This work was supported by AI101263 from the National Institutes of Health (T.V.B), and by a grant from The Hartwell Foundation (M.L.M). M.L.M. is a member of the Duke Comprehensive Cancer Center.

NOTE: Toutes les procédures expérimentales impliquant l&#39;utilisation de rats ont été effectuées conformément aux protocoles approuvés par le soin et l&#39;utilisation des animaux Comité de l&#39;Université Duke. 1. Préparation de la canule d&#39;intubation trachéale <ol><li> Coupez l&#39;extrémité de l&#39;aiguille d&#39;une aiguille de 14 G angiocathether 2 pouces avec une pince coupante. </li><li> Pincez la lumière de l&#39;aiguille fermé avec une pince à bec effilé. </li><li> Couper la section pincé vers le bord de la partie ouverte-lumière avec une pince coupante, puis couper les bords des deux côtés de la fin restant à 30-45 ° angle au bord de l&#39;aiguille. </li><li> Placez la partie inférieure de 3 à 4 mm de la canule dans la zone ouverte de pinces à joint coulissant droites et tirez vers le haut jusqu&#39;à une courbe à la hausse légère a été formé à la fin. NOTE: Cela facilitera diriger le cathéter dans la trachée (Figure 1). </li><li> Utilisez du papier abrasif fin pour lisser les bords à l&#39;extrémité de la canule. </li><li&gt; Retour l&#39;instrument émoussé dans la tubulure de angiocathéter. Assurez-vous que le tube est légèrement plus courte que l&#39;aiguille émoussée. </li></ol> Procédures 2. Pré-chirurgicales <ol><li> Mettre en place le site pour la procédure en plaçant un microscope opératoire réglé à un grossissement de 10X sur la zone d&#39;exploitation. </li><li> Placez un coussin chauffant sur la zone d&#39;exploitation et couvrir avec un tampon absorbant propre. </li><li> Configuration d&#39;un rythme cardiaque et un moniteur d&#39;oxygénation du sang à proximité de la zone d&#39;opération chirurgicale. </li><li> Peser le rat pour les médicaments posologie en fonction du poids (par exemple, l&#39;analgésie postopératoire, antibiotiques, anticorps ou de lymphocytes ozone). NOTE: typique rats adultes pèsent entre 350 à 450 g. </li><li> Sedate le rat vaporisé avec 3% d&#39;isoflurane-O 2 (3 L / min) en utilisant une chambre d&#39;induction évacué vers un système d&#39;évacuation des gaz d&#39;anesthésie déchets contenant du charbon activé. Laisser 5 min pour induction de l&#39;anesthésie pour fournir une anesthésie profonde nécessaire à l&#39;intubation endotrachéale. </li><li> Administrer Carprofène ou Meloxicane (4,4 mg / kg) sous-cutanée chez le rat avant de procéder à l&#39;intubation et la chirurgie. </li><li> Utilisez une tondeuse électrique se raser le cou et la poitrine du rat anesthésié avant l&#39;intubation. </li></ol> 3. intubation <ol><li> Préparer la configuration d&#39;intubation en frottant une petite quantité de lubrification (par exemple, gel KY) sur l&#39;extrémité de la canule d&#39;intubation. Allumer le ventilateur à commencer l&#39;écoulement de gaz à travers le tube isoflurane du connecteur. </li><li> Transférer le rat de l&#39;appareil d&#39;intubation et suspendre le rat sur ​​la barre métallique par ses dents d&#39;incisive supérieure (figure 2A). NOTE: Certains appareils d&#39;intubation peuvent être achetés en ligne. Le modèle présenté ici est fait maison en utilisant une feuille acrylique moulée autour des sections d&#39;un morceau lombaire 2 x 4 pouces utilisant une torche au butane, et des renforts sont attachés avec du chloroforme. Le bar (ou fil métallique) est fixé par des trous de forage de plus de ruban de masquage mis sur le side de l&#39;acrylique pour prévenir l&#39;éclatement ou de fissuration. </li><li> Trans-éclairer le cou du rat par le positionnement d&#39;une source de lumière souple à haute intensité de 1 à 2 cm de la surface ventrale du cou (figure 2B). </li><li> Utilisez une paire de pinces étudiants modèle standard de tirer la langue doucement vers le haut et sur le côté des dents du bas. Saisir la langue entre le pouce et l&#39;index d&#39;une main tout en plaçant la surface intérieure d&#39;un côté plat de la pince contre l&#39;extrémité inférieure de la langue. Appuyez sur le ventre pour exposer l&#39;épiglotte et l&#39;ouverture du larynx (figure 2C). </li><li> Visualiser les cordes vocales et guider la retroussé, extrémité émoussée de la canule d&#39;intubation en avant à travers la glotte ouverte dans la trachée jusqu&#39;à ce que le moyeu de la angiocathéter touche les incisives. </li><li> Retirez le stylet de métal, et fixer le tube d&#39;anesthésie à l&#39;ouverture de angiocathéter pour commencer la ventilation à l&#39;isoflurane. NOTE: Le flo typique d&#39;oxygènetaux w pour le ventilateur est de 3 L / min avec 3% d&#39;isoflurane. <ol><li> Régler le ventilateur à une vitesse de 60 respirations / min dans un mode de ventilateur volumique contrôlée qui permet d&#39;obtenir une pression de ~ 12 à 14 mmHg. Utilisez une pression expiratoire positive (PEEP) de 3 cm H 2 O. </li></ol></li><li> Observez l&#39;expansion de la paroi thoracique bilatérale en synchronisation avec le ventilateur pour assurer un placement correct de la sonde endotrachéale. </li><li> Appliquer pommade ophtalmique vétérinaire recommandé aux yeux du rat pour prévenir la sécheresse sous anesthésie. </li><li> Appliquer vétérinaire pommade sur les yeux du rat pour empêcher sec sous anesthésie. </li><li> Fixer la sonde endotrachéale à la tête du rat avec une bande de ruban de tissu. Assurer la connexion entre la sonde endotrachéale et le tube d&#39;entrée anesthésique. </li><li> Fixez l&#39;oxygénation du sang et moniteur de fréquence cardiaque au pied du rat et de commencer la surveillance. Confirmez anesthésie adéquate en confirmant qu&#39;il n&#39;y a pas de réaction àe-pincement. </li></ol> 4. thoracotomie et Thymectomie <ol><li> Suivez technique aseptique standard pour l&#39;ensemble de la procédure. <ol><li> Nettoyez la zone et d&#39;exploitation table de travail et désinfecter avec une solution d&#39;éthanol à 70%. </li><li> Utiliser des gants chirurgicaux stériles pendant la procédure, et l&#39;autoclave tous les instruments et les matériaux utilisés lors de la procédure. </li><li> Appliquer providone-iode à toute la région de la poitrine et laisser sécher. Ensuite, nettoyer la surface de la peau avec 70% d&#39;éthanol sur de la gaze. Couvrir le rat avec une pellicule de plastique transparent découper un trou pour exposer le champ opératoire stérile. </li></ol></li><li> Identifier la fourchette sternale dans la région thoracique supérieure. Faire une incision médiane 2 cm à travers la peau à partir de 2 à 3 mm au-dessus de la fourchette sternale et se étendant distalement ligne médiane le long du sternum aide de ciseaux Shea épointées. </li><li> Effectuez une sternotomie 1,5 cm médian de la fourchette sternale utiliser à nouveau des ciseaux Shea épointées. KEEP le bord inférieur de la paire de ciseaux juste sous le sternum et avance lentement. </li><li> Insérez un petit écarteur Alm juste en dessous du sternum séparé et se ouvrent pour révéler les muscles de la sangle pré-trachéale (sterno et les muscles sternothyroid). Séparer les muscles de la sangle pré-trachéales utilisant une pince Graefe contondants. NOTE: A ce stade, la trachée peut être vu, et le tube d&#39;intubation doit être visualisé intérieur de la trachée. </li><li> Placez les dents de la petite enrouleur Alm sous les muscles de la sangle et séparés sternum. Ouvrez l&#39;enrouleur pour exposer la face supérieure du thymus. </li><li> Utilisez pince fine Dumont pour libérer les bords latéraux du tissu de thymus et exposer les lobes thymiques inférieurs. </li><li> Tirez doucement le thymus supérieurement dans le site de l&#39;incision ouverte, en prenant soin d&#39;éviter tout contact avec la veine cave, sous-clavière et carotide navires supérieurs, et de minimiser les perturbations de la plèvre délicat entre le thymus et les poumons. </li><li> Comme til thymique vaisseaux sont disséqués et révélé, utiliser des micro-ciseaux pour les diviser. Utilisez un coton-tige pour maintenir la pression sur les navires pour l&#39;hémostase si nécessaire. </li><li> Livrer les lobes thymiques inférieurs dans l&#39;incision, puis lyser fortement les attaches postérieures. Retirez le thymus intact et inspecter soigneusement le thymus retiré pour toutes les sections manquantes. Remarque: Les petits ganglions lymphatiques thoraciques peuvent être visualisées entourant le thymus et sont souvent difficiles à différencier de tissu thymique. Ceux-ci seront rondes noeuds discrets avec une apparence similaire à tissu thymique mais ne seront pas en continuité avec le thymus. </li><li> Retirer l&#39;écarteur Alm et fermer les muscles sterno et sternothyoid avec deux interrompu 5-0 sutures Maxon. </li><li> Ajouter 2 gouttes de grade chirurgical adhésif tissulaire à base de cyanoacrylate pour les sutures à sceller le médiastin sous ventilation à pression positive. NOTE: Ce sera de diminuer l&#39;incidence de pneumothorax et l&#39;hématome. </li><li> Fermez le sternum avec deux interrompu 4-0 sutures de soie sur une aiguille de coupe. Insérez l&#39;aiguille à travers des espaces entre les nervures, en prenant soin de guider l&#39;aiguille juste sous le sternum afin d&#39;éviter la couche de muscle sous-jacent. </li><li> Fermez la couche de la peau avec un exécutant 4-0 Nylon suture. Cesser l&#39;isoflurane à ce stade de raccourcir la période de récupération de l&#39;anesthésie post-opératoire. </li><li> Nettoyez le site d&#39;incision et la peau environnante avec de la gaze saline humidifié. </li><li> Appliquer plusieurs gouttes de bupivicaïne (0,25%) pour l&#39;anesthésie locale, suivie de 1-2 gouttes de colle cyanoacrylate sur l&#39;incision pour sceller l&#39;incision. </li><li> Continuer à ventiler jusqu&#39;à ce que le rat montre des signes de l&#39;effort respiratoire indépendant et commence à se déplacer ses extrémités. Extuber le rat et lui permettre de récupérer sous étroite surveillance dans une cage placée sur un coussin chauffant. </li><li> Administrer buprénorphine (0,01 à 0,05 mg / kg) sous-cutanée chez le rat immédiatement post-opératoire et répéter tous les 8-12 h pour 48 h post-opératoire (normalement 5 doses au total). À 24 et 48 h post-opératoire, administrer ou Carprofène Meloxicane (4,4 mg / kg) par voie sous cutanée (trois doses totales, y compris la dose pré-opératoire). </li><li> Continuer à surveiller jusqu&#39;à ce que le rat se déplace autour de la cage. Au moindre signe de détresse respiratoire, effectuez re-intubation et re-exploration de la poitrine. <ol><li> Ne pas laisser un animal sans surveillance tant qu&#39;il a repris conscience suffisante pour maintenir décubitus sternal. </li><li> Ne retournez pas un animal qui a subi une intervention chirurgicale à la compagnie d&#39;autres animaux jusqu&#39;à guérison complète. </li></ol></li><li> Retirez les sutures de la couche de la peau 1 semaine post-opératoire. </li></ol>

<ol>
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The authors have nothing to disclose.

Le présent protocole pour thymectomie complète fournit une approche minimalement invasive avec une incision de fermeture à trois couches avec l&#39;application de la colle chirurgicale conçue pour minimiser les complications. L&#39;élimination complète du thymus a été démontré par la perte de cellules T naïves et par coloration IHC de tissu lymphoïde médiastinale pour la cytokératine. La procédure de thymectomie de rat adulte a été compliquée par des taux de mortalité alla...

Depuis le début des années 1960, le thymus a été reconnu pour son rôle critique dans le développement de la tolérance immunologique centrale. Rongeur thymectomie se est avérée être une procédure indispensable dans la définition du rôle du thymus dans la différenciation des lymphocytes, une auto-tolérance, et immunotolérance dans le cadre de la transplantation d&#39;allogreffes et les métastases tumorales. Retrait du thymus de rat permet études impliquant l&#39;épuisement des lymphocytes T ou le transfert adoptif de populations de lymphocytes T définis sans la ré-émergence de cellules T naïves indigènes. Thymectomies néonatale chez les rongeurs peuvent être accomplies en utilisant une technique d&#39;aspiration à des résultats fiables 1. Chez les rats adultes, cette technique est associée à un taux de mortalité d&#39;environ 20% et se traduit souvent par une thymectomie deux incomplet. Pour atteindre constamment thymectomie complète chez les rats adultes, l&#39;exposition ouverte du médiastin à travers une sternotomie médiane est nécessaire. Toutefois, cetteprocédure est associée à des complications qui incluent les blessures de la trachée, l&#39;hémorragie et de pneumothorax conduisant à un taux de mortalité global allant de 1,5 à 6% 2- 4. Au cours des deux dernières décennies amélioration des techniques de Thymectomie ont diminué complications périopératoires et ont amélioré les taux de survie. L&#39;intubation endotrachéale permet une ventilation à pression positive a diminué les taux de pneumothorax 5. Méthodes d&#39;intubation précédemment décrits plage de l&#39;exposition ouverte à la trachée à des méthodes moins invasives utilisant la visualisation des cordes vocales directe. Les complications associées à la procédure d&#39;intubation trachéale comprennent les blessures, vocal rupture du cordon, intubation oesophagienne involontaire, et l&#39;hémorragie résultant de ponction cardiaque ou une lacération de la veine cave supérieure. En outre, près des lobes thymiques inférieurs à la plèvre peut entraîner un pneumothorax. Nous décrivons ici une technique dethymectomie à travers une incision de 2 cm mini-invasive de la peau suite à une méthode simple de l&#39;intubation endotrachéale en utilisant un angiocathéter émoussé-end et l&#39;éclairage de la trachée transcutanée. La thymectomie implique une sternotomie 1,5 cm et d&#39;une fermeture à trois couches avec application de colle chirurgicale pour sceller le médiastin et de minimiser l&#39;incidence de l&#39;hémorragie et de complications respiratoires. Cette méthode permet de manière fiable dans la thymectomie complète comme le montre la disparition de CD4 + et CD8 + des cellules T naïves suivant la thymectomie et l&#39;absence de tissu thymique de coloration IHC. Temps opératoires et la mortalité péri-opératoire sont réduits au minimum.

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Name of Reagent/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:152px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">2&#34; 14-gauge angiocatheter</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Operating microscope</td>
			<td style="width:117px;">Zeiss</td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Warming pad</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Heart rate and blood oxygenation monitor for rodents with foot sensors</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-8010, ST1 72-8098 (Rat foot sensor)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Intubation apparatus (plastic with metal bar at the top)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;">See Figure 2&#160;</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:283px;">Small animal anesthesia system with induction box, isoflurane tank and O2 tank</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-6420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:283px;">Small animal ventilator with tubing</td>
			<td style="width:117px;">CWE</td>
			<td style="width:152px;">12-02000 (ventilator)and 12-04000 (external valve assembly for mice/rats)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High-intensity Fiber-Optic Illuminator</td>
			<td style="width:117px;">Dolan Jenner</td>
			<td style="width:152px;">EEG 2823M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Student standard pattern forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">91100-16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine straight scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14060-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt-tipped Shea scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14105-12</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Small Alm retractor (for sternum)</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">17008-07</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt Graefe forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11050-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine Dumont forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5-0 Maxon sutures</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">4-0 Silk sutures (with cutting needle)</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">6-0 Nylon suture</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cyanoacrylate glue (Endermil)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Lubrication gel</td>
			<td style="width:117px;">Akorn Animal Health</td>
			<td style="width:152px;">NDC 17478-162-35</td>
			<td></td>
		</tr>
	</tbody>
</table>

complete thymectomy in adult rats with non-invasive endotracheal intubation

Thymectomie néonatale chez les rongeurs est une procédure établie et fiable pour les études immunologiques. Toutefois, chez les rats adultes, les complications de l&#39;hémorragie et de pneumothorax perturbation pleural peuvent entraîner un taux de mortalité significatif. Ce protocole est une méthode simple de thymectomie de rat qui utilise un mini-sternotomie et l&#39;intubation endotrachéale. Intubation est réalisée avec une méthode non-invasive et facilement reproductible et permet une ventilation à pression positive pour empêcher un pneumothorax et une voie aérienne contrôlée qui permet suffisamment de temps pour la dissection minutieuse thymus de minimiser les perturbations pleural. Une incision sternale 1,5 cm diminue contact avec les vaisseaux médiastinaux et la plèvre, tout en offrant une visualisation complète du thymus. Après exposition du médiastin, le thymus est éliminé par dissection mousse sous grossissement. La cavité pleurale est ensuite scellée par la fermeture de suture des muscles trachéaux pré-suivi par l&#39;application de la colle chirurgicale. Lethorax est alors fermé par la fermeture de suture du sternum, suivie d&#39;une fermeture de suture de la peau. Tous thymectomies étaient complets comme en témoigne immunohistochimique (IHC) coloration du tissu médiastinale, et l&#39;absence de lymphocytes T naïfs par cytométrie de flux, et la procédure a eu un taux de survie de 96%. Cette méthode convient lorsque thymectomie complet avec un minimum de complications est souhaitée pour d&#39;autres études immunologiques chez les rats athymiques adultes.

Cette procédure a été réalisée sur des rats Lewis adultes (n = 26). La durée de fonctionnement moyenne était de 15 ± 3 minutes. Il n&#39;y avait aucune mortalité peropératoire. Le temps moyen d&#39;intubation - de placement de rat sur l&#39;appareil d&#39;intubation de l&#39;établissement de ventilation - était de 45 ± 5 s. 24 rats avaient un rétablissement complet de l&#39;opération sans aucun signe de difficultés respiratoires ou une hémorragie en jour post-opératoire (POD) 14. Un rat difficultés re...

Watch this Scientific Journal Video about Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation at JoVE.com

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

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 ...

complete-thymectomy-adult-rats-with-non-invasive-endotracheal

Research

JoVE Journal

Immunology and Infection

16.4K vues.  Duke University Medical Center. 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. 

Vidéo: Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

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 ...

Recherche

Immunologie et infection

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 ...

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 ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia de culture du système nerveux central néonatale et de l&#39;adulte

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological 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 ...

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 ...

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

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

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

Une méthode non-invasive et technique non-intensif pour Induction et phénotypage of Experimental pneumonie bactérienne chez les souris

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

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non invasif<em&gt; In Vivo</em&gt; Imagerie optique à fluorescence d&#39;une activité MMP inflammatoire à l&#39;aide d&#39;un agent d&#39;imagerie fluorescente activable

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Analyse des Lymphocytes extravasation L&#39;utilisation d&#39;un<em&gt; In vitro</em&gt; Modèle de la barrière hématoencéphalique humaine

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Une manière non invasive d&#39;isoler et de phénotype des cellules de la conjonctivite

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...