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).

Alle experimenten werden uitgevoerd in overeenstemming met de Animal Welfare Act en de US Public Health Service Beleid inzake Humane zorg en het gebruik van proefdieren na toetsing door de Animal Care en gebruik Comite van de NIEHS. 1. Bereiding van K. pneumoniae Cultuur Let op: Voer alle stappen in een bioveiligheid van niveau 2 (BSL2) kap of andere BSL2 aangewezen gebied en afval per instituut BSL2 richtlijnen negeren. <ol><li> Voor schorsing groei v...

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Muismodellen van bacteriële longontsteking, samen met gen-targeting en in vivo biologische en farmacologische interventies, zijn kritische inzichten in de pulmonaire afweer reactie. Grote vooruitgang is geboekt in het bijzonder in ons begrip van de chemokines en adhesie moleculen die de aanwerving van neutrofielen regeren tot de geïnfecteerde luchtruim 10,11. In vivo modellen van longontsteking, in tegenstelling tot de cel-gebaseerde of alternatieve benaderingen, hebben ook belangrijke inz...

De gemeenschap verworven longontsteking blijft de belangrijkste oorzaak van de dood van een infectie in de VS, met weinig algehele verandering in sterftecijfers in de afgelopen 40 jaar, ondanks verbeteringen in de vaccinatie en antibiotische strategieën 1,2. Ondanks het gebrek aan duidelijk vooruitgang op de volksgezondheid niveau de laatste jaren dramatisch vooruitgang geboekt in ons begrip van de moleculaire en cellulaire pathogenese van longontsteking, met veel van deze vooruitgang mogelijk gemaakt door het gebruik van muizenmodellen van longontsteking. De genetische traceerbaarheid van de muis, hebben de gelijkenis van de muizen en menselijke immuunsysteem, en de enorme hoeveelheid-muizen gerichte immunologische reagentia die in de handel verkrijgbaar zijn geworden samen vergemakkelijkt snelle vooruitgang van het veld. Muismodellen van bacteriële longontsteking in de literatuur beschreven zijn algemeen wordt aangevoerd uit vier technische routes pathogeen inoculatie: i) aërosolvorming; ii) intranasal levering; iii) perorale levering; en iv) chirurgische intratracheale injectie (dwz tracheotomie) 3. Alle besmettingswegen hebben voor- en nadelen 3. Vooral relatieve blootstelling van de bovenste luchtwegen, mogelijke vermenging van oronasale flora, vereisten voor algemene anesthesie, variabiliteit van de aan de distale long inoculum, lobaire verdeling van de geleverde pathogenen, technische eisen deskundigheid en procedurele morbiditeit variëren sterk tussen deze benaderingen. Veel gebruikte perorale besmetting technieken omvatten endotracheale (translaryngeal) infusen via ofwel een &#39;blind&#39; (niet zichtbaar) aanpak, of in direct larynx visualisatie 3-5. Beide werkwijzen, terwijl robuust, vereisen grote scholing en voeren ook het risico van trauma aan de bovenste luchtwegen. In dit rapport beschrijven we een technisch non-intensieve, niet-invasieve, goedkope en snelle methode van perorale besmetting, whierbij bacteriën (Klebsiella pneumoniae in de verstrekte voorbeeld) gepipetteerd in de oropharynx van een verdoofde muis om de longen wordt geleverd via aspiratie (dat wil zeggen, inhalatie). Wij en anderen hebben de aspiratiepneumonie techniek met succes 6-9 gebruikt. Deze veelzijdige en makkelijk te leren long-levering werkwijze kan worden uitgebreid tot het leveren van meer extra niet-bijtende middelen aan de longen, met inbegrip van cytokinen en andere proteïnen, pathogeen geassocieerde moleculen (bijvoorbeeld lipopolysaccharide), cellen (dwz adoptieve overdracht), en toxinen (bijvoorbeeld bleomycine). Naast het bespreken van belangrijke technische overwegingen, we beschrijven ook een geïntegreerde, kwantitatieve benadering voor de beoordeling van de daaropvolgende gastheer reactie op longontsteking, met inbegrip van downstream meting van de bacteriële klaring (dwz, kiemgetal [CFU] kwantificering in de longen en perifere organen) en leukocyten accumulatie in het luchtruim.

<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

Hoewel de gemeenschap verworven pneumonie blijft een groot probleem voor de volksgezondheid, hebben muismodellen van bacteriële longontsteking recent gefaciliteerd significante preklinische vooruitgang in ons begrip van de onderliggende moleculaire en cellulaire pathogenese. In vivo muismodellen vastleggen van de geïntegreerde fysiologie en veerkracht van de afweer reactie in een manier die niet onthuld door alternatieve, vereenvoudigde ex vivo benaderingen. Verscheidene werkwijzen zijn beschreven in de literatuur voor intrapulmonale inoculatie van bacteriën bij muizen, met inbegrip aërosolvorming, intranasale afgifte, perorale endotracheale canule onder blinde omstandigheden en gevisualiseerd en transcutane endotracheale canule. Alle methoden relatieve voordelen en beperkingen. Hierin beschrijven we in detail een niet-invasieve, technisch niet-intensieve, goedkope en snelle werkwijze voor intratracheale afgifte van bacteriën die aspiratie (bijvoorbeeld inhalatie) gaat door de muisvan besmettelijke inoculum gepipetteerd in de oropharynx terwijl onder narcose. Deze methode kan worden gebruikt voor pulmonaire afgifte van een groot aantal niet bijtend biologische en chemische agentia, en is relatief eenvoudig te leren, zelfs voor laboratoria met een minimale eerdere ervaring met pulmonale procedures. Naast een beschrijving aspiratie pneumonie methode wij ook stap voor stap procedures voor het testen van de volgende in vivo pulmonaire aangeboren immuunrespons van de muis, in het bijzonder werkwijzen voor het kwantificeren van bacteriële verwijdering en de cellulaire immuunreactie van de geïnfecteerde luchtwegen. Deze geïntegreerde en eenvoudige benadering longontsteking evaluatie maakt een snelle en grondige evaluatie van het effect van genetische en omgevingsfactoren manipulaties op pulmonale aangeboren immuniteit.

C57BL / 6-muizen werden geïnfecteerd met 2000 CFU K. pneumoniae 43.816 (serotype 2) via oropharyngeal inademing in de longen. Bij deze dosis muizen beginnen meestal klinische symptomen vertonen 12-24 uur na infectie zoals lethargie, gegolfde vacht en gewichtsverlies van 5-10% (Figuur 2A). Binnen 48-72 uur na infectie veel van de muizen tonen ziektesymptomen en morbiditeit die typisch wordt voorafgegaan door een gemiddeld 20% gewichtsverlies en resulteert in geb...

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

Verscheidene werkwijzen zijn beschreven in de literatuur voor het modelleren van bacteriële pneumonie bij muizen. Hierin beschrijven we een niet-invasieve, goedkope, snelle werkwijze voor het induceren via aspiratie pneumonie (bijvoorbeeld inhalatie) van een bacterieel inoculum gepipetteerd in de orofarynx. Downstream methoden voor de beoordeling van de pulmonaire aangeboren immuunrespons zijn ook gedetailleerd.

Een niet-invasieve en Technisch Niet-intensieve methode voor Inductie en fenotypering van experimentele bacteriële longontsteking in Muizen

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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Niet-invasieve beeldvorming van Leukocyte Homing en migratie In vivo</em

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

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

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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

Niet-invasieve beeldvorming van de aangeboren immuunrespons in een zebravis larven Model van<em&gt; Streptococcus iniae</em&gt; Infectie

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