Name: reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis
Uploaded: 2012-02-22T12:00:00
Description: Watch this Scientific Journal Video about Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis at JoVE.com

Dette arbeidet ble støttet av NIH tilskudd K08NS065157 (til TAF) I tillegg Penn Center for Muskel-og skjelettplager, Award Number P30AR050950 fra National Institute of Arthritis, støttet Muskel og Skin Diseases dette arbeidet (TAF og Steven S. Scherer). Til slutt støttet Shriners Pediatric Research Center Seed finansiering (TAF) dette arbeidet. Vi ønsker å takke Dr. Young-Jin Son for i utgangspunktet å demonstrere hele mount prosedyren og Amy A. Kim for bistand henne i å produsere de skissene av Figur 1.

1. Dyr fag 1.1. Behandling <ol><li> Alle dyr prosedyrer skal utføres med godkjenning av den lokale institusjonens Animal Care og etisk komité og i samsvar med bruk og komiteen og National Institutes of Health retningslinjer, med tiltak for å redusere smerte og ubehag. </li><li> Våre musene ble plassert i henhold til temperatur-kontrollerte forhold på en 12-timers revers lys og mørke-syklusen, mens fôret mus Chow og vann ad libitum. </li><li> For studier av voksen foryngelse, skal mus v...

<ol>
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Methods. 188, 71-75 (2010).</li><li>Berg, A., Zelano, J., Cullheim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrin+G-2+ligand+mRNA+is+downregulated+in+spinal+motoneurons+after+sciatic+nerve+lesion.">Netrin G-2 ligand mRNA is downregulated in spinal motoneurons after sciatic nerve lesion.</a> Neuroreport. 21, 782-785 (2010).</li><li>Girolami, E. I., Bouhy, D., Haber, M., Johnson, H., David, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+potential+role+of+SOCS1+and+SOCS3+in+Wallerian+degeneration+in+injured+peripheral+nerve.">Differential expression and potential role of SOCS1 and SOCS3 in Wallerian degeneration in injured peripheral nerve.</a> Exp. Neurol. 223, 173-182 (2010).</li><li>Hossain-Ibrahim, M. K., Rezajooi, K., Stallcup, W. B., Lieberman, A. 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Vi har presentert en metode for å få en pålitelig komplett hoftenervene forelsket med presis merking av trengselen nettstedet. Som tidligere nevnt, er isjiasnerven knuse en felles modell av perifer nerveskade hos mus og rotter. Selv om hver metode av trengselen har sine fordeler og ulemper, fant vi denne metoden produsert et komplett forelsket som ble lett markert med et minimum av spesialutstyr (f.eks spesielle klemmer osv.). Crush Metoder Den vanligste instrume...

<table border="1"><tbody><tr><td> Navn (beskrivelse / mengde) </td><td> Leverandør </td><td> Katalog nummer </td></tr><tr><td> Mini Clipper med nr. 0000 Blade </td><td> Roboz </td><td> RC-5903 </td></tr><tr><td> Nair Hair Remover (9 oz.) </td><td> Kirke og Dwight Co, Inc. </td><td> N / A </td></tr><tr><td> Betadine Kirurgisk Scrub (1 gallon) </td><td> Fisher Scientific </td><td> 19066452 </td></tr><tr><td> Ophthalmic salve (1 oz.) </td><td> Fisher Scientific </td><td> 19082795 </td></tr></tbody></table> Kirurgisk Verktøy: <table border="1"><tbody><tr><td> Navn (beskrivelse / mengde) </td><td> Leverandør </td><td> Katalog nummer </td></tr><tr><td> FST 250 Hot perle sterilisator </td><td> Fine Science Verktøy </td><td> 18000-45 </td> &lt;/ Tr&gt; <tr><td> Iris Sakser (11 cm lang) </td><td> World presisjonsinstrumenter </td><td> 500216 </td></tr><tr><td> Potts-Smith tang (Straight, 18cm, 1x2 tenner) </td><td> Fine Science Verktøy </td><td> 11024-18 </td></tr><tr><td> McPherson-Vännäs Sakser (5 mm blad) </td><td> World presisjonsinstrumenter </td><td> 14 124-G </td></tr><tr><td> Dumont # 5 tang - Dumoxel Standard Tip </td><td> Fine Science Verktøy </td><td> 11252-30 </td></tr><tr><td> Dumont # 5/45 tang - Dumoxel Standard Tip </td><td> Fine Science Verktøy </td><td> 11251-35 </td></tr><tr><td> Ultra Fine Hemostats (rett; Glatte innvendige flater) </td><td> Fine Science Verktøy </td><td> 13020-12 </td></tr><tr><td> Pulverisert Activated Carbon (500 g) </td><td> Fisher Scientific </td><td> C272-500 </td></tr><tr><td> Størrelse 6-0 suturer med C-1 Needle (Steril, Silk, Black, Flettede,Ikke-absorberbare; 18 &quot;lengde, Box av 36) </td><td> Roboz </td><td> SUT-1073-1011 </td></tr><tr><td> Reflex Clip applier (for 9 mm klipp) </td><td> World presisjonsinstrumenter </td><td> 500345 </td></tr><tr><td> 9mm rustfritt stål Reflex Klipp (100/box) </td><td> World presisjonsinstrumenter </td><td> 500346 </td></tr></tbody></table> Animal Care: <table border="1"><tbody><tr><td> Navn (beskrivelse / mengde) </td><td> Leverandør </td><td> Katalog nummer </td></tr><tr><td> 0,9% natriumklorid Injection (uten konserveringsmiddel, 20 ml) </td><td> Hospira </td><td> 0409-4888-20 </td></tr><tr><td> Komplett Homeothermic Blanket system med fleksibel Probe (Medium, 115 VAC, 60 Hz) </td><td> Harvard Apparatus </td><td> 507222F </td></tr></tbody></table> Kirurgisk Platform og haker: (Kjøpes på lokal jernvarehandel) <table border="1"><tbody><tr><td> Navn (beskrivelse /) </td><td> Antall </td></tr><tr><td> Rustfritt stål Platform (Milled; ~ 12 &quot;x12&quot; x1 / 8 &quot;) </td><td> 1 </td></tr><tr><td> Button Magneter (Eksempel: Eclipse E825) </td><td> 3 </td></tr><tr><td> Rustfrie bolter (3 &quot;Long; Diameter Bestemmes av Button magneter) </td><td> 3 </td></tr><tr><td> Rustfrie nøtter (størrelsen er tilpasset Bolter) </td><td> 9 </td></tr><tr><td> Gummistrikk (Light Tension) </td><td> 3 </td></tr><tr><td> Insect Pins (begge ender bøyd i Hooks) </td><td> 3 </td></tr></tbody></table> Semi-Thins: <table border="1"><tbody><tr><td> Navn (beskrivelse / mengde) </td><td> Leverandør &lt;/ Strong&gt; </td><td> Katalog nummer </td></tr><tr><td> Paraformaldehyde (1 kg) </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Sodium Dinatriumhydrogenfosfat vannfri (500 g, brukes til å forberede fosfatbuffer) </td><td> Fisher Scientific </td><td> S375-500 </td></tr><tr><td> Natriumfosfat Monobasic vannfri (1 kg; brukes til å forberede fosfatbuffer) </td><td> Fisher Scientific </td><td> AC38987-0010 </td></tr><tr><td> Glutaraldehyd (50%; 10 x 10 ml) </td><td> Ted Pella, Inc. </td><td> 18431 </td></tr><tr><td> Osmium Tetroxide (4% vandig, 10 x 10 ml) </td><td> Ted Pella, Inc. </td><td> 18465 </td></tr><tr><td> Propylene oxide (450 ml) </td><td> Ted Pella, Inc. </td><td> 18601 </td></tr><tr><td> Embed 812 (Kit, for harde blokker / høy bildekontrast) </td><td> Elektron mikroskopi Sciences </td><td> 14120 </td></tr><tr><td> Toluidine Blå (25 g) </td><td> Ted Pella, Inc. </td><td> 19451 </td></tr></tbody></table> Hele Mount Muskel Forberedelse og Immunhistokjemi: <table border="1"><tbody><tr><td> Navn (beskrivelse / mengde) </td><td> Leverandør </td><td> Katalog nummer </td></tr><tr><td> Paraformaldehyde (1 kg) </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Sodium Dinatriumhydrogenfosfat vannfri (500 g, brukes til å forberede fosfatbuffer) </td><td> Fisher Scientific </td><td> S375-500 </td></tr><tr><td> Natriumfosfat Monobasic vannfri (1 kg; brukes til å forberede fosfatbuffer) </td><td> Fisher Scientific </td><td> AC38987-0010 </td></tr><tr><td> 10% BSA Diluent / Blokkering Solution (200 ml) </td><td> Kirkegaard &amp; Perry Laboratories, Inc. </td><td> 50-61-00 </td></tr><tr><td> Triton X-100 (100 ml) </td&gt; <td> Dot Vitenskapelig Incorporated </td><td> 9002-93-1 </td></tr><tr><td> Glysin, 98% (1 kg) </td><td> Fisher Scientific </td><td> AC12007-0010 </td></tr><tr><td> Tissue-Tek Cryo-oktober Compound (Case of 12, 4 unse flasker) </td><td> Fisher Scientific </td><td> 14-373-65 </td></tr><tr><td> Sylgard DOW 170 (£ 2 Kit) </td><td> Fisher Scientific </td><td> NC9492579 </td></tr><tr><td> Stainless Steel Insect Pins, Size 1 (100/pkg) </td><td> Fine Science Verktøy </td><td> 26001-40 </td></tr><tr><td> Tetramethylrhodamine-A-Bungarotoxin (0,5 mg) </td><td> Sigma-Aldrich </td><td> T0195-.5MG </td></tr><tr><td> Monoklonalt antistoff mot SMI-312 (0,1 ml) </td><td> Covance </td><td> SMI-312R </td></tr><tr><td> Monoklonalt antistoff mot SV2 (0,1 ml) </td><td> Utviklingsstudier hybridoma Bank (DSHB) </td><td> SV2 </td></tr><tr><td> Rabbit Polyklonal antistoff mot GAP-43 </td><td> Novus Biologicals </td><td> NB300-143 </td></tr><tr><td> Flourescein Konjugert Goat anti-mus IgG, Fcγ Underklasse en spesifikk </td><td> Jackson ImmunoResearch </td><td> <a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=115-095-205&amp;Attributes=Yes&amp;Quantity=1">11 5-095-205</a> </td></tr><tr><td> DyLight 649 Konjugert Donkey Anti-Rabbit IgG </td><td> Jackson ImmunoResearch </td><td> <a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=711-495-152&amp;Attributes=Yes&amp;Quantity=1">711-495-152</a> </td></tr><tr><td> 4 &#39;,6-diamidino-2-phenylindole (DAPI, dilactate, 10 mg) </td><td> Invitrogen </td><td> D3571 </td&gt; </tr><tr><td> Vectashield Montering Medium (10 ml) </td><td> Vector Laboratories </td><td> H-1000 </td></tr><tr><td> Superfrost Plus objektglass (hvit; Størrelse: 75 x 25 mm, pakke med 144) </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Fisherfinest Premium Cover Briller (Størrelse: 40 x 22 mm; pakke med 1 oz.) </td><td> Fisher Scientific </td><td> 12-548-5C </td></tr></tbody></table>

reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis

Regeneration i det perifere nervesystemet (PNS) er mye studert både for dens relevans for menneskelig sykdom og for å forstå den robuste regenerativ respons montert av PNS nevroner dermed muligens belysning av svikt i CNS regenerering en. Hoftenervene forelsket (axonotmesis) er en av de vanligste modellene av perifer nerveskade hos gnagere 2. Knusing avbryter alle axoner men Schwann celle basal lamellene er bevart slik at gjenfødelse er optimal 3,4. Dette gjør at undersøkeren å studere nettopp muligheten for en voksende axon å samhandle med både Schwann cellen og basal lameller 4. Rotter har generelt vært de foretrukne dyremodeller for eksperimentell nerve klem. De er allment tilgjengelig og deres lesioned isjiasnerven gir en rimelig tilnærming til menneskelig nerve lesjoner 5,4. Selv mindre i størrelse enn rotte nerve, har musen nerve mange lignende kvaliteter. Likevel, det viktigste enormte-modellene er stadig mer verdifull på grunn av store tilgjengeligheten av transgene linjer gjør det nå mulig for en detaljert disseksjon av de enkelte molekyler kritiske for nerve 6 gjenfødelse, 7. Tidligere etterforskere har brukt flere metoder for å produsere en nerve forelsket eller skade inkludert enkle vinklede tang, kjølt tang, hemostatic tang, vaskulære klemmer, og etterforsker utformede klemmer 8,9,10,11,12. Etterforskerne har også brukt ulike metoder for merking skade området inkludert sutur, karbon partikler og fluoriserende perler 13,14,1. Vi beskriver vår metode for å få et reproduserbart komplett hoftenervene knuse med presis og vedvarende merking av trengselen-området med en fin hemostatic tang og påfølgende karbon forelsket stedet merking. Som en del av vår beskrivelse av isjiasnerven trengselen prosedyre har vi også inkludert en relativt enkel metode for muskel hel montering vi bruker til senere kvantifisere foryngelse.

Watch this Scientific Journal Video about Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis at JoVE.com

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

I denne rapporten beskriver vi en metode for å knuse mus hoftenervene. Denne metoden bruker lett tilgjengelige hemostatic pinsett og lett og reproduserbart produserer komplette hoftenervene knuse. I tillegg beskriver vi en metode for å forberede muskler hele mounts egnet for analyse av nerve foryngelse etter hoftenervene knuse.

Reproduserbar Mouse hoftenervene Crush og påfølgende vurdering av Regeneration av Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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