Este trabalho foi suportado pelas seguintes subsídios para Eva Sevick: NIH R01 CA128919 e NIH R01 HL092923.

Todos os estudos com animais foram realizados de acordo com as normas da Universidade do Texas Health Science Center (Houston, TX), Departamento de Medicina Comparativa e Centro de Imagem Molecular, após análise e aprovação do protocolo pelo seu respectivo Animal Care Institucional e Comitê de Uso (IACUC) ou Comitê de Bem-Estar Animal (CTA). 1. Preparação de Animais 24 horas antes da imagem Os passos a seguir deve ser feito (conforme necessário) um dia antes de imagem linfática ocorre. <ol><li> Lugar animal em uma caixa de indução e tranqüilo com isoflurano. </li><li> Uma vez que o animal se encontra num estado de anestesia profunda (monitorizado com dedo do pé pinch-manobra), animal sedado lugar sobre uma almofada de tecido / fluff e nariz posição em um cone de nariz ligada a gás isofluorano. </li><li> Clipe todo o cabelo / pele (se houver) em torno da área a ser trabalhada. </li><li> Aplicar depilatório agente (NAIR) para a área cortada e deixá-lo ón a pele por até 3 min. </li><li> Limpe suavemente todo agente depilatório com gaze, quente e úmido ou toalha de papel. </li><li> Lave a pele com água morna e seque a área gentilmente com gaze ou toalha de papel. </li><li> Permitir que os animais se recuperar em uma almofada de aquecimento ou sob uma lâmpada de calor, e voltar para sua gaiola. </li></ol> 2. Dia da Imagem <ol><li> Reconstituir agente de imagem com água esterilizada, em seguida, dilua com estéril, normal (0,85%) de solução salina para obter 645 uM (5 μg/10 ul) para ICG ou 200 uM (6,8 ul μg/10) para CABD-IRDye800. Mantenha em soluções escuro condições de uso e dentro de 6 horas de reconstituição. </li><li> Lugar animal em uma caixa de indução e tranqüilo com isoflurano. </li><li> Uma vez que o animal se encontra num estado de anestesia profunda (monitorizado com dedo do pé pinch-manobra), animal sedado lugar no seu lado sobre uma almofada de tecido / fluff e nariz posição em um cone de nariz ligada a gás isofluorano. </li><li> Desligue as luzes (de modo que o quarto éescuro). Se necessário, uma luz de halogéneo pequena mesa pode ser usada para uma pequena quantidade de luz para ver injecções. </li><li> Utilizando uma seringa de insulina com uma agulha de 31-gauge, injectar ul ID 5 a 50 ul de ICG ou CABD-IRDye800 no aspecto dorsal de cada pata traseira e / ou no lado esquerdo e direito da base da cauda, ​​dependendo a área de interesse (ver Discussão). Cada dose injectada pode variar 0,083-1,25 mg / kg (ICG) ou 0,113-1,7 mg / kg (CABD-IRDye800). Os volumes de injecção irá variar com a estirpe de animais e no local de injecção. Para ratinhos atímicos, o volume de injecção pode ser 5 (pata traseira) ul ou 10 ul (base da cauda). Se um animal não está sob o sistema de imagem para a injecção (s), colocar o animal no âmbito do sistema de imagem imediatamente após o injecção (ões). </li><li> Se nenhuma absorção do corante é visto nos vasos linfáticos, passo 2,5 terá de ser repetido conforme necessário por protocolo animal. </li><li> Uma vez linfáticos são vistos, cobrir o local da injecção com fita isolante preta ou preta papor. </li><li> Adquirir imagens linfáticos por até 1 hora usando V + + software e um animal pequeno, NIRF sistema de imagem. (Os animais são sedados com isoflurano e respiração são monitoradas enquanto as imagens estão adquirindo.) Embora pequeno animal, imagers NIRF estão comercialmente disponíveis, nós utilizamos um personalizado, pequenos animais NIRF sistema de imagens composto por um diodo laser 785-nm (1005-9mm-78503 , intenso, North Brunswick, NJ) equipada com uma lente asférica (C24TME-B, Thorlabs, Newton, NJ), o difusor (ED1-C20, Thorlabs) e do filtro (LD01-785/10-25, Semrock, Rochester, NY ) para criar um campo de excitação uniforme que ilumina o animal a uma taxa de fluência incidente de menos de 1,4 mW por centímetro quadrado 10. Um elétron multiplicando cobrado-coupled device (EMCCD, PhotonMax512, Princeton Instruments, Trenton, NJ) sistema de câmera com duas 830 nm filtros (AND11333, Andover Corp, Salem, NH) e um de 28 mm lente Nikkor (1992, Nikon, Melville, NY) é usado para capturar imagens linfáticos com integração times de 200 ms para imagens dinâmicas e 800 mseg para imagiologia estático 5. Veja a Figura 1 para configuração do sistema, a tabela para obter detalhes adicionais de cada componente, e da discussão para uma breve discussão sobre as propriedades imager chave. </li><li> Permitir que os animais se recuperar em uma almofada de aquecimento ou sob uma lâmpada de calor e voltar para sua gaiola, ou eutanásia. </li><li> Analisar imagens usando ImageJ ou software MATLAB. Veja Figura 6. </li></ol>

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Autores têm nada a revelar, mas alguns autores são listados em uma patente.

Nós usamos um costume, de pequenos animais NIRF sistema de imagem para capturar imagens de vasos linfáticos marcados em camundongos. Para construir filmes do movimento da linfa, 300 ou mais imagens são coletadas. Para a análise funcional do sistema linfático de filmes, ROIs dois ou mais são manualmente desenhadas ao longo de um vaso linfático. As dimensões da ROIs são mantidas constantes para cada navio e são de aproximadamente o diâmetro do vaso. Enquanto a resolução espacial animal inteiro pode descrever ...

<table border="1"><tbody><tr><td> Soluções, reagentes, equipamentos e </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Indocianina verde (ICG) </td><td> Patheon Italia SPA </td><td> NDC 25431-424-02 </td><td> Reconstituir a 645 uM (5 uL μg/10) </td></tr><tr><td> Domínio Albumina cíclica Binding (CABD) </td><td> Bachem </td><td> Personalizado </td><td> Reconstituir a 200 uM (6,8 μg/10 uL) </td></tr><tr><td> IRDye800 </td><td> Li-COR </td><td> IRDye 800CW </td><td> Reconstituir acordo com as instruções do fabrico; conjugar com CABD em concentrações equilmolar </td></tr><tr><td> Água 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> Lojas locais </td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td> Imaging System (componentes abaixo) </td><td> Centro de Imagem Molecular </td><td> N / A </td><td> Custom-construído em nossos laboratórios. </td></tr><tr><td> Elétron-multiplicação charge-coupled device câmera (EMCCD) </td><td> Princeton Instruments, Trenton, NJ </td><td> Photon Max 512 </td><td></td></tr><tr><td> Nikon lente da câmera </td><td> Nikon Inc., Melville, NY </td><td> Modelo n º 1992, Nikkor 28 milímetros </td><td></td></tr><tr><td> Filtro óptico </td><td> Andover Corp, Salem, NH </td><td> ANDV11333 </td><td> Dois filtros passa-banda 830.0/10.0 nm são utilizadas em frente da lente </td></tr><tr><td> 785-nm laser diodo </td><td> Ltd intensa, North Brunswick, NJ </td><td> 1005-9MM-78503 </td><td> 500 mW de potência óptica </td></tr&gt; <tr><td> Óptica de colimação </td><td> Thorlabs, Newton, NJ </td><td> C240TME-B </td><td> Colima saída do laser antes do filtro de limpeza </td></tr><tr><td> Limpeza do filtro </td><td> Semrock, Inc., Rochester, NY </td><td> LD01-785/10-25 </td><td> Remove emissão laser na banda de fluorescência </td></tr><tr><td> Difusor óptico </td><td> Thorlabs, Newton, NJ </td><td> ED1-C20 </td><td> Difunde a laser sobre o animal </td></tr><tr><td> V + + </td><td> Digital Óptica, Browns Bay, Auckland, Nova Zelândia </td><td> Versão 5.0 </td><td> Software usado para controlar o sistema de câmera e gravar as imagens para o computador. <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td></tr><tr><td></td><td></td><td></td><td> Software analítico Qualquer um dos pacotes de software que se seguem podem ser utilizados para análise de imagem </td></tr><tr><td> ImageJ </td><td> Instituto Nacional detitui de Saúde, Bethesda, MD </td><td> Versão mais atual disponível </td><td> Freeware disponível no <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> Versão 2008a ou posterior </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

O sistema vascular linfático é um componente importante do sistema circulatório, que mantém a homeostase de fluidos, proporciona vigilância imune, e medeia a absorção de gordura no intestino. No entanto, apesar de sua função crítica, há relativamente pouco entendimento de como o sistema linfático se adapta para servir estas funções na saúde e na doença 1. Recentemente, demonstramos a capacidade de arquitetura dinamicamente imagem linfática e linfa &quot;bombear&quot; ação em indivíduos normais, bem como em pessoas que sofrem de disfunção linfática usando a administração traço de um infravermelho próximo fluorescente corante (NIRF) e um costume, Gen III- intensificou imagens do sistema 2-4. NIRF imagem mostrou mudanças dramáticas na arquitetura linfática e função com a doença humana. Ainda não está claro como essas mudanças ocorrem e novos modelos animais estão sendo desenvolvidas para elucidar a sua base genética e molecular. Neste protocolo, apresentamos NIRF linfático, sMall animais imagiologia 5,6 utilizando verde de indocianina (ICG), um corante que tem sido utilizado há 50 anos em humanos 7, e um corante NIRF marcado albumina domínio de ligação cíclica (CABD-IRDye800) péptido que se liga preferencialmente de rato e humano 8 albumina . Aproximadamente 5,5 vezes mais brilhante do que ICG, Abd-IRDye800 tem um perfil semelhante linfático e depuração pode ser injectada em doses menores do que os sinais de ICG para realizar NIRF suficientes para imagiologia 8. Porque se ligam CABD-IRDye800 ICG e de albumina no espaço intersticial 8, que tanto pode representar o transporte de proteínas activas para dentro e para dentro dos vasos linfáticos. Intradérmica (ID) injecções (ul 5-50) de ICG (645 uM) ou CABD-IRDye800 (200 fiM) em solução salina são administrados para o aspecto dorsal de cada pata traseira e / ou no lado esquerdo e direito da base do cauda de um rato anestesiado com isoflurano-. A concentração do corante, resultando em que o animal é 83-1,250 ug / kg para o ICG ou 113-1,700 mg / kg paraAbd-IRDye800. Imediatamente após as injeções, imagem funcional linfática é realizada por até 1 hora usando um personalizado, pequenos animais NIRF sistema de imagem. Resolução espacial animal inteiro pode descrever vasos linfáticos fluorescentes de 100 microns ou menos, e as imagens de estruturas até 3 cm de profundidade podem ser adquiridos 9. As imagens são adquiridas usando V + software + e analisados ​​usando ImageJ ou software MATLAB. Durante a análise, as regiões consecutivos de interesse (ROIs) abrangendo o diâmetro do vaso inteiro são atraídos ao longo de um vaso linfático dado. As dimensões de cada ROI são mantidas constantes para um dado navio e intensidade NIRF é medido para cada ROI para avaliar quantitativamente &quot;pacotes&quot; de linfa em movimento através dos vasos.

Exemplo de NIRF imagem linfática em Ratos Quando ICG ou CABD-IRDye800 é injectado ID na base da cauda de um rato normal, a vasculatura linfática entre o local da injecção na base da cauda e do nó de linfa inguinal (LN) deve ser imediatamente visualizado. Pouco tempo após a injecção (de alguns segundos a minutos), o vaso linfático entre o LN LN inguinal e axilar deve ser visualizado, como visto na Figura 2. Uma vez que os vasos linfáticos em ratinhos variar de animal ...

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

Recentemente técnicas de imagem desenvolvidos utilizando fluorescência no infravermelho próximo (NIRF) pode ajudar a elucidar a função do sistema linfático desempenha na metástase do cancro, a resposta imune, reparação de feridas, e outras doenças associadas linfático.

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 ...

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

Research

JoVE Journal

Immunology and Infection

16.0K visualizações.  University of Texas Health Science Center-Houston. Recentemente técnicas de imagem desenvolvidos utilizando fluorescência no infravermelho próximo (NIRF) pode ajudar a elucidar a função do sistema linfático desempenha na metástase do cancro, a resposta imune, reparação de feridas, e outras doenças associadas linfático.

Vídeo: 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.

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

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

Imunologia e Infecção

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.

Preparação de rato Imunogénio Pituitária para a indução de hipofisite Auto-Imune 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.

Uso de uma armadilha óptica de Estudos de Interações Hospedeiro-Patógeno para imagens de células dinâmica ao vivo

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.

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 ...

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.

Imagem intravital do Linfonodo Popliteal Rato

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.

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

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

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

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

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

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

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

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

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.

Timectomia completo em ratos adultos com não-invasiva entubação endotraqueal

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

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

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

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

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

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

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

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

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

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

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

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