Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.
Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.
Det overordnede mål med denne metode er at isolere rygmarven samt at identificere og isolere DRG 1, 2. Hydraulisk ekstrudering af rygmarven er en betydeligt hurtigere metode end den traditionelle måde at rygmarven isolering ved laminektomi, dvs ved at bryde ryghvirvlerne en ad gangen, og denne fremgangsmåde reducerer risikoen for vævsskader forårsaget af dissektion proces 3, 4 . DRG kan være vanskelige at identificere. Korrekt identifikation er meget vigtigt for væv analyse, fx efter iskiasnerven skade. Ved nummerering af DRG efter deres lokalisering i forhold til costae, kan DRG identificeres konsekvent.
Væv isoleret ved de teknikker påvist her, kan anvendes til en bred vifte af analyser herunder Western blotting 5,6, qPCR 7 og immunhistokemisk farvning 8.
Ved identificeringen DRG, er det vigtigt at tage hensyn til, at antallet af rygmarv segmenter vides at variere med dyrets art og stamme 9, 10, 11. Fordelene i udøvelse af denne fremgangsmåde i mus er det store antal genetisk modificerede varianter til rådighed, og de relativt lave boligudgifter. Fordelene ved at anvende rotter er den relativt høje væv udbytte og hvis der involverer nerveskader, vil proceduren blive lempet med størrelse.
Hvis rygsøjlen forstyrres, fx ved cervikal dislokation, vil rygmarven split under ekstrudering. Hvis rygmarven ikke kan ekstruderes, kan rygsøjlen trimmes lidt i begge ender og ekstruderingen forsøg kan gentages. Hvis der er behov hele rygmarven til yderligere analyse, dvs. bestående af den cervikale udvidelse samt den lumbale udvidelsen bør rygsøjlen kun beskåret lidt. Hvis der kun er brug columna udvidelsen til yderligere analyse, bør rygmarven trimmes ifølge den foreliggende protokol. Rygraden skal rettes ud ved hjælp fingerspidserne til at lette ekstrudering.
Den protokol for gnavere i alle aldre og er her eksemplificeret af en voksen mus (8 uger), en mus pup (5 dage) og en voksen rotte (10 uger). Afhængigt af dyreart og stamme, kan antallet af thorax og lumbal sektioner variere 9, </sup> 10, 11. Afhængigt af de proteiner, som skal analyseres, voksne gnavere kan perfunderet med enzym-inhiberende opløsninger før eutanasi. Hvis der udføres et forsøg med ensidig nerveskade, kan rygmarven opdeles i ipsilaterale og kontralaterale sider ved hjælp ultrafine pincet umiddelbart efter ekstrudering. Endvidere kan hver side opdeles i dorsale og ventrale sider. Vævet kan derefter behandles til yderligere analyser, f.eks Western blotting.
Transcardial perfusion-fiksering med PFA før rygmarv ekstrudering bør undgås, da dette gør rygmarven ikke-fleksibel og forhindrer rygmarv hydrauliske ekstrudering. Hydraulisk rygmarv ekstrudering vil rive de dorsale rødder. For forsøg, hvor vedhæftede dorsale rødder er nødvendige, anbefales laminektomi. Endvidere er spinal meninges tabt ved hydraulisk ekstrudering, som kan undgås ved standard laminektomi <sup class = "xref"> 3.
Hydraulisk ekstrudering af rygmarven og DRG isolation kunne også udføres ved anvendelse oxygeneret kunstig cerebrospinalvæske (ACSF) i stedet for iskold PBS. Brugen af ACSF tillader bevarelse af det isolerede væv i en bedre fysiologisk miljø, hvilket er særlig vigtigt i tilfælde af efterfølgende elektrofysiologiske optagelser 12, 13. Et alternativ til ACSF kunne være PBS indeholdende 1 g / l glucose til generering af primær DRG-kulturer 14.
Hydraulisk ekstrudering af rygmarven er en betydeligt hurtigere metode end den traditionelle måde at rygmarven isolering ved laminektomi, reducere væv-handling tid, og derfor mindske risikoen for skader protein. Perfusion med et fiksativ forud for isolering af rygmarven ved laminektomi kan reducere risikoen for vævsskader under dissektion og under den endelige fjernelse afrygmarv fra rygraden. Men vævsfiksering udelukker dens anvendelse på analyser såsom Western blotting. Hydrauliske ekstrudering udbytter strukturelt ubeskadiget væv 3 egnet til en bredere vifte af analyser.
Konsekvent identifikation af DRG kan være vanskelig. Dette er imidlertid væsentligt for væv analysen, f.eks efter iskiasnerven skade. Ved nummerering af DRG efter deres lokalisering i forhold til costae, kan DRG identificeres konsekvent. Farvning af rygmarven væv samt af DRG kan optimeres ved at udføre de illustrerede vævsbehandling variationer skitseret i protokol trin 7.
The authors have nothing to disclose.
We would like to acknowledge David Kiel, Aarhus University, for filming and editing. VirtualDub software was used for processing of microscope video sequences. We thank the Danish Research Institute of Translational Neuroscience – DANDRITE, Nordic EMBL Partnership, for access to equipment and the Aarhus University Research Foundation, EU 7FP project PAINCAGE, Det Frie Forskningsrad (DFF) and The Lundbeck Foundation for funding.
Bonn scissors, extra fine, straight, 8.5 cm | F.S.C. (Fine Science Tools) | # 14084-08 | For cutting open the spinal cord prior to isolation of DRGs (adult mouse, adult rat); Other manufacturer may be used |
Centrifuge | Eppendorf | # 5427 R | For centrifugation of homogenized tissue |
Cryostat microtome | Leica Biosystems | # CM 3050 S | For cutting the embedded tissue prior to staining; Other model and manufacturer may be used |
Forceps, Dumont, # 3 | F.S.C. (Fine Science Tools) | # 11231-30 | For handling of spinal cord after extrusion; Other manufacturer/type may be used |
Forceps, Dumont, # 5 | F.S.C. (Fine Science Tools) | # 11252-20 | For isolation of DRGs (adult mouse, adult rat, pup); Other manufacturer may be used |
Isoflurane (furane) IsoFlo Vet 100 % | Abbott | # 002185 | For euthanization; Other manufacturer may be used; CAUTION: toxic |
Iso-pentane GPR rectapur | VWR chemicals | # 24872.298 | For snap-freezing of tissue; Other manufacturer may be used; CAUTION: toxic |
Microtome | Leica Biosystems | # RM 2155 | For sectioning of paraffin embedded tissue; Other model and manufacturer may be used |
Paraffin wax pellets | Sigma-Aldrich | # 76243 | For paraffin embedding; Other manufacturer may be used |
Paraffin tissue embedding station | Leica Biosystems | # EG1160 | For paraffin embedding of tissue for later sectionning; Other model and manufacturer may be used |
Pellet pestel, motor cordless | Sigma-Aldrich | # Z359971-1EA | For mechanical homogenization of isolated tissue prior to Western blotting; Other manufacturer may be used |
Petri dish, 35 mm | Thermo Fischer Scientific | # 121V | For storage of isolated DRGs; Other manufacturer and size may be used |
Petri dish, 100 mm | Sigma-Aldrich | # P7741 | For spinal cord extrusion; Other manufacturer and size may be used |
Phosphatase inhibitor, Phosstop | Sigma-Aldrich | # 04906845001 | Phosphatase inhibitor cocktail for addition to TNE-lysis buffer if needed; Other manufacturer may be used |
Pipette tip, 1-200 μl, no filter | Sarstedt | # 70.1189.105 | For hydraulic extrusion; Other manufacturer may be used |
Protease inhibitor, Complete | Sigma-Aldrich | # 05892791001 | Protease inhibitor cocktail for addition to TNE-lysis buffer if needed; Other manufacturer may be used |
RNAlater solution | Sigma-Aldrich | # R0901 | For RNA stabilization for storage; Other manufacturer may be used |
Rneasy Protect Mini KiT | Qiagen | # 74124 | For RNA isolation; Other manufacturer may be used |
Scalpel; Swann-Morton surgical blade no. 11 | Swann-Morton | # REF0203 | For isolation of spinal cord lumbar area; Other manufacturer/type may be used |
Scissors, straight, type 3, 25 mm cutting edge | Bochem | # 4070 | For isolation of spine; Other manufacturer/type may be used |
Scissors, 130 mm cutting edge | Hounisen | # 1902.0130 | For isolation of spinal cord (adult rat) |
Spring scissors, straight, 8 mm cutting edge | F.S.C. (Fine Science Tools) | # 15009-08 | For cutting open the spinal column prior to isolation of DRGs (pup) and for isolation of DRGs (adult mouse, adult rat, pup); Other manufacturer may be used |
Standard syringes, 2.5 ml, 5 ml, 10 ml | Terumo | # SS02SE1; # SS05SE1; # SS10SE1 | For hydraulic extrusion; Other manufacturer may be used |
Stereomicroscope MZ12.5 with objective 1x and eyepiece 10x | Leica | # 10446370 | Other manufacturer/type may be used |
Sterile PBS | GIBCO | # 10010-015 | For hydraulic extrusion; Can also be made according to standard protocols |
Syringe needle 23 G x 1", 0.6 x 25 mm | Terumo Neolus | # NN-2325R | For hydraulic extrusion in pups; Other manufacturer/type may be used |
Syringe needle 18 G x 1 1/2, 1.2 x 40 mm | Terumo Neolus | # NN-1838S | Alternative to pipette tip for hydraulic extrusion in adult mice; Other manufacturer may be used |
Tissue-Tek | Sakura | # 4583 | Tssue embedding material for later cryosectionning |
TNE-lysis buffer | VWR chemicals | # 10128-582 | For tissue lysis prior to Western blotting; Other manufacturer may be used; Can also be made according to standard protocols |