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).

Toutes les expériences ont été effectuées en conformité avec la Loi sur la protection des animaux et de la politique US Public Health Service sur Humane soin et l&#39;utilisation des animaux de laboratoire après examen par le soin des animaux et l&#39;utilisation du Comité des NIEHS. 1. Préparation de K. pneumoniae Culture Attention: Effectuer toutes les étapes d&#39;un niveau de biosécurité 2 (NSB2) hotte ou autre zone désignée de NSB2 et jet...

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Les modèles murins de la pneumonie bactérienne, en partenariat avec le ciblage de gènes et dans les interventions biologiques et pharmacologiques in vivo, ont fourni des informations critiques dans la réponse de la défense de l&#39; hôte pulmonaire. De grands progrès ont été accomplis en particulier dans notre compréhension des chimiokines et des molécules d&#39;adhésion qui régissent le recrutement des neutrophiles à l&#39;espace aérien infecté 10,11. Modèles in vivo</e...

Pneumonie communautaire demeure la principale cause de décès par infection aux Etats - Unis, avec peu de changements dans les taux de mortalité au cours des 40 dernières années , en dépit des améliorations en matière de vaccination et des stratégies antibiotiques 1,2. Malgré l&#39;absence de progrès sensibles au niveau de la santé publique, au cours des dernières années, des progrès considérables ont été réalisés dans notre compréhension de la pathogenèse moléculaire et cellulaire de la pneumonie, avec un grand nombre de ces avancées rendues possibles par l&#39;utilisation de modèles de souris de l&#39;infection pulmonaire. La traçabilité génétique de la souris, la similitude du murin et le système immunitaire humain et la vaste gamme de réactifs immunologiques murins ciblés qui sont devenus disponibles dans le commerce ont ainsi facilité des progrès rapides du champ. Des modèles de souris de la pneumonie bactérienne décrite dans la littérature ont généralement compté sur l&#39;un des quatre itinéraires techniques pour pathogène inoculation: i) aérosolisation; ii) intrlivraison anasal; iii) la livraison par voie orale; et iv) l&#39; injection intra - trachéale chirurgicale (ie, trachéotomie) 3. Toutes les voies d&#39;infection ont des avantages et des inconvénients 3. En particulier l&#39;exposition, relative des voies respiratoires supérieures, le potentiel pour le mélange de la flore oronasaux, les exigences pour l&#39;anesthésie générale, la variabilité de l&#39;inoculum livré au poumon distal, la distribution lobaire des agents pathogènes livrés, les exigences d&#39;expertise technique, et de la morbidité procédurale varient considérablement entre ces approches. Communément techniques utilisées d&#39;infection pérorales comprennent endotrachéale (translaryngée) canulation soit par un (non visualisées) approche «aveugle», ou sous visualisation directe laryngé 3-5. Les deux méthodes, tandis que robuste, nécessitent une formation et portent aussi le risque de traumatisme des voies respiratoires supérieures. Dans le présent rapport, nous décrivons une méthode techniquement non intensive, non-invasive, peu coûteuse et rapide de l&#39;infection par voie orale, wdit bactéries (Klebsiella pneumoniae dans l&#39;exemple fourni) pipetés dans l&#39;oropharynx d&#39;une souris anesthésiée sont livrés aux poumons par aspiration (inhalation). Nous et d&#39; autres ont utilisé la technique de la pneumonie par aspiration avec succès 6-9. Cette méthode poumon-livraison polyvalent et facile à apprendre peut être étendue à la prestation de nombreux agents non caustiques supplémentaires pour les poumons, y compris les cytokines et d&#39; autres protéines, des molécules d&#39;agents pathogènes associés (par exemple, lipopolysaccharide), les cellules ( par exemple, le transfert adoptif), et les toxines (par exemple, la bléomycine). En plus de discuter des considérations techniques importantes, nous décrivons également une approche intégrée, quantitative pour évaluer la réponse de l&#39; hôte à la suite de la pneumonie, y compris la mesure en aval de la clairance bactérienne (c. -à- unité formant colonie [CFU] quantification dans les poumons et périphériques organes) et leucocytaire l&#39;accumulation dans l&#39;espace aérien.

<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

Bien que la pneumonie communautaire reste un problème majeur de santé publique, des modèles murins de la pneumonie bactérienne ont récemment facilité les avancées précliniques importantes dans notre compréhension de la pathogenèse cellulaire et moléculaire sous - jacent. Modèles in vivo de souris capturent la physiologie intégrée et de la résilience de la réponse de défense de l&#39; hôte en d&#39; une manière non révélée par, ex vivo approches simplifiées alternatives. Plusieurs procédés ont été décrits dans la littérature pour l&#39;inoculation des bactéries intra-pulmonaire chez la souris, y compris la pulvérisation en aérosol, l&#39;administration intranasale, par voie buccale endotrachéale canulation dans des conditions «aveugle» et visualisé et transcutanée endotrachéale canulation. Toutes les méthodes ont leurs mérites et les limites relatives. Ici, nous décrivons en détail un procédé non invasif sur le plan technique non intensif, peu coûteuse et rapide pour la délivrance intratracheale de bactéries qui implique l&#39; aspiration ( par exemple, inhalation) , par la sourisd&#39;un inoculum infectieux pipeté dans l&#39;oropharynx sous anesthésie générale. Cette méthode peut être utilisée pour l&#39;administration pulmonaire d&#39;une grande variété d&#39;agents biologiques et chimiques non-caustique, et il est relativement facile à apprendre, même pour les laboratoires ayant une expérience minimale préalable avec les procédures pulmonaires. En plus de décrire la méthode de pneumonie par aspiration, nous fournissons également des procédures étape par étape pour le dosage de l&#39;subséquente in vivo la réponse immunitaire innée pulmonaire de la souris, en particulier, des méthodes pour quantifier la clairance bactérienne et la réponse immunitaire cellulaire des voies respiratoires infectées. Cette approche intégrée et simple d&#39;évaluation de la pneumonie permet une évaluation rapide et robuste de l&#39;effet des manipulations génétiques et environnementaux sur l&#39;immunité innée pulmonaire.

C57BL / 6 ont été infectées avec 2000 UFC de K. 43816 pneumoniae (sérotype 2) par aspiration de l&#39; oropharynx dans les poumons. A cette dose, les souris commencent généralement à montrer des symptômes cliniques post-infection 12-24 h , y compris la léthargie, fourrure ébouriffée, et la perte de poids de 5 à 10% (figure 2A). Dans 48-72 h post-infection, la plupart des souris montrent des symptômes de la maladie et de la morbidité qui es...

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

Plusieurs procédés ont été décrits dans la littérature pour la modélisation de la pneumonie bactérienne chez les souris. Nous décrivons ici un peu coûteux, un procédé rapide et non invasif destiné à induire une pneumonie par aspiration ( par exemple, inhalation) , d&#39;un inoculum bactérien pipeté dans l&#39;oropharynx. méthodes en aval pour l&#39;évaluation de la réponse immunitaire innée pulmonaire sont également détaillées.

Une méthode non-invasive et technique non-intensif pour Induction et phénotypage of Experimental pneumonie bactérienne chez les souris

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.

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

Induction de murin inflammation intestinale par transfert adoptif de effecteur CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Haute</sup&gt; T cellules dans des souris immunodéficientes

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

Imagerie non invasive de la réponse immunitaire innée dans un modèle de poisson zèbre larves de<em&gt; Streptococcus iniae</em&gt; Infection

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