Laser nanosurgery af cerebellare Axoner<em> In Vivo</em
Summary
To-foton billeddannelse, koblet til laser nanodissection, er nyttige værktøjer til at studere degenerative og regenerative processer i centralnervesystemet med subcellulær opløsning. Denne protokol viser, hvordan til at mærke, billede, og dissekere enkelt klatring fibre i den cerebellare cortex in vivo.
Abstract
Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.
Introduction
Axonal transection følge af mekanisk skade, giftige fornærmelse eller neurodegenerative sygdomme er normalt efterfulgt af degeneration af den distale del af Axon, der er løsrevet fra cellen kroppen 10-13. Med nogle få undtagelser 2,7,14,15, afhuggede axoner i CNS af voksne dyr er som regel i stand til at aktivere en genvækst program 16.
Lidt er kendt om real-time dynamik degenerative begivenheder på celleniveau og subcelleniveauet. Udviklingen af nye strategier til begrænsning af neuronal skade og fremme neuronal genvækst kræver som et første skridt, der præciserer den mekanisme, som ualmindeligt sårede neuronale celler degenerere og regenerere. Denne undersøgelse er mest direkte behandlet ved at overvåge dynamikken i en enkelt neuron in vivo. Mens den ene-foton fluorescens billeddannelse teknikker er begrænset af intens spredning af synligt lys, to-foton excitation når dybe kortikale lag i live mus med subcellulær opløsning 3,4,17. Drage fordel af transgene mus, hvor fluorescerende proteiner er selektivt udtrykt i subpopulationer af neuroner 18-20 har TPF mikroskopi blevet anvendt til udforskning af synaptisk plasticitet og axonforlængende under udviklingen in vivo 21,22. T han evne ualmindeligt beskadigede neuroner til regrow efter skade kan undersøges ved kobling in vivo overvågning af to-foton billeddannelse med en model af skade specifikt rettet til Axon af interesse. Multi-foton absorption af femtosekund pulser er blevet brugt til at forstyrre enkelt dendritter eller endda enkelte pigge 5,23. Desuden er denne skade paradigme tillader skæring enkelt axonale grene uden at forstyrre kontakte dendritceller 6. Inden for rammerne af dissekere de funktioner, der giver specifik neuronal befolkning til at regenerere deres axoner engang beskadigede, cerebellare klatring fibre (CFS) er en nyttig model since de bevarer bemærkelsesværdige plastiske egenskaber efter skaden, selv i voksne dyr 24,25. For nylig, langsigtet billeddannelse CFS viste, at disse axoner er i stand til fornybare i de dage, der følger laser axotomi 6.
Denne protokol beskriver, hvordan man mærke olivocerebellar neuroner og deres axonforlængende gennem anterograd sporing. Når neuroner af interesse fluorescens-mærkede, kan de blive overvåget gentagne gange på vilkårlige tidspunkter for uger eller måneder under et kranie vindue. Proceduren for at dissekere en enkelt axonale grene ved hjælp af laser axotomi in vivo vil derefter blive vist.
De teknikker, der præsenteres her giver ny indsigt i den mekanisme af axonal remodeling in vivo, og kan bidrage til udviklingen af terapeutiske strategier til at begrænse neuronal degeneration og fremme axonal genvækst.
Protocol
Representative Results
Discussion
Denne protokol viser, hvordan at mærke neuroner i ringere oliven med et fluorescerende farvestof. Efterfølgende metode til at udføre en kraniel vindue på cerebellar cortex beskrevet. Denne teknik giver optisk adgang til den terminale del af olivocerebellar neuroner, klatring fibre. Desværre, resultatet af både mærkning og craniotomy kirurgi er ganske lav, selv i hænderne på dygtige operatører (normalt 1 ud af 3 mus er mærket, og 1 ud af 3 kranie vinduer forbliver klar efter 1-2 uger).
<p class="jove_conte…Divulgations
The authors have nothing to disclose.
Acknowledgements
We would like to thank Erica Lorenzetti for technical assistance on the injections and Irene Costantini for making figure 1. The research leading to these results has received funding from LASERLABEUROPE (Grant 284464, European Commission’s Seventh Framework Programme). This research project has also been supported by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX and by Italian Ministry of Health in the framework of the “Stem Cells Call for Proposals.” This work is part of the research activities of the European Flagship Human Brain Project and has been carried out in the framework of the International Center of Computational Neurophotonics foundation supported by “Ente Cassa di Risparmio di Firenze”.
Materials
| Lab standard stereotaxic, rat and mouse | Stoelting | 51670 | |
| Borosilicate glass with filament | Sutter Instrument Inc | BF100-50-10 | |
| Germinator 500 (Glass bead sterilizer) | Roboz | ||
| Microinjection dispense system | Picospritzer | ||
| Small diameter round cover glass, #1 thickness, 3 mm, 100 pack (CS-3R) | Warner Instruments | 64-0720 | |
| Ti:Sapphire laser, 120 fs width pulses, 90 MHz repetition rate | Coherent | Chameleon | |
| Spongostan, haemostatic sponge | Ferrosan | MS0005 | |
| Galvanometric mirrors | GSI Lumonics | VM500+ | |
| Objective | Olympus | XLUMPLFLN 20XW | |
| Piezoelectric stage | Physik Instrumente | P-721 | |
| Photomultiplier modules | Hamamatsu Photonics | H7710-13 | |
| LabVIEW System Design Software | National Instruments | ||
| Voren, 1 mg/ml (dexamethasone-21- isonicotinate) | Boheringer Ingelheim | ||
| Rymadil (carprofen) | Pfizer | ||
| Lidocaine clorohydrate 2% | ATI | ||
| Alexa488 dextran | Life Technologies | D22910 |
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