Pesquisa no laboratório de Bragonzi foi financiado pela Fundação de Fibrose Cística italiano (CFaCore) e UE-F7-2009-223670. Parte deste trabalho foi realizado em ALEMBIC, um laboratório de microscopia avançada, e mouse histopatologia foi realizada na Unidade de Anatomia Patológica (Instituto Científico San Raffaele).

1. As bactérias que se preparam para a infecção crônica (três e dois dias antes Rato Challenge) <ol><li> Selecione o P. apropriado aeruginosa para ser testado. </li><li> Inocule uma ansa de P. aeruginosa a partir de uma -80 ° C a uma cultura de placa de agar de tripticase de soja (TSA) e incubar a 37 ° C durante a noite. </li><li> Escolha de uma única colónia e inocular em 5 ml de Caldo de Soja Trypticase (TSB) num encaixe de casquilho tubo de 15 ml e incubar a 37 ° C durante a noite numa incubadora com agitação a 200 rpm. </li></ol> 2. Incorporação bactérias em ágar Beads para a infecção crônica (um dia antes da infecção) <ol><li> Tomar uma pequena alíquota da cultura bacteriana durante a noite, dilui-se a 1:50 em tampão fosfato salino (PBS) e medir a densidade óptica (DO) a 600 nm. </li><li> Dilui-se a cultura durante a noite de bactérias por adição de 2 OD em um novo tubo de pressão com tampa contendo 20 ml de TSB fresco. </li><li> Incubar a 37 ° C durante cerca de 3 -4 h até à fase logarítmica em uma incubadora com agitação a 200 rpm, até um total de 10-15 OD é atingido. </li><li> No entanto, preparar o TSA, feito de TSB com 1,5% de ágar, autoclave e equilibrar-se a 50 ° C em um banho de água. Equilibrar a 150 ml de óleo mineral pesado preautoclaved em um balão de Erlenmeyer a 50 ° C em um banho de água. </li><li> Uma vez que P. aeruginosa alcança a fase log, recolher as células bacterianas por centrifugação a 2.700 xg durante 15 min a 4 ° C e desprezar o sobrenadante. </li><li> Ressuspender o sedimento de bactérias em 1 ml de PBS estéril e vórtice completamente para ressuspender completamente o sedimento. </li><li> Misturar 1 ml de suspensão bacteriana com 9 ml de líquido de TSA pré-equilibradas a 50 ° C. </li><li> Adicionar 10 ml TSA-p aeruginosa mistura de óleo mineral pesado (pré-aquecida a 50 ° C) e agita-se imediatamente, durante 6 minutos à temperatura ambiente. A agitação deve produzir um vórtice visível no óleo. </li><li> Arrefece-se a mistura a 4 ° C, agitou-se à sp mínimoeed durante 35 minutos (Figura 1). </li><li> Repousar a mistura de ágar-esferas de óleo em gelo durante mais 20 min. </li><li> Transferir os grânulos de agar-em 50 ml de tubos Falcon e centrifugar a 2700 x g durante 15 min a 4 ° C. </li><li> Exacta remover o óleo mineral e lavagem com PBS estéril, seis vezes, tal como descrito no passo 2.11. Depois de três lavagens, os grânulos podem ser peletizadas por gravidade em vez de usar a centrífuga. Após a última lavagem, ressuspender as contas em ágar-20-30 ml PBS. </li><li> Tomar uma aliquota de pérolas (cerca de 0,5 ml) e assepticamente homogeneizar. </li><li> Tomar 100 ul dos grânulos homogeneizados e diluídos em 900 ul de PBS estéril. Diluir em série 1:10 até 10 -6. </li><li> Placa de diluição em série em placas de TSA, incluindo a amostra não diluída até 10 -6 e incubar as placas a 37 ° C. </li><li> Meça o diâmetro do talão usando um microscópio de luz invertida em vários campos. O diâmetro do grânulo deve estar entre 100-200 mM ( <strong&gt; Figura 1). </li><li> Armazenar as pérolas durante a noite a 4 ° C. </li></ol><img alt="Figura 1" fo:content-width="6in" fo:src="/files/ftp_upload/51019/51019fig1highres.jpg" src="/files/ftp_upload/51019/51019fig1.jpg" width="500px" /> Figura 1. Visão geral da infecção preparação e mouse contas agar. P. aeruginosa células são ressuspensas em 1 ml de PBS e adicionado a 9 ml de líquido de TSA (50 ° C). Esta mistura é adicionada a 150 ml de óleo mineral pesado a 50 ° C num balão e agitou-se a alta velocidade durante 6 min à temperatura ambiente. Quando o frasco é arrefecido a 4 ° C com uma agitação lenta durante 35 min, o agar solidifica criação de grânulos, e bactérias presentes na mistura são incorporados os grânulos de agar. Detalhe de um grânulo de agar contendo as células bacterianas é mostrado (A). Depois de remover o óleo mineral com várias lavagens utilizando PBS esterilizado, a suspensão de agar-esferas está pronto fou inoculação nos pulmões de camundongos por uma injeção intratraqueal (B). <a href="https://www.jove.com/files/ftp_upload/51019/51019fig1highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada</a> . 3. Ratos Challenge com ágar-contas Declaração de Ética: Este protocolo e experimentação siga as orientações do cuidado com os animais e comitê de ética do Instituto Científico San Raffaele. <ol><li> Contar o número de unidades formadoras de colónias (CFU) nas placas de TSA para determinar o número de UFC / ml na suspensão de agar-esferas. Dilui-se os grânulos de agar-com PBS esterilizado a 2-4 x 10 7 UFC / ml para atingir um inoculo óptimo de 1-2 x 10 6 em 50 ul. </li><li> Anestesiar C57Bl/6NCr (20-22 g, 6-8 semanas de idade) com cetamina ratos macho (50 mg / ml) e xilazina (5 mg / ml) em 0,9% de NaCl administrada a um volume de 0,002 ml / g de peso corporal pela injecção intraperitoneal. NOTA: A anestesia é considerada adeqliação formal quando o animal permanece ainda em silêncio, não responde a estímulos externos, e tem coração constante e as taxas respiratórias. </li><li> Posicione o mouse na posição supina. Desinfetar o brasão do mouse com etanol 70%. </li><li> Expor a traqueia por um corte vertical da pele e entubar a traqueia com uma agulha estéril, flexível 22 L 0,9 milímetros x 25 mm IV cateter, mantendo a atenção para remover o estilete, enquanto se deslocam para dentro da traqueia. Insira o cateter não muito profundo na traquéia. Pare antes de chegar à carina (bifurcação). </li><li> Tomar imediatamente um volume de 50 mL de suspensão de esferas agar por uma seringa de 1 ml e anexá-lo ao cateter. Empurrar suavemente o êmbolo da seringa, permitindo que os grânulos a serem implantados no pulmão. Feche a incisão usando clipes de sutura. </li><li> Colocar o animal em uma almofada de aquecimento até que esteja totalmente acordado. </li></ol> 4. Ratos Avaliação <ol><li> Observe os ratos diariamente para sinais clínicos, incluindo qualidade da pelagem, postura,deambulação e estado de hidratação. Monitorar o peso corporal por dia. Ratos que perdem ≥ 20% do peso corporal devem ser sacrificados. </li><li> Siga o ponto 5 para coleta de lavado broncoalveolar (LBA) e Pontos de 6 ou 7 para a recolha de pulmões ea análise da CFU total, a análise histológica, a resposta inflamatória em termos de contagem total e diferencial de células no LBA, análise de citocinas e mieloperoxidase (MPO) atividade. </li></ol> 5. Coleção LBA e Análise <ol><li> Eutanásia dos ratos por inalação de CO2. </li><li> Posicione o mouse na posição supina. Desinfetar o brasão do mouse com etanol 70%. </li><li> Expor a traquéia e caixa torácica por um corte vertical da pele. Expor os pulmões, cortando o diafragma. </li><li> Insira um fio de sutura sob a traquéia usando uma pinça e entubar a traqueia com uma estéril, flexível 22 g 0,9 milímetros x 25 mm cateter IV. Puxe as duas pontas do fio de sutura para vincular o cacath para a traquéia e nó a linha ao redor da traquéia. </li><li> Tome um volume de 1 ml de RPMI 1640, utilizando uma seringa de 1 ml e anexá-lo ao cateter. Empurrar o êmbolo da seringa, para lavar os pulmões e imediatamente recuperar o líquido, armazenando-o num tubo de 15 ml. NOTA: Se as citocinas estão a ser analisadas, adicionar inibidores da protease para RPMI 1640. </li><li> Repita este passo três vezes com um total de 3 ml de RPMI. A partir de agora, guarde o LBA no gelo. Vá para a etapa 6 para a coleta e análise dos pulmões. NOTA: Por favor, note que nem todos o líquido será recuperado (2,8 ml no máximo). </li><li> Para a quantificação de bactérias presentes no fluido BAL, a amostra numa pequena alíquota (300 ul), diluído em série 1:10 em PBS estéril, a placa em placas de TSA e incubar a 37 ° C durante a noite. </li><li> Contagem total de células utilizando um microscópio óptico de luz invertido diluindo uma alíquota do fluido de BAL de 1:2 com solução Tuerk numa câmara de contagem de células Burker. </li><li> Centrifugar a remaining LBA a 330 xg durante 8 minutos a 4 ° C. Tome-se o sobrenadante para análise de citocinas ELISA, armazenando-se a -80 ° C. Siga os passos de 5,10-5,13 para contagem celular diferencial por cytospin. </li><li> Se o sedimento é vermelho, a lise dos eritrócitos ressuspensão do sedimento em 250-300 mL de tampão de lise de RBC diluído 1:10 em água destilada ultra-puro durante 3 minutos. Neutraliza-se com 2 ml de PBS e centrifugar a 330 xg durante 8 minutos a 4 ° C. </li><li> Descartar o sobrenadante e ressuspender o pellet em RPMI 10% de soro fetal bovino (FBS). Utilizar um volume que vai proporcionar 1 x 10 6 células / ml, com base no número total de células. </li><li> Colocar as lâminas de microscópio e os filtros em ranhuras apropriadas no cytospin com os filtros de cartão de frente para o centro do cytospin. Pipetar 150 l de cada amostra para os poços apropriados de citospina e centrifugar numa citocentrífuga a 300 xg durante 5 min. </li><li> Mancha slides Romanowsky coloração utilizando um kit comercial, de acordo com a mainstruções de nufacturer e 12 como descrito anteriormente. Siga os passos de 5,14-5,17 para análise da atividade da MPO. </li><li> Centrifuga-se o volume restante de BAL a 380 xg por 5 min a 4 ° C. Descartar o sobrenadante e ressuspender o sedimento em 250 ul de cloreto de hexadeciltrimetilamônio 0,5% em água destilada ultra-pura a lisar as células. A suspensão pode ser congelada a -20 ° C durante vários dias antes da realização do ensaio. </li><li> Centrifugar a 16.000 xg durante 30 min a 4 ° C e usa-se o sobrenadante para realizar ensaio de MPO em placas de 96 poços, adicionando a amostra em duplicado a cada poço e, se necessário, também diluições adequadas da amostra. </li><li> Adicionar a cada amostra nos poços num volume igual de 3,3 &#39;, 5,5&#39;-tetrametilbenzidina (TMB) como um substrato para a peroxidase. Permitir que a reação ocorra no escuro por pelo menos 5 minutos e até que não haja um maior desenvolvimento na cor. </li><li> Parar a reacção por adição de 2 MH 2 SO 4 umª medir o OD a 450 nm. O valor OD será diretamente proporcional à atividade da peroxidase. </li></ol> 6. Medição da carga bacteriana no pulmão e citocinas Análise <ol><li> Imediatamente após a coleta de LBA, pulmões especiais de consumo a partir do mouse, lavá-los em PBS estéril, lóbulos separados, colocá-los em um tubo de fundo redondo com 2 ml de PBS estéril e armazenar no gelo. NOTA: Se as citocinas estão a ser analisadas, adicionar inibidores da protease a PBS, para os tubos. </li><li> Assepticamente homogeneizar pulmões, levar uma pequena alíquota (300 ul) do homogenato, serialmente diluídas 1:10 em PBS, em placas de TSA placa e incubar a 37 ° C durante a noite. Por favor note que a carga total das vias aéreas bacteriana será a soma dos UFCs encontrados em amostras de LBA e pulmão. </li><li> Centrifuga-se o homogeneizado a 16.000 xg, permanecendo durante 30 min a 4 ° C. Leve o sobrenadante para análise de citocinas ELISA, e armazenar a -80 ° C. </li></ol> 7. O exame histológico &lt;ol&gt; <li> Realizar análise histológica somente nos pulmões em que LBA não foi coletado, para preservar aspecto de pulmão e propriedades. </li><li> Eutanásia o mouse por inalação de CO 2. </li><li> Expor a cavidade torácica de um corte vertical da pele, expor os pulmões, cortando o diafragma. </li><li> Pulmões Especiais de Consumo, lavá-los em PBS, separe os lobos, colocá-los em um tubo contendo formol 5-10 ml a 10% neutro tamponado (formaldeído 4%) e armazenar a 4 ° C ao abrigo da luz. </li><li> Pulmões Incorporar em parafina, usando procedimentos padrão. </li><li> Corte 5 mm de espessura utilizando um micrótomo. </li><li> Stain lâminas com hematoxilina e eosina e cobrir as lâminas com uma lamela, de acordo com os procedimentos padrão. </li><li> Examinar as lâminas usando um microscópio de campo claro invertido e adquirir imagens ligando o microscópio para uma câmera. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

As etapas críticas do P. preparação aeruginosa-esferas e desafio do mouse são relatados abaixo. A cepa P. aeruginosa utilizado para ratos desafio é crítica. A mortalidade, infecção crônica ou depuração podem diferir significativamente dependendo da cepa bacteriana utilizada para o desafio. O P. aeruginosa RP73 tensão clínico foi preferido em relação a cepa de laboratório de referência PAO1 uma vez que resultou em uma taxa de morta...

A fibrose cística (FC) é uma doença genética causada por mutações no transmembrana da fibrose cística condutância gene regulador (CFTR). Este gene codifica para um canal de cloreto expressa na membrana da maior parte das células epiteliais. Destruição bronquiectasia, o entupimento do muco e do parênquima causada principalmente por infecções por Pseudomonas aeruginosa, progressivamente, induzir doença pulmonar grave e mortalidade na maioria dos pacientes com FC 1. Entendimento patogênese CF e um maior desenvolvimento de novas terapias confiar em modelo animal com traços característicos da CF. Vários ratinhos geneticamente modificados, para o gene de CFTR, foram gerados, mas limitações na capacidade destas espécies para recapitular doença pulmonar CF-like e várias outras alterações de órgãos observados em pacientes com FC foi amplamente documentado 2. Desenvolvimento de infecção é um dos principais desafios na CF modelo animal. Os cl literaturainiciais sugerem que uma infecção crónica que durou mais do que um mês pode ser conseguido somente se os ratinhos são inoculados com bactérias incorporados num agente imobilizante tais como agar, agarose, ou algas alginato 3-5. Estes agentes imobilizantes proporcionar as condições microaeróbico / anaeróbias, que permitem que as bactérias crescem na forma de microcolónias, de forma semelhante para o crescimento no muco de doentes CF 6. Este modelo de infecção crônica leva à persistência da bactéria nos pulmões, causando inflamação das vias respiratórias e danos 7. No entanto, dependendo do método utilizado, a estirpe bacteriana e da dose inoculada nos pulmões, a percentagem de ratinhos infectados crónicas e a carga bacteriana nos pulmões recuperado em diferentes pontos de tempo pode variar consideravelmente. Em particular, o principal desafio para a infecção crônica a longo prazo continua a ser a baixa carga bacteriana por P. aeruginosa e o baixo percentual de semanas ratos infectados após o desafio, indicando that células bacterianas são progressivamente apuradas pelo anfitrião. Ao selecionar o P. aeruginosa RP73 tensão clínico de uma coleção de CF isola 8 obtivemos êxito baixa mortalidade, as lesões mais graves, e alta porcentagem de infecção crônica com uma carga bacteriana estável até um mês em ratos C57Bl/6NCrl. Este artigo detalha a metodologia para a incorporação de P. aeruginosa nos grânulos de agar; temos de camundongos infectados por instilação intratraqueal, medido a carga bacteriana nos pulmões e citocinas, recolhidos do fluido BAL e realizado o exame histológico. No geral, este protocolo vai ajudar os pesquisadores na abordagem de questões fundamentalmente importantes na patogênese 8,9 e teste de novas terapias contra o P. aeruginosa infecção crônica 10,11.

<table border="0" cellpadding="0" cellspacing="0" width="605">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Bacto Tryptic Soy Broth</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">211823</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Difco Agar, granulated</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">214510</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Heavy mineral oil</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">330760-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">S-(+)-Ketamine hydrochloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">K1884</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine hydrochloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">X1251</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:272px;">1 ml syringe 25 G 5/8&#39;&#39;&#160;0.5 x 16 mm</td>
			<td style="width:191px;">PIC</td>
			<td style="width:143px;">3071250300350</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Catheter 22GA 0.9 x 25 mm</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">381223</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Graefe Forceps - 0.5 mm Tips Curved</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">11152-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Scissors, Iris, 11 cm, straight</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:143px;">501758</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Suture clips</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">12040-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Suture thread</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">18020-40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RPMI 1640</td>
			<td style="width:191px;">Lonza</td>
			<td style="width:143px;">BE12-167F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Complete protease inhibitor cocktail</td>
			<td style="width:191px;">Roche</td>
			<td style="width:143px;">11836145001</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:272px;">Fast-Read 102 Burker disposable chamber</td>
			<td style="width:191px;">Biosigma</td>
			<td style="width:143px;">390497</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Tuerk solution</td>
			<td style="width:191px;">Fluka</td>
			<td style="width:143px;">93770</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RBC lysis buffer</td>
			<td style="width:191px;">Biolegend</td>
			<td style="width:143px;">420301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fetal bovine serum</td>
			<td style="width:191px;">Lonza</td>
			<td style="width:143px;">DE14-801F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">EZ cytofunnel</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">A78710021</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Superfrost ultra plus microscope slides</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">J3800AMNZ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Diff-Quik Romanowsky staining set</td>
			<td style="width:191px;">Medion Diagnostics</td>
			<td style="width:143px;">130832</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">Hexadecyltrimethylammonium chloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">52366-10G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">96-well EIA/RIA plate</td>
			<td style="width:191px;">Costar</td>
			<td style="width:143px;">3590</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">3,3&#8217;,5,5&#8217;- tetramethylbenzidine</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">T8665-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sulfuric acid</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">320501-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">10% neutral buffered formalin</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-01005Q</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Harris haematoxylin non Papanicolau</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-M06004</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Eosin plus alcoholic solution</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-M11007</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
<td>[header]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;"></td>
			<td style="width:191px;"></td>
			<td style="width:143px;"></td>
<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Shaking incubator</td>
			<td style="width:191px;">Amerex Instruments</td>
			<td style="width:143px;">Steady Shake 757</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Water bath</td>
			<td style="width:191px;">Grant</td>
			<td style="width:143px;">SUB14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Homogenizer</td>
			<td style="width:191px;">Ystral</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Precision balance</td>
			<td style="width:191px;">KERN</td>
			<td style="width:143px;">440-47N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cytocentrifuge</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">A78300003</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Low Cost Heating Pad</td>
			<td style="width:191px;">2biol</td>
			<td style="width:143px;">LCHP</td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:272px;">Homogenization probe</td>
			<td rowspan="2" style="width:191px;">Ystral</td>
			<td style="width:143px;">2366931(great)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:143px;">2366925(small)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted optical microscope</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:143px;">Axioplan2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Camera (microscope)</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:143px;">Axiocam MRc5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Rotary microtome</td>
			<td style="width:191px;">Leica</td>
			<td style="width:143px;">RM2255</td>
		</tr>
	</tbody>
</table>

long term chronic pseudomonas aeruginosa airway infection in mice

Um modelo do rato de infecção crônica das vias aéreas é um elemento essencial na fibrose cística (FC) de pesquisa, embora haja uma série de preocupações sobre o próprio modelo. Fases precoces da infecção e inflamação tem sido amplamente estudado utilizando o modelo de rato de agar-grânulos de Pseudomonas aeruginosa, embora apenas alguns relatórios têm-se centrado na infecção crónica a longo prazo in vivo. O principal desafio para a infecção crónica a longo prazo continua a ser a baixa carga bacteriana por P. aeruginosa e o baixo percentual de semanas ratos infectados após o desafio, indicando que as células bacterianas são progressivamente apuradas pelo anfitrião. Este artigo apresenta um método para a obtenção de infecção crônica eficiente a longo prazo em camundongos. Este método baseia-se na incorporação do p estirpes clínicas aeruginosa no agar-beads in vitro, seguida por instilação endotraqueal em ratos C57Bl/6NCrl. Infecção pulmonar bilateral está associada a SEVeral mensuráveis ​​de leitura-outs incluindo perda de peso, mortalidade, infecção crônica, e na resposta inflamatória. O P. aeruginosa RP73 tensão clínico foi preferido em relação a cepa de laboratório de referência PAO1 uma vez que resultou em uma taxa de mortalidade mais baixa comparativamente, as lesões mais graves, e infecção crônica superior. P. colonização aeruginosa pode persistir no pulmão por mais de três meses. Patologia do pulmão murino se assemelha ao de pacientes com FC com doença pulmonar crônica avançada. Este modelo murino imita mais de perto o curso da doença humana e pode ser utilizado tanto para os estudos sobre a patogénese e para a avaliação de novas terapias.

Quando o protocolo é feito corretamente, o P. aeruginosa ágar-contas vai medir entre 100-200 mM e podem ser observados com um microscópio de luz invertida pipetando um pequeno volume da suspensão de ágar-esferas em um slide. Células bacterianas individuais são visíveis nos grânulos de agar, como mostrado em detalhe na Figura 1. A escolha de P. aeruginosa estirpe utilizada na preparação de agar-pérolas é crítica. Figura 2 e <st...

Watch this Scientific Journal Video about Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice at JoVE.com

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Descreve-se o método de ágar-esferas de estabelecer infecção das vias aéreas persistente a longo prazo crônica Pseudomonas aeruginosa no modelo de mouse.

Crônica de Longo Prazo<em&gt; Pseudomonas aeruginosa</em&gt; Infecção das vias aéreas em ratos

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

long-term-chronic-pseudomonas-aeruginosa-airway-infection-in-mice

Research

JoVE Journal

Immunology and Infection

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

22.4K visualizações.  San Raffaele Scientific Institute.  Descreve-se o método de ágar-esferas de estabelecer infecção das vias aéreas persistente a longo prazo crônica Pseudomonas aeruginosa no modelo de mouse. 

Vídeo: Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-cultura de Modelos Pseudomonas aeruginosa Biofilmes Grown no Live Células Airway Humanos

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Imunologia e Infecção

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa e Saccharomyces cerevisiae Biofilme em Células de Fluxo

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Isolamento de RNA Pseudomonas aeruginosa Colonizar o trato gastrointestinal Murino

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de meio de escarro Artificial para testar a eficácia antibiótica contra<em&gt; Pseudomonas aeruginosa</em&gt; Em condições mais relevantes para o Pulmão Fibrose Cística

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa Lesão Pulmonar Induzida Modelo

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

Venha para o lado da luz: In Vivo monitoramento de infecções por Pseudomonas aeruginosa biofilme em feridas crônicas em um modelo murino Hairless diabética

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Métodos para detecção de citotóxicos amiloides seguir infecção de pulmonar células endoteliais por Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

A longo prazo no Vivo controle da dinâmica celular inflamatória dentro drosófila pupas

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Cultura de Variante de Pequena Colônia de Pseudomonas aeruginosa e Quantitação de seu Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Um modelo de inoculação retardada da infecção crônica de pseudomonas aeruginosa ferida

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...