Name: a non-invasive and technically non-intensive method for induction and phenotyping of experimental bacterial pneumonia in mice
Uploaded: 2016-09-28T14:00:00
Description: Watch this Scientific Journal Video about A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice at JoVE.com

This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (Z01 ES102005).

Todos los experimentos se llevaron a cabo de conformidad con la Ley de Bienestar Animal y la Política de Servicio de Salud Pública de Estados Unidos sobre la Integridad Personal Cuidado y Uso de Animales de Laboratorio después de la revisión por el Cuidado de Animales y el empleo del NIEHS. 1. Preparación de K. pneumoniae Cultura Precaución: Realizar todos los pasos del 2 campana (BSL2) nivel de bioseguridad u otra área designada BSL2 y desechar los...

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Los modelos murinos de neumonía bacteriana, se asociaron con la orientación de genes y en las intervenciones biológicas y farmacológicas in vivo, han proporcionado una perspectiva crítica en la respuesta de defensa del huésped pulmonar. Grandes avances se han hecho en particular en nuestra comprensión de las quimiocinas y moléculas de adhesión que rigen el reclutamiento de neutrófilos al espacio aéreo infectada 10,11. En modelos in vivo de neumonía, a diferencia...

Neumonía adquirida en la comunidad sigue siendo la causa principal de muerte por infección en los EE.UU., con poco cambio en las tasas de mortalidad en los últimos 40 años a pesar de las mejoras en la vacunación y estrategias de antibióticos 1,2. A pesar de la falta de progresos importantes en el ámbito de la salud pública, en los últimos años dramáticos avances se han hecho en nuestra comprensión de la patogénesis molecular y celular de la neumonía, con muchos de estos avances hechos posibles por el uso de modelos de ratón de infección pulmonar. La trazabilidad genética del ratón, la similitud de la murina y el sistema inmunológico humano, y la gran variedad de reactivos inmunológicos murinos-dirigida que se han convertido en disponible en el mercado en su conjunto han facilitado el rápido progreso del campo. Los modelos de ratón de la neumonía bacteriana descrita en la literatura en general, se han basado en una de las cuatro rutas técnicas para la inoculación de patógenos: i) aerosolización; ii) intrAnasal entrega; iii) la entrega por vía oral; y iv) la inyección intratraqueal quirúrgico (es decir, traqueotomía) 3. Todas las vías de infección tienen ventajas y desventajas 3. En particular, la exposición, relativa de la vía aérea superior, el potencial para la mezcla de la flora oronasales, los requisitos para la anestesia general, la variabilidad del inóculo entregado al pulmón distal, distribución lobular de los patógenos entregados, los requisitos de pericia técnica, y la morbilidad de procedimiento varían ampliamente entre éstos enfoques. Comúnmente usadas técnicas de infección perorales incluyen endotraqueal canulación (translaríngea), ya sea a través de un enfoque &quot;a ciegas&quot; (no visualizado), o bajo visualización directa de la laringe 3-5. Ambos métodos, mientras robusto, requieren una formación sustancial y también conllevan un riesgo de traumatismo en la vía aérea superior. En el presente informe se describe un método técnicamente no intensiva, no invasiva, barata y rápida de la infección por vía oral, wdeclara bacterias (Klebsiella pneumoniae en el ejemplo proporcionado) pipetearon en la orofaringe de un ratón anestesiado se entregan a los pulmones a través de aspiración (es decir, la inhalación). Nosotros y otros han utilizado la técnica de la neumonía por aspiración con éxito 6-9. Este método de pulmón de entrega versátil y fácil de aprender puede extenderse a la entrega de muchos agentes no cáusticos adicionales a los pulmones, incluyendo citoquinas y otras proteínas, moléculas asociados a patógenos (por ejemplo, lipopolisacáridos), células (es decir, la transferencia adoptiva), y toxinas (por ejemplo, bleomicina). Además de discutir las consideraciones técnicas importantes, como la descripción de un enfoque integrado, cuantitativo para evaluar la respuesta del huésped después de la neumonía, incluyendo la medición aguas abajo de la eliminación de bacterias (es decir, unidades formadoras de colonias [UFC] cuantificación de los órganos pulmonares y periféricos) y leucocitos acumulación en el espacio aéreo.

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Klebsiella pneumoniae, serotype 2</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:119px;">43816</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tryptic soy broth</td>
			<td style="width:169px;">Becton Dickenson</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel Safelet IV Catheters, 18G x 1 1/4&#34;</td>
			<td>Claflin Medical Equipment</td>
			<td style="width:119px;">MEDC-031122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Solution 1</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Solution 2</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-952</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Fixative</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-929</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">27&#189; gauge tuberculin syringes</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">14-826-87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lithium heparin plasma collectors</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">2675187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">L-shaped disposable spreaders</td>
			<td style="width:169px;">Lab Scientific</td>
			<td style="width:119px;">DSC</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:243px;">1x PBS, pH 7.4</td>
			<td style="width:169px;">prepared in-house</td>
			<td style="width:119px;">n/a</td>
			<td>Distilled water (5 L), NaCl (40 g), KCl (1 g), Na2HPO4 (5.75 g), KH2PO4 (1 g)&#160;&#160;&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:243px;">ACK lysis buffer</td>
			<td style="width:169px;">prepared in-house</td>
			<td style="width:119px;">n/a</td>
			<td>NH4Cl (4.145 g), KHCO3 (0.5 g), EDTA (18.6 mg), bring up to 500 ml with distilled water and pH to 7.4</td>
		</tr>
	</tbody>
</table>

a non-invasive and technically non-intensive method for induction and phenotyping of experimental bacterial pneumonia in mice

Aunque la neumonía adquirida en la comunidad sigue siendo un problema importante de salud pública, los modelos murinos de neumonía bacteriana han facilitado recientemente importantes avances preclínicos en nuestra comprensión de la patogénesis molecular y celular subyacente. En vivo modelos de ratón capturan la fisiología integrada y capacidad de recuperación de la respuesta de defensa del huésped en de una manera no reveló por enfoques alternativos, simplificados ex vivo. Varios métodos han sido descritos en la literatura para la inoculación intrapulmonar de bacterias en ratones, incluyendo la aerosolización, la administración intranasal, la canulación endotraqueal peroral en condiciones &quot;ciego&quot; y visualizados, y la canulación endotraqueal transcutánea. Todos los métodos tienen ventajas relativas y limitaciones. En este documento, se describe en detalle un método no invasivo, técnicamente no intensiva, barato y rápido para la entrega intratraqueal de bacterias que consiste en la aspiración (es decir, inhalación) por el ratónde un inóculo infeccioso pipeteó en la orofaringe, mientras que bajo anestesia general. Este método se puede utilizar para la administración pulmonar de una amplia variedad de agentes biológicos y químicos no cáusticos, y es relativamente fácil de aprender, incluso para los laboratorios con experiencia previa mínima con procedimientos pulmonares. Además de describir el método de la neumonía por aspiración, también ofrecemos procedimientos paso a paso para ensayar la posterior in vivo la respuesta inmune innata pulmonar del ratón, en particular, los métodos para cuantificar la eliminación de bacterias y la respuesta inmune celular de las vías respiratorias infectadas. Este enfoque integrado y sencillo de evaluación neumonía permite la evaluación rápida y robusta de los efectos de las manipulaciones genéticas y ambientales en la inmunidad innata pulmonar.

C57BL / 6 ratones fueron infectados con 2000 UFC de K. pneumoniae 43816 (serotipo 2) a través de la aspiración orofaríngea en los pulmones. A esta dosis, los ratones normalmente comienzan a mostrar síntomas clínicos 12-24 horas después de la infección, incluyendo letargo, pelo erizado, y la pérdida de peso del 5-10% (Figura 2). Dentro de 48-72 horas después de la infección, muchos de los ratones muestran síntomas de la enfermedad y la morbilidad que p...

Watch this Scientific Journal Video about A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice at JoVE.com

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

Varios métodos han sido descritos en la literatura para el modelado de la neumonía bacteriana en ratones. En este documento, se describe un método no invasivo, de bajo costo, rápida para inducir neumonía a través de aspiración (es decir, inhalación) de un inóculo bacteriano pipeteó en la orofaringe. métodos aguas abajo para la evaluación de la respuesta inmune innata pulmonar también se detallan.

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 ...

The overall goal of this experimental protocol is to provide a non-invasive and technically non-intensive procedure for inducing bacterial pneumonia in mice and the subsequent quantification of the in vivo host defense response in the lungs. This method can help answer key questions in the fields of infectious disease and immunology, about the genes and molecular pathways that control the host response to infection. The main advantage of this lung infection method is its technical simplicity.Which allows even laboratories with little expertise in pulmonary procedures to study the pulmonary innate immune response. In a biosafety level two facility, thaw a glycerol stock of K.pneumoniae and transfer one milliliter of the bacteria into a 500 milliliter culture flask containing 50 milliliters of Tryptic Soy Broth. Incubate the suspension culture overnight at 37 degrees Celsius and 200 to 225 RPM.The next morning, dilute one to five milliliters of the bacterial culture in a new flask containing 50 milliliters of Tryptic Soy Broth, and incubate the culture, in the shaker, at 37 degrees Celsius for an additional 2.5 hours to achieve log phase. At the end of the second incubation, transfer one milliliter of bacteria from the second culture to a 1.5 milliliter tube for centrifugation. Remove the supernatant without disturbing the palette and gently resuspend the bacteria in one milliliter of sterile saline for three separate washes.After the third wash, resuspend the bacteria in another milliliter of sterile saline, and set up log-wise serial dilutions of the inoculum. Measure one milliliter of the one to 10 and one to 100 dilutions in disposable cuvettes, in duplicate, to determine the OD600 of the washed K.pneumoniae. An OD600 of 1.0 is roughly equivalent to four to seven times 10 to the eighth CFUs per milliliter.After calculating the concentration of the bacteria in each dilution, resuspend the desired inoculum CFU dose in 50 to 60 microliters of sterile saline per eight to 12 week old recipient animal. To deliver the bacteria to the animals, draw up the dosing inoculum into a P200 pipette tip, and place the anesthetized mouse in a semi-recumbent supine position, suspended by the maxillary incisors, from a rubber band stretched between the pegs of a slanted Plexiglas board. Using a pair of blunt, non-ridged forceps, gently pull the tongue to the side and deposit the dose into the animal's oral cavity taking care to avoid inducing trauma to the tongue or the oral pharynx.Keeping the tongue retracted, gently occlude the nose with a gloved finger, until the house inhales two to three times. When no liquid is visible in the oral cavity, transfer the mouse from the inoculation board to its cage, in the supine position, to prevent blockage of the nares while the mouse is recovering. Make a longitudinal cut just below the sternum.Then, lifting the sternum with forceps, nick the diaphragm, allowing the lung to fall back into the chest cavity. Continue the incision up through the chest cavity on either side of the vasculature and up through the neck. When the trachea is visible, carefully cut through the middle of the neck and push the tissue to the sides to avoid damaging the surrounding vasculiture.Now nick the trachea about one quarter of the way down from the head, and caudally insert a cannula attached to a one milliliter syringe pre-loaded with one milliliter of PBS into the trachea. Push the volume into the lungs, slowly, allowing all of the lobes to inflate. Then pull the volume back out with the syringe and pool the collected washes in a 15 milliliter tube, on ice.At the appropriate experimental end point, collect the blood from the left ventricle and transfer it into a heparinized tube to avoid clotting. Then harvest all of the lung lobes in a 15 milliliter tube containing five milliliters of PBS, and the spleen in a 15 milliliter tube containing two milliliters of PBS. Next, use individual, disposable homogenizers per sample, to macerate the tissues on ice, followed by serial dilution of the tissue slurries.Plate the dilutions, in duplicate, on separate sides of a single TSA plate, and allow the samples to, briefly, dry. Then invert the plates and culture the samples in a static 37 degrees Celsius incubator overnight, averaging the bacterial colonies from both sides of the plate and from each dilution the next morning. At 2000 CFU of K.pneumoniae, mice, typically, begin to demonstrate clinical symptoms 12 to 24 hours post infection.Within 48 to 72 hours, many of the animals exhibit symptoms of illness and morbidity that are typically preceded by an average of 20 percent weight loss. An LD50 dose, however, allows the detection of both the relatively increased and decreased survival within the experimental group, and may be preferred for longer term studies. Lower respiratory tract infection with K.pneumoniae is characterized by a robust influx of leukocytes into the airway within six hours of infection, that peaks by 24 hours and is mostly represented by neutrophils.Cytokines are detectable in the bronchoalveolar lavage fluid at both 24 and 48 hours post infection with K.pneumoniae, and provide insight into the lung microenvironment and its immune self-recruitment in response to bacterial invasion. The bacterial load in the lung, blood, and spleen, also dramatically increases between 24 and 48 hours of infection. Once mastered, the oropharyngeal aspiration method of lung infection can be done in just one to two minutes per mouse if it is performed properly.While attempting this procedure, it is important to remember not to use an excessive aspiration volume. A 50 to 60 microliter volume typically works well for an eight to 12 week old mouse. Following this procedure, a variety of methods can be performed on the lung and peripheral tissues to allow the quantification of the immune response in pathogen clearance.After watching this video you should have a good understanding of how to induce experimental bacterial pneumonia via oropharyngeal pathogen delivery and to determine the pathogen burden in mice. Don't forget that working with microbes can be hazardous and that basic BSL-2 precautions and practices should always be taken while preforming this procedure.

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

De imagen no invasivas de Homing leucocitos y Migración In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Investigación

Inmunología e Infección

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Preparación de inmunógeno ratón hipófisis para la inducción de la hipofisitis autoinmune experimental

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

La inducción de la hipofisitis autoinmune experimental en ratones SJL

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Using Luciferase to Image Bacterial Infections in Mice

Luciferasa utilizando a las infecciones bacterianas de la imagen en ratones

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

A Method for Labeling Vasculature in Embryonic Mice

Un método para etiquetar los Vasos sanguíneos en embriones de ratones

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

La imagen no invasiva de la candidiasis diseminada en larvas de pez cebra

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

No invasiva imágenes ópticas de la vasculatura linfática de un ratón

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Modelo no invasiva de neuropatogénico<em&gt; Escherichia coli</em&gt; La infección en Neonatología Rata

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

La inducción de la inflamación intestinal murino por transferencia adoptiva de Efector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; T células en ratones inmunodeficientes

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Imagen no invasiva de la respuesta inmune innata en un modelo de larvas de pez cebra de<em&gt; Streptococcus iniae</em&gt; Infección

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...