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 th....

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 ....

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