Vaskulær støbning af voksne og tidlige postnatal mus lunger til Micro-CT Imaging
Summary
Formålet med denne teknik er ex vivo visualisering af lungearteriale netværk af tidlige postnatal og voksne mus gennem lungeinflation og injektion af en radio-uigennemsigtig polymer-baseret forbindelse via lungearterien. Potentielle anvendelser for støbt væv er også drøftet.
Abstract
Blodkar danner indviklede netværk i 3-dimensionelle rum. Derfor er det vanskeligt visuelt at forstå, hvordan vaskulære netværk interagerer og opfører sig ved at observere overfladen af et væv. Denne metode giver et middel til at visualisere den komplekse 3-dimensionelle vaskulære arkitektur i lungen.
For at opnå dette indsættes et kateter i lungepulsåren, og vaskulaturen skylles samtidig af blod og forstørres kemisk for at begrænse resistens. Lungerne er derefter oppustet gennem luftrøret ved et standardtryk og polymerforbindelsen infunderes i den vaskulære seng ved en standard strømningshastighed. Når hele arterielt netværk er fyldt og lov til at helbrede, lunge vaskulaturen kan visualiseres direkte eller afbildet på en mikro-CT (μCT) scanner.
Når det udføres med succes, kan man sætte pris på lungearterie netværk i mus lige fra tidlige postnatal aldre til voksne. Derudover, mens demonstreret i lungearterie seng, denne metode kan anvendes på enhver vaskulær seng med optimeret kateter placering og slutpunkter.
Introduction
Fokus for denne teknik er visualisering af lungearterial arkitektur ved hjælp af en polymer-baseret forbindelse i mus. Mens omfattende arbejde er blevet udført på systemiske vaskulære senge såsom hjerne, hjerte og nyre1,2,3,4,5, mindre information er til rådighed om forberedelse og påfyldning af lungearterial netværk. Formålet med denne undersøgelse er derfor at uddybe tidligerearbejde 6,,7,8 oggive en detaljeret skriftlig og visuel reference, at efterforskerne nemt kan følge for at producere billeder i høj opløsning af lungearterietræet.
Mens der findes mange metoder til mærkning og billeddannelse lunge vaskulatur, såsom magnetisk resonans imaging, ekkokardiografi, eller CT angiografi9,10, mange af disse modaliteter undlader at tilstrækkeligt fylde og / eller fange de små fartøjer, begrænse omfanget af, hvad der kan studeres. Metoder som seriel sektionsopdeling og genopbygning giver høj opløsning , men er tids-/arbejdskrævende11,12,13. Omgivende bløddelsintegritet er kompromitteret i traditionel korrosionsstøbning10,13,14,15,16. Selv dyrs alder og størrelse bliver faktorer, når de forsøger at indføre et kateter eller, opløsningen mangler. Polymerindsprøjtningsteknikken fylder derimod arterierne til kapillærniveau, og når den kombineres med μCT, giver den mulighed for uovertruffen opløsning5. Prøver fra muselunger så unge som postnatal dag 14 er blevet støbt8 og behandlet i løbet af få timer. Disse kan scannes på ubestemt tid, eller endda sendes til histologisk forberedelse / elektronmikroskopi (EM) uden at gå på kompromis med den eksisterende bløde væv17. De vigtigste begrænsninger ved denne metode er de indledende omkostninger ved CT-udstyr/software, udfordringer med nøjagtig overvågning af intravaskulært tryk og manglende evne til at indsamle data i længderetningen hos det samme dyr.
Dette papir bygger på eksisterende arbejde for yderligere at optimere lungearterie injektion teknik og skubbe alder / størrelse relaterede grænser ned til postnatal dag 1 (P1) for at give slående resultater. Det er mest nyttigt for teams, der ønsker at studere arteriel vaskulære netværk. Derfor giver vi ny vejledning til kateterplacering/stabilisering, øget kontrol over påfyldningshastighed/volumen og fremhæver bemærkelsesværdige faldgruber for øget støbningssucces. Resulterende kaster kan derefter bruges til fremtidig karakterisering og morfologisk analyse. Måske endnu vigtigere, dette er den første visuelle demonstration, at vores viden, der fører brugeren gennem denne indviklede procedure.
Protocol
Representative Results
Discussion
Udført korrekt, denne metode giver slående billeder af lungearterial netværk, giver mulighed for sammenligning og eksperimenter i gnaver modeller. Flere kritiske skridt undervejs sikrer succes. For det første skal efterforskerne heparinisere dyret i den forberedende fase for at forhindre blodpropper i at danne i lungevaskulaturen og kamre i hjertet. Dette giver mulighed for fuldstændig arteriel transit af polymer sammensatte. For det andet, når punktering af mellemgulvet og fjerne brystkassen, passe på at beskytte…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev delvist støttet af NHLBI Intramural Research Program (DIR HL-006247). Vi vil gerne takke NIH Mouse Imaging Facility for vejledning i billederhvervelse og analyse.
Materials
| 1cc syringe | Becton Dickinson | 309659 | |
| 20ml Glass Scintillation Vials | Fisher | 03-340-25P | |
| 30G Needle | Becton Dickinson | 305106 | |
| 50mL conical tubes | Cornin | 352098 | For sample Storage and scanning |
| 60cc syringe | Becton Dickinson | 309653 | |
| 7-0 silk suture | Teleflex | 103-S | |
| Analyze 12.0 Software | AnalyzeDirect Inc. | N/A | Primary Software |
| Amira 6.7 Software | Thermo Scientific | N/A | Alternative Sofware |
| CeramaCut Scissors 9cm | Fine Science tools | 14958-09 | |
| Ceramic Coated Curved Forceps | Fine Science tools | 11272-50 | |
| CO2 Tank | Robert's Oxygen Co. | n/a | |
| Dual syringe pump | Cole Parmer | EW-74900-10 | |
| Dumont Mini-Forceps | Fine Science tools | 11200-14 | |
| Ethanol | Pharmco | 111000200 | |
| Formalin | Sigma – Life Sciences | HT501128 | |
| Gauze | Covidien | 441215 | |
| Hemostat | Fine Science tools | 13013-14 | |
| Heparin (1000USP Units/ml) | Hospira | NDC 0409-2720-01 | |
| Horos Software | Horos Project | N/A | Alternative Sofware |
| induction chamber | n/a | n/a | |
| Kimwipe | Fisher | 06-666 | fiber optic cleaning wipe |
| Labelling Tape | Fisher | 15966 | |
| Magnetic Base | Kanetec | N/A | |
| Micro-CT system | SkyScan | 1172 | |
| Microfil (Polymer Compound) | Flowech Inc. | Kit B – MV-122 | 8 oz. of MV compound; 8 oz. of diluent; MV-Curing Agent |
| Micromanipulator | Stoelting | 56131 | |
| Monoject 1/2 ml Insulin Syringe | Covidien | 1188528012 | |
| Octagon Forceps Straight Teeth | Fine Science tools | 11042-08 | |
| Parafilm | Bemis company, Inc. | #PM999 | |
| PE-10 tubing | Instech | BTPE-10 | |
| Phospahte buffered Saline | BioRad | #161-0780 | |
| Ring Stand | Fisher | S13747 | Height 24in. |
| Sodium Nitroprusside | sigma | 71778-25G | |
| Steel Plate | N/A | N/A | 16 x 16 in. area, 1/16 in thick |
| Straight Spring Scissors | Fine Science tools | 15000-08 | |
| SURFLO 24G Teflon I.V. Catheter | Santa Cruz Biotechnology | 360103 | |
| Surgical Board | Fisher | 12-587-20 | This is a converted slide holder |
| Universal 3-prong clamp | Fisher | S24280 | |
| Winged Inf. Set 25X3/4, 12" Tubing | Nipro | PR25G19 | |
| Zeiss Stemi-508 Dissection Scope | Zeiss | n/a |
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