Reproducerbar Mouse iskiasnerven Crush og efterfølgende vurdering af Regeneration af Whole Mount Muskel Analyse
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name (description/quantity) | Supplier | Catalogue Number |
| Mini Clipper with No. 0000 Blade | Roboz | RC-5903 |
| Nair Hair Remover (9 oz.) | Church and Dwight Co., Inc. | N/A |
| Betadine Surgical Scrub (1 gallon) | Fisher Scientific | 19066452 |
| Ophthalmic Ointment (1 oz.) | Fisher Scientific | 19082795 |
Surgical Tools:
| Name (description/quantity) | Supplier | Catalogue Number |
| FST 250 Hot bead sterilizer | Fine Science Tools | 18000-45 |
| Iris Scissors (11 cm long) | World Precision Instruments | 500216 |
| Potts-Smith Forceps (Straight; 18cm; 1×2 Teeth) | Fine Science Tools | 11024-18 |
| McPherson-Vannas Scissors (5 mm blades) | World Precision Instruments | 14124-G |
| Dumont #5 Forceps – Dumoxel Standard Tip | Fine Science Tools | 11252-30 |
| Dumont #5/45 Forceps – Dumoxel Standard Tip | Fine Science Tools | 11251-35 |
| Ultra Fine Hemostats (Straight; Smooth Inside Surfaces) | Fine Science Tools | 13020-12 |
| Powdered Activated Carbon (500 g) | Fisher Scientific | C272-500 |
| Size 6-0 Sutures with C-1 Needle (Sterile, Silk, Black, Braided , Non-absorbable; 18” Length; Box of 36) | Roboz | SUT-1073-11 |
| Reflex Clip Applier (for 9 mm clips) | World Precision Instruments | 500345 |
| 9mm Stainless Steel Reflex Clips (100/box) | World Precision Instruments | 500346 |
Animal Care:
| Name (description/quantity) | Supplier | Catalogue Number |
| 0.9% Sodium Chloride Injection (Preservative Free; 20 mL) | Hospira | 0409-4888-20 |
| Complete Homeothermic Blanket System with Flexible Probe (Medium, 115 VAC, 60 Hz) | Harvard Apparatus | 507222F |
Surgical Platform and Retractors:
(purchased at local hardware store)
| Name (description/) | Quantity |
| Stainless Steel Platform (Milled; ~12”x12”x1/8”) | 1 |
| Button Magnets (Example: Eclipse E825) | 3 |
| Stainless Steel Bolts (3” Long; Diameter Determined by Button Magnets) | 3 |
| Stainless Steel Nuts (Sized to Fit Bolts) | 9 |
| Rubber Bands (Light Tension) | 3 |
| Insect Pins (Both Ends Bent to Form Hooks) | 3 |
Semi-Thins:
| Name (description/quantity) | Supplier | Catalogue Number |
| Paraformaldehyde (1 kg) | Sigma-Aldrich | P6148-1KG |
| Sodium Phosphate Dibasic Anhydrous (500 g; Used to Prepare Phosphate Buffer) | Fisher Scientific | S375-500 |
| Sodium Phosphate Monobasic Anhydrous (1 kg; Used to Prepare Phosphate Buffer) | Fisher Scientific | AC38987-0010 |
| Glutaraldehyde (50%; 10 x 10 mL) | Ted Pella, Inc. | 18431 |
| Osmium Tetroxide (4% Aqueous, 10 x 10 mL) | Ted Pella, Inc. | 18465 |
| Propylene Oxide (450 mL) | Ted Pella, Inc. | 18601 |
| Embed 812 (Kit, for hard blocks/high image contrast) | Electron Microscopy Sciences | 14120 |
| Toluidine Blue (25 g) | Ted Pella, Inc. | 19451 |
Whole Mount Muscle Preparation and Immunohistochemistry:
| Name (description/quantity) | Supplier | Catalogue Number |
| Paraformaldehyde (1 kg) | Sigma-Aldrich | P6148-1KG |
| Sodium Phosphate Dibasic Anhydrous (500 g; Used to Prepare Phosphate Buffer) | Fisher Scientific | S375-500 |
| Sodium Phosphate Monobasic Anhydrous (1 kg; Used to Prepare Phosphate Buffer) | Fisher Scientific | AC38987-0010 |
| 10% BSA Diluent/Blocking Solution (200 mL) | Kirkegaard & Perry Laboratories, Inc. | 50-61-00 |
| Triton X-100 (100 mL) | Dot Scientific Incorporated | 9002-93-1 |
| Glycine, 98% (1 kg) | Fisher Scientific | AC12007-0010 |
| Tissue-Tek Cryo-OCT Compound (Case of 12; 4 oz bottles) | Fisher Scientific | 14-373-65 |
| Sylgard DOW 170 (2 lb. Kit) | Fisher Scientific | NC9492579 |
| Stainless Steel Insect Pins, Size 1 (100/pkg) | Fine Science Tools | 26001-40 |
| Tetramethylrhodamine-A-Bungarotoxin (0.5 mg) | Sigma-Aldrich | T0195-.5MG |
| Mouse Monoclonal Antibody Against SMI-312 (0.1 mL) | Covance | SMI-312R |
| Mouse Monoclonal Antibody Against SV2 (0.1 mL) | Developmental Studies Hybridoma Bank (DSHB) | SV2 |
| Rabbit Polyclonal Antibody Against GAP-43 | Novus Biologicals | NB300-143 |
| Flourescein Conjugated Goat Anti-Mouse IgG, Fcγ Subclass 1 Specific | Jackson ImmunoResearch | 115-095-205 |
| DyLight 649 Conjugated Donkey Anti-Rabbit IgG | Jackson ImmunoResearch | 711-495-152 |
| 4′,6-diamidino-2-phenylindole (DAPI, dilactate; 10 mg) | Invitrogen | D3571 |
| Vectashield Mounting Medium (10 mL) | Vector Laboratories | H-1000 |
| Superfrost Plus Microscope Slides (White; Size: 75 x 25 mm; Pack of 144) | Fisher Scientific | 12-550-15 |
| Fisherfinest Premium Cover Glasses (Size: 40 x 22 mm; Pack of 1 oz.) | Fisher Scientific | 12-548-5C |
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