Laser nanosurgery van Cerebellar Axonen<em> In Vivo</em
Summary
Twee-foton beeldvorming, gekoppeld aan laser nanodissection, zijn nuttige hulpmiddelen om degeneratieve en regeneratieve processen te bestuderen in het centrale zenuwstelsel met subcellulaire resolutie. Dit protocol laat zien hoe u een label, afbeelding en ontleden enkele klimmen vezels in de cerebellaire schors 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
Axonale doorsnijding gevolg van mechanische schade, toxische insult of neurodegeneratieve ziekten wordt meestal gevolgd door degeneratie van het distale gedeelte van de axon die wordt losgemaakt van het cellichaam 10-13. Op enkele uitzonderingen na 2,7,14,15, gescheiden axonen in het CZS van volwassen dieren zijn meestal niet in staat om een hergroei programma 16 te activeren.
Er is weinig bekend over de real-time dynamiek van degeneratieve gebeurtenissen op cellulair en subcellulair niveau. De ontwikkeling van nieuwe strategieën ter beperking van neuronale schade en het bevorderen van neuronale hergroei vereist, als eerste stap, verduidelijken het mechanisme waarmee singulier gewonde neuronale cellen degenereren en regenereren. Deze studie is het meest rechtstreeks door volgen van de dynamiek van een neuron in vivo. Terwijl de een-foton fluorescentie beeldvormende technieken worden beperkt door intense verstrooiing van zichtbaar licht, twee-foton excitatie diepe corticale lagen in li bereiktve muizen met subcellulaire resolutie 3,4,17. Gebruikmakend van transgene muizen waarin fluorescente proteïnen selectief uitgedrukt in subpopulaties van neuronen 18-20, is TPF microscopie toegepast op de exploratie van synaptische plasticiteit en axonale verlenging tijdens de ontwikkeling in vivo 21,22. D vermogen van singulier beschadigde neuronen teruggroeien na een blessure kan worden onderzocht door het koppelen in vivo controle door twee-foton beeldvorming met een model van letsel specifiek gericht op de axon van belang. Multi-foton absorptie van femtoseconde pulsen is gebruikt om enkele dendrieten of zelfs enkele stekels 5,23 verstoren. Bovendien is deze blessure paradigma laat snijden enkele axonale vertakkingen zonder verstoring van het contacteren van dendriet 6. In het kader van het ontleden van de functies die specifieke neuronale populatie laat hun axonen eenmaal beschadigd, cerebellaire klimvezels (CF) regenereren is een bruikbaar model siNVU ze behouden opmerkelijke plastische eigenschappen na een blessure, zelfs bij volwassen dieren 24,25. Onlangs langdurige beeldvorming van CF toonde aan dat deze axons in staat regrowing in de dagen laser axotomie 6 volgen.
Dit protocol beschrijft hoe olivocerebellair neuronen en hun axonen rek labelen door anterograde tracing. Zodra de neuronen van belang fluorescent gelabeld zijn, kunnen ze herhaaldelijk worden gecontroleerd op willekeurige tijdstippen gedurende weken of maanden onder een craniale venster. De procedure enkele axonale vertakkingen ontleden laser axotomie in vivo wordt dan geïllustreerd.
De hier gepresenteerde technieken bieden nieuwe inzichten in het mechanisme van axonale remodeling in vivo en kan helpen de ontwikkeling van therapeutische strategieën om neuronale degeneratie te beperken en te bevorderen axonale hergroei.
Protocol
Representative Results
Discussion
Dit protocol laat zien hoe de neuronen van de inferieure olijf labelen met een fluorescerende kleurstof. Vervolgens wordt de methode craniaal venster uitvoeren op de cerebellaire cortex beschreven. Deze techniek biedt optische toegang tot het eindgedeelte van olivocerebellair neuronen, het klimmen vezels. Helaas, de uitkomst van zowel de etikettering en craniotomy operatie is vrij laag, zelfs in de handen van geschoolde operatoren (meestal 1 op 3 muizen is gelabeld, en 1 op de 3 craniale ramen helder blijft na 1-2 weken…
Divulgaciones
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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