inflammatory cytokines that accentuate lung injury.

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit β and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine 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.

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

In this study, a non-invasive endotracheal instillation technique is demonstrated to develop ALI in rats using a basic laryngoscope to ensure precise LPS delivery to the lungs. The LPS is instilled via an endotracheal tube using a laryngoscope. The magnitude of lung injury is evaluated at multiple time points.

Non-Invasive Endotracheal Administration of Lipopolysaccharide to Induce Acute Lung Injury in Rodents

Learn to generate pulmonary neutrophilia using aerosolized lipopolysaccharide in mice. This method enables reproducible assessment of neutrophil recruitment and lung inflammation through bronchoalveolar lavage analysis.

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Explore More Videos