Este trabalho foi apoiado por números conceder R01AI055001 e R01AI082127 de NIAID a NRM e Award Fellowship SREB Dissertação de JLB.

1. Fluoresceína rotulados dextran e GSL I-isolectin B 4 injeções veia facial para rotular vasculatura embrionárias <ol><li> Prepare FITC-dextran (50ug/mL) em tampão fosfato salino (PBS) ou GSL 1 - isolectin B 4 (20ug/200uL) em PBS em um tubo Eppendorf 1,5 ml e aquecer a 37 ° C. Adicionar 100uL de estoque 1,25 mM Fast Green / PBS para a solução FITC-dextran (volume total de 1ml) e 180uL de estoque 1,25 mM Fast Green / PBS a um GSL - isolectin B 4 (200uL volume total), de modo que a solução é visivelmente azul. </li><li> E14.5-E18.5 dissecar embriões e saco vitelino juntos, deixando o talo alantóide (artéria e veia umbilical) intacta. </li><li> Transferência de embriões para um prato novo Petrie (60 X 15 mm) e mergulha-as na PBS em temperatura ambiente. </li><li> Posição do embrião para fornecer uma visão sagital da cabeça / face. Use uma pinça de dissecação micro suavemente para entender o embrião na cabeça. </li><li> Usando uma agulha 30G, injetar 50uL FITC-dextran (50ug/mL) ou GSL 1 - isolectin B 4 (20ug em 200uL PBS) na veia facial apontando a agulha para a parte traseira da cabeça. </li><li> Quando a tintura é visível na veia umbilical, retire a agulha e separar o embrião do talo alantóide (artéria e veia umbilical). </li><li> Após as injeções, permitem embrião permanecer em PBS à temperatura ambiente por 2-3 minutos para que o corante circula por todo o embrião. </li></ol> 2. Todo-mount análise da vascularização da pele <ol><li> Depois de permitir que o corante para circular por todo o embrião, retire amostras de pele das regiões dos membros, costas e estômago, etc 8,9. </li><li> Lavar amostra de pele no frio PBS, e fixar em 4% PFA / PBS por 2 horas 8,9. Lavar 3 vezes durante 10 minutos cada, em frasco com 2 ml clara 4mL frio PBS. </li><li> Colocar a amostra de pele em um microscópio de slides e adicione 100 ml de montagem de mídia para cada slide e uma tampa de vidro. </li><li> Permitir slides para secar em uma área de armazenamento. </li><li> Vá para a Etapa seção &quot;Aquisição de Imagem&quot; 2. </li></ol> 3. Multi-cor rotulagem de timo e vascularização do coração e células perivascular para criosecções (Continue da Seção 1, Passo 7) <ol><li> Embrião &#39;Flash congelar &quot;todo em nitrogênio líquido. Embriões podem ser e armazenadas a -80 ° C até a análise. </li><li> Alternativamente, dissecar timo, enxágüe em 4 ° C PBS, e fixar em 2 mL paraformaldeído 4% (PFA) / PBS por 2 horas. Lavar 3 vezes durante 10 minutos no frio PBS, thymi lugar em outubro, e congelar e armazenar até usar a -80 ° C. </li><li> Para cryosectioning, espalhados em outubro &#39;block&#39; uma seção e montar o embrião ou órgãos dissecados / tecidos para corte. </li><li> Corte de tecido congelado em 10 mM de espessura e recolher em slides. </li><li> Fix seções em acetona por 5-10 minutos. Lavar 3 vezes em TBS frio. </li><li> Bloco em 10% de soro de burro / TBS em uma câmara de umidade em temperatura ambiente. </li><li> Incubar seções de 1 hora durante a noite com 100 ul de anticorpos primários em câmara úmida a 4 ° C: neste exemplo, usamos ratos anti-rato-CD31 (1:100) ao endotélio rótulo, e de cabra anti-rato PDGFR β- (1:100) para rotular as células perivascular. É útil para cobrir slides com tiras cortadas individualmente Parafilm para garantir que o anticorpo é uniformemente distribuídos em toda a seção. </li><li> Após a incubação com o anticorpo primário, lave seções 3 vezes em TBS frio. Incube com 100 ul de anticorpos secundários apropriada por 30 minutos mínimo. </li><li> Lavar 3 vezes em TBS frio. Adicione 100 ml de montagem de mídia para cada slide e uma tampa de vidro. </li><li> Permitir slides para secar em uma área de armazenamento. </li><li> Vá para a seção &quot;Aquisição de Imagem&quot;. </li></ol> 4. Multi-cor rotulagem de timo vascularização e células perivascular para seções vibratome (Continue da Seção 1, Passo 7) <ol><li> Dissecar lobos do timo, do embrião e enxaguar em água fria PBS. </li><li> Fix timo em 4% PFA / PBS em temperatura ambiente por 2 horas. </li><li> Lavar em PBS-Triton X (0,15%) 3 vezes, 10 minutos e thymi lugar em um cartucho de plástico pequeno e submergir em 4% baixo derreter agarose / PBS (~ 4 ° C). Timo deve estar em contato com a parte inferior do cartucho. </li><li> Permitir agarose para solidificar em gelo (3-5 minutos). Use uma lâmina de barbear para cortar o excesso de agarose. Adicionar cola no bloco vibratome e aderir ao bloco de amostra. </li><li> Adicionar frio PBS para vibratome banho-maria até que a amostra ea lâmina estão imersos. </li><li> Definir a velocidade e amplitude (amplitude de alta e baixa velocidade moderada é ideal para o soft seções timo). A amplitude deve ser reduzida se as seções quebrar devido à agitação em excesso. </li><li> Corte de 50 seções um. </li><li> Usando um pincel, recolher seções em uma microplaca de 24 poços no frio PBS. </li><li> Seções de bloco em 500 l de soro de burro de 10% em PBS-Triton X (0,15%) por 30 minutos. </li><li> Seções incubar por 8 horas durante a noite com o anticorpo primário, tais como anti-CD31 e anti-PDGFR-β, Em uma coberta de microplacas de 24 poços. </li><li> Lavar 3 vezes em PBS-Triton X (0,15%) sobre um total de 8 horas a 4 ° C. </li><li> Seções de bloco no soro de burro de 10% em PBS-Triton X (0,15%) por 30 minutos. </li><li> Incubar seções de oito horas durante a noite a 4 ° C com anticorpos secundários apropriado. </li><li> Lavar 3 vezes em PBS-Triton X (0,15%) sobre um total de 8 horas a 4 ° C. </li><li> Re-fix amostras em PFA 4% / PBS por 30 minutos no gelo. </li><li> Lavar 3 vezes em PBS-Triton X (0,15%) mais de 30 minutos no gelo. </li><li> Desidratar as amostras através de um classificado MeOH / PBS-Triton X série: 25% MeOH, MeOH 50%, 75% MeOH, MeOH e 100% menos 10 minutos para cada etapa. Substituir MeOH 100% com MeOH fresco depois de 10 minutos e incubar por 1 hora em temperatura ambiente. </li><li> Em um recipiente de vidro, misture Babb (Benzyl Álcool: benzoato de benzila) em uma proporção de 1:2. Combine Babb com MeOH para uma concentração final de Babb 50% e MeOH 50%. Incubar a amostra em Babb: MeOH por 10-15 minutos. </li><li> Transferência da amostra para um recipiente de vidro com 100% de Babb e incubar por 10-15 minutos ou até eliminado, em temperatura ambiente. </li><li> Preencha deslize depressão (0,7 milímetros de profundidade) com Babb 100% frescos e de transferência de amostra para o lado. Adicionar tampa de vidro (n º 1.5) e selar com 2-3 camadas de unha polonês. Permitir unha polonês para endurecer no escuro à temperatura ambiente, em seguida, armazenar amostras a 4 ° C. Nota: Os slides devem ser completamente selados antes da aquisição da imagem confocal. As imagens devem ser adquiridas dentro de 12-24 horas, como corantes fluorescentes podem desaparecer em Babb. </li><li> Vá para a seção &quot;Aquisição de Imagem&quot;. </li></ol> 5. Aquisição de imagem <ol><li> Imagem 10 mM cortes congelados com um microscópio confocal utilizando o Plano de Apochromat-20X/0.8 objetivo (512 X 512 pixels) com 488 - (FITC-dextran/GSL 1 - isolectin B 4), 543 -, e as linhas 633-nm laser. </li><li> Adquirir confocal z-seções da pele montagem inteira e 50 mm de agarose-embedded seções usando o Plano Apochromat-10X/0.4 objetivo (512 X 512 pixels) com 488 - (FITC-dextran/GSL 1 - isolectin B 4), 543 -, e 633 nm linhas laser. Série Z-seções devem ser coletadas sequencialmente em 1-micron para cada respectivo canal. </li><li> Reconstruir série Z-seções usando Zeiss Axiovision 4.6 ou software de análise de imagem. </li></ol> 6. Resultados representativos: Rotulagem eficiente da vasculatura embrionárias é fundamental para avaliar os defeitos dos vasos sanguíneos em ratos embrionárias. A Figura 1 mostra a rotulagem específica de E16.5 vasos sanguíneos timo (1A-B) e co-rotulagem com CD31 (1B), além da coloração dos ventrículos direito e esquerdo (1E-F), respectivamente. A GSL I-B 4 isolectin protocolo para criosecções conforme descrito nas seções 1, 3 e 5 foi usado nesses experimentos. Todo-mount rotulagem da rede navio pele sangue em ratos E16.5, utilizando os protocolos descritos nos pontos 1, 2 e 5 é mostrado na Figura 1C-D. <img alt="Figura 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> Figura 1 Legend. FITC GSL I - isolectin B 4 injeções em veia facial E16.5 embriões de camundongos. a. Cryosection do timo embrionárias após a injecção. b. direta de CD31 co-rotulagem com B isolectin 4. c. e d. Whole-mount da injeção embrionárias da pele vasculatura seguinte. e. e f. Cryosection embrionárias de coração e. (à direita ventrículo) f. (ventrículo esquerdo) após a injecção.

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Todo o monte e PECAM-1 (CD31) coloração em cortes são os métodos convencionais para a rotulagem da vasculatura embrionárias em camundongos. Estes métodos requerem o uso de imunofluorescência direta e / ou indireta, e detergentes para permeabilizar o tecido mouse. Isso prova a ser um processo bastante oportuna. Aqui, nós empregamos FITC-dextran ou isolectin B 4 injeções veia facial diretamente rótulo a vasculatura embrionárias, eliminando a exigência de rotulagem passos de anticorpos. Além disso,...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> FITC-dextran </td><td> Sigma </td><td> FD150S-1G </td></tr><tr><td> Fluoresceína rotulados GSL 1 - B isolectin 4 </td><td> Vector Laboratories </td><td> FL-1201 </td></tr><tr><td> Verde rápido </td><td> Biomedicals MP </td><td> 195178 </td></tr><tr><td> PFA </td><td> Fluka </td><td> 76240 </td></tr><tr><td> Soro fetal bovino </td><td> Atlanta Biologicals </td><td> S11550 </td></tr><tr><td> Composto Temperatura ideal de corte (OCT </td><td> VWR </td><td> 25608-930 </td></tr><tr><td> Acetona </td><td> JT Baker </td><td> 9006-33 </td></tr><tr><td> Donkey Serum </td><td> Jackson </td><td> 017-000-121 </td></tr><tr><td> rato anti-CD31 do mouse, </td><td> BD PharMingen </td><td> 558736 </td></tr><tr><td> cabra anti-rato PDGFR β- </td><td> R &amp; D Systems </td><td> AF1042 </td></tr><tr><td> donkey anti-CD31 rato Alexa 647 (Invitrogen) </td><td> Biolegend </td><td> 102516 </td></tr><tr><td> burro anti-cabra Alexa 594 (Invitrogen) </td><td> Invitrogen </td><td> A11058 </td></tr><tr><td> Triton X -100 </td><td> Sigma-Aldrich </td><td> X-100 </td></tr><tr><td> Baixo derreter agarose / PBS </td><td> Sigma-Aldrich </td><td> A9414-25G </td></tr><tr><td> Metanol </td><td> Fisher Scientific </td><td> A413-4 </td></tr><tr><td> Álcool benzílico </td><td> Acros Scientific </td><td> 148390010 </td></tr><tr><td> Benzoato de benzila </td><td> Acros Scientific </td><td> 105860010 </td></tr><tr><td> Slides depressão </td><td> Fisher Scientific </td><td> S175201 </td></tr><tr><td> Fluorogel </td><td> Microscopia Eletrônica de Ciências </td><td> 17985-10 </td></tr><tr><td> Cobertura de vidro (22x22) -1,5 </td><td> Thermo Scientific </td><td> 152222 </td></tr><tr><td> Zeiss LSM 510 Meta Microscópio Confocal </td><td> Zeiss </td><td></td></tr><tr><td> Micro pinça de dissecação </td><td> Roboz </td><td> RS-5135 </td></tr><tr><td> Parafilm No. OM992 </td><td> Fisher Scientific </td><td> 13-374-16 </td></tr><tr><td> 12 e 24 microplacas bem </td><td> Scientific Evergreen </td><td> 222-8044-01F </td></tr><tr><td> Superfrost / Plus Lâminas de Microscopia </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> 4mL frascos claros </td><td> Científica Nacional </td><td> B7800-2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

O estabelecimento de uma rede funcional dos vasos sanguíneos é uma parte essencial da organogênese, e é necessário para a função do órgão ideal. Por exemplo, na formação adequada vascularização do timo e padronização é essencial para a entrada timócito no órgão e saída de células T maduras para a periferia. O arranjo espacial dos vasos sanguíneos no timo é dependente de sinais a partir do microambiente local, ou seja, células epiteliais tímicas (TEC). Vários relatórios recentes sugerem que a interrupção desses resultados no timo sinais de sangue 1,2 defeitos navio. Estudos anteriores descreveram técnicas usadas para rotular os 1,2 vasculatura neonatal e adulto timo. Nós demonstramos aqui uma técnica para a rotulagem dos vasos sanguíneos no timo embrionárias. Este método combina o uso de FITC-dextran ou Griffonia (Bandeiraea) Simplicifolia Lectina I (GSL 1 - isolectin B 4) injeções veia facial e coloração de anticorpos CD31 para identificar timo estruturas vasculares e PDGFR-β para rotular tímica perivascular mesênquima 3-5. A opção de usar ou seções criosecções vibratome também é fornecido. Este protocolo pode ser usado para identificar defeitos do timo vascular, que é crítico para a definição dos papéis dos TEC derivadas moléculas na formação do timo dos vasos sanguíneos. Como o método de rótulos toda a vasculatura, ele também pode ser usado para analisar as redes vascular em vários órgãos e tecidos em todo o embrião, incluindo pele e do coração 60-10.

Watch this Scientific Journal Video about A Method for Labeling Vasculature in Embryonic Mice at JoVE.com

A Method for Labeling Vasculature in Embryonic Mice

Este artigo descreve um método para a rotulagem de pele embrionária e vasos sanguíneos do timo.

Um método para Etiquetagem Vascularização no embrião do rato

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

a-method-for-labeling-vasculature-in-embryonic-mice

Research

JoVE Journal

Immunology and Infection

15.5K visualizações.  University of Georgia. Este artigo descreve um método para a rotulagem de pele embrionária e vasos sanguíneos do timo.

Vídeo: A Method for Labeling Vasculature in Embryonic Mice

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

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 ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizando Vírus da Dengue por meio Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

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 ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D Multimodalidade de imagens de<em&gt; Citrobacter rodentium</em&gt; Infecções em Ratos

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

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 ...

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 ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Um método para a medição da função da glândula salivar em ratos

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Trombose venosa profunda induzida por estase em camundongos monitorado por ultra-sonografia de alta frequência

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

Um método de nanopartículas prata para atenuar a síndrome de Atresia biliar em ratos

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...