Les auteurs tiennent à remercier Laura Diaz, Kevin Phillips, Willa A. Hsueh, et le roi C. Li pour leur soutien utiles commentaires et technique dans le développement de cette méthode.

1. Préparation des animaux et de Traitement par le froid <ol><li> Localiser et inspecter un 4 ° C chambre froide qui a été approuvé pour recevoir des souris de laboratoire. </li><li> Cages pour animaux de pré-refroidissement pendant la nuit dans la chambre froide. Les cages sont assemblés sans nourriture et la litière, mais avec une bouteille d&#39;eau. </li><li> Dans la matinée de la journée expérimentale, les souris lieu un par un dans chacune des cages pré-réfrigérés à intervalles de 30 min. Chaque souris individuellement en cage devrait rester dans la chambre froide pendant près de 4 heures avant d&#39;être transporté au laboratoire d&#39;imagerie. S&#39;assurer que les souris sont à jeun mais avec un accès à l&#39;eau. </li><li> À 4 h après le transport d&#39;un animal traitement par le froid à la fois toutes les 30 minutes au laboratoire d&#39;imagerie. Ceci peut être obtenu en remplissant un récipient en polystyrène avec de la glace et à placer une cage de boîtier pré-refroidi au-dessus de la glace à l&#39;intérieur de la boîte. Librement placer le couvercle sur la boîte en polystyrène. </li></ol> 2. Configuration Micro-PET/CT imagerie de flux de travail <p class=&quot;Jove_content&quot;&gt; Dans ce protocole d&#39;imagerie micro-PET/CT est atteint avec la TEP Siemens Inveon dédié (PDEF) Système et multimodalité Inveon (MM) système (CT / SPECT) en mode connecté. L&#39;animal est placé de l&#39;entrée MM, balayée par le premier scanner de repères anatomiques, et ensuite déplacé vers le centre de la MEPF pour une acquisition statique F18 PET. Afin de permettre à l&#39;ordinateur hôte pour mener à bien ces tâches séquentielles automatiquement, ce qui suit &quot;workflow&quot; est programmé avec le milieu de travail Acquisition Inveon (AIF) logiciel avant la séance d&#39;imagerie réelle. <ol><li> Acquisition CT: Pour l&#39;ensemble du corps tomodensitométrie, régler le courant à 500 uA, la tension à 80kV, temps d&#39;exposition à 200 ms, et 240 marches pour 240 ° de rotation. Pour détecteur de rayons X, sélectionnez la résolution à «faible grossissement système&quot; avec 78 mm Imagerie en mode axial et un lit simple. Sélectionnez «reconstruction en temps réel&quot; en utilisant le &quot;Common Cone-Beam reconstruction&quot; méthode pour que les pourparlers PC hôte avecun véritable ordinateur dédié reconstruction de temps (Cobra) pour lancer la tâche. </li><li> Acquisition d&#39;émission PET: Set 600 sec (10 min) pour &quot;temps de cycle fixe&quot; dans le &quot;acquérir par le temps&quot; option. Sélectionnez F-18 comme «isotope étude&quot; et utilisez 350-650 keV comme «niveau d&#39;énergie». </li><li> Histogramme des émissions PET: Set &quot;cadre dynamique» comme «noir» pour traiter les données comme une image pendant toute la durée de balayage pour atteindre statique. Sélectionnez &quot;3D&quot; comme type histogramme et choisissez &quot;aucune correction de dispersion&quot;. </li><li> Reconstruction TEP: Utilisez 2D Maximisation Commandé Attente sous-ensemble (OSEM2D) que l&#39;algorithme de reconstruction. </li></ol> 3. L&#39;injection de FDG <ol><li> Commander un paquet clinique de 18 F-FDG (10 mCi) d&#39;un fournisseur régional de son arrivée au laboratoire d&#39;imagerie ~ 30 min avant l&#39;injection programmée premier. Suivre les procédures de sécurité de l&#39;Institut pour recevoir et étudier le paquet contenant materia radioactifsls (RAM). </li><li> Avec la protection offerte par un bouclier L-bloc de dessus de table, partie aliquote du FDG et faire des dilutions de sérum physiologique stérile. La concentration d&#39;activité du FDG diluée devrait être disponible à 200-300 μCi/100 ul pour chaque injection. Aspirer la solution de FDG dans une seringue de 1 ml avec 26G 1/2 pouces aiguille, et de mesurer la radioactivité de la seringue en entier avec un calibreur de dose. </li><li> Injecter l&#39;animal qui est tout simplement transporté de la chambre froide (voir l&#39;étape 1.4) avec 100 pi de solution de FDG par voie intrapéritonéale (ip) de route. Noter le temps d&#39;injection. Mesurer la radioactivité résidu de la seringue à nouveau avec le calibreur de dose. </li><li> Placez l&#39;animal à l&#39;intérieur d&#39;une cage froide refroidisseur polystyrène maintenues avec de la glace. Incuber l&#39;animal au froid (~ 4 ° C) pendant 1 heure pour la fixation du FDG. </li><li> Calculer l&#39;activité injectée FDG pour chaque souris par la formule suivante: Injecté activité (Ci) = activité dans la seringue avant l&#39;injection- L&#39;activité dans la seringue après l&#39;injection </li></ol> 4. Micro-PET/CT Imaging <ol><li> L&#39;imagerie micro-PET/CT commence 1 heure après l&#39;injection du FDG ou 5 heures après le traitement par le froid. Placez l&#39;animal dans une chambre d&#39;induction de l&#39;anesthésie avec l&#39;isoflurane 3% en oxygène. </li><li> Une fois l&#39;anesthésie est induite, l&#39;animal est déplacé sur une pastille micro-CT (lit pour animaux) avec sa tête reposant dans un masque cône qui fournit en permanence l&#39;isoflurane (2%) à un débit de 2 L / min. Un coussin chauffant électrique (BioVet, m2m Imaging Corp) est placé sous l&#39;animal pour aider à maintenir la température du corps. </li><li> Faites glisser l&#39;animal à l&#39;entrée du scanner MM, activer le &quot;laser&quot; icône de la barre d&#39;outils, et utilisez la commande touchpad pour déplacer le lit de sorte que la poitrine de l&#39;animal est à la croisée des lignes laser horizontales et verticales. Dans le &quot;alignement laser&quot; fenêtre, sélectionnez &quot;Type de la première analyse&quot; comme la tomodensitométrie, et «Acquisition TEP inclus dans le workflow&quot; comme option. </li><li> Ouvrez la fenêtre &quot;Vue scout&quot; fenêtre et acquérir une vision scout radiographie radiographie. Utiliser AIF pour ajuster la position du lit animal de façon que le champ de vue du centre CT est situé dans le centre du corps de la souris (foie). Répétez cette étape si nécessaire. </li><li> Démarrez le &quot;workflow&quot; créé à l&#39;étape 2. Lorsque des options s&#39;affiche, sélectionnez une appropriée 3D PET-CT matrice de transformation de fichier à utiliser en reconstruction tomographique, et choisissez un fichier normalisation récemment créé pour la reconstruction TEP sans correction d&#39;atténuation. L&#39;AIF va alors commencer CT et TEP séquentiellement comme programmés. </li><li> Après l&#39;ensemble du workflow est terminé, ce qui peut prendre 20-25 minutes, brièvement évaluer la qualité de l&#39;acquis images TEP et CT avec le VM ASIPro, un logiciel d&#39;analyse micro-TEP co-installé avec AIF. Archiver les données d&#39;imagerie à un périphérique de stockage de données ou de transférer les données à travers le réseau à un poste de travail après l&#39;analyse d&#39;imagerie (voir l&#39;étape 5) pour une analyse plus approfondie. </li><li> ReleaSE l&#39;animal à partir des systèmes d&#39;imagerie et le placer à une cage de boîtier propre à l&#39;alimentation normale et d&#39;approvisionnement en eau pour sa récupération à la température ambiante. Les systèmes sont maintenant prêts pour le prochain animal dans la file d&#39;attente. Notez le soin et la manipulation des animaux de post-imagerie doit se conformer aux règlements de l&#39;Institut en ce qui concerne &quot;la manipulation des animaux de laboratoire injectés avec de la mémoire&quot;. A noter également que utilisé aiguilles / seringues, les tampons absorbants, gants, et toute la literie et les matières fécales doivent être considérés comme des déchets radioactifs, et manipulés conformément à la réglementation pertinentes institut RAM élimination des déchets. </li></ol> 5. Analyse post-imagerie <ol><li> Ouvrir la recherche Inveon milieu de travail (IRW) logiciel (Siemens) et importer manuellement CT et PET ensembles de données. Co-registre images TEP et CT dans &quot;Enregistrement&quot; fenêtre à l&#39;aide des outils de l &#39;«analyse générale&quot; la fonction, et sous la rubrique «Revue&quot; fenêtre de s&#39;assurer un alignement parfait entre les images TEP et CT dans les 3 dimensions. </li><li>De «région d&#39;intérêt (ROI) Quantification&quot; fenêtre, avec les références fournies par les images CT co-enregistrés, d&#39;identifier les meilleures techniques disponibles dans la région interscapulaire du cou, le plus prédominant inductible par le froid BAT chez la souris adulte, et d&#39;en tirer volume d&#39;intérêt (VOI) des MTD sur l&#39;ensemble des données TEP. Sélectionnez &quot;Intensité Voxel» comme «Type de Quantification&quot; et enregistrer la radioactivité moyenne dans le VOI en Bq / ml. Un facteur d&#39;étalonnage qui convertit coups / sec en Bq / ml a été établi préalablement par la numérisation d&#39;un fantôme à la radioactivité connue. </li><li> La fixation du FDG dans la MTD est quantifié par la dose injectée pourcentage par gramme de tissu (% ID / g) avec correction de décroissance. La dose injectée est le résultat de l&#39;étape 3.5, cependant, converti en becquerel (Bq) Unité (1 pCi = 37.000 Bq), nous supposons que 1 ml de tissu est égal à 1 g. </li></ol>

<ol>
	<li>Cypess, A. M., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+importance+of+brown+adipose+tissue+in+energy+homeostasis.">The role and importance of brown adipose tissue in energy homeostasis.</a> Curr. Opin. Pediatr. 22, 478-484 (2010).</li><li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+function+and+physiological+significance.">Brown adipose tissue: function and physiological significance.</a> Physiol. Rev. 84, 277-359 (2004).</li><li>Collins, S., Surwit, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-adrenergic+receptors+and+the+control+of+adipose+tissue+metabolism+and+thermogenesis.">The beta-adrenergic receptors and the control of adipose tissue metabolism and thermogenesis.</a> Recent Prog. Horm. Res. 56, 309-328 (2001).</li><li>Ouellet, V., 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=Outdoor+temperature,+age,+sex,+body+mass+index,+and+diabetic+status+determine+the+prevalence,+mass,+and+glucose-uptake+activity+of+18F-FDG-detected+BAT+in+humans.">Outdoor temperature, age, sex, body mass index, and diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-detected BAT in humans.</a> J. Clin. Endocrinol. Metab. 96, 1115-1125 (2011).</li><li>Cypess, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> N. Engl. J. Med. 360, 1509-1517 (2009).</li><li>Fruhbeck, G., Becerril, S., Sainz, N., Garrastachu, P., Garcia-Velloso, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAT:+a+new+target+for+human+obesity.">BAT: a new target for human obesity.</a> Trends Pharmacol. Sci. 30, 387-396 (2009).</li><li>Cypess, A. M., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+fat+as+a+therapy+for+obesity+and+diabetes.">Brown fat as a therapy for obesity and diabetes.</a> Curr. Opin. Endocrinol. Diabetes Obes. 17, 143-149 (2010).</li><li>Virtanen, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> N. Engl. J. Med. 360, 1518-1525 (2009).</li><li>van Marken Lichtenbelt, W. D., 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=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> N. Engl. J. Med. 360, 1500-1508 (2009).</li><li>Cohade, C., Mourtzikos, K. A., Wahl, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="USA-Fat":+prevalence+is+related+to+ambient+outdoor+temperature-evaluation+with+18F-FDG+PET/CT.">"USA-Fat": prevalence is related to ambient outdoor temperature-evaluation with 18F-FDG PET/CT.</a> J. Nucl. Med. 44, 1267-1270 (2003).</li><li>Garcia, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+brown+fat+2-deoxy-2-[F-18]fluoro-D-glucose+uptake+by+controlling+environmental+temperature+prior+to+positron+emission+tomography+scan.">Reduction of brown fat 2-deoxy-2-[F-18]fluoro-D-glucose uptake by controlling environmental temperature prior to positron emission tomography scan.</a> Mol. Imaging Biol. 8, 24-29 (2006).</li><li>Saito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+incidence+of+metabolically+active+brown+adipose+tissue+in+healthy+adult+humans:+effects+of+cold+exposure+and+adiposity.">High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.</a> Diabetes. 58, 1526-1531 (2009).</li><li>Agrawal, A., Nair, N., Baghel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+for+reduction+of+brown+fat+uptake+on+FDG+PET.">A novel approach for reduction of brown fat uptake on FDG PET.</a> Br. J. Radiol. 82, 626-631 (2009).</li><li>Soderlund, V., Larsson, S. A., Jacobsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+FDG+uptake+in+brown+adipose+tissue+in+clinical+patients+by+a+single+dose+of+propranolol.">Reduction of FDG uptake in brown adipose tissue in clinical patients by a single dose of propranolol.</a> Eur. J. Nucl. Med. Mol. Imaging. 34, 1018-1022 (2007).</li><li>Zhao, J., Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermogenesis+is+beta3-+but+not+beta1-adrenergically+mediated+in+rat+brown+fat+cells,+even+after+cold+acclimation.">Thermogenesis is beta3- but not beta1-adrenergically mediated in rat brown fat cells, even after cold acclimation.</a> Am. J. Physiol. 275, 2002-2011 (1998).</li><li>Rowland, D. J., Garbow, J. R., Laforest, R., Snyder, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registration+of+[18F]FDG+microPET+and+small-animal+MRI.">Registration of [18F]FDG microPET and small-animal MRI.</a> Nucl. Med. Biol. 32, 567-5672 (2005).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Dans cette étude, une méthode d&#39;imagerie micro-PET/CT-based a été développé pour la détection des activités de BAT chez la souris adulte qui nécessite simplement un traitement par le froid et une injection de disponible dans le commerce 18 F-FDG. L&#39;ensemble de la procédure peut se faire en un jour à la suite d&#39;un traitement et d&#39;imagerie séquence qui commence toutes les 30 minutes jusqu&#39;à ce que tous les animaux sont traités et imagée. Dans les conditions expérimentales dé...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Micro-PET/CT Imaging System </td><td> Siemens Medical Solutions USA, Inc </td><td> Inveon dédié PET Système et multimodalité Inveon CT / SPECT système (amarré) </td><td></td></tr><tr><td> Propranolol </td><td> Sigma </td><td> P0884 </td><td></td></tr><tr><td> BRL 37344 </td><td> Sigma </td><td> B169 </td><td></td></tr><tr><td> 18 F-FDG </td><td> Cyclotope Inc </td><td></td><td></td></tr><tr><td> Les souris mâles C57BL/6J </td><td> Jackson Laboratory </td><td> 000664 </td><td> 3-4 mois </td></tr></tbody></table>

functional imaging of brown fat in mice with 18f-fdg micro-pet/ct

Le tissu adipeux brun (BAT) diffère de tissu adipeux blanc (WAT) par son emplacement discret et une couleur brun-rouge due à la vascularisation riche et haute densité des mitochondries. BAT joue un rôle majeur dans la dépense énergétique et non frissons thermogenèse chez les mammifères nouveau-nés ainsi que les adultes 1. BAT-thermogenèse induite est fortement régulée par le système nerveux sympathique, principalement par l&#39;intermédiaire des récepteurs adrénergiques β 2, 3. Des études récentes ont montré que les activités de BAT chez les adultes humains sont corrélés négativement avec l&#39;indice de masse corporelle (IMC) et les paramètres de diabétiques d&#39;autres 4-6. BAT a donc été proposé comme une cible potentielle pour la thérapie anti-obesity/anti-diabetes mettant l&#39;accent sur ​​la modulation de la balance énergétique 6-8. Alors que plusieurs froides défi à base de tomographie par émission de positons (TEP) méthodes ont été établies pour détecter BAT humaine 9-13, il ya pratiquement pas de protocole standardisé pour l&#39;imagerie et QUANTIFication des MTD dans de petits modèles animaux tels que des souris. Nous décrivons ici une méthode d&#39;imagerie robuste TEP / TDM pour l&#39;évaluation fonctionnelle des MTD chez la souris. Brièvement, des souris C57BL/6J adultes étaient traités à froid à jeun pour une durée de 4 heures avant d&#39;avoir reçu une dose de 18 F-fluorodésoxyglucose (FDG). Les souris ont été resté dans le froid pendant une heure après l&#39;injection supplémentaire FDG, puis scannées avec un système micro-PET/CT animal dévoué mais peu nombreux. Les images TEP acquises ont été co-enregistré avec les images CT pour les références anatomiques et analysés pour la fixation du FDG dans la zone BAT interscapulaire de présenter l&#39;activité BAT. Ce standard de traitement à froid et le protocole d&#39;imagerie a été validé par des activités de test MTD lors des interventions pharmacologiques, par exemple, l&#39;activation BAT supprimée par le traitement de la β-adrénergique antagoniste propranolol 14, 15, ou l&#39;activation BAT renforcée par agoniste β3 BRL37344 16. La méthode described ici peuvent être appliqués pour cribler des médicaments / composés qui modulent l&#39;activité BAT, ou pour identifier les gènes / voies qui sont impliqués dans le développement de BAT et de la réglementation dans plusieurs études précliniques et de base.

Un exemple de l&#39;imagerie micro-PET/CT des MTD souris est illustré à la figure 1. Alors que l&#39;imagerie CT fournit des informations anatomiques, l&#39;imagerie TEP code de la distribution et de la quantité de 18 F-FDG dans tout le corps. Ces données d&#39;imagerie peuvent être regardées séparément (1A et 1B), fusionnée (1C), ou résulter d&#39;une fonctionnalité 3D comme projection d&#39;intensité maximale (MIP, 1D). Avec l&#39;aide d&#39;un outil d&#39;imagerie 3D, un volume...

Watch this Scientific Journal Video about Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT at JoVE.com

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Une méthode d&#39;imagerie fonctionnelle du tissu adipeux brun de la souris (BAT) est décrit dans lequel froide stimulée par l&#39;absorption de 18F-fluorodésoxyglucose (FDG) dans la MTD est non-invasive évaluée avec un protocole standardisé micro-PET/CT. Cette méthode est robuste et sensible pour détecter des différences dans les activités de BAT dans des modèles murins.

Imagerie fonctionnelle de la graisse brune chez les souris avec FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

functional-imaging-of-brown-fat-in-mice-with-18f-fdg-micro-petct

Research

JoVE Journal

Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

19.0K vues.  The Methodist Hospital Research Institute, Houston. Une méthode d'imagerie fonctionnelle du tissu adipeux brun de la souris (BAT) est décrit dans lequel froide stimulée par l'absorption de 18F-fluorodésoxyglucose (FDG) dans la MTD est non-invasive évaluée avec un protocole standardisé micro-PET/CT. Cette méthode est robuste et sensible pour détecter des différences dans les activités de BAT dans des modèles murins.

Vidéo: Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Test de transition clair / foncé pour souris

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Recherche

Biologie

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Étiquetage CSEh et hMSCs d&#39;oxyde de fer nanoparticules pour les non-invasives dans le suivi in ​​vivo avec IRM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Étiquetage des cellules souches avec des colorants fluorescents pour les non-invasive de détection avec l&#39;imagerie optique

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Imagerie fonctionnelle avec des renforts, Eyetracking, et en surveillance physiologique

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Elevated Maze Plus pour Souris

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Assemblée, de chargement, et l&#39;alignement d&#39;une cellule échantillon ultracentrifugation analytique

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

Une méthode alternant le modèle traditionnel de la NASA hindlimb déchargement chez la souris

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Imagerie optique basée sur des photodiodes pour l’enregistrement de la dynamique des réseaux avec une résolution mononeurone chez les invertébrés non transgéniques

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

Isolement et différenciation des préadipocytes primaires blancs et bruns de souris nouveau-nées

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Visualisation et quantification des tissus adipeux bruns et beiges chez la souris à l’aide de l’imagerie micro-PET/MR [18F]FDG

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...