Name: hydraulic extrusion of the spinal cord and isolation of dorsal root ganglia in rodents
Uploaded: 2017-01-22T15:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents at JoVE.com

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.

All animals were handled in full compliance with Danish and European regulations. Permission number: 2012-15-2934.
1. Preparation of the Syringe for Hydraulic Extrusion of the Spinal Cord
<ol>
	<li>For an adult rat, use a 10 mL syringe. For an adult mouse, use a 5 mL syringe. For pups, use a 2.5 mL syringe.</li>
	<li>Adjust a non-filter pipette tip universally suitable for 2-200 &#181;L pipettes by trimming the large end of the pipette tip until it fits firmly to the syringe. Place the adjusted pipette tip on the syringe. For pups, use a 23 G or similar needle instead of an adjusted pipette tip.</li>
	<li>Aspirate ice-cold sterile PBS and leave the syringe on ice until further use.</li>
</ol>
2. Euthanasia
<ol>
	<li>Do not perform cervical dislocation, as this will disturb the spinal vertebrae rendering extrusion impossible.</li>
	<li>For adult rat/mouse, euthanize using gas such as CO2 or isoflurane. These substances are harmful. Perform euthanasia in a fume hood and handle the substances according to institution guidelines. (CAUTION: CO2: H280, P410, P403; isoflurane: H361d, P260, P281, P280)
	<ol>
		<li>To ensure that the rodent is dead, ensure absence of reflex by pinching a paw with tweezers. Decapitate the rodent using large scissors. For pups, euthanize by decapitation.</li>
	</ol>
	</li>
</ol>
3. Isolation of the Spinal Column
<ol>
	<li>Sprinkle water onto the fur to control the hair.</li>
	<li>Using a pair of scissors, cut open the fur along the spinal column in a distal direction and isolate the spinal column by cutting on both sides along the spinal column past the pelvic bone. Cut the spinal cord distally to the pelvic bone with a pair of scissors. 
	NOTE: The rostral-caudal axis is denoted as the proximal-distal axis throughout the protocol.
	<ol>
		<li>Trim the spinal column using a pair of scissors to avoid the proximal-most and distal-most areas as these may render the spinal column too S-shaped for successful spinal cord extrusion. Ensure that the spinal cord is visible at both ends. If the spinal cord is not visible, trim the spinal column with scissors until the spinal cord becomes visible.</li>
	</ol>
	</li>
</ol>
4. Hydraulic Extrusion of the Spinal Cord
<ol>
	<li>Place a Petri dish (100 mm in diameter) filled with sterile PBS on ice.</li>
	<li>Straighten the spinal column by applying an index finger on the bent part (proximal-most end) of the spinal column, thereby straightening the spinal column as much as possible.
	<ol>
		<li>For pups, avoid bending the spinal column, as the vertebrae will be disrupted rendering spinal cord extrusion impossible.</li>
	</ol>
	</li>
	<li>Insert the pipette tip (23 G needle for pups, alternatively 18 G needle for adult mice) at the distal-most end of spinal column. If inserted correctly, the tip is stabilized in the spinal cavity.</li>
	<li>Ensure that the spinal column is straightened as much as possible. Apply steady pressure, and extrude the spinal cord into the Petri dish on ice. Ensure that the spinal cord is kept moist on ice by keeping it in PBS until further handling.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55226/55226fig1.jpg" /> 
Figure 1: Representative spinal cords. Hydraulically extruded spinal cords. Left to right: adult rat, adult mouse, mouse pup. Lumbar enlargements marked by box. <a href="http://cloudfront.jove.com/files/ftp_upload/55226/55226fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Identification of Dorsal Root Ganglia (DRGs)
<ol>
	<li>Split the spinal column in two equal longitudinal parts using scissors and place the split spinal column under the microscope.</li>
	<li>Identify the T13 vertebra segment by locating the attachment site of the distal-most costae on the spinal column paying attention not to confuse the costae with the nearby intercostal nerves. The distal-most costae are attached to the proximal part of the T13 vertebra segment and the T13 DRG is located distally to vertebra T13 and proximally to vertebra L1.</li>
	<li>Identify concomitant DRGs in the distal direction. For an adult rat, concomitant DRGs appear white with clearly visible dorsal and ventral roots. For an adult mouse, concomitant DRGs appear white with clearly visible dorsal and ventral roots. For pups, concomitant DRGs appear as clear spheres.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55226/55226fig2.jpg" /> 
Figure 2: Identification of DRGs in spinal column after extrusion of spinal cord. The spinal column has been split allowing visualization of DRGs as indicated by arrows. Exemplified by adult rat and mouse pup. <a href="http://cloudfront.jove.com/files/ftp_upload/55226/55226fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Isolation of DRGs
<ol>
	<li>Number Petri dishes (30 mm in diameter) according to the specific DRGs to be isolated, e.g. L3, L4, L5. Fill the Petri dishes with ice-cold sterile PBS and place them on ice.</li>
	<li>Using micro scissors, cut dorsal and ventral roots as close to the DRGs as possible to release them while avoiding damaging the DRGs.</li>
	<li>With the tip of closed tweezers, gently scoop out DRGs and transfer them to the corresponding numbered Petri dishes.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55226/55226fig3.jpg" /> 
Figure 3: Representative isolated DRGs. Left to right: adult rat, adult mouse, mouse pup. <a href="http://cloudfront.jove.com/files/ftp_upload/55226/55226fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. Processing of Tissues for Further Analysis
<ol>
	<li>Western blotting
	<ol>
		<li>Isolate the tissue area of interest, e.g. the spinal cord lumbar enlargement. If needed, separate the spinal cord into ipsilateral and contralateral sides and further into dorsal and ventral horns.</li>
		<li>Place the tissue in lysis buffer such as TNE lysis buffer (10 mM Tris-base, 150 mM NaCl, 1 mM EDTA, 1% NP40 in double distilled H2O) containing appropriate enzyme inhibitors on ice.</li>
		<li>Homogenize the tissue for approximately 30 s on ice using a mechanical grinding pestle.</li>
		<li>Centrifuge for 15 min at 16,000 x g at 4 &#176;C and use the supernatant for further processing according to standard protocols5,6 or keep the supernatant at -20 &#176;C until further use.</li>
	</ol>
	</li>
	<li>RNA-analysis
	<ol>
		<li>Isolate the tissue of interest.</li>
		<li>Immediately place the tissue in RNA stabilization solution to stabilize the RNA for storage.</li>
		<li>Isolate RNA according to standard protocols7.</li>
		<li>Convert RNA to cDNA and perform qPCR according to standard protocols7.</li>
	</ol>
	</li>
	<li>Immunohistochemistry
	<ol>
		<li>For fresh-frozen tissue (applicable to spinal cord):
		<ol>
			<li>Prepare dry ice powder by pulverizing dry ice cubes to an amount corresponding to two tablespoons of powder. Cover dry ice cubes in a cloth and pulverize the cubes using a hammer. Place silver foil (approximately 5 cm x 5 cm) on a layer of dry ice cubes and cover the silver foil with dry ice powder.</li>
			<li>Place the spinal cord area selected for further analyses on the dry ice powder using tweezers, let it snap-freeze, and transfer the spinal cord to a cryo tube. Keep it on dry ice at all times or store it at -80 &#176;C until further use.</li>
			<li>Transfer the spinal cord to a cryostat (-20 &#176;C). If needed, trim the spinal cord inside the cryostat. Place the spinal cord in embedding material inside the cryostat in order to attach it to the specimen disc.</li>
			<li>Cut the spinal cord in the desired thickness, e.g. 5 &#181;m, and collect on glass plates. Heat the glass plates with a finger immediately prior to tissue collection for better attachment. Keep any leftover tissue in cryo tubes at -80 &#176;C until further use.</li>
			<li>Leave the sections on glass plates inside the cryostat until ready for post-fixing.</li>
			<li>Post-fix the sections by immersion into 4% PFA for 10 min at room temperature.</li>
			<li>Perform staining according to standard protocols8.</li>
		</ol>
		</li>
		<li>Fixed tissue for cryo sectioning (applicable to spinal cord and DRGs)
		<ol>
			<li>Fix spinal cord and DRGs in 4% PFA (2 h for spinal cord, 1.5 h for mouse DRGs and 4 h for rat DRGs) at 4 &#176;C (place spinal cord in horizontal position to prevent it from fixing in a bent position).</li>
			<li>Transfer the tissue to 25% w/v sucrose overnight at 4 &#176;C. The tissue can be stored in 25% w/v sucrose supplemented with a few droplets of 10% sodium azide to prevent microbial contamination at 4 &#176;C until further use. If needed, trim the spinal cord to make up the lumbar enlargement only on a silicone board or similar.</li>
			<li>Using the tip of a paper towel, aspirate excess sucrose solution from the tissue.</li>
			<li>Fill a cryo mold halfway with embedding material. Push any bubbles to the sides of the cryo mold with tools such as closed tweezers.</li>
			<li>Place the tissue on the embedding material using tweezers and ensure the desired tissue orientation.</li>
			<li>Fill the cryo mold completely with embedding material and push any bubbles as far away from the tissue as possible (e.g. by using closed tweezers).</li>
			<li>Fill a Petri dish (100 mm in diameter) with 2-methylbutane. This substance is harmful. Perform this step in a fume hood and handle the substance according to institution guidelines (CAUTION: H224, H304, H336, H411, P210, P280, P273, P301, P331, P304/P340, P309/P310).</li>
			<li>Place the Petri dish on a layer of dry ice and add two dry ice cubes to the Petri dish. Place the cryo mold in the Petri dish and allow the embedding material to freeze completely.</li>
			<li>Keep the embedded tissue on dry ice at all times or at -80 &#176;C wrapped in Parafilm until further processing.</li>
			<li>Cut the tissue on a cryostat (-20 &#176;C) in the desired thickness, e.g. 5 &#181;m.</li>
			<li>Perform staining according to standard protocols8.</li>
		</ol>
		</li>
		<li>Fixed tissue for paraffin-embedding (applicable to spinal cord and DRGs)
		<ol>
			<li>Fix spinal cord and DRGs in 4% PFA (2 h for spinal cord, 1.5 h for mouse DRGs and 4 h for rat DRGs) at 4 &#176;C (place spinal cord in horizontal position to prevent it from fixing in a bent position).</li>
			<li>Transfer the tissue to 25% w/v sucrose overnight at 4 &#176;C. The tissue can be stored in 25% w/v sucrose supplemented with a few droplets of 10% sodium azide to prevent microbial contamination at 4 &#176;C until further use.</li>
			<li>Isolate the tissue area of interest, e.g. the spinal cord lumbar enlargement.</li>
			<li>Fill an embedding mold halfway with paraffin.</li>
			<li>Cool down the embedding mold briefly by placing it on the cooling area of the embedding machine to harden the paraffin slightly. This allows for better tissue positioning.</li>
			<li>Place the tissue in the desired orientation in the embedding mold. Fill the embedding mold with paraffin and cool immediately.</li>
			<li>Place the paraffin-containing embedding mold at 4 &#176;C overnight to let it harden completely and remove the paraffin block containing the tissue from the embedding mold.</li>
			<li>Cut sections on a microtome in the desired thickness, e.g. 5 &#181;m.</li>
			<li>Perform staining according to standard protocols8.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

If the spinal column is disturbed, e.g. by cervical dislocation, the spinal cord will split during extrusion. If the spinal cord cannot be extruded, the spinal column may be trimmed slightly at both ends and the extrusion attempt can be repeated. In case the entire spinal cord is needed for further analysis, i.e. consisting of the cervical enlargement as well as the lumbar enlargement, the spinal column should only be trimmed slightly. If only the lumbar enlargement is needed for further analysis, the spinal cord should be trimmed according to the present protocol. The spinal column should be straightened using the fingertips to ease extrusion.
The protocol is applicable to rodents of all ages and is here exemplified by an adult mouse (8 weeks), a mouse pup (5 days) and an adult rat (10 weeks). Depending on animal type and strain, the number of thoracic and lumbar sections may vary9,10,11. Depending on the proteins to be analyzed, adult rodents may be perfused with enzyme-inhibiting solutions prior to euthanasia. If performing an experiment involving one-sided nerve injury, the spinal cord can be split into ipsilateral and contralateral sides using ultra-fine tweezers immediately after extrusion. Further, each side can be split into dorsal and ventral sides. The tissue can then be processed for further analyses, e.g. Western blotting.
Transcardial perfusion-fixation with PFA prior to spinal cord extrusion should be avoided, as this renders the spinal cord non-flexible and prevents spinal cord hydraulic extrusion. Hydraulic spinal cord extrusion will tear off the dorsal roots. For experiments where attached dorsal roots are necessary, laminectomy is recommended. Furthermore, spinal meninges are lost by hydraulic extrusion, which can be avoided by standard laminectomy3.
Hydraulic extrusion of the spinal cord and DRG isolation could also be performed using oxygenated artificial cerebrospinal fluid (ACSF) instead of ice-cold PBS. The use of ACSF allows preservation of the isolated tissue in a better physiological environment, which is particularly important in case of subsequent electrophysiological recordings12,13. An alternative to ACSF could be PBS containing 1 g/L glucose for generation of primary DRG cultures14.
Hydraulic extrusion of the spinal cord is a significantly faster method than the traditional way of spinal cord isolation by laminectomy, reducing tissue-handling time and therefore decreasing the risk of protein damage. Perfusion with a fixative prior to isolating the spinal cord by laminectomy may reduce the risk of tissue damage during dissection and during final removal of the spinal cord from the spinal column. However, tissue fixation precludes its applicability to analyses such as Western blotting. Hydraulic extrusion yields structurally undamaged tissue3 suitable for a broader range of analyses.
Consistent identification of DRGs may be difficult. However, this is essential for tissue analysis, e.g. following sciatic nerve injury. By numbering the DRGs according to their localization relative to the costae, DRGs can be identified consistently. Staining of spinal cord tissue as well as of DRGs may be optimized by performing the illustrated tissue treatment variations outlined in protocol step 7.

The overall goal of this method is to isolate the spinal cord as well as to identify and isolate DRGs1,2. Hydraulic extrusion of the spinal cord is a significantly faster method than the traditional way of spinal cord isolation by laminectomy, i.e. by breaking the spinal vertebrae one at a time, and this method reduces the risk of tissue damage caused by the dissection process3,4. DRGs may be difficult to identify. Correct identification is highly important for tissue analysis, e.g. following sciatic nerve injury. By numbering the DRGs according to their localization relative to the costae, DRGs can be identified consistently.
Tissue isolated by the techniques demonstrated here can be applied to a broad range of analyses including Western blotting5,6, qPCR7 and immunohistochemical staining8.
When identifying DRGs, it is important to take into account that the number of spinal cord segments is known to vary with animal type and strain9,10,11. The advantages in performing this method in mice are the high number of genetically modified variants available and the relatively low housing expenses. The advantages in using rats are the relatively high tissue yield and if involving nerve injuries, the procedure will be eased with size.

<table><tbody><tr><td>Bonn scissors, extra fine, straight, 8.5 cm</td><td>F.S.C. (Fine Science Tools)</td><td># 14084-08</td><td>For cutting open the spinal cord prior to isolation of DRGs (adult mouse, adult rat); Other manufacturer may be used</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td># 5427 R</td><td>For centrifugation of homogenized tissue</td></tr><tr><td>Cryostat microtome</td><td>Leica Biosystems</td><td># CM 3050 S</td><td>For cutting the embedded tissue prior to staining; Other model and manufacturer may be used</td></tr><tr><td>Forceps, Dumont, # 3</td><td>F.S.C. (Fine Science Tools)</td><td># 11231-30</td><td>For handling of spinal cord after extrusion; Other manufacturer/type may be used</td></tr><tr><td>Forceps, Dumont, # 5</td><td>F.S.C. (Fine Science Tools)</td><td># 11252-20</td><td>For isolation of DRGs (adult mouse, adult rat, pup); Other manufacturer may be used</td></tr><tr><td>Isoflurane (furane) IsoFlo Vet 100%</td><td>Abbott</td><td># 002185</td><td>For euthanization; Other manufacturer may be used; CAUTION: toxic</td></tr><tr><td>Iso-pentane GPR rectapur</td><td>VWR chemicals</td><td># 24872.298</td><td>For snap-freezing of tissue; Other manufacturer may be used; CAUTION: toxic</td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td># RM 2155</td><td>For sectioning of paraffin embedded tissue; Other model and manufacturer may be used</td></tr><tr><td>Paraffin wax pellets</td><td>Sigma-Aldrich</td><td># 76243</td><td>For paraffin embedding; Other manufacturer may be used</td></tr><tr><td>Paraffin tissue embedding station</td><td>Leica Biosystems</td><td># EG1160</td><td>For paraffin embedding of tissue for later sectionning; Other model and manufacturer may be used</td></tr><tr><td>Pellet pestel, motor cordless</td><td>Sigma-Aldrich</td><td># Z359971-1EA</td><td>For mechanical homogenization of isolated tissue prior to Western blotting; Other manufacturer may be used</td></tr><tr><td>Petri dish, 35 mm</td><td>Thermo Fischer Scientific</td><td># 121V</td><td>For storage of isolated DRGs; Other manufacturer and size may be used</td></tr><tr><td>Petri dish, 100 mm</td><td>Sigma-Aldrich</td><td># P7741</td><td>For spinal cord extrusion; Other manufacturer and size may be used</td></tr><tr><td>Phosphatase inhibitor, Phosstop</td><td>Sigma-Aldrich</td><td># 04906845001</td><td>Phosphatase inhibitor cocktail for addition to TNE-lysis buffer if needed; Other manufacturer may be used</td></tr><tr><td>Pipette tip, 1-200 &amp;mu;L, no filter</td><td>Sarstedt</td><td># 70.1189.105</td><td>For hydraulic extrusion; Other manufacturer may be used</td></tr><tr><td>Protease inhibitor, Complete</td><td>Sigma-Aldrich</td><td># 05892791001</td><td>Protease inhibitor cocktail for addition to TNE-lysis buffer if needed; Other manufacturer may be used</td></tr><tr><td>RNAlater solution</td><td>Sigma-Aldrich</td><td># R0901</td><td>For RNA stabilization for storage; Other manufacturer may be used</td></tr><tr><td>Rneasy Protect Mini KiT</td><td>Qiagen</td><td># 74124</td><td>For RNA isolation; Other manufacturer may be used</td></tr><tr><td>Scalpel; Swann-Morton surgical blade no. 11</td><td>Swann-Morton</td><td># REF0203</td><td>For isolation of spinal cord lumbar area; Other manufacturer/type may be used</td></tr><tr><td>Scissors, straight, type 3, 25 mm cutting edge</td><td>Bochem</td><td># 4070</td><td>For isolation of spine; Other manufacturer/type may be used</td></tr><tr><td>Scissors, 130 mm cutting edge</td><td>Hounisen</td><td># 1902.0130</td><td>For isolation of spinal cord (adult rat)</td></tr><tr><td>Spring scissors, straight, 8 mm cutting edge</td><td>F.S.C. (Fine Science Tools)</td><td># 15009-08</td><td>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</td></tr><tr><td>Standard syringes, 2.5 mL, 5 mL, 10 mL</td><td>Terumo</td><td># SS02SE1; # SS05SE1; # SS10SE1</td><td>For hydraulic extrusion; Other manufacturer may be used</td></tr><tr><td>Stereomicroscope MZ12.5 with objective 1X and eyepiece 10X</td><td>Leica</td><td># 10446370</td><td>Other manufacturer/type may be used</td></tr><tr><td>Sterile PBS</td><td>GIBCO</td><td># 10010-015</td><td>For hydraulic extrusion; Can also be made according to standard protocols</td></tr><tr><td>Syringe needle 23 G x 1&quot;, 0.6 x 25 mm</td><td>Terumo Neolus</td><td># NN-2325R</td><td>For hydraulic extrusion in pups; Other manufacturer/type may be used</td></tr><tr><td>Syringe needle 18 G x 1 1/2, 1.2 x 40 mm</td><td>Terumo Neolus</td><td># NN-1838S</td><td>Alternative to pipette tip for hydraulic extrusion in adult mice; Other manufacturer may be used</td></tr><tr><td>Tissue-Tek</td><td>Sakura</td><td># 4583</td><td>Tssue embedding material for later cryosectionning</td></tr><tr><td>TNE-lysis buffer</td><td>VWR chemicals</td><td># 10128-582</td><td>For tissue lysis prior to Western blotting; Other manufacturer may be used; Can also be made according to standard protocols</td></tr></tbody></table>

hydraulic extrusion of the spinal cord and isolation of dorsal root ganglia in rodents

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.

Hydraulically extruded spinal cords display a smooth undamaged surface shown in Figure 1. The lumbar enlargement can easily be identified as the thickened area of the spinal cord, boxed in Figure 1. The dorsal and ventral sides of the spinal cord can be identified by eye according to the morphology. In Figure 1, the ventral side is facing the viewer, identifiable by one clear midline along the entire spinal cord. Two broader lines along the spinal cord allow identification of the dorsal side. At the distal-most end, the cauda equina may sometimes remain attached in adult rats and adult mice.
Individual DRGs can only be identified while located in the spinal column, Figure 2. Following isolation, Figure 3, the individual DRGs cannot be identified by appearance. If needed, it is therefore important to keep them clearly separated upon isolation. After isolation, the spinal cord and DRGs can be processed and stained as outlined in step 7.3 and represented in Figure 4. Figure 4a shows an immunohistochemical staining of a DRG section where the neurons and their surrounding satellite glial cells are clearly visible. By correct identification of the DRG it is possible to analyze the effect of sciatic nerve injuries on the soma of the injured neuron as well as on their surrounding satellite glial cells. Figure 4b shows a Nissl staining of a hydraulically extruded spinal cord where the tissue is intact as reflected by the smooth edges of the section.
<img alt="Figure 4" src="/files/ftp_upload/55226/55226fig4.jpg" /> 
Figure 4: Representative tissue sections. A. Immunohistochemical staining of DRG section. B. Nissl stained section of hydraulically extruded spinal cord. <a href="http://cloudflare.jove.com/files/ftp_upload/55226/55226fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents at JoVE.com

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

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 ...

hydraulic-extrusion-spinal-cord-isolation-dorsal-root-ganglia

Nanyang Technological University

Research

JoVE Journal

Neuroscience

47.8K Views.  Aarhus University. 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. 

Video: Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

Author Spotlight: In Vitro and In Vivo Models to Enhance Chronic Pain Treatment Efficacy

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

Bioengineering

Harvesting DRG from Rat Spine: A Procedure to Extract Dorsal Root Ganglion from the Spine of a Rat Model

Source: Schmidt, L. B. et al. <a href="https://www.jove.com/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the extraction of dorsal root ganglion or DRG from a harvested rat spine for downstream applications.

Zbieranie DRG z kręgosłupa szczura: procedura ekstrakcji zwoju korzenia grzbietowego z kręgosłupa modelu szczura

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
...

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

Assays & techniques

Spinal Cord Extraction: A Protocol to Isolate Murine Spinal Cord for In Vitro Studies

Source: Na&#39;ara, S. et al. <a href="https://www.jove.com/video/52990" target="_blank">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model.</a> J. Vis. Exp. (2016)
In this video, we describe a step-by-step protocol to isolate spinal cord from a mouse model for in vitro studies.

Ekstrakcja rdzenia kręgowego: protokół izolacji mysiego rdzenia kręgowego do badań in vitro

Source: Na&#39;ara, S. et al. In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016)
In this video, we ...

In vivo studies

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

Source: Tsui, Y, et al. <a href="https://app.jove.com/v/55794">Hypoxic Preconditioning of Marrow-derived Progenitor Cells as a Source for the Generation of Mature Schwann Cells.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure for isolating dorsal root ganglion (DRG) neurons from rat embryos and co-culturing them with Schwann cell precursors to generate mature Schwann cells. The isolated DRGs are processed to isolate neurons and glial cells. The isolated cells are cultured to purify the neurons, which are then co-cultured with Schwann cell-like cells (SCLCs) in a co-culture medium. Interaction with the DRG neurons leads to the maturation of SCLCs into Schwann cells.

Izolacja neuronów DRG i kokultura z prekursorami komórek Schwanna w celu wytworzenia komórek Schwanna

Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons
1. Preparation of purified rat DRG neurons

	Autoclave all dissection tools ...

Neuronal Culture Techniques

Co-culture and Interaction Studies

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app.jove.com/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

Izolacja nerwowych komórek progenitorowych łodygi z obszaru okołokomorowego rdzenia kręgowego dorosłego szczura

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...

Primary Neuronal Culture

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

Source: Lin, Y., et al. <a href="https://app.jove.com/v/57569">Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for isolating and culturing dorsal root ganglia (DRG) cells from the rat spinal column. DRGs excised from the spinal column are treated with enzymes and mechanically dissociated to obtain a single-cell suspension. The cells are cultured in a medium containing inhibitors and growth factors that limit glial cell proliferation and promote neuronal growth, respectively.

Ustanowienie pierwotnej hodowli komórek zwojów korzenia grzbietowego z kręgosłupa szczura

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Collect Lumbar DRG from ...