Este trabajo fue apoyado por las siguientes subvenciones: a Eva Sevick NIH R01 CA128919 y NIH R01 HL092923.

Todos los estudios con animales se realizaron de conformidad con las normas de la Universidad de Texas Health Science Center (Houston, TX), Departamento de Medicina Comparada, y el Centro de Imagen Molecular después de la revisión y aprobación del protocolo por el cuidado de su respectivo Animales institucional y el empleo Comisión (IACUC) o Animal Welfare Committee (AWC). 1. Preparación de los animales 24 horas antes de la Radiología Los siguientes pasos se debe hacer (si es necesario) el día antes de exponer linfático lleva a cabo. <ol><li> Lugar animal en una caja de inducción con isoflurano y sosegado. </li><li> Una vez que el animal se encuentra en un estado de anestesia profunda (monitorizado con la maniobra de dedo del pie-pinch), animal sedado lugar en un cojín de pañal / pelusa y la nariz posición en un cono nasal conectada a gas isoflurano. </li><li> Clip todo el pelo / piel (si los hay) alrededor de la zona a explorar. </li><li> Aplicar agente depilatorio (Nair) a la zona rasurada y se deja on de la piel para un máximo de 3 minutos. </li><li> Suavemente limpie todo agente depilatorio con una gasa húmeda y caliente o una toalla de papel. </li><li> Lave suavemente la piel con agua tibia y seque suavemente el área con una gasa o toalla de papel. </li><li> Deje que los animales puedan recuperarse en una almohadilla eléctrica o bajo una lámpara de calor, y volver a su jaula. </li></ol> 2. Día de Imágenes <ol><li> Agente de formación de imágenes reconstituir con agua estéril, luego se diluye con estéril, normal (0,85%) de solución salina para alcanzar 645 mM (5 g/10 l) durante ICG o 200 mM (6,8 g/10 l) durante CABD-IRDye800. Mantener las soluciones en la oscuridad condiciones y uso dentro de las 6 horas de la reconstitución. </li><li> Lugar animal en una caja de inducción con isoflurano y sosegado. </li><li> Una vez que el animal se encuentra en un estado de anestesia profunda (controlado con la maniobra de punta-pinch), animal sedado lugar de costado sobre una plataforma de pañales / pelusa y la nariz posición en un cono de nariz conectada a gas isoflurano. </li><li> Apague las luces (por lo que la habitación esoscuro). Si es necesario, una luz de escritorio halógeno pequeña se puede utilizar para una pequeña cantidad de luz para ver inyecciones. </li><li> Usando una jeringa de insulina con una aguja de calibre 31-, inyectar ID 5 l a 50 l de ICG o CABD IRDye800-en la cara dorsal de cada pata trasera y / o en el lado izquierdo y derecho de la base de la cola, dependiendo el área de interés (véase la discusión). Cada dosis inyectada puede variar desde 0,083 hasta 1,25 mg / kg (ICG) o 0.113 a 1.7 mg / kg (CABD-IRDye800). Los volúmenes de inyección puede variar con la cepa animal y sitio de la inyección. Para los ratones atímicos, el volumen de inyección puede ser de 5 l (pata trasera) o 10 l (base de la cola). Si el animal no está en el sistema de formación de imágenes para la inyección (s), colocar al animal en el sistema de imágenes inmediatamente después de la inyección (s). </li><li> Si no se observa absorción de colorante en los vasos linfáticos, paso 2,5 tendrá que repetirse según sea necesario de acuerdo al protocolo de animales. </li><li> Una vez que se observan vasos linfáticos, cubra el sitio de la inyección con cinta eléctrico negro o negro paper. </li><li> Adquirir imágenes linfáticos de hasta 1 hora con V + software + y un animal pequeño, NIRF sistema de imágenes. (Los animales son sedados con isoflurano y las respiraciones se supervisan mientras que las imágenes están adquiriendo.) Mientras animal pequeño, generadores de imágenes NIRF están comercialmente disponibles, utilizamos un servicio personalizado, pequeños animales NIRF sistema de imagen que consiste en un diodo láser 785-nm (1005-9mm-78503 , intenso, North Brunswick, NJ) equipado con una lente asférica (C24TME-B, Thorlabs, Newton, NJ), difusor (ED1-C20, Thorlabs) y filtro (LD01-785/10-25, Semrock, Rochester, NY ) para crear un campo de excitación uniforme que ilumina el animal en una tasa de fluencia incidente de menos de 1,4 mW por centímetro cuadrado 10. Un electrón multiplicando dispositivo de carga acoplada (EMCCD, PhotonMax512, Princeton Instruments, Trenton, NJ) sistema de cámara con dos 830-nm filtros (AND11333, Andover Corp., Salem, NH) y una lente de 28-mm Nikkor (1992, Nikon, Melville, NY) se utiliza para capturar imágenes linfáticos con integración times de 200 ms para imágenes dinámicas y estáticas ms 800 para imágenes 5. Véase la figura 1 para la configuración del sistema, la tabla para obtener más detalles de cada componente, y el debate para una breve discusión de las propiedades imager clave. </li><li> Deje que los animales puedan recuperarse en una almohadilla eléctrica o bajo una lámpara de calor y volver a su jaula, o la eutanasia. </li><li> Analizar imágenes utilizando ImageJ o software MATLAB. Véase la Figura 6. </li></ol>

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Gastroenterol. 13, 192-218 (2007).</li><li>Schramm, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cervical+lymph+node+preparation:+a+novel+approach+to+study+lymphocyte+homing+by+intravital+microscopy.">The cervical lymph node preparation: a novel approach to study lymphocyte homing by intravital microscopy.</a> Inflammation research : official journal of the European Histamine Research Society. 55, 160-167 (2006).</li><li>Hall, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+prostate+cancer+lymph+node+metastases+with+a+multimodality+contrast+agent.">Imaging prostate cancer lymph node metastases with a multimodality contrast agent.</a> Prostate. 72, 129-146 (2012).</li><li>Zhu, B., Sevick-Muraca, E. 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Los autores no tienen nada que revelar, pero algunos autores se muestran en una patente.

Utilizamos una costumbre, un animal pequeño NIRF sistema de imagen para capturar imágenes de los vasos linfáticos marcadas en ratones. Para construir películas de movimiento linfático, 300 o más imágenes se recogen. Para el análisis funcional de los vasos linfáticos de las películas, dos o más regiones de interés son manualmente trazada a lo largo de un vaso linfático. Las dimensiones de las ROIs se mantienen constantes para cada buque y son aproximadamente el diámetro del vaso. Mientras que la resolución...

<table border="1"><tbody><tr><td> Soluciones, reactivos y equipo </td><td> Empresa </td><td> Número de Catálogo </td><td> Comentarios </td></tr><tr><td> Indocianina verde (ICG) </td><td> Patheon Italia SPA </td><td> NDC 25431-424-02 </td><td> Reconstituir a 645 mM (5 g/10 l) </td></tr><tr><td> Cíclica del dominio de unión de albúmina (CABD) </td><td> Bachem </td><td> Costumbre </td><td> Reconstituir a 200 micras (6,8 g/10 l) </td></tr><tr><td> IRDye800 </td><td> Li-COR </td><td> IRDye 800CW </td><td> Reconstituir de acuerdo con las instrucciones del fabricante; conjugar con CABD a concentraciones equilmolar </td></tr><tr><td> Agua estéril </td><td> Hospira, Inc., Lake Forest, IL </td><td> NDC 0409-4887-10 </td><td></td></tr><tr><td> NAIR </td><td> Church &amp; Dwight Co.,Inc. </td><td> Tiendas Locales </td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td> Imaging System (componentes a continuación) </td><td> Centro de Imagen Molecular </td><td> N / A </td><td> Custom-construido en nuestros laboratorios. </td></tr><tr><td> Multiplicador de electrones dispositivo de carga acoplada (EMCCD) de la cámara </td><td> Princeton Instruments, Trenton, NJ </td><td> Photon Max 512 </td><td></td></tr><tr><td> Nikon lente de la cámara </td><td> Nikon Inc., Melville, NY </td><td> Modelo N º 1992, Nikkor 28mm </td><td></td></tr><tr><td> Filtro óptico </td><td> Andover Corp., Salem, NH </td><td> ANDV11333 </td><td> Dos filtros paso banda 830.0/10.0 nm se utilizan en la parte frontal del objetivo </td></tr><tr><td> 785-nm láser de diodo </td><td> Intenso Ltd, North Brunswick, NJ </td><td> 1005-9MM-78503 </td><td> 500 mW de salida óptica </td></tr&gt; <tr><td> Óptica de colimación </td><td> Thorlabs, Newton, NJ </td><td> C240TME-B </td><td> Colima salida del láser antes de filtro de limpieza </td></tr><tr><td> Clean-up filtro </td><td> Semrock, Inc., Rochester, NY </td><td> LD01-785/10-25 </td><td> Elimina emisión láser en la banda de fluorescencia </td></tr><tr><td> Difusor óptico </td><td> Thorlabs, Newton, NJ </td><td> ED1-C20 </td><td> Difunde el láser sobre el animal </td></tr><tr><td> V + + </td><td> Digital Óptica, Browns Bay, Auckland, Nueva Zelanda </td><td> Versión 5.0 </td><td> Software utilizado para controlar el sistema de cámara y guardar las imágenes a la computadora. <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td></tr><tr><td></td><td></td><td></td><td> Software analítico Cualquiera de los siguientes paquetes de software se puede utilizar para análisis de imagen </td></tr><tr><td> ImageJ </td><td> Instituto Nacional detutos de Salud, Bethesda, MD </td><td> Versión más reciente disponible </td><td> Programas de dominio público disponible en <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> MATLAB </td><td> MathWorks, Natick, MA </td><td> Versión 2008a o más tarde </td><td> <a href="http://www.mathworks.com/" target="_blank">http://www.mathworks.com/</a> </td></tr></tbody></table>

non-invasive optical imaging of the lymphatic vasculature of a mouse

El sistema linfático vascular es un componente importante del sistema circulatorio que mantiene la homeostasis de fluidos, proporciona la vigilancia inmune, y media la absorción de grasas en el intestino. Sin embargo, a pesar de su función crítica, existe una comprensión relativamente poco de cómo funciona el sistema linfático se adapta a servir a estas funciones en la salud y la enfermedad 1. Recientemente, hemos demostrado la capacidad de la arquitectura dinámicamente imagen linfático y los ganglios &quot;bombeo&quot; acción en sujetos humanos normales, así como en las personas que padecen disfunción linfática mediante la administración de un rastro en el infrarrojo cercano fluorescente (NIRF) colorante y una costumbre, Gen III- intensificado sistema de imágenes 2-4. NIRF imágenes mostraron cambios dramáticos en la arquitectura linfático y la función con la enfermedad humana. Aún no está claro cómo se producen estos cambios y nuevos modelos animales se están desarrollando para dilucidar su base genética y molecular. En este protocolo, se presenta linfático NIRF, salameda animal de formación de imágenes utilizando 5,6 verde de indocianina (ICG), un colorante que se ha utilizado durante 50 años en los seres humanos 7, y un NIRF marcada con colorante cíclico dominio de unión de albúmina (CABD-IRDye800) péptido que se une preferentemente a la albúmina humana y de ratón 8 . Aproximadamente 5,5 veces más brillante que ICG, Abd-IRDye800 tiene un perfil linfático autorización similar y se puede inyectar en dosis más pequeñas de ICG para lograr suficientes señales NIRF para formación de imágenes 8. Debido a que ambos se unen CABD-IRDye800 ICG a la albúmina y en el espacio intersticial 8, ambas pueden representar el transporte de proteínas en activo y dentro de los vasos linfáticos. Intradérmica (ID) inyecciones (l 5-50) de ICG (645 mM) o CABD-IRDye800 (200 mM) en solución salina y se administra a la cara dorsal de cada pata trasera y / o el lado izquierdo y derecho de la base de la cola de un ratón isoflurano anestesia. La concentración de colorante resultante en el animal es 83-1,250 mg / kg de ICG o 113-1,700 mg / kg paraCABD-IRDye800. Inmediatamente después de las inyecciones, las imágenes linfático funcional se lleva a cabo durante un máximo de 1 h usando un personalizado, un animal pequeño NIRF sistema de formación de imágenes. Resolución espacial Whole animales pueden representar los vasos linfáticos fluorescentes de 100 micrones o menos, y las imágenes de las estructuras de hasta 3 cm de profundidad pueden ser adquiridos 9. Las imágenes se adquieren mediante V + software + y analizados mediante el software ImageJ o MATLAB. Durante el análisis, regiones consecutivas de interés (ROIs) que abarca todo el diámetro del recipiente se dibujan a lo largo de un vaso linfático dado. Las dimensiones de cada retorno de la inversión se mantienen constantes para un buque determinado y la intensidad NIRF se mide para cada ROI para evaluar cuantitativamente &quot;paquetes&quot; de linfa en movimiento a través de los vasos.

Ejemplo de imágenes NIRF linfático en ratones Cuando ICG o CABD IRDye800-ID se inyecta en la base de la cola de un ratón normal, el conjunto de vasos linfáticos entre el sitio de la inyección en la base de la cola y el ganglio linfático inguinal (LN) debe ser visualizado inmediatamente. Poco después de la inyección (unos pocos segundos a minutos), el vaso linfático entre el LN inguinal y el LN axilar debe ser visualizado como se ve en la Figura 2. Dado que los vasos li...

Watch this Scientific Journal Video about Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse at JoVE.com

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

Recientemente desarrollados utilizando técnicas de imagen de fluorescencia en el infrarrojo cercano (NIRF) puede ayudar a dilucidar el papel del sistema linfático juega en la metástasis del cáncer, la respuesta inmune, la reparación de heridas, y otras enfermedades asociadas linfático.

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 ...

non-invasive-optical-imaging-of-the-lymphatic-vasculature-of-a-mouse

Research

JoVE Journal

Immunology and Infection

16.0K vistas.  University of Texas Health Science Center-Houston. Recientemente desarrollados utilizando técnicas de imagen de fluorescencia en el infrarrojo cercano (NIRF) puede ayudar a dilucidar el papel del sistema linfático juega en la metástasis del cáncer, la respuesta inmune, la reparación de heridas, y otras enfermedades asociadas linfático.

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

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.

De imagen no invasivas de Homing leucocitos y Migración 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 ...

Investigación

Inmunología e Infección

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Preparación de inmunógeno ratón hipófisis para la inducción de la hipofisitis autoinmune experimental

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

El uso de una trampa óptica para el estudio de interacciones huésped-patógeno de imágenes dinámicas de células vivas

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

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 ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagen intravital del nodo linfático poplíteo ratón

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

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.

La imagen no invasiva de la candidiasis diseminada en larvas de pez cebra

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

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 no invasiva de neuropatogénico<em&gt; Escherichia coli</em&gt; La infección en Neonatología Rata

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

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

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.

La timectomía completo en ratas adultas con no invasiva La intubación endotraqueal

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

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.

Imagen no invasiva de la respuesta inmune innata en un modelo de larvas de pez cebra de<em&gt; Streptococcus iniae</em&gt; Infección

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.

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 ...