A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Summary

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Abstract

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site — demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Introduction

The central nervous system contains a subpopulation of self-renewing, multipotent stem cells that have the capacity to give rise to all the different mature neural cell types^1,2,3,4. These neural stem cells (NSCs) reside in specialized niches in the brain and spinal cord and can be activated following injury to proliferate, migrate, and differentiate into mature neural cells. NSCs and their progeny have been shown to migrate to the injury site in cortical injury models^5,6. In the brain, NSCs have been shown to migrate from the lateral ventricles to the site of injury where they differentiate into astrocytes that contribute to glial scar formation⁷. In the spinal cord, however, few studies have been done to ask if these same endogenous NSCs can be harnessed to promote recovery following spinal cord injury. Indeed, there is currently a debate as to whether activation of the stem cell pool in the spinal cord requires a direct physical damage of the periventricular niche lining the central canal⁸ or if the damage to the spinal cord parenchyma (leaving the stem cell niche intact) is sufficient to activate endogenous NSCs⁹.

A number of spinal cord injury (SCI) models have been used to study the pathophysiology of acute and chronic injury. These models have also been used to test potential therapies to treat SCI through neuroprotection, immunomodulation, and developing cell transplantation/replacement strategies^10,11,13. Current models include compression and/or contusion injuries, which cause large-scale functional deficits as well as extensive lesions and cavitations in the cord^14,15. Resultant glial scars can span several spinal segments along with the majority of the width/circumference of the spinal cord¹⁶. Thus, while these models are clinically relevant, they afford significant challenges to studying the response of endogenous NSCs following injury. There are chemical models of injury that can be adapted to cause milder forms of injury that can spare the central canal¹⁷. However, these types of injury focus on the demyelination associated with SCI and are not clinically relevant models for the physical and/or mechanical damage associated with traumatic SCI.

To address the limitations of current injury models, we have adapted a needle track minimal SCI model, originally developed in the rat⁹, for the application in an adult mouse model. Our adapted injury model can create a consistent lesion of the dorsolateral region of the mouse spinal cord and spare the central canal at the level(s) of injury. The advantage of this model is that it permits the study of NSC kinetics following injury and their potential radial migration to the site of injury. The use of a mouse model also permits the use of transgenic mice that allow lineage tracking of endogenous NSCs and their progeny following injury. The properties of NSCs can further be assessed using a modified form of the in vitro neurosphere assay that is introduced in this protocol.

The neurosphere assay is an in vitro colony-forming assay that permits the isolation of NSCs in the presence of mitogens. At clonal plating densities, individual NSCs proliferate to give rise to free-floating spherical colonies of cells that are comprised of a small subpopulation of NSCs and a vast majority of progenitors^18,19. In our protocol, we demonstrate the isolation of two distinct, lineally-related NSCs from the periventricular region of the spinal cord — under baseline conditions and following our minimal SCI model. Definitive neural stem cells (dNSCs) express nestin and the glial fibrillary acidic protein (GFAP) and are grown in the presence of epidermal growth factor (EGF), fibroblast growth factor (FGF), and heparin (together termed EFH)²⁰. These dNSCs are rare in the naive spinal cord, giving rise to very few neurospheres in vitro. However, we show that dNSCs are activated following minimal SCI, expanding the numbers of neurospheres isolated from the periventricular region²¹. Primitive neural stem cells (pNSCs) are upstream of dNSCs in the neural stem cell lineage. pNSCs are exceedingly rare, express low levels of the pluripotency marker Oct4, and are leukemia inhibitory factor (LiF) responsive²². pNSCs do not form neurospheres when isolated from the adult mouse spinal cord due to the presence of myelin basic protein (MBP) in primary cultures; however, pNSC neurospheres can be isolated from MBP deficient mice and their numbers are expanded following injury — similar to dNSCs²¹. Finally, we show that dNSC-derived neurospheres can be isolated from the site of injury at early times following minimal SCI. These findings demonstrate that our injury model and assays can assess the activation characteristics of periventricular NSCs such as their ability to proliferate and migrate in response to injury.

Protocol

This protocol was approved by the Animal Care Committee at the University of Toronto and is in accordance with the "Guide to the Care and Use of Experimental Animals" (2^nd Edition, Canadian Council on Animal Care, 2017).

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 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.
17. Apply antibiotic ointment to the top of the superficial sutured skin to prevent postoperative infection using the tip of a cotton swab. Administer post-operative analgesic of 0.1 mg/kg of Buprenorphine subcutaneously along with fluids (1 mL of lactated Ringer's solution).
18. Turn off the isoflurane vaporizer and oxygen. Remove the mouse from the stereotaxic device and place in a clean cage with no bedding.
19. Place the mouse in the cage about 30 cm away from a heat lamp for recovery from anesthesia and monitor behavior following waking. The mouse should wake up within 5–10 min and have the hind-limb function with possible paresis and/or tail weakness upon waking.
20. Monitor mouse behavior over the next few days and provide proper post-operative care and analgesics (i.e., mash, lactated Ringer's solution fluid subcutaneous, 0.1 mg/kg buprenorphine 2x daily subcutaneously for 2–3 days, and proper weight monitoring) in accordance to local animal facility regulations and on an individual basis as recovery may vary.

2. Neurosphere Assay Dissection

NOTE: Follow proper sterile tissue culture procedures throughout.

1. Enzymatic Solution Preparation
   1. Add 32 mL of Earle's Balanced Salt Solution (EBSS) to the albumin ovomucoid inhibitor containing glass bottle and gently vortex until dissolved.
   2. Add 5 mL of EBSS to a pre-portioned papain glass vial and place in a 37 °C water bath to dissolve. The solution will become clear (Solution 1).
   3. Take 500 µL of EBSS and add to the pre-portioned DNase I glass vial to obtain a concentration of 1 mg/mL (Solution 2). Mix very gently by tilting the vial back and forth and add 250 µL of this mixture to Solution 1.
      NOTE: All solutions made (Solution 1 and 2) are enough for processing of two pieces of the tissue. If processing more pieces of tissue, accommodate with more solution prep.
2. Tissue Preparation and Dissection
   1. Place a laboratory wipe soaked in isoflurane into the mouse cage and allow 2 - 3 min for vapors to completely anesthetize mouse. Following knockout, remove the mouse from the cage and confirm the absence of a toe-pinch reflex.
   2. Perform cervical dislocation with a blunt metal object (i.e., metal cage card) to euthanize the animal. Spray the euthanized animal from its neck to mid back generously with 70% ethanol.
   3. At the base of the skull, use scissors to cut off the head and discard into a bio bag. Make a midline incision in the skin along the entire length of the back to expose the muscle and vertebral bone contour (Figure 2A).
   4. Lift the medial aspect of each scapula (i.e., shoulder blade — identified by overlying fat pad) and use scissors to cut away soft tissue (between scapulae and vertebrae). Separate fully to expose the underlying vertebral column.
   5. Following the curvature of the spinal column, insert the same pair of scissors into the upper thoracic aperture and isolate the vertebral column by cutting away attached ribs, muscle, and internal organs (Figure 2B).
   6. Insert scissors into the caudal vertebral foramen/opening and cut the intervertebral joints on either side of the laminae (dorsal surface). Take particular care to not damage the intact cord. Continue this process until the desired level and/or length of the cord is exposed.
   7. Carefully cut the spinal nerve(s) extending laterally from the cord prior to transferring the spinal cord tissue into a Petri dish with regular artificial cerebrospinal fluid. Keep the sample on ice (Figure 2C).
   8. Under a dissection microscope, cut the spinal cord tissue to include ~2 mm rostral and caudal to the injury site. Use two pairs of forceps to gently separate the cord into 2 halves longitudinally along the dorsal and ventral fissures (Figure 2C').
   9. With microscissors, cut out the injured dorsolateral white matter. Place the piece into a 15 mL conical tube.
   10. Holding one half of the spinal cord down with forceps, tease and remove white matter using another pair of fine forceps along the rostrocaudal extent of the spinal cord to isolate the desired periventricular region. Repeat for the other half of the spinal cord.
   11. Place pieces of periventricular tissue into a separate 15 mL conical tubes. Use microscissors and gently mince the tissue against the walls of the conical tubes.
       NOTE: Tissue Dissociation Modifications are from Papain Dissociation System Protocol²³ and previously described tissue preparation procedures²⁴.
   12. Add 2.5 mL of new Solution 1 (step 2.1.3) to the tissue sample and place on a rocker at 37 °C for 30 min.
   13. Gently triturate the tissue in solution with a 1,000 µL pipette tip. Centrifuge the cloudy cell suspension at 300 x g for 5 min at RT.
   14. Prepare Solution 3 with 2.7 mL of Earle's Balanced Salt Solution, 300 µL of ovomucoid inhibitor solution (step 2.1.1) and 150 µL of the DNase I solution (step 2.1.3).
   15. Discard the supernatant from step (step 2.2.13) and resuspend in 1.5 mL of Solution 3 from the previous step (step 2.2.14).
   16. Layer this new cell suspension (step 2.2.15) on top of 5 mL of ovoimucoid inhibitor solution (step 2.1.1) in a new 15 mL conical tube to create a discontinuous density gradient. Centrifuge at 300 x g for 5 min at RT.
   17. Discard supernatant and resuspend in 2 mL of neurobasal media. Centrifuge at 300 x g for 3 min at RT.
   18. Discard supernatant and resuspend in 1 mL of neurobasal media. Filter the suspension using a 20 µm cell strainer followed by 4 mL of media for a total volume of 5 mL.
3. Plating
   1. Prepare culture media for neurosphere growth by supplementing neurobasal media with epidermal growth factor (20 ng/mL) / fibroblast growth factor (20 ng/mL)/ heparin (2 µg/mL) [for definitive NSCs] or leukemina inhibitory factor (10 ng/mL) [for primitive NSCs]. Add 10 mL of either media into a 25 mL tissue culture flask.
   2. Use a hemocytometer and Trypan blue dye to calculate the number of cells in suspension from (step 2.2.18)²⁵. Once calculated, add cells to tissue culture flasks prepared in (step 2.3.1) to a clonal density of 10 cells/µL and place tissue culture flask with added cells into the incubator for 24 h (37 °C, 95% humidity, 5% CO₂).
      NOTE: An example of the counting technique is as follows: Take 10 µL of the cell suspension and mix with 10 µL of Trypan blue dye. Add 10 µL of this mixture into a hemocytometer. Under a 10X light microscope, count the numbers of live cells and sum all cells counted within 4 of the 4 x 4 quadrants. Use the formula: 100,000/[(X cells/4) x 2 x 10 x 1,000] to give the volume of cell suspension to be plated in each 25 mL tissue culture flask to ensure clonal density (10 cells/µL).
   3. After 24 h, gently swirl the flask and pour contents into a 15 mL conical tube. Centrifuge at 300 x g for 5 min at RT.
   4. Discard the supernatant and re-suspend the cells in 1–2 mL of fresh neurobasal media.
   5. Repeat step 2.3.2 to count the number of cells in suspension and plate cells at clonal density in a 24-well tissue culture plate containing 500 µL each of their respective mitogen supplemented media (EFH for definitive NSC growth and LIF for primitive NSC growth).
      NOTE: Use the adapted formula (5,000/[X cells/4) x 2 x 10 x 1,000) to calculate the volume of the cell suspension to be plated in each well containing 500 µL of media.
   6. Place plates containing cells in the incubator (37 °C, 95% humidity, 5% CO₂) to grow for 7 days.
4. Counting
   1. Following 7 days in culture, remove plates and view under a light microscope. Quantify neurospheres by counting spherical colonies that are >80 µm in diameter for dNSCs cultures and >50 µm diameter for pNSCs cultures.
      NOTE: If desired, neurospheres can be further passaged²⁶ or differentiated. If cells are no longer needed, dispose by adding accelerated hydrogen peroxide to the wells (approx. 1 mL) for 20 min so that the color of the medium turns yellow. Then discard.

Results

Following surgery, the mice should experience minimal motor deficits which may include tail and possible hind-limb paresis for up to 24 h. After this time, the mice should experience no hind-limb paralysis and/or paresis and minimal changes in gait.

Figure 3 shows representative results from the neurosphere assay 5 days following the minimal spinal cord injury. The absolute numbers of dNSC-derived n...

Discussion

During the surgical procedure, there are a few critical steps where the researcher should pay particular attention to in order to obtain optimal outcomes and minimize variability between animals. Care must be taken with the inhaled anesthesia (isoflurane) during surgery as the anesthetic has been shown to have neuroprotective effects with prolonged exposure²⁷. Accordingly, when studying the regenerative capacity of the spinal cord following injury, make an effort to perform the surgery as quickly ...

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, 1 x 2 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
Buprenorphine			or any appropirate sanctioned my animal care facility
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
30 G Needles	Becton Dickinson	305106	For Injury
25 G Needles	Becton Dickinson	305122	For Drug injections
1 mL Syringes	Becton Dickinson	3090659	for drug injections
3 mL 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
Polysporin ointment	amazon.ca	102051
Isoflurane Vaporizer	VetEquip	901806
15 mL conical tubes	ThermoFisher		Any appropriate
Petri Dishes	ThermoFisher		any appropriate
Trypan Blue	ThermoFisher		Any
Hemocytometer	ThermoFisher		Any appropriate
Centrifuge	ThermoFisher		Any appropriate
Standard Dissection Tools	Fine Science Tools
Dissection Microscope	Zeiss	Stemi 2000
Counting Microscope	Olympus	CKX41
Neural Basal-A Medium	Invitrogen	10888-022
B27	Invitrogen	17404-044
Penicillin- Streptomycin	Gibco	15070
L- Glutamine	Gibco	25030
DMEM	Invitrogen	12100046
F12	Invitrogen	12700075
30% Glucose	Sigma	G6152	1 M, 9.01 g in 100 mL dH2O
1 M Glucose
7.5% NaHCO3	Sigma	S5761	155 mM, 1.30 g in 100 mL dH2O
155 mM NaHCO3
1 M HEPES	Sigma	H3375	23.83 g in 100 mL dH2O
Apo-Transferrin	R&D Systems	3188-AT
Putrescine	Sigma	P7505
Insulin	Sigma	I5500
Selenium	Sigma	S9133
Progesterone	Sigma	P6149
Papain Dissociation System	Worthington Biochemical Corporation	PDS	1 vial of papain can be used for 2 samples
Epidermal Growth Factor	Invitrogen	PMG8041	Powder reconstituted with 1mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer
Fibroblast Growth Factor	Invitrogen	PHG0226	Powder reconstituted with 0.5 mL Hormone Mix and aliquoted into 20 μL vials to be stored in freezer
Heparin	Sigma	H3149
Leukemia Inhibitory Factor	In House
Trypan Blue
Hemocytometer
24 well Plates	NUNC
2 M NaCl	Sigma	S5886	11.69 g in 100 mL dH2O
1 M KCL	Sigma	P5405	7.46 g in 100 mL dH2O
1 M MgCl2	Sigma	M2393	20.33 g in 100 mL dH2O
108 mM CaCl2	Sigma	C7902	1.59 g in 100 mL dH2O