<p class="jove_content"&gt; Os estudos atuais são suportados pelo National Heart, Lung, and Blood Institute Grant R01-HL0921, R01-DK083385 e R01HL098294 para HK Eltzschig, o 1K08HL102267-01 a T. Eckle e Fundação para a Educação Anestesia e Bolsas de Investigação para T. Eckle e HK Eltzschig, e da American Heart Association para Grant T. Eckle e Eltzschig HK e Deutsche Forschungsgemeinschaft bolsa de pesquisa (DFG) para M. Koeppen.</p

Observações gerais: Todas as operações devem ser realizadas em um microscópio de dissecação vertical (Olympus, SZX10 com Z-Axis Mensagem Crank com STU2 Fique lança Stand) e usando um coagulador cirúrgica (11). Os grupos experimentais devem ser combinados da melhor forma possível na idade e peso para garantir a comparabilidade dos resultados. Administração de temperatura, pressão arterial anestesia, e fluido deve ser estável por toda parte. 1. Anestesia e preparação traquéia <ol><li> Use camundongos C57BL / 6 que são pelo menos 10 semanas de idade e tem um corpo de 22-25 g. Induzir a anestesia com pentobarbital sódico na dose de um ip 70 mg / kg peso corporal (6). Manter a anestesia com cerca de 10 mg / kg / h de sódio pentobarbital. Seja cauteloso com overdose uma vez que este pode reduzir significativamente a pressão arterial. Re-dose de pentobarbital - mesmo depois de horas-pode levar a um aumento severo dos níveis plasmáticos. </li><li> Após indução da anestesia, os ratos segura em posição supina com as extremidades superiores e inferiores anexado à mesa com uma fita e uma sutura presa aos tornozelos. Faça o mesmo para a cabeça, usando os dentes. Restrição suficiente é importante para uma intubação de sucesso e uma cirurgia bem controlada. Antes da cirurgia, cobrir o mouse com óleo mineral para reduzir o risco de alergia cabelo mouse. A fim de assegurar que a temperatura corporal permanece estável cobrir os ratos com filme de alimentos disponíveis comercialmente. </li><li> Camundongos colocar em uma mesa aquecida com temperatura controlada (RT, Effenberg, Munique, Alemanha) com uma sonda termômetro retal ligado ao controlador de retorno térmico para manter a temperatura corporal a 37 ° C. </li><li> Expor a traquéia cirurgicamente. Dissecar laterais e dorsal da traquéia do tecido conjuntivo e coloque duas fio de seda 3,0 cirúrgico (aparelho Harvard, EUA) a cada 10 cm de comprimento por baixo da traquéia. As suturas devem ser de aproximadamente 1 cm. </li><li> Cuidadosamente inciso traquéia 3-4 mm abaixo da laringe entre duas cartilagens circular usando uma tesoura McPherson-Vannas (8 cm, lâmina reta; Instrumentos de Precisão World, EUA). Certifique-se de não causar um sangramento, já que isso pode confundir os parâmetros de resultados. </li><li> Realizar uma intubação traqueal com cânula de polietileno blunt (INSYTE 22g, Beckton Dickinson, EUA). Insira a ponta da cânula de polietileno em um ângulo de 85 graus na traquéia. Em seguida, incline a cânulas por isso está de acordo com a luz traqueal. Lentamente avanço do tubo mais abaixo da traquéia até a ponta da cânula desapareceu na abertura do tórax. Fixar o tubo nesta posição com o fio de seda dois cirúrgica colocada dorsal da traquéia (ver 1.4). </li></ol> 2. Técnica de ventilação mecânica da lesão pulmonar induzida <ol><li> Ligue o tubo a um ventilador. Para induzir a lesão pulmonar usamos uma pressão técnica de ventilação controlada usando um C 900 Servo da Siemens (DRE Veterinária, EUA). Animais serão ventiladas com pico de pressão inspiratória de 45 mbar, freqüência de 80 respirações / min e uma pressão positiva expiratória final de 0-3 mbar com uma FiO 2 = 1,0. A inspiração à relação de expiração deve ser de 1:1. Apesar do fato de que o C 900 Servo é construído como ventilador para os seres humanos, a sua utilização em um ambiente de ventilação pressão controlada excelentes trabalhos para a ventilação de camundongos. </li><li> Monitor de freqüência cardíaca com ECG (por exemplo, Hewlett Packard, B blingen, Alemanha). Certifique-se que a freqüência cardíaca não cai abaixo de 400. Deve-se ver uma mudança do eixo do coração para a direita, quando a ventilação mecânica é instituído como um sinal de aumento da pressão arterial pulmonar decorrente de um aumento na pressão intratorácica. Se o rato se desenvolve bradicardia, verifique a temperatura ea dose do anestésico / concentração. Xylacin / cetamina induz anestesia um coração coração de 250 / min e não é recomendada. </li><li> Aplique uma reposição de líquidos adequada. Uma infusão com solução salina 0,1 ml / hora através de um cateter arterial ou venosa deve ser realizada antes da ventilação e garantir o arquivamento venosa suficiente. Devido às pressões de ventilação alta o retorno venoso para o coração é prejudicada, o que pode levar a uma queda crítica da pressão arterial média. Além disso, uma solução salina em bolus de 500 mL pode ser dado ip antes da cirurgia. </li><li> Colocar um cateter na artéria carótida para a gravação contínua da pressão arterial (27). Fixe o braço para o corpo antes de começar a dissecção da artéria. A artéria carótida é exposta através de dissecção romba da musculatura paratraqueal. Após a exposição ainda mais cuidado e evitar de qualquer trauma tecidual (particular do nervo vago), um cateter é inserido dentro do vaso usando duas suturas e uma braçadeira pequena (37). Isto irá expor um longo segmento da artéria. Coloque uma ligadura no final da artéria carótida. Anexar um maior aperto para o final da sutura para obter ª tensão ou fixarsutura e para a mesa usando fita. Coloque outro sutura ao redor da artéria e dissecar a artéria até o fim distal. Aqui, colocar uma braçadeira de pequeno porte. Use uma tesoura para cortar micro de uma pequena abertura na diagonal dentro da artéria. Segure a abertura com uma pinça fina (Dumont, WPI) e avançar o cateter de tamanho apropriado com as mãos / fórceps. Faça um nó com o seu segundo e segura de sutura da artéria. Solte a braçadeira e avançar o cateter ainda mais. Fixe o cateter com vários nós e fita. Alternativamente, o cateter de artéria carótida pode ser realizado no final do experimento para coletar amostras de sangue arterial para gasometria. </li></ol> 3. Recuperação de amostras de tecido Após 3 horas de ventilação mecânica, serão colhidas amostras para avaliar a extensão da lesão pulmonar. Recomendamos coletar lavado brochnoalveolar (BAL), sangue arterial e no tecido pulmonar. <ol><li> Obter LBA, no final do experimento. Depois de aprofundamento da anestesia, lave o tubo traqueal com 1 ml solução salina tamponada com fosfato (PBS). O fluido deve permanecer na traquéia / pulmão por três segundos antes que ele é a recuperação através da seringa conectada. O LBA é snap-congelados em nitrogênio líquido e armazenadas a -80 ° C para análise posterior. Estar ciente de que o volume pode ser recuperado significativamente menor do que 1 ml. </li><li> Realizar a análise de gases sanguíneos, no final do experimento. A fim de fazer isso, uma incisão deve ser feita logo abaixo do esterno. Segure o esterno com uma pinça e extensão da incisão ao longo da caixa torácica. Em seguida, o diafragma é incisão nas bordas e é cortado a partir as costelas. Agora há uma visão aberta para o mais baixo da abertura do tórax. Levantar o esterno até com uma pinça e abrir o tórax por cortes longos no lado direito e esquerdo (como laterais possível) para que a parede do tórax anterior completa está ligado-alas. Isto deve ser feito, enquanto o animal ainda é experimental ventilação mecânica. O ventrículo esquerdo é perfurado usando um 27 ½ G agulha e análise do sangue arterial é realizada utilizando o Sistema i-STAT (Abbott, EUA). Se a gasometria arterial deve ser realizada, uma BAL não pode ser obtida, uma vez que este será um fator de confusão significativa para os resultados. </li><li> Em alternativa ao método acima, as amostras de sangue arterial podem ser coletadas através do cateter de artéria carótida. No entanto, isto deve ser feito antes do LBA é obtido. </li><li> Especiais de consumo os pulmões em bloco, puxando-se o coração e cortar a traquéia. Ter um pedaço de tecido pronto para absorver o sangue, de modo que o sítio cirúrgico é visível. Puxe o coração na direção do abdômen e corte cuidadosamente ao longo da espinha para mobilizar todos os órgãos do tórax. Corte a aorta, remover os órgãos torácicos e coloque-os sobre uma mesa limpa cirúrgico. </li><li> Cortar o coração e os vasos principais da amostra de tecido. Certifique-se que não há tecido do timo ainda ligado ao pulmão. Separar os pulmões com uma tesoura e coloque em tubos individuais e encaixe-congelante. Armazenar a -80 ° C para análise posterior. </li></ol> 4. Medição de lesão pulmonar Recomendamos a utilização dos parâmetros seguintes resultados para avaliar a extensão da lesão pulmonar: Realizar um ELISA albumina (Bethyl Laboratories, EUA) e um mieloperoxidase (MPO) ELISA (Hycult Biotecnologia, EUA) para avaliar a extensão da disfunção de barreira e a quantidade de inflamatória células no LBA. Realizar um ELISA MPO também formam o tecido pulmonar. Se wet-a-seco proporção deve ser medida não obtemos LBA e da circulação pulmonar não é lavada (ver 3.3). Medir o peso dos pulmões após a excisão. Em seguida, os pulmões são liofilizadas por 48 h e no tecido pulmonar é medido novamente. Então a relação de wet-a-seco é medido em mg de água por mg de tecido seco (5). <img src="/files/ftp_upload/2525/2525fig1.jpg" alt="Figura 1" /> Figura 1. Teor de proteínas na BAL em resposta a VILI. Camundongos foram anestesiados com pentobarbital, ventilação mecânica foi instituída e os ratos foram ventilados com pressão controlada configurações (pressão inspiratória de 45 mbar, positiva ao final da exspiratory pressão de 3 mbar, 100% inspirado concentração de oxigênio). Após 0, 1, 2 e 3 horas de ventilação BAL foi colhida eo teor de proteína foi quantificado através de um ensaio de ácido bicinchoninic (ensaio BCA). A mudança relativa do teor de proteínas é mostrado normalizado para 0 horas de ventilação (n = 4 por grupo, * indica p &lt;0,05 comparado ao grupo controle, média ± SEM)

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD39/ectonucleoside+triphosphate+diphosphohydrolase+1+provides+myocardial+protection+during+cardiac+ischemia/reperfusion+injury.">CD39/ectonucleoside triphosphate diphosphohydrolase 1 provides myocardial protection during cardiac ischemia/reperfusion injury.</a> Circulation. 116, 1784-1794 (2007).</li><li>Kuhlicke, J., Frick, J. S., Morote-Garcia, J. C., Rosenberger, P., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+Inducible+Factor+(HIF)-1+Coordinates+Induction+of+Toll-Like+Receptors+TLR2+and+TLR6+during+Hypoxia.">Hypoxia Inducible Factor (HIF)-1 Coordinates Induction of Toll-Like Receptors TLR2 and TLR6 during Hypoxia.</a> PLoS ONE. 2, e1364-e1364 (2007).</li><li>Morote-Garcia, J. C., Rosenberger, P., Kuhlicke, J., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1-dependent+repression+of+adenosine+kinase+attenuates+hypoxia-induced+vascular+leak.">HIF-1-dependent repression of adenosine kinase attenuates hypoxia-induced vascular leak.</a> Blood. 111, 5571-5580 (2008).</li><li>Morote-Garcia, J. C., Rosenberger, P., Nivillac, N. M., Coe, I. R., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-dependent+repression+of+equilibrative+nucleoside+transporter+2+attenuates+mucosal+inflammation+during+intestinal+hypoxia.">Hypoxia-inducible factor-dependent repression of equilibrative nucleoside transporter 2 attenuates mucosal inflammation during intestinal hypoxia.</a> Gastroenterology. 136, 607-618 (2009).</li><li>Reutershan, J., Vollmer, I., Stark, S., Wagner, R., Ngamsri, K. C., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+and+inflammation:+CD39+and+CD73+are+critical+mediators+in+LPS-induced+PMN+trafficking+into+the+lungs.">Adenosine and inflammation: CD39 and CD73 are critical mediators in LPS-induced PMN trafficking into the lungs.</a> FASEB J. 23, 473-482 (2009).</li><li>Schingnitz, U., Hartmann, K., Macmanus, C. F., Eckle, T., Zug, S., Colgan, S. P., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+the+A2B+adenosine+receptor+dampens+endotoxin-induced+acute+lung+injury.">Signaling through the A2B adenosine receptor dampens endotoxin-induced acute lung injury.</a> J Immunol. 184, 5271-5279 (2010).</li><li>Thompson, L. F., Eltzschig, H. K., Ibla, J. C., Van De Wiele, C. J., Resta, R., Morote-Garcia, J. C., Colgan, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+for+ecto-5'-nucleotidase+(CD73)+in+vascular+leakage+during+hypoxia.">Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia.</a> J. Exp. Med. 200, 1395-1405 (2004).</li><li>Aherne, C. M., Kewley, E. M., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+resurgence+of+A2B+adenosine+receptor+signaling.">The resurgence of A2B adenosine receptor signaling.</a> Biochim Biophys Acta. , (2010).</li><li>Eckle, T., Koeppen, M., Eltzschig, H. 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O presente estudo descreve uma técnica de realizar ventilador da lesão pulmonar induzida em camundongos. Este modelo demonstra lesão altamente reprodutíveis, devido à ventilação de alta pressão. Investigadores que consideram estudar lesão pulmonar aguda em ratos podem beneficiar deste modelo.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Pentobarbital sódico (Plus Fatal) </td><td> Vortech Pharmaceutical Ls, Ltd, </td><td> VPL 9372 </td><td> 4mg/ml em solução salina </td></tr><tr><td> INSYTE 22 G </td><td> Beckton Dickinson </td><td> n / a </td><td></td></tr><tr><td> Sutura, de seda 4,0 </td><td> Harvard Apparatus </td><td> 517698 </td><td></td></tr><tr><td> Sutura, Prolene 8,0 </td><td> Ethicon, EUA </td><td> M8739 </td><td> reutilizáveis </td></tr><tr><td> Siemens 900 ° C </td><td> DRE Veterinária, EUA </td><td> # 336 </td><td> remodelado </td></tr><tr><td> microscópio de dissecação (SZX10) </td><td> Olimpo </td><td> n / a </td><td> considerar a distância de trabalho generosa </td></tr><tr><td> Tabela de aquecimento </td><td> Rt, Effenberger, Alemanha </td><td> n / a </td><td> único fornecedor e único </td></tr><tr><td> Dispositivo de pressão arterial </td><td> Cyber-Sense, Inc </td><td> BPM02 </td><td></td></tr><tr><td> STAT I </td><td> Abbott </td><td> n / a </td><td></td></tr></tbody></table>

pressure controlled ventilation to induce acute lung injury in mice

Modelos murinos são amplamente utilizados para investigar lesões agudas de diferentes sistemas dos órgãos (1-34). Lesão pulmonar aguda (LPA), que ocorre com a ventilação mecânica prolongada, contribui para a morbidade e mortalidade da doença crítica, e estudos sobre novos alvos farmacológicos ou genéticos são áreas de intensa investigação (1-3, 5, 8, 26, 30, 33 -36). ALI é definido pelo início agudo da doença, o que leva a não-cardíacas edema pulmonar e insuficiência pulmonar subseqüente de troca gasosa (36). Nós desenvolvemos um modelo murino de ALI usando uma ventilação com pressão controlada para induzir o ventilador da lesão pulmonar induzida (2). Para este fim, os camundongos C57BL / 6 são anestesiados e uma traqueostomia é realizada seguida de indução da LPA através de ventilação mecânica. Ratos são ventilados em um ambiente com pressão controlada com um pico de pressão inspiratória de 45 mbar por 1-3 horas. Como parâmetros de resultado, edema pulmonar (wet-a-seco ratio), conteúdo líquido broncoalveolar albumina, fluido broncoalveolar e conteúdo mieloperoxidase pulmonar tecidos e as trocas gasosas pulmonares são avaliados (2). Usando esta técnica nós poderíamos mostrar que suficientemente induz inflamação pulmonar aguda e pode distinguir entre diferentes grupos de tratamento ou genótipos (1-3, 5). Portanto, esta técnica pode ser útil para pesquisadores que buscam mecanismos moleculares envolvidos na ALI usando uma abordagem genética em camundongos com o gene-alvo exclusão.

Watch this Scientific Journal Video about Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice at JoVE.com

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Um modelo murino para o ventilador lesão pulmonar induzida é uma ferramenta importante para estudar uma lesão pulmonar aguda<em&gt; In vivo</em&gt;. Aqui, nós relatamos uma aplicáveis ​​fácil<em&gt; In situ</em&gt; Modelo de lesão pulmonar aguda de alta pressão de ventilação mecânica para induzir insuficiência aguda do pulmão.

Ventilação a pressão controlada para induzir lesão pulmonar aguda em ratos

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

pressure-controlled-ventilation-to-induce-acute-lung-injury-in-mice

Research

JoVE Journal

Medicine

18.2K visualizações.  University of Colorado. Um modelo murino para o ventilador lesão pulmonar induzida é uma ferramenta importante para estudar uma lesão pulmonar aguda In vivo. Aqui, nós relatamos uma aplicáveis ​​fácil In situ Modelo de lesão pulmonar aguda de alta pressão de ventilação mecânica para induzir insuficiência aguda do pulmão.

Vídeo: Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

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.

induzida por lavagem de surfactante Esgotamento em porcos como modelo da Síndrome da Angústia Respiratória Aguda (SARA)

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

Medicina

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Um método melhorado para rápida de intubação da traqueia em Ratos

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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 ...

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 ...

Imunologia e Infecção

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.

Visualizando adaptações celulares pulmonares durante a lesão pulmonar aguda de ozônio e LPS induzidas por LPS

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

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

Microscopia de Fluorescência De Largura Intravital de Microcirculação Pulmonar em Lesão Pulmonar Aguda Experimental Usando um Sistema de Imagem Estabilizada a Vácuo

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

Um Modelo de Rato de Intubação Orotraqueal e Cirurgia de Reperfusão de Isquemia Pulmonar Ventilada

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

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. ...

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can be exploited to quantify pulmonary physiology, including ventilation, restricted diffusion (alveolar-airspace size), and gas exchange. In humans, it has been used to evaluate disease severity and progression in a variety of pulmonary disorders and is approved for clinical use in the United States and United Kingdom. Beyond its clinical applications, the ability of 129Xe MRI to noninvasively assess pulmonary pathophysiology and provide spatially resolved information is valuable for preclinical research. Among animal models, mice are the most widely used due to the accessibility of genetically modified disease models. Here, 129Xe MRI is promising as a minimally invasive, radiation-free, and sensitive technique to longitudinally monitor lung disease progression and therapy response (e.g., in drug discovery). This technique can extend to preclinical applications by incorporating an MRI-triggered, free-breathing apparatus or mechanical ventilator to deliver gas. Here, we describe the steps and provide checklists to ensure robust data collection and analysis, including creating a thermally polarized xenon gas phantom for quality control, optimizing polarization, animal handling (sedation, intubation, ventilation, and care for mice), and protocols for ventilation, restricted diffusion, and gas exchange data. While preclinical 129Xe MRI can be applied in various animal models (e.g., rats, pigs, sheep), this protocol focuses on mice due to the challenges posed by their small anatomy, which are balanced by their affordability and the availability of many disease models.

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can ...

Bioengenharia

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial infusion of oleic acid, infusion of endotoxins or bacteria, cecal ligation and puncture, various pneumonia models, lung ischemia/reperfusion models and, of course, surfactant depletion models, among others. Surfactant depletion produces a rapid, reproducible deterioration of pulmonary gas exchange and hemodynamics and can be induced in anesthetized pigs using repeated lung lavages with 0.9% saline (35 mL/kg body weight, 37 &#176;C). The surfactant depletion model supports investigations with standard respiratory and hemodynamic monitoring with clinically applied devices. But the model suffers from a relatively high recruitability and ventilation with high airway pressures can immediately reduce the severity of the injury by reopening atelectatic lung areas. Thus, this model is not suitable for investigations of ventilator regimes that use high airway pressures. A combination of surfactant depletion and injurious ventilation with high tidal volume/low positive end-expiratory pressure (high Tv/low PEEP) to cause ventilator induced lung injury (VILI) will reduce the recruitability of the resulting lung injury. The advantages of a timely induction and the possibility to perform experimental research in a setting comparable to an intensive care unit are preserved.

Surfactant Depletion Combined with Injurious Ventilation Results in a Reproducible Model of the Acute Respiratory Distress Syndrome ARDS

A combination of surfactant washout using 0.9% saline (35 mL/kg body weight, 37 &#176;C) and high tidal volume ventilation with low PEEP to cause moderate ventilator induced lung injury (VILI) results in experimental acute respiratory distress syndrome (ARDS). This method provides a model of lung injury with low/limited recruitability to study the effect of various ventilation strategies for extended periods.

Esgotamento do surfactante combinado com ventilação prejudicial resulta em um modelo reprodutível da Síndrome do Sofrimento Respiratório Agudo (ARDS)

Various animal models exist to study the complex pathomechanisms of the acute respiratory distress syndrome (ARDS). These models include pulmo-arterial ...