Ce travail a été soutenu par NIH R01-3155-53502 et de l&#39;Université de Washington-Pfizer accord de recherche biomédicale.

1. La préparation des animaux: Ce protocole utilise adulte femelle adulte LysM-eGFP souris, dans lequel les neutrophiles et les macrophages sont marqués par fluorescence par l&#39;expression de eGFP 1. <ol class="lalpha"><li> Étiquetage des vaisseaux sanguins: Pour visualiser les vaisseaux sanguins, 15μl de points quantiques non-ciblées (655nm) sont dilués dans 100 ul de PBS et injecté par voie intraveineuse chez la souris 10-30 min avant l&#39;imagerie. </li><li> Anesthésie: <ol><li> Placez la souris dans la chambre de l&#39;induction de l&#39;anesthésie par inhalation système de vétérinaires (Noble Service médical, Inc). </li><li> Réglez le vaporisateur à 3-4% isoflurane et le transporteur 100% du débit d&#39;oxygène gazeux taux à ~ 1 L / min. Afin d&#39;éviter un surdosage accidentel d&#39;anesthésie, de surveiller étroitement l&#39;animal jusqu&#39;à ce que le réflexe de pieds à l&#39;arrière de pincement est perdu. </li><li> L&#39;anesthésie est maintenue à 1,5 ~ 2% isoflurane et peaufiné lors de l&#39;imagerie afin de minimiser les artefacts de respiration si nécessaire. Le rythme respiratoire de la souris est surveillée attentivement tout au long de la séance d&#39;imagerie afin d&#39;assurer un avion adéquat d&#39;anesthésie. </li><li> Puralube une pommade vétérinaire est appliqué sur les yeux pour éviter le dessèchement. </li><li> La souris est placée sur un 36 ° C régulée coussin chauffant (Braintree scientifique) pendant les injections et l&#39;imagerie et à prévenir l&#39;hypothermie. </li><li> Pour prévenir la déshydratation, la souris est donné 100 ul de PBS SC toutes les 1-2 heures. </li></ol></li><li> Offrir un stimulus inflammatoire pour induire le recrutement des leucocytes: <ol><li> Placez la souris sur la scène la chirurgie et le coussinet plantaire tige avec de l&#39;éthanol à 70%. </li><li> Plier l&#39;aiguille d&#39;une seringue à insuline ~ 90 degrés près de la conique pour faciliter l&#39;injection et d&#39;améliorer la précision de la profondeur de livraison. </li><li> Charger la seringue avec 0,5 pg de E.coli bioparticules dilué dans 1 microlitre PBS. Cela peut être fait dans le couvercle d&#39;un tube de centrifugeuse pour rendre plus facile à charger les gouttelettes. </li><li> Tenir le pied avec une pince courbée et insérer l&#39;aiguille doucement à travers la peau avec le biseau vers le haut jusqu&#39;à ce qu&#39;il soit juste en dessous de la peau. </li><li> Injecter lentement et maintenir en place pendant 5 secondes pour éviter tout retour rincer. </li></ol></li></ol> 2. Configuration scénique d&#39;imagerie: La plate-forme d&#39;imagerie se compose d&#39;une plaque d&#39;aluminium avec un trou circulaire coupé par le centre. Un couvercle en verre grande est collée sur le bas de la plaque et un joint torique en caoutchouc est collé sur le dessus pour créer une chambre étanche renfoncés pour la patte arrière. La plaque d&#39;aluminium est chauffé à 36 ° C au moyen de deux éléments de chauffage fixé au sommet de la plaque. <ol class="lalpha"><li> Placez la souris sur la scène d&#39;imagerie pré-chauffée et maintenir la température centrale du corps de la souris en utilisant le pavé de 36 ° C le réchauffement. </li><li> Fixez la patte de la lamelle de la chambre de l&#39;imagerie avec un mince film de colle. </li><li> Laver la patte à deux reprises pour enlever tous les débris flottants de colle, puis remplir la chambre avec du PBS frais (préchauffé à 36 ° C). </li></ol> Les souris peuvent être imagées pour un maximum de 4 heures sans comprenant la viabilité de l&#39;animal. Le pied peut être doucement libérée avec une petite quantité d&#39;acétone et de souris repris pour les études d&#39;imagerie longitudinales. Toutefois, dans ce cas, nous recommandons la période d&#39;imagerie à &lt;2 heures et 24-48 heures d&#39;attente entre les séances afin de minimiser le risque de surdose d&#39;anesthésie ou de déshydratation. 3. Acquisition d&#39;imagerie Le microscope à deux photons (microscope 2P) utilisé dans ce protocole est un sur-mesure à double laser, vidéo-système de taux comprenant un microscope droit. Le système est équipé de deux Ti: Sapphire lasers, quatre de front Bi-alcali PMT pour les acquisitions simultanées canal 3 et 4 et d&#39;un objectif 20x immersion dans l&#39;eau (l&#39;Olympe, 20x/0.95 NA). <ol class="lalpha"><li> Tune le Caméléon XR Ti: saphir laser (Coherent Inc) à 890-900nm. </li><li> Utilisez 480nm et 560nm miroirs dichroïques (SemRoc) pour séparer trois canaux: le bleu (&lt;480 nm, la deuxième génération d&#39;harmoniques du signal), vert (480-560nm, LysM-eGFP neutrophiles positifs) et rouge (&gt; 560 nm, quantum dot vaisseaux sanguins étiquetés ). Sinon, le filtre 560nm peut être remplacé par un filtre 570nm pour distinguer E. bioparticules coli (576nm bioparticules apparaîtra en orange) à partir des points quantiques 655nm (apparaîtra rouge profond). </li><li> Abaisser avec précaution l&#39;objectif dans le PBS et se concentrer sur un navire de l&#39;orteil. </li><li> Avec la vitesse de fonctionnement d&#39;acquisition d&#39;images à la vidéo-taux (30f/sec) et en utilisant le signal de second harmonique émanant de collagène des tissus comme un repère, rapidement enquête sur les tissus (à la vitesse d&#39;acquisition vidéo-taux) pour localiser un vaisseau sanguin d&#39;intérêt (un avec les visibles lysM-eGFP neutrophiles). </li><li> Réglez le gain sur la PMT pour optimiser la séparation des couleurs et minimiser la quantité de laser requise pour obtenir un signal suffisamment sur fond (soitAttention à ne pas saturer l&#39;image!). </li><li> Une fois que la région centre d&#39;intérêt se situe, commencent imagerie time-lapse. Time-lapse imagerie peut être effectuée avec deux différentes résolutions temporelles. Pour rouler imagerie cellulaire et de l&#39;adhérence et en temps réel dans les vaisseaux sanguins, nous acquérons 30f/sec à un simple plan z. Pour documenter l&#39;extravasation des cellules et la migration dans le parenchyme nous effectuons 3D imagerie time-lapse. Un protocole d&#39;acquisition typiques consistera en moyenne 15 images pour chaque étape et Z-acquisition 21 2.5μm séquentielle z-étapes pour un volume de 200 à 225 50 microns, à intervalles de 30 sec. </li><li> Une fois les fichiers de données sont stockés sur le serveur de laboratoire, des logiciels ou des Imaris Volocity peut être utilisé pour effectuer le rendu multi-dimensionnelle et de la cellule de suivi 2, 3. </li></ol> 4. Les résultats représentatifs 1.Real-temps (30f/sec) imaginant des neutrophiles rouler le long des vaisseaux à 200x. Laps de temps d&#39;imagerie montre des neutrophiles, en vert, rouler le long de l&#39;endothélium des vaisseaux sanguins environ 10 minutes après l&#39;infection par la bactérie Listeria monocytogenes, représentée en rouge. Les fibres de collagène dans le tissu conjonctif apparaissent en bleu. <a href="http://www.jove.com/files/ftp_upload/2062/2062_Miller_Movie1.mov">S&#39;il vous plaît cliquer ici pour voir la vidéo.</a> 2.Les-lapse imagerie 3D de la dynamique de recrutement des neutrophiles à 200x. Typiques recrutement des neutrophiles de la circulation et la migration à travers les tissus enflammés est montré. <a href="http://www.jove.com/files/ftp_upload/2062/2062_Miller_Movie2.mov">S&#39;il vous plaît cliquer ici pour voir la vidéo.</a>

<ol>
	<li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li><li>Zinselmeyer, B. H., Lynch, J. N., Zhang, X., Aoshi, T., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-rate+two-photon+imaging+of+mouse+footpad+-+a+promising+model+for+studying+leukocyte+recruitment+dynamics+during+inflammation.">Video-rate two-photon imaging of mouse footpad - a promising model for studying leukocyte recruitment dynamics during inflammation.</a> Inflamm Res. 57, 93-96 (2008).</li><li>Zinselmeyer, B. H., Dempster, J., Wokosin, D. L., Cannon, J. J., Pless, R., Parker, I., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+16.+Two-photon+microscopy+and+multidimensional+analysis+of+cell+dynamics.">Chapter 16. Two-photon microscopy and multidimensional analysis of cell dynamics.</a> Methods Enzymol. 461, 349-378 (2009).</li><li>Gray, D. F., Jennings, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergy+in+experimental+mouse+tuberculosis.">Allergy in experimental mouse tuberculosis.</a> Am Rev Tuberc. 72, 171-195 (1955).</li><li>Lin, A., Loughman, J. A., Zinselmeyer, B. H., Miller, M. J., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+S+inhibits+neutrophil+recruitment+during+the+early+stages+of+Streptococcus+pyogenes+infection.+Infect+Immun.">Streptolysin S inhibits neutrophil recruitment during the early stages of Streptococcus pyogenes infection. Infect Immun.</a> 77, 5190-5201 (2009).</li><li>Smith, C. J., Zhang, Y., Koboldt, C. M., Muhammad, J., Zweifel, B. S., Shaffer, A., Talley, J. J., Masferrer, J. L., Seibert, K., Isakson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+analysis+of+cyclooxygenase-1+in+inflammation.">Pharmacological analysis of cyclooxygenase-1 in inflammation.</a> Proc Natl Acad Sci U S A. 95, 13313-13318 (1998).</li><li>Wipke, B. T., Allen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+neutrophils+in+the+initiation+and+progression+of+a+murine+model+of+rheumatoid+arthritis.">Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis.</a> J Immunol. 167, 1601-1608 (2001).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc Res. 5, 384-394 (1973).</li><li>Mempel, T. R., Scimone, M. L., Mora, J. R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">in vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr Opin Immunol. 16, 406-417 (2004).</li><li>Miller, M. J., Wei, S. H., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+T+cell+trafficking+examined+in+vivo+with+intravital+two-photon+microscopy.">Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.</a> Proc Natl Acad Sci U S A. 100, 2604-2609 (2003).</li></ol>

Toutes les expériences ont été réalisées selon des protocoles approuvés par le Comité des études animales de l&#39;Université Washington à St Louis.

Dans ce protocole vidéo nous montrent les procédures d&#39;imagerie non-invasive 2P du recrutement des leucocytes en réponse à l&#39;inflammation. Le coussinet plantaire est un site classique pour l&#39;étude de l&#39;inflammation physiologiques telles que la maladie d&#39;allergie, d&#39;infection et auto-immunes 4-7. 2P imagerie fournit une image détaillée des étapes distinctes dans les voies de trafic de leucocytes, de laminage et d&#39;adhérence dans les vaisseaux san...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Butler Animal Health Supply</td>
<td>029405</td>
<td>&nbsp;</td>
<tr>
<td>655nm non-targeted quantum dots</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Q21021MP</td>
<td>&nbsp;</td>
<tr>
<td>Tetramathylrhodamine conjugated E. coli BioParticles </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>E2862</td>
<td>&nbsp;</td>
<tr>
<td>Listeria monocytogenes (Lm)</td>
<td>&nbsp;</td>
<td>Reference 2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Quick gel</td>
<td>&nbsp;</td>
<td>Duro</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Puralube Vet Ointment </td>
<td>&nbsp;</td>
<td>Pharmaderm Animal Health</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>08290-3284-38</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Thermo Scirntific</td>
<td>SH30256.02</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

non-invasive imaging of leukocyte homing and migration in vivo

À deux photons (2P) est une technique de microscopie à haute résolution d&#39;imagerie qui a été largement adaptée par les biologistes. La valeur de la microscopie 2P est qu&#39;il fournit de riches informations sur les comportements spatio-temporelle des cellules dans les tissus intacts et dans des souris vivantes. Le recrutement des leucocytes jouent un rôle important dans la défense de l&#39;hôte contre l&#39;infection et quand incontrôlée, peut contribuer à des maladies inflammatoires et autoimmunes. Etudier le recrutement des leucocytes in vivo est un défi technique, car les cellules se déplacent rapidement dans les vaisseaux situés en profondeur dans les tissus diffusion de la lumière. À ce jour, la plupart des études d&#39;imagerie intravitale nécessitent une préparation chirurgicale pour exposer les vaisseaux sanguins et des tissus. Pour éviter les dommages tissulaires et l&#39;inflammation induite par la chirurgie elle-même, ici, nous décrivons une approche non invasive d&#39;imagerie à cellule unique qui peut être utilisée pour étudier le trafic des leucocytes dans le coussinet plantaire de souris et des phalanges. Nous discutons des aspects techniques de notre préparation imagerie 2P et marcher le lecteur à travers une expérience typique de la configuration initiale à la collecte de l&#39;exécution et des données.

Watch this Scientific Journal Video about Non-invasive Imaging of Leukocyte Homing and Migration in vivo at JoVE.com

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Ici, nous décrivons un non-invasive à deux photons approche microscopique (2P) pour l&#39;étude des leucocytes homing dans le coussinet plantaire de la souris. Nous discutons des aspects techniques de notre préparation imagerie des tissus et de marcher le lecteur à travers une expérience typique de la configuration initiale à la collecte de l&#39;exécution et des données.

L&#39;imagerie non invasive des Homing leucocytes et des migrations 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 ...

non-invasive-imaging-of-leukocyte-homing-and-migration-in-vivo

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

12.7K vues.  Washington University in St. Louis. Ici, nous décrivons un non-invasive à deux photons approche microscopique (2P) pour l'étude des leucocytes homing dans le coussinet plantaire de la souris. Nous discutons des aspects techniques de notre préparation imagerie des tissus et de marcher le lecteur à travers une expérience typique de la configuration initiale à la collecte de l'exécution et des données.

Vidéo: Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

Les analyses concurrentielles Homing d&#39;étude Gut tropisme migration des lymphocytes T

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Recherche

Immunologie et infection

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Adhésion leucocytaire imagerie de l&#39;endothélium vasculaire au Haute pression intraluminale

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

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.

L&#39;imagerie non invasive de la candidose disséminée chez les larves poisson zèbre

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

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent imagerie bactérienne<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

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

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Non invasive d&#39;imagerie optique du système vasculaire lymphatique d&#39;une souris

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

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.

Modèle non-invasive de neuropathogènes<em&gt; Escherichia coli</em&gt; Maladies infectieuses chez le rat nouveau-né

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.

Thymectomie complète chez des rats adultes avec les non-invasive intubation endotrachéale

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.

Imagerie non invasive de la réponse immunitaire innée dans un modèle de poisson zèbre larves 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 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Interactions Microscopie intravitale des leucocytes endothéliaux et plaquettes-leucocytes dans mésentériques veines chez les souris

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Imagerie CD4 T Cellule interstitielle Migration dans les Derme Enflammé

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...