Name: intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation
Uploaded: 2016-03-02T15:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

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 los experimentos se realizaron de acuerdo con las directrices del Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Baylor Research Institute. 1. El cultivo no tipificable de Haemophilus influenzae (NTHi) y preparar el inóculo <ol><li> Placa NTHi en una placa de agar chocolate y mantener la placa boca abajo en un incubador de CO2 humidificado durante la noche a 37 ° C y 5% de CO2. El siguiente día, cultivar las bacterias en caldo de infusión de corazón cerebro. A continuación, añadir 1 ml del caldo en un tubo estéril de 1,5 ml. </li><li> Centrifugar a 4000 xg durante 5 min a temperatura ambiente. A continuación, descartar el sobrenadante y resuspender el sedimento en 1 ml de PBS estéril 1x. Repita este paso una vez. </li><li> Transferir 100 l de solución de re-suspendido en 900 l de 1x PBS y se mide la absorbancia del cultivo diluido en la longitud de onda de 600 nm en un espectrofotómetro. </li><li> Determinar la viabilidad y unidad de formación de coloniass (UFC) del inóculo cultivando diluciones 1:10 en serie en placas de agar de chocolate y se incubó durante la noche a 37 ° C y 5% de CO 2. </li><li> Sobre la base de la viabilidad y las UFC determinada a partir del paso anterior, ajustar el inóculo a una concentración final de 20 x 10 6 CFU / ml. </li></ol> 2. intra-traqueal (IT) La instilación <ol><li> Inyectar el ratón por vía intraperitoneal (ip) con 0,1 ml / 10 g de peso corporal de ratón con un + solución de 150 mg / ml de ketamina 20 mg / ml de xilazina. El procedimiento debe realizarse en la campana con una fuente de luz para mantener caliente el animal. </li><li> Pellizcar el espacio interdigital del ratón para verificar que el ratón se anestesia adecuada. </li><li> Coloca el ratón sobre su espalda. Afeitar el área ventral del cuello y desinfectar con clorhexidina y alcohol etílico al 70%. </li><li> Levantar la piel de la parte ventral superior del cuello con unas pinzas de dientes de rata. Hacer una pequeña incisión (0,5-1,0 cm) por encima del timo utilizando un bisturíy diseccionar cuidadosamente el músculo para exponer la tráquea. <ol><li> Usando una jeringa de 1 ml con una aguja de 27 G, inyectar lentamente 0,05 ml de aire, seguido por 0,05 ml NTHi obtenidos a partir de la Sección 1 o solución salina como control, seguido de otro 0,05 ml de aire. Mantener animales vertical para alrededor de 1 min. </li></ol></li><li> Cerrar la incisión con pegamento quirúrgico. Mantenga el ratón lateralmente reclinada y caliente durante unos 10 minutos. </li></ol> 3. Colección BALF <ol><li> Sacrificar el ratón por dislocación cervical 24 horas después de la infección. </li><li> Abra la cavidad abdominal del ratón con tijeras anatómicas y cortar la aorta ventral a sangrar. Exponer la parte ventral del pulmón. </li><li> Exponer la tráquea y intubar con un catéter de calibre 20. Inculcar 0,8 ml de tampón de BALF (1x PBS, 0,1% de dextrosa, 10 U / ml de heparina) y recoger el lavado por aspiración suave. Repita este procedimiento tres veces. </li><li> Contar el número total de células en 100 l de suspensión de BALF usando un hemocytometros bajo un aumento de 10X con tinción de azul de tripano. </li><li> Preparar en otro portaobjetos para la tinción de Giemsa modificado. Alícuota de 100 l de BALF en los pocillos apropiados de la citospina y centrifugar a 500 g durante 5 min. Secar los portaobjetos durante 10 min y teñir las células con tinción de Giemsa modificado de acuerdo con los protocolos del fabricante. </li><li> Utilizar las células restantes para su posterior análisis, tales como células activadas por la clasificación de fluorescencia (FACS), microscopía confocal, la expresión génica y western blot 11-13. </li></ol> 4. Preparación Histopatología <ol><li> Para el análisis histopatológico, infectar a los ratones con NTHi como se describe en el paso 2.4. Después de 24 horas de la infección, sacrificar los animales por dislocación cervical. Cortar abrir la cavidad torácica con tijeras anatómicas para exponer los pulmones y la tráquea como se ha mencionado antes. A continuación, insertar el catéter de calibre 20 en la tráquea. </li><li> Llenar los pulmones con solución de agarosa al 2% caliente preparado en formalina al 4%. Una vez elpulmones están completamente inflados, lugar de hielo en la parte superior de los pulmones para solidificar la solución de agarosa. </li><li> A continuación, retire con cuidado el tapón torácica y lo puso en una solución de 50 ml de formalina al 4% en PBS 1x hasta que se procesa para (H &amp; E) secciones de hematoxilina y eosina. </li></ol> 5. FACS tinción de las células en el BALF <ol><li> Poner 1 x 10 5 células del líquido BALF en una placa de titulación de micro de fondo en V de 96 pocillos. </li><li> Lavar las células con 300 l de PBS frío 1x y centrifugado a 4.000 xg durante 3 min a 4 ° C. </li><li> Teñir las células con solución de tinción de células en 100 l de PBS 1x (1: 1000 dilución) y mantener a temperatura ambiente durante 15 min. </li><li> Lavar las células con 300 l de 1x PBS frío a 4000 xg durante 3 min a 4 ° C. </li><li> Mantener células en 100 l de tampón de FACS de lavado [tampón de lavado (1x PBS, 2% de FBS, 1 mM EDTA, 0,1% de azida sódica] que contiene 1: 100 diluido Fc-Block durante 15 min a 4 ° C. </li><li> centrifugar las células a 4.000 xg durante 3 min y deseche supernatant. </li><li> Las células se tiñen con 100 l de cóctel tinción de la superficie (dilución 1: 100) en tampón de lavado FACS durante 30 min a 4 ° C. </li><li> Lavar las células tres veces con 300 l de tampón de lavado FACS a 4.000 xg durante 3 min a 4 ° C. </li><li> Fijar las células en 100 l de 4% de paraformaldehído durante 15 min a temperatura ambiente. </li><li> Lavar las células dos veces con 300 l de 1x PBS a 400 xg durante 3 min a 4 ° C y volver a suspender en 300 l de tampón de lavado FACS. </li><li> Hacer controles de tinción de un solo color utilizando citometría de flujo perlas. <ol><li> Añadir una gota de perlas en la placa de microlitro de fondo en V de 96 pocillos y se lava con 200 l de 1x PBS. </li><li> Añadir 1 l de anticuerpo de tinción de 100 l de 1x PBS y los granos y se incuba a temperatura ambiente durante 5 min. </li><li> Lávese las perlas dos veces con 300 l de PBS 1x. </li></ol></li><li> Analizar los controles manchados de células individuales y por citometría de flujo 14. </li></ol> 6. La homogeneización del tejido pulmonar para aislar ARN <ol><li> Después de la infección, se recoge una pequeña pieza de pulmón (20-40 mg) y mantenerlo en la estabilización de ARN reactivo a 4 ° C durante la noche. A continuación, almacenarlo a -80 ° C hasta que el siguiente paso. </li><li> Lavar los tejidos del pulmón en frío PBS 1x para deshacerse del reactivo de estabilización de ARN. </li><li> Coloque el tejido en un tampón que contiene 1,5 ml de lisis tubo de centrífuga (1 ml de tampón de lisis + 10 l β-mercaptoetanol). </li><li> Picar el tejido en trozos pequeños con unas tijeras de punta y homogeneizar usando una mano de mortero de pellets de motor sin cuerda de 20-40 seg. </li><li> Mantenga el lisado en hielo durante 5 min y proceder con el paso 7.1. </li></ol> 7. Aislamiento de ARN del pulmón <ol><li> Se centrifuga el lisado a 18.000 xg durante 3 min. Eliminar el sobrenadante con la pipeta y transferir cuidadosamente a un nuevo tubo de 1,5 ml. </li><li> Añadir 1 volumen de etanol al 70% al lisado. Mezclar inmediatamente por pipeteo y esperar 2-4 minutos. </li><li> Transferir hasta 700 l de lisado a una columna giratoria colocadaen un tubo de recogida de 2 ml. </li><li> Se centrifuga a 9.600 g durante 15 seg y desechar el flujo a través. </li><li> Añadir 350 l tampón de lavado riguroso y se centrifuga a 9.600 g durante 15 seg. Desechar el flujo continuo. </li><li> Añadir 80 l de DNasa I directamente a la membrana de la columna de centrifugación y mantenerlo a temperatura ambiente durante 15 min. </li><li> Añadir 350 l de tampón de lavado astringente a la columna de centrifugación y centrifugar a 9600 xg durante 15 seg. Desechar el flujo continuo. </li><li> Añadir 500 l de tampón de lavado ARN y se centrifuga a 9.600 g durante 15 seg. Desechar el flujo continuo. </li><li> Añadir 500 l de tampón de ARN de lavado y centrifugado a 9.600 xg durante 2 min. Desechar el flujo continuo. </li><li> Colocar la columna de centrifugación en un nuevo tubo de recogida de 2 ml y se centrifuga a 9.600 g durante 1 min. Desechar el flujo continuo. </li><li> Añadir 30-50 l de agua libre de RNasa directamente a la columna de centrifugación y se centrifuga a 9.600 g durante 1 min. ARN almacenar a -80 ° C. </li><li> Realizar análisis RNAseq 14. </li></ol>

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The authors have nothing to disclose.

En este documento, se describe un procedimiento único y mínimamente invasivo para inocular los pulmones de ratones con un patógeno pulmonar bacteriana. Se demuestra que este procedimiento puede ser usado para estudiar la función de genes diferentes que utilizan ratones que son deficientes en genes de vías de señalización inflamatorias. Este procedimiento también se puede utilizar para estudiar las respuestas inflamatorias a infecciones pulmonares víricas y fúngicas. Las ventajas de este procedimiento con respe...

La inflamación es un mecanismo inmunológico fundamental en la defensa contra agentes infecciosos. Promueve la erradicación de patógenos y la reparación del tejido dañado. También facilita la recuperación a un estado normal y saludable 1. Sin embargo, la inflamación dysregulated a menudo conduce a enfermedades inflamatorias crónicas 2. Inflamación de las vías respiratorias es un disparador inicial para diferentes enfermedades pulmonares tales como enfermedad pulmonar obstructiva crónica (EPOC), asma y fibrosis pulmonar 3. El Haemophilus influenzae no tipificable (no encapsulada) (NTHi) se asocia con enfermedades inflamatorias superior e inferior pulmonares crónicas 4,5. Es la especie dominante aisladas de las vías respiratorias inferiores de los niños y adultos con 4,6,7 enfermedad pulmonar obstructiva crónica. La respuesta inflamatoria después de la infección NTHi se caracteriza por la regulación positiva de citoquinas proinflamatorias (tales como TNF e IL-1β), y está mediada bY por mitógenos proteína quinasa activada (MAPK) y el factor nuclear-kB (NF-kB) a través de los receptores tipo toll (TLR) 8. Los modelos de ratón son herramientas muy útiles para el análisis de la patología subyacente de la enfermedad inflamatoria pulmonar debido a la disponibilidad de las diferentes líneas de genes deficientes. Varios métodos han sido utilizados para inocular bacterias vivas / atenuadas y productos bacterianos, incluyendo la instilación intranasal y 9,10 inhalación en forma de aerosol. Aquí, demostramos la instilación intratraqueal. Aunque se utiliza con menos frecuencia, este enfoque es más eficiente y altamente reproducible debido a la entrega directa de inóculo en el tracto respiratorio 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

A continuación, describimos un procedimiento detallado de manera eficiente y entregar directamente Haemophilus influenzae en las vías respiratorias inferiores de los ratones. Demostramos el procedimiento para la preparación de H. inóculo influenzae, la instilación intratraqueal de H. influenzae en el pulmón, la recogida de fluido de lavado bronco-alveolar (BALF), el análisis de las células inmunes en el BALF, y el aislamiento de ARN para análisis de expresión génica diferencial. Este procedimiento puede ser usado para estudiar la respuesta inflamatoria pulmonar a cualquier bacteria, virus u hongos. instilación traqueal directa se prefiere sobre todo sobre los procedimientos intranasal o inhalación de aerosol, ya que ofrece más eficiente el inóculo bacteriano en el tracto respiratorio inferior con menos ambigüedad.

Instilación intratraqueal resultó en un marcado aumento del número de leucocitos en el BALF (Figura 1A, panel izquierdo) de la instalación con solución salina. El análisis recuento diferencial de los leucocitos mostró claramente el aumento de la infiltración de neutrófilos (Figura 1, panel derecho). El análisis FACS de las células en el BALF confirmó además el aumento del número de neutrófilos (Figura 1B). El análisis his...

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.

Administración intra-traqueal de<em&gt; Haemophilus influenzae</em&gt; En modelos de ratón para estudiar las vías respiratorias inflamación

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 vistas.  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. 

Video: 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.

No quirúrgico instilación intratraqueal de ratones con el análisis de los pulmones y ganglios linfáticos de drenaje del pulmón por citometría de flujo

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

Investigación

Inmunología e Infección

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.

No Invasiva Intratraqueal Intubación para Estudiar la Patología y Fisiología de pulmón de ratón

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.

La utilización de la orofaringe Intratraqueal PAMP Administración y lavado broncoalveolar para evaluar el anfitrión respuesta inmune en ratones

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.

Administración intranasal de vacunas contra la influenza recombinante en modelos de ratón quimérico para el Estudio de la mucosa Inmunidad

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.

El estudio de las comunidades microbianas<em&gt; En Vivo</em&gt;: Un Modelo de anfitrión mediada por la interacción entre<em&gt; Candida Albicans</em&gt; Y<em&gt; Pseudomonas Aeruginosa</em&gt; En las vías respiratorias

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.

La intubación mediada intratraqueal (IMIT) instilación: A, Sistema de Entrega de pulmón-específica no invasiva

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.

Un método no invasivo y técnicamente no intensivos para la inducción y Fenotipificación Experimental de neumonía bacteriana en ratones

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.

Un método mejorado para la rápida intubación de la tráquea en ratones

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.

Proyección de imagen de interacción de células en Mucosa traqueal durante la infección del Virus de la Influenza usando microscopia Intravital de dos fotones

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.

Evaluación de las respuestas del huésped-patógeno y eficacia de la vacuna en ratones

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

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