spinal cord extraction: a protocol to isolate murine spinal cord for in vitro studies

Spinal Cord Extraction: A Protocol to Isolate Murine Spinal Cord for In Vitro Studies

After euthanizing the animal in a CO2 chamber, soak the fur with 70% ethanol and allow it to dry. Pin the mouse in a prone position and then make a midline incision extending craniocaudally from the posterior neck to the lumbar spine.
Next, use forceps to raise the dermal flaps bilaterally and expose the subcutaneous tissue. Palpate the mouse skull until the craniocervical junction is identified. Use scissors angled perpendicular to the animal to cut through the cervical muscles and spine at the craniocervical junction. Then, dissect the spine caudally and use scissors to perform a complete caudal transection at the fifth lumbar vertebrae.
Next, position the mouse in a supine position and, after making an incision along the midline from the neck to the abdomen, retract the skin laterally and then cut open the peritoneum.
Cranially, open the chest wall with scissors. After removing the peritoneal and retroperitoneal organs, use forceps and a surgical blade to cut the ribs, leaving approximately 5 millimeters of ribs protruding from the vertebral column. Remove the vertebral column from the rest of the body and wash twice with cold PBS.

spinal-cord-extraction-protocol-to-isolate-murine-spinal-cord-for

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

In vivo studies

EoE Sub Articles

eoe sub articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Harvesting the Spinal Cord
<ol>
	<li>Euthanize the mouse using a CO2&#160;chamber. Avoid cervical dislocation as it might cause damage to the ganglion roots due to shear forces. From here on, perform all steps under sterile conditions.</li>
	<li>Soak the animal with 70% ethanol. This step is important for sterilization and for preventing the hair from dragging through the inner organs.</li>
	<li>Leave the mouse to dry from ethanol. Prevent any contact of ethanol with the inner organs due to neurotoxicity.</li>
	<li>After drying the mouse, position it using pins in the prone position (face down). Place the pins on each hindlimb and forelimb.</li>
	<li>Using forceps, make a midline incision extending craniocaudally from the posterior neck to the lumbar spine. Next, raise dermal flaps bilaterally using forceps and expose subcutaneous tissue.</li>
	<li>Palpate mouse skull until reaching the craniocervical junction. Using scissors perpendicular to the animal, cut through the cervical muscles and spine at the craniocervical junction (the 7th&#160;cervical vertebrae), using heavy forceps. Dissect the spine caudally and excise the spine with heavy forceps until reaching the lumbar level (hind limbs level).</li>
	<li>Perform a complete caudal transection at the lumbar spine level (the 5th&#160;lumbar vertebrae) using heavy forceps.</li>
	<li>Roll the mouse over (face up). There is no need to cover the incision since the DRG is extracted from lower vertebral levels and not the cervical.</li>
	<li>Cut down at the midline from the neck to the abdomen, retract the skin laterally and then cut to open the peritoneum.</li>
	<li>Remove the inner organs inside the peritoneum (liver, spleen, pancreas, stomach, and intestine)&#160;en bloc. Next, remove the retroperitoneal organs (i.e., kidneys and pancreas). Cranially, open the chest wall.</li>
	<li>Using forceps and a surgical blade, cut the ribs, leaving 5 mm of ribs bilaterally away from the vertebral column. At this point, the vertebral column is separated from the rest of the body.</li>
	<li>Wash the vertebral column from blood with cold (4 &#176;C) fresh phosphate-buffered saline (PBS) twice.</li>
</ol>

<table><tbody><tr><td>Forceps&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F4142</td><td></td></tr><tr><td>Surgical blade&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>Z309036</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S3271</td><td></td></tr><tr><td>35 mm Petri dishes, glass bottom&amp;nbsp;</td><td>de groot&amp;nbsp;</td><td>60-627860</td><td></td></tr><tr><td>70% ethanol&amp;nbsp;</td><td>Sigma</td><td></td><td></td></tr><tr><td>Cold PBS&amp;nbsp;</td><td>Biological industries&amp;nbsp;</td><td>02-023-1A</td><td></td></tr></tbody></table>

Source: Na&#39;ara, S. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52990" target="_blank">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model.</a> J. Vis. Exp. (2016)
In this video, we describe a step-by-step protocol to isolate spinal cord from a mouse model for in vitro studies.

Source: Na&#39;ara, S. et al. In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016)
In this video, we ...

For spinal cord extraction, begin with a euthanized mouse in the prone position. Spray ethanol to prevent any potential contamination from fur.
Cut through the midline from the head to the tail and open the skin to reveal the underlying tissue. Examine the mouse&#8217;s skull to locate the craniocervical junction that connects the skull to the cervical spine.
Next, incise the craniocervical junction perpendicularly to sever the connection between the head and neck. Dissect the spine till the fifth lumbar vertebra. Subsequently, cut the spine at the fifth lumbar vertebra to sever the connection from the lower vertebral levels.
Now, turn the mouse to the supine position. Slit the peritoneum and the chest wall to expose both the abdominal and thoracic cavity. Remove the peritoneal and retroperitoneal organs to access the vertebral column.
Cut the ribs to separate the vertebral column from the rest of the body. Last, rinse the isolated spinal cord with a cold saline solution to wash away any blood contaminants.

2.8K visualizações. Extração da medula espinhal: um protocolo para isolar a medula espinhal murina para estudos in vitro

Spinal Cord Extraction: A Protocol to Isolate Murine Spinal Cord for In Vitro Studies

Transcrição

Spinal Cord Extraction: A Protocol to Isolate Murine Spinal Cord for In Vitro Studies