Name: a drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system
Uploaded: 2018-05-05T14:30:00
Description: Watch this Scientific Journal Video about A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System at JoVE.com

Vi takker Jessica Goldshteyn for teknisk support. Arbejde i sangen lab er finansieret af NIH grant R00NS088211, og den intellektuelle og udviklingsforstyrrelser Research Center (IDDRC) nye Program udvikling Award.

1. forberedelse af kultur plader og flasker
<ol><li>Forberedelse af druesaft agar plader<ol>
<li>Tilsættes 10 g agar pulver, 200 mL drue saft og 192 mL ddH2O i et bægerglas og mikroovn i ca 4-5 min, omrøring periodisk indtil agaren er fuldstændig opløst.</li>
		<li>I et stinkskab, køle ned løsningen til ca. 60 ° C. Tilføje 4,2 mL 95% ethanol og 4,0 mL iseddike. Justere det samlede volumen af løsningen på 400 mL med ddH2O. Mix godt.</li>
		<li>For hver 35-mm plade, tilføje omkring 2-3 m...

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Når oprettelsen af fly krydser, kan antallet af hunner og hanner bruges variere afhængigt af genotyper og antallet af larver behov for konkrete eksperimenter. For WT fluer bruger typisk tværs 10 tæver og 5 hanner. Vinduet indsamling kan blive indsnævret, afhængigt af nøjagtigheden af larver alder påkrævet. For eksempel, vil en 2-h samling periode give larver af en mere homogen befolkning. I dette tilfælde vil ved hjælp af 20 eller mere jomfruelige hunner for at oprette Kors hjælpe give tilstrækkelige æg. Ud...

Axoner manglende evne til at regenerere efter en skade på centralnervesystemet (CNS), kan føre til permanent handicap hos patienter, og spiller også en rolle i de irreversible neurologiske underskud i neurodegenerative sygdomme1,2 ,3,4,5. CNS miljø, samt den iboende vækst evne af neuroner, bestemmer, om axoner er i stand til at regenerere efter traumer. Ekstracellulære faktorer fra oligodendrocyte, astroglial og fibroblastic kilder har vist sig at hæmme neuronal vækst4,6,7,8, men fjernelsen af disse molekyler kun tillader begrænset spiring5. Iboende regenerering signaler kan påvirke regenerativ succes5,9 og repræsenterer potentielle terapeutiske mål, men disse processer er stadig ikke veldefineret på molekylært niveau. Stigninger i trofiske faktor signalering eller afskaffelse af endogene bremser, som pt fosfatase10, kan resultere i cytoskeletale regenerering under visse omstændigheder. Kombinationer af forskellige metoder anset for at være individuelt effektiv også kun levere begrænset samlede recovery til dato11,12,13,14. Derfor, der er desperat behov for at identificere yderligere veje for målrettet terapi. Ud over indledningen af axon genvækst, om og hvordan axoner re-wire til de korrekte mål, reform synapse specificitet, og opnå funktionel genopretning er vigtige ubesvarede spørgsmål.
Sammenfattende er aktuelle forståelse af maskinen dikterer axon regenerering stadig meget fragmentariske. En del af problemet er tekniske vanskeligheder med at studere axon regenerering i pattedyr i real-time, en tilgang, der er dyrt, tidskrævende og udfordrende til at gennemføre storstilet genetiske skærme. Drosophila melanogaster, på den anden side har vist sig for at være en usædvanlig kraftfuld system til studiet af komplekse biologiske spørgsmål. Frugt flue har været medvirkende til at definere gener og signalering veje, der er påfaldende bevaret i mennesker og har været en vellykket model for studiet af menneskelige lidelser, såsom neurodegenerative sygdomme, gennem de store molekylær genetik værktøjer rådighed til at manipulere gen funktion15. Bananfluer er navnlig anses for at være et ideelt værktøj for opdagelsen af gener involveret i neurale skade og genvækst15,16. Flere flyve neurale skade modeller er blevet udviklet, herunder voksen-hoved eller larve ventrale nerve ledningen (VNC) stikkende med nåle, larve VNC eller nerve crush med pincet, larve neuron laser axotomy, olfaktoriske receptor neuron fjernelse, explants hjerneskade, og perifere nerve læsion af wing fratrædelsesgodtgørelse15,17,18,19,20,21,22,23. Spændende, har seneste arbejde udnytte Drosophila skade modeller avancerede vores forståelse af de cellulære reaktionsveje og genetiske bruges af nervesystemet til at reagere på neurale skader, hvoraf nogle har vist sig at være bevaret i pattedyr24 ,25. Igen, dette understreger nytten af denne model organisme for at identificere nye mekanismer af neurale reparation.
Beskrevet her er en to-foton laser-baseret Drosophila larve sensoriske neuron skade model. En to-foton laser blev første gang brugt til at skære axoner i zebrafisk i vivo i 200326. I samme år blev den første laser dendriotomy udført i Drosophila ved hjælp af en pulserende kvælstof laser27. Kort efter bruges flere C. elegans labs femtosekund lasere til at oprette modeller af axon regenerering28. I 2007, Wu og kolleger sammenlignet og rapporteret forskelle mellem laser skader i C. elegans induceret af forskellige typer af lasere29. I 2010, var axon regenerering efter laser axotomy først vist sig at forekomme i Drosophila30. Bygger på denne omfattende laser skade litteratur, har vi udviklet en flyve neurale skade model ved hjælp af to-foton laser, som giver præcise induktion af skade til målrettede websteder med minimal undertrykkelse af netbårne af nærliggende væv, giver en relativt ren system til at studere både de indre og ydre egenskaber af neuroregeneration med enkelt-celle opløsning. Specifikt, vi har etableret et sæt skade metoder for dendritiske arborization (da) sensoriske neuroner i både det perifere nervesystem (PNS) og CNS. Da neuroner kan inddeles i fire forskellige klasser adskiller sig primært ved deres dendrit forgrening kompleksiteter: klasse I til IV31. Vores offentliggjort arbejde viser, at da neuron regenerering ligner pattedyr skade modeller på fænotypiske og molekylært niveau: da neuroner vise specifikke regenerering klasseegenskaber med klasse IV men ikke klasse I eller III da neuroner udstiller regenerering i den PNS; klasse IV da neuron axons regenerere håndfast i periferien, men deres regenerativ potentiale reduceres dramatisk i CNS, der dermed ligner dorsalrods ganglion (DRG) neuroner i pattedyr; forbedre mTOR aktivitet via Yvonne_h_p forbedrer sletning eller Akt overekspression axon regenerering i flyve CNS19. Utilizing denne skade model, vi har udøvet genetiske skærme og har identificeret RNA forarbejdning enzymet Rtca som et evolutionært bevarede hæmmende faktor for axon regenerering, forbinder axon skade til cellulært stress og RNA ændring20 .
I de præsenterede paradigme, er skaden fremkaldt via laser axotomy/dendriotomy af larve klasse IV eller III da neuroner, mærket af ppk-CD4-tdGFP eller 19-12-Gal4, UAS-CD4-tdGFP, repo-Gal80, henholdsvis. Skaden, der er udført på 2nd til 3rd instar larver på ca 48-72 h efter æglægning (h AEL). Til PNS er axotomy læsion målrettet til del af axon ~ 20-50 µm fra cellen organ, for CNS axotomy til et område af ~ 20 µm i diameter på commissure krydset i VNC, og dendriotomy til de primære dendritiske gren punkter. Den samme neuron er afbildet på 8-24 timer efter skade (AI) at bekræfte komplet transection, og 48-72 h AI at vurdere regenerering. Gennem time-lapse Konfokal imaging, kan degeneration og regeneration af individuelle axoner/dendritter, der er blevet skadet i vivo følges over tid.

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a drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system

Genvækst kapacitet af beskadigede neuroner styrer neuroregeneration og funktionel genopretning efter nervesystem traumer. I de seneste par årtier, er blevet identificeret forskellige indre og ydre hæmmende faktorer involveret i begrænsning af axon regenerering. Men blot at fjerne disse hæmmende signaler er utilstrækkelig for vellykket regenerering, der angiver eksistensen af yderligere lovgivningsmæssige maskiner. Drosophila melanogaster, frugtflue, deler evolutionært bevarede gener og signalering veje med hvirveldyr, herunder mennesker. Ved at kombinere de kraftfulde genetiske værktøjskasse af fluer med to-foton laser axotomy/dendriotomy, beskriver vi her Drosophila sensoriske neuron-dendritiske arborization (da) neuron skade model som en platform for systematisk screening for roman regenerering regulatorer. Kort, omfatter dette paradigme a) udarbejdelsen af larver, b) læsion induktion til dendrite(s) eller axon(s) ved hjælp af en to-foton laser, c) levende Konfokal imaging efter skade og d) data analyse. Vores model giver meget reproducerbare skade af enkelt mærket neuroner, axoner og dendrites af veldefinerede neuronal undertyper, i både perifere og centrale nervesystem.

Da neuroner Vis differential regenerering potentielle mellem de perifere og centrale nervesystem, samt klasse specificitet. Dette giver unikke muligheder for at screene for romanen faktorer, der er nødvendige for axon regenerering (ved hjælp af klasse IV PNS skade), såvel som dem, der er hæmmende for regenerering (med klasse IV CNS skade og klasse III PNS skade).
Axon regenerering i PNS
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Watch this Scientific Journal Video about A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System at JoVE.com

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Vi præsenterer her, en protokol bruger Drosophila sensoriske neuron - dendritiske arborization (da) neuron skade model, som kombinerer i vivo live imaging, to-foton laser axotomy/dendriotomy og kraftfuld flyve genetiske værktøjskassen, som en platform for screening potentielle initiativtagere og hæmmere af neuroregeneration.

En Drosophila In Vivo skade Model til at studere Neuroregeneration i perifere og centrale nervesystem

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

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