The authors have no acknowledgements.

NOTA: Tutte le procedure sugli animali sono state eseguite con l&#39;approvazione della Cura e uso Comitato Animal Istituzionale (IACUC) dell &#39;Università di Cincinnati ed in conformità con la guida per la cura e l&#39;uso di animali da laboratorio dal National Institutes of Health (NIH Publication No . 85-23, Revised 1996). 1. Euthanize e sterilizzare il mouse <ol><li> Posizionare il mouse in un set contenente una dose supratheraputic di isoflurano e permettono di inalare a effetto. Una volta che il mouse è eutanasia, rimuoverlo dalla scatola. </li><li> Cervicale dislocate come un secondo mezzo di eutanasia. </li><li> Sterilizzare la superficie esterna del mouse dalla pulizia del animale con etanolo al 70%. </li></ol> 2. Identificazione e isolamento di tre diversi Depositi adipose <ol><li> Brown Tessuto adiposo (BAT) Isolamento: <ol><li> Assicurarsi che il pelo è bagnato dalla pulizia alcol per facilitare il taglio attraverso la epidermico e dermico Layers. </li><li> Posizionare il mouse in posizione supina con la schiena contro il tavolo. </li><li> Afferra la pelle appena sotto il diaframma con una pinza, ascensore e incidere con le forbici. </li><li> Tagliare trasversalmente attorno alla circonferenza del mouse per esporre il peritoneo. </li><li> Rivela il deposito BAT a forma di farfalla dal degloving la metà superiore del mouse. Tenere le appendici inferiori e dell&#39;addome in una mano e tirando la pelle verso la testa. </li><li> Orientare il mouse, in modo che sia posizionato prono sul tavolo. Fare attenzione a non contaminare il deposito a vista con i capelli. </li><li> Strumenti chirurgici pulite e cambiamento per un nuovo paio di guanti. </li><li> Individuare le scapole e deposito corrispondente. Rimuovere con cautela qualsiasi adiposo bianco superficiale in cima la farfalla e poi sezionare la farfalla di interscapolare grasso bruno. Fare attenzione a evitare il muscolo strettamente associato con il grasso marrone. NOTA: L&#39;uso di un microscopio dissezione è raccomandato per la rimozione del white adiposo, così come nella separazione del adiposo marrone dal scapole. </li><li> Rimuovere deposito di grasso e trasferire in una provetta 2 ml. </li><li> Se RNA o proteina deve essere estratta, congelare il tessuto mediante immersione in azoto liquido e conservare a -80 ° C. Congelare il campione in una sola volta per evitare il degrado. Se coltura, coprire il tessuto in DMEF-12 e impostare su ghiaccio fino a che tutti i campioni vengono raccolti per la cultura (info supplementare). </li></ol></li><li> Isolamento di sottocutaneo tessuto adiposo (SQ), un bianco Tessuto adiposo Depot (WAT): <ol><li> Mettere su un nuovo paio di guanti per non contaminare tra depositi di grasso. </li><li> Rivela il inguinale, depositi SQ triangolari da DE-guanto la metà inferiore del mouse. Tenere le appendici superiori e torace in una mano e tirando la pelle verso i piedi con l&#39;altra mano. </li><li> Orientare il mouse in posizione supina facendo attenzione a non contaminare il deposito a vista con i capelli. </li><li> Clean surgicastrumenti l e passare a un nuovo paio di guanti. </li><li> Sezionare con cura i triangoli di grasso SQ. Fare attenzione a non contaminare il campione con il muscolo, la vicina di grasso, ghiandole mammarie o tagliando le navi e contaminare il campione di sangue. NOTA: L&#39;uso di un microscopio dissezione è consigliata se i confini non sono chiaramente definiti. </li><li> Rimuovere depositi di grasso e trasferire a 2 ml microprovette. </li><li> Se RNA o proteina deve essere estratta, congelare il tessuto mediante immersione in azoto liquido e conservare a -80 ° C. Congelare il campione in una sola volta per evitare il degrado. </li><li> Per la colorazione, correggere o incorporare in ottobre Se coltura, coprire il tessuto in DMEF-12 e impostare su ghiaccio fino a che tutti i campioni vengono raccolti per la cultura (info supplementare). </li></ol></li><li> Isolamento di gonadica Fat un tessuto adiposo viscerale (VAT) e Bianco Tessuto adiposo (WAT) Depot: <ol><li> Mettere su un nuovo paio di guanti per non contaminare tra depositi di grasso. </li><li> Con scissors, tagliate il peritoneo trasversalmente, direttamente sotto il diaframma. Tagliare il peritoneo dal diaframma alla metà del retto coronale per esporre gli organi addominali. </li><li> Individuare i testicoli o delle ovaie e identificare il tessuto adiposo bianco attaccato, noto come il adiposo epididimale nei maschi o adiposo gonadica nelle femmine. </li><li> Strumenti chirurgici puliti e cambiare per un nuovo paio di guanti </li><li> Sezionare con cura entrambi i depositi di grasso dell&#39;epididimo dal testicoli, epididimi, e vasi deferenti. Oppure, se femmina, sezionare accuratamente entrambi cuscinetti di grasso gonadici dalle ovaie. </li><li> Rimuovere i depositi di grasso e trasferirli a 2 tubi ml microcentrifuga. </li><li> Se RNA o proteina deve essere estratta, congelare il tessuto mediante immersione in azoto liquido e conservare a -80 ° C. Congelare il campione in una sola volta per evitare il degrado. </li><li> Per la colorazione, correggere o incorporare in ottobre Se coltura, coprire il tessuto in DMEF-12 e impostare su ghiaccio fino a che tutti i campioni vengono raccolti per la cultura (info supplementare). </li&gt; </ol></li></ol> 3. Isolamento di Perivascolare Tessuto adiposo (PVAT) <ol><li> Isolamento del Cuore: <ol><li> Disporre il mouse in posizione supina con appendici superiori e inferiori estesi verso l&#39;esterno. </li><li> Appendici protetta tramite nastro chirurgico. </li><li> Dopo aver posizionato il mouse, come riportato sopra, creare tensione sollevando il processo xifoideo con una pinza. Tagliare orizzontalmente attraverso la membrana, esponendo la parte inferiore della cavità toracica. </li><li> Pur mantenendo la tensione, sollevando il processo xifoideo, tagliare la gabbia toracica superiormente verso la testa, appena a lato dello sterno. </li><li> Sollevare la gabbia toracica appena inferiore alla clavicola, e tagliare lungo la lunghezza inferiore della clavicola verso il cavo ascellare in entrambe le direzioni. La cavità toracica e il suo contenuto (cuore, polmoni, ecc) ora dovrebbero essere chiaramente visibili. </li><li> Pulire la cavità toracica di sangue estraneo e fluido usando ga sterileuze di assorbire il liquido. Se gli organi di raccolta o navi sono sicuri profusa (info supplementare). </li><li> Una volta che la superficie è eliminato da fluido, tagliare i vasi bronchi e connessi per rimuovere i polmoni, permettendo una migliore esposizione del cuore. </li></ol></li><li> Localizzazione e Asportazione di aortica Perivascolare Tessuto adiposo (PVAT): <ol><li> Rimuovere i seguenti organi per identificare meglio le parti inferiori della aorta: fegato, stomaco, milza, pancreas, intestino, e colon. </li><li> Prima iniziare con l&#39;identificazione dello stomaco e dell&#39;esofago. Tagliare dell&#39;esofago alla giunzione gastro-esofageo per liberare lo stomaco dal corpo. </li><li> Avanti, individuare l&#39;intestino e mesentere circostante. Tagliare superficialmente attraverso mesentere, in quanto si trova molto vicino alla parte renale dell&#39;aorta, poi &quot;eseguire l&#39;intestino.&quot; </li><li> Tagliare il colon liberare il più vicino al retto possibile. Così, liberando lo stomaco, intestino e colon dal mouse. </li><li> Respostare lo stomaco, intestino, colon, pancreas e milza tagliando attraverso il mesentere Collegamento e vasi. Il pancreas e la milza dovrebbe venire gratis con lo stomaco, intestino e colon. </li><li> Rimuovere il fegato tagliando attraverso le vene epatiche e allegando mesentere, rimuovere tutti i lobi. </li><li> Tagliare via lo strato di grasso viscerale e circondano i reni. Lasciare i reni allegate all&#39;aorta in vivo per servire come marcatori geografiche per diversi segmenti dell&#39;aorta. </li><li> Risciacquare la zona con 1x PBS sterile e rimuovere tutto il fluido per assorbimento con una garza sterile. </li><li> Utilizzando micro-forbici e micro-pinza, separare l&#39;aorta dal suo attaccamento dorsale per la colonna vertebrale e il suo attaccamento ventrale all&#39;esofago. </li><li> Isolare l&#39;aorta seguendo e staccando l&#39;aorta la lunghezza dell&#39;aorta discendente dall&#39;origine nel cuore alla biforcazione della regione iliaca. </li><li> Identificare e isolare i vasi succlavi. Isolare questi vasidal collo alla radice aortica per esporre meglio la giunzione aortica e la radice nel cuore. </li><li> Rimuovere il timo poi tagliare l&#39;arteria brachiocefalico, l&#39;arteria carotide comune sinistra, e succlavia sinistra, permettendo il movimento del cuore. </li><li> Con l&#39;aiuto di un microscopio dissezione, visualizzare il tessuto adiposo (PVAT) strato perivascolare che circonda l&#39;aorta. </li><li> Facendo molta attenzione a non schiacciare o spremere il PVAT, tirare delicatamente il modo PVAT dall&#39;aorta con micro-pinza. Tagliare delicatamente il fissaggio del PVAT all&#39;aorta con micro-forbici a partire regione toracica appena superiore al punto in cui si trova il diaframma. NOTA: Si consiglia un microscopio a dissezione. </li><li> Ripetere questo processo l&#39;intera lunghezza dell&#39;aorta, terminando a regione aortica sottorenale, che si trova appena superiore alla biforcazione iliaca del vaso aortico. </li><li> Se aortico PVAT è desiderato, utilizzato lo stesso metodo per rimuovere il PVAT dalla minore curvature dell&#39;arco. </li><li> Posizionare i campioni PVAT in 2 ml provetta (s). </li><li> Se RNA o proteina deve essere estratta, congelare il tessuto mediante immersione in azoto liquido e conservare a -80 ° C. Congelare il campione in una sola volta per evitare il degrado. Se coltura, coprire il tessuto in DMEF-12 e impostare su ghiaccio fino a che tutti i campioni vengono raccolti per la cultura (info supplementare). NOTA: ulteriori depositi adiposi da considerare se interessati in una completa analisi depot adiposo includono: retroperitoneale, mesenterica, omentale, pericardico e poplitea. </li></ol></li></ol>

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The authors declare that they have no competing financial interests or other conflicts of interest.

L&#39;obesità può portare a una grande serie di morbilità e la piena comprensione del ruolo che svolge adiposo non è pienamente compreso; ricerca pertanto continuato nel campo della adiposo è necessaria. I modelli animali, in particolare i modelli murini sono ideali per la ricerca iniziale nella progressione delle malattie e la sperimentazione di potenziali trattamenti farmaceutici. Utilizzando questi modelli, isolamento precisa e escissione di depositi adiposi è uno strumento estremamente importante e necessario ...

Adiposa fatto un&#39;apparizione notevole sotto i riflettori dei media, a causa della notevole aumento di obesità negli ultimi decenni del 20 ° secolo. L&#39;obesità colpisce attualmente più di un terzo degli adulti e il 17% di bambini e adolescenti negli Stati Uniti (US) 1. Spanning tutti i gruppi etnici, ricerca statistica che circonda l&#39;epidemia di obesità ha mostrato che i neri non ispanici hanno il tasso aggiustato per età più alta di obesità (49,5%) rispetto ai messicani americani (40,4%), tutti gli ispanici (39,1%), e non bianchi ispanici (34,3%) 2. L&#39;effetto economico di obesità è anche una preoccupazione crescente per il sistema sanitario. Nel 2012, è stato stimato che il costo medico annuale delle cure per l&#39;obesità negli Stati Uniti nel 2005 è stato di 190,2 miliardi dollari, quasi il 21% del bilancio complessivo di spesa medica. Purtroppo, l&#39;obesità infantile è stata stimata essere responsabile a $ 14 miliardi di dollari in costi medici diretti da solo. Statisticamente, è stato determinato che il costo medio di medicaindividui con obesità è stato $ 2741 superiore di un anno rispetto a quelli senza questa morbosità 3-5. L&#39;obesità è un importante fattore di rischio per una serie di condizioni, quali: diabete di tipo 2, dislipidemia, malattie cardiovascolari, cancro, disturbi muscolari e scheletrici infiammazione cronica. L&#39;obesità è profondamente legata alla patogenesi della sindrome metabolica e di altre malattie croniche 6-8. Con tali connessioni drastiche e negativi tra obesità e altre patologie concomitanti, la ricerca scientifica si è concentrata l&#39;attenzione per capire meglio l&#39;epidemia attuale e le diverse e cardine ruoli adiposo. Storicamente, il tessuto adiposo è stato considerato irrilevante ed è stato considerato solo come un semplice tessuto di riempimento. Attualmente, adiposo ha dimostrato di giocare molti ruoli essenziali nella funzione del corpo in: metabolismo, regolazione ormonale, l&#39;infiammazione, la protezione e l&#39;isolamento 9. Il tessuto adiposo è composto principalmente daadipociti, ma contiene anche periciti, le cellule endoteliali, monociti, macrofagi e cellule staminali pluripotenti 8. Il tessuto adiposo è distribuito in tutto il corpo in depositi distinti. I principali depositi si trovano subdermally, per via sottocutanea, intramuscolare, e visceralmente 10. Depositi adiposi hanno dimostrato di avere deposito specifici profili metabolici, che hanno mostrato una predisposizione specifica depot di obesità e disturbi correlati 8. Tradizionalmente, il tessuto adiposo è stata classificata in due tipi principali: tessuto adiposo bianco (WAT) e tessuto adiposo bruno (BAT); anche se recente letteratura indica le presenze di un terzo gruppo battezzato brite o beige adiposo 11. Il tessuto adiposo è stato dimostrato di avere colori diversi, morfologie, funzioni metaboliche, le caratteristiche biochimiche e modelli genetici di espressione 10. Gli adipociti in WAT hanno un unico, grande goccia di lipidi e di quantità variabili di mitocondri. WATsi trova in località prevalentemente sottocutaneo e viscerale del corpo. Funzioni WAT principalmente come un sito di stoccaggio di energia e di protezione d&#39;organo. Gli adipociti in BAT hanno una morfologia multiloculare e mitocondri abbondanti. BAT si trova principalmente nel collo e grandi vasi sanguigni del torace, nonché le scapole 12. BAT funzioni principalmente a comportamenti dispendio energetico che regolano la termogenesi 7. Adiposo Brite o beige ha dimostrato di condividere una morfologia analogo ed espressione BAT, ma è stato trovato per essere originato da adipociti bianchi 11. Il metodo chirurgico descritto in questo manoscritto fornisce ai ricercatori con la capacità di analizzare diversi effetti che fattori quali: l&#39;ambiente, i prodotti farmaceutici, e la genetica, hanno sulla adiposo; così come il ruolo adiposo benefico o dannoso gioca nella malattia e la salute generale. Inoltre, fornendo un modo per identificare e isolare diversi tipi di tessuto adiposopermettere una migliore comprensione delle relazioni biochimiche e le differenze tra depositi. Questo può aiutare a determinare il rapporto tra posizione, la funzione, e tipi di grassi nel corpo. Questo metodo descritto realizza questo fornendo i mezzi per la visualizzazione lordo, analisi di espressione genica, analisi di espressione della proteina, l&#39;esame istologico, e l&#39;isolamento di linee cellulari primarie per studi in vitro. Attualmente non ci sono molti articoli che forniscono informazioni sul comportamento metabolico dei diversi depositi adiposi, nonché le loro sedi anatomiche; ma non forniscono un metodo di approfondimento su come localizzare in particolare, di identificare e isolare questi depositi. Questo metodo fornisce una tecnica chirurgica precisa che permette l&#39;isolamento di più depositi con una quantità minima di dissezione e la contaminazione rispetto ad altri metodi progettati per l&#39;isolato di uno o due depositi 13-14. L&#39;obiettivo di questo protocollo è quello di fornireun metodo preciso per l&#39;identificazione e l&#39;isolamento di diversi tipi di depositi di grasso di molteplici posizioni anatomiche.

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		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane</td>
			<td>Med-Vet International&#160;</td>
			<td>#RXISO-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">70% Ethanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:125px;">07-678-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEF-12</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:125px;">D-6421</td>
			<td>Warm in waterbath before putting on tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">2 mL microcentrifuge tubes</td>
			<td style="width:175px;">Midsci</td>
			<td style="width:125px;">MCT-200-C-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Phosphate buffered saline</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:125px;">P5368-10PAK</td>
			<td></td>
		</tr>
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localization, identification, and excision of murine adipose depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Identificazione e localizzazione delle adiposo sottocutaneo inguinale, interscapolare adiposo bruno, viscerale adiposo epididimale (figura 1), nonché la aortico adiposo perivascolare arco, toracica adiposo aortica, suprarenal adiposo aortica e dell&#39;aorta infrarenale adiposo (Figura 2) è stata ottenuta con successo usando il metodo chirurgico descritto. L&#39;esame istologico e la differenziazione tra i campioni BAT e Wat sono stati positivamente valutati usando ematossilina e eosi...

Watch this Scientific Journal Video about Localization, Identification, and Excision of Murine Adipose Depots at JoVE.com

Localization, Identification, and Excision of Murine Adipose Depots

Grazie al collegamento drastica e negativa tra obesità e altre patologie concomitanti, la ricerca sul adiposo giochi di ruolo nella malattia e la salute generale è garantito. Presentiamo un protocollo per l&#39;isolamento e l&#39;escissione di depositi adiposi consentano lo studio della adiposo usare in situ e metodi in vitro.

Localizzazione, identificazione, e l&#39;escissione dei Depositi Murine adipose

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

localization-identification-and-excision-of-murine-adipose-depots

Research

JoVE Journal

Medicine

40.9K Views.  University of Cincinnati College of Medicine.  Grazie al collegamento drastica e negativa tra obesità e altre patologie concomitanti, la ricerca sul adiposo giochi di ruolo nella malattia e la salute generale è garantito. Presentiamo un protocollo per l'isolamento e l'escissione di depositi adiposi consentano lo studio della adiposo usare in situ e metodi in vitro. 

Video: Localization, Identification, and Excision of Murine Adipose Depots

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Una tecnica semplificata per<em&gt; In situ</emEscissione&gt; della cornea e l&#39;eviscerazione del tessuto retinico dal globo oculare umano

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Ricerca

Medicina

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Cerenkov Luminescenza Imaging di interscapolare Brown Tessuto adiposo

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Isolamento e Asportazione di Murine Aorta; Una tecnica versatile nello studio delle malattie cardiovascolari

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Umano Brown adipose Depositi tessuti segmentato automaticamente da Positron Emission Tomography / tomografia computerizzata e registrati Risonanza Magnetica Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Incapsulamento termogenico preadipocytes al trapianto nel tessuto adiposo Depositi tessuti

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

Valutazione della funzione microvascolare di tessuto adiposo umano mediante videomicroscopia

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Vessel-sparing Excision and Primary Anastomosis

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short bulbar or posterior urethral strictures, urethroplasty by excision and primary anastomosis (EPA) is recommended. As EPA only requires the excision of the narrowed segment and the surrounding spongiofibrosis, a full-thickness transection of the corpus spongiosum, as performed in the traditional transecting EPA (tEPA), is usually unnecessary. Jordan et al. introduced the idea of a vessel-sparing approach in 2007, aiming to reduce surgical trauma, especially to the dual arterial blood supply of the urethra, and, thus, potentially reducing the risk of postoperative erectile dysfunction or glans ischemia. This approach could also be beneficial for subsequent urethral interventions such as redo urethroplasty using a free graft, in which a well-vascularized graft bed is imperative. Nevertheless, these potential benefits are only assumptions as prospective studies comparing the functional outcome of both techniques with validated questionnaires are currently lacking. Moreover, vessel-sparing EPA (vsEPA) should at least be able to provide similar surgical outcomes as tEPA. The aim of this paper is to give an elaborate, step-by-step overview of how to manage patients with isolated short bulbar or posterior urethral strictures with vsEPA. The main objective of this manuscript is to outline the surgical technique and to report the representative surgical outcome. A total of 117 patients were managed according to the described protocol. The analysis was performed on the entire patient cohort and on the bulbar (n = 91) and posterior (n = 26) vsEPA group separately. Success rates were 93.4% and 88.5% for the bulbar and posterior vsEPA, respectively. To conclude, vsEPA, as outlined in the protocol, provides excellent success rates with low complication rates for isolated short bulbar and posterior urethral strictures.

Here, we present an elaborate and efficient protocol to treat isolated short bulbar or posterior urethral strictures with vessel-sparing excision and primary anastomosis.

Nave-con parsimonia l'asportazione e l'anastomosi primaria

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

Localizzazione di noduli transtoracici di navigazione elettromagnetica per chirurgia toracica minimamente invasiva

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

Murine Appendectomy Modello di Cronica Colite Associata Cancro Colorettale da Localizzazione precisa di Patch Caecal

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Modello di trapianto di Murine (ISWAT) del tessuto adiposo sottocutaneo inguinale (ISWAT)

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...