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 arbejde blev støttet af NIH tilskud K08NS065157 (til TAF) Dertil kommer, at Penn Center for Muskel-og skeletbesvær, Award nummer P30AR050950 fra National Institute of Arthritis, led, muskler og hud sygdomme støttet dette arbejde (TAF og Steven S. Scherer). Endelig Shriners Pediatric Research Center Seed finansiering (TAF) støttede dette arbejde. Vi vil gerne takke Dr. Young-Jin Son for først at vise hele mount proceduren og Amy A. Kim for sin bistand til at producere skitser i figur 1.

1. Dyreindivider 1,1. Behandling <ol><li> Alle dyr procedurer bør udføres med godkendelse af den lokale institutionens Animal Care and Ethics Udvalg og i overensstemmelse med den anvendelse og Udvalget og National Institutes of Health retningslinjer, med foranstaltninger for at mindske smerter og ubehag. </li><li> Vores mus blev huset under temperatur-kontrollerede betingelser på en 12-timers revers lys og mørke cyklus, medens fodret mus foder og vand ad libitum. </li><li> For undersøgel...

<ol>
	<li>Pan, Y. A., Misgeld, T., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neurotoxic+and+neuroprotective+agents+on+peripheral+nerve+regeneration+assayed+by+time-lapse+imaging+in+vivo.">Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo.</a> J. Neurosci. 23, 11479-11488 (2003).</li><li>Magill, C., Tong, A., Kawamura, D., Hayashi, A., Hunter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinnervation+of+the+tibialis+anterior+following+sciatic+nerve+crush+injury:+A+confocal+microscopic+study+in+transgenic+mice.">Reinnervation of the tibialis anterior following sciatic nerve crush injury: A confocal microscopic study in transgenic mice.</a> Exp. 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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. R., Anderson, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+axonal+regeneration+in+the+central+and+peripheral+nervous+systems+of+the+NG2-deficient+mouse.">Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse.</a> BMC Neurosci. 8, 80-80 (2007).</li><li>Thornton, M. R., Mantovani, C., Birchall, M. A., Terenghi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+N-CAM+and+N-cadherin+expression+in+axotomized+and+crushed+rat+sciatic+nerve.">Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve.</a> J. Anat. 206, 69-78 (2005).</li><li>Beer, G. M., Steurer, J., Meyer, V. 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Vi har præsenteret en metode til at opnå en pålidelig komplet iskiasnerven crush med præcis mærkning af knuse webstedet. Som tidligere nævnt ischiasnerven knuse er en almindelig model af perifer nerveskade i mus og rotter. Selv om hver metode til at knuse har sine fordele og ulemper, fandt vi denne metode produceres et komplet crush, der var let markeret med et minimum af specialudstyr (fx særlige klemmer osv.). Crush Metoder Den mest almindelige instrument a...

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~ 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> Bolte af rustfrit stål (3 &quot;Long; Diameter Bestemt af Button magneter) </td><td> 3 </td></tr><tr><td> Rustfrit stål nuts (størrelse, der passer Bolte) </td><td> 9 </td></tr><tr><td> Rubber Bands (Light Tension) </td><td> 3 </td></tr><tr><td> Insekt Pins (Begge ender bøjet til dannelse af kroge) </td><td> 3 </td></tr></tbody></table> Semi-thins: <table border="1"><tbody><tr><td> Navn (beskrivelse / mængde) </td><td> Leverandør &lt;/ Strong&gt; </td><td> Katalognr </td></tr><tr><td> Paraformaldehyd (1 kg) </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Dibasisk natriumphosphat-anhydrat (500 g; anvendes til fremstilling af phosphatpuffer) </td><td> Fisher Scientific </td><td> S375-500 </td></tr><tr><td> Monobasisk natriumphosphat Vandfri (1 kg, anvendes til fremstilling af phosphatpuffer) </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> Osmiumtetroxid (4% vandig, 10 x 10 ml) </td><td> Ted Pella, Inc. </td><td> 18465 </td></tr><tr><td> Propylenoxid (450 ml) </td><td> Ted Pella, Inc. </td><td> 18601 </td></tr><tr><td> Embed 812 (Kit, for hårde blokke / høj billedkvalitet kontrast) </td><td> Electron Microscopy Sciences </td><td> 14120 </td></tr><tr><td> Toluidine Blue (25 g) </td><td> Ted Pella, Inc. </td><td> 19451 </td></tr></tbody></table> Hele Mount Muskel Forberedelse og Immunhistokemi: <table border="1"><tbody><tr><td> Navn (beskrivelse / mængde) </td><td> Leverandør </td><td> Katalognr </td></tr><tr><td> Paraformaldehyd (1 kg) </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Dibasisk natriumphosphat-anhydrat (500 g; anvendes til fremstilling af phosphatpuffer) </td><td> Fisher Scientific </td><td> S375-500 </td></tr><tr><td> Monobasisk natriumphosphat Vandfri (1 kg, anvendes til fremstilling af phosphatpuffer) </td><td> Fisher Scientific </td><td> AC38987-0010 </td></tr><tr><td> 10% BSA fortyndingsmiddel / blokerende opløsning (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 Videnskabelig Incorporated </td><td> 9002-93-1 </td></tr><tr><td> Glycin, 98% (1 kg) </td><td> Fisher Scientific </td><td> AC12007-0010 </td></tr><tr><td> Tissue-Tek Cryo-oktober Forbindelse (Sag af 12, 4 oz 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> Rustfrit stål Insekt Pins, størrelse 1 (100/pkg) </td><td> Fine Science Tools </td><td> 26001-40 </td></tr><tr><td> Tetramethylrhodamin-A-Bungarotoxin (0,5 mg) </td><td> Sigma-Aldrich </td><td> T0195-.5MG </td></tr><tr><td> Muse-monoklonalt antistof mod SMI-312 (0,1 ml) </td><td> Covance </td><td> SMI-312R </td></tr><tr><td> Monoklonalt museantistof mod SV2 (0,1 ml) </td><td> Developmental Studies Hybridoma Bank (DSHB) </td><td> SV2 </td></tr><tr><td> Rabbit Polyklonal Antistof mod GAP-43 </td><td> Novus Biologicals </td><td> NB300-143 </td></tr><tr><td> Flourescein konjugeret gede-anti-muse-IgG, Fcy underklasse en specifik </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 konjugeret æsel anti-kanin-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-phenylindol (DAPI, dilactate, 10 mg) </td><td> Invitrogen </td><td> D3571 </td&gt; </tr><tr><td> Vectashield monteringsmedium (10 ml) </td><td> Vector Laboratories </td><td> H-1000 </td></tr><tr><td> SuperFrost Plus Microscope Slides (White, Størrelse: 75 x 25 mm, Pakke med 144) </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Fisherfinest Premium dækglasset (Størrelse: 40 x 22 mm, Pakke med 1 ounce). </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 nervesystem (PNS) er bredt undersøgt både for dets relevans for sygdom hos mennesker og til at forstå den robuste regenerative respons monteret af PNS-neuroner og derved muligvis belyser svigt i CNS regenerering 1. Iskiasnerven knuse (axonotmesis) er en af de mest almindelige modeller af perifer nerveskade i gnavere 2. Knusning afbryder alle axoner, men Schwann-celler basal laminae er bevaret, således at regenerering er optimal 3,4. Dette tillader undersøgeren for at studere præcist evnen af en voksende axon at interagere med både Schwann-celle-og basal lag 4. Rotter har generelt været de foretrukne dyremodeller for eksperimenterende nerve crush. De er bredt tilgængelige, og deres læderede iskiasnerven giver en rimelig tilnærmelse af de menneskelige nerve læsioner 5,4. Selvom mindre i størrelse end rotter nerve, har musen nerve mange lignende kvaliteter. Vigtigst er dog, aftalememorandae modeller er stadig mere værdifuld på grund af den brede tilgængelighed af transgene linier giver nu mulighed for en detaljeret dissektion af de enkelte molekyler kritiske for nerve regenerering 6, 7. Tidligere forskere har anvendt flere metoder til at producere en nerve crush eller skade, herunder simple vinklet pincet, kølet pincet, hæmostatiske pincet, vaskulære klemmer, og investigator-designet skruetvinger 8,9,10,11,12. Efterforskere har også brugt forskellige metoder til mærkning skaden site, herunder sutur, kulpartikler og fluorescerende perler 13,14,1. Vi beskriver vores metode til at opnå en reproducerbart komplet iskiasnerven crush med præcise og vedvarende mærkning af knuse-site med en fin hæmostatiske pincet og efterfølgende kulstof crush-site mærkning. Som en del af vores beskrivelse af iskiasnerven crush procedure, vi har også en forholdsvis simpel metode til muskel hele montere vi bruger til efterfølgende at kvantificere regenerering.

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 rapport beskriver vi en metode til at knuse musen iskiasnerven. Denne metode bruger let tilgængelige hæmostatiske pincet og let og reproducerbart producerer komplet iskiasnerven crush. Herudover beskriver vi en fremgangsmåde til fremstilling af muskel hele underlag egnet til analyse af nerveregenerering efter iskiasnerven knuse.

Reproducerbar Mouse iskiasnerven Crush og efterfølgende vurdering af Regeneration af Whole Mount Muskel Analyse

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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