For the present study, the animal experiments were approved by UCSF Institutional Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory animals. Adult, 8-12-week-old C57BL/6 male and female mice (in-house bred) were used here.
1. DRG sample preparation
<ol>
	<li>Anesthetize the mice with 2.5% Avertin (see Table of Materials). Ensure adequate anesthesia by the lack of response to painful stimulation. Perfuse the animals transcardially with 1x phosphate-buffered saline (PBS) followed by 4% formaldehyde, as previously described14,15,16.</li>
	<li>Dissect DRG tissue from the spinal cord, as previously described14,15,16.</li>
	<li>Post-fix the dissected DRGs in 4% formaldehyde for 3 h at room temperature.</li>
	<li>Put the DRGs in 30% sucrose in PBS at 4 &#176;C overnight.</li>
	<li>Preparing the base optimal cutting temperature (OCT)
	<ol>
		<li>Add the OCT compound (see Table of Materials) to cover the top surface of the aluminum block and freeze at -20 &#176;C for 5-10 min (Figure 1A).</li>
		<li>Cut off the top of the OCT using the cryostat (see Table of Materials) at 30 &#181;m thickness until its surface is flat as the base OCT (Figure 1B).</li>
		<li>Make a mark at the bottom (six o&#39;clock position) of the base OCT to mark its orientation, before taking the aluminum block off the cryostat (Figure 1C).</li>
		<li>In the following steps, keep the mark at the six o&#39;clock position to maintain the same orientation, which can lead to obtaining more sections of the DRG sample. 
		NOTE: If the orientation is lost and the DRG sample is cut at a different angle, the DRG sample cannot be cut completely from top to bottom, which will leave some samples left out on the base OCT and wasted, as the base OCT will be encountered while trying to cut the DRG sample (Figure 1C).</li>
	</ol>
	</li>
	<li>Performing OCT embedment of DRGs
	<ol>
		<li>Dry the DRG tissue before putting it on the OCT.
		<ol>
			<li>Before adding the DRG onto the block, ensure to remove the 30% sucrose/PBS solution around the sample.</li>
			<li>Dry the DRG sample by placing it on a dry Petri dish (Figure 2A) and moving it from one location to another two or three times with dry tweezers (Figure 2B).</li>
			<li>Dry the tweezers with lint-free tissue before moving another DRG sample (Figure 2C).</li>
		</ol>
		</li>
		<li>Put the dry DRG onto the base OCT.
		<ol>
			<li>With dry tweezers, place the dry DRG onto the base OCT at the 12 o&#39;clock position (Figure 1D).</li>
			<li>Keep at least 5 mm between the upper edge of the DRG and the upper edge of the base OCT.</li>
			<li>Put the dorsal part of the DRG at the 12 o&#39;clock position and the ventral part at the six o&#39;clock position (Figure 1D).</li>
		</ol>
		</li>
		<li>Embed the DRG tissue with OCT.
		<ol>
			<li>Add more OCT to cover the entire DRG sample in a round or oval shape, with the DRG in the center (Figure 1E).</li>
			<li>Ensure the upper edge of the OCT covers the DRG at the upper edge of the base OCT (Figure 1F), as this makes it easier to transfer the section onto the glass slide (Figure 3A). 
			NOTE: If the cover OCT is in the middle of the base OCT, the upper edge of the base OCT will block the glass slide to collect the section (Figure 3B).</li>
			<li>Freeze the cover OCT and DRG sample at -20 &#176;C for another 5 min (Figure 1G). Do not trim the OCT around the DRG sample.</li>
			<li>Cut off the top of the cover OCT at a 30 &#181;m thickness until the DRG is visible.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Cryosectioning of the DRG
<ol>
	<li>Change the cryostat section thickness to 12 &#181;m to cut DRG sections onto the slide (Figure 1H).</li>
	<li>Hold the OCT section at the bottom with a small paintbrush cooled to -20 &#176;C. 
	NOTE: It is important not to cut the whole section completely, but to leave 1-3 mm uncut (Figure 1I).</li>
	<li>Use the end of a room-temperature tweezer to gently touch the bottom (six o&#39;clock position) of the section so that it sticks to the platform surface (Figure 1J). This prevents the section from curving back in a roll, which makes it difficult to collect the section on the slide. 
	NOTE: When the bottom of the section sticks to the platform surface, and the top of the section still connects with the cover OCT, the section is in a flat shape (Figure 1K), making it much easier to collect the section on the slide.</li>
	<li>Take a charged glass slide (see Table of Materials) and slowly place the slide over the section. As soon as the section starts to stick to the slide, gently pull the slide backward (Figure 1L).</li>
	<li>Follow the same process to get more DRG sections onto the slide without overlapping the sections (Figure 1M). Ensure that a new DRG section is not placed over the OCT of another DRG section (Figure 4), as such a section cannot stay well on the slide and can be washed away during the immunochemistry staining process.</li>
</ol>
3. Nissl staining of the DRG section on the glass slide
<ol>
	<li>Wash the sections for 10 min in PBS plus 0.1% Triton X-10. Then, wash the sections twice with PBS.</li>
	<li>Apply a 1:300 Nissl stain (see Table of Materials) to the slide and incubate for 20 min at room temperature.</li>
	<li>Wash the section with PBS plus 0.1% Triton X-100. Then, wash the sections twice with PBS for 5 min, and then for 2 h at room temperature.</li>
	<li>Apply 4&#8242;,6-diamidino-2-phenylindole (DAPI) fluoromount-G (see Table of Materials) to cover the sections with a coverslip.</li>
</ol>

Mouse Dorsal Root Ganglion Cryosectioning

<ol>
	<li>Guan, Z., 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=Injured+sensory+neuron-derived+CSF1+induces+microglial+proliferation+and+DAP12-dependent+pain.">Injured sensory neuron-derived CSF1 induces microglial proliferation and DAP12-dependent pain.</a> Nature Neuroscience. 19 (1), 94-101 (2016).</li><li>Yu, X., 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=Dorsal+root+ganglion+macrophages+contribute+to+both+the+initiation+and+persistence+of+neuropathic+pain.">Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain.</a> Nature Communications. 11 (1), 264 (2020).</li><li>Costa, F. A. L., Moreira Neto, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+glial+cells+in+sensory+ganglia:+its+role+in+pain.">Satellite glial cells in sensory ganglia: its role in pain.</a> Brazilian Journal of Anesthesiology. 65 (1), 73-81 (2015).</li><li>Noguri, T., Hatakeyama, D., Kitahashi, T., Oka, K., Ito, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profile+of+dorsal+root+ganglion+neurons:+study+of+oxytocin+expression.">Profile of dorsal root ganglion neurons: study of oxytocin expression.</a> Molecular Brain. 15 (1), 44 (2022).</li><li>Su, P. P., Zhang, L., He, L., Zhao, N., Guan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neuro-immune+interactions+in+chronic+pain:+implications+for+clinical+practice.">The role of neuro-immune interactions in chronic pain: implications for clinical practice.</a> Journal of Pain Research. 15, 2223-2248 (2022).</li><li>Esposito, M. F., Malayil, R., Hanes, M., Deer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+characteristics+of+the+dorsal+root+ganglion+as+a+target+for+neuromodulation.">Unique characteristics of the dorsal root ganglion as a target for neuromodulation.</a> Pain Medicine. 20, S23-S30 (2019).</li><li>Chen, X. J., Sun, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+circuit+mechanisms+of+itch.">Central circuit mechanisms of itch.</a> Nature Communications. 11 (1), 3052 (2020).</li><li>Guan, Z., Hellman, J., Schumacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contemporary+views+on+inflammatory+pain+mechanisms:+TRPing+over+innate+and+microglial+pathways.">Contemporary views on inflammatory pain mechanisms: TRPing over innate and microglial pathways.</a> F1000Research. , (2016).</li><li>Boadas-Vaello, P., 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=Neuroplasticity+of+ascending+and+descending+pathways+after+somatosensory+system+injury:+reviewing+knowledge+to+identify+neuropathic+pain+therapeutic+targets.">Neuroplasticity of ascending and descending pathways after somatosensory system injury: reviewing knowledge to identify neuropathic pain therapeutic targets.</a> Spinal Cord. 54 (5), 330-340 (2016).</li><li>Guha, D., Shamji, 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=The+dorsal+root+ganglion+in+the+pathogenesis+of+chronic+neuropathic+pain.">The dorsal root ganglion in the pathogenesis of chronic neuropathic pain.</a> Neurosurgery. 63, 118-126 (2016).</li><li>Shorrock, H. K., 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=UBA1/GARS-dependent+pathways+drive+sensory-motor+connectivity+defects+in+spinal+muscular+atrophy.">UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy.</a> Brain. 141 (10), 2878-2894 (2018).</li><li>Sleigh, J. N., 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=Trk+receptor+signaling+and+sensory+neuron+fate+are+perturbed+in+human+neuropathy+caused+by+Gars+mutations.">Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.</a> Proceedings of the National Academy of Sciences. 114 (16), E3324-E3333 (2017).</li><li>Rubio, M. A., Herrando-Grabulosa, M., Gaja-Capdevila, N., Vilches, J. J., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+somatosensory+neuron+involvement+in+the+SOD1(G93A)+mouse+model.">Characterization of somatosensory neuron involvement in the SOD1(G93A) mouse model.</a> Scientific Reports. 12 (1), 7600 (2022).</li><li>Sleigh, J. N., West, S. J., 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+video+protocol+for+rapid+dissection+of+mouse+dorsal+root+ganglia+from+defined+spinal+levels.">A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels.</a> BMC Research Notes. 13 (1), 302 (2020).</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 Research Notes. 9, 82 (2016).</li><li>Perner, C., Sokol, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dissection+and+culture+of+murine+dorsal+root+ganglia+neurons+to+study+neuropeptide+release.">Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.</a> STAR Protocols. 2 (1), 100333 (2021).</li><li>Haberberger, R. V., Barry, C., Matusica, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+dorsal+root+ganglion+neuron+cell+lines.">Immortalized dorsal root ganglion neuron cell lines.</a> Frontiers in Cellular Neuroscience. 14, 184 (2020).</li><li>Pokhilko, A., Nash, A., Cader, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+transcriptional+signatures+of+neuropathic+pain.">Common transcriptional signatures of neuropathic pain.</a> Pain. 161 (7), 1542-1554 (2020).</li><li>Martin, S. L., Reid, A. J., Verkhratsky, A., Magnaghi, V., Faroni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+dorsal+root+ganglia+following+peripheral+nerve+injury:+roles+in+inflammation,+cell+death+and+nociception.">Gene expression changes in dorsal root ganglia following peripheral nerve injury: roles in inflammation, cell death and nociception.</a> Neural Regeneration Research. 14 (6), 939-947 (2019).</li><li>Miller, R. J., Jung, H., Bhangoo, S. K., White, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+and+chemokine+regulation+of+sensory+neuron+function.">Cytokine and chemokine regulation of sensory neuron function.</a> Handbook of Experimental Pharmacology. (194), 417-449 (2009).</li><li>Neto, 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=Axonal+outgrowth,+neuropeptides+expression+and+receptors+tyrosine+kinase+phosphorylation+in+3D+organotypic+cultures+of+adult+dorsal+root+ganglia.">Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia.</a> PLoS One. 12 (7), e0181612 (2017).</li><li>Nascimento, A. I., Mar, F. M., Sousa, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intriguing+nature+of+dorsal+root+ganglion+neurons:+Linking+structure+with+polarity+and+function.">The intriguing nature of dorsal root ganglion neurons: Linking structure with polarity and function.</a> Progress in Neurobiolology. 168, 86-103 (2018).</li><li>Middleton, S. J., Perez-Sanchez, J., Dawes, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+sensory+afferent+compartments+in+health+and+disease.">The structure of sensory afferent compartments in health and disease.</a> Journal of Anatomy. 241 (5), 1186-1210 (2022).</li><li>Nguyen, M. Q., von Buchholtz, L. J., Reker, A. N., Ryba, N. J., Davidson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-nucleus+transcriptomic+analysis+of+human+dorsal+root+ganglion+neurons.">Single-nucleus transcriptomic analysis of human dorsal root ganglion neurons.</a> eLife. 10, e71752 (2021).</li><li>Usoskin, D., 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=Unbiased+classification+of+sensory+neuron+types+by+large-scale+single-cell+RNA+sequencing.">Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.</a> Nature Neuroscience. 18 (1), 145-153 (2015).</li><li>Hunt, M. A., 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=DRGquant:+A+new+modular+AI-based+pipeline+for+3D+analysis+of+the+DRG.">DRGquant: A new modular AI-based pipeline for 3D analysis of the DRG.</a> Journal of Neuroscience Methods. 371, 109497 (2022).</li></ol>

The authors have nothing to disclose.

This protocol provides an easy step-by-step procedure for cryostat sectioning of the mouse DRG to obtain high-quality DRG sections on slides reliably.There are four critical steps in this protocol. First, the DRG sample and the tweezers must be dry before putting the DRG sample onto the base OCT. Any liquid surrounding the DRG sample will form an ice shell around it, resulting in DRG sections separating from the OCT and curving up. Second, if the aluminum block does not have a mark, or if the base OCT covers the mark, it...

The dorsal root ganglion (DRG) contains the primary sensory neurons, the tissue macrophages, and the satellite cells that surround the primary sensory neurons1,2,3,4. It is a key anatomic structure in processing innocuous and noxious signals, and plays critical roles in pain, itch, and various peripheral nerve disorders5,6,7,8,9,10,11,12,13. Although several methods have been developed to dissect DRG tissue from the mouse spinal cord14,15,16, cryosectioning of the DRG tissue remains challenging as the DRG tissue is quite small, and cryostat sections of DRG samples tend to curve into rolls, making it difficult to properly transfer the cryostat sections onto glass slides. However, proper cryosectioning of the DRG tissue is crucial for immunohistochemistry studies and the structure of DRG sensory neurons17,18,19,20,21,22,23. Moreover, as single-cell RNA sequencing results have revealed the remarkable heterogeneity of DRG sensory neurons in both humans24 and mice25, proper cryosectioning of DRG tissue is critical for investigating the functional role of different DRG cells in various physiological and pathological conditions.
Although the tissue-clearing technique has been applied to investigate the 3D reconstruction of the DRG26 as an alternative technique of cryosectioning the DRG, the tissue-clearing technique is time- and labor-consuming. In comparison, cryosectioning of the DRG is quick and relatively easy to perform, and thus it remains to be a key technique for immunohistochemistry and structure studies of the DRG and other regions of the central nervous system. However, obtaining high-quality, intact, and flat cryostat sections onto glass slides remains to be a challenge in neuroscience research because of the tiny sample size of tissues, like the DRG and certain brain regions, and there is no article describing the optimal protocol at this point for cryosectioning small-sized tissue samples, such as mouse DRGs.
This protocol provides an easy, step-by-step technique for cryostat sectioning of the mouse DRG to reliably obtain as many high-quality DRG sections on the slides for subsequent DRG studies. While specifically designed for cryosectioning DRG samples, this technique can potentially be used for cryosectioning various other tissues with a small sample size.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Avertin</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-25G</td>
			<td>Anesthetize animal</td>
		</tr>
		<tr>
			<td>Epredia&#160;Cryotome Cryostat Cryocassettes, 25 mm dia. Crosshatched</td>
			<td>Fisherbrand</td>
			<td>1910</td>
			<td>Hold the OCT section at the bottom&#160;</td>
		</tr>
		<tr>
			<td>Ergo Tweezers</td>
			<td>Fisherbrand</td>
			<td>S95310</td>
			<td>Using the end of a tweezer to gently touch the bottom (6 o&#8217;clock) of the section so that it sticks to the platform surface to prevent the section from curving back in a roll&#160;</td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>1255015</td>
			<td>To collect the DRG section&#160;</td>
		</tr>
		<tr>
			<td>Marking pens</td>
			<td>Fisherbrand</td>
			<td>133794</td>
			<td>&#160;Mark the orientation of base OCT</td>
		</tr>
		<tr>
			<td>Scigen&#160;Tissue-Plus O.C.T. Compound</td>
			<td>Fisherbrand</td>
			<td>&#160;23730571</td>
			<td>Embedding medium for frozen tissue specimens to ensure optimal cutting temperature (O.C.T.).</td>
		</tr>
	</tbody>
</table>

a procedure for mouse dorsal root ganglion cryosectioning

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of inflammatory and neuropathic pain, itch, as well as other peripheral neurological conditions. However, it remains a challenge to consistently obtain high-quality, intact, and flat cryostat sections onto glass slides because of the tiny sample size of the DRG tissue. So far, there is no article describing an optimal protocol for DRG cryosectioning. This protocol presents a step-by-step method to resolve the frequently encountered difficulties associated with DRG cryosectioning. The presented article explains how to remove the surrounding liquid from the DRG tissue samples, place the DRG sections on the slide facing the same orientation, and flatten the sections on the glass slide without curving up. Although this protocol has been developed for cryosectioning the DRG samples, it can be applied for the cryosectioning of many other tissues with a small sample size.

The current study collected about 16 continuous, high-quality DRG sections from one mouse L4 DRG. The obtained sections were without any distortion. Figure 1 depicts the step-by-step procedure for the cryosectioning. The removal of extra liquid from the tissue sections is shown in Figure 2. The process of OCT embedment of the tissues is highlighted in Figure 3. Figure 4 shows the proper placement of the...

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Author Spotlight: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning  

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...

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Neuroscience

5.8K Views.  Capital Medical University. Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

Video: Author Spotlight: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning  

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 ...

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 ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

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 ...

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 ...

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 ...

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 ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...