Name: pseudomonas aeruginosa induced lung injury model
Uploaded: 2014-10-29T15:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about Pseudomonas aeruginosa Induced Lung Injury Model at JoVE.com

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

As experiências com animais foram aprovados pelo Comitê de Cuidados Animal e Comitês de Biossegurança Institucional da Universidade de Illinois, em Chicago. NOTA: Todos os procedimentos que envolvem pseudomonas deve ser realizada com Nível de Biossegurança 2 práticas (BSL2), que incluem mas não estão limitados a: máscara, protecção para os olhos, vestido ou macacão, luvas e duplas. Trabalhar em certificado de Biossegurança Gabinete. Trate instrumentos em contato com bactérias com desinfetante à base de lixívia ou cloro dióxido de carbono. Use uma caixa selada para amostras de transporte. 1. P. aeruginosa Cultura e Crescimento <ol><li> Loja P. aeruginosa PA103 como um estoque bacteriana em uma tampa de rosca frasco de congelação a -80 ° C. </li><li> Colocar o frasco em um rack em uma caixa com 70% de etanol molhada toalhas de papel e levar para o laboratório de Nível de Biossegurança 2 (BSL2). </li><li> Streak as bactérias em placas de agar de sangue de ovelha e crescer a 37 ° C durante ~ 15 horas numa incubadora. </li&gt; <li> Em um capuz BSL2, arranhar as bactérias da placa com bactérias loop e ressuspender em 5 ml de PBS. </li><li> Armazenar o estoque, a 4 ° C por até 3 meses. No entanto, determinar o título de cada 2 semanas. </li><li> Serialmente dilua as bactérias em PBS (geralmente de 1:10 a 1:10 4 7) e para fora da placa de diluição conhecido em placas de agar de sangue de ovelha. </li><li> Incubar as placas durante ~ 15 h. Contagem das colónias e calcular unidade formadora de colônia (UFC) para determinar a concentração de bactérias. </li><li> Ressuspender as concentrações apropriadas (~ 5 x 10 3 CFU / ul) em 0,5 ml de PBS em 1,5 ml estéreis criotubos com tampa de rosca. Vedar as cryovials e colocá-los em um rack cryovial em toalhas de papel carregado desinfetantes em uma caixa de encaixe da tampa. </li><li> Transportar a caixa para BSL2 biotério. Uma vez lá, abra a caixa no armário BSL2. </li></ol> 2. P. aeruginosa instilação <ol><li> Realize assepticamente cirurgia sobrevivência (luvas estéreis, instrumentos esterilizadose as técnicas assépticas). Use um campo estéril para fornecer uma superfície de trabalho para (autoclavados) instrumentos cirúrgicos estéreis. Esterilizar instrumentos usando um esterilizador quente talão entre cada procedimento instilação traqueal. </li><li> Pesar ratinhos antes da anestesia. Anestesiar ratos com cetamina (100 mg / kg), xilazina (5 mg / kg), em 0,1-0,2 ml de PBS por via intraperitoneal (ip). Determinar a eficácia do anestésico por não responsividade ao dedo do pé pitada. Use uma pomada veterinário sobre os olhos para evitar a secura sob anestesia. </li><li> Contenha camundongos em uma placa cirúrgica no gabinete BSL2. </li><li> Identificar a área para o corte para baixo. Raspar nesta área e preparar a pele usando alternando álcool e iodo povidona compressas 3 vezes. </li><li> Trate a área da incisão, com anestesia local (lidocaína), pois esta anestesia é suficiente para uma pequena cirurgia, como uma incisão na pele para acessar a traqueia. </li><li> Adicione uma pequena incisão (aproximadamente 5 mm) na linha média do pescoço, e usar romba forceps para mover suavemente o músculo para o acesso à traquéia. Expor a traquéia por dissecção cirúrgica. </li><li> Desenhe solução bactérias dentro de uma seringa descartável 1 ml com agulha 27 G. Para cada rato, administrar 20-30 ul de bactérias na concentração apropriada (até 10 5 CFU cada ratinho). </li><li> Insira a agulha na traquéia. Injectar solução lentamente na traqueia. </li><li> Assegure-se que os animais suspiros, que normalmente indica que a solução tenha atingido no pulmão. </li><li> Fechar a ferida com suturas de monofilamento estéreis (6-0), sob condições assépticas. </li><li> Use 0,1 mg / kg de buprenorfina por injecção subcutânea como analgesia no pós-operatório para controle da dor pós-cirúrgica. </li><li> Assegure-se que o tempo necessário desde a indução da anestesia inicial à incisão de fecho é inferior a 15 min. </li><li> Após a injeção, descarte de seringas e agulhas em um recipiente de risco biológico farelos apropriado. </li><li> Trate os instrumentos cirúrgicos com DISI base de dióxido de cloronfectant durante 15 minutos, enxaguar e voltar para o laboratório para esterilização e reutilização quando necessário. </li><li> Casa dos ratos individualmente em gaiolas limpas em ambiente quente. </li><li> Confira no animal a cada 30 minutos até que ele recobra a consciência e começa a se mover; e, posteriormente, em intervalos de 12 h para os primeiros 3 dias pós-cirurgia. </li><li> Manter os ratinhos na instalação de BSL2 animais ao longo da experiência. Certifique-se de que somente o tecido contido na caixa selada deixarem a instalação BSL2 animal. Se os ratinhos expressam qualquer um dos comportamentos moribundos (definida como dificuldade respiratória, letargia, insuficiência de deambulação em resposta à estimulação suave) em qualquer momento do estudo, os ratinhos eutanásia por inalação de CO 2 a partir de uma fonte mineral seguido por deslocamento cervical. </li><li> Euthanize o assunto experimental por sangria sob anestesia. <ol><li> Recolhe tecido pulmonar de ratos submetidos a eutanásia. Realizar esses procedimentos em uma capa BSL2. Se necessário, transferir a amostra usando tubos vedados colocados em um rack em toalhas de papel carregado desinfetantes em uma caixa de tampa snap para posterior processo. </li></ol></li></ol>

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The authors have nothing to disclose.

O modelo de lesão pulmonar rato Pseudomonas que descrevemos aqui imita todo o processo de inflamação, lesão pulmonar, reparo e resolução que ocorrem após a lesão pulmonar aguda ou pneumonia. Ele tem vantagens únicas comparando com vários outros modelos de lesão em que é clinicamente relevante e relativamente seguro e fácil de manusear. O passo crítico no processo é que a solução de injecção de bactérias tem de ser muito lento. Se a injecção for demasiado rápido, os rat...

Os pulmões são expostos a agentes patogénicos ambientais e são susceptíveis a inflamação e lesão 1-3. Durante condições patológicas como pneumonia ou Síndrome da Angústia Respiratória Adulto (SARA), patógenos, bem como fatores inflamatórios liberados por leucócitos induzir lesão e morte das células alveolares 1-3. É importante para o desenvolvimento de modelos animais de lesão pulmonar aguda para facilitar o estudo de lesões da patologia, bem como mecanismo de reparação. Atualmente, a maioria das pessoas usam hiperóxia e bleomicina rato induzida modelos de lesão pulmonar 4. No entanto, os mecanismos de hiperoxia causado um prejuízo não são o mesmo que a maioria das lesões pulmonares comuns que ocorrem durante a pneumonia ou ARDS 5. Bleomicina lesão aguda induzida é raro em um contexto clínico 4. Aqui nós relatamos um modelo de lesão pulmonar mouse usando a injeção intra-traqueal de P. aeruginosa 6,7. Este modelo é clinicamente relevante, e imita a processes que acontecem após pneumonia 8. Como um patógeno oportunista, nosocomial de indivíduos imunocomprometidos, P. aeruginosa normalmente infecta o trato pulmonar, urinário, queimaduras, feridas, e também causa outras infecções sanguíneas 6. A bactéria liberta virulência fator exotoxina A, multiplicar e desencadear respostas imunes 6. Administração intra-trachael de P. aeruginosa reflecte a situação em exposição humana para as bactérias que causam a pneumonia e a patologia é provável que seja diferente do modelo de lesão pulmonar induzida por vírus da gripe H1N1 recentemente relatado 9. Desde P. aeruginosa é um agente patogénico oportunista, é relativamente seguro de manusear em comparação com alguns dos agentes patogénicos mais virulentas. Aqui utilizou-se a injecção intra-traqueal para administrar as bactérias porque observou-se que este método introduzido mais bactérias na região distal alvéolos do pulmão em comparação comalguns outros procedimentos, tais como a utilização de um cateter através da boca. Em comparação com outros modelos de lesão pulmonar aguda, o P. aeruginosa modelo descrito é apropriado para o estudo de lesão pulmonar induzida por bactérias e por inflamação excessiva. Ao contrário de outros modelos animais que utilizam P. aeruginosa a indução da sepse 10,11, aqui vamos usar a injeção intra-traqueal destas bactérias para induzir localizada lesão pulmonar aguda.

<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

A fim de estudar a lesão pulmonar aguda humana e pneumonia, é importante para desenvolver modelos animais para imitar várias características patológicas da doença. Aqui nós desenvolvemos um modelo de lesão pulmonar rato pela injeção intra-traqueal de bactérias Pseudomonas aeruginosa (P. aeruginosa ou PA). Utilizando este modelo, fomos capazes de mostrar a inflamação pulmonar na fase inicial da lesão. Além disso, alveolar leakiness barreira epitelial foi observada por análise de lavagem broncoalveolar (BAL); e morte celular alveolar foi observada por ensaio de Tunel usando tecido preparado a partir de pulmões lesionados. Numa fase posterior, após lesão, observou-se proliferação de células necessário para o processo de reparação. A lesão foi resolvida 7 dias a partir do início do P. injecção aeruginosa. Este modelo imita o curso seqüencial de inflamação pulmonar, lesão e reparação durante pneumonia. Este modelo animal clinicamente relevante é adequado para estudar patologia, mecanismo de reparação, fepois lesão pulmonar aguda, e também pode ser utilizado para testar potenciais agentes terapêuticos para esta doença.

A partir 24-72 h pós P. injecção aeruginosa, celularidade aumentada foi observada em secções de pulmão (Figura 1A-D). O pulmão começou a se recuperar a partir de 96 horas pós lesão (Figura 1E). Aos 7 dias pós-P. aeruginosa, alvéolos morfologia normal foi restaurado pela maior parte (Figura 1F). Seções usando pulmonares coloração Tunnel preparado em 24 horas pós P. aeruginosa mostrou a morte celular em células em alv?...

Watch this Scientific Journal Video about Pseudomonas aeruginosa Induced Lung Injury Model at JoVE.com

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 Lesão Pulmonar Induzida Modelo

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

Research

JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model

18.4K visualizações.  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. 

Vídeo: 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.

Crônica de Longo Prazo<em&gt; Pseudomonas aeruginosa</em&gt; Infecção das vias aéreas em ratos

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 ...

Imunologia e Infecção

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.

Um método para gerar Pulmonar Neutrofilia Usando Aerosolized 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.

Estudar comunidades microbianas<em&gt; In Vivo</em&gt;: Um modelo de interação mediada por anfitrião entre<em&gt; Candida Albicans</em&gt; E<em&gt; Pseudomonas aeruginosa</em&gt; Nas Airways

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.

Um Modelo Pneumonia robusta em imunocompetentes Roedores para avaliar antibacteriano Eficácia contra<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; ou<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.

Um modelo de mouse transiente Transientized IL-8 para o<em&gt; In Vivo</em&gt; Monitoramento de Longo Prazo de Respostas Inflamatórias

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.

Abrir traqueostomia ácido gástrico aspiração Modelo murino de lesão pulmonar aguda Resultados em Maximal aguda não-letal 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 ...

Medicina

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.

Um modelo de lesão pulmonar aguda autolimitada por instilação unilateral de ácido intra-brônquico

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.

Um modelo de inoculação retardada da infecção crônica de pseudomonas aeruginosa ferida

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 Co-Cultura 3D infectada de células epiteliais e macrófagos bronquiais na interface ar-líquida para avaliação pré-clínico de anti-infecciosos

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.

Induzindo lesão pulmonar aguda em camundongos por instilação de lipopolissacárido intratraqueal direto

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

Pseudomonas aeruginosa Lesão Pulmonar Induzida Modelo

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