This work was supported by National Institutes of Health grants HL105947-01 (YL), HL07829-16 (AM), HL090152 (AM).

Die Tierversuche wurden von der Tierpflege Ausschuss und Institutional Biosafety Ausschüsse der Universität von Illinois in Chicago genehmigt. HINWEIS: Maske, Augenschutz, Kleid oder Overall und Doppelhandschuhe: Alle Verfahren, die Pseudomonas sollte mit biologischen Sicherheitsstufe 2 (BSL2) Praktiken, die umfassen, sind aber nicht beschränkt durchgeführt werden. Arbeiten Sie in zertifizierten Sicherheitswerkbank. Gönnen Instrumente in Kontakt mit Bakterien mit Bleichmittel oder Chlordioxid Desinfektionsmittel. Verwenden Sie eine versiegelte Verpackung für den Transport Proben. 1. P. aeruginosa Kultur und Wachstum <ol><li> Shop P. aeruginosa PA103 als Bakterienbestand in einer Schraubkappe Kryoröhrchen bei -80 ° C. </li><li> Legen Sie das Fläschchen in einem Rack in einem Kasten mit 70% Ethanol benetzt Papierhandtücher und zu ergreifen, um die biologischen Sicherheitsstufe 2 (BSL2) Labor. </li><li> Streak die Bakterien auf Schafblut-Agar-Platten wachsen bei 37 ° C für ~ 15 Stunden in einem Inkubator. </li&gt; <li> In einer BSL2- Haube, kratzen die Bakterien von der Platte mit Bakterien Schleife und resuspendieren in 5 ml PBS. </li><li> Bewahren Sie die Lager bei 4 ° C für bis zu 3 Monate. Allerdings bestimmen die Titer alle 2 Wochen. </li><li> Seriell zu verdünnen, die Bakterien in PBS (in der Regel von 1:10 4 bis 1:10 7) und die Platte des bekannten Verdünnung auf Schafblut-Agar-Platten. </li><li> Inkubieren Sie die Platten für ~ 15 Std. Graf Kolonien und berechnen Colony Forming Unit (CFU) nach Bakterienkonzentration zu bestimmen. </li><li> Resuspendieren der entsprechenden Konzentrationen (~ 5 x 10 3 KbE / ul) in 0,5 ml PBS in 1,5 ml steriler Schraubkappe Kryoröhrchen. Dichten Sie die Kryoröhrchen und legen Sie sie in einem Kryoröhrchen Rack auf Desinfektionsmittel beladene Papierhandtücher in einem Schnappdeckel-Box. </li><li> Transportieren Sie das Kästchen, um BSL2- Tieranlage. Dort angekommen, öffnen Sie die Box in der BSL2- Schrank. </li></ol> 2. P. aeruginosa Instillation <ol><li> Führen Überleben Chirurgie aseptisch (sterile Handschuhe, sterile InstrumenteUnd aseptischer Techniken). Verwenden Sie eine sterile Abdeckung, eine Arbeitsfläche für sterile (autoklaviert) chirurgische Instrumente bereitzustellen. Sterilisieren Instrumente mit einer Heiß Perle Sterilisator zwischen jeder Instillation Verfahren. </li><li> Wiegen Mäusen vor Betäubung. Mäuse anästhesiert mit Ketamin (100 mg / kg), Xylazin (5 mg / kg) in 0.1 - 0.2 ml PBS intraperitoneal (ip). Bestimmung der Wirksamkeit der Betäubung durch Nichtreaktionsfähigkeit auf Zehenklemm. Verwenden Sie einen Tierarzt Salbe auf die Augen bis zur Trockene unter Narkose zu verhindern. </li><li> Rain Mäuse auf einer chirurgischen Bord in BSL2- Schrank. </li><li> Identifizieren Sie den Bereich für den Schnitt nach unten. Rasur dieses Gebiet und bereiten die Haut mit Wechselstrom Alkohol und Povidoniod Tupfer 3 mal. </li><li> Behandlung der Schnittbereich mit einem Lokalanästhetikum (Lidocain), da dies der Anästhesie ist ausreichend für eine kleinere chirurgische Eingriffe, wie einen Hautschnitt, um die Luftröhre gelangen. </li><li> Machen Sie einen kleinen Schnitt (ca. 5 mm) an der Mittellinie des Halses, und benutzen stumpfe forceps sanft bewegen die Muskeln für den Zugriff auf die Luftröhre. Setzen Sie die Luftröhre durch chirurgische Dissektion. </li><li> Zeichnen Bakterienlösung in eine 1 ml Einwegspritze mit 27 G-Nadel. Für jede Maus, verabreichen 20-30 ul Bakterien bei der entsprechenden Konzentration (bis zu 10 5 CFU je Maus). </li><li> Setzen Sie die Nadel in die Luftröhre. Injizieren Lösung langsam in den Luftröhren. </li><li> Stellen Sie sicher, dass die Tier keucht, die normalerweise an, dass Lösung in die Lunge erreicht. </li><li> Schließen Sie die Wunde mit sterilem Nahtmaterial (6-0 Monofilament) unter aseptischen Bedingungen. </li><li> Verwenden Sie 0,1 mg / kg Buprenorphin durch subkutane Injektion als post-op Analgesie zu postoperativen Schmerzen zu kontrollieren. </li><li> Stellen Sie sicher, dass die Zeit von der ersten Narkoseeinleitung erforderlichen Schließung Schnitt weniger als 15 min. </li><li> Nach der Injektion entsorgen Spritzen und Nadeln in einem geeigneten Biohazard Behälter für scharfe Gegenstände. </li><li> Behandeln Sie die chirurgischen Instrumente mit Chlordioxid basierend DISInfectant für 15 min, spülen und zum Labor für die Sterilisation und Wiederverwendung wie nötig. </li><li> Haus die Mäuse einzeln in saubere Käfige bei warmen Umgebung. </li><li> Überprüfen Sie auf dem Tier alle 30 Minuten, bis er das Bewusstsein wiedererlangt und beginnt sich zu bewegen; und später in 12 Stunden Intervallen für die ersten 3 Tage nach der Operation. </li><li> Halten die Mäuse in der Tier BSL2 Einrichtung während des Experiments. Stellen Sie sicher, dass nur in versiegelten Kiste enthaltene Gewebe verlässt das Tier BSL2- Möglichkeiten. Wenn die Mäuse exprimieren einem der moribund Verhaltensweisen (definiert als Atemnot, Lethargie, Versagen in Reaktion auf sanfte Stimulierung ambulate) an einem Punkt in der Studie euthanize die Mäuse durch CO 2 Inhalation aus einem Flaschenquelle gefolgt durch zervikale Dislokation. </li><li> Euthanize die Versuchspersonen durch Ausbluten unter Narkose. <ol><li> Sammeln Lungengewebe von Mäusen getötet. Führen Sie diese Verfahren in einem BSL2 Kapuze. Falls erforderlich, überweisen Sie den Proben mit verschlossenen Röhrchen in einem rac platziertk on Desinfektions beladene Papierhandtücher in einem Schnappdeckel-Box für weitere Prozess. </li></ol></li></ol>

<ol><li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+acute+respiratory+distress+syndrome%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li>
<li>Matthay, M. A., Ware, L. B., Zimmerman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+acute+respiratory+distress+syndrome%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li>
<li>Shimabukuro, D. W., Sawa, T., Gropper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Injury+and+repair+in+lung+and+airways%5BTitle%5D)+AND+%22Crit+Care+Med%22%5BJournal%5D)">Injury and repair in lung and airways.</a> Crit Care Med. 31 (8), 524-531 (2003).</li>
<li>Wansleeben, C., Barkauskas, C. E., Rock, J. R., Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cells+of+the+adult+lung%3A+Their+development+and+role+in+homeostasis%2C+regeneration%2C+and+disease%5BTitle%5D)+AND+%22Wiley+Interdiscip+Rev+Dev+Biol%22%5BJournal%5D)">Stem cells of the adult lung: Their development and role in homeostasis, regeneration, and disease.</a> Wiley Interdiscip Rev Dev Biol. 2, 131-148 (2013).</li>
<li>Singleton, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamin+2+and+c-abl+are+novel+regulators+of+hyperoxia-mediated+nadph+oxidase+activation+and+reactive+oxygen+species+production+in+caveolin-enriched+microdomains+of+the+endothelium%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Dynamin 2 and c-abl are novel regulators of hyperoxia-mediated nadph oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium.</a> J Biol Chem. 284, 34964-34975 (2009).</li>
<li>Sadikot, R. T., Blackwell, T. S., Christman, J. W., Prince, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogen-host+interactions+in+pseudomonas+aeruginosa+pneumonia%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Pathogen-host interactions in pseudomonas aeruginosa pneumonia.</a> Am J Respir Crit Care Med. 171, 1209-1223 (2005).</li>
<li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Foxm1+mediates+the+progenitor+function+of+type+ii+epithelial+cells+in+repairing+alveolar+injury+induced+by+pseudomonas+aeruginosa%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Foxm1 mediates the progenitor function of type ii epithelial cells in repairing alveolar injury induced by pseudomonas aeruginosa.</a> J Exp Med. 208, 1473-1484 (2011).</li>
<li>Kurahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis+of+septic+shock+in+pseudomonas+aeruginosa+pneumonia%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Pathogenesis of septic shock in pseudomonas aeruginosa pneumonia.</a> J Clin Invest. 104, 743-750 (1999).</li>
<li>Kumar, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distal+airway+stem+cells+yield+alveoli+in+vitro+and+during+lung+regeneration+following+h1n1+influenza+infection%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Distal airway stem cells yield alveoli in vitro and during lung regeneration following h1n1 influenza infection.</a> Cell. 147, 525-538 (2011).</li>
<li>Delano, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sepsis+induces+early+alterations+in+innate+immunity+that+impact+mortality+to+secondary+infection%5BTitle%5D)+AND+%22J+Immunology%22%5BJournal%5D)">Sepsis induces early alterations in innate immunity that impact mortality to secondary infection.</a> J Immunology. 186, 195-202 (2011).</li>
<li>Lange, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+murine+model+of+sepsis+following+smoke+inhalation+injury%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">A murine model of sepsis following smoke inhalation injury.</a> Biochem Biophys Res Commun. 391 (3), 1555-1560 (2010).</li>
<li>Kobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+chemokines+in+neutrophil+biology%5BTitle%5D)+AND+%22Front+Biosci%22%5BJournal%5D)">The role of chemokines in neutrophil biology.</a> Front Biosci. 13, 2400-2407 (2008).</li>
<li>Matute-Bello, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+acute+lung+injury%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">Animal models of acute lung injury.</a> Am J Physiol Lung Cell Mol Physiol. 295, L379-L399 (2008).</li>
<li>Sadikot, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+immunomodulation+of+the+nf-kappab+pathway+in+airway+epithelium+impacts+host+defense+against+pseudomonas+aeruginosa%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Targeted immunomodulation of the nf-kappab pathway in airway epithelium impacts host defense against pseudomonas aeruginosa.</a> J Immunol. 176, 4923-4930 (2006).</li>
<li>Pinto, I. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+an+experimental+model+of+neutrophilic+pulmonary+response+induction+in+mice%5BTitle%5D)+AND+%22J+Pneumologia%22%5BJournal%5D)">Development of an experimental model of neutrophilic pulmonary response induction in mice.</a> J Pneumologia. 29, 213-214 (2003).</li>
</ol>

The authors have nothing to disclose.

Die Pseudomonas-Maus Lungenverletzung Modell, das wir beschreiben imitiert den gesamten Prozess der Entzündung, Lungenverletzung, Reparatur und Auflösung, die nach einer akuten Lungenschädigung oder Lungenentzündung auftreten. Es hat einzigartige Vorteile im Vergleich mit einigen anderen Verletzungen Modelle, dass es klinisch relevanten und relativ sicher und einfach zu handhaben. Der kritische Schritt in dem Verfahren ist, dass die Injektion von Bakterienlösung muss sehr langsam. Wenn ...

Lungen Umweltkrankheitserreger ausgesetzt und sind anfällig für Entzündungen und Verletzungen 1-3. Während pathologischen Zuständen, wie Lungenentzündung oder Adult Respiratory Distress Syndrome (ARDS), Krankheitserreger sowie Entzündungsfaktoren von Leukozyten freigesetzt induzieren Verletzungen und Tod von Alveolarzellen 1-3. Es ist wichtig zu Tiermodellen von akutem Lungenversagen entwickeln, um die Untersuchung der Pathologie von Verletzungen sowie Mechanismus der Reparatur zu erleichtern. Gegenwärtig sind die meisten Leute benutzen Hyperoxie und Bleomycin induzierten Mauslungenverletzung Modelle 4. Jedoch sind die Mechanismen der Hyperoxie Schädigung verursacht sind nicht die gleichen wie die häufigste Lungenverletzungen, die bei Lungenentzündung oder ARDS 5 auftreten. Bleomycin induzierte akute Verletzung ist selten im klinischen Kontext 4. Eine Maus Lungenverletzung Modell mit intratracheale Injektion von P. Hier berichten wir aeruginosa 6,7. Dieses Modell ist klinisch relevant, und ahmt die prozesse, die folgende Lungenentzündung 8 geschehen. Als opportunistische, nosokomiale Erreger der immungeschwächten Personen, P. aeruginosa infiziert in der Regel die Lungenwege, der Harnwege, Verbrennungen, Wunden, und verursacht auch andere Blutinfektionen 6. Die Bakterien setzen Virulenzfaktor Exotoxin A, multiplizieren und lösen Immunreaktionen 6. Intra-trachael Verabreichung von P. aeruginosa spiegelt die Situation in der menschlichen Exposition gegenüber den Bakterien, die Lungenentzündung verursachen und die Pathologie ist wahrscheinlich anders als die vor kurzem berichtet, Influenza-Virus H1N1 induzierten Lungenschädigung Modell 9 zu sein. Da P. aeruginosa ist ein opportunistisches Pathogen, das relativ sicher im Vergleich zu einigen der virulenten Pathogenen handhaben ist. Hier verwendeten wir intratracheale Injektion, um die Bakterien zu verabreichen, weil wir festgestellt, dass dieses Verfahren eingeführt mehr Bakterien in den distalen Bereich der Alveolen der Lunge im Vergleich mitEinige andere Verfahren wie die Verwendung eines Katheters über den Mund. Verglichen mit anderen akuten Lungenverletzung Modelle, die P. hier beschriebenen aeruginosa Modell eignet sich für das Studium Lungenschädigung durch Bakterien und durch übermäßige Entzündung induziert. Im Gegensatz zu anderen Tiermodellen, die P. verwenden aeruginosa Sepsis 10,11 induzieren, hier verwenden wir intratracheale Injektion dieser Bakterien, um lokalisierte akute Lungenschädigung zu induzieren.

<table><tbody><tr><td>Anesthetic: Ketamin, xylazine, lidocaine, buprenorphine</td><td></td><td></td><td>pharmaceutical grade</td></tr><tr><td>27 G needle</td><td>Fisher</td><td>1482648</td><td></td></tr><tr><td>syringe</td><td>Fisher</td><td>14823434</td><td>1 ml</td></tr><tr><td>scissors</td><td>Fine Science tools</td><td></td><td></td></tr><tr><td>forceps</td><td>Fine Science tools</td><td></td><td></td></tr><tr><td>suture&amp;nbsp;</td><td>Fisher</td><td>19-037-526</td><td></td></tr><tr><td>Eye gauge, glove, gown</td><td></td><td></td><td></td></tr><tr><td>Biosafety Cabinet</td><td></td><td></td><td></td></tr><tr><td>chlorine dioxide based disinfectant</td><td></td><td></td><td>Clidox</td></tr><tr><td>sheep blood agar plates&amp;nbsp;</td><td>Medex supply</td><td>HL-1160</td><td></td></tr></tbody></table>

pseudomonas aeruginosa induced lung injury model

Um die menschliche akute Lungenschädigung oder Lungenentzündung zu untersuchen, ist es wichtig, Tiermodelle zu entwickeln, um verschiedene pathologische Merkmale dieser Krankheit zu imitieren. Hier haben wir eine Maus Lungenverletzung Modell durch intratracheale Injektion von Bakterien Pseudomonas aeruginosa (P. aeruginosa oder PA) entwickelt. Mit diesem Modell konnten wir Lungenentzündung in der frühen Phase der Verletzung zeigen. Zusätzlich wurde Alveolarepithel- Sperr Undichtigkeit durch Analysieren bronchoalveoläre Lavage (BAL) beobachtet; und alveoläre Zelltod wurde durch TUNEL-Assay mit Gewebe aus verletzten Lunge vorbereitet beobachtet. Zu einem späteren Phase nach der Verletzung, beobachteten wir die Zellproliferation für den Reparaturvorgang erforderlich. Die Verletzung wurde von 7 Tagen ab der Einleitung des P. gelöst aeruginosa Injektion. Dieses Modell imitiert die sequentielle Verlauf Lungenentzündung, Verletzungen und Reparatur während einer Lungenentzündung. Diese klinisch relevanten Tiermodell eignet sich für das Studium der Pathologie, Mechanismus der Reparatur, fach akuter Lungenverletzung, und kann auch verwendet werden, um potentielle therapeutische Mittel für diese Krankheit zu testen.

Ab 24 bis 72 Stunden nach P. aeruginosa Injektion erhöhte Zellularität wurde in Lungenschnitten beobachtet (1A-D). Die Lunge begonnen, von 96 Stunden nach der Verletzung (1E) erholen. 7 Tage nach P. aeruginosa, normalen Alveolen Morphologie wurde weitgehend wiederhergestellt (1F). Tunnel Färbung mit Lungenabschnitte bei 24 h nach P. vorbereitet aeruginosa zeigte Zelltod in Alveolen Zellen (1G-I). Um den Reparaturpr...

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Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa induzierte Lungenschädigung-Modell

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

pseudomonas-aeruginosa-induced-lung-injury-model

Universidad de Cartagena UDEC

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Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model

18.5K Aufrufe.  University of Illinois at Chicago. We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant. 

Serie: Pseudomonas aeruginosa Induced Lung Injury Model

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Langzeitchronik<em&gt; Pseudomonas aeruginosa</em&gt; Airway-Infektion bei Mäusen

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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 ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studieren Mikrobielle Gemeinschaften<em&gt; In-vivo-</em&gt;: Ein Modell von Host-vermittelte Interaktion zwischen<em&gt; Candida albicans</em&gt; Und<em&gt; Pseudomonas aeruginosa</em&gt; In den Atemwegen

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

A Robust Pneumonia Modell in intaktem Immunsystem Nagetiere zur Bewertung antibakterieller Wirksamkeit gegen<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; oder<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Ein IL-8 transientes transgenisiertes Mausmodell für die<em&gt; In vivo</em&gt; Langzeitüberwachung von entzündlichen Reaktionen

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Offene Tracheotomie Magensäure Aspiration Mausmodell der akuten Lungenschädigung Ergebnisse in Maximal Akute nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Medizin

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

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

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

Ein Modell der selbstbegrenzten akuten Lungenverletzung durch einseitige Intra-Bronchialsäure-Instillation

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

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Ein verzögertes Impfmodell der chronischen Pseudomonas aeruginosa Wundinfektion

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. aeruginosa Infizierte 3D-Co-Kultur von Bronchialepithelzellen und Makrophagen an der Luft-Flüssig-Schnittstelle zur präklinischen Bewertung von Anti-Infektivika

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

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. ...

Pseudomonas aeruginosa induzierte Lungenschädigung-Modell

Zusammenfassung

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