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 os experimentos foram realizados de acordo com o Animal Welfare Act e da Política de Serviços de Saúde Pública dos EUA na Humane Cuidado e Uso de Animais de Laboratório após revisão pelo Animal Care e Use Committee do NIEHS. 1. Preparação de K. Cultura pneumoniae Cuidado: Executar todas as etapas de um 2 (BSL2) capô nível de segurança biológica ou outro BSL2 área designada e descartar resíduos por diretrizes BSL2 instituto. </...

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Modelos murinos de pneumonia bacteriana, em parceria com o gene alvo e nas intervenções biológicas e farmacológicas in vivo, têm fornecido insights críticos sobre a resposta de defesa do hospedeiro pulmonar. Grandes avanços foram feitos, em particular, no nosso entendimento das quimiocinas e moléculas de adesão que regulam o recrutamento de neutrófilos para o espaço aéreo infectada 10,11. Modelos in vivo de pneumonia, ao contrário de abordagens baseadas em células ou a...

Pneumonia adquirida na comunidade continua a ser a principal causa de morte por infecção em os EUA, com pouca mudança global nas taxas de mortalidade nos últimos 40 anos, apesar de melhorias na vacinação e estratégias de antibióticos 1,2. Apesar da falta de progresso perceptível ao nível da saúde pública, nos últimos anos avanços dramáticos foram feitos em nosso entendimento da patogênese molecular e celular de pneumonia, com muitos desses passos em frente possibilitadas pelo uso de modelos de ratos com infecção pulmonar. A rastreabilidade genética do rato, a semelhança do murino e sistema imunológico humano, e a vasta gama de reagentes imunológicos alvo de murino que se tornaram disponíveis comercialmente, em conjunto, facilitou o progresso rápido do campo. mouse modelos de pneumonia bacteriana descrita na literatura em geral invocada uma das quatro rotas técnicas de inoculação: i) aerosolization; ii) intrentrega anasal; iii) a entrega por via oral; e iv) a injecção intratraqueal cirúrgica (ou seja, traqueotomia) 3. Todas as vias de infecção têm vantagens e desvantagens 3. Em particular, a exposição, relativa das vias aéreas superiores, o potencial para a mistura da flora oronasal requisitos para anestesia geral, a variabilidade do inoculo entregue ao pulmão distai, distribuição lobar dos agentes patogénicos entregues, os requisitos de peritagem técnica, e morbidade processual variam amplamente entre estes abordagens. Comumente técnicas de infecção por via oral usados incluem endotraqueal (translaríngea) canulação, quer através de uma abordagem &quot;cega&quot; (não visualizado), ou sob visualização direto da laringe 3-5. Ambos os métodos, ao mesmo tempo robusta, requer treinamento substancial e também carregam risco de trauma na via aérea superior. No presente relatório, nós descrevemos um método tecnicamente não-intensivo, não-invasivo, barato e rápido da infecção por via oral, wdeclara bactérias Klebsiella pneumoniae (no exemplo apresentado) pipetada para a orofaringe de um rato anestesiado são entregues para os pulmões por meio de aspiração (isto é, por inalação). Nós e outros autores utilizaram a técnica de pneumonia aspirativa com êxito 6-9. Este método de pulmão de entrega versátil e facilmente aprendido pode ser estendido para a entrega de muitos agentes não cáustico adicionais para os pulmões, incluindo citoquinas e outras proteínas, moléculas associadas a agentes patogénicos (por exemplo, lipopolissacarídeo), células (isto é, a transferência adoptiva), e toxinas (por exemplo, bleomicina). Além de discutir as considerações técnicas importantes, nós também descrevem uma abordagem integrada, quantitativa para avaliar a resposta do hospedeiro após a pneumonia, incluindo a medição a jusante da depuração bacteriana (ou seja, unidades formadoras de colônia [UFC] quantificação nos órgãos do pulmão e periféricos) e leucócitos acumulação no espaço 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

Embora a pneumonia adquirida na comunidade continua sendo um grande problema de saúde pública, modelos murinos de pneumonia bacteriana recentemente facilitada avanços pré-clínicos significativos em nossa compreensão da patogênese celular e molecular subjacente. Modelos in vivo rato capturar a fisiologia integrada e resiliência da resposta de defesa do hospedeiro em uma forma não revelada por, simplificado vivo abordagens alternativas ex. Vários métodos foram descritos na literatura para a inoculação de bactérias intrapulmonar em ratinhos, incluindo o tratamento com aerossóis, administração intranasal, endotraqueal canulação perorai sob condições &quot;cegas&quot; e visualizadas, e canulação endotraqueal transcutânea. Todos os métodos têm méritos e limitações relativas. Aqui, nós descrevemos em pormenor um método não-invasivo, tecnicamente não intensiva, de baixo custo, e para a entrega rápida intratraqueal de bactérias que envolve a aspiração (isto é, por inalação) pelo mousede um inoculo infeccioso pipetados para a orofaringe, enquanto sob anestesia geral. Este método pode ser utilizado para a administração pulmonar de uma grande variedade de agentes químicos e biológicos não cáustico, e é relativamente fácil de aprender, mesmo para laboratórios com o mínimo de experiência anterior com procedimentos pulmonares. Além de descrever o método de pneumonia por aspiração, nós também fornecemos procedimentos passo-a-passo para ensaio subsequente in vivo resposta imune inata pulmonar do rato, em particular, métodos de quantificação de apuramento bacteriana e resposta imune celular das vias aéreas infectado. Esta abordagem integrada e simples para avaliação da pneumonia permite a avaliação rápida e robusta do efeito de manipulações genéticas e ambientais sobre a imunidade inata pulmonar.

Ratinhos C57BL / 6 foram infectados com 2,000 CFU de K. pneumoniae 43816 (serótipo 2) por meio de aspiração da orofaringe para dentro dos pulmões. Nesta dose, os ratos normalmente começam a mostrar sintomas clínicos 12-24 h pós-infecção, incluindo letargia, pele enrugadas, e perda de peso de 5-10% (Figura 2A). Dentro de 48-72 horas após a infecção, muitos dos murganhos apresentam sintomas de doença e de morbilidade que é tipicamente precedido por u...

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

Vários métodos foram descritos na literatura para a modelagem de pneumonia bacteriana em murganhos. Aqui, nós descrevemos um método de baixo custo, rápida não-invasiva para induzir pneumonia por aspiração (isto é, por inalação) de um inoculo bacteriano pipetados para a orofaringe. métodos a jusante para avaliação da resposta imune inata pulmonar também são detalhados.

Um método não-invasivo e tecnicamente não-intensivo para indução e fenotipagem of Experimental pneumonia bacteriana em ratos

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

Imagem não invasivo do imunitário inatas Response em um modelo de peixe-zebra larval 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 ...