harvesting dorsal root ganglion from rat spine

Harvesting Dorsal Root Ganglion from Rat Spine

Clean the dorsal skin of a euthanized rat with 70% ethanol and use scissors to remove the spine. Separate the cervical, thoracic, and lumbar regions, and place the spine sections in a 10-centimeter culture dish with PBS to keep the tissue samples hydrated.
Using delicate bone scissors, open the thoracic and cervical vertebral bones in the dorsal and ventral aspects to separate the spine into two lateral halves, and place the tissue sections into a clean 10-centimeter dish with fresh PBS. Using forceps, gently detach the spinal cord from the vertebral bones to visualize the dorsal root ganglions.
Placing the tips of a pair of fine forceps held under each ganglion, grasp the tissue and pull it from the bone cavity in which it is lodged. Immediately after harvesting, place each ganglion into DMEM culture medium supplemented with 2% Penicillin and Streptomycin or Pen/Strep and 10% Fetal Bovine Serum or FBS to help prevent bacterial contamination of the nerve tissues.

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JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

Assays & techniques

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

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Harvest and Preparation of DRGs (estimated timing: 2 h, day 8)
NOTE: Experiments with mice and rats require approval from the IACUC. In some countries, the use of chicken eggs also requires approval.
<ol>
	<li>Get six to seven week-old Sprague Dawley rats (~200 g in weight) to extract DRGs. 
	NOTE: One rat should yield ~40 cervical and thoracic DRGs. Mouse DRG also integrates in the CAM; however, the conditions for this species need to be optimized independently.</li>
	<li>In a laminar flow cabinet, harvest DRGs from cervical and thoracic regions following the protocol for mouse DRG extraction published elsewhere. Follow Figure 1 for orientation on how to harvest DRGs.
	<ol>
		<li>Euthanize the rat by cardiac puncture after administration of Ketamine/ Xylazine injected intraperitoneally. Clean the rat skin with 70% ethanol and remove the rat spine using a scissor. Do not perform cervical dislocation because this would damage the cervical DRGs.</li>
		<li>Separate the cervical, thoracic, and lumbar regions with the same scissor, following the schematic anatomic representation and gross images provided in the Figure 1A-D. Place the spine sections in a 10 cm culture dish with 1X PBS to keep tissues wet.</li>
		<li>With a delicate bone scissor, open the vertebral bones in the dorsal and ventral aspects, separating the spine in two lateral halves (Figure 1E-F).&#160; Place the tissue sections in a clean 10 cm dish with fresh 1X PBS.</li>
		<li>Using forceps, gently detach the spinal cord from the vertebral bones to visualize the DRGs (Figure 1G).</li>
		<li>With fine forceps held underneath each DRG, grasp it and pull it out from the bone cavity in which it is lodged. Do not hold the DRG directly because this will cause tissue damage. Do not trim the axon bundles from the DRG (Figure 1H). 
		NOTE: Avoid using lumbar DRGs since these have reduced integration in the CAM. For DRG region location, follow the schematic illustration and gross anatomic images in Figure 1A-D.</li>
	</ol>
	</li>
	<li>Immediately after harvesting, place each DRG into DMEM culture medium supplemented with 2% Penicillin/Streptomycin (Pen/Strep) and 10% heat-inactivated Fetal Bovine Serum (FBS) to help prevent bacterial contamination of the DRGs. Group all DRGs in the same 6 cm culture dish with 4 mL of culture medium.</li>
	<li>After harvesting all DRGs, transfer them to a new culture dish with DMEM culture medium supplemented with 2% Pen/Strep plus 10% FBS and containing 1.25 &#181;g/mL of red fluorescent dye. Incubate for 1 h in the cell culture incubator.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PBS (1x) pH 7.4</td>
			<td>Gibco</td>
			<td># 10010-023</td>
			<td>Phosphate Buffered Saline</td>
		</tr>
		<tr>
			<td>Sprague Dawley rats (females)</td>
			<td>Charles River laboratories</td>
			<td>Strain code: 001</td>
			<td>6-7 weeks old (190-210g in weight)</td>
		</tr>
		<tr>
			<td>Dumont # 5 fine forceps</td>
			<td>Fine Science Tools (FST)</td>
			<td># 11254-20</td>
			<td>Used to harvest DRG</td>
		</tr>
		<tr>
			<td>Extra fine Graefe forceps, curved</td>
			<td>Fine Science Tools (FST)</td>
			<td># 11151-10</td>
			<td>Used to graft DRG onto the CAM on day 8 and to harvest CAM tissue on day 17</td>
		</tr>
		<tr>
			<td>Extra fine Graefe forceps, straight</td>
			<td>Fine Science Tools (FST)</td>
			<td># 11150-10</td>
			<td>Used to graft DRG onto the CAM on day 8 and to harvest CAM tissue on day 17</td>
		</tr>
		<tr>
			<td>DMEM (1x)</td>
			<td>Gibco</td>
			<td># 11965-092</td>
			<td>Dulbeecco`s Modified Eagle Medium</td>
		</tr>
		<tr>
			<td>CellTracker Red CMTPX fluorescent dye</td>
			<td>Life Technologies</td>
			<td># C34552</td>
			<td>Reconstitute 50&#181;g in 40&#181;L of DMSO and stock at -20oC. Use 1&#181;L of stock solution/mL of culture medium</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td># 15140-122</td>
			<td>10,000 Units/mL Penicilin, 10,000 &#181;g/mL Streptomycin</td>
		</tr>
		<tr>
			<td>HI FBS</td>
			<td>Gibco</td>
			<td># 10082-147</td>
			<td>Heat-inactivated Fetal Bovine Serum</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Bioreagents</td>
			<td># BP231-100</td>
			<td>Dimethyl Sulfoxide</td>
		</tr>
	</tbody>
</table>

harvesting drg from rat spine: a procedure to extract dorsal root ganglion from the spine of a rat model

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

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20476/20476fig1.jpg" /> 
Figure 1: DRG extraction on day 8. A. Rat schematic illustrating the anatomical location of the spine. B. Diagram of the rat vertebrae configuration showing different body regions; green for cervical, dark blue for thoracic, orange for lumbar and light blue for sacral vertebrae. C-D. Ventral aspect of the rat spine after surgical exci...

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

The dorsal root ganglia or DRG are clusters of cell bodies of sensory neurons lodged within the vertebrae on either side of the spinal cord. Their primary function is to transmit sensory neuronal signals from the peripheral nervous system to the central nervous system.
To isolate the dorsal root ganglia, begin by taking a freshly harvested rat spine. Dissect the spine to separate the cervical, thoracic, and lumbar regions. Keep the spine sections in a&#160; suitable buffer to ensure tissue hydration.
Now, position the thoracic section with its dorsal side facing up. Divide the section into two lateral halves. Next, secure the dissected half of the spine segment, medial side up. Gently detach the bisected spinal cord - a long, tubular structure - from the segmented vertebral bones of the section to reveal the underlying DRG.
The DRG appear as distinct white masses with dorsal and ventral roots embedded within the spinal canal. With fine forceps, carefully pull out the DRG to dislodge it from the spinal canal. Finally, transfer the harvested DRG into a suitable media for further downstream applications.

harvesting-drg-from-rat-spine-procedure-to-extract-dorsal-root

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

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Harvesting Dorsal Root Ganglion from Rat Spine