The authors have declared no conflict of interest.

The overall protocol requires 2 days, including the first day of sepsis induction and isolation of BAL fluid from the animal, and the second day for exosomes isolation from the mouse BALF. All procedures have been reviewed and approved by the Institutional Animal Care and Use Committee at Atlanta VA medical center.
1. Mouse Acute Septic Lung Injury Model
<ol>
	<li>Use 6 - 8 weeks- old male wild-type C57BL/6J mice (weight 20 - 22 g) for the animal model, and perform all procedures using aseptic techniques and instruments. Autoclave all surgical instruments, including forceps and scissors for 20 min at 121 &#176;C. &#160;</li>
	<li>Randomly distribute mice into experimental and control group. Restrain mice on a surgical board. Treat mice with Escherichia coli lipopolysaccharide (LPS, 15 mg/kg) by intraperitoneal route. According to the individual body weight, dilute appropriate LPS dosages to 0.1 mL PBS per mouse, then inject LPS solution using a 1 mL syringe with 27 G needle and inject an equivalent volume of PBS to control animals. &#160;&#160;</li>
	<li>Keep mice in the animal BSL2 facility, which consists of clean cages at appropriate room temperature.</li>
	<li>24 h later, euthanize mice with CO2 inhalation.</li>
	<li>Select the surgical area in the center of mouse neck. Make a small incision (approximately 5 mm), and gently separate muscles for the access to the trachea using blunt forceps until the trachea is exposed.</li>
	<li>Withdraw mouse BAL fluid with 10 mL sterile syringe with a 27 G needle. First, gently, insert the needle into the trachea and inject 1 mL of sterile PBS slowly into the trachea, then draw BALF into a 3 mL disposable syringe with a 27 G needle. Upon completion of the procedure, dispose of syringes and needles in biohazard sharps container.</li>
	<li>Treat surgical instruments with chlorine dioxide, as described previously by Chauret et al.27, and then sterilize all surgical instruments by autoclaving for 20 min at 121 &#176;C.</li>
</ol>
2. &#160;Exosomes Isolation-serial Ultra-centrifugation and Characterization
<ol>
	<li>Collect mouse BAL fluid in 15 mL conical tubes. To harvest alveolar cells, centrifuge samples for 10 min at 1,000 g, at 4 &#176;C.</li>
	<li>Transfer the supernatant to new 15 mL conical tubes, then centrifuge samples for 30 min at 4,000 x g at 4 &#176;C to remove cell debris.</li>
	<li>Collect the supernatant in ultracentrifuge tubes and centrifuge samples for 60 min at 10,000 x g at 4 &#176;C to remove larger cellular particles.</li>
	<li>Transfer the supernatant to new conical tubes, then filter the samples through 0.2 &#181;m syringe-filter to remove remaining larger particles.</li>
	<li>Collect samples in ultracentrifuge tubes, and balance tubes. Centrifuge samples for 2 h at 140,000&#160;x&#160;g at 4&#176;C to pellet the exosomes. 
	Note: The size of exosomes varies 20 - 200 nm. 0.2 &#181;m filter is used for removing larger particles that are more than 200 nm in size.</li>
	<li>Discard the supernatants gently. Dissolve the pellet with 100 &#181;L PBS or lysis solution, and transfer exosomes samples to 1.5 mL tubes, and store at -80 &#176;C until use. 
	Note: Transmission electron microscope is recommended to verify isolated exosomes.</li>
	<li>Follow the standard TEM sample processing protocol28. Analyze exosomes samples using transmission electron microscopy, and take images, scale bar 200 nm.</li>
</ol>
3. Preparation of miRNA and Performing miRNA- q-RT-PCR
Note: miRNA-q-RT-PCR is recommended to analyze expression level of miRNAs.
<ol>
	<li>Follow standard miRNAs isolation protocol and make sure to use an equal amount of purified RNAs in single-strand reverse transcription24.</li>
	<li>Follow the standard real-time PCR protocol to detect exosomal microRNA level in BAL fluid. Use specific real-time PCR primers against miR-155, miR-146a, and U6 SnRNA. Run PCR reaction with the quantitative PCR apparatus and normalize the expression values to internal control U6 snRNA. 
	Note: Each group consists of triplicate samples24.</li>
</ol>
4. Exosomes Labeling and Purification
<ol>
	<li>Suspend exosomes pellet with 100 &#181;L suspension solution in a 1.5 mL polypropylene tube, then add 0.4 &#181;L of the exosomes dye into the other tube with 100 &#181;L of the suspension solution.</li>
	<li>Quickly add exosomes in the solution to the dye solution and mix it thoroughly by pipetting. Keep samples for 5 min at room temperature, protect from light.</li>
	<li>Put one exosome spin column in a 1.5 mL elution tube. Transfer the solution mixture to the center of the exosome spin column.</li>
	<li>Spin the column for 2 min at 800 x g at room temperature.</li>
	<li>Discard the column and store the eluted sample at -20 &#176;C until use.</li>
</ol>
5. Validation of Exosomes Up Taken by Recipient Cells
<ol>
	<li>Seed 1 x 104 MLE 12 /cell in an 8-well chamber slide, and culture cells at 37 &#176;C, 5% CO2.</li>
	<li>Add 10 &#181;L fluorescent-labeled exosomes to the cells. Incubate for 1 h at 37 &#176;C.</li>
	<li>Remove culture media and wash cells with PBS, then fix cells with 1% paraformaldehyde for 10 mins at room temperature.</li>
	<li>Mount the slide in mount medium with DAPI.</li>
	<li>Analyze samples through a fluorescence microscope and take images, 40X magnification.</li>
</ol>
6. Verification of Interaction between Exosomes and Recipient Cells
<ol>
	<li>Seed 2 x 105 MLE12/well in a 12-well culture plate containing appropriate culture medium, and culture cells at 37 &#176;C, 5% CO2.</li>
	<li>Add 20 &#181;g exosomes from mouse BAL fluid to MLE12 cells and set up replicates per experimental group.</li>
	<li>24 h later, wash cells with PBS and add 600 &#181;L lysis buffer into well. Gently shake the plate and collect lysis samples in 1.5 mL tube with the pipette.</li>
	<li>Follow the standard total RNA isolation protocol and cDNA reverse transcription protocol, complete the single-strand cDNA reverse transcription29.</li>
	<li>Follow the standard real-time PCR protocol to detect mRNA expression level in recipient cells29.&#160;Use specific real-time PCR primers against TNF-&#945;, IL-6, and ZO-1. Run PCR reactions with the quantitative PCR apparatus and normalize the expression values to the internal control GAPDH. 
	Note: Each group consists of triplicate samples.</li>
</ol>

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This work was supported by VA Merit Review 2I01BX001786-06A1 funding to RS.

Mouse models of diseases are commonly used to evaluate the physiological function of specific genes and to reduce the cost of experimentation2. The acute septic lung injury described here mimics inflammatory response seen in humans with ARDS. This model is relevant to investigate the molecular pathogenesis, development of biomarkers and to test potential new therapies5.
Co-culture systems are relevant for in vivo studies in the context o...

ALI and ARDS are the life-threatening forms of respiratory failure with severe hypoxemia caused by non-cardiogenic pulmonary edema which affects approximately 1 million people worldwide annually1. The etiology of ARDS includes direct injury to the lungs from infections or aspiration and a variety of indirect insults. Over the last decade there has been an increased understanding of the molecular pathogenesis of ARDS, however, specific targeted treatments for ARDS are yet to be developed2,3.
Several animal models of acute lung injury have been developed which provide a bridge for translating experimental therapies to human studies4,5. Commonly used models include local installation of oleic acid, bacteria, LPS, and bleomycin. Other approaches include ischemia-reperfusion, cecal ligation puncture, mechanical ventilation-induced stretch injury, hyperoxia or systemic administration of bacteria and LPS5. These models provide a useful biological system to test clinical hypotheses and for the development of potential therapies. To simulate human ARDS, animal models should reproduce inflammation and acute injury to the epithelial and endothelial cells with defects in barrier function in the lungs.
Exosomes are membrane vesicles with 20 - 200 nm in diameter, whose molecular content contains proteins, DNA, RNA, and lipids, and facilitate inter-cellular communications in tissue microenvironment via molecular composition transfer. Exosomes are secreted by multiple types of cells, such as endothelial cells, epithelial cells, smooth muscle cells and tumor cells, and exist in human body fluid. Studies indicate that exosomes regulate cross-talk between immune cells and stromal cells during infectious and sterile inflammatory diseases, and their abnormal release appears to be regulated by various natural and experimental stimuli during physiological and pathological processes6. Such communication network may play an important role in the pathogenesis of lung diseases and may affect pathophysiological progression7,8. As 18 - 22 nucleotide non-coding RNAs, miRNAs exist in both tissue and body fluids, plasma, sera, and modulate mRNA expression at post-translational level9,10.
Packaged miRNAs in exosomes influence differentiation and function of multiple types of cells, and excessive levels are associated with a variety of diseases, including cancer, lung diseases, obesity, diabetes, and cardiovascular disease11,12,13,14,15,16. Entry into the recipient cells and shuttling of exosomal miRNAs facilitate intercellular communications modifying the hemostasis of microenvironment17,18. Acute lung-injury is a complex processes, involving multiple cell types with extensive intercellular communications through exosomes8. miR-155 and miR-146a share the common transcriptional regulatory mechanism and contribute to the inflammatory response and the immune tolerance19,20. Recent studies indicate that both modulate inflammatory response via exosomal miRNAs shuttling between immune cells21. However, the molecular mechanisms underlying modulatory effects of exosomal miRNAs on alveolar response to endotoxin remain unclear, undoubtedly the potential clinical relevance and translational implication merit further investigation.
Co-culture models are being employed to define the interaction of the specific cell types in the complex environment, such as inflammation and cancer22,23. These platforms provide an alternative strategy to interrogate cross talk between cell types particularly for immune and structural cells.
Intra-tracheal, aerosolized, intraperitoneal or systemic administration of LPS are widely used to induce experimental lung injury24,25,26, and have shown to induce epithelial and endothelial permeability defects. Here we use intraperitoneal LPS to induce a septic model of acute lung injury in mice. Within 24 h of intraperitoneal administration of LPS, permeability defects are induced in the lungs with recruitment of inflammatory cells. Further, we show that exosomes from BAL contain miRNA-155 and miR-146a, and exosomes from BAL fluid induce pro-inflammatory cytokines expression in recipient epithelial cells, including IL-6 and TNF-&#945;. These data are the first to show that exosomal miRNAs are secreted in BAL in this model of acute septic lung injury.

<table><tbody><tr><td>lipopolysaccharide</td><td>Sigma-Aldrich</td><td>Escherichia coli 055:B5</td><td></td></tr><tr><td>PBS pH7.2(1X)</td><td>Life technologies</td><td>20012-027</td><td></td></tr><tr><td>mirVana miRNA isolation kit</td><td>Thermo Fisher</td><td>449774</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Thermo Fisher</td><td>4369016</td><td></td></tr><tr><td>mmu-miR-155 (002571) primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>has-miR-146a (000468) primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>U6snRNA primer</td><td>Thermo Fisher</td><td>4427975</td><td></td></tr><tr><td>TNF-&amp;alpha;(Mm00443258_m1 ) primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>IL-6 (Mm00446190_m1&amp;nbsp;&amp;nbsp; )primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>ZO-1 (Mm00493699_m1&amp;nbsp; )primer</td><td>Thermo Fisher</td><td>4331182</td><td></td></tr><tr><td>minimal essential medium (MEM)</td><td>Thermo Fisher</td><td>11095-072</td><td></td></tr><tr><td>Trysin-EDTA solution(0.05%)</td><td>Thermo Fisher</td><td>25300054</td><td></td></tr><tr><td>Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&amp;nbsp;</td><td>Sigma-Aldrich</td><td>MINI67-1KT</td><td></td></tr><tr><td>Exosome Spin Columns (MW3000)</td><td>Thermo Fisher</td><td>4484449</td><td></td></tr><tr><td>Beckman Coulter Optima L-100XP ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>L-100XP</td><td></td></tr><tr><td>Ultra-clear centrifuge tubes</td><td>Beckman Coulter</td><td>344058</td><td></td></tr><tr><td>Applied Biosystems 7500 Fast Real-Time PCR System</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>transmission electron microscopes (TEM)</td><td>JEOL Ltd</td><td>120 kV TEM</td><td></td></tr><tr><td>Olympus BX41 microscope</td><td>Olympus</td><td>BX41</td><td></td></tr></tbody></table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high morbidity and mortality. The molecular pathogenesis of ALI is being better defined; however, because of the complex nature of the disease molecular therapies have yet to be developed. Here we use a lipopolysaccharide (LPS) induced mouse model of acute septic lung injury to delineate the role of exosomes in the inflammatory response. Using this model, we were able to show that mice that are exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid from the lungs that are packaged with miRNA and cytokines which regulate inflammatory response. Further using a co-culture model system, we show that exosomes released from macrophages disrupt expression of tight junction proteins in bronchial epithelial cells. These results suggest that 1) cross talk between innate immune and structural cells through the exosomal shuttling contribute to the inflammatory response and disruption of the structural barrier and 2) targeting these miRNAs may provide a novel platform to treat ALI and ARDS.

To induce septic lung injury, mice were treated with intraperitoneal LPS (15 mg/kg). Within 24 h of LPS administration, neutrophilic influx was seen in the lungs, as shown in Figure 1A. Mouse BAL fluid was drawn, followed by isolation and purification of exosomes. Morphology of BALF exosomes was confirmed by electron transmission microscopy (Figure 1B).
Expression of se...

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Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

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.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

bronchoalveolar-lavage-exosomes-lipopolysaccharide-induced-septic

Research

JoVE Journal

Genetics

10.8K Views.  Atlanta VAMC. Muizen blootgesteld aan intraperitoneaal LPS afscheiden exosomes in Broncho-alveolaire lavage (BAL) vloeistof die zijn ingepakt met miRNAs. Met behulp van een systeem co cultuur tonen we dat exosomes vrijgegeven in de BAL vloeistof expressie van strakke junction eiwitten in bronchiale epitheliale cellen verstoren en verhogen van expressie van pro-ontsteking cytokinen die long letsel accentueren.

Video: Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

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

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

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 host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Immunology and Infection

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.

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 ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

Medicine

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

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

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

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

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

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

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 &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

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.

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 ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

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.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

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

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

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

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

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

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

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