Questo lavoro è stato sostenuto dalle concessioni dal National Institutes of Health (NIH) a SSH (R01-DK078640), del Howard Hughes Medical Institute (HHMI) attraverso il HHMI / Vanderbilt Certificate Program University di Medicina Molecolare per TJW (GRDOT56006779), la Vanderbilt Diabetes Research and Training Center (P30-DK020593) e la Digestive Disease Research Center Vanderbilt (P30-DK058404) fornendo servizi di base.

1. Preparare Cannula <ol><li> Scaldare un 1-pollice lungo tratto di PE10 tubi con la punta delle dita e allungare in modo che il tubo diventa sottile. Nota: la dimensione del recipiente o condotto da incannulata determinerà il grado di stiramento richiesto. La cannula deve essere ben teso per via biliare getta, ma può essere richiesto solo per essere moderatamente allungato per adattarsi all&#39;interno della vena porta più grande. </li><li> Tagliare il tubo allungato in diagonale per generare una punta smussata. </li><li> Tagliare il bordo opposto del tubo per creare un segmento 5-6 pollici. </li><li> Inserire un contatore 32, ago ipodermico ½ pollice nel non smussato bordo del tubo. </li></ol> 2. Preparare Siringa con PBS <ol><li> Riempire una siringa da 3 ml con PBS. </li><li> Avvitare la siringa l&#39;ago ipodermico con annessa tirata PE10 tubi precedentemente preparato. </li><li> Siringa Flick e spingere lo stantuffo della siringa per assicurare che PBS ha riempito l&#39;ago e il tubo enon vi siano bolle d&#39;aria nella siringa. </li><li> Posizionare la siringa con PBS in una siringa porta-a forma di plastilina. Questo supporto manterrà la siringa ferma mentre il tubo viene inserito nel condotto. </li></ol> 3. Altro Set-up <ol><li> Pesare 0,1 g di catalizzatore in un piccolo contenitore, ad esempio, un pozzetto di una piastra a 24 pozzetti. </li><li> Tirare 1 resina ml in una siringa da 3 ml. Nota: quantità relative di resina e catalizzatore può essere regolato per alterare il tempo di indurimento. Diminuendo quantità di catalizzatore può, tuttavia, aumentare gettato ritiro e portare ad un cast meno desiderabile. Nota: quando si maneggia resina, prendere le dovute precauzioni per evitare il contatto con la resina o l&#39;inalazione dei fumi di resina. Indossare sempre guanti ed eseguire le operazioni che comportano resina in una zona ben ventilata, come una cappa di laboratorio </li><li> Tagliare un pezzo di 5,0 sutura di seta di circa 5 centimetri per essere utilizzati come una legatura. </li></ol> 4. Preparare mouse <ol&gt; <li> Sacrificio topo adulto. Appoggiare il mouse sul dorso e di aprire la cavità addominale. </li><li> Lay mouse sulla dissezione piattaforma portata. Posizionare a 15 ml tubo conico perpendicolarmente sotto il mouse per aumentare la visibilità e l&#39;accessibilità al fegato. </li><li> Bagnare un batuffolo di cotone con PBS e utilizzarlo per capovolgere il fegato verso l&#39;alto e lontano da voi per esporre la visione dorsale del fegato con la vena porta extraepatico o dotto biliare comune. </li><li> Per i calchi della vena porta, è possibile impedire la coagulazione del sangue svuotando la vena con PBS temperatura ambiente. Inserire un ago di una siringa da 3 ml riempita con PBS e inserire l&#39;ago nella vena portale extraepatica più lontano possibile dal fegato, come si può. Spingere delicatamente attraverso PBS nella vena porta. Fai un nick nella vena comune cardinale a drenare il sangue. Usare un tampone di cotone bagnato al fegato massaggio, migliorando il drenaggio del sangue. Questo passaggio non è raccomandato per il dotto biliare e potrebbe non essere necessario per tutti i sistemi vascolari. Nota: questo passaggio può anche essere performed con 4% paraformaldeide invece di PBS. Uso di paraformaldeide al 4% può aumentare l&#39;integrità vascolare e risultato in un cast più precisa, in particolare di piccole strutture vascolari. </li></ol> 5. Incannulare vena portale extraepatica o dotto biliare comune <ol><li> Utilizzare pinze curve per passare la legatura chirurgica sutura sotto la vena porta o dotto biliare, circa ¼ di pollice dal fegato. </li><li> Legare la sutura in un nodo sciolto comune. NON stringere il nodo. </li><li> Se si utilizza un sistema aperto, può essere utile per legare e stringere una legatura all&#39;altra estremità del sistema. Se avete aperto la vena comune cardinale per drenare il sangue dalla vena porta, legare una legatura intorno ad esso per aumentare la pressione all&#39;interno della vena porta e impedire la fuoriuscita di resina nella vena epatica centrale. </li><li> Con le forbici a molla per fare un piccolo taglio nella vena porta o dotto biliare, circa ¼ di pollice sotto il nodo di sutura. Questo cut deve penetrare non più di metà del diametro della vena porta o dotto biliare. </li><li> Utilizzando # 5 forcipe, tenere la vena porta o dotto biliare nel sito del taglio e inserire estremità smussata del tubo nel dotto biliare. </li><li> Spingere punta del tubo oltre il pre-legato legatura e verso il fegato. </li><li> Test per la precisione incannulazione iniettando PBS attraverso la cannula e guardare per vedere se entra nella vena porta o dotto biliare. </li><li> Serrare la legatura di tenere la cannula in posizione. </li></ol> 6. Resina della vena porta o dotto biliare intraepatico <ol><li> Aggiungi pre-misurata 1 resina ml di pre-misurato 0,1 g di catalizzatore e mescolare accuratamente. Evitare la formazione di bolle d&#39;aria. </li><li> Disegnare resina-catalizzatore miscela nella siringa 3 ml. Rimuovere le bolle d&#39;aria delicatamente sfogliando la siringa ed espellere le bolle. Nota: per la colata di strutture di piccole dimensioni, può essere utile usare una camera a vuoto per rimuovere le bolle d&#39;aria molto piccole da the resina e migliorare la qualità del cast. Concedere tempo per eseguire questa operazione, è necessario diminuire il catalizzatore / resina rapporto da quella suggerita per rallentare resina polimerizzabile. </li><li> Accuratamente sostituire PBS contenente siringa con resina contenente siringa, lasciando l&#39;ago 32 attaccato ad cannula. </li><li> Lentamente e spingere delicatamente resina nella vena porta o dotto biliare finché la resistenza aumenta e il fegato è pieno. </li><li> Utilizzare tampone di cotone bagnato di massaggiare delicatamente fegato e favorire ancor di riempimento. </li><li> Rimuovere cannula dalla struttura e rapidamente serrare la legatura per evitare perdite di resina. </li><li> Lasciare il mouse per disteso a temperatura ambiente per 20 minuti, mentre le cure in resina, quindi rimuovere fegato. </li><li> Procedere sia con compensazione o macerazione del fegato. </li></ol> 7. Fegato trasparente per la visualizzazione in situ <ol><li> Fissare il fegato in 4% paraformaldeide notte a 4 ° C, a dondolo. </li><li> Lavare il fegatoin PBS per almeno quattro ore, a dondolo. A questo punto, è possibile separare i lobi del fegato se lo si desidera. </li><li> Disidratare in 50% metanolo e quindi in metanolo 100% per almeno quattro ore ciascuna, dondolo a temperatura ambiente. </li><li> Deselezionare il fegato in una soluzione di alcool benzilico 01:02 e benzil benzoato (BABB) per almeno 24 ore, dondolo. Tempi più lunghi di lavaggio può essere necessaria. Se il fegato non si risolve completamente dopo 2-3 giorni, tornare al 100% di metanolo per 24 ore e ripetere BABB compensazione. </li><li> Fegato Immergere in BABB in un piatto di vetro Petri per l&#39;imaging in situ. </li></ol> 8. Macerare tessuto epatico <ol><li> Rimuovere fegato di topo e messo in una provetta da 50 ml con acqua. Attendere 1 ora per la polimerizzazione resina completo. Lobi del fegato separati se lo si desidera. </li><li> Versare l&#39;acqua e passare fegato di una bottiglia di vetro. Immergere nel 15% KOH. Lasciare per una notte a temperatura ambiente. Non rock. </li><li> Versare con cautela fuori KOH e lavare fuso in acqua distillata. Nota: il cast è very fragile e deve essere maneggiato con estrema cura. Evitare di interrompere il cast durante l&#39;aggiunta o la rimozione di liquido dal tubo. </li><li> Quando in resina è pulito, appoggiare delicatamente su un Kimwipe ad asciugare, poi trasferirla in un contenitore da memorizzare. </li></ol> 9. Risultati rappresentativi Un cast successo della vena porta o dotto biliare mostrerà una rete continua di rami progressivamente minori estendono dal ilo alla periferia fegato. La figura 1 mostra una vena portale espressi come visualizzato in situ dopo BABB compensazione. Figura 1A mostra il cast del intero lobo epatico sinistro e la forma e le dimensioni del getto. Figura 1B mostra una vista ravvicinata del getto stesso, dimostrando l&#39; penetrazione della resina nelle più piccole rami della vena porta alla periferia fegato. La figura 2 mostra un cast vena che ha subito KOH mAceration. Figura 2A mostra l&#39;intero lobo sinistro del fegato e figura 2B è un primo piano di piccoli rami periferici. Questa tecnica è stata applicata con successo al dotto biliare intraepatica e pubblicato utilizzando sia il BABB compensazione e tecniche di macerazione KOH 1,2,7. Problemi comuni includono incomplete riempie, bolle nella resina, e troppopieno in sistemi o tessuti circostanti. Figura 3 mostra come riconoscere un riempimento incompleto (Figura 3A), bolle (Figura 3A), e un overflow di resina dalla vena porta alla vena centrale (Figura 3B). Tutte le immagini sono scattate con una Leica MZ 16 stereoscopio FA e QImaging RETIGA fotocamera 4000R. <img alt="Figura 1" src="/files/ftp_upload/4272/4272fig1.jpg" /> Figura 1. BABB nulla osta di resina di sinistra della vena porta epatica lobo. Resina A. presenta una struttura gerarchica ramificata che si estende dal ilo alla periferia del fegato. Resina appare di colore giallo all&#39;interno azzurrato lobo epatico. B. Un primo piano mostra che il riempimento resina si estende alle filiali di piccole dimensioni nella periferia fegato. Resina è biancastra con fegato cancellato. Barra di scala = 1 mm. <img alt="Figura 2" src="/files/ftp_upload/4272/4272fig2.jpg" /> Figura 2. Tissue-macerata in resina di sinistra della vena porta epatica lobo. Resina A. cast di tutto lobo epatico mostra molti rami di varie dimensioni. B. Close-up mostra piccoli rami in periferia di fegato. Sfumatura comparsa di piccoli rami rappresenta resina di riempimento di spazi sinusoidali e possono o non possono essere visibili su calchi in resina e aumenterà la densità apparente ramo se presente. Resina è di colore bianco. Barra di scala = 1 mm. Figura 3. Problemi comuni con calchi in resina. A. Un riempimento incompleto della vena portale lobo sinistro è evidente da alcuni o tutti i rami del cast non riescono a estendere alla periferia del fegato. Questo cast visualizza anche le bolle, visibile come rotture nella colata continua (freccia). A causa della vicinanza della vena porta e rami venosi centrali in alcuni luoghi B., la resina vena porta spesso entrare e riempire la vena centrale. Questo può essere riconosciuta quando vi sono due distinti rami ilari (frecce) e due strutture ramificate che hanno modelli diversi e sovrapposizioni. Discriminante tra errori tecnici e veri alterazioni morfologiche può essere fatto in due modi. Primo, la struttura colata deve essere confrontata con la struttura prevista sulla base di 2-dimensionale o di altri metodi. In secondo luogo, è importante effettuare diverse repliche, e quando possibile, quantificare il structure ed eseguire analisi statistica per determinare la riproducibilità e se vi è una alterazione morfologica statisticamente significativa tra e all&#39;interno sperimentali diversi e bracci di controllo.

<ol>
	<li>Sparks, E. E., Perrien, D. S., Huppert, K. A., Peterson, T. E., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defects+in+hepatic+Notch+signaling+result+in+disruption+of+the+communicating+intrahepatic+bile+duct+network+in+mice.">Defects in hepatic Notch signaling result in disruption of the communicating intrahepatic bile duct network in mice.</a> Dis. Model Mech. 4, 359-367 (2011).</li><li>Vanderpool, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+interactions+between+hepatocyte+nuclear+factor-6+and+notch+signaling+regulate+mouse+intrahepatic+bile+duct+development+in+vivo.">Genetic interactions between hepatocyte nuclear factor-6 and notch signaling regulate mouse intrahepatic bile duct development in vivo.</a> Hepatology. 55, 233-243 (2012).</li><li>Meyer, E. P., Ulmann-Schuler, A., Staufenbiel, M., Krucker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+morphology+and+3D+architecture+of+brain+vasculature+in+a+mouse+model+for+Alzheimer's+disease.">Altered morphology and 3D architecture of brain vasculature in a mouse model for Alzheimer's disease.</a> Proceedings of the National Academy of Sciences. 105, 3587-3592 (2008).</li><li>Van Steenkiste, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+corrosion+casting:+analyzing+wall+shear+stress+in+the+portal+vein+and+vascular+abnormalities+in+portal+hypertensive+and+cirrhotic+rodents.">Vascular corrosion casting: analyzing wall shear stress in the portal vein and vascular abnormalities in portal hypertensive and cirrhotic rodents.</a> Lab Invest. 90, 1558-1572 (2010).</li><li>Dickie, R., Cormack, M., Semmler-Behnke, M., Kreyling, W. G., Tsuda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+pulmonary+lymphatics+in+immature+lungs.">Deep pulmonary lymphatics in immature lungs.</a> Journal of Applied Physiology. 107, 859-863 (2009).</li><li>Kelly, D. M., McEntee, G. P., McGeenery, K. F., Fitzpatrick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvasculature+of+the+pancreas,+liver,+and+kidney+in+cerulein-induced+pancreatitis.">Microvasculature of the pancreas, liver, and kidney in cerulein-induced pancreatitis.</a> Archives of Surgery. 128, 293-295 (1993).</li><li>Sparks, E. E., Huppert, K. A., Brown, M. A., Washington, M. K., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+regulates+formation+of+the+three-dimensional+architecture+of+intrahepatic+bile+ducts+in+mice.">Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.</a> Hepatology. 51, 1391-1400 (2010).</li><li>Carlson, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+expression+of+constitutively+active+Notch4+elicits+reversible+arteriovenous+malformations+in+adult+mice.">Endothelial expression of constitutively active Notch4 elicits reversible arteriovenous malformations in adult mice.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 9884-9889 (2005).</li><li>Hemmeryckx, B., Emmerechts, J., Bovill, E. G., Hoylaerts, M. F., Lijnen, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ageing+on+the+murine+venous+circulation.">Effect of ageing on the murine venous circulation.</a> Histochem. Cell Biol. , (2012).</li><li>Hossler, F. E., Olson, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvasculature+of+the+nasal+salt+gland+of+the+duckling,+Anas+platyrhynchos:+quantitative+responses+to+osmotic+adaptation+and+deadaptation+studied+with+vascular+corrosion+casting.">Microvasculature of the nasal salt gland of the duckling, Anas platyrhynchos: quantitative responses to osmotic adaptation and deadaptation studied with vascular corrosion casting.</a> J. Exp. Zool. 254, 237-247 (1990).</li><li>Hossler, F. E., West, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venous+valve+anatomy+and+morphometry:+studies+on+the+duckling+using+vascular+corrosion+casting.">Venous valve anatomy and morphometry: studies on the duckling using vascular corrosion casting.</a> Am. J. Anat. 181, 425-432 (1988).</li><li>Hossler, F. E., Monson, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+blood+supply+of+intrinsic+lymph+nodes+in+the+wall+of+the+rabbit+urinary+bladder--studies+with+light+microscopy,+electron+microscopy,+and+vascular+corrosion+casting.">Structure and blood supply of intrinsic lymph nodes in the wall of the rabbit urinary bladder--studies with light microscopy, electron microscopy, and vascular corrosion casting.</a> Anat. Rec. 252, 477-484 (1998).</li><li>Hossler, F. E., Monson, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+unique+elastic+sheath+surrounding+the+vesicular+arteries+of+the+rabbit+urinary+bladder--studies+of+the+microvasculature+with+microscopy+and+vascular+corrosion+casting.">Evidence for a unique elastic sheath surrounding the vesicular arteries of the rabbit urinary bladder--studies of the microvasculature with microscopy and vascular corrosion casting.</a> Anat. Rec. 252, 472-476 (1998).</li><li>Hossler, F. E., Kao, 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=Microvasculature+of+the+urinary+bladder+of+the+dog:+a+study+using+vascular+corrosion+casting.">Microvasculature of the urinary bladder of the dog: a study using vascular corrosion casting.</a> Microsc. Microanal. 13, 220-227 (2007).</li><li>Wischgoll, T., Choy, J. S., Kassab, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+morphometry+and+branching+angles+of+porcine+coronary+arterial+tree+from+CT+images.">Extraction of morphometry and branching angles of porcine coronary arterial tree from CT images.</a> Am J Physiol. Heart. Circ. Physiol. 297, H1949-H1955 (2009).</li><li>Hossler, F. E., Douglas, J. E., Douglas, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+morphometry+of+myocardial+capillaries+studied+with+vascular+corrosion+casting+and+scanning+electron+microscopy:+a+method+for+rat+heart.">Anatomy and morphometry of myocardial capillaries studied with vascular corrosion casting and scanning electron microscopy: a method for rat heart.</a> Scan Electron Microsc. , 1469-1475 (1986).</li><li>Hossler, F. E., Douglas, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Corrosion+Casting:+Review+of+Advantages+and+Limitations+in+the+Application+of+Some+Simple+Quantitative+Methods.">Vascular Corrosion Casting: Review of Advantages and Limitations in the Application of Some Simple Quantitative Methods.</a> Microsc. Microanal. 7, 253-264 (2001).</li><li>Mondy, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+of+corrosion+casts+for+use+in+the+computer-aided+design+of+microvasculature.">Micro-CT of corrosion casts for use in the computer-aided design of microvasculature.</a> Tissue Eng. Part C Methods. 15, 729-738 (2009).</li><li>Masyuk, T. V., Ritman, E. L., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Assessment+of+the+Rat+Intrahepatic+Biliary+System+by+Three-Dimensional+Reconstruction.">Quantitative Assessment of the Rat Intrahepatic Biliary System by Three-Dimensional Reconstruction.</a> The American Journal of Pathology. 158, 2079-2088 (2001).</li><li>Masyuk, T. V., Ritman, E. L., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+Artery+and+Portal+Vein+Remodeling+in+Rat+Liver:+Vascular+Response+to+Selective+Cholangiocyte+Proliferation.">Hepatic Artery and Portal Vein Remodeling in Rat Liver: Vascular Response to Selective Cholangiocyte Proliferation.</a> The American Journal of Pathology. 162, 1175-1182 (2003).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo descritto esempi specifici di come la vena porta e intraepatiche sistemi dotti biliari del fegato può essere fusa, ma questa tecnica può essere applicata a qualsiasi altro sistema duttale o vascolare con lievi modifiche. Il lavoro precedente ha dimostrato la fattibilità di questa tecnica in più specie, tra cui mouse, 1,2,7-9 anatroccolo 10,11, 12,13 coniglio, cane 14, e maiale 15, e in molti organi, tra cui ghiandola nasale 14, cuore 16, 12,13...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> PE10 tubi in polietilene </td><td> Fisher Scientific </td><td> 1417012P </td><td></td></tr><tr><td> 5-0 chirurgico di seta nera intrecciata </td><td> Roboz chirurgico </td><td> SUT-15-1 </td><td></td></tr><tr><td> Steriject 32 G x 13 mm ago </td><td> Air-tite </td><td> TSK3213 </td><td></td></tr><tr><td> Primavera forbici </td><td> Strumenti Scienza Belle </td><td> 15000-08 </td><td></td></tr><tr><td> Numero 5 pinza </td><td> Strumenti Scienza Belle </td><td> 91150-20 </td><td></td></tr><tr><td> Mercox II Kit </td><td> Ladd ricerca </td><td> 21247 </td><td></td></tr><tr><td> Alcool benzilico </td><td> Fisher Scientific </td><td> 1816-1804 </td><td> Oolo necessario per BABB-compensazione </td></tr><tr><td> Benzoato di benzile </td><td> MP Biomedicals, LLC </td><td> 154839 </td><td> Richiesto solo per BABB-compensazione </td></tr><tr><td> Tampone fosfato salino (PBS) </td><td></td><td></td><td></td></tr><tr><td> Modellazione di argilla </td><td></td><td></td><td></td></tr><tr><td> Scala </td><td></td><td></td><td></td></tr><tr><td> Laboratorio di forbici </td><td></td><td></td><td></td></tr><tr><td> 15 ml tappo in polipropilene tubi </td><td></td><td></td><td></td></tr><tr><td> 4% paraformaldahyde </td><td></td><td></td><td></td></tr><tr><td> Idrossido di potassio al 15% (KOH) </td><td></td><td></td><td> Richiesto solo per la macerazione </td></tr><tr><td> Lametta </td><td></td><td></td><td></td></tr><tr><td> 100% metanolo </td><td></td><td></td><td></td></tr><tr><td> 3 ml luer lock siringa </td><td></td><td></td><td></td></tr></tbody></table>

3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system

Di organi, l&#39;architettura corretta delle strutture vascolari e duttale è indispensabile per una corretta funzione fisiologica, e la formazione e il mantenimento di queste strutture è un processo altamente regolato. L&#39;analisi di questi complessi, tre strutture tridimensionali è notevolmente dipendeva sia 2-dimensionale esame in sezione o su studi iniezione tintura. Queste tecniche, tuttavia, non sono in grado di fornire una rappresentazione completa e quantificabile delle strutture duttali o vascolari sono destinati a chiarire. In alternativa, la natura del 3-dimensionale calchi in resina plastica genera un&#39;istantanea permanente del sistema ed è un romanzo e tecnica ampiamente utile per la visualizzazione e la quantificazione tre strutture tridimensionali e reti. Un vantaggio fondamentale del sistema di colata di resina è la capacità di determinare la intatte e collegate, o comunicare, struttura di un vaso sanguigno o condotto. La struttura vascolare e di ducreti mentali sono essenziali per la funzione degli organi, e questa tecnica ha il potenziale per aiutare studio delle reti vascolari e duttale in diversi modi. Colata di resina può essere utilizzata per analizzare la morfologia normale e l&#39;architettura funzionale di una struttura luminale, identificare cambiamenti morfogenetici sviluppo, e scoprire differenze morfologiche dell&#39;architettura tessuti normali tra stati e malattie. Lavoro precedente ha utilizzato resina colata a studiare, per esempio, difetti architettoniche e funzionali nel sistema biliare intraepatica topo condotto che non si sono riflesse in 2-dimensionale analisi della struttura 1,2, alterazioni vascolare cerebrale di un modello di malattia di Alzheimer mouse 3 , anomalie della vena porta nel portale topi ipertesi e cirrotici 4, passi di sviluppo in maturazione ratto linfatico tra polmoni immaturi e adulti 5, immediati cambiamenti microvascolari nel fegato di ratto, del pancreas e del rene in risposta a al danno chimico6. Presentiamo qui un metodo di generazione di un 3-dimensionale di resina di una rete vascolare del mouse o duttale specificamente dedicato alla vena porta e dotto biliare intraepatica. Questi calchi possono essere visualizzati tramite compensazione o macerazione del tessuto e possono quindi essere analizzati. Questa tecnica può essere applicata a qualsiasi sistema vascolare o duttale e sarebbe direttamente applicabile a qualsiasi studio indagare lo sviluppo, la funzione, la manutenzione, o lesioni di un 3-dimensionale struttura duttale o vascolare.

Watch this Scientific Journal Video about 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System at JoVE.com

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

Un metodo di visualizzare e quantificare il 3-dimensionale struttura vena porta mouse o dotto biliare intraepatica è descritto. Questa tecnica resina può anche essere applicata ad altri sistemi duttali o vascolare e permette<em&gt; In situ</em&gt; Visualizzazione o la quantificazione di architettura intatta comunicare un sistema.

3-Dimensional Casting resina e imaging di Vena mouse portale o sistema biliare intraepatica Duct

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

3-dimensional-resin-casting-imaging-mouse-portal-vein-or-intrahepatic

Research

JoVE Journal

Medicine

17.1K Views.  Vanderbilt University. Un metodo di visualizzare e quantificare il 3-dimensionale struttura vena porta mouse o dotto biliare intraepatica è descritto. Questa tecnica resina può anche essere applicata ad altri sistemi duttali o vascolare e permette In situ Visualizzazione o la quantificazione di architettura intatta comunicare un sistema.

Video: 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modellazione e imaging 3-Dimensional Invasion cella Collettiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Ricerca

Medicina

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Bile Duct Ligation in topi: Induzione infiammatoria epatica Lesioni e fibrosi da colestasi ostruttiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Utilizzando Multi-fluorurati biliari acidi e<em&gt; In Vivo</em&gt; Risonanza Magnetica misurare Bile Acid Trasporti

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

Overaccumulation ileectomy-indotta della Bile nell'intestino del Mouse

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Modello del ratto della partizione del fegato associando i e legatura della vena portale per procedura di messa in scena epatectomia (ALPPS)

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Imaging perfusione Doppler laser nell'hindlimb del mouse

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Un modello murino per la pancreatite cronica tramite infusione di TNBS del dotto biliare

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Ridurre al minimo il sanguinamento della vena porta post-infusione durante il trapianto di isole intraepatiche nei topi

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

DUCT: Double Resin Casting seguito da Tomografia Micro-Computerizzata per Analisi 3D del Fegato

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

Dotto biliare extraepatico e dissezione della cistifellea nei neonati di topo di nove giorni

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...