AR, XH e PE foram apoiados pela concessão do NIH R01HL140398 e uma bolsa da Fundação Gilson Longenbaugh. A DEMM e a KK foram apoiadas pelas bolsas DO NIH R01HL136333 e R01HL134880 (KYK), e uma bolsa da Helis Medical Research Foundation. A DEMM também é apoiada pela Howard Hughes Medical Institute (HHMI) Gilliam Fellowship for Advanced Study. A PE também conta com o apoio do Programa de Bolsas de Pesquisa em Ciências da Saúde Ambiental de Precisão NIEHS T32 ES027801. O JC e o MF são apoiados pelo Tobacco Research Funds do Departamento de Epigenética e Carcinogênese Molecular e pelo Centro de Epigenética (Prêmio Acadêmico para MF) da MD Anderson. FK e YZ são apoiados pelas bolsas NIH R01 ES029442-01 e R01 AI135803-01, bem como a concessão do Mérito VA CX000104. Este projeto foi apoiado pelo Núcleo de Citometria e Triagem Celular da Baylor College of Medicine com financiamento do CPRIT Core Facility Support Award (CPRIT-RP180672), do NIH (CA125123 e RR024574), e da assistência de Joel M. Sederstrom.

Todos os animais envolvidos nos experimentos e no desenvolvimento dessa técnica estão sob nosso protocolo de uso animal aprovado pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) e pelas instituições Baylor College of Medicine e MD Anderson que são credenciadas pela Associação de Avaliação e Acreditação de Cuidados Com Animais Laboratoriais (AAALAC).
1. Construção do aparelho
<ol><li>Montagem do compressor de ar com o sistema da válvula.
	<ol>
<li>Conecte os medidores de fluxo (dois 15 L/min com barra Y e 2 decolagens de energia) ao regulador de pressão em miniatura usando um encaixe de mamilo adaptador masculino de 1/8 polegada. Certifique-se de usar a fita de vedação do fio em todas as extremidades roscadas.</li>
		<li>Conecte o regulador de pressão montado com o medidor de fluxo ao instrumento médico do compressor de ar usando o seguinte: um mamilo hexaxado de 1/8 polegada na saída de ar do compressor, um encaixe de acoplamento rosqueado de 1/8 polegadas e um encaixe de mamilo adaptador masculino de 1/8 polegada que se conecta ao regulador de pressão.</li>
		<li>Instale o conector farpado giratório de oxigênio em cada (4) medidor de fluxo.</li>
		<li>Instale um adaptador masculino na saída de ar superior do compressor de ar (peça incluída com o instrumento médico do compressor de ar).</li>
	</ol></li>
	<li>Montagem de câmaras de exposição (fazer 4 unidades)
	<ol>
<li>Corte um tubo de cloreto de polivinil clorado de 3/4 polegadas (CPVC) em oito segmentos de 4 polegadas.</li>
		<li>Insira cada segmento em um encaixe CPVC de cotovelo de 3/4 polegadas 90° e conecte o lado de encaixe do cotovelo a um adaptador masculino CPVC de 3/4 polegadas de diâmetro. Deve haver oito segmentos de CPVC, cada um ligado a um encaixe de cotovelo CPVC e um adaptador masculino CPVC.</li>
		<li>Faça dois furos (1 1/4 polegada de diâmetro) em lados opostos mais distantes um do outro de um recipiente hermético de 8,5 L (11,25 x 7,75 x 6 polegadas) com uma tampa (ver Câmara de exposição Figura 1). O posicionamento dos orifícios deve ser centrado de cima para baixo e da esquerda para a direita.</li>
		<li>Insira as laterais roscadas do adaptador masculino CPVC montado antes em cada orifício nos recipientes.</li>
		<li>Do interior do recipiente, conecte uma tampa CPVC de 3/4 polegadas do outro lado (entrada de fumaça de câmara) e um adaptador feminino de irrigação de gotejamento CPVC de 3/4 polegadas de um lado (saída de fumaça da câmara).</li>
		<li>Faça cinco furos de 3 mm na parte superior da tampa CPVC da saída de fumaça da câmara em um padrão de quincunx (chuveiro). Isso permitirá que a fumaça do cigarro entre na câmara com maior velocidade e garante que ela se espalhe uniformemente dentro da câmara em todas as direções.</li>
	</ol></li>
	<li>Montagem de câmaras de cigarro (compõe até 4 unidades de exposição murina)
	<ol>
<li>Pegue uma rolha de borracha de um orifício (tamanho do fabricante 8,5) e insira um conector Y farpado de 1/4 polegada no lado mais largo e um encaixe reto de barbeado (abertura de 8 mm) no lado mais estreito. O cigarro será colocado aqui durante o procedimento de fumar (pedestal de cigarro).</li>
		<li>Conecte uma extremidade de um tubo de vinil de grau médico de 12 polegadas a um dos conectores farpados no conector Y ligado à rolha de borracha e a outra extremidade a um encaixe de 1/4 polegada e insira o lado oposto deste encaixe em uma rolha de borracha de um orifício (tamanho do fabricante 1).</li>
		<li>Em outra rolha de borracha (tamanho do fabricante 8,5), insira um conector de tubo reto de 1/4 polegada no lado mais largo da rolha e conecte a extremidade externa do encaixe a um tubo de vinil de grau médico de 7 pés.</li>
		<li>Conecte as duas estruturas de rolha de borracha montadas antes nas etapas 1.3.1-1.3.3 a um cilindro de vidro de 8 polegadas x 1,75 polegadas de um tubo de drenagem de vidro de laboratório.</li>
	</ol></li>
	<li>Sistema de controle da válvula
	<ol>
<li>O sistema é controlado por uma abertura rítmica e fechamento de válvulas solenoides que simulam a inalação (puffing) de fumaça de cigarro e ar limpo. O sistema que controla as válvulas solenoides foi projetado comercialmente (ver Tabela de Materiais).</li>
	</ol></li>
	<li>Montando todos os componentes juntos (ver Figura 1)
	<ol>
<li>Monte quatro válvulas solenoides nas laterais do sistema de controle da válvula usando fixadores de 1 polegada.</li>
		<li>Conecte as válvulas solenoides ao sistema de controle da válvula seguindo as instruções do fabricante.</li>
		<li>Conecte um conector reto roscado de 10 a 32 (M) ao escape ("EXH") na válvula solenoide e um adaptador de porta rosqueado nas conexões "IN" e "OUT" da mesma válvula solenoide.</li>
		<li>Conecte o medidor de fluxo ligado ao compressor à válvula solenoide através da conexão "OUT" usando uma tubulação de vinil de grau médico de 7 pés.</li>
		<li>Conecte o tubo de vinil de 7 pés montado com a rolha de borracha na etapa 1.3.3. no conector "IN" na válvula solenoide.</li>
		<li>Insira a pequena rolha de borracha da câmara de cigarro na entrada de fumaça da câmara.</li>
		<li>Conecte a válvula solenoide à segunda conexão do conector Y farpado no pedestal de cigarro montado na etapa 1.3.1.</li>
	</ol></li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61793/61793fig01.jpg"> Figura 1: Esquema das conexões do nosso WBIS para exposição à CS. Esta figura demonstra como todos os componentes são montados para formar um aparelho de trabalho. A figura mostra apenas uma câmara de fumantes montada dos quatro que a máquina é capaz de operar. <a href="https://www.jove.com/files/ftp_upload/61793/61793fig01large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
2. Exposição à fumaça do cigarro
ATENÇÃO: Evite a exposição em segunda e terceira mão à fumaça do cigarro. As câmaras de cigarro e exposição devem ser usadas dentro de um Gabinete de Segurança Biológica de Fluxo De Fluxo De Fluxo Classe II B2. Epi adequado deve ser usado durante a realização dos experimentos de exposição à fumaça (ou seja, máscaras, luvas, rede de cabelo, vestido).
<ol><li>
Definição de pressão e fluxo de ar
	<ol>
<li>Uma vez que todos os componentes sejam montados como mostrado na Figura 1,ligue o compressor de ar e espere que o alarme de segurança seja desligado por conta própria.</li>
		<li>Ajuste a pressão do compressor de ar para 40-50 psi girando o botão sobre o regulador de pressão.</li>
		<li>Ajuste o fluxo de ar do compressor de ar para 5 L usando o medidor de fluxo.</li>
		<li>Ligue o controlador da válvula.</li>
		<li>Ajuste o temporizador digital no controlador da válvula para o modo operacional PULSE-C (mostrado no display como "Pu-c") pressionando a tecla SET/LOCK enquanto segura a tecla UP no primeiro dígito do temporizador. Em seguida, pressione a tecla UP até que o modo Pu-c seja atingido. Pressione a tecla RESET para definir o modo de operação exibido (ou seja, Pu-C) como o modo de trabalho.</li>
		<li>Pressione o SET/LOCK para alterar o temporizador 1 (mostrado no visor como "T1").</li>
		<li>Pressione as teclas para cima ou para baixo para definir T1 a 20 s.</li>
		<li>Pressione o SET/LOCK para alterar o temporizador 2 (mostrado no visor como "T2").</li>
		<li>Pressione as teclas para cima ou para baixo para definir T2 a 3 s. NOTA: As etapas 2.1.5 a 2.1.9 são adaptadas para serem usadas com o temporizador específico (ver Tabela de Materiais). Para obter instruções adicionais sobre outros usos para este produto, consulte o manual do usuário correspondente.</li>
	</ol>
</li>
	<li>
Tratamento de fumaça de cigarro NOTA: Este sistema permite o uso de 1-4 câmaras de exposição murina ao mesmo tempo.<ol>
<li>Ligue o compressor de ar e espere o alarme de segurança desligar sozinho.</li>
		<li>Ligue o controlador da válvula.</li>
		<li>Transfira 5 camundongos para cada uma das quatro câmaras de exposição com tampas removíveis herméticas com um volume de 8,5 L. Coloque as quatro câmaras de exposição com ratos dentro de um Gabinete de Segurança Biológica de Fluxo De Fluxo Classe II Tipo B2.</li>
		<li>Dentro do armário de segurança biológica de fluxo laminar, acenda um cigarro e insira o cigarro dentro da câmara de cigarros. Use cigarros comercialmente disponíveis que contenham 15 mg/cig tar e 1,1 mg/cig nicotina8 em comparação com os cigarros de pesquisa Kentucky 3RF4 (9,5 mg/cig tar e 0,73 mg/cig nicotina)9.</li>
		<li>Ligue as válvulas do controlador da válvula que correspondem às câmaras que estão atualmente em uso. A exposição é dividida em 2 fases: (T1) o ar limpo é bombeado para a câmara de exposição por 20 s e (T2) o fluxo de ar faz com que o cigarro queime e a fumaça da câmara de cigarro seja bombeada para a câmara de exposição por 3 s. Deixe o cigarro queimar completamente até chegar ao filtro.<ol>
<li>Ajuste as configurações do temporizador para executar uma média de ~10 puffs/cigarro durante um período de ~4 minutos. Observe que o temporizador e o sistema são facilmente personalizáveis para melhorar ou diminuir o regime de dosagem de CS de acordo com as necessidades de pesquisa dos pesquisadores.</li>
		</ol>
</li>
		<li>Remova o filtro de cigarro e descarte-o colocando a bituca de cigarro em um copo de vidro com água para apagar a chama e umedecer o odor.</li>
		<li>Certifique-se de que a câmara de cigarros está fechada novamente e sem um cigarro. Deixe a máquina bombear ar limpo por 10 minutos. É de extrema importância manter o monitoramento constante dos animais vertebrados expostos à SC. Este regime de exposição é otimizado para 5 camundongos fêmeas com mais de 9 semanas de idade por câmara de exposição.</li>
		<li>Repita os passos 2.3.4 a 2.3.7 três vezes para um total de 4 cigarros por câmara por dia. Esse procedimento se repete 5 dias por semana enquanto o pesquisador precisar de seus experimentos.</li>
		<li>Remova os camundongos das câmaras de exposição de volta às suas gaiolas correspondentes.</li>
		<li>Desligue o controlador da válvula e o compressor de ar.</li>
		<li>Remova a exposição e as câmaras de cigarro e lave com água e sabão para remover qualquer resíduo de piche.</li>
		<li>Deixe as câmaras secarem completamente antes de usá-las novamente.</li>
	</ol>
</li>
</ol>

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+gd+T+cells+in+cigarette+smoke+and+influenza+infection.">Protective role of gd T cells in cigarette smoke and influenza infection.</a> Nature Mucosal Immunology. 11, 834-908 (2018).</li><li>Kim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+induces+intestinal+inflammation+via+a+Th17+cell-neutrophil+axis.">Cigarette smoke induces intestinal inflammation via a Th17 cell-neutrophil axis.</a> Frontiers in Immunology. 10, 1-11 (2019).</li><li>Lu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-22+inhibits+the+histone+deacetylase+HDAC4+to+promote+T+H+17+cell+-+dependent+emphysema.">The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote T H 17 cell - dependent emphysema.</a> Nature Immunology. 16, 1185-1194 (2015).</li><li>Hendrix, A. Y., Kheradmand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Role of Matrix Metalloproteinases in Development, Repair, and Destruction of the Lungs. Progress in Molecular Biology and Translational Science. 148, (2017).</li><li>Siggins, R. W., Hossain, F., Rehman, T., Melvan, J. N., Welsh, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+alters+the+hematopoietic+stem+cell+niche.">Cigarette smoke alters the hematopoietic stem cell niche.</a> Med Sci. 2, 37-50 (2014).</li><li>Kheradmand, F., You, R., Gu, B. H., Corry, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+and+DNA+cleavage+promote+lung+inflammation+and+emphysema.">Cigarette smoke and DNA cleavage promote lung inflammation and emphysema.</a> Transactions of the American Clinical and Climatological Association. 128, 222-233 (2017).</li><li>Ha, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Menthol+attenuates+respiratory+irritation+and+elevates+blood+cotinine+in+cigarette+smoke+exposed+mice.">Menthol attenuates respiratory irritation and elevates blood cotinine in cigarette smoke exposed mice.</a> PLoS ONE. , 1-16 (2015).</li><li>Moreno-Gonzalez, I., Estrada, L. 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Aqui fornecemos as informações necessárias para a construção de um aparelho para WBIS de ratos para CS. Após a instalação do sistema, é extremamente importante que os pesquisadores calibram o sistema com base na dose entregue de nicotina ou cotinina em animais. O aparelho contém um temporizador e medidores de pressão que podem ser usados para ajustar o volume do puff de cigarro, a frequência do puff, o período combinado de exposição à fumaça e os intervalos de descanso que os animais recebem entre cada c...

O tabagismo continua sendo uma das principais causas de doenças evitáveis nos EUA, apesar do declínio constante do número de adultos fumantes de cigarros nos últimos 50-60 anos1. É amplamente conhecido que o tabagismo está ligado a múltiplas doenças dos pulmões e do sangue, incluindo doença pulmonar obstrutiva crônica (DPOC), um grupo de doenças que inclui enfisema e bronquite crônica2,3,4. De acordo com o Center for Disease Control (CDC), em 2014, a DPOC foi a terceira principal causa de morte nos Estados Unidos, com mais de 15 milhões de americanos sofrendo dessa doença5.
CS também tem sido recentemente associado a um maior risco de desenvolver hematopoiese clonal (CH)6,7, uma condição na qual uma única célula-tronco hematopoiética produz desproporcionalmente uma grande porcentagem do sangue periférico de uma pessoa. Este achado indica uma conexão potencial entre o tabagismo e a função da medula óssea. Dadas as implicações de saúde generalizadas e altamente significativas da CS e dado que os modelos murinos de doenças são uma pedra angular do progresso na pesquisa biomédica, é útil desenvolver sistemas eficientes e acessíveis para o modelo CS em camundongos.
Aqui, fornecemos um guia passo-a-passo para a construção de um sistema acessível para o tratamento e estudo dos efeitos in vivo da SC sobre enfisema pulmonar e homeostase de medula óssea. A montagem deste equipamento não exige que o usuário tenha conhecimento especializado e, portanto, permite a montagem DIY.

<table>
	<tbody>
		<tr>
			<td>1 in fastener</td>
			<td>Lowes</td>
			<td>756990</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in Barbed Y connector</td>
			<td>VWR</td>
			<td>89093-282</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in straight tubing connector</td>
			<td>VWR</td>
			<td>62866-378</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 hex nipple</td>
			<td>Lowes</td>
			<td>877221</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 in threaded coupling fitting</td>
			<td>Lowes</td>
			<td>877208</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 in threaded male adapter nipple fitting</td>
			<td>Lowes</td>
			<td>877243</td>
			<td></td>
		</tr>
		<tr>
			<td>10/32 (M) threaded straight connector</td>
			<td>Bimba</td>
			<td>EB60</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in 90-degree elbow CPVC fitting</td>
			<td>Lowes</td>
			<td>22643</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in chlorinated polyvinyl chloride (CPVC) pipe</td>
			<td>Lowes</td>
			<td>23814</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in CPVC cap</td>
			<td>Lowes</td>
			<td>23773</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in CPVC Drip irrigation female adapter</td>
			<td>Lowes</td>
			<td>194629</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in diameter CPVC male adapter</td>
			<td>Lowes</td>
			<td>23766</td>
			<td></td>
		</tr>
		<tr>
			<td>8.5 L airtight container with lid (11.25in x 7.75in x 6 in)</td>
			<td>Komax</td>
			<td>N/A</td>
			<td>Listed as &#34;Komax Biokips Large Bread Box | (280-oz) Large Storage Container&#34;</td>
		</tr>
		<tr>
			<td>Glass drain tube (1.75 in diameter x 8 in length)</td>
			<td>KIMAX</td>
			<td>6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isonic Solenoid Valves</td>
			<td>Bimba</td>
			<td>V2A02-AW1</td>
			<td></td>
		</tr>
		<tr>
			<td>Marlboro Red 100&#39;s</td>
			<td>Marlboro</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen swivel barbed connector</td>
			<td>Global Medical Solutions</td>
			<td>RES002</td>
			<td></td>
		</tr>
		<tr>
			<td>Panasonic Timer LT4H-W</td>
			<td>Panasonic</td>
			<td>LT4HW</td>
			<td>Item was built-in the valve controller by Shepherd Controls &#38; Associates</td>
		</tr>
		<tr>
			<td>Pressure regulator</td>
			<td>Allied Electronics and Automation</td>
			<td>70600552</td>
			<td>Also listed as &#34;Norgren R07-100-RGKA&#34;</td>
		</tr>
		<tr>
			<td>Rubber stopper # 1 (one hole)</td>
			<td>VWR</td>
			<td>59581-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber stopper # 8.5 (one hole)</td>
			<td>VWR</td>
			<td>59581-389</td>
			<td></td>
		</tr>
		<tr>
			<td>Scireq inExpose system</td>
			<td>Scireq and Emka Technologies</td>
			<td>N/A</td>
			<td>Commercial system used for comparison with our DIY WBIS</td>
		</tr>
		<tr>
			<td>Straight barbed fitting (8mm opening)</td>
			<td>VWR</td>
			<td>10028-872</td>
			<td></td>
		</tr>
		<tr>
			<td>Thread Sealant tape</td>
			<td>Lowes</td>
			<td>1184243</td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded port adaptor</td>
			<td>Bimba</td>
			<td>P1SA1</td>
			<td></td>
		</tr>
		<tr>
			<td>Timeter Aridyne 2000 Medical Air Compressor</td>
			<td>MFI Medical</td>
			<td>AHC-TE20</td>
			<td></td>
		</tr>
		<tr>
			<td>Timeter flowmeter</td>
			<td>Allied Healthcare Products</td>
			<td>15006-03YP2</td>
			<td>Also listed as &#34;Puritan Air Meter&#34;</td>
		</tr>
		<tr>
			<td>Valve Control system</td>
			<td>Shepherd Controls and Associates</td>
			<td>N/A</td>
			<td>Company custom designed the valve control system for this model.</td>
		</tr>
		<tr>
			<td>Vinyl pipes</td>
			<td>Vitality Medical</td>
			<td>RES3007</td>
			<td></td>
		</tr>
	</tbody>
</table>

cigarette smoke exposure in mice using a whole-body inhalation system

Cerca de 14% dos adultos nos Estados Unidos foram reportados a fumar cigarros em 2018. Os efeitos da fumaça do cigarro (SC) nos pulmões e nas doenças cardiovasculares têm sido amplamente estudados, no entanto, o impacto da SS em outros tecidos e órgãos, como sangue e medula óssea, permanece incompletamente definido. Encontrar o sistema apropriado para estudar os efeitos da CS em roedores pode ser proibitivamente caro e exigir a compra de sistemas disponíveis comercialmente. Assim, pretendemos construir um sistema acessível, confiável e versátil para estudar os efeitos patológicos da CS em camundongos. Esta configuração de exposição à inalação do corpo inteiro (WBIS) imita a respiração e o inchaço dos cigarros, alternando a exposição ao CS e ao ar limpo. Aqui mostramos que este sistema "faça você mesmo" (DIY) induz inflamação das vias aéreas e enfisema pulmonar em camundongos após 4 meses de exposição à fumaça de cigarro. Os efeitos da inalação do corpo inteiro (AIC) da CS sobre as células hematopoiéticas e progenitoras (HSPCs) na medula óssea usando este aparelho também são mostrados.

Uma das principais marcas da exposição à CS é o enfisema que é caracterizado pelos danos e destruição de sacos de ar (alvéolos) no pulmão. Assim, os experimentos iniciais se concentraram na capacidade do sistema DIY de provocar alterações enfisemasas nos pulmões de camundongos fêmeas após repetida exposição do corpo inteiro à SC. O regime de dosagem de CS foi escolhido com base em nossas publicações anteriores em que utilizamos o sistema DIY descrito aqui para tratar camundongos com CS e estudar a fisi...

Watch this Scientific Journal Video about Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System at JoVE.com

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Este protocolo demonstra o estudo dos efeitos fisiofisiológicos da fumaça do cigarro (SC) com um sistema de exposição de inalação total (WBI) construído internamente. Este sistema pode expor animais a CS sob condições repetíveis controladas para pesquisa de efeitos mediados pela CS em enfisema pulmonar e hematopoiese.

Exposição à fumaça de cigarro em camundongos usando um sistema de inalação de corpo inteiro

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

cigarette-smoke-exposure-in-mice-using-a-whole-body-inhalation-system

Research

JoVE Journal

Immunology and Infection

6.1K visualizações.  Baylor College of Medicine. Este protocolo demonstra o estudo dos efeitos fisiofisiológicos da fumaça do cigarro (SC) com um sistema de exposição de inalação total (WBI) construído internamente. Este sistema pode expor animais a CS sob condições repetíveis controladas para pesquisa de efeitos mediados pela CS em enfisema pulmonar e hematopoiese.

Vídeo: Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Usando imagens Bioluminescent para Investigar sinergismo entre Streptococcus pneumoniae E Vírus da Influenza A em Ratos infantil

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Imunologia e Infecção

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Desenvolvimento de um marcador negativo selecionáveis ​​para Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Inferência genotípica do HIV-1 Tropismo Usando de base populacional de Seqüenciamento V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Usando a luciferase Infecções bacterianas Imagem em camundongos

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia cultura do Sistema Nervoso Central Neonatal e Adulto

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Visualização de IL-expressando-22 Linfócitos Usando ratos repórter

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Indução de paralisia e lesão do sistema Visual em camundongos por específicos de células T para Neuromielite Óptica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Camundongos NOG humanizados para exposição intravaginal ao HIV e tratamento da infecção pelo HIV

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Estudando efeitos da fumaça do cigarro na infecção por pseudomonas em células epiteliais pulmonares

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infecção recorrente do trato urinário de Escherichia coli desencadeada por gardnerella vaginalis exposição bexiga em camundongos

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...