Die Autoren haben keinen Interessenkonflikt erklärt.

Das gesamte Protokoll erfordert 2 Tage, unter anderem am ersten Tag der Sepsis Induktion und Absonderung von BAL-Flüssigkeit aus der Tier- und am zweiten Tag exosomen Isolation von der Maus BALF. Alle Verfahren wurden überprüft und durch die institutionelle Animal Care and Use Committee an Atlanta VA medical Center genehmigt.
1. akute septische Lunge Verletzungen Mausmodell
<ol><li>Verwenden Sie 6 bis 8 Wochen - alte männliche Wildtyp C57BL/6J Mäuse (20-22 g Gewicht) für das Tier zu modellieren, und führen Sie alle Verfahren mit aseptischen Techniken und Instrumente. Autoklav alle chirurgischen Instrumente, einschließlich Pinzetten und Scheren für 20 min bei 121 ° C.</li>
	<li>Verteilen Sie Mäuse in experimentellen und Kontrollgruppe nach dem Zufallsprinzip. Mäuse auf eine chirurgische Board zurückhalten. Behandeln Sie Mäuse mit Escherichia coli Lipopolysaccharid (LPS, 15 mg/kg), intraperitoneale Weg. Verdünnen Sie nach dem individuellen Körpergewicht entsprechende LPS Dosierungen bis 0,1 mL PBS per Mausklick, dann injizieren Sie LPS-Lösung mit einer 1 mL Spritze mit 27 G Nadel und Spritzen Sie gleichwertigem Umfang von PBS zu Kontrolltieren.</li>
	<li>Halten Sie Mäuse in der tierischen BSL2-Anlage, bestehend aus sauberen Käfigen angemessene Raumtemperatur.</li>
	<li>24 Stunden später einschläfern Mäuse mit CO2 Einatmen.</li>
	<li>Wählen Sie den OP-Bereich in der Mitte der Maus Hals. Machen Sie einen kleinen Schnitt (ca. 5 mm) und trennen Sie vorsichtig Muskeln für den Zugriff auf die Luftröhre mit stumpfen Pinzette, bis die Luftröhre ausgesetzt ist.</li>
	<li>Maus-BAL-Flüssigkeit mit 10 mL sterile Spritze mit einer 27 G Nadel zurückziehen. Zuerst sanft, Stechen Sie die Nadel in die Luftröhre und Spritzen Sie 1 mL sterile PBS langsam in die Luftröhre, dann ziehen Sie BALF in eine Einweg-Spritze 3 mL mit einer 27 G Nadel. Nach Abschluss des Verfahrens Spritzen und Nadeln im Biohazard Sharps Container entsorgen.</li>
	<li>Chirurgische Instrumente mit Chlordioxid, zu behandeln, wie zuvor beschrieben von Chauret Et al. 27, und Sterilisieren Sie alle chirurgischen Instrumente durch Autoklavieren für 20 min bei 121 ° C.</li>
</ol>(2) Exosomen Isolation-seriell Ultra-Zentrifugation und Charakterisierung
<ol><li>Maus-BAL-Flüssigkeit in 15 mL konische Röhrchen zu sammeln. Alveoläre Zellen ernten, Zentrifugieren Sie Proben für 10 min bei 1.000 g bei 4 ° C.</li>
	<li>Übertragen Sie den überstand auf neue 15 mL konische Röhrchen, dann Zentrifugieren Sie Proben für 30 min bei 4.000 x g bei 4 ° C, Zelle Ablagerungen zu entfernen.</li>
	<li>Sammeln des Überstands in Ultrazentrifuge Tuben und Zentrifuge Proben 60 min bei 10.000 x g bei 4 ° C, größere zelluläre Partikel zu entfernen.</li>
	<li>Den überstand auf neue konische Rohre übertragen, dann die Proben durch 0,2 µm Spritze-Filter zu entfernen, bleiben größere Partikel zu filtern.</li>
	<li>Proben in Ultrazentrifuge Rohre und Rohre zu balancieren. Zentrifugieren Sie Proben für 2 h bei 140.000 x g bei 4° C zu die exosomen Pellets. Hinweis: Die Größe der exosomen variiert 20-200 nm. 0,2 µm-Filter dient zur Entfernung von größerer Partikeln, die mehr als 200 nm Größe.</li>
	<li>Entsorgen Sie die Überstände sanft. Das Pellet mit 100 µL PBS oder Lyse-Lösung und Transfer exosomen Proben zu 1,5 mL Röhrchen lösen und bei-80 ° C bis zur Verwendung aufbewahren. Hinweis: Transmissions-Elektronenmikroskop wird empfohlen, um isolierte exosomen zu überprüfen.</li>
	<li>Folgen Sie der Standardprobe TEM Verarbeitung Protokoll28. Analysieren von exosomen Proben mit Transmissions-Elektronenmikroskopie und nehmen Sie Bilder, Skala bar 200 nm.</li>
</ol>3. Vorbereitung der MiRNA und darstellende MiRNA-Q-RT-PCR
Hinweis: MiRNA-Q-RT-PCR wird empfohlen, Ausdruck von MiRNAs analysieren.
<ol><li>Folgen Sie standard-MiRNAs Isolierung Protokoll und achten Sie darauf, eine gleiche Menge von gereinigtem RNAs in Einzel-Strang reversen Transkription24.</li>
	<li>Folgen Sie das Echtzeit-PCR Standardprotokoll zur Erkennung von Exosomal RNA Ebene in BAL-Flüssigkeit. Verwenden Sie spezifische Echtzeit-PCR-Primer vor MiR-155, MiR-146a und U6 SnRNA. Laufen Sie PCR-Reaktion mit dem quantitativen PCR-Gerät und Normalisieren Sie die Ausdruckswerte zur internen Kontrolle U6 SnRNA. Hinweis: Jede Gruppe besteht aus dreifacher Proben24.</li>
</ol>4. Exosomen Etikettier- und Reinigung
<ol><li>Aussetzen Sie exosomen Pellet mit 100 µL Aussetzung Lösung in einem 1,5 mL Polypropylen Röhrchen, dann fügen Sie 0.4 µL der exosomen Farbstoff in das andere Rohr mit 100 µL der Aussetzung-Lösung.</li>
	<li>Schnell die Farbstofflösung exosomen in der Lösung hinzu und mischen Sie es gründlich durch pipettieren. Beispiele für 5 min bei Zimmertemperatur aufbewahren, vor Licht schützen.</li>
	<li>Setzen Sie eine exosom Spin Spalte in einem 1,5 mL Elution Rohr. Übertragen Sie die Lösung-Mischung in die Mitte der Spalte exosom Spin.</li>
	<li>Drehen Sie die Spalte für 2 min bei 800 X g bei Raumtemperatur.</li>
	<li>Verwerfen Sie die Spalte und speichern Sie die eluierten Probe bei-20 ° C bis zu seiner Verwendung zu.</li>
</ol>5. Validierung von Exosomen Up Empfängerzellen getroffenen
<ol><li>Samen Sie 1 x 104 MLE 12 /cell in einem 8-Well-Kammer-Folie und Zellkulturen bei 37 ° C, 5 % CO2.</li>
	<li>Fügen Sie 10 µL Leuchtstoff-Label exosomen zu den Zellen. 1 h bei 37 ° c inkubieren</li>
	<li>Entfernen Sie, Kultur, Medien und waschen Sie Zellen mit PBS, dann befestigen Sie Zellen mit 1 % Paraformaldehyd für 10 Minuten bei Raumtemperatur.</li>
	<li>Montieren Sie die Folie in Mount Medium mit DAPI.</li>
	<li>Analysieren Sie Proben durch ein Fluoreszenzmikroskop und nehmen Sie Bilder, 40 X Vergrößerung.</li>
</ol>6. Überprüfung der Interaktion zwischen Exosomen und Empfängerzellen
<ol><li>Samen 2 x 105 MLE12/Brunnen in einer Kultur 12-Well-Platte mit geeigneten Nährmedium und Zellkulturen bei 37 ° C, 5 % CO2.</li>
	<li>MLE12 Zellen 20 µg exosomen aus Maus BAL Flüssigkeit hinzufügen und Einrichten von Wiederholungen pro Versuchsgruppe.</li>
	<li>24 h später Zellen mit PBS waschen und Hinzufügen von 600 µL Lyse Puffer in Brunnen. Schütteln Sie die Platte und Lyse Proben in 1,5 mL-Tube mit der Pipette.</li>
	<li>Folgen Sie den gesamten RNA Isolierung Standardprotokoll und cDNA reversen Transkription Protokoll, vervollständigen Sie die Einzel-Strang cDNA reversen Transkription29.</li>
	<li>Folgen Sie Real-Time PCR Standardprotokoll zur Erkennung von mRNA Ausdruck Ebene in empfängerzellen29. Verwenden Sie spezifische Echtzeit-PCR-Primer gegen TNF-α, IL-6 und ZO-1. Laufen Sie PCR-Reaktionen mit dem quantitativen PCR-Gerät und Normalisieren Sie die Ausdruckswerte der internen Kontrolle GAPDH. Hinweis: Jede Gruppe besteht aus dreifacher Proben.</li>
</ol>

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Diese Arbeit wurde von VA Verdienst Beitrag 2I01BX001786-06A1 Finanzierung RS unterstützt.

Mausmodelle von Krankheiten werden üblicherweise verwendet, um die physiologische Funktion bestimmter Gene bewerten und zur Reduzierung der Kosten des Experimentierens2. Die akute septische Lungenversagen beschrieben hier imitiert Entzündungsreaktion bei Menschen mit ARDS gesehen. Dieses Modell ist relevant, die molekulare Pathogenese, Entwicklung von Biomarkern zu untersuchen und mögliche neue Therapien5zu testen.
Kokultur Systeme sind releva...

ALI und ARDS sind die lebensbedrohliche Formen der respiratorische Insuffizienz mit schweren Hypoxämie, verursacht durch nicht-kardiogenen Lungenödem betrifft etwa 1 Million Menschen weltweit pro Jahr1. Die Ätiologie des ARDS umfasst direkte Verletzung zu den Lungen von Infektionen oder Aspiration und eine Vielzahl von indirekten Beleidigungen. Im letzten Jahrzehnt gab es ein besseres Verständnis der molekularen Pathogenese des ARDS, gezielten Spezialbehandlungen für ARDS sind jedoch noch zu2,3entwickelt.
Verschiedenen Tiermodellen der akuten Lungenversagen sind entwickelt worden, die eine Brücke bieten, für die Übersetzung von experimentellen Therapien auf Studien am Menschen4,5. Häufigsten verwendeten Modelle verfügen über lokale Installation von Ölsäure, Bakterien, LPS und Bleomycin. Andere Ansätze sind Ischämie-Reperfusion, cecal Ligatur Punktion, mechanische Lüftung-induzierte Stretch Verletzungen, hyperoxie oder systemischen Verabreichung von Bakterien und LPS5. Diese Modelle bieten ein nützliches biologische System um klinische Hypothesen zu testen und für die Entwicklung von möglichen Therapien. Um menschliche ARDS zu simulieren, sollten Tiermodellen Entzündungen und akute Verletzung der epithelialen und endothelial Zellen mit defekten in Barriere-Funktion in der Lunge reproduzieren.
Exosomen sind membranvesikel mit 20-200 nm im Durchmesser, deren molekulare Inhalt Lipide, Proteine, DNA und RNA enthält, und Inter Mobilfunk in Gewebe Mikroumgebung über Molekulare Zusammensetzung Transfer erleichtern. Exosomen sind mehrere Arten von Zellen, wie endothelial Zellen, Epithelzellen, glatten Muskelzellen und Tumor-Zellen sezerniert und im menschlichen Körper Flüssigkeit vorhanden. Studien zeigen, dass exosomen Übersprechen zwischen Immunzellen und Stromazellen Zellen bei Infektions- und sterilen entzündlichen Erkrankungen regulieren und ihre abnorme Release erscheint durch die verschiedenen natürlichen und experimentellen Stimuli während physiologische geregelt werden und pathologische Prozesse6. Solchen Kommunikationsnetz kann eine wichtige Rolle in der Pathogenese von Lungenerkrankungen und pathophysiologischen Fortschreiten7,8beeinträchtigen. So 18-22 Nukleotid nicht-kodierende RNAs, MiRNAs vorhanden im Gewebe und Körperflüssigkeiten, Plasma, Serum und modulieren mRNA Expression am Post-translationalen Ebene9,10.
Verpackte MiRNAs in exosomen zu beeinflussen, Differenzierung und Funktion von mehreren Arten von Zellen und übermässigem sind verbunden mit einer Vielzahl von Krankheiten, einschließlich Krebs, Lungenkrankheiten, Fettleibigkeit, Diabetes und Herz-Kreislauf-Erkrankungen11, 12,13,14,15,16. Einstieg in den empfängerzellen und pendelt von Exosomal MiRNAs erleichtern interzelluläre Kommunikation ändern die Blutstillung Mikroumgebung17,18. Akutes Lungenversagen ist eine komplexe Prozesse, bei denen mehrere Zelltypen mit umfangreichen interzelluläre Kommunikation durch exosomen8. MiR-155 und MiR-146a Teilen der gemeinsamen transkriptionelle Regulierungsmechanismus und dazu beitragen, die entzündliche Reaktion und die Immuntoleranz19,20. Aktuelle Studien zeigen, dass sowohl entzündliche Reaktion über Exosomal MiRNAs pendeln zwischen Immunzellen21modulieren. Jedoch die molekularen Mechanismen, die modulierende Effekte der Exosomal MiRNAs alveoläre Reaktion auf Endotoxin bleiben unklar, zweifellos die potenzielle klinische Relevanz und Translationale Implikation Verdienst weitere Untersuchungen.
Kokultur Modelle werden eingesetzt, um die Interaktion der spezifischen Zelltypen im komplexen Umfeld, wie Entzündungen und Krebs22,23zu definieren. Diese Plattformen bieten eine alternative Strategie zur Nebensprechen zwischen Zelltypen besonders für immun- und strukturelle Zellen zu befragen.
Intra-trachealen, Aerosol, intraperitoneal oder systemische Gabe von LPS sind weit verbreitet, experimentelle Lunge Verletzungen24,25,26zu induzieren und haben gezeigt, dass die Durchlässigkeit von epithelialen und endotheliale induzieren Mängel. Hier verwenden wir intraperitoneale LPS, um septische Modellcharakter für die akute Lungenschädigung bei Mäusen zu induzieren. Innerhalb 24 h nach intraperitonealer Gabe von LPS induziert werden Permeabilität Defekte in der Lunge bei der Einstellung von Entzündungszellen. Darüber hinaus zeigen wir, dass exosomen aus BAL MiRNA-155 und MiR-146a enthalten, und exosomen von BAL-Flüssigkeit Pro-inflammatorischen Zytokinen Ausdruck im Empfänger Epithelzellen induzieren, wie IL-6 und TNF-α. Diese Daten sind erstmals zeigen, dass Exosomal MiRNAs sind in diesem Modell der akuten septischen Lungenschädigung in BAL abgesondert.

<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

Akute Lungenschädigung (ALI) und akute Atemnotsyndrom (ARDS) repräsentieren eine heterogene Gruppe von Lungenkrankheiten, die nach wie vor eine hohe Morbidität und Mortalität haben. Die molekulare Pathogenese von ALI wird besser definiert; jedoch aufgrund der Komplexität der Krankheit müssen Molekulare Therapien erst noch entwickelt werden. Hier verwenden wir ein Lipopolysaccharid (LPS) induzierte Mausmodell der akuten septischen Lungenschädigung, um die Rolle der exosomen in der entzündlichen Reaktion zu beschreiben. Mit diesem Modell konnten wir zeigen, dass Mäuse, die intraperitoneale LPS ausgesetzt sind, exosomen in Broncho-alveoläre Lavage (BAL) Flüssigkeit aus der Lunge absondern, die verpackt sind, mit MiRNA und Zytokine die entzündliche Reaktion zu regulieren. Weiter mit kokultur Modellsystem, zeigen wir, dass exosomen veröffentlicht von Makrophagen engen Kreuzung Proteinexpression in bronchialen Epithelzellen stören. Diese Ergebnisse deuten darauf hin, dass (1) Übersprechen zwischen angeborenen immun- und strukturelle Zellen durch die Exosomal pendelt beitragen, die entzündliche Reaktion und Störung der strukturelles Hindernis und (2) diese MiRNAs targeting kann eine neuartige Plattform zur Behandlung von ALI und ARDS.

Septische Lungenschädigung induzieren, wurden Mäuse mit intraperitonealer LPS (15 mg/kg) behandelt. Innerhalb von 24 h LPS Verwaltung wurde neutrophile Zustrom in der Lunge gesehen, wie in Figur 1Adargestellt. Maus-BAL-Flüssigkeit wurde gezogen, gefolgt von Isolierung und Reinigung der exosomen. Morphologie der BALF exosomen bestätigte Transmissions-Elektronen-Mikroskopie (Abbildung 1 b).
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Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Alvéolaire Lavage Exosomen in Lipopolysaccharid-induzierte septischen Lungenschädigung

Mäusen intraperitoneal LPs absondern exosomen in Broncho-alveoläre Lavage (BAL) Flüssigkeit, die mit MiRNAs verpackt sind. Ein Kokulturen-System verwenden, zeigen wir, dass exosomen veröffentlicht in der BAL-Flüssigkeit engen Kreuzung Proteinexpression in bronchialen Epithelzellen stören und Ausdruck von Pro-inflammatorischen Zytokinen, die Lungenschädigung betonen zu erhöhen.

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 Aufrufe.  Atlanta VAMC. Mäusen intraperitoneal LPs absondern exosomen in Broncho-alveoläre Lavage (BAL) Flüssigkeit, die mit MiRNAs verpackt sind. Ein Kokulturen-System verwenden, zeigen wir, dass exosomen veröffentlicht in der BAL-Flüssigkeit engen Kreuzung Proteinexpression in bronchialen Epithelzellen stören und Ausdruck von Pro-inflammatorischen Zytokinen, die Lungenschädigung betonen zu erhöhen.

Serie: 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.

Die Nutzung der Oropharyngeale Intratracheale PAMP Verwaltung und bronchoalveoläre Lavage, um die Immunantwort in Mäusen Bewerten

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

Forschung

Immunologie und Infektion

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.

Verfahren zur Erzeugung von Lungen Neutrophilie Mit aerosolisiertes Lipopolysaccharide

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.

Bronchoalveoläre Lavage von murinen Lungen zur Analyse der entzündlichen Zellinfiltration

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.

Lavage-induzierte Surfactant Depletion in Schweine als Modell des 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 ...

Medizin

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.

Isolierung der extrazellulären Vesikel aus murinen alvéolaire Lavage Fluid eine Ultrafiltration Zentrifugation Technik

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).

Ölsäure-Injektion bei Schweinen als Modell für 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 ...

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.

Visualisierung von lungenzellulären Anpassungen während kombinierter Ozon- und LPS-induzierter akuter Lungenverletzungen bei Der Maus

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.

Mausmodell des Ölsäure-induzierten akuten Atemnotsyndroms

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.

Induzieren akute Lungenschädigung bei Mäusen durch direkte intratracheale Lipopolysaccharid-Instillation

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.

<meta charset="utf8"></head><body>Bronchoalveoläre Lavage und Ölsäure-Injektion bei Schweinen als Double-Hit-Modell für das akute Atemnotsyndrom 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.

Bronchoalveoläre Lavage und Ölsäure-Injektion bei Schweinen als Double-Hit-Modell für das akute Atemnotsyndrom (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% ...