The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility &#8221;Two-Photon Imaging&#8221;, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.

REMARQUE: Les expériences ont été effectuées en conformité avec la législation allemande régissant les études animales suivantes le «Guide pour le soin et l&#39;utilisation des animaux de laboratoire de (la publication NIH, 8e édition, 2011) et la directive 2010/63 / UE sur la protection des animaux utilisé à des fins scientifiques (Journal officiel de l&#39;Union européenne, 2010). Autorisation officielle a été accordée dans le soin des animaux et l&#39;utilisation bureau gouvernemental (LANUV Nordrhein-Westfalen, Recklinghausen, Allemagne). NOTE: Les mesures qui peuvent être omis pour l&#39;imagerie à court terme (par exemple, des piles Snap Shots 3-D ou aussi courte durée time-lapse microscopie) sont marquées d&#39;un astérisque (*) pour réduire le temps de préparation et de simplifier le protocole chirurgical. L&#39;imagerie peut également être réalisée sans un contrôle étendu de surveillance et de la circulation, si nécessaire, cependant, le temps de survie sera nettement réduite. 1. Configuration de Microscope et pré-chirurgie Préparation (5-10 kmn) <ol><li> système de commutateur (microscope, puissance Lab, laser, coussin chauffant, chambre climatique, web cam, dispositif de pompe de la seringue, un respirateur). </li><li> Connectez O 2 offre à l&#39;isoflurane vapeur et régler le niveau isoflurane à 1,5% en volume (v / v), se connecter à un petit appareil respiratoire des animaux. <ol><li> Pour court imagerie terme, maintenir l&#39;anesthésie à l&#39;isoflurane seulement après l&#39;induction initiale de l&#39;anesthésie ip (voir l&#39;étape 2.2). Ensuite, utilisez 2 Vol% (v / v) isoflurane, et de garder le temps d&#39;imagerie en dessous de 2 h pour garantir stable microcirculation du foie. </li></ol></li><li> Réglez la respiration à 120-140 cycles / min respiratoires avec un volume de 150 à 200 pi. </li><li> Mettre en place l&#39;étape d&#39;agarose de la souris. Préparer 100 ml de 3% (p / v) d&#39;agarose, le verser dans un plat (environ 10 cm x 15 cm base) dans un angle de 40 °. </li><li> Mettre en place la zone de travail chirurgical recueillir l&#39;instrumentation nécessaire. Pour prévenir l&#39;infection microbienne, la désinfection d&#39;instruments en utilisant une solution à 1% du Forte Sekusept S (9,9% p / v), Glutaral 9,8% p / v, de formaldéhyde pendant 5 min avant l&#39;expérience (figure 1A). </li><li> Obtenir des composants restants: désinfectant de la peau (par exemple, une solution de la povidone iodée 10%), l&#39;étape du foie (piles et pont), une solution d&#39;agarose (3% (p / v) dans du PBS), la pompe à seringue avec 5% (p / v) d&#39;une solution de glucose (G-5), la pompe à seringue avec une solution anesthésique I (Kétamine 0,1 mg / ml, xylazine 0,5 mg / ml, 5 pg de fentanyl / ml), des tampons de coton, le n-butyl-2-Cyanoacrylat et 1x PBS. Mettez plat stade agarose dans la chambre d&#39;incubation pour le préchauffage. </li></ol> 2. trachéotomie (10-15 min) <ol><li> Utilisez souris C57BL / 6 dans la maison de l&#39;élevage pesant 25-28 g. Maison des souris dans des conditions exempts d&#39;organismes pathogènes spécifiques en conformité avec les lignes directrices FELASA, dans un environnement à température et humidité contrôlées avec un / 12 h cycle d&#39;obscurité 12 heures de lumière. Permettre aux animaux un accès gratuit à un régime standard de souris (Sniff standard rongeurs Diète) et de l&#39;eau ad libitum. </li><li> Anesthésier la souris par initial intraperitoneale (ip) d&#39;injection de 250 ul d&#39;une solution anesthésique de kétamine II (0,1 mg / ml, xylazine 1 mg / ml, la buprénorphine 10 ug / ml). <ol><li> * Pour l&#39;entretien lors de l&#39;imagerie intravitale, administrer en continu la solution anesthésique I par injection ip continu (Kétamine 0,1 mg / ml, xylazine 0,5 mg / ml, le fentanyl 5 ug / ml) en utilisant un appareil de pompage à seringue à un débit de 0,2 ml / h dans par inhalation d&#39;isoflurane combinaison avec 0,5% en volume (v / v). </li><li> Vérifiez réflexes après 5 min (par exemple pincée de coussinet plantaire avec une pince), désinfecter la peau pour la trachéotomie, commencer la préparation si la souris est en état ​​d&#39;anesthésie chirurgicale tolérante. </li><li> Appliquer une crème de protection oculaire (par exemple, Dexpanthenol 50 mg / g) pour empêcher la cornée de sécher (figure 1B). </li></ol></li><li> Fixer la souris sur la table de préparation exposer face ventrale avec du ruban adhésif pour les extrémités; overstretch doucement le cou, fixer dans cette position, par exemple., en utilisant une bande de caoutchouc accroché to les incisives. <ol><li> Désinfectez la peau et de la fourrure en utilisant une solution d&#39;iode povidone, en appliquant un désinfectant avec un coton-tige. </li></ol></li><li> Effectuer la découpe de la peau initial (0,5 à 1 cm de longueur) directement sous le menton (figure 1C) <ol><li> Disséquer soigneusement le tissu conjonctif entre les glandes salivaires (figure 1D). </li><li> Détachez avec soin ouverte musculaire tube entourant la trachée (figure 1E, F). </li></ol></li><li> Placez fil chirurgical sous la trachée pour plus tard la fixation du tube de ventilation (figure 1G). <ol><li> Ouvrir la trachée par des micro-ciseaux entre les anneaux cartilagineux effectuant une incision en forme de T (figure 1H) </li></ol></li><li> Placez le tube de ventilation dans la trachée à travers l&#39;incision (~ 0,5 cm). Pour pousser le tube avant, saisir extrémité caudale avec de petites pinces anatomiques et avancer doucement le tube dans la trachée (Figure 1I) </li><li> Fixer tube avec fil chirurgical (par exemple, </em&gt;, 5-0) avec un fil placé à l&#39;étape 2.5 un nœud autour du tube intérieur de la trachée; effectuer seconde fixation crânienne du tube à la peau pour éviter réciproquement des accidentelle (figure 1J, K). </li><li> coupe du joint avec de la colle de tissu (par exemple, le n-butyl-2-Cyanoacrylat) (Figure 1L). </li><li> Fixer le tube à la tête avec du ruban adhésif. </li></ol> 3. laparotomie (15-20 min) <ol><li> Placez la souris sur coussin chauffant pour prévenir l&#39;hypothermie. Rasez abdomen, retirer délicatement les poils de la peau. Désinfectez la peau et de la fourrure rasée en utilisant une solution de povidone iode. </li><li> Effectuer une petite couper la peau en dessous du sternum avec des ciseaux chirurgicaux (figure 2A). Prolonger la coupe latéralement sous les côtes des deux côtés, cautériser tous les vaisseaux sanguins visibles pour prévenir les saignements. </li><li> Effectuer avec précaution une petite coupure à la ligne blanche en dessous du sternum, ouverture du péritoine (figure 2B). Prolonger la coupe des deux côtés utilisant cauterization pour prévenir les saignements (figure 2C, D). </li><li> Placez la souris dans un plat de scène agarose (figure 2E). Placez des piles de hauteur appropriée des deux côtés de la souris (en général de 12 à 14 des lamelles) (figure 2F). </li><li> Placez capteur de déclenchement respiratoire sous le haut du dos pour synchroniser le microscope avec la respiration. Activer unité de déclenchement (figure 2G). </li><li> Placez fil chirurgical (5-0) à travers le sternum se rétracter nervures (Figure 2I). </li><li> Découpez soigneusement ligament reliant le foie et le diaphragme (ligament falciforme) ainsi que le foie et tractus gastro-intestinal avec des ciseaux chirurgicaux courbes. Couper le ligament falciforme jusqu&#39;à l&#39;aorte (figure 2J). </li></ol> 4. Configuration de l&#39;échantillon (10-15 min) <ol><li> Placez la souris sur le côté droit avec un angle de 45 ° pour un accès facile à gros lobe du foie. </li><li> Ajouter pont en scène sous les côtes, couvrant la abdominalecavité. Fabrication d&#39;un pont en utilisant une lamelle standard avec revêtement adhésif de bande pour couvrir les arêtes vives (Figure 2K). </li><li> Placez gros lobe du foie sur scène. Glisser soigneusement double roulement sonde de stylet ou une spatule rembourrée dessous du foie et tenir le haut de l&#39;organe en utilisant un coton-tige humide ou tissu humide. Soulevez lobe sur la diapositive et appliquer glisser en douceur. Lobe peut être plié ou plié. Fournir des soins supplémentaire pour que la manipulation douce du foie (Figure 2L). </li><li> Placez piquets de soutien latéral, par exemple, des tas de petites lamelles (20 mm x 20 mm), d&#39;environ la même hauteur du lobe du foie à côté de lui pour soutenir lamelle. </li><li> Passer grande lamelle (24 mm x 50 mm) sur le lobe du foie. Veiller à ce que lamelle est orienté aussi horizontale que possible (Figure 2M). NOTE: La lamelle doit être en contact avec le tissu sans presser. Vérifiez les signes visibles de la circulation sanguine avec facultés affaiblies (tissu blanc). Si MICROCIRCulation est perturbé, ajouter soutenir davantage enjeux. </li><li> * Placez deux cathéters intra-péritonéales indépendants (Figure 2N) pour l&#39;anesthésie à long terme et l&#39;application G-5. Installez cathéters latéralement dans le bas-ventre à côté des membres postérieurs. REMARQUE: Les cathéters intra-péritonéales peuvent être self-made en connectant 27 G aiguilles avec un tube de silicone souple (Figure 3). </li><li> * Aiguille Fixate avec une boucle de fil chirurgical 5-0 sur la peau pour éviter tout déplacement accidentel. </li><li> * Joindre pompe seringue avec une solution d&#39;anesthésie I cathéter 1, réglage du débit à 0,2 ml / h; attacher pompe seringue avec le G-5 à cathéter 2, débit réglé à 0,1 ml / h. </li></ol> 5. Suivi de la souris <ol><li> * Lieu électrodes ECG dans les extrémités avant et arrière (figure 4A). </li><li> * Fixer le tube d&#39;expiration de CO 2 capteur de niveau. </li><li> Ajouter capteur de température externe connecté au pad thermique (figure 4C). </li></ol> 6. Embedding et fixation tissulaire (5-10 min) <ol><li> Préparer 100 ml de 3% (p / v) d&#39;agarose dans du PBS 1X. Incorporer foie lorsque la température est à 41 ° C en utilisant une seringue de 5 ml et une aiguille 18 G (figure 5A). </li><li> Verser le reste d&#39;agarose sur lamelle couvre-objet et autour de la souris (figure 5B, C). Attendez jusqu&#39;à ce que l&#39;agarose est complètement gélatinisé. </li><li> Enlever l&#39;excès d&#39;agarose en utilisant la spatule Heidemann, la préparation d&#39;un Viewfield suffisamment grande pour balayer le lobe hépatique préparé (figure 5D, E). </li></ol> 7. Imaging <ol><li> Transférer la souris dans la chambre climatique de microscope. </li><li> Ajouter 50 à 100 ml de 1 x PBS (préchauffé à 37 ° C). </li><li> Couvrir le plat de l&#39;échantillon pour éviter l&#39;évaporation (figure 5F). </li><li> * Diminution par inhalation d&#39;isoflurane à 0,5% en volume (v / v). </li><li> * Lancer un logiciel de surveillance, l&#39;enregistrement ECG, la fréquence cardiaque et de CO 2 expiratoire. </li><li> Lancer imagerie: ouvrir laser volets, identifier les champs de vue de l&#39;intérêt, définissent la limite supérieure et inférieure pour Z-piles, et commencer l&#39;enregistrement laps de temps. Réajuster Z-piles si nécessaire pour corriger Z-dérive (par ex. En raison de changements dans la pression sanguine ou les fluctuations de température, Figure (5G). REMARQUE: Après la fin de la période de formation d&#39;image ou si la circulation ou l&#39;anesthésie des animaux devient instable, sacrifier les souris par dislocation cervicale (sans réveil de l&#39;anesthésie). </li></ol>

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K., 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=NKT-associated+hedgehog+and+osteopontin+drive+fibrogenesis+in+non-alcoholic+fatty+liver+disease.">NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.</a> Gut. 61 (9), 1323-1329 (2012).</li><li>McDonald, B., 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=Interaction+of+CD44+and+hyaluronan+is+the+dominant+mechanism+for+neutrophil+sequestration+in+inflamed+liver+sinusoids.">Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.</a> J Exp Med. 205 (4), 915-927 (2008).</li><li>Egen, J. 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The authors disclose no con&#64258;ict of interests.

Le but de notre étude était de développer une méthode très standardisé, stable et reproductible pour l&#39;imagerie intravitale TPLSM du foie. Imagerie intravitale en général a donné de précieuses indications sur le comportement cellulaire dans des conditions réelles suivantes homing et l&#39;interaction des différentes populations de leucocytes dans le développement, l&#39;homéostasie et la maladie. Cependant, la position anatomique peu difficile du foie, en raison de laquelle respiratoire et le mouvement...

La visualisation des cellules et des fonctions cellulaires dans des organes entiers, voire des organismes entiers a été d&#39;un grand intérêt pour les plus de 50 ans, y compris presque toutes les parties du corps 2. Par conséquent, certaines études antérieures déjà employés imagerie intravitale du foie 3,4. Cependant, plusieurs limitations existent à jour concernant stable à long terme imagerie à haute résolution des tissus du foie. En raison de la position anatomique du foie en contact étroit avec la membrane et le tractus gastro-intestinal 5, le problème le plus commun pour l&#39;imagerie microscopique intravitale mouvement est due à la respiration et, dans une moindre mesure, péristaltique de l&#39;intestin 6. En comparaison à d&#39;autres organes solides, la chirurgie du foie est particulièrement difficile. En raison de la structure microvasculaire dense, manipulation chirurgicale peut entraîner des lésions hémorragiques massifs, troubles de la microcirculation 7 et également l&#39;activation de résident immune cellules telles que des cellules de Kupffer 8. Par conséquent, la fixation mécanique du tissu telle que publiée ailleurs 6,9 est susceptible d&#39;interférer avec l&#39;imagerie par microscopie intravitale. Dans un foie en bonne santé, de 10 à 15% du volume total de sang réside dans le système vasculaire du foie, et l&#39;organe reçoit l&#39;ordre de 25% de la production globale cardiaque 10, rendant l&#39;organe très sensible aux variations de la circulation (par exemple, les fluctuations de la pression artérielle ). Par conséquent, les perturbations dans le flux sanguin hépatique due à par exemple, contrainte de cisaillement, de déplacement, de blessure par une manipulation excessive des tissus ou la circulation centralisée seront conduire à des altérations artificielles dans leucocytes comportement migratoire, troubles de l&#39;oxygénation du foie et donc également des dommages au foie, affecter les réponses immunitaires du foie ainsi comme conservation d&#39;organes et de la durée de vie globale de l&#39;animal. Études microscopiques étaient fondés sur km à épifluorescence intravitalecroscopy, mais plusieurs contraintes techniques telles que le blanchiment photo et une faible profondeur de pénétration limiter l&#39;utilisation de cette technique pour le long terme imagerie du foie 4,11,12. Avec le développement de la microscopie multiphotonique dans les années 1990, les limites de la photo blanchiment ou la profondeur de pénétration ont principalement été résolus, que cette nouvelle méthode était techniquement capable d&#39;effectuer des études d&#39;imagerie dans pratiquement tous les organes dans des situations de la vie réelle de 13 à 15. Toutefois, les principaux défis à relever en ce qui concerne l&#39;imagerie du foie étaient: mouvements de respiration, autofluorescence des tissus du foie, de fixation flux sanguin inchangée dans les sinusoïdes hépatiques, et l&#39;imagerie particulièrement stable pour des périodes de plusieurs heures 16 plus longues. Bien que plusieurs études ont abordé la fonction et la migration des différents leucocytes dans le foie, par exemple, 17 cellules NKT 18-20, les cellules T 21,22, les macrophages du foie 23,24 ou 25 neutrophiles, long terme multiphotonique mimagerie icroscopy ne avait pas encore été établie avec succès, une tâche encore plus difficile chez les animaux atteints de maladie hépatique aiguë ou chronique en raison des dommages existants et la susceptibilité donc supérieur à 26 autres dommages. Toutefois, le suivi du comportement migratoire et la fonction cellulaire de leucocytes dans le foie en temps réel permet de nouvelles perspectives dans leur rôle particulier dans l&#39;homéostasie du foie et la maladie 27. Le CXCR6 des récepteurs de chimiokines est exprimé sur plusieurs sous-ensembles de lymphocytes, y compris les cellules tueuses naturelles (NK), les cellules NKT et certaines populations de cellules T 18,28. Des études antérieures sur des souris ont indiqué que CXCR6 et son CXCL16 de ligand apparenté peuvent contrôler les patrouilles des cellules NKT sur sinusoïdes du foie lors de l&#39;homéostasie. Par conséquent, l&#39;utilisation de souris CXCR6.gfp (portant un knock-in de la protéine verte fluorescente [GFP] dans le locus CXCR6) a été décrite pour étudier la migration des lymphocytes dans divers organes tels que le cerveau 29et aussi le foie 18,20, montrant l&#39;infiltration de cellules CXCR6.gfp augmenté sur l&#39;inflammation. Avec la méthode prévue dans cette étude, il était possible de suivre ces processus sur une longue période de temps dans des conditions stables. La procédure basée multiphotonique intravitale permis imagerie qui était très reproductible avec une perturbation minimale de l&#39;animal et l&#39;organe; optimisé pour la survie des animaux à long terme par une surveillance importante suivie d&#39;un contrôle étroit de la respiration et de la circulation; et très flexible et facile à adopter aussi à d&#39;autres organes parenchymateux tels que le rein ou de la rate.

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Anesthetics</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td style="width:293px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Buprenorphine</td>
			<td style="width:263px;">Essex Pharma</td>
			<td style="width:219px;">997.00.00</td>
			<td>Analgeticum, 0.1 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fentanyl</td>
			<td style="width:263px;">Rotex Medica</td>
			<td style="width:219px;">charge: 30819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fluovac anesthesia system</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td style="width:219px;">34-1030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glucose 5%</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td style="width:263px;">Eickemeyer</td>
			<td style="width:219px;">4802885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isoflurane</td>
			<td style="width:263px;">Forene Abbott</td>
			<td style="width:219px;">B 506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isotonic (0.9%) NaCl solution</td>
			<td style="width:263px;">DeltaSelect GmbH</td>
			<td style="width:219px;">PZN 00765145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ketamin 10%</td>
			<td style="width:263px;">ceva</td>
			<td style="width:219px;">Charge: 36217/09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xylazin 2%</td>
			<td style="width:263px;">medistar</td>
			<td style="width:219px;">Charge: 04-03-9338/23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Consumable supplies</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">20ml Syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250ml Erlenmeyer flask</td>
			<td style="width:263px;">Schott Duran</td>
			<td>21 226 36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">25mL Beaker 2x</td>
			<td style="width:263px;">Schott Duran</td>
			<td style="width:219px;">50-1150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">2ml syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl suture</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:219px;">V7980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:263px;">Bayer Vital GmbH</td>
			<td style="width:219px;">6029009.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Change-A-Tip Deluxe High-Temp Cautery Kit</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">18010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Gauze swabs</td>
			<td style="width:263px;">Fuhrmann GmbH</td>
			<td style="width:219px;">32014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cover Slip 24x50mm</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Durapore silk tape</td>
			<td style="width:263px;">3M</td>
			<td style="width:219px;">1538-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Feather disposable scalpel</td>
			<td style="width:263px;">Feather</td>
			<td style="width:219px;">02.001.30.011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fine Bore Polythene Tubing 0,58mm ID</td>
			<td style="width:263px;">Smiths medical</td>
			<td style="width:219px;">800/100/200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Histoacryl</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;">1050052</td>
			<td>5x 0,5ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Leukoplast</td>
			<td style="width:263px;">BSN Medical Inc.</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microscope Slides</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Poly-Alcohol Haut&#8230;farblos Antisepticum</td>
			<td style="width:263px;">Antiseptica GmbH</td>
			<td style="width:219px;">72PAH200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 18 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">304622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 27 3/4 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">4657705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue paper</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Instruments</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amalgam burnisher 3PL</td>
			<td style="width:263px;">Gatz</td>
			<td style="width:219px;">0110?</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blair retractors (4 pronged (blunt)) x2</td>
			<td style="width:263px;">Storz&#38;Klein</td>
			<td style="width:219px;">S-01134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont No.7 forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">91197-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps curved x1</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11151-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps straight x2</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11050-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heidemann spatula HD2</td>
			<td style="width:263px;">Stoma</td>
			<td style="width:219px;">2030.00</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:411px;">Needle holder Mathieu</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">12010-14</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Scissor</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14074-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Semken forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11008-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors curved</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14029-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors straight</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Standard pattern forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas spring scissors</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">15000-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td style="width:263px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">ECG Trigger Unit</td>
			<td style="width:263px;">Rapid Biomedical</td>
			<td style="width:219px;">3000003686</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Minivent Typ 845</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td>73-0043</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Multiphoton microscope Trimscope I</td>
			<td style="width:263px;">LaVision</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Perfusor Compact</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">PowerLab 8/30 8 channel recorder</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;">PL3508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled heating pad</td>
			<td style="width:263px;">Sygonix</td>
			<td style="width:219px;">26857617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature sensor</td>
			<td style="width:263px;">comercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled System for Microscopes -Cube&#38;Box</td>
			<td style="width:263px;">Life Imaging Services</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
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long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice

<html> inflammation du foie en tant que réponse à une blessure est un processus hautement dynamique impliquant l&#39;infiltration des sous-types distincts de leucocytes, y compris les monocytes, les neutrophiles, les sous-ensembles de cellules T, les cellules B, les cellules tueuses naturelles (NK) et des cellules NKT. Microscopie intravitale du foie pour le suivi de la migration des cellules immunitaires est particulièrement difficile en raison des exigences élevées en ce qui concerne la préparation des échantillons et la fixation, la résolution optique et la survie des animaux à long terme. Pourtant, la dynamique des processus inflammatoires ainsi que des études d&#39;interaction cellulaires pourraient fournir des informations cruciales pour mieux comprendre l&#39;initiation, la progression et la régression de la maladie inflammatoire du foie. Par conséquent, une méthode très sensible et fiable a été créé pour étudier la migration et cellule-cellule-interactions de cellules immunitaires différentes dans le foie de souris sur de longues périodes (environ 6 h) par intravitale deux photons microscopie à balayage laser (TPLSM) en combinaison avec des soins intensifs surveillance. ent &quot;&gt; Le procédé proposé comprend une préparation douce et la fixation stable du foie avec une perturbation minimale de l&#39;organe; imagerie intravitale à long terme en utilisant multicolore multiphotonique microscopie avec pratiquement pas de photoblanchiment ou des effets phototoxiques sur une période de temps allant jusqu&#39;à 6 heures, ce qui permet un suivi de sous-ensembles de leucocytes spécifiques et des conditions de formation d&#39;image stable en raison de la surveillance des paramètres vitaux extensif de souris et de stabilisation de la circulation, la température et l&#39;échange de gaz. Pour étudier la migration des lymphocytes à une inflammation du foie CXCR6.gfp souris knock-in ont été soumis à l&#39;imagerie du foie intravitale dans des conditions de base et après des lésions du foie aiguë et chronique induite par injection intraperitoneale (s) de tétrachlorure de carbone (CCl 4). CXCR6 est un récepteur de chimiokine exprimée sur les lymphocytes, principalement sur tueuses naturelles T (NKT) -, Natural Killer (NK) - et des sous-ensembles de lymphocytes T telles que les cellules T CD4 mais aussi asso muqueusesATED invariant des cellules (MAIT) T 1. Suivant le modèle migratoire et le positionnement de CXCR6.gfp + cellules immunitaires permis un aperçu détaillé dans leur changement de comportement lors d&#39;une lésion du foie et donc leur implication potentielle dans la progression de la maladie.

Pour valider notre approche de TPLSM intravitale, nous avons soumis GFP / + souris CXCR6 à l&#39;imagerie intravitale TPLSM. Les souris ont été soit laissées non traitées comme témoins ou de référence soumis à une injection intraperitoneale unique de tétrachlorure de carbone (CCl 4) pour induire des dommages au foie aiguë 20. Les séquences vidéo ont été prises sur une période de temps de 2 à 5 h, et les cellules ont été retracés au fil du ...

Watch this Scientific Journal Video about Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice at JoVE.com

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

À long terme intravitale microscopie multiphotonique imagerie des cellules immunitaires dans sains et malades du foie en utilisant des souris CXCR6.Gfp Reporter

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

long-term-intravital-multiphoton-microscopy-imaging-immune-cells

Research

JoVE Journal

Immunology and Infection

11.8K vues.  RWTH University-Hospital Aachen. Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Vidéo: Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sept étapes pour Stellate Cellules

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Recherche

Immunologie et infection

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagerie intravitale du Thymus souris utilisant la 2-microscopie biphotonique

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Chronique à long terme<em&gt; Pseudomonas aeruginosa</em&gt; Infection des voies respiratoires chez les souris

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Très Résolu microscopie intravitale Striped-éclairage de centres germinaux

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Microscopie intravitale imagerie du foie suivante<em&gt; Leishmania</em&gt; Infection: Une évaluation de l&#39;hémodynamique hépatiques

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

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 ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analyse de<em&gt; Yersinia enterocolitica</em&gt; Effecteur translocation dans les cellules hôtes à l&#39;aide de bêta-lactamase effecteur Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imagerie neutrophiles et des monocytes dans les veines mésentérique par Microscopie intravitale sur des souris anesthésiées en temps réel

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Imagerie intravitale de neutrophiles Amorçage Utilisation de IL-1ß conduit promoteur-DsRed Souris Reporter

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Imagerie de Mycobacterium tuberculosis chez la souris avec la journaliste Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...