Gli autori desiderano ringraziare Laura Diaz, Kevin Phillips, Willa A. Hsueh, e il re C. Li per il loro sostegno utili osservazioni e tecnico per lo sviluppo di questo metodo.

1. Animal Preparazione e trattamento a freddo <ol><li> Individuare e ispezionare una stanza fredda 4 ° C, che è stato approvato per l&#39;alloggiamento di topi di laboratorio. </li><li> Pre-chill gabbie per gli animali durante la notte in cella. Le gabbie sono assemblate senza mangime e le lettiere, ma con una bottiglia d&#39;acqua. </li><li> Al mattino del giorno sperimentale, topi posto uno per uno in ciascuna delle pre-raffreddate gabbie a intervalli di 30 min. Ogni topo singolarmente in gabbia dovrebbe stare in cella per circa 4 ore prima di essere trasportato al laboratorio di imaging. Assicurarsi che i topi sono il digiuno, ma con accesso all&#39;acqua. </li><li> Alle 4 di trasporto dopo il trattamento a freddo ora un animale alla volta ogni 30 minuti al laboratorio di imaging. Questo può essere ottenuto mediante riempimento di un contenitore di polistirolo con ghiaccio e mettendo un pre-raffreddata gabbia di alloggiamento sopra il ghiaccio all&#39;interno della scatola. Liberamente posizionare il coperchio della scatola di polistirolo. </li></ol> 2. Setup Micro-PET/CT Imaging Workflow <p class=&gt; &quot;Jove_content&quot; In questo micro-PET/CT protocollo di imaging è ottenuto con il Inveon Siemens Dedicated PET (dPET) Sistema e multimodalità Inveon (MM) Sistema (CT / SPECT) nella modalità ancorata. L&#39;animale viene posto dall&#39;ingresso MM, con la prima scansione CT per riferimenti anatomici, e poi scivolare verso il centro del dPET per una statica F18 acquisizione PET. Al fine di consentire al computer host di svolgere questi compiti in sequenza automaticamente, il seguente &quot;flusso di lavoro&quot; viene programmato con il posto di lavoro Inveon Acquisition (IAW) il software prima della sessione di imaging attuale. <ol><li> Acquisizione CT: per un intero corpo TC, impostare la corrente a 500 uA, tensione a 80kV, tempo di esposizione a 200 msec, e 240 passaggi per 240 ° di rotazione. Per il rivelatore di raggi X, selezionare la risoluzione a &quot;ingrandimento di basso&quot; con il 78 mm assiale campo dell&#39;imaging e la modalità di un letto singolo. Selezionare &quot;ricostruzione in tempo reale&quot; con il &quot;Common Cone-Beam ricostruzione&quot; metodo in modo che i PC colloqui di accoglienza conun apposito reale ricostruzione al computer tempo (Cobra) per avviare l&#39;attività. </li><li> Acquisizione PET Emissione: Situato a 600 sec (10 min) per &quot;tempo di ciclo fisso&quot; in opzione &quot;acquisto dal tempo&quot;. Selezionare F-18 come &quot;isotopo studio&quot; e l&#39;uso 350-650 keV come &quot;livello di energia&quot;. </li><li> PET Istogramma Emissione: Set &quot;telaio dinamico&quot; come &quot;nero&quot; per i dati di processo come una cornice per tutta la durata per ottenere la scansione statica. Selezionare &quot;3D&quot; come tipo di istogramma e scegliere &quot;nessuna correzione dispersione&quot;. </li><li> PET Ricostruzione: Usa 2D Ordered Subset Expectation Maximization (OSEM2D) come algoritmo di ricostruzione. </li></ol> 3. L&#39;iniezione di FDG <ol><li> Ordinare un pacchetto clinico di 18 F-FDG (10 mCi) da un venditore regionale per il suo arrivo al laboratorio di imaging ~ 30 minuti prima dell&#39;iniezione programmato prima. Seguire le procedure di sicurezza dell&#39;istituto per ricevere ed esaminare il pacchetto contenente materia radioattivals (RAM). </li><li> Con la protezione fornita da un L-block scudo tavolo, l&#39;aliquota FDG e fare diluizioni con soluzione salina sterile. La concentrazione di attività diluita di FDG dovrebbe essere disponibile a 200-300 μCi/100 ul per ogni iniezione. Aspirare la soluzione FDG in una siringa da 1 ml con 26G 1/2 pollici aghi, e misurare la radioattività della siringa intera con un calibratore di dose. </li><li> Iniettare l&#39;animale che è appena trasportato dalla camera fredda (vedi punto 1.4) con 100 ml di soluzione FDG via intraperitoneale (ip) route. Registrare il tempo di iniezione. Misurare la radioattività residuo della siringa di nuovo con il calibratore dose. </li><li> Mettere l&#39;animale torna alla gabbia fredda all&#39;interno di un dispositivo di raffreddamento polistirolo mantenuto con ghiaccio. Incubare l&#39;animale in freddo (~ 4 ° C) per 1 ora per la captazione di FDG. </li><li> Calcolare la dose iniettata FDG per ciascun topo con la seguente formula: Iniettata attività (uCi) = attività in siringa prima dell&#39;iniezione- Attività in siringa dopo l&#39;iniezione </li></ol> 4. Micro-PET/CT Imaging <ol><li> L&#39;imaging micro-PET/CT inizia 1 ora dopo l&#39;iniezione di FDG o 5 ore dopo il trattamento a freddo. Mettere l&#39;animale in una camera di induzione dell&#39;anestesia con il 3% isoflurano in ossigeno. </li><li> Una volta che l&#39;anestesia è indotta, l&#39;animale viene spostato su un micro-CT pellet (letto animale) con la testa appoggiata in una maschera cono che eroga in continuo isoflurano (2%) ad una portata di 2 L / min. Un pad riscaldamento elettrico (BIOVET, m2m Imaging Corp.) è posta sotto l&#39;animale per aiutare a mantenere la temperatura corporea. </li><li> Far scorrere l&#39;animale verso l&#39;ingresso dello scanner MM, attivare l&#39;icona &quot;laser&quot; dalla barra degli strumenti, e utilizzare il controllo touchpad per spostare il letto in modo che il petto dell&#39;animale è alla croce di linee laser orizzontali e verticali. Nella &quot;Laser Allinea&quot; finestra selezionare &quot;primo tipo di scansione&quot; come TAC, e &quot;Acquisizione PET incluso nel flusso di lavoro&quot; come option. </li><li> Aprire il &quot;View Scout&quot; finestra e acquisire una visione esploratore radiografia radiografia. Utilizzare IAW per regolare la posizione del letto animale in modo che il centro del campo di vista di CT si trova nel centro del corpo del mouse (fegato). Ripetere l&#39;operazione se necessario. </li><li> Avviare il &quot;flusso di lavoro&quot; stabilito al punto 2. Quando le opzioni pop-up, selezionare un appropriato 3D PET-CT trasformazione di file di matrice da utilizzare in CT ricostruzione, e scegliere un file di normalizzazione di recente creato per la ricostruzione PET senza la correzione dell&#39;attenuazione. La IAW inizierà CT e PET in sequenza come programmato. </li><li> Dopo l&#39;intero flusso di lavoro è completato, che può prendere 20-25 min, brevemente valutare la qualità delle immagini acquisite CT e PET con la VM ASIPro, un micro-PET software di analisi co-installato con IAW. Archiviare i dati di imaging di un dispositivo di memorizzazione dati o trasferire i dati attraverso la rete a una workstation analisi post di imaging (vedere il punto 5) per ulteriori analisi. </li><li> Release l&#39;animale dai sistemi di imaging e posizionarlo a una gabbia di alloggiamento pulito con cibo normale e acqua per il suo recupero a temperatura ambiente. I sistemi sono ora pronti per l&#39;animale successivo nella coda. Nota: la cura e la gestione del post-immagini animali dovrebbe seguire dell&#39;istituto norme in materia di &quot;gestione degli animali da laboratorio iniettati con RAM&quot;. Si noti inoltre che gli aghi usati / siringhe, i pad assorbenti, guanti, biancheria da letto e tutto e la materia fecale devono essere considerati come rifiuti radioattivi, e trattati secondo le norme dell&#39;istituto pertinenti RAM di smaltimento di rifiuti. </li></ol> 5. Post-analisi di imaging <ol><li> Apri ricerca Inveon Workplace (IRW) software (Siemens) e importare manualmente sia TC e dati PET set. Co-registrazione TC e immagini PET nella finestra &quot;Registrazione&quot; utilizzando gli strumenti con il &quot;generale analisi&quot; la funzione, e sotto la finestra &quot;Review&quot; assicurarsi un perfetto allineamento tra TC e immagini PET in tutte le 3 dimensioni. </li><li>Da &quot;regione di interesse (ROI) Quantificazione&quot; finestra, con i riferimenti forniti dal co-registrate le immagini TAC, identificare BAT alla regione interscapolare del collo, il più predominante freddo inducibile BAT in topi adulti, e disegnare il volume di interesse (VOI) di BAT sul set di dati PET. Selezionare &quot;Intensità Voxel&quot; come &quot;Tipo Quantificazione&quot; e registrare la radioattività media all&#39;interno del VOI come Bq / ml. Un fattore di calibrazione che converte i conteggi / sec di Bq / ml è stato precedentemente stabilito mediante la scansione di un fantasma con radioattività nota. </li><li> Captazione di FDG nel BAT è quantificato come dose percentuale iniettata per grammo di tessuto (% ID / g) con correzione decadimento. La dose iniettata è il risultato del Passaggio 3,5, tuttavia, convertito becquerel (Bq) Unità (1 uCi = 37.000 Bq); assumiamo che 1 ml di tessuto pari a 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>

Nessun conflitto di interessi dichiarati.

In questo studio un metodo di imaging micro-PET/CT-based è stato sviluppato per rilevare attività BAT in topi adulti che richiede soltanto un trattamento a freddo e una iniezione di commercialmente disponibile 18 F-FDG. L&#39;intera procedura può essere fatto in un giorno dopo un trattamento e sequenza di immagini che parte ogni 30 minuti fino a quando tutti gli animali vengono trattati e ripreso. Nelle condizioni sperimentali descritte, un totale di 10 topi (o due gruppi di 5 topi) possono essere testati ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Micro-PET/CT Imaging System </td><td> Siemens Medical Solutions USA, Inc. </td><td> Inveon dedicato PET e di sistema Inveon multimodalità CT / SPECT del sistema (inserito) </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> Mice C57BL/6J Maschio </td><td> Jackson Laboratory </td><td> 000664 </td><td> 3-4 mesi vecchio </td></tr></tbody></table>

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

Tessuto adiposo bruno (BAT) si differenzia dal tessuto adiposo bianco (WAT) per la sua posizione discreta e un colore marrone-rosso a causa della vascolarizzazione ricca e alta densità di mitocondri. BAT svolge un ruolo importante nel dispendio energetico e non brividi termogenesi nei mammiferi neonati e gli adulti 1. BAT-mediata termogenesi è altamente regolata dal sistema nervoso simpatico, prevalentemente attraverso i recettori adrenergici β 2, 3. Recenti studi hanno dimostrato che le attività BAT negli adulti umani sono negativamente correlati con l&#39;indice di massa corporea (BMI) e altri parametri diabetici 4-6. BAT è stato quindi proposto come un potenziale bersaglio per la terapia anti-obesity/anti-diabetes focalizzazione sulla modulazione di bilancio energetico 6-8. Mentre diverse freddi sfida basati tomografia a emissione di positroni (PET) sono stabiliti metodi per rilevare BAT 9-13 umana, non vi è essenzialmente alcun protocollo standard per l&#39;imaging e quantificazione delle BAT in modelli animali di piccole dimensioni come i topi. Qui descriviamo un robusto PET / CT metodo di imaging per la valutazione funzionale della BAT nei topi. In breve, i topi adulti C57BL/6J sono stati trattati a freddo in condizioni di digiuno per una durata di 4 ore prima di aver ricevuto una dose di 18 F-Fluorodeoxyglucose (FDG). I topi sono stati rimasta al freddo per una ulteriore ora dopo l&#39;iniezione FDG, e poi scansionato con un piccolo animale sistema dedicato micro-PET/CT. Le immagini PET acquisite erano co-registrate con le immagini TC per i riferimenti anatomici e analizzati per la captazione di FDG nella zona BAT interscapolare per presentare l&#39;attività BAT. Questo trattamento standardizzato freddo e protocollo di imaging è stato convalidato attraverso attività BAT test durante interventi farmacologici, per esempio, l&#39;attivazione BAT soppressa dal trattamento di β-adrenergico antagonista propranololo 14, 15, o l&#39;attivazione BAT potenziata β3 agonisti BRL37344 16. Il metodo described qui può essere applicato per lo screening per i farmaci / composti che modulano l&#39;attività BAT, o per identificare i geni / pathways che sono coinvolti nello sviluppo BAT e regolazione in vari studi preclinici e di base.

Un esempio di imaging micro-PET/CT di BAT mouse viene mostrato in Figura 1. Mentre l&#39;imaging CT fornisce informazioni anatomiche, il PET codifica la distribuzione e la quantità di 18 F-FDG tutto il corpo. Questi dati di imaging possono essere visualizzati separatamente (1A e 1B), fusa (1C), o risultare in una funzione 3D come proiezione massima intensità (MIP, 1D). Con l&#39;aiuto di uno strumento di imaging 3D, un volume di interesse (VOI), qui la regione interscapolare BAT (indicato d...

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

Un metodo di imaging funzionale di tessuto adiposo bruno topo (BAT) è descritto in cui freddo stimolato l&#39;assorbimento di 18F-fluorodesossiglucosio (FDG) in BAT è non invasivo valutata con un protocollo standardizzato micro-PET/CT. Questo metodo è robusto e sensibile per rilevare le differenze nelle attività BAT in modelli murini.

Imaging funzionale del grasso bruno nei topi con 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 Views.  The Methodist Hospital Research Institute, Houston. Un metodo di imaging funzionale di tessuto adiposo bruno topo (BAT) è descritto in cui freddo stimolato l'assorbimento di 18F-fluorodesossiglucosio (FDG) in BAT è non invasivo valutata con un protocollo standardizzato micro-PET/CT. Questo metodo è robusto e sensibile per rilevare le differenze nelle attività BAT in modelli murini.

Video: 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.

Luce / buio test di transizione per i Topi

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, ...

Ricerca

Biologia

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.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la RM

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.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con 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 ...

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.

Imaging funzionale con rinforzo, eyetracking e monitoraggio fisiologico

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.

Elevata Maze Plus per topi

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.

Assemblaggio, carico, allineamento e di una cella analitica del campione Ultracentrifuga

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.

Un metodo alternant al tradizionale modello della NASA arti posteriori di scarico in topi

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.

Imaging ottico basato su fotodiodi per la registrazione di dinamiche di rete con risoluzione a singolo neurone in invertebrati non transgenici

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.

Isolamento e differenziazione dei preadipociti bianchi e marroni primari dai topi appena nati

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.

Visualizzazione e quantificazione dei tessuti adiposi marroni e beige nei topi utilizzando [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 ...