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 suppo...

<ol>
	<li>Johansson, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+neural+stem+cell+in+the+adult+mammalian+central+nervous+system.">Identification of a neural stem cell in the adult mammalian central nervous system.</a> Cell. 96 (1), 25-34 (1999).</li><li>McKay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+central+nervous+system.">Stem cells in the central nervous system.</a> Science. 276 (5309), 66-71 (1997).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+neural+stem+cells.">Mammalian neural stem cells.</a> Science. 287 (5457), 1433-1438 (2000).</li><li>Temple, S., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+adult+mammalian+central+nervous+system.">Stem cells in the adult mammalian central nervous system.</a> Current Opinion in Neurology. 9 (1), 135-141 (1999).</li><li>Zhang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+neural+stem+cells+contribute+to+stroke-induced+neurogenesis+and+neuroblast+migration+toward+the+infarct+boundary+in+adult+rats.">Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats.</a> Journal Of Cerebral Blood Flow And Metabolism. 24 (4), 441-448 (2004).</li><li>Komitova, M., Mattsson, B., Johansson, B. B., Eriksson, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enriched+environment+increases+neural+stem/progenitor+cell+proliferation+and+neurogenesis+in+the+subventricular+zone+of+stroke-lesioned+adult+rats.">Enriched environment increases neural stem/progenitor cell proliferation and neurogenesis in the subventricular zone of stroke-lesioned adult rats.</a> Stroke. 36 (6), 1278-1282 (2005).</li><li>Faiz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neural+stem+cells+from+the+subventricular+zone+give+rise+to+reactive+astrocytes+in+the+cortex+after+stroke.">Adult neural stem cells from the subventricular zone give rise to reactive astrocytes in the cortex after stroke.</a> Cell Stem Cell. 17 (5), 624-634 (2015).</li><li>Ren, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ependymal+cell+contribution+to+scar+formation+after+spinal+cord+injury+is+minimal,+local+and+dependent+on+direct+ependymal+injury.">Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury.</a> Science Reports - UK. 7, (2017).</li><li>Mothe, A. J., Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+migration,+and+differentiation+of+endogenous+ependymal+region+stem/progenitor+cells+following+minimal+spinal+cord+injury+in+the+adult+rat.">Proliferation, migration, and differentiation of endogenous ependymal region stem/progenitor cells following minimal spinal cord injury in the adult rat.</a> Neuroscience. 131 (1), 177-187 (2005).</li><li>Thuret, S., Moon, L. D., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+interventions+after+spinal+cord+injury.">Therapeutic interventions after spinal cord injury.</a> Nature Reviews Neuroscience. 7 (8), 628-643 (2006).</li><li>Bethea, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemically+administered+interleukin-10+reduces+tumor+necrosis+factor-alpha+production+and+significantly+improves+functional+recovery+following+traumatic+spinal+cord+injury+in+rats.">Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats.</a> Journal of Neurotrauma. 16 (10), 851-863 (1999).</li><li>Donnelly, D. J., Popovich, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+its+role+in+neuroprotection,+axonal+regeneration+and+functional+recovery+after+spinal+cord+injury.">Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury.</a> Experimental Neurology. 209 (2), 378-388 (2008).</li><li>Cummings, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cells+differentiate+and+promote+locomotor+recovery+in+spinal+cord-injured+mice.">Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice.</a> Proceedings of the National Academy of Sciences USA. 102 (39), 14069-14074 (2005).</li><li>Beattie, M. S., Hermann, G. E., Rogers, R. C., Bresnahan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+models+of+spinal+cord+injury.">Cell death in models of spinal cord injury.</a> Progress in Brain Research. 137, 37-47 (2002).</li><li>Metz, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+the+weight-drop+contusion+model+in+rats:+a+comparative+study+of+human+spinal+cord+injury.">Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord injury.</a> Journal of Neurotrauma. 17 (1), 1-17 (2000).</li><li>Faulkner, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+astrocytes+protect+tissue+and+preserve+function+after+spinal+cord+injury.">Reactive astrocytes protect tissue and preserve function after spinal cord injury.</a> Journal of Neuroscience. 24 (9), 2143-2155 (2004).</li><li>Cheriyan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury+models:+a+review.">Spinal cord injury models: a review.</a> Spinal Cord. 52 (8), 588-595 (2014).</li><li>Deleyrolle, L. P., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+expansion,+and+differentiation+of+adult+Mammalian+neural+stem+and+progenitor+cells+using+the+neurosphere+assay.">Isolation, expansion, and differentiation of adult Mammalian neural stem and progenitor cells using the neurosphere assay.</a> Neural Cell Transplantation: Methods and Protocols. , 91-101 (2009).</li><li>Singec, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nature Methods. 3 (10), (2006).</li><li>Mignone, J. L., Kukekov, V., Chiang, A. S., Steindler, D., Enikolopov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+and+progenitor+cells+in+nestin-GFP+transgenic+mice.">Neural stem and progenitor cells in nestin-GFP transgenic mice.</a> The Journal of Comparative Neurology. 469 (3), 311-324 (2004).</li><li>Xu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+basic+protein+regulates+primitive+and+definitive+neural+stem+cell+proliferation+from+the+adult+spinal+cord.">Myelin basic protein regulates primitive and definitive neural stem cell proliferation from the adult spinal cord.</a> Stem Cells. 35 (2), 485-496 (2017).</li><li>Sachewsky, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primitive+neural+stem+cells+in+the+adult+mammalian+brain+give+rise+to+GFAP-expressing+neural+stem+cells.">Primitive neural stem cells in the adult mammalian brain give rise to GFAP-expressing neural stem cells.</a> Stem Cell Reports. 2 (6), 810-824 (2014).</li><li>Mothe, A., Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem/progenitor+cells+from+the+periventricular+region+of+the+adult+rat+and+human+spinal+cord.">Isolation of neural stem/progenitor cells from the periventricular region of the adult rat and human spinal cord.</a> Journal of Visualized Experiments. (99), (2015).</li><li>Absher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemocytometer+counting.">Hemocytometer counting.</a> Tissue Culture. , 395-397 (1973).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> Journal of Visualized Experiments. (45), (2010).</li><li>Xiong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconditioning+with+isoflurane+produces+dose-dependent+neuroprotection+via+activation+of+adenosine+triphosphate-regulated+potassium+channels+after+focal+cerebral+ischemia+in+rats.">Preconditioning with isoflurane produces dose-dependent neuroprotection via activation of adenosine triphosphate-regulated potassium channels after focal cerebral ischemia in rats.</a> Anesthesia and Analgesia. 96 (1), 233-237 (2003).</li><li>Metz, G. A., Merkler, D., Dietz, V., Schwab, M. E., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+testing+of+motor+function+in+spinal+cord+injured+rats.">Efficient testing of motor function in spinal cord injured rats.</a> Brain Research. 883 (2), 165-177 (2000).</li><li>Hamers, F. P., Koopmans, G. C., Joosten, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CatWalk-assisted+gait+analysis+in+the+assessment+of+spinal+cord+injury.">CatWalk-assisted gait analysis in the assessment of spinal cord injury.</a> Journal of Neurotrauma. 23 (3-4), 537-548 (2006).</li></ol>

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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			<td style="width:201px;">Fine Science Tools</td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Dissection Microscope</td>
			<td style="width:201px;">Zeiss</td>
			<td style="width:197px;">Stemi 2000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Counting Microscope</td>
			<td style="width:201px;">Olympus</td>
			<td style="width:197px;">CKX41</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Neural Basal-A Medium</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:197px;">10888-022</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">B27</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:197px;">17404-044</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Penicillin- Streptomycin</td>
			<td style="width:201px;">Gibco</td>
			<td align="right" style="width:197px;">15070</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">L- Glutamine</td>
			<td style="width:201px;">Gibco</td>
			<td align="right" style="width:197px;">25030</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">DMEM</td>
			<td style="width:201px;">Invitrogen</td>
			<td align="right" style="width:197px;">12100046</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">F12</td>
			<td style="width:201px;">Invitrogen</td>
			<td align="right" style="width:197px;">12700075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">30% Glucose</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">G6152</td>
			<td>1M- 9.01g in 100mL dH2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">1M Glucose</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">7.5% NaHCO3</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">S5761</td>
			<td>155mM- 1.30g in 100mL dH2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">155mM NaHCO3</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">1M HEPES</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">H3375</td>
			<td>23.83 g in 100mL dH2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Apo-Transferrin</td>
			<td style="width:201px;">R&#38;D Systems</td>
			<td style="width:197px;">3188-AT</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Putrescine&#160;</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">P7505</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Insulin</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">I5500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Selenium</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">S9133</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Progesterone</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">P6149</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Papain Dissociation System&#160;</td>
			<td style="width:201px;">Worthington Biochemical Corporation</td>
			<td style="width:197px;">PDS</td>
			<td>1 vial of papain can be used for 2 samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Epidermal Growth Factor</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:197px;">PMG8041</td>
			<td>Powder reconstituted with 1mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Fibroblast Growth Factor</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:197px;">PHG0226</td>
			<td>Powder reconstituted with 0.5mL Hormone Mix and aliquoted into 20uL vials to be stored in freezer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Heparin</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">H3149</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Leukemia Inhibitory Factor</td>
			<td style="width:201px;">In House</td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Trypan Blue</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Hemocytometer</td>
			<td style="width:201px;"></td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">24 well Plates</td>
			<td style="width:201px;">NUNC</td>
			<td style="width:197px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">2M NaCl</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">S5886</td>
			<td>11.69g in 100mL dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">1M KCL</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">P5405</td>
			<td>7.46g in 100mL dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">1M MgCl2</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">M2393</td>
			<td>20.33g in 100mL dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">108mM CaCl2</td>
			<td style="width:201px;">Sigma&#160;</td>
			<td style="width:197px;">C7902</td>
			<td>1.59g in 100mL dH2O</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 ...

This method can help answer questions related to the role of neural precursor cells in regenerative medicine. Specifically, we can ask how injury affects the behavior of neural precursor cells. The main advantage of this technique is that it provides an approach to study the effect of spinal cord injury on endogenous neural precursor cell kinetics.Visual demonstration of these techniques are critical. As the surgical and dissection steps requires precision, and can be difficult to learn, although they can be perfected with practice. Before beginning the procedure, use hair clippers to remove the fur from the dorsum of an anesthetized mouse from the mid back to the neck and ears.After securing the mouse in a stereotactic device, place four to five pieces of rolled gauze beneath the abdomen, while lightly pulling on the base of the tail to straighten out the body and spine. Push the thoracic gauze roll rostrally from the mouse abdomen toward the upper thorax to support the thoracic spine. And use laboratory tape to secure the tail and limbs in a star-like orientation.After disinfecting the exposed skin with sequential 70%ethanol and povidone iodine swabs, use a number 10 scalpel blade to make a vertical incision parallel to the longitudinal axis of the animal, from the midpoint of both shoulder blades, to the curvature of the thoracic spine. Retract the skin to expose the soft tissue and the spinal column contour. And identify the lower border of the super scapular fat pad.Then, use the scalpel to carefully but forcefully cut along both sides of the vertebral T5 to T8 and nine bones, to detach the back muscle tendons from the spinal column. Reposition retractors so that the teeth of the retractors are inserted into the incision site on either side of the spine. And expand each retractor to adjust the exposure, until the spine is sufficiently elevated without putting too much strain on the retracted muscle layers.Under a surgical microscope, carefully clean the residual muscle and other soft tissue overlying the spine, to expose the vertebral bone. Clasping the spinous process of the vertebrae with toothed forceps, and moving the forceps slightly up and down to identify the vertebrae that will be removed. Next, insert one tip of a pair of curved blunted scissors into either side of the exposed intervertebral foramen, caudal to the vertebral lamina to be excised.And cut the connecting intervertebral joints bilaterally. Then, lift the lamina upwards and cut off the upper attachment of the lamina to isolate and remove the bone. When cutting away and removing the vertebral lamina, take care to always angle the scissors upwards so not as to damage the underlying spinal cord.With the dorsal midline veins serving as a landmark, insert the 45 degree bent shaft of a 30 gage needle tip, bevel side up, one millimeter deep into the dorsal lateral surface of the spinal cord. And approximately 0.5 millimeters lateral to either side of the midline. Move the needle about two millimeters from the caudal to rostral direction, so that the entire length of the bevel is inserted into the cord.Then, retrace the path of the entry to remove the needle. To close the wound, remove the retractor and use a 6-0 absorbable suture to join the back muscles on either side of the injury along the midline. And a 4-0 silk suture to close the overlying skin.To isolate the neurospheres, make a midline incision in the skin along the entire length of the back to expose the muscle and vertebral bone contour. Lift the medial aspect of each scapula and use scissors to cut away the soft tissue between the scapula and vertebrae. When the underlying vertebral column has been exposed, insert the scissors into the upper thoracic aperture, following the curvature of the spinal column, and cut away the attached ribs, muscle and internal organs to isolate the vertebral column.Insert the scissors into the caudal vertebral foramen, and cut the intervertebral joints on either side of the laminae, taking care to not damage the intact cord. When the appropriate length of the cord is exposed, carefully cut any spinal nerves extending laterally from the cord, before transferring the spinal cord tissue into a Petri dish of regular, artificial cerebral spinal fluid on ice. Under a dissection microscope, cut the spinal cord tissue to include about two millimeters of tissue rostral and caudal to the injury site, and use two pairs of fine forceps to gently separate the cord into two longitudinal halves, along the dorsal and ventral fissures.Use the micro scissors to cut out the injured dorsal lateral white matter. And place the injured tissue into a 15 milliliter conical tube. Holding one half of the spinal cord with the forceps, tease and remove the uninjured white matter with two pairs of forceps along the rostral caudal extent of the spinal cord, to isolate the appropriate periventricular region.After isolating the injured white matter and associated periventricular regions from the other half of the spinal cord in the same manner, place the pieces of periventricular tissue into individual 15 milliliter conical tubes. And use the micro scissors to gently mince the tissue against the walls of the tubes. Once the tissue digestion procedure has been completed, and the single cell suspension has been obtained, set up a neurosphere culture by adding 10 milliliters of the appropriate medium into one 25 milliter tissue culture flask per cell condition.And add the isolated neural stem cells up to a clonal density of 10 cells per microliter for their culture in a cell culture incubator. After 24 hours, gently swirl the flasks and decant the contents into individual 15 milliliter conical tubes for centrifugation. Resuspend the pellets in one to two milliliters of fresh neural basal medium for counting.And plate the cells at clonal density in one 24 well tissue culture plate per condition, containing 500 microliters of the appropriate corresponding mitogen supplemented medium per well. Then, grow the neurospheres in the cell culture incubator for seven days. Five days following the minimal spinal cord injury, the absolute numbers of definitive neural stem cell derived neurospheres is greater than the number of primitive neural stem cell derived neurospheres.Definitive neurospheres exhibit a larger diameter and a large dark center. While primitive neurospheres are smaller in size and more tightly packed. Minimal spinal cord injury induces a significant increase in neurospheres grown from the central canal at the level of injury, compared to a relatively modest increase at the central canal, rostral to the level of injury.Definitive neurospheres can also be generated from the lesioned white matter at the level of the injury. A region that does not contain neurosphere forming cells in laminectomy alone animals. A similar increase in primitive neural stem cells is observed post injury, with the exception of primitive neurospheres at the white matter at the level of the lesion site.Once mastered, the surgical technique can be performed in under 40 minutes. And the tissue dissection and isolation can be performed under 30 minutes, if performed properly. Following the surgical procedure, depending on the animal strain, additional methodologies such as immunohistochemistry and lineate shocking, can be used to answer additional questions such as proliferation kinetics, and self fate of the neural precursor cells after injury.After watching this video, you should have a good understanding of how to utilize the neurosphere assay to assess neural stem cell activation, following spinal cord injury.

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Research

JoVE Journal

Neuroscience

9.8K Views.  University of Toronto. This method can help answer questions related to the role of neural precursor cells in regenerative medicine. Specifically, we can ask how injury affects the behavior of neural precursor cells. The main advantage of this technique is that it provides an approach to study the effect of spinal cord injury on endogenous neural precursor cell kinetics.Visual demonstration of these techniques are critical. As the surgical and dissection steps requires precision, and can be difficult to lear...