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).

Tutti gli esperimenti sono stati eseguiti in conformità con la legge sul benessere degli animali e la politica US Public Health Service su Humane cura e l&#39;uso di animali da laboratorio dopo la revisione dalla cura degli animali e del Comitato utilizzo del NIEHS. 1. Preparazione di K. Cultura pneumoniae Attenzione: eseguire tutti i passaggi del 2 (BSL2) cappuccio o altra area designata BSL2 livello di biosicurezza e gettare i rifiuti secondo l...

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Modelli murini di polmonite batterica, collaborato con gene targeting e negli interventi biologiche e farmacologiche in vivo, hanno fornito spunti critici nella risposta difesa ospite polmonare. Grandi progressi sono stati fatti, in particolare, nella nostra comprensione delle chemochine e molecole di adesione che regolano il reclutamento dei neutrofili allo spazio aereo infetto 10,11. Modelli in vivo di polmonite, a differenza di approcci basati su celle o alternativi, hanno anche ...

Polmonite acquisita in comunità rimane la principale causa di morte per infezioni negli Stati Uniti, con poca variazione complessiva dei tassi di mortalità nel corso degli ultimi 40 anni, nonostante i miglioramenti nella vaccinazione e le strategie di antibiotici 1,2. Nonostante la mancanza di progressi sensibili a livello della salute pubblica, in anni recenti progressi drammatici sono stati fatti nella comprensione della patogenesi molecolare e cellulare di polmonite, con molti di questi passi avanti reso possibile dall&#39;utilizzo di modelli murini di infezione polmonare. La trattabilità genetica del topo, la somiglianza del murino e sistema immunitario umano, e la vasta gamma di reagenti immunologici murini mirati che si sono resi disponibili in commercio hanno facilitato insieme rapido progresso del campo. modelli murini di polmonite batterica descritti in letteratura sono generalmente invocato uno dei quattro percorsi tecnici per l&#39;inoculazione dei patogeni: i) aerosol; ii) intrconsegna Anasal; iii) la consegna per via orale; e iv) iniezione intratracheale chirurgica (cioè, tracheotomia) 3. Tutte le vie di infezione presentano vantaggi e svantaggi 3. In particolare, l&#39;esposizione relativa delle vie aeree superiori, possibilità di mescolanza di flora oronasali, i requisiti per l&#39;anestesia generale, la variabilità della inoculo consegnato al polmone distale, la distribuzione lobare dei patogeni consegnati, i requisiti di perizia tecnica, e la morbilità procedurali variano ampiamente tra questi approcci. Comunemente tecniche di infezione orale per utilizzati includono incannulamento endotracheale (translaringea) sia tramite un approccio &#39;blind&#39; (non visualizzato), o sotto la visualizzazione della laringe diretta 3-5. Entrambi i metodi, mentre robusti, richiedono una formazione sostanziale e portano anche il rischio di traumi per le vie aeree superiori. Nella presente relazione, si descrive un metodo tecnicamente non intensivo, non invasivo, economico, e la rapida delle infezioni per via orale, wdichiara batteri Klebsiella pneumoniae (nell&#39;esempio fornito) pipettati in orofaringe di un mouse anestetizzato vengono consegnati ai polmoni tramite aspirazione (vale a dire, per inalazione). Noi e altri abbiamo utilizzato la tecnica polmonite ab ingestis successo 6-9. Questo metodo polmone recapito versatile e di facile apprendimento può essere esteso alla consegna di molti agenti non corrosivi aggiuntivi ai polmoni, quali citochine e altre proteine, molecole associati ai patogeni (ad esempio, lipopolisaccaride), cellule (cioè, trasferimento adottivo), e le tossine (ad esempio, bleomicina). Oltre a discutere importanti considerazioni tecniche, abbiamo anche descriviamo un approccio integrato, quantitativa per valutare la successiva risposta dell&#39;ospite alla polmonite, compresa la misurazione a valle della clearance batterica (ad esempio, unità formanti colonia [CFU] quantificazione negli organi polmone e periferici) e dei leucociti accumulo nello spazio aereo.

<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

Anche se polmonite acquisita in comunità rimane un importante problema di salute pubblica, modelli murini di polmonite batterica hanno recentemente facilitato significativi progressi preclinici nella nostra comprensione della patogenesi cellulare e molecolare di base. In vivo modelli murini catturare la fisiologia integrata e la resilienza della risposta di difesa ospitante un modo non rivela, ex vivo approcci semplificati alternativi. Diversi metodi sono stati descritti in letteratura per l&#39;inoculazione intrapolmonare di batteri nei topi, compresi aerosolizzazione, consegna intranasale, perorale cannulazione endotracheale in condizioni &#39;ciechi&#39; e visualizzate, e transcutanea cannulazione endotracheale. Tutti i metodi hanno meriti relativi e limitazioni. Qui, descriviamo in dettaglio un metodo non invasivo, tecnicamente non intensivo, poco costoso, e rapido per la consegna intratracheale di batteri che coinvolge l&#39;aspirazione (cioè, inalazione) per il mousedi un inoculo infettiva pipettati in orofaringe, mentre in anestesia generale. Questo metodo può essere utilizzato per la consegna polmonare di un&#39;ampia varietà di agenti biologici e chimici non corrosivi, ed è relativamente facile da imparare, anche per laboratori con minima esperienza precedente con procedure polmonari. Oltre a descrivere il metodo di polmonite ab ingestis, forniamo anche procedure passo-passo per saggiare la successiva in vivo polmonare risposta immunitaria innata del mouse, in particolare, i metodi per quantificare la clearance batterica e la risposta immunitaria cellulare delle vie aeree infetto. Questo approccio integrato e semplice valutazione polmonite consente di valutazione rapida e robusta dell&#39;effetto delle manipolazioni genetiche e ambientali upon polmonare immunità innata.

C57BL / 6 topi sono stati infettati con 2.000 UFC di K. pneumoniae 43816 (sierotipo 2) tramite aspirazione orofaringea nei polmoni. A questa dose, i topi di solito iniziano a mostrare sintomi clinici dopo l&#39;infezione 12-24 ore tra cui letargia, pelliccia arruffata, e perdita di peso del 5-10% (Figura 2A). All&#39;interno di post-infezione 48-72 ore, molti dei topi mostrano sintomi della malattia e morbilità che in genere viene preceduto da una media di perd...

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

Diversi metodi sono stati descritti in letteratura per modellare la polmonite batterica nei topi. Qui, descriviamo un non invasivo, economico, rapido metodo per indurre la polmonite tramite aspirazione (vale a dire, l&#39;inalazione) di un inoculo batterico pipettati in orofaringe. metodi di valle per la valutazione della risposta immunitaria innata polmonare sono anche dettagliata.

Un metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

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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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 ...

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

Imaging non invasiva della risposta immunitaria innata in un modello di Zebrafish larvale<em&gt; Streptococcus iniae</em&gt; Infezione

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