This work was supported by AI101263 from the National Institutes of Health (T.V.B), and by a grant from The Hartwell Foundation (M.L.M). M.L.M. is a member of the Duke Comprehensive Cancer Center.

NOTA: Todos os procedimentos experimentais que envolvam a utilização de ratos foram feitas de acordo com os protocolos aprovados pelo cuidado e uso Comitê da Universidade de Duke Animal. 1. Preparação da intubação traqueal Cannula <ol><li> Corte a ponta da agulha de um 2 polegadas 14 G angiocathether agulha com alicate de corte. </li><li> Comprima o lúmen da agulha fechado com alicate de bico. </li><li> Corte a parte comprimida para baixo à extremidade da porção de lúmen aberto com alicates de corte, e depois cortar as extremidades de ambos os lados da extremidade remanescente a 30 - 45 ° ângulo da borda da agulha. </li><li> Coloque a parte inferior 3 - 4 mm de comprimento da cânula para a área aberta do alicate deslizamento-conjunta rectas e puxar para cima até uma ligeira curva para cima foi formado no final. NOTA: Este irá facilitar a dirigir o cateter para dentro da traqueia (Figura 1). </li><li> Use lixa fina para alisar as arestas na extremidade da cânula. </li><li&gt; Devolva o instrumento rombo na tubulação angiocateter. Certifique-se de que o tubo é ligeiramente mais curto do que a agulha embotada. </li></ol> Procedimentos 2. Pré-cirúrgicos <ol><li> Configure o local para o procedimento, colocando um microscópio cirúrgico definido como aumento de 10x sobre a área de operação. </li><li> Coloque uma almofada de aquecimento na área de operação e cubra com um pano absorvente limpo. </li><li> Configurar um monitor de frequência cardíaca e oxigenação do sangue perto do campo cirúrgico. </li><li> Pesar o rato para sistemas baseados em peso medicação dosagem (por exemplo, a analgesia pós-operatória, os antibióticos, ou anticorpo linfócitos esgotando). NOTA: Típico ratos adultos pesam entre 350-450 g. </li><li> Sedar o rato vaporizado com 3% de isoflurano-O2 (3 L / min) usando uma câmara de indução ventilada para um sistema de eliminação de resíduos de gás anestésico contendo carvão activado. Permitir 5 min para a indução anestésica para fornecer anestesia profunda necessária para entubação endotraqueal. </li><li> Administrar carprofeno ou Meloxicane (4,4 mg / kg) por via subcutânea a ratos antes de prosseguir com a intubação e cirurgia. </li><li> Use cortador elétrico para fazer a barba do pescoço e peito do rato anestesiado antes da intubação. </li></ol> 3. Intubation <ol><li> Preparar a configuração intubação por fricção uma pequena quantidade de lubrificante (por exemplo, gel KY) na extremidade da cânula de intubação. Ligar o ventilador para iniciar o fluxo de gás através do tubo de isoflurano conector. </li><li> Transferir o rato para o aparelho de intubação e suspender o rato sobre a barra de metal por sua dentes incisivos superiores (Figura 2A). NOTA: Alguns aparelhos de intubação pode ser comprado online. O modelo mostrado aqui é caseiro usando uma folha de acrílico moldado em torno de seções de uma polegada pedaço 2 x 4 lombar usando um maçarico de butano, e reforços são presos com clorofórmio. O bar (ou fio de metal) é presa por fazer furos mais de fita adesiva colocados no side do acrílico para impedir uma fragmentação ou fissuras. </li><li> Trans-iluminar o pescoço do rato por posicionamento de uma fonte de luz de alta intensidade flexível 1 - 2 cm da superfície ventral do pescoço (Figura 2B). </li><li> Utilizar um par de fórceps estudante padrão normalizados para puxar a língua suavemente para cima e para o lado dos dentes de baixo. Aderência a língua entre o polegar eo dedo indicador de uma mão, enquanto coloca a superfície interior plana de um lado da pinça contra a extremidade inferior da língua. Pressione ventralmente para expor a epiglote e a abertura da laringe (Figura 2C). </li><li> Visualize as cordas vocais e orientar o arrebitado final, embotada da cânula de intubação anteriormente através da glote aberta na traquéia até o centro da angiocateter toca os incisivos. </li><li> Retirar o estilete metal, e conectar o tubo de anestesia para a abertura angiocateter para início da ventilação com isoflurano. NOTA: O flo típico de oxigêniotaxa de w para o ventilador é de 3 L / min, com 3% de isoflurano. <ol><li> Definir o ventilador a uma taxa de 60 respirações / min, em um modo de ventilador de volume controlado que atinge uma pressão de ~ 12 - 14 mmHg. Use uma pressão expiratória final positiva (PEEP) de 3 cm H 2 O. </li></ol></li><li> Observar a expansão da parede torácica bilateral em sincronia com o ventilador para assegurar a colocação adequada do tubo endotraqueal. </li><li> Aplicar recomenda-veterinário pomada para os olhos do rato para evitar o ressecamento e sob anestesia. </li><li> Aplicar veterinário pomada nos olhos do rato para evitar a secura e sob anestesia. </li><li> Prenda o tubo endotraqueal para a cabeça do rato com uma tira de fita de tecido. Garanta uma ligação sólida entre o tubo endotraqueal e a tubulação ingresso anestésico. </li><li> Fixe a oxigenação do sangue e monitor cardíaco para o pé do rato e iniciar o monitoramento. Confirme anesthetization adequada ao confirmar que não há reação ao quee-pinch. </li></ol> 4. Thoracotomy e Timectomia <ol><li> Siga técnica asséptica padrão para a totalidade do processo. <ol><li> Limpe a área e operacional a mesa de trabalho e desinfectar com uma solução de etanol a 70%. </li><li> Use luvas cirúrgicas estéreis durante o procedimento, e autoclave todos os instrumentos e materiais utilizados durante o procedimento. </li><li> Aplicar providone-iodo para a área de todo o peito e deixe-a secar. Em seguida, limpar a superfície da pele com etanol a 70% em gaze. Cubra o rato com filme plástico transparente cortar um buraco para expor o campo operatório estéril. </li></ol></li><li> Identificar fúrcula na região torácica superior. Faça uma incisão na linha média 2 centímetros através da pele a partir 2-3 mm acima da fúrcula e estendendo-se distalmente linha média ao longo do esterno com uma tesoura Shea atraumáticas. </li><li> Realize uma sternotomy 1,5 centímetros mediana de fúrcula novamente usando uma tesoura Shea atraumáticas. KEEP da borda inferior das tesouras apenas por baixo do esterno e avançar lentamente. </li><li> Insira um pequeno afastador Alm logo abaixo do esterno separadas e aberto para revelar o pré-traqueal músculos cinta (esterno e músculos esternotireóideo). Separe os músculos cinta pré-traqueais usando uma pinça Graefe contundentes. NOTA: Neste ponto, a traqueia pode ser visto, o tubo de intubação e deve ser visualizada no interior da traqueia. </li><li> Coloque os dentes do pequeno afastador Alm debaixo dos músculos cinta separadas e esterno. Abra o retractor para expor a face superior do timo. </li><li> Use uma pinça Dumont finas para libertar as arestas laterais do tecido do timo e expor os lóbulos inferiores do timo. </li><li> Puxe o timo suavemente superiormente para o local da incisão aberta, tendo o cuidado de evitar o contacto com as veias cavas, subclávia e carótida vasos superiores, e para minimizar a interrupção do revestimento pleural delicado entre o timo e os pulmões. </li><li> Como tele thymic vasos são dissecados e revelou, usar micro-tesoura para dividi-los. Use um cotonete para manter a pressão sobre os vasos para hemostasia, se necessário. </li><li> Entregar os lobos do timo mais baixos na incisão e, em seguida, lisar acentuadamente os anexos posteriores. Retire o timo intacta e inspecione cuidadosamente o timo removido para quaisquer seções desaparecidas. Nota: Pequenas linfonodos torácicos pode ser visualizado em torno do timo e são muitas vezes difíceis de diferenciar de tecido do timo. Estes serão os nós redondas discretas com uma aparência semelhante a tecido tímico, mas não será em continuidade com o timo. </li><li> Retire o afastador Alm e fechar os músculos esterno hioideo e sternothyoid com dois interrompido 5-0 Maxon. </li><li> Aplique 2 gotas de aplicação de cola grau cirúrgico através das suturas para selar o mediastino sob ventilação com pressão positiva. NOTA: Isto irá diminuir a incidência de pneumotórax e hematoma. </li><li> Feche a sternum com dois interrompido fio de seda 4-0 em uma agulha de corte. Insira a agulha através de espaços entre as costelas, tendo o cuidado para guiar a agulha logo abaixo do esterno para evitar a camada muscular subjacente. </li><li> Fechar a camada de pele com uma sutura de nylon 4-0 execução. Descontinuar o isoflurano neste momento para encurtar o período de recuperação da anestesia pós-operatória. </li><li> Limpe o local da incisão e pele circundante com gaze embebida em soro fisiológico. </li><li> Aplique várias gotas de bupivicaína (0,25%) para a anestesia local, seguido por 1 - 2 gotas de cola de cianoacrilato sobre a incisão para selar a incisão. </li><li> Continuar a ventilar até o rato mostra sinais de esforço respiratório independente e começa a se mover suas extremidades. Extubação o rato e permitir que ele se recuperar sob observação em uma jaula colocada sobre uma almofada de aquecimento. </li><li> Administrar Buprenorfina (0,01-0,05 mg / kg) por via subcutânea ao rato imediatamente no pós-operatório e repetir a cada 8-12 horas para 48 horas post-operatório (normalmente 5 doses no total). Aos 24 e 48 horas de pós-operatório, administrar Carprofen ou Meloxicane (4,4 mg / kg) por via subcutânea (3 doses totais, incluindo a dose pré-operatório). </li><li> Continue a acompanhar até que o rato está se movendo ao redor da gaiola. Em quaisquer sinais de desconforto respiratório, realizar re-intubação e re-exploração do peito. <ol><li> Não deixe um animal sem supervisão até que ele recuperou a consciência suficiente para manter decúbito esternal. </li><li> Não devolva um animal que passou por uma cirurgia para a companhia de outros animais até que esteja totalmente recuperado. </li></ol></li><li> Retirar as suturas da camada da pele uma semana de pós-operatório. </li></ol>

<ol>
	<li>Hard, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymectomy+in+the+neonatal+rat.">Thymectomy in the neonatal rat.</a> Lab. Anim. 9 (2), 105-110 (1975).</li><li>Kobayashi, E., 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=A+technique+for+complete+thymectomy+in+adult+rats.">A technique for complete thymectomy in adult rats.</a> J. Immunol. Methods. 1759 (94), 0 (1994).</li><li>Delrivi&#232;re, L., Gibbs, P., Kobayashi, E., Kamada, N., Gianello, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+technique+of+complete+thymectomy+in+adult+rats+without+tracheal+intubation.">New technique of complete thymectomy in adult rats without tracheal intubation.</a> Microsurgery. 18 (1), 6-8 (1998).</li><li>Walker, K. G., Jacques, B. C., Tweedle, J. R., Bradley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simpler+technique+for+open+thymectomy+in+adult+rats.">A simpler technique for open thymectomy in adult rats.</a> J. Immunol. 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The authors have nothing to disclose.

O presente protocolo para thymectomy completa fornece uma abordagem minimamente invasiva com um fecho de incisão de três camadas com aplicação de cola cirúrgica projetado para minimizar as complicações. A remoção completa do timo foi demonstrado pela perda de células T naive e por IHC coloração do tecido linfóide do mediastino para citoqueratina. O procedimento de thymectomy rato adulto tem sido complicada por taxas de mortalidade variando de 1,5 - 20% devido a complicações pe...

Desde o início da década de 1960, o timo foi reconhecido o papel crítico no desenvolvimento da tolerância imunológica central. Roedor timectomia provou ser um processo essencial para definir o papel do timo na diferenciação de linfócitos, a auto-tolerância, e imunotolerância na definição de transplante aloenxerto e metástase do tumor. A remoção do timo do rato permite a estudos que envolvem a depleção de células T ou a transferência adoptiva de populações de células T definidos, sem o reaparecimento de células T naive nativas. Timectomias em roedores neonatais pode ser realizada utilizando uma técnica de sucção com resultados fiáveis ​​1. Em ratos adultos, esta técnica está associada com uma taxa de mortalidade de 20% e, frequentemente, resulta em uma timectomia 2 incompleta. Para alcançar consistentemente thymectomy completo em ratos adultos, é necessária a exposição aberta do mediastino por esternotomia mediana. No entanto, esteprocedimento está associada a complicações que incluem lesão traqueal, hemorragia e pneumotórax levando a uma taxa global de mortalidade variando 1,5-6% 2- 4. Ao longo das duas últimas décadas melhorias nas técnicas de timectomia diminuíram complicações perioperatórias e melhoraram as taxas de sobrevivência. A entubação endotraqueal permitindo a ventilação com pressão positiva diminuiu as taxas de pneumotórax 5. Métodos de intubação anteriormente descrito gama de exposição aberta ao traquéia para métodos menos invasivos, utilizando a visualização direta das cordas vocais. As complicações associadas ao procedimento de intubação traqueal incluem lesão, a ruptura das cordas vocais, intubação esofágica não intencional, e hemorragia decorrente da punção cardíaca ou laceração da veia cava superior. Além disso, a proximidade dos lobos do timo mais baixos para o revestimento pleural podem resultar em pneumotórax. Aqui nós descrevemos uma técnica dethymectomy através de um minimamente invasiva dois centímetros da pele incisão na sequência de um método simples de entubação endotraqueal usando um angiocateter embotados-end e iluminação traqueal transcutânea. O thymectomy envolve um 1,5 centímetros sternotomy e um encerramento de três camadas com aplicação de cola cirúrgica para selar o mediastino e minimizar a incidência de hemorragia e complicações respiratórias. Este método resulta de forma fiável, timectomia completa, como evidenciado pelo desaparecimento das células T ingénuas CD4 + e CD8 + a seguir a timectomia e a ausência de tecido do timo de coloração IHC. Tempos operatórios e mortalidade peri-processual são mantidos a um mínimo.

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Name of Reagent/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:152px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">2&#34; 14-gauge angiocatheter</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Operating microscope</td>
			<td style="width:117px;">Zeiss</td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Warming pad</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Heart rate and blood oxygenation monitor for rodents with foot sensors</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-8010, ST1 72-8098 (Rat foot sensor)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Intubation apparatus (plastic with metal bar at the top)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;">See Figure 2&#160;</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:283px;">Small animal anesthesia system with induction box, isoflurane tank and O2 tank</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-6420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:283px;">Small animal ventilator with tubing</td>
			<td style="width:117px;">CWE</td>
			<td style="width:152px;">12-02000 (ventilator)and 12-04000 (external valve assembly for mice/rats)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High-intensity Fiber-Optic Illuminator</td>
			<td style="width:117px;">Dolan Jenner</td>
			<td style="width:152px;">EEG 2823M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Student standard pattern forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">91100-16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine straight scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14060-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt-tipped Shea scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14105-12</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Small Alm retractor (for sternum)</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">17008-07</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt Graefe forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11050-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine Dumont forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5-0 Maxon sutures</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">4-0 Silk sutures (with cutting needle)</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">6-0 Nylon suture</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cyanoacrylate glue (Endermil)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Lubrication gel</td>
			<td style="width:117px;">Akorn Animal Health</td>
			<td style="width:152px;">NDC 17478-162-35</td>
			<td></td>
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complete thymectomy in adult rats with non-invasive endotracheal intubation

Timectomia em roedores neonatais é um procedimento estabelecido e confiável para estudos imunológicos. No entanto, em ratos adultos, complicações de hemorragia e pneumotórax de ruptura pleural pode resultar numa taxa de mortalidade significativa. Este protocolo é um método simples de thymectomy rato que utiliza um mini-esternotomia e entubação endotraqueal. Intubação é realizado com um método não-invasivo e de fácil reprodução e permite a ventilação com pressão positiva para evitar pneumotórax e uma via aérea controlada que permite tempo suficiente para a dissecção timo cuidado para minimizar a interrupção pleural. A 1,5 centímetros incisão esternal diminui o contato com os vasos do mediastino e pleura, enquanto ainda fornece a visualização completa do timo. Após a exposição do mediastino, o timo é removido por dissecção romba sob ampliação. O espaço pleural é então selado por fecho de sutura dos músculos pré-traqueal, seguido pela aplicação de cola cirúrgica. Otórax é então fechada por sutura de encerramento do esterno, seguido por fecho de sutura da pele. Todos os timectomias estavam completos como evidenciado pela imuno-histoquímica (IHQ) coloração de tecidos do mediastino, e ausência de células T naive por citometria de fluxo, e que o processo tinha uma taxa de sobrevivência de 96%. Esse método é apropriado quando a timectomia completa com o mínimo de complicações é desejado para mais estudos imunológicos em ratos atímicos adultos.

Este procedimento foi realizado em ratos Lewis adultos (n = 26). O tempo médio de operação foi de 15 ± 3 min. Não houve mortalidade intra-operatória. O tempo de intubação média - de colocação de rato em cima do aparelho de intubação para o estabelecimento da ventilação - foi de 45 ± 5 segundos. 24 ratos tiveram uma recuperação completa da operação, sem evidência de dificuldades respiratórias ou hemorragia através dia de pós-operatório (DPO) 14. Um rato desenvolvido dificuldade respiratória em P...

Watch this Scientific Journal Video about Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation at JoVE.com

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Timectomia completo em ratos adultos com não-invasiva entubação endotraqueal

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

complete-thymectomy-adult-rats-with-non-invasive-endotracheal

Research

JoVE Journal

Immunology and Infection

16.4K visualizações.  Duke University Medical Center. Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described. 

Vídeo: Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Não-invasiva por imagem do Homing leucócitos e Migração In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Imunologia e Infecção

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Não-invasivos de imagem de candidíase disseminada em larvas do peixe

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

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

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Não-invasiva de Imagem Óptico da vasculatura linfática de um rato

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

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 ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Modelo não-invasiva da neuropatogênicos<em&gt; Escherichia coli</em&gt; Infecção no Rato Neonatal

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

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

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

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

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagem não invasivo do imunitário inatas Response em um modelo de peixe-zebra larval de<em&gt; Streptococcus iniae</em&gt; Infection

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

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

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

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non-invasive<em&gt; In Vivo</em&gt; Fluorescência de imagem óptico de Inflamatória MMP Atividade Usando um Agente de Imaging Activatable fluorescente

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análise de Linfócitos extravasamento Usando um<em&gt; In Vitro</em&gt; Modelo do Sangue-Cérebro humano Barreira

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Uma maneira não invasiva de isolar e células de fenótipo da Conjuntiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...