Retrograd perfusion og påfyldning af Mouse koronar kar som forberedelse til Micro Computed Tomography Imaging
Summary
Visualisering af koronarkar er afgørende for at fremme vores forståelse af hjerte-kar-sygdomme. Her beskriver vi en metode til perfusion murine koronar kar med en røntgenfast silikonegummi (Microfil), som forberedelse til mikro-Computed Tomography (μCT) billeddannelse.
Abstract
Visualisering af vaskulaturen bliver stadig vigtigere for forståelsen af mange forskellige sygdomstilstande. Mens flere teknikker eksisterer for billedbehandling kar, få er i stand til at visualisere det vaskulære netværk som en helhed og samtidig udvide til en resolution, der omfatter mindre skibe 1,2. Desuden er mange vaskulære støbeteknikker ødelægge det omgivende væv, hvilket forhindrer yderligere analyse af prøven 3-5. En metode, der omgås disse spørgsmål, er mikro-Computed Tomography (μCT). μCT billeddannelse kan scanne i opløsninger <10 um, er i stand til at producere 3D rekonstruktion af det vaskulære netværk, og efterlader vævet intakt til efterfølgende analyse (f.eks histologi og morfometri) 6-11. Men, billedbehandling fartøjer ved ex vivo μCT metoder kræver, at fartøjer fyldt med en røntgenfast stof. Som sådan er den nøjagtige gengivelse af vaskulaturen fremstilles ved μCT billeddannelse betingetpålidelige og fuldstændige fyldning af skibene. I denne protokol, beskriver vi en teknik til at fylde mus koronarkar i forberedelse til μCT billeddannelse.
To dominerer teknikker eksisterer til fyldning af koronar vaskulaturen: in vivo via en kanyle og retrograd perfusion af aorta (eller en gren af aortabuen) 12-14 eller ex vivo via en Langendorff perfusionssystem 15-17. Her beskriver vi pt de vivo aorta kanylering metode, der er specielt designet til at sikre fyldning af alle fartøjer. Vi anvender en lav viskositet røntgenabsorberende stof kaldet Microfil som kan perfundere gennem mindste fartøjer for at fylde hele kapillærer, såvel som både arterielle og venøse side af det vaskulære netværk. Skibe perfunderet med buffer med en tryksat perfusionssystem, og derefter fyldt med Microfil. At sikre, at Microfil fylder mindre højere modstand fartøjer vi ligere de store grene emanating fra aorta, som afleder Microfil ind i blodpropper. Når påfyldning er fuldført, for at forhindre den elastiske beskaffenhed af hjertevæv fra presse Microfil af nogle kar, vi ligere tilgængelige store vaskulære udgangssteder umiddelbart efter fyldning. Derfor er vores teknik optimeret til fuldstændig fyldning og maksimal retention af fyldstof, muliggør visualisering af hele koronar vaskulære netværk – arterier, kapillærer og vener ens.
Protocol
Discussion
Hjertevæv har en meget høj metabolisk behov, og derfor kræver en konstant tilførsel af næringsstoffer og oxygen fra blodet leveret af koronar vaskulatur. Sygdomme i koronarkar, som aftager koronar funktion på grund af skibet stenose og blokering, kan føre til hypoxi og iskæmi, og sætte de berørte patienter med risiko for myokardieinfarkt og uoprettelig skade på hjertemusklen. En bedre forståelse af den syge tilstand af disse fartøjer er nødvendig, og afgørende for vores evne til at studere koronarkar er v…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi takker Dr. Kelly Stevens til indledende forsøg i protokollen, Dr. Michael Simons, Dr. Kip Hauch, og medlemmer af begge deres laboratorier til generel diskussion.
Dette arbejde er støtte fra NIH tilskud HL087513 og P01 HL094374.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| 1 ml syringes | Becton Dickinson | BD-309602 | |
| 1/2cc insulin syringes with permanently attached 29G ½’ needles | Becton Dickinson | BD-309306 | |
| 2″ x 2″ Gauze pads | Med101store.com | SKU 2208 | |
| 24G ¾” Angiocath IV catheter | Becton Dickinson | BD-381112 | |
| 26G ½”gauge needles | Becton Dickinson | BD-305111 | |
| Adenosine | Sigma | A9251 | 1g/L in PBS for Vasodilation Buffer (with Papaverine) |
| Angled Graefe Forceps | Fine Science Tools | 11052-10 | |
| Cotton-tipped applicators: 6″ non-sterile | Cardinal Health | C15055-006 | |
| Curved Surgical Scissors | Fine Science Tools | 14085-09 | |
| Dissecting stereoscope and light source | Nikon | NA | NA |
| Dissecting Tray, 11.5 x 7.5 inches | Cole-Parmer | YO-10915-12 | Filled with tar for pinning down the mouse |
| Fine Curved Forceps | Aesculap | FD281R | Need two |
| Heparin, 5000 U/ml stock | APP Pharmaceuticals LLC | NDC 63323-047-10 | 1:100 dilution in water |
| KCl | Fisher | P217 | Saturated solution in H2O |
| Ketamin (Ketaset), 100 mg/ml stock | Fort Dodge, Overland Park, KS, USA | NDC 0856-2013-01 | Mixed as 130 mg/kg body weight, with Xylazine in 0.9% saline |
| Microfil | Flow Tech | MV-122 (yellow). Other color options are also available. | Mix 1:1 by weight, with 10% by volume of curing agent. Prepare just before injection, and vortex to ensure it is well mixed |
| Non-sterile Suture: 6-0, braided silk | Harvard Apparatus | 723287 | |
| Papaverine | American Regent Inc. | NDC 0517-4010-01 | 4mg/L in PBS for Vasodilation Buffer (with Adenosine) |
| Paraformaldehyde | Sigma | P6148 | Prepared as 4% solution |
| Perfusion Apparatus | See figure 2 | ||
| Spring Scissors | Fine Science Tools | 15018-10 | |
| Xylazine (Anased), 20 mg/gl stock | Lloyd Labs | NADA #139-236 | Mixed as 8.8 mg/kg body weight, with Ketamin in 0.9% saline |
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