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.
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.
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 types1,2,3,4. 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 models5,6. 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 strategies10,11,13. Current models include compression and/or contusion injuries, which cause large-scale functional deficits as well as extensive lesions and cavitations in the cord14,15. 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 progenitors18,19. 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.
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" (2nd 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).
2. Neurosphere Assay Dissection
NOTE: Follow proper sterile tissue culture procedures throughout.
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 neurospheres (grown in EFH) is greater than the numbers of pNSC-derived neurospheres (grown in LIF) after injury (Figure 3A and 3B, respectively). Higher power images of a definitive neurosphere (Figure 3C) and a primitive neurosphere (Figure 3D) reveal differences in the appearance of these distinct colonies with definitive neurospheres being larger in diameter (≥80 µm) and usually having a large dark center. Primitive neurospheres are smaller in size (≥50 µm) and the cells are more tightly packed. Representative numbers of neurospheres arising from different parts of the injured cord are seen in Figure 3E (definitives) and 3F (primitives). Figure 3E shows that minimal SCI causes a significant increase in neurospheres grown from the central canal at the level of injury compared to a relatively modest increase at the rostral non-injured cord.
Interestingly, definitive neurospheres can be generated from the site of lesion, a region which does not contain neurosphere forming cells in laminectomy alone mice. Figure 3F demonstrates a similar increase in pNSCs post-injury with the exception of pNSC neurospheres at the lesion site. Hence, only dNSCs are able to migrate to the site of injury over this time course post-injury.
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 thoracic support arch placed underneath. (C) A picture of the mouse with a vertical skin incision exposing the muscular layer and the spinal column contour. (D) A picture of the mouse body after muscular incisions and exposure and isolation of the spine with retractors. Note taut muscle layers with a lack of tearing. (E) A picture of the isolated mouse spine with muscle layers removed, showing the bony surface of the vertebra (3 segments). This also shows the spinous process and possibly the intervertebral joint and lamina of middle vertebra to be removed. (F) A picture of the visible spinal cord post-laminectomy. Labeled is the dorsal midline vein. (G) A picture of the mouse with bilateral needle track injuries on either side of dorsal midline vein. There is a close-up insert of the 45° bent shaft of 30 G needle. Please click here to view a larger version of this figure.
Figure 2: Spinal Cord Dissection. (A) A picture of the decapitated mouse with the midline skin incision to expose the back muscle and the vertebral bone contour (B) A picture of the isolated vertebral column, showing rostral and caudal orientation. The second picture shows the cord isolation and the third shows the isolated spinal cord in a Petri dish. (C) A graphical representation of the fine dissection of the spinal cord, where different segments (periventricular, injury area) are removed and separated for culturing. Please click here to view a larger version of this figure.
Figure 3: Representative results from neurosphere assay following minimal spinal cord injury. (A) The numbers of dNSC-derived neurospheres (grown in EFH) cultured from the injured spinal cord is greater than (B) the number of pNSC-derived neurospheres (grown in LIF) when plated at equal density. (C, D) Higher power images of "typical" neurospheres in (C) dNSC cultures and (D) pNSC cultures. (E) Minimal needle tract injury results in significant increases in the numbers of dNSC-derived neurospheres from the central canal at the level of injury as well as the central canal rostral to the injury, and from the injury site. (F) pNSC-derived neurospheres cannot be isolated from the uninjured cord but can be isolated following injury from the central canal (at the level of the injury and from the rostral central canal). Error bars represent standard error of the mean. Please click here to view a larger version of this figure.
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 and efficiently as possible to prevent confounding variables. Maintaining the same isoflurane exposure time per mouse will reduce variability. The breathing rate of the mouse should be monitored throughout the surgery and should not be too slow (less than 1 breath every 2–3 s) or heavily labored (i.e., gasping). The maintenance dose of anesthesia can be lowered from 2–3% to prevent death due to prolonged anesthesia with the note of mice that received the lower dosing.
During the surgery, take extra care when performing muscular incisions on either side of the vertebral column. Ensure that the cuts are deep, and the blade is angled medially so that the edge of the blade rests against the bony vertebrae during muscular incision. If the blade is angled outwards, there is the possibility of excessive bleeding from vascular cuts. The researcher should also pay extra attention when performing the laminectomy to avoid deep angling of the scissors which will damage the spinal cord, causing unwanted tissue damage and functional deficits. The appropriate control for these experiments is a "laminectomy only" group (no injury), which will enable a comparison of the activation of NSCs attributed to the needle track injury as opposed to that which can result from the laminectomy only. We have shown that laminectomy alone can result in a small, albeit insignificant increase in NSC activation as revealed by an increase in neurosphere numbers from the periventricular region at the level of the lesion21. Laminectomy alone does not cause increases in neurosphere numbers from the periventricular region rostral or caudal to the laminectomy and no neurospheres are found in cultures of white matter isolated at the level of the laminectomy. Additionally, when performing the laminectomy, take care to remove the dorsal lamina in one piece to permit wide exposure of the cord. This will (1) allow sufficient access to perform the minimal needle track injury of the dorsolateral spinal cord, and (2) prevent segments of bone from being left behind and causing secondary damage following closure and post-recovery movement of the mouse. If the entire lamina is not taken as one piece, or segments of sharp bone protruding on either side of the laminectomy are observed, use instruments (such as toothed forceps and curved scissors) to remove these fragments prior to suturing.
The researcher should be extremely careful when applying sutures to the muscular layer during the surgical closure. One suture (double-knotted) should be placed caudal to the level of laminectomy/injury so that the suture lies on top of the intact vertebral bone. This is to prevent any secondary damage that may result from the muscular suture being in contact with the exposed cord when the mouse moves after recovery. Furthermore, the muscular suture caudal to the laminectomy/injury acts as a landmark for where the SCI was performed when isolating the spinal cord for analysis. Care should be taken when dissecting out the injured spinal cord area to avoid compromising the structure of the injured region so that it can remain recognizable during fine dissection under the dissecting microscope. In regard to the neurosphere assay, it is important to perform the mechanical trituration of cell pellets gently to avoid the production of air bubbles that can increase cell death. pNSC-derived neurospheres are even more sensitive to debris heavy, growth conditions — excessive trituration and prolonged exposure in enzymatic solutions — relative to dNSC cultures. pNSC derived spheres are more compact and smaller than dNSCs. Given the rarity of pNSCs, we recommend isolating at least 240,000 cells per sample.
The minimal SCI model is ideal for studying the cellular events following injury (such as activation of endogenous NSCs) but it does not permit the study of functional impairments. As noted previously, mice that recover from the needle track injury experience no notable behavioral deficits that persist and as such, the mice cannot be evaluated for the effectiveness of therapeutic interventions designed to improve functional outcomes. One important aspect of regenerative medicine is that a treatment should not only promote tissue repair (which can be evaluated using this model, in combination with the neurosphere assay, lineage tracing and immunohistochemistry/immunofluorescence) but should also demonstrate relevant functional improvement using behavioral paradigms where applicable. A number of behavioral tasks used to test functional outcomes in thoracic models of injury such as the foot fault test and the Basso, Beattie, Breshnan (BBB) Open Field Locomotor Scale28, are not sensitive enough to detect measurable deficits in our minimal SCI model. To overcome this shortcoming, one can make use of more sensitive digital scoring systems (e.g., CatWalk) which measures multiple parameters of gross and fine hind-limb locomotion parameters including gait analyses, which may detect the minimal deficits resulting from the minimal injury model29.
This method could also be adapted to study endogenous NSC activation as a potential therapy in models of cervical spinal cord injury. Cervical SCI is the most clinically relevant model and we propose that adapting this injury model to higher vertebral segments would provide insight into whether there are regional variations in the response of NSCs and their progeny (kinetics, migration, differentiation) and whether the lesion would result in more profound and measurable functional deficits. The currently used murine cervical injury models (such as transection, clip compression and/or contusion) require intensive post-operative care including the need to manually express bladders and constantly monitor the breathing of the mouse. Adapting the minimal injury model to the cervical spinal cord may reduce the mortality, morbidity and post-operative care associated with other cervical SCI models as well as permit one to examine the effectiveness of drugs/small molecules and/or rehabilitation outcomes on neural recovery. Our injury model can also be adapted to create a minimal injury in different parts of the cord (i.e., dorsal or lateral columns) and/or larger injuries with deeper penetration and/or the use of larger sized needles (smaller gauge). This allows the researcher to control/manipulate the type and size of the lesion and thus evaluate cellular and functional/behavioral recovery accordingly.
The minimal injury model in mice permits the use of transgenic animal models. The mouse models enable labeling of endogenous stem cells and/or progenitors prior to the injury. This can allow the researcher to track the fate of these pre-labeled cells following injury and evaluate neural precursor cell proliferation, migration, and differentiation following injury — potentially contributing to neural repair.
The authors have nothing to disclose.
This work is funded by the Krembil Foundation (operating grant CMM). WX was the recipient of the Carlton Marguerite Smith student award. NL received an Ontario Graduate Scholarship.
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, 1×2 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 | |
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 |
Polysporin ointment | amazon.ca | 102051 | |
Isoflurane Vaporizer | VetEquip | 901806 | |
15mL 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 | 1M- 9.01g in 100mL dH2O |
1M Glucose | |||
7.5% NaHCO3 | Sigma | S5761 | 155mM- 1.30g in 100mL dH2O |
155mM NaHCO3 | |||
1M HEPES | Sigma | H3375 | 23.83 g in 100mL 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 0.5mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer |
Heparin | Sigma | H3149 | |
Leukemia Inhibitory Factor | In House | ||
Trypan Blue | |||
Hemocytometer | |||
24 well Plates | NUNC | ||
2M NaCl | Sigma | S5886 | 11.69g in 100mL dH2O |
1M KCL | Sigma | P5405 | 7.46g in 100mL dH2O |
1M MgCl2 | Sigma | M2393 | 20.33g in 100mL dH2O |
108mM CaCl2 | Sigma | C7902 | 1.59g in 100mL dH2O |