Questo lavoro è stato supportato da i seguenti aiuti a Eva Sevick: NIH R01 CA128919 e NIH R01 HL092923.

Tutti gli studi sugli animali sono stati eseguiti in conformità con le norme della University of Texas Health Science Center (Houston, TX), Dipartimento di Medicina comparativa, e Centro di Imaging Molecolare dopo l&#39;esame e l&#39;approvazione del protocollo da parte loro rispettivi Cura degli animali e uso Comitato Istituzionale (IACUC) o il benessere degli animali Committee (AWC). 1. Preparazione del 24 Hr Animali Prima di Imaging La procedura che segue deve essere fatto (se necessario) il giorno prima di imaging linfatico avviene. <ol><li> Inserire animale in una scatola di induzione e sedare con isoflurano. </li><li> Una volta che l&#39;animale è in uno stato di anestesia profonda (monitorato con toe-pinch manovra), animale sedato posto su un pannolino / fluff pad naso e la posizione in un cono collegato gas isoflurano. </li><li> Agganciare tutti i capelli / pelliccia (se del caso) intorno alla zona da acquisire. </li><li> Applicare un agente depilatoria (NAIR) per l&#39;area ritagliata e lasciare on la pelle fino a 3 min. </li><li> Pulire delicatamente fuori tutte agente depilatoria con il caldo, garza umida o un tovagliolo di carta. </li><li> Lavare delicatamente la pelle con acqua tiepida e asciugare delicatamente la zona con una garza o un tovagliolo di carta. </li><li> Consentire agli animali di recuperare su una piastra elettrica o sotto una lampada di calore, e tornare alla loro gabbia. </li></ol> 2. Giorno di Imaging <ol><li> Agente di imaging Ricostituire con acqua sterile, quindi diluire con sterile, normale (0,85%) soluzione fisiologica per ottenere 645 pM (5 μg/10 pl) per ICG o 200 mM (6.8 μg/10 pl) per &#39;Abd-IRDye800. Conservare le soluzioni al buio condizioni e l&#39;uso entro 6 ore dalla ricostituzione. </li><li> Inserire animale in una scatola di induzione e sedare con isoflurano. </li><li> Una volta che l&#39;animale è in uno stato di anestesia profonda (monitorato con toe-pinch manovra), animale sedato posto su un lato su un pannolino / fluff pad naso e la posizione in un cono collegato gas isoflurano. </li><li> Spegni le luci (quindi la camera èscuro). Se necessario, una piccola luce alogena scrivania può essere utilizzato per una piccola quantità di luce per vedere iniezioni. </li><li> Utilizzando una siringa da insulina con un ago di calibro 31, iniettare ID 5 pl a 50 pl di ICG o CabD-IRDye800 nel dorso di ciascuna zampa posteriore e / o sul lato sinistro e destro della base della coda, a seconda l&#39;area di interesse (vedi Discussione). Ogni dose iniettata può variare 0,083-1,25 mg / kg (ICG) o 0,113-1,7 mg / kg (CabD-IRDye800). Volumi di iniezione varia con il ceppo animale e sito di iniezione. Per topi atimici, il volume di iniezione può essere 5 microlitri (zampa posteriore) o 10 microlitri (base della coda). Se animale non è sotto il sistema di imaging per l&#39;iniezione (s), collocare l&#39;animale sotto il sistema di imaging immediatamente dopo l&#39; iniezione (s). </li><li> Se non si vede l&#39;assorbimento colorante nei vasi linfatici, passo 2,5 dovrà essere ripetuta, se necessario per protocollo animale. </li><li> Una volta che si vedono vasi linfatici, coprire il sito di iniezione con nastro isolante nero o nero paper. </li><li> Acquisire le immagini linfatiche per un massimo di 1 ora con V + software + e un piccolo animale, NIRF sistema di imaging. (Gli animali vengono sedati con isoflurano e respirazione sono monitorati mentre le immagini stanno acquisendo.) Mentre per piccoli animali, imager NIRF sono disponibili in commercio, ci avvaliamo di un personalizzato, piccolo sistema animale NIRF immagini costituito da un 785-nm diodo laser (1005-78503-9mm , intenso, North Brunswick, NJ) dotato di una lente asferica (C24TME-B, Thorlabs, Newton, NJ), diffusore (ED1-C20, Thorlabs), e filtro (LD01-785/10-25, Semrock, Rochester, NY ) per creare un campo uniforme di eccitazione che illumina l&#39;animale a un tasso incidente fluenza inferiore a 1,4 mW per centimetro quadrato 10. Un elettrone moltiplicando charged-coupled dispositivo (EMCCD, PhotonMax512, Princeton Instruments, Trenton, NJ) sistema di telecamere con due 830-nm (filtri AND11333, Andover Corp., Salem, NH) e da 28 mm, obiettivo Nikkor (1992, Nikon, Melville, NY) viene utilizzato per catturare le immagini linfatici con integrazione times di 200 msec per immagini dinamiche e 800 msec per l&#39;imaging statico 5. Vedere la Figura 1 per la configurazione del sistema, la tabella per ulteriori dettagli di ogni componente, e la discussione per una breve discussione delle proprietà imager chiave. </li><li> Consentire agli animali di recuperare su una piastra elettrica o sotto una lampada di calore e ritornano nella loro gabbia, o eutanasia. </li><li> Analizzare le immagini utilizzando ImageJ o software MATLAB. Vedere la Figura 6. </li></ol>

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Imaging Biol. , (2011).</li><li>Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+lymph+function.">Quantitative imaging of lymph function.</a> Am. J. Physiol. Heart Circ. Physiol. 292, 3109-3118 (2007).</li><li>Kwon, S., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+lymphatic+imaging+in+tumor-bearing+mice.">Functional lymphatic imaging in tumor-bearing mice.</a> J. Immunol. Methods. 360, 167-172 (2010).</li><li>Karlsen, T. V., McCormack, E., Mujic, M., Tenstad, O., Wiig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+quantification+of+lymph+flow+in+mice+and+rats+by+imaging+depot+clearance+of+near-infrared+albumin.">Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 391-401 (2012).</li><li>Zhou, Q., Wood, R., Schwarz, E. M., Wang, Y. J., Xing, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+lymphatic+imaging+demonstrates+the+dynamics+of+lymph+flow+and+lymphangiogenesis+during+the+acute+versus+chronic+phases+of+arthritis+in+mice.">Near-infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute versus chronic phases of arthritis in mice.</a> Arthritis Rheum. 62, 1881-1889 (2010).</li><li>Adams, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+lymphatic+function+improvement+after+advanced+pneumatic+compression+device+treatment+of+lymphedema.">Direct evidence of lymphatic function improvement after advanced pneumatic compression device treatment of lymphedema.</a> Biomed. Opt. Express. 1, 114-125 (2010).</li><li>Tan, I. C., 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=Assessment+of+lymphatic+contractile+function+after+manual+lymphatic+drainage+using+near-infrared+fluorescence+imaging.">Assessment of lymphatic contractile function after manual lymphatic drainage using near-infrared fluorescence imaging.</a> Arch. Phys. Med. Rehabil. 92, 756-764 (2011).</li><li>Lapinski, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RASA1+maintains+the+lymphatic+vasculature+in+a+quiescent+functional+state+in+mice.">RASA1 maintains the lymphatic vasculature in a quiescent functional state in mice.</a> J. Clin. Invest. 122, 733-747 (2012).</li><li>Maus, E. 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=Near-infrared+fluorescence+imaging+of+lymphatics+in+head+and+neck+lymphedema.">Near-infrared fluorescence imaging of lymphatics in head and neck lymphedema.</a> Head Neck. 34, 448-453 (2012).</li><li>Galanzha, E. I., Tuchin, V. V., Zharov, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+small+animal+mesentery+models+for+in+vivo+flow+cytometry,+dynamic+microscopy,+and+drug+screening.">Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening.</a> World J. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+excitation+leakage+and+maximizing+measurement+sensitivity+for+molecular+imaging+with+near-infrared+fluorescence.">Minimizing excitation leakage and maximizing measurement sensitivity for molecular imaging with near-infrared fluorescence.</a> J. Innovat. Opt. Health Sci. 4, 301-307 (2011).</li></ol>

Gli autori non hanno nulla da rivelare, ma alcuni autori sono elencati su un brevetto.

Usiamo una consuetudine, piccolo sistema animale NIRF immagini per catturare immagini di vasi linfatici etichettati nei topi. Per costruire film di circolazione linfatico, 300 o più immagini sono raccolte. Per l&#39;analisi funzionale dei vasi linfatici da film, ROI due o più sono disegnata a mano lungo un vaso linfatico. Le dimensioni della ROI sono mantenute costanti per ciascun recipiente e sono approssimativamente il diametro del vaso. Mentre risoluzione animale intero territorio può rappresentare vasi linfatici ...

<table border="1"><tbody><tr><td> Solutions, reagenti e attrezzature </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Verde indocianina (ICG) </td><td> Patheon Italia SPA </td><td> NDC 25431-424-02 </td><td> Ricostituire a 645 pM (5 μg/10 mL) </td></tr><tr><td> Ciclico dominio di legame (&#39;Abd) </td><td> Bachem </td><td> Costume </td><td> Ricostituire a 200 pM (6.8 μg/10 mL) </td></tr><tr><td> IRDye800 </td><td> Li-COR </td><td> IRDye 800CW </td><td> Ricostituire secondo le istruzioni del produttore; coniugare con &#39;Abd a concentrazioni equilmolar </td></tr><tr><td> Acqua sterile </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> Negozi locali </td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td> Imaging System (componenti sotto) </td><td> Center for Molecular Imaging </td><td> N / A </td><td> Custom-costruito nei nostri laboratori. </td></tr><tr><td> Electron moltiplicatore di Charge-Coupled Device (EMCCD) fotocamera </td><td> Princeton Instruments, Trenton, NJ </td><td> Photon Max. 512 </td><td></td></tr><tr><td> Nikon obiettivo </td><td> Nikon Inc., Melville, NY </td><td> Modello n 1992, Nikkor 28 millimetri </td><td></td></tr><tr><td> Filtro ottico </td><td> Andover Corp., Salem, NH </td><td> ANDV11333 </td><td> Due filtri passa-banda 830.0/10.0 nm vengono utilizzati davanti alla lente </td></tr><tr><td> 785-nm laser a diodi </td><td> Intenso Ltd, North Brunswick, NJ </td><td> 1005-9MM-78503 </td><td> 500 mW di uscita ottica </td></tr&gt; <tr><td> Ottiche collimanti </td><td> Thorlabs, Newton, NJ </td><td> C240TME-B </td><td> Collima uscita laser prima filtro di pulitura </td></tr><tr><td> Clean-up filtro </td><td> Semrock, Inc., Rochester, NY </td><td> LD01-785/10-25 </td><td> Rimuove emissione laser in banda di fluorescenza </td></tr><tr><td> Diffusore ottico </td><td> Thorlabs, Newton, NJ </td><td> ED1-C20 </td><td> Diffonde il laser sull&#39;animale </td></tr><tr><td> V + + </td><td> Ottica digitale, Browns Bay, Auckland, Nuova Zelanda </td><td> Versione 5.0 </td><td> Software utilizzato per controllare il sistema di telecamere e di salvare le immagini al computer. <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td></tr><tr><td></td><td></td><td></td><td> Software analitica Uno dei seguenti pacchetti software può essere utilizzato per l&#39;analisi delle immagini </td></tr><tr><td> ImageJ </td><td> Istituto Nazionale dituti di salute, Bethesda, MD </td><td> Una versione più aggiornata </td><td> Gratuito disponibile a <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> Versione 2008a o successiva </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

Il sistema vascolare linfatico è un componente importante del sistema circolatorio che mantiene l&#39;omeostasi fluido, fornisce la sorveglianza immunitaria, e media l&#39;assorbimento di grassi nell&#39;intestino. Eppure, nonostante la sua funzione critica, non vi è la comprensione relativamente poco di come il sistema linfatico si adatta a servire queste funzioni in materia di salute e malattia 1. Recentemente, abbiamo dimostrato la capacità di architettura dinamica linfatica immagine e linfa azione &quot;pompaggio&quot; in soggetti umani normali e anche a persone che soffrono di disfunzione linfatica mediante somministrazione traccia di un vicino infrarosso fluorescente (NIRF) colorante e un costume, Gen III- intensificata sistema di imaging 2-4. NIRF di imaging hanno mostrato cambiamenti drammatici in architettura linfatica e la funzione con la malattia umana. Non è ancora chiaro come questi cambiamenti si verificano e di nuovi modelli animali che sono in fase di sviluppo per chiarire la loro base genetica e molecolare. In questo protocollo, presentiamo linfatico NIRF, small animale Imaging 5,6 utilizzando verde indocianina (ICG), un colorante che è stato usato per 50 anni nell&#39;uomo 7, e un colorante marcato NIRF ciclico dominio di legame (CabD-IRDye800) peptide che si lega preferenzialmente topo e albumina umana 8 . Circa 5,5 volte più luminoso di ICG, &#39;Abd-IRDye800 ha un profilo simile gioco linfatico e può essere iniettato in piccole dosi di ICG per ottenere segnali NIRF sufficienti per l&#39;imaging 8. Poiché sia bind &#39;Abd-IRDye800 e ICG all&#39;albumina nello spazio interstiziale 8, entrambi possono descrivere trasporto attivo le proteine ​​in e nei vasi linfatici. Intradermica (ID) iniezioni (microlitri 5-50) di ICG (645 uM) o CabD-IRDye800 (200 pM) in soluzione salina sono somministrati al dorso di ciascuna zampa posteriore e / o il lato sinistro e destro della base del coda di un topo anestetizzato-isoflurano. La concentrazione di colorante conseguente animale è 83-1,250 mcg / kg per ICG o 113-1,700 mg / kg per&#39;Abd-IRDye800. Subito dopo le iniezioni, l&#39;imaging funzionale linfatico è condotto per un massimo di 1 ora con un personalizzato, piccolo sistema animale NIRF imaging. Risoluzione animale intero territorio può rappresentare vasi linfatici fluorescenti di 100 micron o meno, e le immagini di strutture fino a 3 cm di profondità possono essere acquisite 9. Le immagini sono acquisite in V + software + e analizzati utilizzando il software ImageJ o MATLAB. Durante l&#39;analisi, consecutivi regioni di interesse (ROI) comprende l&#39;intero diametro nave sono disegnati lungo un vaso linfatico dato. Le dimensioni di ogni ROI sono mantenute costanti per un determinato peschereccio e intensità NIRF è misurato per ciascuna ROI per valutare quantitativamente &quot;pacchetti&quot; di linfa si muove attraverso le navi.

Esempio di Imaging linfatico NIRF in Mouse Quando ICG o CabD IRDye800-ID viene iniettato alla base della coda di un topo normale, la vascolarizzazione linfatica tra il sito di iniezione alla base della coda e il linfonodo inguinale (LN) dovrebbe essere immediatamente visualizzata. Poco dopo l&#39;iniezione (pochi secondi o minuti), il vaso linfatico tra la LN inguinale e ascellare LN deve essere visualizzato come visibile in figura 2. Poiché i linfatici nei topi variano da un ...

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

Tecniche di imaging di recente sviluppate utilizzando fluorescenza nel vicino infrarosso (NIRF) può aiutare a chiarire il ruolo del sistema linfatico gioca in metastasi del cancro, la risposta immunitaria, la riparazione delle ferite, e altre malattie associate linfatico.

Non invasiva di imaging ottico del sistema vascolare linfatico di un mouse

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 Views.  University of Texas Health Science Center-Houston. Tecniche di imaging di recente sviluppate utilizzando fluorescenza nel vicino infrarosso (NIRF) può aiutare a chiarire il ruolo del sistema linfatico gioca in metastasi del cancro, la risposta immunitaria, la riparazione delle ferite, e altre malattie associate linfatico.

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.

Non invasiva di imaging di homing dei leucociti e la migrazione 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 ...

Ricerca

Immunologia e infezioni

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.

Preparazione di Immunogeno mouse ipofisari per l&#39;induzione di ipofisite autoimmune sperimentale

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.

L&#39;uso di una trappola ottica per lo Studio delle interazioni ospite-patogeno per Dynamic Imaging Live Cell

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 metodo per l&#39;etichettatura vasi embrionali nei topi

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.

Imaging intravitale del Nodo poplitea Lymph mouse

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.

Non invasiva Imaging di candidosi disseminata nelle larve Zebrafish

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.

Non invasiva Modello di neuropatogeno<em&gt; Escherichia coli</em&gt; L&#39;infezione neonatale nel ratto

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.

Thymectomy completa in ratti adulti con non-invasiva intubazione endotracheale

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.

Imaging non invasiva della risposta immunitaria innata in un modello di Zebrafish larvale<em&gt; Streptococcus iniae</em&gt; Infezione

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 metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

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