To-foton Imaging for Cellulær Dynamics i Mouse Spinal Cord
Summary
En ny ex vivo forberedelse til afbildning af mus rygmarven. Denne protokol giver mulighed for to-foton-billeddannelse af levende cellulære interaktioner i hele rygmarven.
Abstract
Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.
Introduction
Musemodeller for demyelination, herunder eksperimentel autoimmun encephalomyelitis (EAE) og intrakraniel infektion med neuroadapted mus hepatitis virus (MHV), er fremragende værktøjer til at studere molekylære veje og cellulære interaktioner forbundet med sygdommen. De har ført til og støttet effektiviteten af FDA godkendte farmaceutiske behandlinger, primært rettet mod ophør af autoimmunitet og inflammation 1. Men når endogene remyelinering mislykkedes, de aktuelt godkendte behandlinger ikke effektivt reparere demyeliniserede læsioner i centralnervesystemet. Derfor reparation-fokuserede terapier på dette stadium af sygdommen er afgørende for lindring af kroniske symptomer og forbedring af livskvaliteten. For nylig har neurale precursorceller (NPCs) kommer i forgrunden som en potentiel regenerativ terapeutisk modalitet til at målrette områder af inflammation og demyelinisering. Flere undersøgelser har fremhævet evne NPC'ere at inducere endogenoos remyelinering og deltage direkte i remyelinering 2-8. Fordi NPC'ere er involveret i direkte remyelinering, er det bydende nødvendigt at forstå deres kinetik og interaktioner med endogene celler efter transplantation. Efter transplantation, NPC'ere migrere ventralt til områder af hvid substans skader, så rostrally og kaudalt i forhold til transplantation webstedet 5,9. Kinetikken for migration varierer som reaktion på miljømæssige signaler; NPC transplanteret ind i en ikke-beskadiget rygmarv har større hastigheder end NPC transplanteret ind i en beskadiget rygmarv 6. Efter en vandrende periode, overføres NPC'ere formere meget, med en højere hastighed i en beskadiget rygmarven i forhold til en intakt rygmarv 6. Endelig er de fleste af NPC differentiere til oligodendrocytter og iværksætte direkte remyelinering 4,6,9.
Den demyeliniserede læsion er kompleks og kan indeholde en forskelligartet population af celler på forskellige stadier af enctivation. For eksempel kan en aktiv dissemineret sklerose (MS) læsion omfatter en væsentlig population af aktiverede T-celler, M1 mikroglia og M1 makrofager, men en kronisk tavs MS læsion kan bestå primært af reaktive astrocytter med få inflammatoriske celler 10-13. På grund af mangfoldigheden af effektorceller, to-foton (2P) billeddannelse i musemodeller for demyelinisering er et yderst nyttigt redskab til at hjælpe med at forstå de lokale cellulære interaktioner inden læsionen. I MS og mange ekstensivt udnyttede MS forskningsmodeller, er de fleste af læsioner placeret på den ventrale side af rygmarven, en region utilgængeligt for intravital 2P billeddannelse på grund af læsion dybde og det høje indhold af rygmarven lipid. For at omgå disse problemer og undersøgelse celle-celle-interaktioner inden læsioner langs den ventrale rygmarv har vi udviklet en simpel ex vivo 2P billeddannelse fremstilling 6.
Denne undersøgelse er en opfølgning af en tidligere metoder publikation, som visteproceduren til omplantning forstærket grønt fluorescerende protein (eGFP) som udviser NPC i rygmarven af mus efter JHMV stamme af MHV-induceret demyelinisering 14. Fem uger gamle mus inficeret med JHMV og transplanteret med eGFP-NPC'ere på thorax niveau 9 på dag 14 efter infektion. Protokollen præsenteres her giver detaljerede trin om, hvordan man udtrække rygmarven, foretage en ex vivo agarose forberedelse, og image transplanteret eGFP-NPC interaktioner med forstærket gult fluorescerende protein (EYFP) som udviser axoner. Mus, der udtrykker EYFP under neuronal-specifikke Thy1 promotoren blev anvendt i denne procedure 15. Kun nogle af axoner udtrykker EYFP, hvilket gør den anvendelig til billeddannelse enkelte axoner. Her viser vi rygmarv fjernet 7 dage efter transplantation; Imidlertid kan rygmarv ekstraheres på noget tidspunkt efter transplantation. Vi viser interaktioner af NPC med beskadigede axoner, kan vores protokol anvendes i kombination med genetiske fluorescerende markøreraf andre celletyper til at undersøge en mangfoldighed af cellulære interaktioner, der forekommer hele mus rygmarven.
Protocol
Representative Results
Discussion
Real-time 2P billeddannelse af intakt væv er forpligtet til at undersøge NPC kinetik og interaktioner efter transplantation ind i demyeliniserede mus rygmarven. Intravital 2P billeddannelse er almindeligt anvendt til at bestemme cellulære dynamik på den dorsale side af rygmarven i levende mus, og er blevet anvendt til at undersøge dorsal demyelinisering i demyeliniserende sygdom 17-19. Men fordi transplanterede NPC'ere migrere til den ventrale hvide substans, der ligger for dybt til billedet på sted…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Isoflurane, USP | Piramal Critical Care, Inc | N/A | |
| Fine scissors | Fine Science Tools | 14060-09 | sharp |
| scalpel blade #10 | Fine Science Tools | 10010-00 | |
| scalpel handle | Fine Science Tools | 10003-12 | |
| Luer rongeurs | Fine Science Tools | 16001-15 | |
| Graefe forceps | Fine Science Tools | 11052-10 | |
| Vannas scissors | Fine Science Tools | 15615-08 | |
| scalpel blade #11 | Fine Science Tools | 10011-00 | |
| RPMI-1640 | Gibco | 12-115F | |
| agarose, low gelling temperature | Sigma | A9414-25G | |
| Parafilm | Fisher Scientific | 13-374-12 | |
| Vetbond (tissue adhesive) | 3M | 1469SB | |
| 22 mm square cover slip | Fisher Scientific | 12-547 | |
| 25x dipping objective, 1.1 NA | Nikon | CFI Apo LWD 25XW | |
| Single inline solution heater | Warner Instruments | 64-0102 | |
| 520 nm single-edge dichroic beam splitter | Semrock | FF520-Di02-25×36 | Brightline |
| 560 nm single-edge dichroic beam splitter | Semrock | FF560-FDi01-25×36 | Brightline |
| photomultiplier tubes | Hamamatsu | R928 | |
| C/L variable-speed tubing pump | Masterflex | 77122-22 | |
| digital thermometer | Comar Instruments | 3501 | |
| Chameleon Ultra Ti:Sapphire laser | Coherent | N/A | |
| Slidebook 6 software | 3i | N/A | |
| Imaris 7.7 software | Bitplane | N/A |
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