Name: a polished and reinforced thinned-skull window for long-term imaging of the mouse brain
Uploaded: 2012-03-07T12:00:00
Description: Watch this Scientific Journal Video about A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain at JoVE.com

Dette arbeidet ble støttet av American Heart Association (Post-doc til ays) og National Institutes of Health (MH085499, EB003832, og OD006831 til DK). Vi takker Beth Friedman og Pablo Blinder for kommentarer til manuskriptet.

1. Forberedelse til kirurgi i <ol><li> Rengjør de kirurgiske verktøy ved sonicating i en blanding av Maxizyme og kirurgisk Milk i en ultrasoniske renere. Autoklaver kirurgiske verktøy før hvert forsøk. </li><li> Sikre at alle nødvendige reagenser og forbruksmateriell er tilgjengelig. En liste av reagenser og forbruksmateriell er gitt i Tabell 2. Reagenser og forbruksmateriell som kommer i kontakt med utsatt vev bør være steril, når det er mulig. </li><li> Indusere anestesi. Typiske bedøvelse egnet...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248, 73-76 (1990).</li><li>Srinivasan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+for+the+quantitative+study+of+cerebrovascular+physiology.">Optical coherence tomography for the quantitative study of cerebrovascular physiology.</a> Journal of Cerebral Blood Flow &#38; Metabolism. 31, 1339-1345 (2011).</li><li>Hu, S., Wang, L. 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To-foton bildebehandling gjennom en PORTS vindu krever overføring gjennom tynnet bein og dura, som demper laserlys og legger optiske avvik på større dyp 8. Men til tross for denne ulempen, kan bildebehandling dybder opp til 250 mikrometer under pial overflaten oppnås med 900 nm eksitasjon. Større bildebehandling dybder kan i prinsippet være mulig med lengre eksitasjonsbølgelengdene 13. En stor fordel med denne metoden er fraværet av kortikale betennelse som kan eksistere forbigående i meto...

<table border="1"><tbody><tr><td> Agent </td><td> Route av levering </td><td> Dose for mus </td><td> Varighet </td><td> Merknader </td><td> Kilde </td><td> Ref Ref </td></tr><tr><td> Pentobarbital (Nembutal) </td><td> IP </td><td> 90 mikrogram / g </td><td> 15-60 min </td><td> Smal sikkerhetsmargin. Arbeid opp til riktig dose av anestesi sakte. Tillegg 10% av induksjon dose som kreves. </td><td> 036093; Butler Schein </td><td> 7 </td></tr><tr><td> Ketamin (Ketaset) blandet med xylazin (Anased) </td><td> IP </td><td> 60 mikrogram / g (K) 10 mikrogram / g (X) (bland i samme sprøyte) </td><td> 20-30 min </td><td> Xylazin er co-injiseres som et muskelavslappende middel og smertestillende. Supplement bare Ketamin på 50% av induksjon dose som kreves. </td><td> (K) 010 177, (X) 033198; Butler Schein </td><td> 7 </td></tr><tr><td> Isofluran (Isothesia) </td><td> Innånding </td><td> 4% mener alveolar konsentrasjon (MAC) for induksjon; 1-2% MAC for vedlikehold </td><td> 4-6 timer. </td><td> Forutsatt i blanding av 70% oksygen og 30% lystgass. Langvarig anestesi fører til langsom bedring. </td><td> 029403; Butler Schein </td><td> 26 </td></tr></tbody></table> Tabell 1. Forslag anestesimidler for å overleve studier. <table border="1"><tbody><tr><td> SAK </td><td> COMPANY </td><td><strong&gt; CATALOG # / MODELL </td></tr><tr><td> Betadine </td><td> Butler Schein </td><td> 6906950 </td></tr><tr><td> Buprenorfin (Buprenex) </td><td> Butler Schein </td><td> 031919 </td></tr><tr><td> Fluorescein isothiocyanate dekstran, 2 MDA molekylvekt </td><td> Sigma </td><td> FD2000S </td></tr><tr><td> Isopropylalkohol </td><td> Fisher </td><td> AC42383-0010 </td></tr><tr><td> Ringer-laktat oppløsning </td><td> Butler Schein </td><td> 009846; </td></tr><tr><td> Lidokain løsning, 2% (v / v) </td><td> Butler Schein </td><td> 002468 </td></tr><tr><td> Saline </td><td> Butler Schein </td><td> 009861 </td></tr><tr><td> Kirurgisk Milk </td><td> Butler Schein </td><td&gt; 014 325 </td></tr><tr><td> Texas Red dekstran, 70 kDa molekylvekt </td><td> Invitrogen </td><td> D1864 </td></tr><tr><td> Maxizyme </td><td> Butler Schein </td><td> 035646 </td></tr><tr><td> Forbruksmateriell </td></tr><tr><td> Carbide grader, 1/2 mm dysestørrelse </td><td> Fin Science Verktøy </td><td> 19007-05 </td></tr><tr><td> Cottoned spissen applikatorer </td><td> Fisher Scientific </td><td> 23-400-100 </td></tr><tr><td> Dekk Glass, nei. 0 tykkelse </td><td> Thomas Scientific </td><td> 6661B40 </td></tr><tr><td> Cyanoakrylat lim </td><td> ND Industries </td><td> 31428 H04308 </td></tr><tr><td> Gass Duster </td><td> Newegg </td><td> N82E16848043429 </td></tr><tr><td> Grip sementpulver </td><td> DENTSPLY </td><td> 675571 </td></tr><tr><td> Grip sement løsemiddel </td><td> DENTSPLY </td><td> 675572 </td></tr><tr><td> Insulin sprøyte, 0,3 ml volum med 29,5 gauge nål </td><td> Butler Schein </td><td> 018384 </td></tr><tr><td> Mutter og bolt for å feste hodet </td><td> Digikey </td><td> Mutter, H723-ND, bolt, R2-56X1/4-ND </td></tr><tr><td> Opthalmic salve </td><td> Butler Schein </td><td> 039886 </td></tr><tr><td> Skalpell blad </td><td> Fisher Scientific </td><td> 12-460-448 </td></tr><tr><td> Skruer, selvborende # 000 </td><td> JI Morris Firma </td><td> FF000CE125 </td></tr><tr><td> Silikon akvarium tetningsmasse </td><td> Perfecto Manufacturing </td><td> 31001 </td></tr><tr><td> Tinn oksid pulver </td><td> Mammas Minerals </td><td> EQT-TINOX </td></tr><tr><td> UTSTYR </td></tr><tr><td> Glass skriftlærd </td><td> Fisher Scientific </td><td> 08-675 </td></tr><tr><td> Dissekere mikroskop </td><td> Carl Zeiss </td><td> OPMI-1 FC </td></tr><tr><td> Elektrisk drevet drill </td><td> Foredom eller Osada </td><td> K.1020 (Foredom) eller EXL-M40 (Osada) </td></tr><tr><td> Elektrisk barbermaskin </td><td> Wahl </td><td> Series 8900 </td></tr><tr><td> Tang, Dumont nei. 55 </td><td> Fin Science Verktøy </td><td> 11255-20 </td></tr><tr><td> Tilbakemelding regulerte varmen pad </td><td> FHC </td><td> 40-90-8 (rektal termistor, 40-90-5D-02; hspiser pad, 40-90-2-07) </td></tr><tr><td> Isofluran vaporizer </td><td> Ohmeda </td><td> IsoTec4 </td></tr><tr><td> Pulsoksimeter </td><td> Starr og biovitenskap </td><td> MouseOx </td></tr><tr><td> Skrutrekker, miniatyr </td><td> Garret Wade </td><td> 26B09.01 </td></tr><tr><td> Stereotaxic ramme </td><td> Kopf Instrumenter </td><td> Modell 900 (med mus anestesi maske og ikke-ruptur øret barer) </td></tr><tr><td> Kirurgiske sakser, butte enden </td><td> Fin Science Verktøy </td><td> 14078-10 </td></tr><tr><td> Ultrasoniske renere </td><td> Fisher Scientific </td><td> 15-335-30 </td></tr></tbody></table> Tabell 2. Liste av spesifikke reagenser, forbruksmateriell og utstyr.

a polished and reinforced thinned-skull window for long-term imaging of the mouse brain

In vivo avbildning av kortikal funksjon krever optisk tilgang til hjernen uten avbrudd av intrakraniell miljøet. Vi presenterer en metode for å danne en polert og forsterket tynnet hodeskallen (porter) vindu i musen hodeskallen som spenner over flere millimeter i diameter og er stabil i flere måneder. Skallen er tynnet 10 til 15 mikrometer i tykkelse med en håndholdt drill for å oppnå optisk klarhet, og deretter kledde med Cyanoakrylat lim og et dekke glass til: 1) gir stivhet, 2) hemmer bein gjenvekst og 3) redusere lysspredning fra uregelmessigheter på beinet overflaten. Siden skallen ikke er brutt, er enhver betennelse som kan påvirke prosessen som studeres sterkt redusert. Imaging dybder opp til 250 mikrometer under kortikale overflate kan oppnås ved å bruke to-foton laser scanning mikroskopi. Dette vinduet er godt egnet til å studere cerebral blodstrøm og cellulær funksjon i både anesteserte og våken forberedelser. Det gir ytterligere opportunity å manipulere celleaktivitet bruke optogenetics eller å forstyrre blodgjennomstrømningen i målrettede skip ved bestråling av sirkulerende fotosensitisere.

Watch this Scientific Journal Video about A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain at JoVE.com

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

Vi presenterer en metode for å danne en avbildning vindu i musen hodeskallen som spenner millimeter og er stabil i flere måneder uten betennelse i hjernen. Denne metoden egner seg godt for longitudinelle studier av blodstrøm, cellulære dynamikk, og celle / vaskulær struktur ved hjelp av to-foton mikroskopi.

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