A Minimal Spinal Cord Injury Model to Study Neural Stem Cell Activation and Migration

Abstract

Source: Lakshman, N., et al. A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury. J. Vis. Exp. (2018).

This video demonstrates the activation and migration of neural stem cells (NSCs) in a mouse spinal cord injury model. To expose the spinal cord, a vertebra from the thoracic region of the spine is removed from an anesthetized mouse. A superficial injury is then created in the white matter of the spinal cord using a bent needle, which preserves the central canal. Following the injury, NSCs surrounding the canal are activated and migrate to the injury site to assist tissue repair.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Minimal Spinal Cord Injury Surgery

NOTE: Prior to surgery make sure that all surgical instruments and materials are sterilized by appropriate methods (Figure 1A).

1. Build a thoracic support arch by rolling up 4–5 squares of gauze and taping them together in the middle to obtain a fixed roll of gauze.
2. Place the mouse in the induction chamber and initiate anesthesia with 5% isoflurane. Confirm the absence of a toe pinch reflex before proceeding, as well as throughout the procedure. As the surgery can take upwards of 30 min to complete, optimize for the minimal isoflurane exposure (5 min at 5% and the remaining time at 2–3%).
3. Transfer the mouse to a nose cone attached to the anesthetic machine at 2–3% isoflurane maintenance dose. Use hair clippers to remove fur from the dorsum of the animal from mid-back up to the neck/ears to expose a wide rectangular area of skin for surgery.
4. Transfer the mouse to a stereotaxic instrument by placing its nose in the attached nose cone and stabilizing the skull with ear bars. Maintain isoflurane at 5% during placement of the ear bars and lower back to 2–3% once the mouse is secured in the stereotaxic device.
5. Administer preoperative analgesic medications intraperitoneally — Meloxicam (2.0 mg/kg). Generously apply eye lubrication onto the open mouse eyes to prevent corneal desiccation when under anesthesia.
6. Place the thoracic support arch underneath the mouse abdomen while straightening out the mouse body and spine by lightly pulling on the base of the tail. Use laboratory labeling tape to secure the tail and all extended limbs in a star-like position. Once secure, push the thoracic support arch rostrally, from the mouse abdomen towards the upper thorax — in order to prop up the thoracic spine (Figure 1B).
7. Prepare a sterile surgical field by disinfecting the fur-clipped skin area prepared in step 1.3 with 70% ethanol, followed by povidone-iodine. Repeat twice. Apply a sterile surgical drape to keep a wide sterile field.
8. Using a #10 scalpel blade, make a vertical incision parallel to the longitudinal axis of the animal from the mid-point of both shoulder blades to the curvature of the thoracic spine. Retract the skin to expose soft tissue and the spinal column contour (Figure 1C).
9. Identify the lower border of the suprascapular fat pad (this demarcates the T4/5 vertebral level). With the same #10 blade, carefully but with force, cut along both sides of the vertebral bone T5–T8/9 to detach the back-muscle tendons from the column.
10. Insert the teeth of retractors into the incision sites on either side of the spine. Adjust the exposure by expanding retractors to sufficiently elevate the spine without putting too much strain on the retracted muscle layers (Figure 1D).
11. Under the surgical microscope, carefully clean the residual muscle and other soft tissue overlying the spine to expose the vertebral bone (Figure 1E). Identify the vertebrae that will be removed by clasping the spinous process of the vertebrae with toothed forceps and moving it slightly up and down.
    NOTE: This should allow identification of intervertebral joints and expose the small openings (intervertebral foramina) underneath the vertebrae of interest.
12. Insert one head of the curved blunted scissors into either side of the exposed intervertebral foramen, caudal to the vertebral lamina to be excised out, and cut the connecting intervertebral joints bilaterally.
13. Lift the lamina upwards and cut off the upper attachment of the lamina to isolate and remove the bone.
    NOTE: This will expose the intact dural sac containing the spinal cord (Figure 1F). There might be excessive bleeding which can be controlled by placing precut 1 cm x 1 cm gauze onto the affected areas and/or wash with sterile phosphate-buffered saline (PBS).
14. With the dorsal midline vein serving as a landmark, insert a 45° bent shaft of a 30 G needle tip (with needle bevel facing upwards) into the dorsolateral surface of the spinal cord (approximately 1 mm deep) at approximately 0.5 mm lateral to either side of the midline.
15. Move the needle ~2 mm from caudal to rostral (parallel to the midline) so that the entire length of the bevel of the needle is inserted into the cord. Remove the needle by retracing via the path of entry (Figure 1G).
    NOTE: There should be no bleeding but swelling of the spinal tissue may be seen at this step.
16. To close the wound, remove the retractor and suture the back muscles on either side of the injury together in the midline using a 6-0 absorbable suture. Use a 4-0 sterile silk suture to subsequently close the overlying skin.

Results

Figure 1: Surgical Procedure. (A) The layout of everything required, numbered for reference. (B) A picture of the mouse in a stereotaxic device with a straightened body, spine and limbs spread out and taped along with thorac...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agricola Retractor	Fine Science Tools	17005-04
Moria Vannas-Wolff Spring Scissors (Curved)	Fine Science Tools	15370-50	Customize when ordering to get blunted tips
Graefe Forceps (Straight, 1x2 Teeth)	Fine Science Tools	11053-10
Extra Fine Graefe Forceps (Curved, Serrated)	Fine Science Tools	11152-10	Or any other forceps for suturing
Hartman Hemostats (Straight)	Fine Science Tools	13002-10	Or any other appropriate for suturing
Scalpel Handle #3	Fine Science Tools	10003-12	Or any other appropriate
Hair clippers	amazon.ca	https://www.amazon.ca/Wahl-Professional-8685-Classic-Clipper/dp/B00011K2BA	or any other appropriate
Stereotaxic instrument	Stoeling	51500	or any other appropriate
Meloxicam			or any appropriate sanctioned by animal care facility
Tears Naturale P.M.	Alcon	https://www.amazon.ca/Alcon-Tear-Gel-Liquid-Eye-Gel/dp/B00HHXGUXE	or any other appropriate
Isoflurane	Baxter International Inc	DIN 02225875	or any other appropriate for anesthesia
Q-tips Cottom Swabs	amazon.ca	https://www.amazon.ca/Q-Tips-Cotton-Swabs-500-Count/dp/B003M5UO6U/ref=pd_lpo_vtph_194_bs_tr_img_1/140-7113119-8364127?_encoding=UTF8&psc=1&refRID=JC16N542KVRF2N62N3DS
Cotton Gauze	Fisher Scientific	13-761-52
30G Needles	Becton Dickinson	305106	For Injury
25G Needles	Becton Dickinson	305122	For Drug injections
1mL Syringes	Becton Dickinson	3090659	for drug injections
3mL Syringes	Becton Dickinson	309657	for fluid injections
4-0 Suture	uoftmedstore.com	2297-VS881	for skin suturing
6-0 Suture	uoftmedstore.com	VS889	for muscle suturing
Standard Dissection Tools	Fine Science Tools
Dissection Microscope	Zeiss	Stemi 2000