Name: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning (Video) | JoVE
Uploaded: 2023-06-09T13:50:00
Description: Watch this Scientific Journal Video about A Procedure for Mouse Dorsal Root Ganglion Cryosectioning at JoVE.com

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 stimulati...

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>

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.

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

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...

Watch this Scientific Journal Video about A Procedure for Mouse Dorsal Root Ganglion Cryosectioning at JoVE.com

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning (Video) | JoVE 

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

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