We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.

Todos os experimentos foram realizados em conformidade com as diretrizes da Comissão Institucional animal Cuidado e Uso (IACUC) do Instituto de Pesquisa Baylor. 1. A cultura não tipificável de Haemophilus influenzae (NTHi) e preparar o inóculo <ol><li> Placa de NTHi numa placa de agar de chocolate e manter a placa invertida numa atmosfera humidificada de CO 2 incubadora durante a noite a 37 ° C e 5% de CO 2. No dia seguinte, a cultura das bactérias em caldo de infusão cérebro coração. Em seguida, adicionar 1 ml de caldo num tubo esterilizado de 1,5 mL. </li><li> Centrifugar a 4.000 xg durante 5 min à temperatura ambiente. Em seguida, descartar o sobrenadante e ressuspender o sedimento em 1 ml de solução estéril de PBS 1x. Repita este passo uma vez. </li><li> Transferir 100 ul da solução de re-suspensa em 900 ul de 1x PBS e medir a absorvência da cultura diluída a 600 nm comprimento de onda num espectrofotómetro. </li><li> Determinar a unidade de viabilidade e de formação de colóniass (CFUs) do inoculo de cultura de diluições em série 1:10 em placas de agar de chocolate e incubando durante a noite a 37 ° C e 5% de CO 2. </li><li> Com base na viabilidade e CFU determinada a partir da etapa anterior, ajustar o inóculo até uma concentração final de 20 x 10 6 CFU / mL. </li></ol> 2. Intra-traqueal (IT) A instilação <ol><li> Injetar o mouse por via intraperitoneal (ip) com 0,1 ml / 10 g de peso corporal do rato com um + 20 mg solução de 150 mg / ml de cetamina / ml de xilazina. O procedimento deve ser feito na capa com uma fonte de luz para manter o animal aquecido. </li><li> Comprima o espaço interdigital do mouse para verificar se o mouse está devidamente anestesiados. </li><li> Posicione o mouse sobre as suas costas. Raspar a área ventral do pescoço e desinfectar com clorexidina e álcool etílico a 70%. </li><li> Levantar a pele da parte ventral superior do pescoço de ratos usando uma pinça dente. Fazer uma pequena incisão (0,5-1,0 cm) acima do timo utilizando um bisturie dissecar cuidadosamente o músculo para expor a traqueia. <ol><li> Usando uma seringa de 1 ml com uma agulha de 27 G, injectar lentamente 0,05 ml de ar, seguido por 0,05 ml de NTHi obtidos a partir de uma secção ou solução salina como controlo, seguida por outra de ar de 0,05 ml. Manter animais vertical por cerca de 1 min. </li></ol></li><li> Fechar a incisão com cola cirúrgica. Mantenha o mouse lateralmente reclinada e quente por cerca de 10 min. </li></ol> 3. Coleção LBA <ol><li> Sacrificar o mouse por cervical deslocamento de 24 horas após a infecção. </li><li> Abrir a cavidade abdominal do rato com uma tesoura anatômicas e cortou a aorta ventral a sangrar. Expor a parte ventral do pulmão. </li><li> Expor a traquéia e entubar com um cateter de calibre 20. Instilar 0,8 ml de tampão LBA (1x PBS, 0,1% de dextrose, 10 U / ml de heparina) e recolher a lavagem por aspiração suave. Repita este procedimento três vezes. </li><li> Contar o número total de células em 100 ul de suspensão de LBA utilizando um hemocytometros em 10x com Trypan Blue coloração. </li><li> Preparar lâminas para coloração Giemsa modificado. Alíquota de 100 uL de BALF nos poços apropriados da Cytospin e centrifugar a 500 xg durante 5 min. Secam-se as lâminas durante 10 minutos e corar as células com corante de Giemsa modificado de acordo com os protocolos do fabricante. </li><li> Use as restantes células para posterior análise, tal como células de fluorescência activada (FACS), microscopia confocal, a expressão do gene e western blot 11-13. </li></ol> 4. Preparação Histopatologia <ol><li> Para análise histopatológica, infectar ratos com NTHi como descrito no passo 2.4. Após 24 horas de infecção, sacrificar os animais por deslocação cervical. Cortar abrir a cavidade torácica, com uma tesoura anatômicas para expor os pulmões e a traqueia, como mencionado antes. Em seguida, insira o cateter calibre 20 na traqueia. </li><li> Encha os pulmões com 2% de solução de agarose quente preparado em formalina a 4%. Uma vez opulmões são totalmente inflado, gelo lugar no topo dos pulmões para solidificar a solução de agarose. </li><li> Em seguida, retire cuidadosamente a ficha torácica e colocá-lo em uma solução de 50 ml de formalina a 4% em 1x PBS até que seja processada para (H &amp; E) seções hematoxilina e eosina. </li></ol> 5. FACS coloração de células no LBA <ol><li> Colocar 1 x 10 5 células do fluido de LBA numa placa de micro titulação de fundo em V de 96 poços. </li><li> Lavar as células com 300 uL de PBS frio 1x e centrifugação a 4000 xg durante 3 minutos a 4 ° C. </li><li> Corar as células com solução de coloração de células em 100 ul de PBS 1x (1: 1000 de diluição) e manter à temperatura ambiente durante 15 min. </li><li> Lavar as células com 300 uL de frio 1x PBS a 4000 xg durante 3 minutos a 4 ° C. </li><li> Manter as células em 100 ul de tampão de lavagem de FACS [tampão de lavagem (1x PBS, 2% de FBS, 1 mM de EDTA, 0,1% de azida de sódio] contendo 1: 100 diluído Fc-bloco durante 15 min a 4 ° C. </li><li> células centrifugar a 4000 xg por 3 min e descartar supernatant. </li><li> As células são coradas com 100 ul de coquetel de coloração da superfície (diluição 1: 100) em tampão de lavagem de FACS durante 30 min a 4 ° C. </li><li> Lave as células três vezes com 300 ul de tampão de lavagem FACS a 4000 xg durante 3 minutos a 4 ° C. </li><li> Fixar as células em 100 uL de paraformaldeído a 4% durante 15 min à temperatura ambiente. </li><li> Lavar as células duas vezes com 300 ul de 1x PBS a 400 xg durante 3 min a 4 ° C e re-suspensão em 300 ul de tampão de lavagem de FACS. </li><li> Tornar os controlos de coloração de cor única, utilizando citometria de fluxo esferas. <ol><li> Adicionar uma gota de grânulos para a placa de microlitro de 96 poços de fundo em V e lava-se com 200 ul de PBS 1x. </li><li> Adicionar 1 ml de anticorpo de coloração a 100 ul de PBS 1x e grânulos e incubar à temperatura ambiente durante 5 min. </li><li> Lavar as pérolas duas vezes com 300 ul de PBS 1x. </li></ol></li><li> Analisar controles e células isoladas coradas por citometria de fluxo 14. </li></ol> 6. A homogeneização de tecido pulmonar para isolar RNA <ol><li> Após a infecção, recolhe um pequeno pedaço de pulmão (20-40 mg) e mantê-lo em RNA estabilizador reagente a 4 ° C durante a noite. Em seguida, armazená-lo a -80 ° C até ao passo seguinte. </li><li> Lavar os tecidos do pulmão em frio 1x PBS para se livrar do reagente de estabilização RNA. </li><li> Colocar o tecido em um tubo de 1,5 ml de centrífuga contendo tampão de lise (1 mL de tampão de lise + 10 ul β-mercaptoetanol). </li><li> Pique o tecido em pequenos pedaços com uma tesoura de pontas e homogeneizar utilizando um pilão pelota do motor sem fio para 20-40 seg. </li><li> Manter o lisado em gelo durante 5 minutos e continue com a etapa 7.1. </li></ol> 7. RNA isolamento do pulmão <ol><li> Centrifuga-se o lisado a 18.000 xg durante 3 min. Remover o sobrenadante por pipetagem e transferir cuidadosamente para um novo tubo de 1,5 mL. </li><li> Adicionar 1 volume de etanol a 70% ao lisado. Misturar imediatamente por pipetagem e esperar por 2-4 min. </li><li> Transferir para 700 ul de lisado a uma coluna de rotação colocadoem um tubo de recolha de 2 ml. </li><li> Centrifugar a 9.600 xg durante 15 seg e descartar o fluxo de passagem. </li><li> Adicionar 350 ul de tampão de lavagem rigorosa e centrifugar a 9600 xg por 15 s. Descartar o fluxo de passagem. </li><li> Adicionar 80 ul de ADNase I directamente para a membrana da coluna de spin e mantê-lo à temperatura ambiente durante 15 min. </li><li> Adicionar 350 mL de buffer rigorosas de lavagem para a coluna de centrifugação e centrifugar a 9600 xg por 15 s. Descartar o fluxo de passagem. </li><li> Adicionar 500 ul de tampão de lavagem de ARN e centrifugar a 9600 xg durante 15 seg. Descartar o fluxo de passagem. </li><li> Adicionar 500 mL de tampão de lavagem RNA e girar a 9.600 g durante 2 min. Descartar o fluxo de passagem. </li><li> Colocar a coluna de centrifugação em um novo tubo de recolha de 2 ml e centrifugar a 9600 x g durante 1 min. Descartar o fluxo de passagem. </li><li> Adicionar 30-50 mL água livre de ARNase directamente para a coluna de centrifugação e centrifugar a 9600 x g durante 1 min. ARN armazenar a -80 ° C. </li><li> Realizar a análise de RNA-Seq 14. </li></ol>

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The authors have nothing to disclose.

Aqui, nós descrevemos um procedimento único e minimamente invasivo para inocular os pulmões de ratinhos com um agente patogénico bacteriano pulmão. Nós demonstramos que este procedimento pode ser utilizado para estudar a função de genes diferentes que utilizam ratinhos que são deficientes em genes de vias de sinalização inflamatórias. Este procedimento também pode ser usado para estudar as respostas inflamatória a infecções pulmonares virais e fúngicas. As vantagens deste processo em relação aos outro...

A inflamação é um mecanismo de defesa imunitário fundamental da contra agentes infecciosos. Ela promove a erradicação do patógeno e reparação de tecidos danificados. Além disso, facilita a recuperação para um estado normal e saudável 1. No entanto, a inflamação desregulado muitas vezes leva a doenças inflamatórias crónicas 2. Inflamação das vias respiratórias é um gatilho inicial para diferentes doenças pulmonares tais como doença pulmonar obstrutiva crónica (DPOC), asma e fibrose pulmonar 3. A influenzae não tipificável (não encapsulado) Haemophilus (NTHi) é associada com doenças inflamatórias superiores e inferiores do pulmão crónicas 4,5. É a espécie dominante isolados das vias aéreas inferiores de crianças e adultos com 4,6,7 doença pulmonar obstrutiva crónica. A resposta inflamatória após a infecção NTHi é caracterizado pela regulação positiva de citocinas pró-inflamatórias (tais como TNF e IL-1β), e é mediada by mitogen activated protein kinase (MAPK) e fator nuclear-kB (NF-kB) através de receptores toll-like (TLR) 8. modelos de rato são ferramentas muito úteis para analisar a patologia subjacente de doença inflamatória do pulmão, devido à disponibilidade de linhas diferentes de genes deficientes. Vários métodos têm sido utilizados para inocular / bactérias vivas atenuadas e produtos bacterianos, incluindo a instilação intranasal e 9,10 inalação na forma de aerossol. Aqui, nós demonstramos a instilação intra-traqueal. Embora seja utilizada menos frequentemente, esta abordagem é mais eficiente e altamente reprodutível devido à entrega directa do inoculo para o tracto respiratório inferior.

<table><tbody><tr><td>Chocolate agar plate</td><td>Fisher Scientific</td><td>CAS50-99-7</td><td></td></tr><tr><td>Dextrose Anhydrous</td><td>Themo Scientific</td><td>R01300</td><td></td></tr><tr><td>Heparin</td><td>Hospira,Inc</td><td>RL-3010</td><td></td></tr><tr><td>Deft quick solution</td><td>Sigma</td><td>GS500-500ML</td><td></td></tr><tr><td>Syringe needle 20/26G&amp;nbsp;</td><td>BD</td><td>(REF305115/175)</td><td></td></tr><tr><td>Iml syringe</td><td>BD</td><td>REF 309602</td><td></td></tr><tr><td>Catheter 20GA</td><td>BD</td><td>REF 381433</td><td></td></tr><tr><td>Dissecting Scissors, straight, 10 cm long</td><td>kentscientific</td><td>INS600393</td><td></td></tr><tr><td>Iris Forceps, serrated, 10cm long</td><td>kentscientific</td><td>INS650915</td><td></td></tr><tr><td>Tweezer #5 Stainless steel, 11cm long</td><td>kentscientific</td><td>INS600095</td><td></td></tr><tr><td>10% Formalin</td><td>Fisher Scientific</td><td>CAS 67-56-1</td><td></td></tr><tr><td>Agarose</td><td>peqlab</td><td>35-1020</td><td></td></tr><tr><td>5ml polystyrene round-bottom tubes</td><td>BD</td><td>REF 352058</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tubes</td><td>Light Labs</td><td>A-7001-R</td><td></td></tr><tr><td>Reasy Mini&amp;nbsp; kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Pellet pestile motor (Tissue homoginizer)</td><td>Sigma</td><td>Z359971-1EA</td><td></td></tr><tr><td>96 well microtiter plates V bottom</td><td>Thermo</td><td>2605</td><td></td></tr><tr><td>1X PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111-42</td><td></td></tr><tr><td>CD45.2-APC</td><td>eBioscience</td><td>17-0454-81</td><td>Working dilution 1:100</td></tr><tr><td>Ly-6G-eFlor 450</td><td>eBioscience</td><td>48-5931-82</td><td>Working dilution 1:100</td></tr><tr><td>BSA</td><td>HyClone</td><td>SH30574.03</td><td></td></tr><tr><td>RBC Lysis Buffer (10X)</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>Live/Dead fixable aqua dead cell stain kit</td><td>Invitrogen</td><td>L-34957</td><td></td></tr><tr><td>EDTA (0.5M)</td><td>lifetechnologies</td><td>15575-020</td><td></td></tr><tr><td>CD16/CD32 FcBlock</td><td>BD</td><td>553142</td><td></td></tr><tr><td>Facs tubes polystyrene round bottom tube</td><td>BD</td><td>352052</td><td></td></tr><tr><td>Formaldehyde</td><td>Polyscience</td><td>4018</td><td></td></tr></tbody></table>

intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation

Aqui, descrevemos um procedimento detalhado de forma eficiente e entregar diretamente Haemophilus influenzae pelas vias respiratórias inferiores de ratos. Nós demonstramos o procedimento para a preparação H. influenzae inóculo, instilação intra-traqueal de H. influenzae para o pulmão, acúmulo de líquido bronco-alveolar lavagem (LBA), análise de células imunes no LBA, e isolamento de RNA para análise de expressão diferencial de genes. Este procedimento pode ser utilizado para estudar a resposta inflamatória do pulmão para qualquer bactérias, vírus ou fungos. A instilação traqueal directa é mais preferido mais de procedimentos de inalação de aerossóis intranasais ou porque mais eficiente proporciona o inoculo bacteriano para o tracto respiratório inferior com menos ambiguidade.

Instilação intra-traqueal resultou num marcadamente aumentada número de leucócitos no LBA (Figura 1A, painel esquerdo) do que a instalação com solução salina. A análise da contagem diferencial dos leucócitos mostrou claramente aumentou a infiltração de neutrófilos (Figura 1, painel da direita). A análise FACS das células no LBA confirmou ainda mais o aumento do número de neutrófilos (Figura 1B). A análise histológica d...

Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Administração intra-traqueal de<em&gt; Haemophilus influenzae</em&gt; No mouse modelos para o Estudo de inflamação das vias aéreas

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

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Immunology and Infection

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

10.1K visualizações.  Baylor Research Institute. We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models. 

Vídeo: Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Não-cirúrgico instilação endotraqueal de camundongos com Análise de Pulmões e Drenagem Linfática Nodes Lung por citometria de fluxo

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Imunologia e Infecção

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

Não invasiva Intratraqueal intubação para estudar a Patologia e Fisiologia do Rato Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Medicina

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

A utilização de orofaringe Intratraqueal Administração PAMP e Lavagem Broncoalveolar para avaliar a resposta imune do hospedeiro em camundongos

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

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

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 ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Intratraqueal (IMIT) instilação mediada por intubação: A não-invasiva, sistema de entrega Lung-specific

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Um método não-invasivo e tecnicamente não-intensivo para indução e fenotipagem of Experimental pneumonia bacteriana em ratos

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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 ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Interação de célula de imagem latente na Mucosa traqueal durante a infecção de vírus de Influenza usando microscopia Intravital dois fotões

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Avaliação das respostas do hospedeiro-patógeno e eficácia da vacina em camundongos

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Administração intra-traqueal de

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