Este trabajo fue apoyado por los números de concesión y R01AI055001 R01AI082127 del NIAID para MRN y Tesis SREB Beca de JLB.

1. Fluoresceína dextrano y GSL I-isolectina B 4 inyecciones vena facial para etiquetar vasculatura embrionaria <ol><li> Prepare FITC-dextrano (50ug/mL) en tampón fosfato salino (PBS) o GSL 1 - isolectina B 4 (20ug/200uL) en PBS en un tubo Eppendorf 1,5 ml y calentar a 37 º C. Añadir 100uL de acciones de 1,25 Fast Green / PBS a la solución de FITC-dextrano (volumen total de 1 ml) y 180uL de acciones de 1,25 Fast Green / PBS a la GSL 1 - isolectina B 4 (200uL volumen total), por lo que la solución es visiblemente azul. </li><li> Diseccionar E14.5 E18.5-embriones y el saco vitelino juntos, dejando el tallo alantoideo (arteria y vena umbilical) se mantienen intactas. </li><li> Transferencia de embriones a un nuevo plato de petri (60 x 15 mm) y sumérgelas en PBS a temperatura ambiente. </li><li> La posición del embrión para proporcionar una vista sagital de la cabeza / cara. El uso de micro pinza de disección para agarrar suavemente el embrión en la cabeza. </li><li> Utilizando una aguja 30G, se inyectan 50 ul FITC-dextrano (50ug/mL) o GSL 1 - isolectina B 4 (20ug en 200uL PBS) en la vena facial, apuntando la aguja hacia la parte posterior de la cabeza. </li><li> Cuando el medio de contraste es visible en la vena umbilical, retire la aguja y por separado el embrión del tallo alantoideo (arteria umbilical y la vena). </li><li> Después de las inyecciones, permiten embrión a permanecer en PBS a temperatura ambiente durante 2-3 minutos para que el colorante circula por todo el embrión. </li></ol> 2. Todo el montaje análisis de la vascularización cutánea <ol><li> Después de permitir que el medio de contraste a circular por todo el embrión, extraer muestras de la piel de las regiones de las extremidades, espalda y estómago, etc 8,9. </li><li> Lavar la piel de la muestra en PBS frío, y fijar en el 4% PFA / PBS durante 2 horas 8,9. Lavar 3 veces durante 10 minutos cada uno en un vial de 4 ml claro con 2 ml de PBS frío. </li><li> Coloque la muestra de piel en un microscopio de diapositivas y añadir 100 ml de montaje de los medios de comunicación en cada diapositiva y una cubierta de vidrio. </li><li> Permitir que se sequen en un área de almacenamiento en la oscuridad. </li><li> Continúe con el Paso sección &#39;Adquisición de imágenes &quot;2. </li></ol> 3. Multi-color etiquetado de timo y los vasos sanguíneos del corazón y las células perivasculares de cryosections (Continúa de la Sección 1, Paso 7) <ol><li> Embrión de todo &#39;Flash congelar en nitrógeno líquido. Los embriones se pueden y se almacenan a -80 ° C hasta su análisis. </li><li> Por otra parte, diseccionar el timo, el enjuague en PBS 4 ° C, y fijar en 2 ml de paraformaldehído al 4% (PFA) / PBS durante 2 horas. Lavar 3 veces durante 10 minutos en PBS frío, lugar thymi en octubre, y congelar y almacenar hasta su uso a -80 ° C. </li><li> Para muestras criostáticas, se extendió en octubre &#39;block&#39; y una sección de montaje en el embrión o los órganos diseccionados / tejidos para el corte. </li><li> Cortar el tejido congelado en 10 micras de espesor y se acumulan en las diapositivas. </li><li> Fix secciones en acetona durante 5-10 minutos. Lavar 3 veces en TBS frío. </li><li> Bloque en el burro 10% de suero / TBS en una cámara húmeda a temperatura ambiente. </li><li> Incubar las secciones de una hora durante la noche-con 100 l de anticuerpos primarios en una cámara húmeda a 4 ° C: en este ejemplo, el uso de rata anti-CD31 de ratón (1:100) al endotelio etiqueta, y de cabra anti-ratón PDGFR-β (1:100) para marcar a las células perivasculares. Es útil para cubrir las diapositivas individualmente cortar tiras Parafilm para asegurarse de que el anticuerpo se extienda uniformemente por toda la sección. </li><li> Después de la incubación con el anticuerpo primario, lave las secciones 3 veces en TBS frío. Incubar con 100 l de anticuerpos secundarios apropiados durante 30 minutos como mínimo. </li><li> Lavar 3 veces en TBS frío. Añadir 100 ml de montaje de los medios de comunicación en cada diapositiva y una cubierta de vidrio. </li><li> Permitir que se sequen en un área de almacenamiento en la oscuridad. </li><li> Pase a la sección &#39;Adquisición de imágenes &quot;. </li></ol> 4. Multi-color de etiquetado de la vasculatura del timo y células perivasculares de las secciones vibratome (Continúa de la Sección 1, Paso 7) <ol><li> Diseccionar los lóbulos del timo del embrión y enjuague con PBS frío. </li><li> Fix timo en el 4% PFA / PBS a temperatura ambiente durante 2 horas. </li><li> Se lavan con PBS-Triton X (0,15%) 3 veces, 10 minutos y thymi lugar en un cartucho de plástico y se sumergen en el 4% de baja fusión de agarosa / PBS (~ 4 ° C). El timo debe estar en contacto con la parte inferior del cartucho. </li><li> Permitir agarosa para solidificar en el hielo (3-5 minutos). Use una hoja de afeitar para cortar el exceso de agarosa. Añadir cola en el bloque de vibratome y adherirse al bloque de la muestra. </li><li> Añadir PBS frío a baño de agua vibratome hasta que la muestra y la hoja están inmersos. </li><li> Ajustar la velocidad y la amplitud (de gran amplitud y baja velocidad moderada es ideal para suaves secciones del timo). La amplitud debe ser reducida si las secciones romper debido a la agitación en exceso. </li><li> Corte secciones de 50 um. </li><li> Usando un pincel, recoger las secciones en una microplaca de 24 pocillos en PBS frío. </li><li> Secciones de bloque en 500 l de suero de burro del 10% en PBS-Triton X (0,15%) durante 30 minutos. </li><li> Secciones se incuban durante 8 horas o toda la noche con el anticuerpo primario, tales como anti-CD31 y anti-PDGFR β-, En una cubierta de 24 pocillos de microplacas. </li><li> Lavar 3 veces con PBS-Triton X (0,15%) sobre un total de 8 horas a 4 ° C. </li><li> Secciones de bloque en el suero de burro del 10% en PBS-Triton X (0,15%) durante 30 minutos. </li><li> Incubar las secciones de 8 horas a la noche a 4 ° C con anticuerpos secundarios apropiados. </li><li> Lavar 3 veces con PBS-Triton X (0,15%) sobre un total de 8 horas a 4 ° C. </li><li> Fijar de nuevo las muestras en el 4% PFA / PBS durante 30 minutos en hielo. </li><li> Lavar 3 veces con PBS-Triton X (0,15%) más de 30 minutos en hielo. </li><li> Deshidratar las muestras a través de una gradual MeOH / PBS Triton X-series: 25% MeOH, 50% MeOH, 75% MeOH, MeOH y 100% a los 10 minutos para cada paso. Reemplazar el 100% de MeOH con MeOH fresco después de 10 minutos y se incuba durante 1 hora a temperatura ambiente. </li><li> En un recipiente de vidrio, mezclar Babb (alcohol bencílico: benzoato de bencilo) en una proporción de 1:2. Combine Babb con MeOH para una concentración final del 50% Babb y el 50% MeOH. Incubar la muestra en Babb: MeOH durante 10-15 minutos. </li><li> Transferir la muestra a un recipiente de vidrio con 100% Babb y se incuba durante 10-15 minutos o hasta que sean borrados, a temperatura ambiente. </li><li> Llena de diapositivas depresión (0,7 mm de profundidad) con Babb fresca 100% y de transferencia de muestras a un lado. Añadir cubierta de vidrio (No. 1.5) y el sello con 2-3 capas de esmalte de uñas. Permita que el esmalte de uñas para endurecer en la oscuridad a temperatura ambiente, luego almacenar la muestra a 4 ° C. Nota: Las diapositivas deben estar completamente cerrados antes de la adquisición de la imagen confocal. Las imágenes deben ser adquiridas en 12-24 horas, como los tintes fluorescentes pueden desaparecer en Babb. </li><li> Pase a la sección &#39;Adquisición de imágenes &quot;. </li></ol> 5. De adquisición de imágenes <ol><li> Imagen 10 micras secciones congeladas con un microscopio confocal con el Plan-Apochromat 20X/0.8 objetivo (512 x 512 píxeles) con 488 - (FITC-dextran/GSL 1 - isolectina B 4), 543 - y 633-nm líneas láser. </li><li> Adquirir confocal z-secciones de la piel y montaje de todo 50 micras de agarosa embebido en secciones con el Plan-Apochromat 10X/0.4 objetivo (512 x 512 píxeles) con 488 - (FITC-dextran/GSL 1 - isolectina B 4), 543 -, y 633-nm líneas láser. De serie Z-secciones deben ser recogidos de forma secuencial en un micrones para cada canal correspondiente. </li><li> Reconstruir serie Z-secciones usando Zeiss Axiovision 4,6 u otro software de análisis de imagen. </li></ol> 6. Los resultados representativos: Etiquetado de la eficiencia de la vasculatura embrionarias es crucial para la evaluación de los defectos de los vasos sanguíneos en embriones de ratones. La figura 1 muestra un etiquetado específico de los vasos sanguíneos del timo E16.5 (1A-B) y el etiquetado de cooperación con CD31 (1B), además de la tinción de los ventrículos derecho e izquierdo (1E-F), respectivamente. El GSL I-isolectina B 4 protocolo para cryosections como se describe en las secciones 1, 3 y 5 se utilizó en estos experimentos. Todo el montaje de etiquetado de la red de vasos sanguíneos en la piel E16.5 ratones, utilizando los protocolos descritos en las secciones 1, 2 y 5 se muestra en la Figura 1C-D. <img alt="Figura 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> Figura 1 Leyenda. FITC GSL I - isolectina B 4 inyecciones en la vena facial E16.5 embriones de ratón. criosección a. de timo embrionario después de la inyección. b. Fusión de CD31 co-etiquetado con isolectina B 4. c. y d. Todo el montaje de la inyección de la piel embrionaria vasculatura siguiente. e. y f. criosección de corazón embrionarias e. (derecha ventrículo) f. (ventrículo izquierdo) después de la inyección.

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No hay conflictos de interés declarado.

Todo el montaje y PECAM-1 (CD31) en las secciones de tinción son los métodos convencionales para el etiquetado de la vasculatura en embriones de ratones. Estos métodos requieren el uso de la inmunofluorescencia directa y / o indirecta, y los detergentes para permeabilizar los tejidos del ratón. Este resulta ser un proceso más oportuna. En este caso, hemos empleado FITC-dextrano o isolectina B 4 inyecciones vena facial para etiquetar directamente la vasculatura embrionaria, eliminando así la necesidad de...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> FITC-dextrano </td><td> Sigma </td><td> FD150S-1G </td></tr><tr><td> Fluoresceína GSL 1 - isolectina B 4 </td><td> Vector Laboratories </td><td> FL-1201 </td></tr><tr><td> Fast Green </td><td> MP Biomedicals </td><td> 195178 </td></tr><tr><td> PFA </td><td> Fluka </td><td> 76240 </td></tr><tr><td> El suero fetal bovino </td><td> Atlanta Productos Biológicos </td><td> S11550 </td></tr><tr><td> Compuesto óptimo de temperatura de corte (octubre </td><td> VWR </td><td> 25608-930 </td></tr><tr><td> Acetona </td><td> JT Baker </td><td> 9006-33 </td></tr><tr><td> Burro suero </td><td> Jackson </td><td> 017-000-121 </td></tr><tr><td> rata anti-ratón CD31, </td><td> BD Pharmingen </td><td> 558736 </td></tr><tr><td> cabra anti-ratón PDGFR-β </td><td> R &amp; D Systems </td><td> AF1042 </td></tr><tr><td> burro anti-CD31 de rata Alexa 647 (Invitrogen) </td><td> Biolegend </td><td> 102516 </td></tr><tr><td> burro de cabra anti-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> Agarosa de bajo derretimiento / 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> El alcohol de bencilo </td><td> Acros Científico </td><td> 148390010 </td></tr><tr><td> Benzoato de bencilo </td><td> Acros Científico </td><td> 105860010 </td></tr><tr><td> Se desliza la depresión </td><td> Fisher Scientific </td><td> S175201 </td></tr><tr><td> Fluorogel </td><td> Microscopía electrónica de Ciencias </td><td> 17985-10 </td></tr><tr><td> Cubierta de vidrio (22x22) -1,5 </td><td> Thermo Scientific </td><td> 152222 </td></tr><tr><td> Zeiss LSM 510 Microscopio Confocal Meta </td><td> Zeiss </td><td></td></tr><tr><td> Micro pinza de disección </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 y 24, así microplacas </td><td> Evergreen Científico </td><td> 222-8044-01F </td></tr><tr><td> Superfrost / Plus Micropreparados </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> 4 ml viales claras </td><td> Nacional de Investigaciones Científicas </td><td> B7800-2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

El establecimiento de una red de vasos sanguíneos funcionales es una parte esencial de la organogénesis, y es necesario para la función del órgano óptimo. Por ejemplo, en la formación de la vasculatura del timo y el patrón adecuado es esencial para la entrada en el órgano de timocitos y células T maduras salida a la periferia. La disposición espacial de los vasos sanguíneos en el timo depende de señales del microambiente local, es decir, las células epiteliales del timo (TEC). Varios informes recientes sugieren que la interrupción de las señales de estos resultados en un recipiente de 1,2 defectos timo sangre. Estudios previos han descrito técnicas que se utilizan para etiquetar los 1,2 timo vasculatura neonatal y de adultos. Se demuestra aquí una técnica para el etiquetado de los vasos sanguíneos en el timo embrionario. Este método combina el uso de FITC-dextrano o Griffonia (Bandeiraea) Simplicifolia lectina I (GSL 1 - isolectina B 4) inyecciones faciales vena y CD31 tinción de anticuerpos para identificar las estructuras vasculares del timo y PDGFR-β a la etiqueta del timo mesénquima perivascular 3-5. La opción de utilizar cryosections o secciones vibratome también se proporciona. Este protocolo se puede utilizar para identificar el timo defectos vasculares, lo cual es crítico para la definición de los roles de TEC-moléculas derivadas del timo en la formación de vasos sanguíneos. Como el método de las etiquetas de todo el sistema vascular, también puede ser utilizado para analizar las redes vasculares en múltiples órganos y tejidos en todo el embrión, incluyendo la piel y el corazón de 6-10.

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

En este artículo se describe un método para el etiquetado de la piel embrionaria y los vasos sanguíneos del timo.

Un método para etiquetar los Vasos sanguíneos en embriones de ratones

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 vistas.  University of Georgia. En este artículo se describe un método para el etiquetado de la piel embrionaria y los vasos sanguíneos del timo.

Video: 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.

Utilizando imágenes bioluminiscentes encargado de investigar la sinergia entre Streptococcus pneumoniae Y el Virus de la Influenza en ratones lactantes

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

Investigación

Inmunología e Infección

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.

Inferencia genotípica del VIH-1 tropismo utilizando la población basada en la secuencia de 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.

Visualización de virus del dengue a través de Alexa Fluor etiquetado

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.

No invasiva imágenes ópticas de la vasculatura linfática de un ratón

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 de imágenes multimodalidad<em&gt; Citrobacter rodentium</em&gt; Las infecciones en ratones

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.

Un método no invasivo y técnicamente no intensivos para la inducción y Fenotipificación Experimental de neumonía bacteriana en ratones

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.

Inducción de parálisis y lesión del sistema Visual en ratones por T las células específicas para el Optica del Neuromyelitis Autoantigen Acuaporina-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.

Un método para la medición de la función de la glándula salival en ratones

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.

Trombosis venosa profunda inducida por estasis en los ratones controlados por sonografía de alta frecuencia

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.

Forma de nanopartículas de plata para paliar el síndrome de Atresia biliar en ratones

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 ...