This research was supported by the following: AHA Award 14SDG20480186 for LC, Subject innovation team of Shanxi University of Chinese Medicine 2019-QN07 for BZ, and Kentucky Spinal Cord and Head Injury Research Trust grant 14-12A for KES and LC.

All the procedures on animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Kentucky, and appropriate care was taken to minimize stress or pain associated with surgery.
1. Preparation of injection catheter and surgical hooks
<ol>
	<li>Construct the injection catheter (Figure 1). Gather necessary materials including: MRE010, MRE025, and MRE050 tubing, 20 G, 26 G and 27 G injection needles (Figure 2A), 600 grit sandpaper, superglue and two-component 5-minute epoxy.
	<ol>
		<li>Cut 20 G and 26 G needles at 1 cm from the needle hub and polish the end on sandpaper (Figure 2B). Flush the needles with 10 mL of double distilled water to clean the needle bore. 
		NOTE: Two different designs (Figure 1) are used. Design 1 has a single connector and is used for injection of solutions or suspensions. Design 2 has 20 G and 26 G Luer lock connectors for injection of cells (20 G needle) and flush of the dead volume (26 G needle) to ensure delivery of the full volume of NSC-containing solution.</li>
	</ol>
	</li>
	<li>Design 1: Insert a 3-4 cm length MRE010 catheter into a 15 cm length MRE025 catheter and secure with superglue.
	<ol>
		<li>Connect the other end of the MRE025 tube to a segment of MRE050 catheter, and secure with superglue. Insert a dulled 20 G needle into the remaining end of the MRE050 catheter and secure with superglue (Figure 1).</li>
		<li>Further reinforce the connection sites with epoxy glue. This catheter design is optimal for injection of reagents (like chemical or drug solutions or other biologics such as cytokines).</li>
	</ol>
	</li>
	<li>Design 2: Insert a 3-4 cm length MRE010 catheter into a 15 cm length MRE025 catheter and secure with superglue.
	<ol>
		<li>Connect the other end of the MRE025 tube to a segment of MRE050 catheter, and secure with superglue. Insert a dulled 20 G needle into the remaining end of the MRE050 catheter and secure with superglue.</li>
		<li>Insert a dulled 26 G needle into the MRE050 tube near the tip of the first needle, following the direction of injection flow, and secure with superglue (Figure 1 and Figure 2C). Reinforce both needles and the segment of MRE050 tube with clear epoxy (Figure 2C). This design allows injection of vehicle solution through needle 2 (26 G) after NSC injection through needle 1 (20 G) to flush the dead volume in the catheter into the brain circulation, achieving more precise control of injection volumes.</li>
		<li>Use a 20 G needle for NSC injection in order to minimize damage to the NSCs, which could adversely affect viability.</li>
	</ol>
	</li>
	<li>After construction, flush the catheters with 10 mL of double distilled water, followed by 70% ethanol, and then soak them in 70% ethanol overnight.</li>
	<li>Before the surgery, remove the catheters from 70% ethanol and flush with 10 mL of sterile PBS, and place them in an autoclaved surgical tool box for storage and transportation.</li>
	<li>Preparation of the surgical hooks
	<ol>
		<li>Cut a 1.5- 2 cm long needle shaft from a 27 G needle, and polish both ends on sandpaper until dull. Then use a small hemostatic clamp to bend the shaft into a hook at one end and a ring-shape at the other end.</li>
		<li>Insert a 10-15 cm long MRE025 catheter through the ring and secure with clear surgical tape (Figure 2D). Make 2 more hooks using the same method.</li>
		<li>Soak all hooks and catheter systems in 70% ethanol until use.</li>
	</ol>
	</li>
</ol>
2. Animal preparation: Delivery, housing, environment adaptation
<ol>
	<li>Male and female C57BL/6 mice (10-12 weeks, n=10/time point) and Wistar rats (10-12 weeks, n=10) were used in this study.</li>
	<li>House them in an environmentally controlled animal vivarium with food and water ad libitum.</li>
	<li>Allow them to adapt to the environment at least 1 week before the stroke surgery. 
	NOTE: One mouse and one rat died at 1 d after stroke surgery and one mouse was euthanized at 3 d post-stroke prior to NSC injection for humane reasons because of severe paralysis.</li>
</ol>
3. Culture of mouse and rat neural stem cells (NSCs)
NOTE: NSCs were isolated and cultured following an established protocol20.
<ol>
	<li>Mouse
	<ol>
		<li>Isolate wildtype (WT) and GFP-labeled NSCs from the E18 embryonic cortex from timed-pregnancy female C57BL/6 mice mated with GFP-positive male mice (B6 ACTb-EGFP). To identify GFP (+) embryos, observe the harvested embryos on a fluorescence microscope using the FITC channel. GFP (+) embryos yield green fluorescence signal while WT embryos show only weak auto-fluorescence (Figure 3A).</li>
	</ol>
	</li>
	<li>Rat
	<ol>
		<li>Isolate NSCs from the subventricular zone (SVZ) of young adult WT rats. Label them with DiI just prior to injection following manufacturer&#8217;s instructions21.</li>
	</ol>
	</li>
	<li>Culture mouse or rat NSCs until they develop into neurospheres, and passage them when the diameter of sphere reaches around 100 &#181;m (Figure 3B). Use the NSCs for injection between passages 3 and 5.</li>
	<li>Verify their stem cell properties using an embryonic stem cell marker panel (Figure 3C).</li>
	<li>On the day of injection, collect NSC spheres and dissociate with the cell detachment solution, suspend in calcium- and magnesium-free PBS to a concentration of 107 cells/mL, and place on wet ice until injection.</li>
</ol>
4. Surgical preparation
<ol>
	<li>Before surgery, mark a dot on the commercial MCAO suture with a silver marker pen at 9 mm (for mouse) or 15 mm (for rat) from the tip for in-surgery reference of insertion length. Autoclave the surgical tools (scissors, forceps) and instruments before each surgery, and heat sterilize them in a glass bead sterilizer between operations.</li>
	<li>Induce anesthesia in animals with 5% isoflurane via inhalation and maintain anesthesia with 1-2% isoflurane. Evaluate the depth of anesthesia through observation of general conditions (breathing pattern, whisker movement, and spontaneous body correction posture), corneal reflex and response to toe pinch.</li>
	<li>Lay animal supine on a heating pad, and prepare the surgical site on the animal by clipping and scrubbing with betadine solution followed by 70% ethanol. Protect the animal&#8217;s eyes from drying by applying ophthalmological ointment (e.g., artificial tear ointment) during surgery.</li>
	<li>Have surgeons thoroughly scrub their hands with a bacteriocidal scrub and wear a mask, sterile gloves, and a clean lab coat.</li>
</ol>
5. Middle cerebral artery occlusion (MCAO) stroke surgery
NOTE: The surgeries to induce ischemic stroke in one hemisphere of mouse or rat are similar in that a suture is introduced into the internal carotid artery (ICA) to occlude blood flow (Figure 4)17,18,19,22. However, the artery selected for suture insertion differs based on the available operation space required for the subsequent stem cell injection. The rat has ample space in the external carotid artery (ECA) segment to permit two separate, sequential surgeries (stroke and NSC injection), but the mouse does not, requiring an alternate approach. Stroke-induced cerebral blood flow changes, brain infarct size and neurological deficits have been reported as in the authors&#8217; previous reports17,18,19.
<ol>
	<li>To induce ischemic stroke, begin both mouse and rat surgeries with a midline incision on the cervical area, and isolation of the left common carotid artery (CCA), ECA and ICA (Figure 4). Exercise caution to not stretch, displace or squeeze the CCA or vagus nerve. Since the selection of artery and surgical steps are different thereafter, MCAO surgery on the mouse and rat will be described separately.</li>
	<li>MCAO surgery on mouse (Figure 4A)
	<ol>
		<li>Place three braided 6-0 nylon sutures under the CCA (Figure 4A, step 1), and make one tight surgical knot to occlude the vessel as far from the bifurcation as possible using the proximal string (Figure 4A, step 2). Trim down the suture ends.</li>
		<li>Make a slipknot at the distal side of CCA (caution: do not over-tighten as it will be released in step 6) and one loose slipknot in between the two tightened knots (Figure 4A, step 2).</li>
		<li>Cut a small incision (~ &#188; - &#8531; of the circumference) close to the proximal knot on the CCA with microscissors (Figure 4A, step 3), and carefully insert the commercial silicone rubber coated 7-0 solid nylon suture (Figure 4A, step 4). Secure this suture with the middle string, tightening sufficiently (Figure 4A, step 5) to ensure no blood leakage from the incision and no movement of the silicone rubber coated nylon filament by the backflow from ICA, while still allowing advancement of the suture toward the ECA with a gentle push by the tweezers.</li>
		<li>Release the upper (distal) slipknot (Figure 4A, step 6) and advance the nylon suture into the ICA until its tip passes the bifurcation for 9 mm (using the silver marker on the suture as a reference). Tighten the upper two slipknots to secure the suture and prevent blood backflow.</li>
		<li>Withdraw the filament 1 h later (Figure 4A, step 7) and ligate the CCA using the middle knot to prevent bleeding (Figure 4A, steps 5-7 in reverse order, final results as seen in step 8). Release the upper knot. Close the wound with 4-0 surgical suture.</li>
	</ol>
	</li>
	<li>MCAO surgery on rat (Figure 4B)
	<ol>
		<li>Place two braided 6-0 nylon sutures under the ECA (Figure 4B, step 1), and make one tight knot at the distal end as far as possible (Figure 4B, step 2).</li>
		<li>Place vessel clips on the ICA and CCA to occlude the arterial blood flow (Figure 4B, step 3). A slipknot can be used as an alternate for a vessel clip.</li>
		<li>Make a small incision on the ECA with microscissors (Figure 4B, steps 3-4), insert a commercial silicone rubber coated 6-0 nylon filament (Figure 4B, step 5), and secure properly with a slipknot on the ECA.</li>
		<li>Release the vessel clip on the ICA, advance the filament into the ICA until the silver marker (15 mm) reaches the bifurcation (Figure 4B, step 6), and then secure the suture with the 2nd knot on the ECA (Figure 4B, step 6).</li>
		<li>After 1 h of ischemia, withdraw this filament and ligate the incision to prevent bleeding (Figure 4B, step 7), remove the vessel clip from CCA (final result as in step 8), and close the wound with 4-0 surgical suture.</li>
	</ol>
	</li>
</ol>
6. Recovery
<ol>
	<li>After stroke surgery, place animals on a heating pad until they fully regain consciousness.</li>
	<li>Provide analgesia via subcutaneous injection. Return animals to their home cages with access to water and food ad libitum.</li>
</ol>
7. Intra-arterial injection
<ol>
	<li>Wash the whole catheter with 70% ethanol and soak overnight until use. Right before the injection, connect the Luer lock of the needle with a sterile syringe, and wash the entire lumen side of the catheter system with 10 mL of sterile PBS.</li>
	<li>Time window and preparation for NSC injection 
	NOTE: Based on experience and reports from other research teams, the timing for NSC injection is crucial for survival of both subjects and exogenous NSCs. In our pilot study, injection of NSCs at early time points (within the first 6 h after reperfusion) led to higher mortality. Thus, we tested later injection time points and determined the time window between 2 d (48 h) to 3 d (72 h) after stroke is safe and tolerable for animals, and is efficient in achieving intraparenchymal distribution of NSCs. Results presented herein are from animals received NSC injection at 3 d after injury.
	<ol>
		<li>Set the syringe pump injection rate at 20 &#181;L/min for mice and 50 &#181;L/min for rats. Excessive speed or duration of the injection can result in systemic volume overload, to which mice are more vulnerable than rats.</li>
		<li>In brief, at 3 d after stroke surgery, anesthetize the animals with isoflurane and lay them supine on a heating pad.</li>
		<li>Reopen the cervical wound and expose the ECA, ICA and CCA again (Figure 5, step 1). As in the stroke surgeries, determine the injection route based on the species. Utilize the CCA for NSC injection in the mouse, and the ECA for the rat23.</li>
	</ol>
	</li>
	<li>Intra-arterial injection through the CCA in mouse
	<ol>
		<li>Place two 6-0 braided nylon sutures under the CCA. Create a loose slipknot with each of them between the bifurcation and lower knots from the previous stroke surgery (Figure 5, step 2).</li>
		<li>Tighten the upper slipknot and then make a small incision above the lower knot (Figure 5, step 3). Insert a MRE010 catheter through the incision (Figure 5, step 4) and secure with the middle knot without blocking the injection flow (Figure 5, step 5). Backflow of blood should be visible in the catheter when releasing the upper knot and adjusting the catheter position.</li>
		<li>Place a vessel clip on the ECA, inject 1 x 106 GFP-NSCs through this catheter at 20 &#181;L/min for 5 min with a syringe pump, followed by a flush with 50-100 &#181;L of PBS at the same speed.</li>
		<li>After injection, ligate the CCA above the incision with the upper slip knot and withdraw the MRE010 catheter (Figure 5, step 6). Tighten and trim the middle knot and the upper knot. Remove the vessel clip from the ECA. Refer to the final image in Figure 5, step 7.</li>
		<li>Close the wound with 4-0 surgical suture.</li>
		<li>After providing adequate recovery on a heating pad and subcutaneous analgesic injection, return animals to their home cage.</li>
	</ol>
	</li>
	<li>Intra-arterial injection through the ECA in rat
	<ol>
		<li>Temporarily occlude the ECA and CCA with vessel clips (Figure 5, step 2).</li>
		<li>Make a small incision at the proximal side of ECA (Figure 5, step 3), insert the MRE010 catheter, and secure with a knot (Figure 5, step 4).</li>
		<li>Remove both vessel clips, inject 5 x 106 NSCs in 100 &#181;L of PBS at 50 &#181;L/min for 2 min, followed by a flush with 50-100 &#181;L of PBS (Figure 5, step 5) at the same speed, using a motorized syringe pump.</li>
		<li>After injection, occlude the CCA and ECA with vessel clips again and ligate the ECA at the proximal side of the second incision after withdrawal of the injection catheter (Figure 5, step 6).</li>
		<li>Remove the two vessel clips (Figure 5, step 7) and close the wound with 4-0 surgical suture.</li>
		<li>After providing adequate recovery on a heating pad and subcutaneous analgesic injection, return animals to their home cage.</li>
	</ol>
	</li>
	<li>Histological assay
	<ol>
		<li>Collect brains from mice and rats that received ischemic stroke followed by injection of NSCs or vehicle solution after euthanasia and intracardiac perfusion with 4% paraformaldehyde at 1 d (mouse and rat), 7 d (mouse) and 30 d (mouse) after injection. Each of these four groups consisted of 5 NSC and 5 vehicle injected animals.</li>
		<li>Fix brains overnight and cryopreserve in 30% sucrose for 3 d.</li>
		<li>Embed the brains into OCT, slice&#160;at 40 &#181;m thickness, and examine the distribution of NSCs after immunostaining with cell specific markers, including glial fibrillary acidic protein (GFAP, astrocytes), Tuj1 (mature neurons), and doublecortin (DCX, immature neurons). 
		NOTE: Because of the lack of a rat strain that expresses GFP, we utilized DiI, a transient fluorescent label, for rat NSCs, which allows only relatively short-term observation. Hence, NSC distribution was only examined at 1 d after stroke in rats.</li>
	</ol>
	</li>
</ol>

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Stem cell therapy for neurological diseases is still at an early exploratory stage. One major issue is there is no established method for sufficient delivery of SCs or NSCs into the brain.
Although exogenous SCs/NSCs can be detected in the brain following intravenous (IV), intraperitoneal (IP) or intraparenchymal/intracerebral injection, each delivery approach has drawbacks. The detectable population within the brain is estimated to be very low with peripheral injection (IV or IP), representin...

Stem cell (SC) therapy holds tremendous potential as a treatment for neurological diseases, including stroke, head trauma and dementia1,2,3,4,5,6. However, an efficient method to deliver exogenous SCs to the diseased brain remains problematic2,6,7,8,9,10,11,12,13. SCs delivered through peripheral delivery routes, including intravenous (IV) or intraperitoneal (IP) injection, are subject to first-pass filtering in the microcirculation, especially in the lung, liver, spleen and muscle8,9,13,14, increasing chances of accumulation of cells in non-target areas. The invasive intracerebral injection method results in localized brain tissue damage and a very restricted distribution of SCs near the injection site2,6,8,14,15,16. We have recently established a catheter-based intra-arterial injection method to deliver exogenous neural SCs (NSCs), which is described here applied in a rodent model of focal ischemic stroke. We induce transient (1 h) ischemia-reperfusion injury in one hemisphere using a silicone rubber coated filament to occlude the left middle cerebral artery (MCA) in the mouse or rat17,18,19. In this model we have reproducibly observed approximately 75-85% depression of cerebral blood flow (CBF) in ipsilateral hemisphere with Laser Doppler or Laser speckle imaging17,19, yielding consistent neurological deficits17,18,19.
For time-saving purposes, the video is set to play at twice the normal speed and routine surgical procedures such as skin preparation and wound closure with suture and the use and setup of the motorized syringe pump are not presented. The method of intra-arterial delivery of NSCs is demonstrated in the context of the middle cerebral artery occlusion (MCAO) model of experimental stroke in rodents. Therefore, we include the transient ischemic stroke procedure in order to later demonstrate how the second surgery, the intra-arterial injection, is performed using the previous surgical site on the same animal. The feasibility of intra-arterial NSC delivery in rodent stroke models is demonstrated by assessing the distribution and survival of exogenous NSCs. The efficacy of NSC therapy to attenuate brain pathology and neurological dysfunction will be reported separately.

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			<td>Invitrogen</td>
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			<td>MRE025 catheter</td>
			<td>Braintree Scientific</td>
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			<td>preparation of injection catheter</td>
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			<td>MRE050 catheter</td>
			<td>Braintree Scientific</td>
			<td>MRE050</td>
			<td>preparation of injection catheter</td>
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			<td>Nu-Tears Ointment</td>
			<td>NuLife Pharmaceuticals</td>
			<td>Nu-Tears Ointment</td>
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			<td>GenieTouch</td>
			<td>surgery</td>
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			<td>Tuj1 antibody</td>
			<td>Millipore</td>
			<td>MAb1637</td>
			<td>immunostaining</td>
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			<td>two-component 5 minute epoxy</td>
			<td>Devcon</td>
			<td>20445</td>
			<td>preparation of injection catheter</td>
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			<td>Vannas spring scissors</td>
			<td>Fine Science Tools</td>
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			<td>Zeiss microscope</td>
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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.

GFP-labeled NSCs were readily detected in the ischemic brain, mostly in the ipsilateral hemisphere, especially in the penumbra and along the injury rim (Figure 6). The examiner was single-blinded during imaging and analysis.
For example, at 1 d after injection, NSCs were detected within the mouse hippocampus. A subset of NSCs showed co-expression of the immature neuron marker DCX in the dentate gyrus even at this early time point (Figure 6A</s...

Watch this Scientific Journal Video about Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia at JoVE.com

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

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 ...

intra-arterial-delivery-neural-stem-cells-to-rat-mouse-brain

Research

JoVE Journal

Neuroscience

10.2K Views.  Shaanxi University of Chinese Medicine. 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.

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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

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 ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.