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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			<td style="width:201px;">ThermoFisher</td>
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			<td>Any appropriate</td>
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			<td height="21" style="height:21px;width:203px;">Centrifuge</td>
			<td style="width:201px;">ThermoFisher</td>
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		<tr height="22">
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			<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>
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			<td height="22" style="height:22px;width:203px;">155mM NaHCO3</td>
			<td style="width:201px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:203px;">1M HEPES</td>
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			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">S5886</td>
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			<td style="width:201px;">Sigma</td>
			<td style="width:197px;">M2393</td>
			<td>20.33g in 100mL dH2O</td>
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		<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>
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</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 ...

a-neurosphere-assay-to-evaluate-endogenous-neural-stem-cell

Research

JoVE Journal

Neuroscience

9.8K 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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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 ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

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 ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...