Name: intratracheal inoculation of fischer 344 rats with francisella tularensis
Uploaded: 2017-09-30T14:00:00.000+00:00
Duration: 6 min 9 s
Description: Watch this Scientific Journal Video about Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis at JoVE.com

Este estudio fue apoyado por la Agencia de reducción de amenaza de defensa (DTRA) bajo contrato HDTRA1-14-C-0116 y el centro de excelencia en genómica de la infección (DOD #W911NF-11-1-0136).

este trabajo se realizó en estricta conformidad con las recomendaciones de la guía para el cuidado y uso de animales de laboratorio de los institutos nacionales de salud. Protocolos animales con roedores fueron aprobados por la Universidad de Texas en San Antonio institucional Animal Care y el Comité uso (IACUC) bajo protocolo MU009(RA).
 1. inóculo de F. tularensis, preparar catéter y tráquea indicador <ol><li>Preparar catéter (20 x 2) <ol><li>corta el 20 x 2 en aguja y la rutina de la punta de la aguja a un blunt y suave acabado con una herramienta giratoria. 
		Nota: La longitud de la aguja blunted debe permitir que la punta metálica rescindir aproximadamente 3 mm en el manguito del catéter cuando se asiente completamente sobre la aguja blunted. La aguja blunted da la rigidez estructural del catéter para permitir la colocación del catéter en la tráquea, y la ausencia de la punta de la aguja blunted que sobresalen del manguito del catéter previene lesiones a la tráquea.</li>
		<li>Limpiar las agujas romas y catéteres con etanol al 70% y esterilizar bajo luz UV de 15 min.</li>
	</ol></li> <li>Preparar tráquea indicador <ol><li>cortar una punta de pipeta de 1000 μL para permitir que la punta para sentarse en una abertura del catéter y formar un sello hermético.</li>
		<li>Quitar un filtro de un lugar dentro de la punta de pipeta de 1000 μl previamente recortada y pipeta μl aerosol barrera 200.</li>
		<li>Asegurar el filtro puede moverse libremente dentro de la punta de la pipeta de 1000 μl cuando el extremo es puntiagudo punta abajo.</li>
	</ol></li> <li>Inóculo de F. tularensis <ol><li>cepa crece F. tularensis durante la noche en placa de agar adecuado a 37 ° C. rascado aproximadamente 100 μl del césped bacteriano de la placa de agar con un estéril inoculación del lazo y utilizar para inocular un 500 mL matraz Erlenmeyer que contenga 250 mL del medio de crecimiento líquido apropiado (para Fn: caldo de soja tríptica (TSB) suplementado con 0,1% L-Cisteína clorhidrato monohidrato) e incubar en agitación toda la noche a 37 ° C. 
		Nota: F. novicida utilizado en el video se cultivaba en una placa de agar (TSA) de soya peptona suplementada con 0,1% L-Cisteína clorhidrato monohidrato.</li>
		<li>Centrifugar el cultivo líquido crecido durante la noche a 4.221 x g a temperatura ambiente durante 10 min y retirar el sobrenadante sin perturbar el pellet de bacterias. 
		PRECAUCIÓN: Por favor, siga la institución &#39; las recomendaciones de bioseguridad s descartar sobrenadante.</li>
		<li>Suavemente Resuspenda el precipitado bacteriano con 25 mL de medio líquido apropiado (Fn: TSB complementado con 0.1% L-Cisteína clorhidrato monohidrato) conteniendo glicerol al 10%, utilizando una pipeta de 25 mL.</li>
		<li>Con una pipeta de 1 mL, alícuota del cultivo bacteriano resuspendido en alícuotas de μl 500 en viales, congelar en un baño de hielo seco/etanol y almacenar a -80 ° C.</li>
		<li>Para determinar la concentración de los frascos de cultivo congelado, sacar dos viales congelados almacenados a-80 ° C, descongelar el hielo y realizar diluciones seriadas en tampón fosfato salino (PBS) seguido de placas en el medio de crecimiento apropiado (Fn: TSA que contiene 0.1% L-cisteína clorhidrato de). Las placas son incubada 24-48 h a 37 ° C, y unidades formadoras de colonias (UFC) se enumeran para calcular el número de células bacterianas dentro de los frascos de cultivo congelado (promedio de dos viales).</li>
		<li>Preparar el inóculo bacteriano por descongelar un vial congelado en el hielo y luego diluir la cultura con PBS a la concentración final de 10 7 CFU/100 μl. El inóculo debe estar en una concentración que producirá la deseada UFC en 100 μl (concentración final de glicerol en el inóculo no puede ser más del 3%, para evitar la asfixia de la rata). Frascos de cultivo congelado almacenados-80 ° C se pueden utilizar hasta seis meses después de la preparación.</li>
	</ol></li></ol> 2. Anestesia de la rata <ol><li>Conecte la cámara de anestesia para el vaporizador de isoflurano funcionamiento de una máquina de anestesia.</li>
	<li>Conectar el gas de barrido del tubo en la cámara de anestesia al gas de barrido sistema.</li>
	<li>Abrir el flujo de oxígeno en la máquina de anestesia a 4 L/min</li>
	<li>Ajustar el vaporizador de isoflurano al 5%.</li>
	<li>Colocar la rata en la cámara de anestesia. 
	Nota: Normalmente utilizamos ratas entre 8-10 semanas de edad (130-180 g); las ratas jóvenes son difíciles de inocular a través de esta técnica debido al pequeño tamaño de la cavidad de la boca.</li>
	<li>Cuando la inducción de la anestesia ha producido, mantener la rata en el 5% isoflurano, 2 L/min de oxígeno al efecto. Esto toma unos 3-10 minutos</li>
	<li>Determinar la profundidad de la anestesia por la calidad y la tasa de respiración y latidos del corazón y la reacción a la estimulación refleja como en la prueba del pellizco del dedo del pie. Profundidad ideal de la anestesia está indicada por la cuenta de 1-1.5 s entre cada respiración.</li>
</ol> 3. La inoculación intratraqueal <ol><li>Quite la rata de la cámara de anestesia y colocar la rata dorsal en el stand de roedores de la intubación. Coloque los dientes de rata al titular para mantener la rata en su lugar. 
	Nota: Si la rata empieza a despertar durante el procedimiento, el catéter puede ser quitado y la rata volvió a la cámara de anestesia para llegar a un plano más profundo de anestesia.</li>
	<li>Utilizar la mano dominante para mover la lengua al mismo lado que la mano de apoyo con fórceps pulgar amplio punto.</li>
	<li>Uso mano y fijar la lengua con el laringoscopio contra la rata &#39; s más bajo quijada y visualizar la tráquea y el esófago del animal. La tráquea se abren y cierran como la rata respira.</li>
	<li>Insertar el catéter con una calibre 20 blunted aguja, preparado en el paso 1.1, en la tráquea. Puede haber ligera resistencia, y la inserción en la tráquea puede ser &#34; lleno de baches &#34; debido al catéter frotando los cartílagos traqueales. Debido a humedad o respiración poco profunda, la tráquea puede ser cubierta por la epiglotis, que impide la visualización de la tráquea. Tocar suavemente el borde del cartílago epiglotis hará que la aleta de cartílago abrir y descubrir la tráquea.</li>
	<li>Quitar la aguja blunted el catéter garantizando el catéter permanece en la tráquea.</li>
	<li>Unos segundos pasar de rata poder respirar por catéter insertado en la tráquea.</li>
	<li>Firmemente Coloque el indicador de la tráquea en el catéter de apertura. Movimiento de la barrera aerosol indicará que el catéter se inserta correctamente en la tráquea.</li>
	<li>Asegurar que la rata está sentando en el stand de intubación que el pecho del animal quede perpendicular al plano del stand intubación.</li>
	<li>Quitar el indicador de tráquea y entregar 100 μl (10 7 UFC) del inóculo con F. tularensis utilizando una pipeta de 200 μL, estar firmemente la punta contra la abertura del catéter.</li>
	<li>Acople una jeringa de tuberculina de punta slip 1 mL llenada de aire y entregar 300 μL de aire para inóculo llegue a los pulmones de la rata.</li>
	<li>Retire el catéter de la tráquea y quitar la rata de la plataforma quirúrgica.</li>
	<li>Permite la rata despertar y volver a la jaula. Asegúrese de que la respiración ha vuelto a la normalidad.</li>
	<li>Determinar la UFC en la F. tularensis entrega de inóculo como se describe en el paso 1.3.5 para confirmar la UFC en la tráquea.</li>
	<li>Repita los pasos 3.1-3.9 en animales no vacunados (ingenuo) con 100 μl PBS en lugar del inóculo.</li>
</ol>

<ol>
	<li>Ellis, J., Oyston, P. C. F., Green, M., Titball, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tularemia.">Tularemia.</a> Clin Microbiol Rev. 15 (4), 631-646 (2002).</li><li>Lyons, C. R., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Francisella+tularensis+infection.">Animal models of Francisella tularensis infection.</a> Ann N Y Acad Sci. 1105, (2007).</li><li>Hutt, J. A., Lovchik, J. A., Dekonenko, A., Hahn, A. C., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Natural+History+of+Pneumonic+Tularemia+in+Female+Fischer+344+Rats+after+Inhalational+Exposure+to+Aerosolized+Francisella+tularensis+subspecies+tularensis+Strain+Schu+S4.">The Natural History of Pneumonic Tularemia in Female Fischer 344 Rats after Inhalational Exposure to Aerosolized Francisella tularensis subspecies tularensis Strain Schu S4.</a> Am J Pathol. 187 (2), 252-267 (2017).</li><li>Ray, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fischer+344+rat+reflects+human+susceptibility+to+Francisella+pulmonary+challenge+and+provides+a+new+platform+for+virulence+and+protection+studies.">The Fischer 344 rat reflects human susceptibility to Francisella pulmonary challenge and provides a new platform for virulence and protection studies.</a> PloS one. 5, e9952 (2010).</li><li>Jemski, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+tularemia:+comparison+of+selected+routes+of+vaccination+in+Fischer+344+rats.">Respiratory tularemia: comparison of selected routes of vaccination in Fischer 344 rats.</a> Infect Immun. 34 (3), 766-772 (1981).</li><li>Wu, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+of+Fischer+344+rats+against+pulmonary+infections+by+Francisella+tularensis+type+A+strains.">Vaccination of Fischer 344 rats against pulmonary infections by Francisella tularensis type A strains.</a> Vaccine. 27 (34), 4684-4693 (2009).</li><li>Mara-Koosham, G., Hutt, J. A., Lyons, C. R., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+contribute+to+effective+vaccination+against+respiratory+infection+by+type+A+Francisella+tularensis+strains.">Antibodies contribute to effective vaccination against respiratory infection by type A Francisella tularensis strains.</a> Infect Immun. 79 (4), 1770-1778 (2011).</li><li>Oyston, P. C., Sjostedt, A., Titball, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tularaemia:+bioterrorism+defence+renews+interest+in+Francisella+tularensis.">Tularaemia: bioterrorism defence renews interest in Francisella tularensis.</a> Nat Rev Microbiol. 2 (12), 967-978 (2004).</li><li>Kingry, L. C., Petersen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+review+of+Francisella+tularensis+and+Francisella+novicida.">Comparative review of Francisella tularensis and Francisella novicida.</a> Front Cell Infect Microbiol. 4, 35 (2014).</li><li>Rohmer, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Francisella+tularensis+genomes+reveals+evolutionary+events+associated+with+the+emergence+of+human+pathogenic+strains.">Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains.</a> Genome Biol. 8 (6), R102 (2007).</li><li>Chu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+attenuated+Francisella+novicida+vaccine+protects+against+Francisella+tularensis+pulmonary+challenge+in+rats+and+non-human+primates.">Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates.</a> PLoS Pathog. 10 (10), e1004439 (2014).</li><li>Signarovitz, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+Immunization+with+Live+Attenuated+Francisella+novicida+U112&#916;iglB+Protects+against+Pulmonary+F.+tularensis+SCHU+S4+in+the+Fischer+344+Rat+Model.">Mucosal Immunization with Live Attenuated Francisella novicida U112&#916;iglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model.</a> PloS one. 7 (10), e47639 (2012).</li><li>Cunningham, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+vaccine+efficacy+by+expression+of+a+TLR5+ligand+in+the+defined+live+attenuated+Francisella+tularensis+subsp.+novicida+strain+U112DiglB::fljB.">Enhancement of vaccine efficacy by expression of a TLR5 ligand in the defined live attenuated Francisella tularensis subsp. novicida strain U112DiglB::fljB.</a> Vaccine. 32 (40), 5234-5240 (2014).</li></ol>

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Las ratas Fischer 344 se está convirtiendo en un importante modelo para tularemia vacuna desarrollo3. Exposición a F. tularensis por vía pulmonar es fundamental para demostrar la eficacia contra las formas arma de F. tularensis, porque éstos son entregados como aerosoles. La inoculación intratraqueal de la rata facilita la exposición de los pulmones de ratas para F. tularensis sin necesidad de aerosoles grandes, costosos y complicados equipos generadores. Todos los...

F. tularensis (Ft) causa la enfermedad humana, tularemia. Cuando las bacterias se adquieren por vía pulmonar, esto conduce a la tularemia neumónica, que tiene alta morbilidad y mortalidad1. F. tularensis se considera a un agente biothreat debido al peligro asociado con las formas aerosol y no existe ninguna vacuna aprobada para uso humano en los Estados Unidos Actualmente está en marcha un esfuerzo intensivo desarrollar vacunas y medidas terapéuticas contra la tularemia neumónica, para proteger a la población humana contra el uso ilícito de este biothreat bacteriana.
Gran parte de la investigación de tularemia se ha centrado en el modelo de ratón, debido a la extrema sensibilidad de los ratones a la infección por F. tularensis y la prevalencia de los reactivos. Sin embargo, ratones han demostrado para ser un modelo difícil para el desarrollo de una vacuna, debido a la dificultad de demostrar la eficacia de la vacuna en este modelo2. Recientemente, las ratas Fischer 344 se ha desarrollado como un modelo para desarrollo de vacuna contra la tularemia3. La sensibilidad de la ratas Fischer 344 a varias subespecies de F. tularensis imita sensibilidad humana4y ratas pueden ser protegidas contra F. tularensis desafío pulmonar por la vacunación con una cepa de vacuna de virus vivos conocida para proteger los seres humanos5,6,7. Porque las ratas Fischer 344 modelos algunas de las características de la infección de F. tularensis de seres humanos, puede ser un modelo muy útil para el desarrollo de una vacuna que protege contra pulmonar F. tularensis exposición.
Una vacuna eficaz debe proteger a los seres humanos contra la exposición pulmonar F. tularensis. La exposición pulmonar probablemente de arma de F. tularensis sería bacterias aerosol inhaladas en los pulmones8. Sin embargo, la generación de aerosol de F. tularensis es peligroso y engorroso y requiere contención y equipo especializado. Una ruta alternativa de la exposición pulmonar en la rata que es tal vez más adaptable para varios laboratorios falta de equipo especializado es a través de inoculación intratraqueal6. Esta técnica utiliza un laringoscopio para la correcta colocación de un catéter dentro de la tráquea de una rata anestesiado. Colocación dentro de la tráquea, en lugar de esófago, es verificada por un dispositivo simple que visualiza el flujo de aire de los pulmones. F. tularensis se entrega posteriormente a los pulmones a través del catéter por la administración con una jeringa, seguida por la introducción de aire en el catéter para asegurar la entrega pulmonar de las bacterias. Por el contrario, lo que indica la inoculación intranasal en ratas hace Jemski5 divulgado previamente que inoculados en ratas Fischer 344 la vía intranasal de F. tularensis no podría cultivarse desde los pulmones hasta la inoculación después de 3 días, no resultado en la entrega directa de las bacterias en los pulmones.
Formas de seleccionar agente de F. tularensis (subsp. deF. tularensis tularensis, F. tularensis subsp. holarctica) requieren procedimientos de contención de bioseguridad nivel 3 (BSL3), que impedirían la videografía. Sin embargo, F. novicida (Fn) está exento de estado seleccione agente debido a su avirulencia en seres humanos sanos y puede ser utilizado con seguridad bajo condiciones de bioseguridad nivel 2 (BSL2)9,10. Por otra parte, Fn sirve como base para vacunas vivas atenuadas que puede proteger contra F. tularensis exposición pulmonar cuando entregado vía intratraqueal inoculación11,12,13. La técnica presentada aquí permite el estudio de las infecciones que ocurren a través de la vía pulmonar utilizando ratas como un modelo para los seres humanos. Esta técnica se puede realizar sin necesidad de equipos especializados de generación de aerosoles. FN fue utilizado para las técnicas filmadas aquí.

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intratracheal inoculation of fischer 344 rats with francisella tularensis

Infección pulmonar con la bacteria Francisella tularensis puede conducir a la enfermedad grave y potencialmente mortal, la tularemia, en seres humanos. Debido a la falta de una vacuna contra la tularemia aprobado para los seres humanos, la investigación se centra en el desarrollo de una vacuna utilizando modelos animales apropiados. Las ratas Fischer 344 ha emergido como un modelo que refleja la susceptibilidad humana a la infección de F. tularensis y por lo tanto es un modelo atractivo para el desarrollo de vacuna contra la tularemia. La inoculación intratraqueal de las ratas Fischer 344 con F. tularensis imita la exposición pulmonar en seres humanos. La entrega exitosa en la tráquea de rata es crítica para la entrega pulmonar. Laringoscopio con iluminación se utiliza para intubar correctamente las tráqueas de las ratas anestesiadas; la colocación correcta dentro de la tráquea está determinada por un simple dispositivo para detectar la respiración. Después de la intubación, la cultura de F. tularensis se entrega en una dosis medida mediante jeringa. Esta técnica normaliza entrega pulmonar de F. tularensis dentro de la tráquea de rata para evaluar la eficacia de la vacuna.

La respuesta humoral a la inoculación intratraqueal de F. tularensis en la rata puede determinarse por análisis enzima-ligado del inmunosorbente (ELISA) contra las bacterias inactivadas por UV, como se describió anteriormente11. Respuesta total de la inmunoglobulina G (IgG) de las ratas Fischer 344 a células enteras inactivadas de bacterias fue evaluado post-intratraqueal inoculación con una cepa atenuada de Fn (inóculo de UFC7 10) en el d...

Watch this Scientific Journal Video about Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis at JoVE.com

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Este protocolo describe intratraqueal inoculaciones de ratas Fischer 344 con tularensis de francisella. Este procedimiento simula la exposición pulmonar de los seres humanos a este agente potencial de biothreat y puede utilizarse para probar la vacuna y la eficacia terapéutica contra la tularemia pulmonar.

La inoculación de Fischer 344 ratas con Francisella tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the ...

intratracheal-inoculation-fischer-344-rats-with-francisella

Research

JoVE Journal

Immunology and Infection

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

7.1K vistas.  University of Texas San Antonio. Este protocolo describe intratraqueal inoculaciones de ratas Fischer 344 con tularensis de francisella. Este procedimiento simula la exposición pulmonar de los seres humanos a este agente potencial de biothreat y puede utilizarse para probar la vacuna y la eficacia terapéutica contra la tularemia pulmonar.

Video: Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

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

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

La inmunoterapia sublingual como alternativa para inducir protección contra las infecciones respiratorias agudas

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

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 ...

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

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

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 ...

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

Método para el aislamiento de Francisella tularensis Membranas externas

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

Biología

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

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

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

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

Un modelo de neumonía en inmunocompetentes robusta roedores para evaluar Eficacia antibacteriana contra<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; o<em&gt; A. baumannii</em

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

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. ...

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.

Inducir lesión pulmonar aguda en ratones por instilación directa de lipopolisacárido intratraal

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

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

Modelo experimental para evaluar la resolución de la neumonía

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

La inoculación de Fischer 344 ratas con Francisella tularensis

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