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

Abstract

Source: Na'ara, S. et al. In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. 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.

Protocol

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

1. Euthanize the mouse using a CO₂ 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.
2. Soak the animal with 70% ethanol. This step is important for sterilization and for preventing the hair from dragging through the inner organs.
3. Leave the mouse to dry from ethanol. Prevent any contact of ethanol with the inner organs due to neurotoxicity.
4. After drying the mouse, position it using pins in the prone position (face down). Place the pins on each hindlimb and forelimb.
5. 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.
6. 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 7^th cervical vertebrae), using heavy forceps. Dissect the spine caudally and excise the spine with heavy forceps until reaching the lumbar level (hind limbs level).
7. Perform a complete caudal transection at the lumbar spine level (the 5^th lumbar vertebrae) using heavy forceps.
8. 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.
9. Cut down at the midline from the neck to the abdomen, retract the skin laterally and then cut to open the peritoneum.
10. Remove the inner organs inside the peritoneum (liver, spleen, pancreas, stomach, and intestine) en bloc. Next, remove the retroperitoneal organs (i.e., kidneys and pancreas). Cranially, open the chest wall.
11. 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.
12. Wash the vertebral column from blood with cold (4 °C) fresh phosphate-buffered saline (PBS) twice.

Materials

Forceps	Sigma-Aldrich	F4142
Surgical blade	Sigma-Aldrich	Z309036
Scissors	Sigma-Aldrich	S3271
35 mm Petri dishes, glass bottom	de groot	60-627860
70% ethanol	Sigma
Cold PBS	Biological industries	02-023-1A