Gli autori non hanno dichiarato alcun conflitto di interessi.

Il protocollo generale richiede 2 giorni, inclusi il primo giorno di induzione di sepsi e l'isolamento del liquido di BAL dall'animale e il secondo giorno per l'isolamento di esosomi dal mouse BALF. Tutte le procedure sono state esaminate e approvate dal comitato di uso Atlanta VA medical center e istituzionali Animal Care.
1. Mouse polmone settica acuta lesioni modello
<ol><li>Utilizzare 6-8 settimane - topi di C57BL/6J wild-type maschi vecchi (peso g 20-22) per l'animale modello ed eseguire tutte le procedure usando tecniche asettiche e strumenti. Autoclave tutti gli strumenti chirurgici, tra cui pinze e le forbici per 20 minuti a 121 ° C.</li>
	<li>Distribuire in modo casuale topi in sperimentale e gruppo di controllo. Trattenga i topi su una tavola chirurgica. Trattamento di topi con il lipopolysaccharide di Escherichia coli (LPS, 15 mg/kg) per via intraperitoneale. Secondo il peso corporeo individuale, diluire appropriati dosaggi di LPS a 0,1 mL di PBS per topo, quindi iniettare la soluzione di LPS usando una siringa da 1 mL con ago 27G e iniettare un volume equivalente di PBS agli animali di controllo.</li>
	<li>Tenere topi nella struttura BSL2 animale, che consiste di gabbie pulite a temperatura appropriata.</li>
	<li>24 ore più tardi, eutanasia topi con inalazione di CO2 .</li>
	<li>Selezionare l'area chirurgica nel centro del collo del mouse. Praticare una piccola incisione (circa 5 mm) e separare delicatamente i muscoli per l'accesso alla trachea con forcipe smussato fino a quando la trachea è esposto.</li>
	<li>Ritirare il liquido di BAL del mouse con siringa sterile da 10 mL con un ago da 27 G. In primo luogo, delicatamente, inserire l'ago nella trachea e iniettare 1 mL di PBS sterile lentamente nella trachea, quindi disegnare BALF in una siringa monouso da 3 mL con ago 27G. Al termine della procedura, smaltire siringhe e aghi in contenitore di sharps di biohazard.</li>
	<li>Trattare gli strumenti chirurgici con biossido di cloro, come descritto in precedenza da Chauret et al. 27e quindi sterilizzare tutti gli strumenti chirurgici in autoclave per 20 minuti a 121 ° C.</li>
</ol>2. esosomi isolamento-serie Ultra-centrifugazione e caratterizzazione
<ol><li>Raccogliere il liquido di BAL del mouse in provette coniche da 15 mL. Per raccogliere le cellule alveolari, centrifugare i campioni per 10 min a 1.000 g, a 4 ° C.</li>
	<li>Trasferire il surnatante nuove provette coniche da 15 mL, quindi centrifugare i campioni per 30 min a 4.000 x g a 4 ° C per rimuovere i detriti cellulari.</li>
	<li>Raccogliere il surnatante in ultracentrifuga tubi e centrifugare i campioni per 60 min a 10.000 x g a 4 ° C per rimuovere le particelle più grandi di cellulare.</li>
	<li>Trasferire il surnatante in nuove provette coniche, quindi filtrare i campioni attraverso 0,2 µm siringa-filtro per rimuovere i restanti particelle più grandi.</li>
	<li>Raccogliere campioni in tubi ultracentrifuga e l'equilibrio di tubi. Centrifugare i campioni per 2 h a 140.000 x g a 4° C a pellet gli esosomi. Nota: La dimensione di esosomi varia 20-200 nanometro. 0,2 µm filtro viene utilizzato per la rimozione di particelle più grandi che sono più di 200 nm in dimensione.</li>
	<li>Scartare i surnatanti delicatamente. Dissolva la pallina con 100 µ l PBS o soluzione di lisi e trasferimento esosomi campioni per provette da 1,5 mL e conservare a-80 ° C fino all'utilizzo. Nota: Microscopio elettronico a trasmissione è consigliabile verificare gli esosomi isolati.</li>
	<li>Seguire il campione TEM standard elaborazione protocollo28. Analisi di campioni di esosomi usando microscopia elettronica di trasmissione e prendere immagini, scala bar 200 nm.</li>
</ol>3. preparazione del miRNA e Performing miRNA-q-RT-PCR
Nota: miRNA-q-RT-PCR si raccomanda di analizzare il livello di espressione dei miRNA.
<ol><li>Seguire il protocollo di isolamento standard Mirna e assicurarsi di utilizzare una quantità uguale di RNA purificato nella trascrizione inversa single-strand24.</li>
	<li>Seguire il protocollo standard di PCR in tempo reale per rilevare il livello di microRNA exosomal in liquido di BAL. Utilizzare specifici iniettori di PCR in tempo reale contro miR-155, miR-146a e SnRNA U6. Eseguire la reazione di PCR con apparato PCR quantitativa e normalizzare i valori di espressione per controllo interno U6 snRNA. Nota: Ogni gruppo è costituito da campioni triplici24.</li>
</ol>4. esosomi etichettatura e purificazione
<ol><li>Sospendere gli esosomi pellet con 100 µ l di soluzione di sospensione in una provetta di polipropilene da 1,5 mL, quindi aggiungere 0,4 µ l della tintura esosomi nell'altro tubo con 100 µ l della soluzione di sospensione.</li>
	<li>Aggiungere esosomi nella soluzione per la soluzione colorante e mescolare accuratamente pipettando rapidamente. Conservare i campioni per 5 min a temperatura ambiente, proteggere dalla luce.</li>
	<li>Mettere una colonna di esosomi spin in una provetta di eluizione da 1,5 mL. Trasferire la miscela di soluzione al centro della colonna di spin di esosomi.</li>
	<li>Girare la colonna per 2 min a 800 x g a temperatura ambiente.</li>
	<li>Scartare la colonna e conservare il campione eluito a-20 ° C fino all'utilizzo.</li>
</ol>5. convalida di esosomi Up preso da cellule recettive
<ol><li>Semi di 1 x 104 MLE 12 prodotto in una diapositiva di 8 pozzetti camera e cellule di coltura a 37 ° C, 5% CO2.</li>
	<li>Aggiungere 10 µ l con etichetta fluorescente esosomi alle celle. Incubare per 1 h a 37 ° C.</li>
	<li>Rimuovere i mezzi di coltura e lavare le cellule con PBS, quindi fissare le cellule con paraformaldeide 1% per 10 minuti a temperatura ambiente.</li>
	<li>Montare il vetrino in un mezzo di montaggio con DAPI.</li>
	<li>Analizzare i campioni attraverso un microscopio a fluorescenza e prendere le immagini, ingrandimento 40x.</li>
</ol>6. Verifica dell'interazione tra esosomi e cellule recettive
<ol><li>Seme 2 x 105 MLE12/pozzetto di una piastra di coltura 12-pozzetti contenenti terreno di coltura appropriato e cellule di coltura a 37 ° C, 5% CO2.</li>
	<li>Aggiungere 20 µ g esosomi dal fluido del mouse BAL MLE12 celle e impostare ripetizioni per gruppo sperimentale.</li>
	<li>24 ore dopo, lavare le cellule con PBS e aggiungere 600 µ l di tampone di lisi in pozzetto. Agitare la piastra delicatamente e raccogliere campioni di lisi in provetta da 1,5 mL con la pipetta.</li>
	<li>Seguire il protocollo di isolamento di RNA totale standard e protocollo di trascrizione d'inversione del cDNA, completare la trascrizione inversa del cDNA del singolo-filo29.</li>
	<li>Seguire il protocollo standard di PCR in tempo reale per rilevare il livello di espressione di mRNA in cellule recettive29. Utilizzare specifici iniettori di PCR in tempo reale contro TNF-α, IL-6 e ZO-1. Eseguire reazioni di PCR con apparato PCR quantitativa e normalizzare i valori di espressione per il controllo interno GAPDH. Nota: Ogni gruppo è costituito da campioni triplici.</li>
</ol>

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Questo lavoro è stato supportato da recensione di merito VA 2I01BX001786-06A1 finanziamenti a RS.

Modelli murini di malattie sono comunemente utilizzati per valutare la funzione fisiologica dei geni specifici e per ridurre il costo della sperimentazione2. La ferita di polmone acuta settica descritta qui imita risposta infiammatoria visto in esseri umani con ARDS. Questo modello è rilevante per studiare la patogenesi molecolare, sviluppo di biomarcatori e per testare potenziali nuove terapie5.
Sistemi di co-coltura sono rilevanti per gli stud...

ALI e ARDS sono le forme pericolose di insufficienza respiratoria con ipossiemia severa causata da edema polmonare non cardiogeno che colpisce circa 1 milione di persone nel mondo ogni anno1. L'eziologia di ARDS include ferita diretta ai polmoni da infezioni o aspirazione e una varietà di insulti indiretti. Nell'ultimo decennio c'è stata una maggiore comprensione della patogenesi molecolare di ARDS, tuttavia, trattamenti specifici mirati per ARDS sono ancora essere sviluppato2,3.
Sono stati sviluppati diversi modelli animali di danno polmonare acuto che forniscono un ponte per la traduzione di terapie sperimentali per studi umani4,5. I modelli comunemente usati includono installazione locale di acido oleico, batteri, LPS e bleomicina. Altri approcci includono ischemia-riperfusione, puntura cecal legatura, lesione elasticizzato indotta da ventilazione meccanica, l'iperossia o somministrazione sistemica di batteri e LPS5. Questi modelli forniscono un sistema biologico utile per testare le ipotesi cliniche e per lo sviluppo di potenziali terapie. Per simulare umano ARDS, modelli animali dovrebbero riprodurre l'infiammazione e lesione acuta alle cellule epiteliali ed endoteliali con difetti nella funzione di barriera nei polmoni.
Gli esosomi sono vescicole di membrana con 20-200 nanometro di diametro, il cui contenuto molecolare contiene proteine, DNA, RNA e lipidi, e facilitano la comunicazione intercellulare nel microambiente del tessuto tramite trasferimento composizione molecolare. Gli esosomi sono secreti da più tipi di cellule, quali cellule endoteliali, cellule epiteliali, cellule muscolari lisce e cellule del tumore e presenti nel fluido del corpo umano. Studi indicano che gli esosomi regolano cross-talk tra le cellule immunitarie e le cellule stromali durante malattie infiammatorie, infettive e sterile, e loro rilascio anormale sembra essere regolata da vari stimoli naturali e sperimentali durante fisiologico e processi patologici6. Tale rete di comunicazione può svolgere un ruolo importante nella patogenesi di malattie polmonari e può influenzare la progressione fisiopatologico7,8. Come 18-22 nucleotidi RNA non codificanti, Mirna esiste nel siero sia dei tessuti e fluidi corporei, al plasma e modula l'espressione di mRNA a livello post-traduzionale9,10.
Mirna confezionati in esosomi influenzano la differenziazione e la funzione di più tipi di cellule, e livelli eccessivi sono associati con una varietà di malattie, compreso il cancro, malattie polmonari, obesità, diabete e malattia cardiovascolare11, 12,13,14,15,16. Entrata in cellule del destinatario e la spola di Mirna exosomal facilitare comunicazioni intercellulari modificando l'emostasi del microambiente17,18. Ferita di polmone acuta è un processo complesso, che prevedono più tipi di cellule con vasta comunicazione intercellulare attraverso esosomi8. miR-155 e miR-146a condividere il comune meccanismo di regolazione trascrizionale e contribuire alla risposta infiammatoria e la tolleranza immunitaria19,20. Gli studi recenti indicano che entrambi modulano la risposta infiammatoria via exosomal Mirna spola tra cellule immunitarie21. Tuttavia, i meccanismi molecolari sottostanti effetti modulatory di Mirna exosomal alveolare risposta all'endotossina rimangono poco chiari, senza dubbio la potenziale rilevanza clinica e traslazionale implicazione merito ulteriore indagine.
Modelli di co-coltura sono impiegati per definire l'interazione dei tipi cellulari specifici nell'ambiente complesso, quali l'infiammazione e cancro22,23. Queste piattaforme forniscono una strategia alternativa per interrogare i cross-talk tra tipi cellulari in particolare per le cellule immuni e strutturali.
Endotracheale, amministrazione aerosolizzato, intraperitoneale o sistemica di LPS sono ampiamente usati per indurre sperimentale del polmone lesioni24,25,26e hanno dimostrato di indurre permeabilità epiteliale ed endoteliale difetti. Qui usiamo LPS intraperitoneale per indurre un modello settico della ferita acuta del polmone in topi. Entro 24 h dalla somministrazione intraperitoneale di LPS, difetti di permeabilità sono indotte nei polmoni con il reclutamento di cellule infiammatorie. Inoltre, mostriamo che esosomi da BAL contengono miRNA-155 e miR-146a, ed esosomi da liquido di BAL inducono l'espressione di citochine pro-infiammatorie in cellule epiteliali destinatario, compreso IL-6 e TNF-α. Questi dati sono il primo a mostrare che i miRNA exosomal vengono secreti nel BAL in questo modello di danno polmonare acuto settico.

<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

Lesioni polmonari acute (ALI) e sindrome da distress respiratorio (ARDS) rappresentano un gruppo eterogeneo di malattie polmonari che continua ad avere un'alta morbosità e mortalità. Patogenesi molecolare delle ALI è essere meglio definiti; Tuttavia, a causa della natura complessa della malattia terapie molecolari devono ancora essere sviluppato. Qui usiamo un modello di topo indotta lipopolysaccharide (LPS) di danno polmonare acuto settica di delineare il ruolo degli esosomi nella risposta infiammatoria. Utilizzando questo modello, siamo stati in grado di dimostrare che i topi che sono esposti a LPS intraperitoneali secernono esosomi nel fluido di lavaggio (BAL) di bronco-alveolare dai polmoni che sono confezionati con miRNA e citochine che regolano la risposta infiammatoria. Ulteriormente utilizzando un sistema di modello di co-coltura, mostriamo che gli esosomi rilasciati dai macrofagi interrompono espressione delle proteine di giunzione stretta in cellule epiteliali bronchiali. Questi risultati indicano che 1) cross-talk tra cellule innate immune e strutturali attraverso la marcia exosomal contribuiscono alla risposta infiammatoria e rottura della barriera strutturale e 2) targeting questi miRNA può fornire una nuova piattaforma per trattare ALI e ARDS.

Per indurre il danno polmonare settico, i topi sono stati trattati con LPS intraperitoneali (15 mg/kg). Entro 24 h dalla amministrazione dei LPS, neutrophilic afflusso è stato visto nei polmoni, come mostrato in Figura 1A. Liquido di BAL del mouse è stato disegnato, seguita da isolamento e purificazione di esosomi. Morfologia di BALF esosomi è stata confermata da microscopia elettronica di trasmissione (Figura 1B).
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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

Il lavaggio broncoalveolare esosomi in lesioni polmonari settici Lipopolysaccharide indotta

Topi esposti a LPS intraperitoneali secernono esosomi nel fluido di lavaggio (BAL) bronco-alveolare che sono confezionati con Mirna. Utilizzando un sistema di co-coltura, indichiamo che esosomi rilasciati nel liquido di BAL perturbare l'espressione delle proteine di giunzione stretta in cellule epiteliali bronchiali ed aumentano l'espressione delle citochine pro-infiammatorie che accentuano la ferita di polmone.

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. Topi esposti a LPS intraperitoneali secernono esosomi nel fluido di lavaggio (BAL) bronco-alveolare che sono confezionati con Mirna. Utilizzando un sistema di co-coltura, indichiamo che esosomi rilasciati nel liquido di BAL perturbare l'espressione delle proteine di giunzione stretta in cellule epiteliali bronchiali ed aumentano l'espressione delle citochine pro-infiammatorie che accentuano la ferita di polmone.

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.

L&#39;utilizzo di orofaringea intratracheale PAMP Amministrazione e lavaggio broncoalveolare per valutare la risposta immunitaria nei topi Host

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

Ricerca

Immunologia e infezioni

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.

Un metodo per generare polmonare Neutrofilia Uso inalato Lipopolisaccaride

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.

Lavaggio broncoalveolare murini di polmoni per analizzare infiammatoria infiltrazione cellulare

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 indotta surfattante esaurimento nei suini come un modello della sindrome da distress respiratorio acuto (ARDS)

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

Medicina

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.

Isolamento delle vescicole extracellulari dal fluido di lavaggio broncoalveolare murino usando una tecnica di centrifugazione di ultrafiltrazione

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).

Iniezione di acido oleico in maiali come modello per la sindrome da Distress respiratorio

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.

Visualizzazione degli adattamenti cellulari polmonari durante l'ozono combinato e la lesione polmonare acuta murina indotta da LPS

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.

Modello murino di sindrome da distress respiratorio acuto indotto da acido oleico

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.

Indurre lesioni polmonari acute nei topi mediante instillazione diretta del lipopolioaccaride intracheale

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% ...