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.
该方法的总的目标是在脊髓隔离以及鉴定和分离的DRG 1,2。脊髓液压挤压是一个比通过椎板脊髓隔离,通过一次破椎骨一个体现的传统方式显著更快的方法,这种方法可以减少组织损伤造成的解剖过程3,4的风险。的DRG可能难以辨认。正确识别是组织分析非常重要, 如下面的坐骨神经损伤。由编号,根据相对于肋脉其本地化的DRG,病种可以一致认定。
组织由这里展示的技术分离可应用于范围广泛的分析,包括Western印迹5,6,7定量PCR和免疫组化染色8。
当识别的DRG,其采取到已知脊髓节段的数目随动物种类和菌株9,10,11,以改变帐户是重要的。在小鼠中进行该方法的优点是高数量的可用遗传修饰的变体和相对低的壳体费用。在用大鼠的优点是相对高的组织的产量和如果涉及神经损伤,该过程将与尺寸有所缓解。
如果脊柱被干扰, 例如 ,通过颈椎脱位,脊髓将挤出期间分割。如果脊髓无法挤出,脊柱可以稍微在两端修剪和挤压尝试可以重复。在情况下,需要进行进一步的分析整个脊髓, 即由所述颈膨大以及腰膨大,脊柱应仅略微修整。如果需要作进一步的分析只腰膨大,脊髓应根据本协议进行修整。脊柱应该用指尖来缓解挤压被拉直。
该协议适用于所有年龄段的啮齿类动物,并通过这里的成年小鼠(8周),鼠标小狗(5天)和成年大鼠(10周)的例子。根据动物种类和菌株,胸椎和腰椎部分的数目可以变化9 </sup> 10,11。根据不同的蛋白质要分析,成年啮齿动物可与安乐死之前酶抑制溶液灌注。如果执行涉及片面神经损伤的实验,脊髓可以分成同侧和对侧侧面采用超细镊子挤压之后立即。此外,每个侧面可以分成背,腹侧面。组织然后可以用于进一步的分析, 例如 Western印迹处理。
穿心灌注固定带之前,脊髓挤出的PFA应该避免,因为这使脊髓非柔性和防止脊髓液压挤出。液压脊髓挤压将撕下背根。对于实验中附后根是必要的,建议椎板切除术。此外,脊膜由液压挤压,这可以通过标准椎板避免丢失<sup系列=“外部参照”> 3。
也可以使用代替冰冷的PBS含氧人工脑脊液(ACSF)执行的脊髓和背根神经节分离的水力挤出。使用ACSF的允许分离的组织保存在一个更好的生理环境,这是在随后的电生理记录12,13的情况下是特别重要的。到ACSF替代可以是含有1克/升葡萄糖用于产生主DRG培养物14的PBS中。
脊髓液压挤压是一个比通过椎板脊髓隔离的传统方式,减少组织处理时间,因此减少蛋白质损坏的风险显著更快的方法。灌注固定剂前分离由椎板脊髓可以减少清扫期间和最终去除的过程中的组织损伤的风险从脊柱脊髓。然而,组织固定排除其适用性分析,如蛋白印迹。液压挤压产量结构未受损组织3适用于更广泛的分析。
按病种付费的一致鉴定可能很困难。然而,这是用于组织分析必需的, 如以下坐骨神经损伤。由编号,根据相对于肋脉其本地化的DRG,病种可以一致认定。脊髓组织的染色以及的DRG可以通过执行协议步骤7中概述的图示组织治疗的变化进行优化。
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 |