Organotypiske skivekulturer at studere oligodendrocyt Dynamics og myelinering
Summary
A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.
Abstract
NG2 udtrykkende celler (polydendrocytes, oligodendrocyt precursorceller) er den fjerde største gliale cellepopulation i det centrale nervesystem. Under embryonale og postnatale udvikling de aktivt formere sig og generere myelinerende oligodendrocytter. Disse celler er almindeligvis blevet undersøgt i primære dissocierede kulturer, neuron cokulturer, og i fast væv. Brug af nyligt tilgængelige transgene muselinjer skive dyrkningssystemer kan anvendes til at undersøge proliferation og differentiering af oligodendrocyt afstamning celler i både grå og hvide substans områder i forhjernen og cerebellum. Slice kulturer fremstilles ud fra tidlige postnatale mus og holdes i kultur i op til 1 måned. Disse skiver kan afbildes flere gange i løbet af kulturen periode at undersøge cellulær adfærd og interaktioner. Denne metode giver visualisering af NG2 celledeling og de skridt, der fører til oligodendrocyt differentiering, samtidig med at en detaljeret analyse af regionens-afhængigeent NG2 celle og oligodendrocyt funktionel heterogenitet. Dette er en stærk teknik, der kan anvendes til at undersøge de interne og eksterne signaler påvirker disse celler over tid i et cellulært miljø, der ligner det, der findes in vivo.
Introduction
Organoytpic skivekulturer af centralnervesystemet har vist sig at være særdeles nyttige til at studere neuron og gliacelle biologi i et semiintact system, 1 – 4. Disse kulturer er relativt enkle at vedtage og fastholde mange fordele ved primære dissocierede cellekulturer såsom manipulation af det ekstracellulære miljø og nem adgang til gentagen langsigtet levende celle billedbehandling og elektrofysiologiske optagelser 5-9. Desuden skivekulturer opretholde 3-dimensionel væv cytoarchitecture, regionalt neurale forbindelse, og de fleste større celletyper er til stede i systemet. Disse egenskaber gør disse kulturer et unikt og praktisk system til at undersøge enkelt celle adfærd og fysiologi med cellulære og miljømæssige interaktioner.
NG2 celler er en population af gliaceller i pattedyrs centralnervesystem der fortsætter med at formere sig og frembringe myelinating oligodendrocytter under embryonisk og postnatal udvikling 10. De er blevet grundigt undersøgt i dissocierede primære cellekulturer, og den seneste udvikling af transgene mus linjer med NG2 celle-specifik ekspression af fluorescerende proteiner har muliggjort in vivo skæbne kortlægning og elektrofysiologiske optagelser i akutte præparater skive. Selv med disse undersøgelser, er lidt om de tidsmæssige dynamik NG2 celleproliferation og oligodendrocyt differentiering. Selv dissocieret cellekultur er almindeligt anvendt til den relative anlæg i farmakologiske og genetiske manipulationer, er det ikke egnet til interrogering funktionelle forskelle af disse celler i forskellige områder af hjernen, især når det er ønskeligt at opretholde forbindelse med det cellulære mikromiljø. Skivekulturer giver et simpelt alternativ, der er modtagelig for farmakologiske manipulationer og er blevet anvendt til at undersøge oligodendrocyt myelinering 11,12, celleular respons efter lysolecithin (LPC) eller antistof induceret demyelinisering 13,14, og induktion af remyelinisering via farmakologisk behandling 15.
En metode til at undersøge og udføre direkte billedvisning og faste væv (eller postfiksering) analyse af NG2 celleproliferation og oligodendrocyt differentiering i organotypiske skive kulturer taget fra både forhjernen og lillehjernen er beskrevet. Dette er en kraftfuld metode, der kan bruges til at studere celle skæbne enkelte NG2 celler efter hovedgruppe 16 og at opdage områdespecifikke og aldersbetingede forskelle i vækstfaktor-induceret NG2 celleproliferation 17. Denne forholdsvis simpel teknik er bredt tilgængelige for yderligere at undersøge celle iboende og / eller miljømæssige mekanismer, der regulerer fysiologi af disse gliaceller og deres reaktion på neuronal aktivitet eller myelin skade.
Protocol
Representative Results
Discussion
Myelinering i det centrale nervesystem er af afgørende betydning for en effektiv neuronal kommunikation og axonal overlevelse 22. NG2 celler kontinuerligt generere myelinerende oligodendrocytter ind i voksenalderen og samtidig opretholde en fastboende befolkning i de fleste områder af hjernen 16,23 – 25. Nogle genetiske og molekylære mekanismer, der regulerer differentieringen af disse celler er blevet beskrevet, men stadig meget at blive opdaget. Organotypiske skive kulturer er et praktis…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by grants from the National Multiple Sclerosis Society (RG4179 to A.N.), the National Institutes of Health (NIH R01NS073425 and R01NS074870 to A.N.) and the National Science Foundation (A.N.). We thank Dr. Frank Kirchhoff (University of Saarland, Homburg Germany) for providing PLPDsRed transgenic mice. We thank Youfen Sun for her assistance in maintaining the transgenic mouse colony.
Materials
| Slice Culture Medium | |||
| Minimum Essential Medium | Invitrogen | 11090 | 50% |
| Hank’s Balanced Salt Solution | Invitrogen | 14175 | 25% |
| HEPES | Sigma-Aldrich | H-4034 | 25mM |
| L-Glutamine | Invitrogen | 25030 | 1mM |
| Insulin | Sigma-Aldrich | I6634 | 1mg/L |
| Ascorbic Acid | Sigma-Aldrich | A-0278 | 0.4mM |
| Horse Serum | Hyclone | SH30074.03 | 25% |
| Titrate to pH 7.22 with NaOH, filter with bottle top filter, and store at 4 degrees Celsius | |||
| Dissection Buffer | |||
| NaCl | Sigma-Aldrich | S3014 | 124mM |
| KCl | Sigma-Aldrich | P5405 | 3.004mM |
| KH2PO4 | Sigma-Aldrich | P5655 | 1.25mM |
| MgSO4 (anhydrous) | Sigma-Aldrich | M7506 | 4.004mM |
| CaCl2 2H2O | Sigma-Aldrich | C7902 | 2mM |
| NaHCO3 | Sigma-Aldrich | S5761 | 26mM |
| D-(+)-Glucose | Sigma-Aldrich | G7021 | 10mM |
| Ascorbic Acid | Sigma-Aldrich | A-0278 | 2.0mM |
| Adenosine | Sigma-Aldrich | A4036 | 0.075mM |
| Filter with bottle top filter and store at 4 degrees Celsius, oxygenate with 95%O2 5%CO2 before use | |||
| Other Reagents and Supplies | |||
| 4-hydroxy tamoxifen (4OHT) | Sigma-Aldrich | H7904 | 100nM |
| Paraformaldehyde (PFA) | EM Sciences | 19200 | 4% |
| L-Lysine | Sigma-Aldrich | L-6027 | 0.1M |
| Sodium Metaperiodate | Sigma-Aldrich | S-1878 | 0.01M |
| Rabbit anti NG2 antibody | Chemicon (Millipore) | AB5320 | 1:500 |
| Mouse anti CC1 antibody | Calbiochem (Millipore) | OP80 | 1:200 |
| Mouse anti Ki67 antibody | Vision Biosystems | NCL-Ki67-MM1 | 1:300 |
| Mouse anti NeuN antibody | Chemicon (Millipore) | MAB377 | 1:500 |
| Species specific secondary antibodies | Jackson Immunoresearch | ||
| Normal Goat Serum (NGS) | 5% | ||
| Triton X-100 | 0.10% | ||
| Mounting medium with DAPI | Vector Labs | H-1200 | |
| Millicell culture membrane inserts 0.45μm pore size | Fisher Scientific | PICM03050 | |
| Bottle top filters | Fisher Scientific | 09-740-25E | |
| Ethanol | |||
| 95% O2 5% CO2 gas mix | |||
| Phosphate Buffered Saline (PBS) | |||
| Sterile six well plates | |||
| Dissecting (hippocampal) or weighing spatulas | |||
| Manual or automatic tissue chopper | |||
| Razor blades | |||
| Disposable transfer pipettes | |||
| 35mm and 60mm sterile Petri dishes | |||
| Stereomicroscope | |||
| Inverted Phase Microscope | |||
| Laminar Flow Hood | |||
| Tissue Culture Incubator | |||
| Inverted Fluorescence Microcope |
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