We would like to thank Andrea Tedeschi for help in setting the initial ChIP experiments in the laboratory and Ricco Lindner
 for his contribution to fine tune the conditions for ChIP. This work was supported by the Hertie Foundation; the Fortune Grant, University of 
 Tuebingen, and the DFG DI 1497/1-1 grants (all granted to Simone Di Giovanni).

1. Sciatic &amp; dorsal column nerve injury
<ol>
 <li>The animal is placed on a surgical towel and underneath it a thermopad is present throughout the procedure keeping the body temperature of the mouse at 37&deg;C. All animals are anesthetized for surgery with a continuous isoflurane/O2 administration. Surgical instruments are autoclaved before the procedure.</li>
 <li>For sciatic injury, both hindquarters are carefully shaved, and depilation is completed with generic hair remover prior to cleansing skin with alcohol followed by Betadyne 3 times.</li>
 <li>The sciatic nerve is exposed by making a 4 mm incision posterior and parallel to the femur at mid-thigh through the skin and by spreading the muscles apart of the biceps femoris with fine forceps. </li>
 <li>The exposed sciatic nerve is then injured , and the skin is closed with two suture clips. </li>
</ol>
NOTE: The same incision is made to expose the sciatic nerve, but the wound is sutured closed leaving the nerve intact for the sham injury.
<ol start="5">
 <li>For dorsal column injury, the back of the mouse is carefully shaved, and depilation is completed with generic hair remover prior to cleansing the skin with alcohol spray/swipe. </li>
 <li>The spinal cord is exposed by making a 2.5 cm incision from about T7 to T13. The skin is spread apart, and the connective tissue is removed along the vertebra from T9 to T11. </li>
 <li>A laminectomy is performed at T10 level</li>
</ol>
NOTE: For most experiments, the laminectomy is considered the Sham injury.
<ol start="8">
 <li>A few drops of Xylocain are added to the tissue to anaesthetize the cord, and the dura mater is removed, paying attention not to touch the spinal cord. </li>
 <li>The dorsal columns are injured and the muscle is sutured close, again paying attention not to touch the spinal cord. Finally, the skin is sutured closed with suture clips. Buprenorphine (0.1 mg/kg body weight) should be administered twice daily for 48 hours or until no longer needed.</li>
</ol>
2. Cross-linking
<ol>
 <li>Dissect L4 and L5 DRG from injured mice after euthanization. Collect a total of 16 DRG into ice-cold HBSS + protease inhibitors cocktail (2 injured and 2 sham DRG from each animal for a total of 4 animals).</li>
 <li>Briefly centrifuge the DRG, then carefully remove the HBSS and add 500 &mu;l of 1% formaldehyde in PBS + protease inhibitors cocktail and incubate the sample for 30 minutes at 37&deg;C.</li>
</ol>
CRITICAL STEP. Use fresh, molecular biology grade formaldehyde.
<ol start="3">
 <li>Add 125 mM of glycine to stop the fixation and incubate for 5 minutes at room temperature.</li>
 <li>Briefly centrifuge, aspirate off buffer, and wash the tissue twice with 500 &mu;l ice-cold PBS + protease inhibitors cocktail.</li>
</ol>
3. Nuclei preparation and chromatin shearing
<ol>
 <li>Aspirate off PBS, add 400 &mu;l of SDS lysis buffer, transfer to a pre-chilled microcentrifuge tube, and disrupt the tissue with approximately 30 strokes with the micropestle.</li>
</ol>
CRITICAL STEP. Lysis and disruption of the tissue are vital for a good yield of crosslinked chromatin.
<ol start="2">
 <li>Sonicate the sample with 8 pulses, 10 second each at 70% output (Bandelin, Sonoplus GM70).</li>
</ol>
CAUTION. The shearing of the chromatin must be optimized for your particular sonication set up and proper fragmentation of the chromatin should be checked before running the actual IP experiment (see section 3).
NOTE. Fragmentation of the chromatin could also be performed by micrococcal nuclease (MNase) digestion. As a rule of thumb, fragmentation by sonication is preferred for fixed tissue, while MNase digestion is favored for native tissue chromatin immunoprecipitation. Cross-linked, sheared chromatin can be stored at -80&deg; C for up to 2 months, but avoid repeated freeze/thaw cycles.
4. Analysis of chromatin digestion (recommended)
<ol>
 <li>Remove 10 &mu;l of your sheared chromatin sample for analysis. Add 200 mM of NaCl to the sample and reverse the cross-link by incubating at 65&deg; C for 2h.</li>
 <li>Purify DNA (see section 8) and run a 1% agarose gel. DNA should be fragmented to a length of approximately 200-1000 bp (Figure 1).</li>
</ol>
CAUTION. Over-fragmentation of chromatin may lead to decreased signal, while incomplete fragmentation will lead to diminished resolution and increased background.
5. Immunoprecipitation
<ol>
 <li>Determine the number of immunoprecipitation reactions (see NOTE below), and divide samples equally into new tubes. Bring the volume of each up to 500 &mu;l with ChIP buffer + protease inhibitors cocktail.</li>
 <li>Remove 5 &mu;l of your diluted sample and transfer into a new tube. This is your 1% input sample and it will be stored at -20&deg; C until needed (Section 7).</li>
 <li>For each immunoprecipitation, add appropriate antibody or normal IgG control and incubate at 4&deg;C overnight with rotation.</li>
</ol>
NOTE. When determining the number of immunoprecipitation, a positive control (e.g. Histone H3 antibody) and a negative control (Normal IgG) should be considered. The quantity of antibody used for each IP varies between antibodies and must be empirically determined. However, it is usually within a range of 2-10 ?g/immunoprecipitation.
<ol start="4">
 <li>To immunoprecipitate your antibody/protein/DNA complex, add 30 &mu;l of ChIP Grade Protein G Magnetic Beads and incubate for 2 h at 4&deg;C with rotation.</li>
</ol>
6. Washing
<ol>
 <li>To pull-down bound chromatin-bead complex, place the tube on the magnetic rack. Wait until the solution is clear and then carefully remove your supernatant.</li>
 <li>Add 1 ml of low salt wash (ChIP buffer) to the beads and incubate at 4&deg;C for 3-5 minutes with rotation, then pull-down beads and aspirate off wash as in step 6.1. Repeat this step for a total of 3 washes.</li>
 <li>Add 1 ml of high salt wash buffer (ChIP buffer+ 350 mM NaCl) to the beads and incubate for 3-5 minutes with rotation, then pull-down beads and aspirate off wash as in step 6.1. </li>
</ol>
7. Elution and reversal of cross-linking
<ol>
 <li>Take your input samples out of the -20&deg;C, add 150 &mu;l of ChIP Elution buffer and set aside at room temperature until Step 7.5.</li>
 <li>Add 150 &mu;l of 1x ChIP Elution buffer for each IP sample from Step 6.3.</li>
 <li>Place the tubes in a thermomixer and elute the chromatin from the beads by incubating samples at 65&deg;C for 30 minutes with gentle vortexing.</li>
 <li>Pull-down beads on the magnetic rack, and carefully transfer the eluted chromatin (supernatant) into new tubes.</li>
 <li>To all tubes, including your input samples, add 200mM of NaCl and 40 &mu;g/reaction of Proteinase K and incubate at 65&deg;C for 2 hours.</li>
</ol>
NOTE. The elution step can be performed at room temperature, too, but it might be not as efficient.
8. DNA recovery
<ol>
 <li>Add 1 volume of Phenol/Chloroform to your samples from Step 7.5 and vortex for 30 seconds.</li>
 <li>Centrifuge at maximum speed in a microcentrifuge at room-temperature for 5 minutes.</li>
 <li>Carefully recover the aqueous (upper) phase into new tubes, add 1 volume of chloroform to the aqueous phase, and vortex for 30 seconds.</li>
 <li>Centrifuge at maximum speed in a microcentrifuge at room-temperature for 5 minutes.</li>
 <li>Carefully recover the aqueous (upper) phase into new tubes, then add 300mM of NaOAc, 20 &mu;g/reaction of glycogen, and 2.5 volumes of ice-cold 100% ethanol.</li>
</ol>
CAUTION. The addition of a carrier, such as the glycogen, is needed to enhance the precipitation of relatively small sized DNA fragments.
<ol start="6">
 <li>Incubate at -80&deg;C for 2 hours to precipitate DNA.</li>
</ol>
NOTE. The precipitation step can also be performed overnight at -20&deg;C.
<ol start="7">
 <li>Centrifuge the samples at maximum speed in a microcentrifuge for 20 minutes at 4&deg;C.</li>
 <li>Carefully remove the supernatant and wash the precipitated DNA pellets with 300 &mu;l of ice-cold 70% ethanol.</li>
 <li>Centrifuge the samples at maximum speed in a microcentrifuge for 10 minutes at 4&deg;C.</li>
 <li>Carefully remove as much supernatant as possible and let the pellets air-dry. </li>
 <li>Resuspend in 20 &mu;l of sterile H2O. Samples are now ready for PCR, or the DNA can be stored at -20&deg;C.</li>
</ol>
9. Representative Results
A representative result of a ChIP experiment in DRGs following sciatic lesion is shown (Figures 1 and 2). First, we show 
fragmented DNA to a length of approximately 200-1000 bp (Figure 1). Second, we demonstrate a PCR signal following ChIP from the proximal promoter 
of the growth associated protein-43 (GAP-43) only upon sciatic nerve injury (lane 4, Figure 2, 5'). No PCR signal is present when the animal 
receives a sham injury only (lane 3), and when using normal IgG serum for the IP (lane 5 and 6). To test for the specificity of the antibody, 
we analyzed the same DNA samples, but used a control primer set which detects a region in the 3'-UTR of our gene of interest where no occupancy is 
expected. PCR signals are absent from all lanes (lanes 3-6, Figure 2, 3'), except for the input signal, representing non-IP DNA samples as standard 
PCR controls (lane 1-2, Figure 2). This ChIP procedure can be performed following either sciatic or spinal dorsal column injury as summarized in 
a schematic (Figure 3).
<img src="/files/ftp_upload/2803/2803fig1.jpg" alt="Figure 1" /> 
Figure 1. Agarose gel of reversed cross-linked DNA that has been sheared by sonication to the proper range of fragment lengths.
<img src="/files/ftp_upload/2803/2803fig2.jpg" alt="Figure 2" /> 
Figure 2. Semi-quantitative PCR results from DRG tissue following sciatic nerve lesion showing that acetylated p53 binds to the GAP-43 proximal promoter region 48 hours after injury to the sciatic nerve. Control PCR from the same DRG tissue show that acetylated p53 does not bind to a control region of DNA located in the 3&#x2019;-untranslated region (UTR) of the GAP-43 gene (S = Sham, I = Injured).
<img src="/files/ftp_upload/2803/2803fig3.jpg" alt="Figure 3" /> 
Figure 3. A general diagram showing the location of the lesion sites on the sciatic nerve, and the dorsal column.

<ol>
	<li>Beirowski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+analysis+of+Wallerian+degeneration+using+restricted+axonal+labelling+in+YFP-H+mice.">Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice.</a> Journal of Neuroscience Methods. 134, 23-35 (2004).</li><li>Carey, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatin immunoprecipitation (ChIP). , (2009).</li><li>Dahl, J. A. <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+micro+chromatin+immunoprecipitation+assay+(&amp;mu;ChIP.">A rapid micro chromatin immunoprecipitation assay (&amp;mu;ChIP.</a> Nat. Protoc. 3, 1032-1045 (2008).</li><li>Haring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.+Plant+Methods.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods.</a> 11, 3-11 (2007).</li><li>Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Makwana, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+successful+peripheral+regeneration.">Molecular mechanisms in successful peripheral regeneration.</a> Febs J. 272, 2628-2638 (2005).</li><li>Tedeschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+p53-CBP/p300+transcription+module+is+required+for+GAP-43+expression,+axon+outgrowth,+and+regeneration.">A p53-CBP/p300 transcription module is required for GAP-43 expression, axon outgrowth, and regeneration.</a> Cell Death and Differentiation. 16, 543-554 (2009).</li><li>Teng, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regeneration+in+adult+CNS+neurons--signaling+molecules+and+pathways.">Axonal regeneration in adult CNS neurons--signaling molecules and pathways.</a> J Neurochem. 96, 1501-1508 (2006).</li></ol>

No conflicts of interest declared.

This protocol provides a method to directly ask about the chromatin environment during axonal regeneration in the adult 
nervous system following axonal injury. It incorporates the DRG injury model with chromatin immunoprecipitation to probe the transcriptional and 
epigenetic environment subsequent to injury to either the PNS or CNS. It is particularly useful for investigators who would like to characterize 
putative binding sites for their favorite transcription factor, and to determine whether the occupancy of these sites occurs in response to injury.
 Epigenetic modification of histones and DNA at these sites can also be simultaneously monitored. A similar protocol can be performed following 
 facial nerve lesion, where the facial nerve nuclei can be dissected from the brainstem and processed by ChIP as we previously reported (Tedeschi 
 et al., 2009).
As typical for chromatin immunoprecipitation assays, two critical steps in the protocol are, (1) efficient fragmentation
 and solubilization of the DNA-protein complex, and (2) the availability of a good immunoprecipitation antibody for your protein of interest. 
 One limitation that might confound these two steps is the low level of starting material. Compared to other structures such as the brain or 
 spinal cord, DRG provide a relatively small amount of tissue. In addition, DRG consists of a mix population of neurons as well as glial cells, all
 of which might have different chromatin environments. This may lead to heterogeneous IP signals; however this caveat is present in most samples 
 taken from the nervous system. A potential solution is to perform ChIP after fluorescent activated cell sorting of transgenically fluorescently 
 labeled DRG neurons, (see for example YFP-H mice, Bogdan et al., 2004), or immuno-isolation of neurons via magnetic beads (Lee et al., 2005). 
 However, these two approaches need to be validated for ChIP in DRGs and will likely need an increased amount of starting material.
 
We have successfully used this method to identify binding sites for several transcription factors and coactivators at the 
promoter of known regeneration associated genes, and we have used both semi-quantitative and quantitative PCR methods to detect the 
immunoprecipitated DNA. One future addition to this protocol could be the use of tiled microarrays following ChIP (ChIP-on-chip), as means to 
detect the final DNA signal. ChIP-on-chip would greatly increase the number of identified transcription sites via a high throughput unbiased 
approach. It could also allow for the study of how two or more transcription factor might cooperatively interact on a genomic level.

<table border="1">
	<tbody>
		<tr>
			<th>Reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>10x ChIP Buffer</td>
			<td>Cell Signaling</td>
			<td>7008</td>
		</tr>
		<tr>
			<td>2x ChIP Elution Buffer</td>
			<td>Cell Signaling</td>
			<td>7009</td>
		</tr>
		<tr>
			<td>ChIP Grade Protein G Magnetic Beads</td>
			<td>Cell Signaling</td>
			<td>9006</td>
		</tr>
		<tr>
			<td>Magna Grip Rack (8 well)</td>
			<td>Millipore</td>
			<td>20-400</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>MERCK</td>
			<td>UN 1888</td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>ROTH</td>
			<td>CP10.1</td>
		</tr>
		<tr>
			<td>10x Glycine Solution</td>
			<td>Cell Signaling</td>
			<td>7005</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Sigma</td>
			<td>G1767</td>
		</tr>
		<tr>
			<td>10x HBSS</td>
			<td>Gibco</td>
			<td>14185</td>
		</tr>
		<tr>
			<td>Histone H3 antibody (rabbit)</td>
			<td>Cell Signaling</td>
			<td>2650</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG</td>
			<td>Cell Signaling</td>
			<td>2729</td>
		</tr>
		<tr>
			<td>Phenol/Chloroform/Isoamyl Alcohol</td>
			<td>ROTH</td>
			<td>A156.1</td>
		</tr>
		<tr>
			<td>Protease Inhibitors Cocktail Tablets</td>
			<td>Roche</td>
			<td>04 693 116 001</td>
		</tr>
		<tr>
			<td>Proteinase K (20 mg/ml)</td>
			<td>Cell Signaling</td>
			<td>10012</td>
		</tr>
		<tr>
			<td>SDS Lysis Buffer</td>
			<td>Upstate</td>
			<td>20-163</td>
		</tr>
	</tbody>
</table>
<table border="1">
	<tbody>
		<tr>
			<th>Equipment needed</th>
		</tr>
		<tr>
			<td>Sonicator</td>
		</tr>
		<tr>
			<td>Micropestle</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury at JoVE.com

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

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 ...

chromatin-immunoprecipitation-from-dorsal-root-ganglia-tissue

Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.1K Views.  University of Tuebingen. 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.

Video: Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

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 ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

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 ...

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.

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 ...

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 ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

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 ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

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 ...