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.

This protocol was approved by the Animal Care Committee at the University of Toronto and is in accordance with the &#34;Guide to the Care and Use of Experimental Animals&#34; (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).
<ol>
	<li>Build a thoracic support arch by rolling up 4&#8211;5 squares of gauze and taping them together in the middle to obtain a fixed roll of gauze.</li>
	<li>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&#8211;3%).</li>
	<li>Transfer the mouse to a nose cone attached to the anesthetic machine at 2&#8211;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.</li>
	<li>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&#8211;3% once the mouse is secured in the stereotaxic device.</li>
	<li>Administer preoperative analgesic medications intraperitoneally &#8212; Meloxicam (2.0 mg/kg). Generously apply eye lubrication onto the open mouse eyes to prevent corneal desiccation when under anesthesia.</li>
	<li>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 &#8212; in order to prop up the thoracic spine (Figure 1B).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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&#8211;T8/9 to detach the back-muscle tendons from the column.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>With the dorsal midline vein serving as a landmark, insert a 45&#176; 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;s solution).</li>
	<li>Turn off the isoflurane vaporizer and oxygen. Remove the mouse from the stereotaxic device and place in a clean cage with no bedding.</li>
	<li>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&#8211;10 min and have the hind-limb function with possible paresis and/or tail weakness upon waking.</li>
	<li>Monitor mouse behavior over the next few days and provide proper post-operative care and analgesics (i.e., mash, lactated Ringer&#39;s solution fluid subcutaneous, 0.1 mg/kg buprenorphine 2x daily subcutaneously for 2&#8211;3 days, and proper weight monitoring) in accordance to local animal facility regulations and on an individual basis as recovery may vary.</li>
</ol>
2. Neurosphere Assay Dissection
NOTE: Follow proper sterile tissue culture procedures throughout.
<ol>
	<li>Enzymatic Solution Preparation
	<ol>
		<li>Add 32 mL of Earle&#39;s Balanced Salt Solution (EBSS) to the albumin ovomucoid inhibitor containing glass bottle and gently vortex until dissolved.</li>
		<li>Add 5 mL of EBSS to a pre-portioned papain glass vial and place in a 37 &#176;C water bath to dissolve. The solution will become clear (Solution 1).</li>
		<li>Take 500 &#181;L 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 &#181;L 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.</li>
	</ol>
	</li>
	<li>Tissue Preparation and Dissection
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Lift the medial aspect of each scapula (i.e., shoulder blade &#8212; 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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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).</li>
		<li>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&#39;).</li>
		<li>With microscissors, cut out the injured dorsolateral white matter. Place the piece into a 15 mL conical tube.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Add 2.5 mL of new Solution 1 (step 2.1.3) to the tissue sample and place on a rocker at 37 &#176;C for 30 min.</li>
		<li>Gently triturate the tissue in solution with a 1,000 &#181;L pipette tip. Centrifuge the cloudy cell suspension at 300 x g for 5 min at RT.</li>
		<li>Prepare Solution 3 with 2.7 mL of Earle&#39;s Balanced Salt Solution, 300 &#181;L of ovomucoid inhibitor solution (step 2.1.1) and 150 &#181;L of the DNase I solution (step 2.1.3).</li>
		<li>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).</li>
		<li>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.</li>
		<li>Discard supernatant and resuspend in 2 mL of neurobasal media. Centrifuge at 300 x g for 3 min at RT.</li>
		<li>Discard supernatant and resuspend in 1 mL of neurobasal media. Filter the suspension using a 20 &#181;m cell strainer followed by 4 mL of media for a total volume of 5 mL.</li>
	</ol>
	</li>
	<li>Plating
	<ol>
		<li>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 &#181;g/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.</li>
		<li>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/&#181;L and place tissue culture flask with added cells into the incubator for 24 h (37 &#176;C, 95% humidity, 5% CO2). 
		NOTE: An example of the counting technique is as follows: Take 10 &#181;L of the cell suspension and mix with 10 &#181;L of Trypan blue dye. Add 10 &#181;L 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/&#181;L).</li>
		<li>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.</li>
		<li>Discard the supernatant and re-suspend the cells in 1&#8211;2&#160;mL of fresh neurobasal media.</li>
		<li>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 &#181;L 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 &#181;L of media.</li>
		<li>Place plates containing cells in the incubator (37 &#176;C, 95% humidity, 5% CO2) to grow for 7 days.</li>
	</ol>
	</li>
	<li>Counting
	<ol>
		<li>Following 7 days in culture, remove plates and view under a light microscope. Quantify neurospheres by counting spherical colonies that are &#62;80 &#181;m in diameter for dNSCs cultures and &#62;50 &#181;m 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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 ...

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&#160;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 &#8212; 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 &#8212; 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.

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instrument</td><td>Stoeling</td><td>51500</td><td>or any other appropriate</td></tr><tr><td>Buprenorphine</td><td></td><td></td><td>or any appropirate sanctioned my animal care facility</td></tr><tr><td>Meloxicam</td><td></td><td></td><td>or any appropriate sanctioned by animal care facility</td></tr><tr><td>Tears Naturale P.M.</td><td>Alcon</td><td>https://www.amazon.ca/Alcon-Tear-Gel-Liquid-Eye-Gel/dp/B00HHXGUXE</td><td>or any other appropriate</td></tr><tr><td>Isoflurane</td><td>Baxter International Inc</td><td>DIN 02225875</td><td>or any other appropriate for anesthesia</td></tr><tr><td>Q-tips Cottom Swabs</td><td>amazon.ca</td><td>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&amp;amp;psc=1&amp;amp;refRID=JC16N542KVRF2N62N3DS</td><td></td></tr><tr><td>Cotton Gauze</td><td>Fisher Scientific</td><td>13-761-52</td><td></td></tr><tr><td>30 G Needles</td><td>Becton Dickinson</td><td>305106</td><td>For Injury</td></tr><tr><td>25 G Needles</td><td>Becton Dickinson</td><td>305122</td><td>For Drug injections</td></tr><tr><td>1 mL Syringes</td><td>Becton Dickinson</td><td>3090659</td><td>for drug injections</td></tr><tr><td>3 mL Syringes</td><td>Becton Dickinson</td><td>309657</td><td>for fluid injections</td></tr><tr><td>4-0 Suture</td><td>uoftmedstore.com</td><td>2297-VS881</td><td>for skin suturing</td></tr><tr><td>6-0 Suture</td><td>uoftmedstore.com</td><td>VS889</td><td>for muscle suturing</td></tr><tr><td>Polysporin ointment</td><td>amazon.ca</td><td>102051</td><td></td></tr><tr><td>Isoflurane Vaporizer</td><td>VetEquip</td><td>901806</td><td></td></tr><tr><td>15 mL conical tubes</td><td>ThermoFisher</td><td></td><td>Any appropriate</td></tr><tr><td>Petri Dishes</td><td>ThermoFisher</td><td></td><td>any appropriate</td></tr><tr><td>Trypan Blue</td><td>ThermoFisher</td><td></td><td>Any</td></tr><tr><td>Hemocytometer</td><td>ThermoFisher</td><td></td><td>Any appropriate</td></tr><tr><td>Centrifuge</td><td>ThermoFisher</td><td></td><td>Any appropriate</td></tr><tr><td>Standard Dissection Tools</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Dissection Microscope</td><td>Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Counting Microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>Neural Basal-A Medium</td><td>Invitrogen</td><td>10888-022</td><td></td></tr><tr><td>B27</td><td>Invitrogen</td><td>17404-044</td><td></td></tr><tr><td>Penicillin- Streptomycin</td><td>Gibco</td><td>15070</td><td></td></tr><tr><td>L- Glutamine</td><td>Gibco</td><td>25030</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>12100046</td><td></td></tr><tr><td>F12</td><td>Invitrogen</td><td>12700075</td><td></td></tr><tr><td>30% Glucose</td><td>Sigma</td><td>G6152</td><td>1 M, 9.01 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>1 M Glucose</td><td></td><td></td><td></td></tr><tr><td>7.5% NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td>155 mM, 1.30 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>155 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>1 M HEPES</td><td>Sigma</td><td>H3375</td><td>23.83 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Apo-Transferrin</td><td>R&amp;amp;D Systems</td><td>3188-AT</td><td></td></tr><tr><td>Putrescine&amp;nbsp;</td><td>Sigma</td><td>P7505</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I5500</td><td></td></tr><tr><td>Selenium</td><td>Sigma</td><td>S9133</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P6149</td><td></td></tr><tr><td>Papain Dissociation System&amp;nbsp;</td><td>Worthington Biochemical Corporation</td><td>PDS</td><td>1 vial of papain can be used for 2 samples</td></tr><tr><td>Epidermal Growth Factor</td><td>Invitrogen</td><td>PMG8041</td><td>Powder reconstituted with 1mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer</td></tr><tr><td>Fibroblast Growth Factor</td><td>Invitrogen</td><td>PHG0226</td><td>Powder reconstituted with 0.5 mL Hormone Mix and aliquoted into 20 &amp;mu;L vials to be stored in freezer</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>Leukemia Inhibitory Factor</td><td>In House</td><td></td><td></td></tr><tr><td>Trypan Blue</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>24 well Plates</td><td>NUNC</td><td></td><td></td></tr><tr><td>2 M NaCl</td><td>Sigma</td><td>S5886</td><td>11.69 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>1 M KCL</td><td>Sigma</td><td>P5405</td><td>7.46 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>1 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M2393</td><td>20.33 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>108 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma&amp;nbsp;</td><td>C7902</td><td>1.59 g in 100 mL dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr></tbody></table>

a neurosphere assay to evaluate endogenous neural stem cell activation in a mouse model of minimal spinal cord 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 &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

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...

Watch this Scientific Journal Video about A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury at JoVE.com

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

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 ...

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Research

JoVE Journal

Neuroscience

9.9K Views.  University of Toronto. 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.

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

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Developmental Biology

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Medicine

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

NPC Neurosphere Assay: An In Vitro Assay to Assess the Migratory Response of Neural Precursor Cells from the Neurosphere

Source: Williams, M. et al. <a href="https://www.jove.com/v/56628/rapid-detection-neurodevelopmental-phenotypes-human-neural-precursor">Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs).</a>&#160;J. Vis. Exp. (2018)
This video demonstrates the neural precursor cell neurosphere migration assay&#8212;an in vitro assay to study the migratory response of neural precursor cells from the neurosphere&#8212;via microscopic analysis.

Test neurosfery NPC: Test in vitro do oceny odpowiedzi migracyjnej neuronalnych komórek prekursorowych z neurosfery

Source: Williams, M. et al. Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs).&#160;J. Vis. Exp. (2018)
This video ...