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>

<ol>
	<li>Malin, S. A., Davis, B. M., Molliver, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+dissociated+sensory+neuron+cultures+and+considerations+for+their+use+in+studying+neuronal+function+and+plasticity.">Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity.</a> Nat Protoc. 2 (1), 152-160 (2007).</li><li>Sleigh, J. N., Weir, G. A., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+step-by-step+dissection+protocol+for+the+rapid+isolation+of+mouse+dorsal+root+ganglia.">A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia.</a> BMC Res Notes. 9, 82 (2016).</li><li>Kennedy, H. S., Jones, C., Caplazi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+standard+laminectomy+with+an+optimized+ejection+method+for+the+removal+of+spinal+cords+from+rats+and+mice.">Comparison of standard laminectomy with an optimized ejection method for the removal of spinal cords from rats and mice.</a> J Histotechnol. 36 (3), 86-91 (2013).</li><li>Meikle, A. D., Martin, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+removal+of+the+spinal+cord.">A rapid method for removal of the spinal cord.</a> Stain Technol. 56 (4), 235-237 (1981).</li><li>Gallagher, S., Chakavarti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblot+analysis.">Immunoblot analysis.</a> J Vis Exp. (16), (2008).</li><li>Ogrean, C., Jackson, B., Covino, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+real-time+PCR+using+the+thermo+scientific+Solaris+qPCR+assay.">Quantitative real-time PCR using the thermo scientific Solaris qPCR assay.</a> J Vis Exp. (40), (2010).</li><li>. Immunohistochemistry Protocol for Parraffin-embedded Tissue Sections - ADVERTISEMENT Available from: <a target="_blank" href="https://www.jove.com/video/5064/immunohistochemistry-protocol-for-paraffin-embedded-tissue-sections">https://www.jove.com/video/5064/immunohistochemistry-protocol-for-paraffin-embedded-tissue-sections</a> (2014)</li><li>Watson, C., Paxinos, G., Puelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Nervous System. , 424-426 (2012).</li><li>Rigaud, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+and+strain+differences+in+rodent+sciatic+nerve+anatomy:+implications+for+studies+of+neuropathic+pain.">Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain.</a> Pain. 136 (1-2), 188-201 (2008).</li><li>Green, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+non-genetic+factors+which+influence+the+type+of+the+skeleton+in+an+inbred+strain+of+mice.">Genetic and non-genetic factors which influence the type of the skeleton in an inbred strain of mice.</a> Genetics. 26 (2), 192-222 (1941).</li><li>Meyer, A., Gallarda, B. W., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+electrophysiology.">Spinal cord electrophysiology.</a> J Vis Exp. (35), (2010).</li><li>Ciglieri, E., Ferrini, F., Boggio, E., Salio, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+in+vitro+morphofunctional+analysis+of+mouse+dorsal+root+ganglia.">An improved method for in vitro morphofunctional analysis of mouse dorsal root ganglia.</a> Ann Anat. , 62-67 (2016).</li><li>Peeraer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+evaluation+of+rat+dorsal+root+ganglion+neurons+as+an+in+vitro+model+for+diabetic+neuropathy.">Pharmacological evaluation of rat dorsal root ganglion neurons as an in vitro model for diabetic neuropathy.</a> J Pain Res. 4, 55-65 (2011).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;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&#34;, 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

Research

JoVE Journal

Neuroscience

47.6K 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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

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 ...