Name: non-invasive optical imaging of the lymphatic vasculature of a mouse
Uploaded: 2013-03-08T13:00:00
Description: Watch this Scientific Journal Video about Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse at JoVE.com

Dette arbejde blev støttet af følgende tilskud til Eva Sevick: NIH R01 CA128919 og NIH R01 HL092923.

Alle dyreforsøg blev udført i overensstemmelse med de standarder, fra University of Texas Health Science Center (Houston, TX), Institut for Sammenlignende Medicin og Center for Molekylær billeddannelse efter gennemgang og godkendelse af protokollen fra deres respektive institutionelle Animal Care og brug Udvalg (IACUC) eller Animal Welfare Committee (AWC). 1. Fremstilling af dyr 24 timer før Imaging Nedenstående trin skal gøres (efter behov) dagen før ly...

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Vi bruger en brugerdefineret, lille dyr NIRF imaging system til at tage billeder af mærkede lymfekar i mus. For at konstruere film af lymfeknuder bevægelse, er 300 eller flere billeder indsamles. For funktionel analyse af lymfekarrene fra film, er to eller flere ROI&#39;er manuelt trukket langs en lymfeknuder fartøj. Dimensionerne af ROIs holdes konstant for hvert fartøj, og er omtrent diameteren af ​​karret. Mens hele dyr rumlig opløsning kan afbilde fluorescerende lymfekar på 100 mikrometer eller derunder, k...

<table border="1"><tbody><tr><td> Solutions, Reagenser og udstyr </td><td> Firma </td><td> Catalog Number </td><td> Kommentarer </td></tr><tr><td> Indocyaningrønt (ICG) </td><td> Patheon Italia SPA </td><td> NDC 25431-424-02 </td><td> Skal rekonstitueres til 645 uM (5 μg/10 uL) </td></tr><tr><td> Cyklisk Albumin bindende domæne (cABD) </td><td> Bachem </td><td> Brugerdefineret </td><td> Skal rekonstitueres til 200 uM (6,8 μg/10 uL) </td></tr><tr><td> IRDye800 </td><td> Li-COR </td><td> IRDye 800CW </td><td> Rekonstituér i overensstemmelse med producentens anvisninger; konjugat med cABD på equilmolar koncentrationer </td></tr><tr><td> Sterilt vand </td><td> Hospira, Inc., Lake Forest, IL </td><td> NDC 0409-4887-10 </td><td></td></tr><tr><td> Nair </td><td> Church &amp; Dwight Co,Inc. </td><td> Lokale Stores </td><td> <a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td></tr><tr><td> Imaging System (bestanddele nedenfor) </td><td> Center for Molekylær billeddannelse </td><td> N / A </td><td> Custom-bygget i vores laboratorier. </td></tr><tr><td> Electron-multiplicere charge-coupled device (EMCCD) kamera </td><td> Princeton Instruments, Trenton, NJ </td><td> Foton Max 512 </td><td></td></tr><tr><td> Nikon kamera linse </td><td> Nikon Inc., Melville, NY </td><td> Model nr. 1992, Nikkor 28mm </td><td></td></tr><tr><td> Optisk filter </td><td> Andover Corp, Salem, NH </td><td> ANDV11333 </td><td> To 830.0/10.0 nm båndpasfiltre anvendes foran linsen </td></tr><tr><td> 785-nm laserdiode </td><td> Intens Ltd, North Brunswick, NJ </td><td> 1005-9MM-78.503 </td><td> 500 mW af optisk udgang </td></tr&gt; <tr><td> Kollimering optik </td><td> Thorlabs, Newton, NJ </td><td> C240TME-B </td><td> Kollimerer laseroutput før oprydning filter </td></tr><tr><td> Clean-up filter </td><td> Semrock, Inc., Rochester, NY </td><td> LD01-785/10-25 </td><td> Fjerner laseremissionstilstand i fluorescensbånd </td></tr><tr><td> Optisk diffuser </td><td> Thorlabs, Newton, NJ </td><td> ED1-C20 </td><td> Diffunderer laseren over dyret </td></tr><tr><td> V + + </td><td> Digital Optik, Browns Bay, Auckland, New Zealand </td><td> Version 5,0 </td><td> Software bruges til at styre kamera system og gemme billeder til computer. <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td></tr><tr><td></td><td></td><td></td><td> Analytisk Software Enten af følgende softwarepakker kan bruges til billedanalyse </td></tr><tr><td> ImageJ </td><td> National Instiningsinstitutter af Health, Bethesda, MD </td><td> Nyeste tilgængelige version </td><td> Freeware tilgængelig på <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> MATLAB </td><td> MathWorks, Natick, MA </td><td> Version 2008a eller nyere </td><td> <a href="http://www.mathworks.com/" target="_blank">http://www.mathworks.com/</a> </td></tr></tbody></table>

non-invasive optical imaging of the lymphatic vasculature of a mouse

Det lymfatiske kar-systemet er en vigtig del af kredsløbet, som opretholder fluid homeostasis, giver immunovervågning, og medierer fedtabsorption i tarmen. Men på trods af sin kritiske funktion, er der forholdsvis ringe forståelse af, hvordan lymfesystemet tilpasser sig betjene disse funktioner i sundhed og sygdom 1. For nylig har vi demonstreret evne til dynamisk billede lymfatisk arkitektur og lymfe &quot;pumpe&quot; handling hos normale forsøgspersoner samt i personer, der lider lymfatisk dysfunktion ved hjælp af spor administration af en nær-infrarødt fluorescerende (NIRF) farvestof og en brugerdefineret, Gen III- intensiveret billeddannelsessystem 2-4. NIRF imaging viste dramatiske ændringer i lymfe arkitektur og funktion med sygdom hos mennesker. Det er fortsat uklart, hvordan disse ændringer sker og nye dyremodeller bliver udviklet for at belyse deres genetiske og molekylære grundlag. I denne protokol, præsenterer vi NIRF lymfatisk, small dyr billeddannelse 5,6 ved hjælp af indocyaningrøn (ICG), et farvestof, der har været anvendt i 50 år hos mennesker 7 og en NIRF farvestof-mærkede cykliske albumin bindingsdomæne (cABD-IRDye800) peptid, der fortrinsvis binder muse-og humant albumin 8 . Omkring 5,5 gange lysere end ICG, cABD-IRDye800 har en lignende lymfatisk clearance profil og kan injiceres i mindre doser end ICG at opnå tilstrækkelige NIRF signaler til billeddannelse 8. Fordi begge cABD-IRDye800 og ICG binder til albumin i det interstitielle rum 8, kan de begge viser aktivt protein transport til og inden lymfevejene. Intradermal (ID) injektion (5-50 pl) af ICG (645 uM) eller cABD-IRDye800 (200 uM) i saltvand indgives til det dorsale aspekt på hver bagpote, og / eller den venstre og højre side af bunden af hale af en isofluran-bedøvede mus. Den resulterende farvestofkoncentration i dyret er 83-1,250 ug / kg til ICG eller 113-1,700 ug / kg tilcABD-IRDye800. Umiddelbart efter injektioner, er funktionel lymfatisk billeddannelse udført i op til 1 time under anvendelse af en tilpasset, lille dyr NIRF imaging system. Hele dyr rumlig opløsning kan afbilde fluorescerende lymfekar på 100 mikrometer eller derunder, og billeder af konstruktioner på op til 3 cm i dybden kan erhverves 9. Billeder er erhvervet ved hjælp af V + + software og analyseres ved hjælp ImageJ eller MATLAB software. Under analyse bliver efterfølgende områder af interesse (ROI&#39;er) omfatter hele fartøj diameter trukket langs en given lymfeknuder fartøj. Dimensionerne for hver ROI holdes konstant for et bestemt fartøj og NIRF intensitet måles for hver ROI til kvantitative vurderinger &quot;pakker&quot; af lymfeknuder bevæger sig gennem fartøjer.

Eksempel på NIRF lymfatisk Imaging i mus Når ICG eller cABD-IRDye800 injiceres ID ved basen af ​​halen med en normal mus, bør det lymfatiske kar mellem injektionsstedet ved haleroden og inguinale lymfeknuder (LN) umiddelbart visualiseres. Kort efter injektionen (nogle få sekunder til minutter), bør lymfekar mellem inguinal LN og axillær LN kan visualiseres som det ses i figur 2. Da lymfevejene i mus varierer fra dyr til dyr, som de gør i mennesker, kan variation i ar...

Watch this Scientific Journal Video about Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse at JoVE.com

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Nyligt udviklede billeddannelsesteknikker hjælp nær-infrarøde fluorescens (NIRF) kan til at belyse den rolle, det lymfatiske system spiller i cancermetastase, immunrespons, sårreparation og andre lymfatiske-associerede sygdomme.

Ikke-invasiv optisk billeddannelse af lymfatisk vaskulatur på en mus

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

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