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.

<table><tbody><tr><td>20 G needle</td><td>Becton &amp;amp; Dickinson</td><td>BD PrecisionGlide 305175</td><td>preparation of injection catheter</td></tr><tr><td>26 G needle</td><td>Becton &amp;amp; Dickinson</td><td>BD PrecisionGlide 305111</td><td>preparation of injection catheter</td></tr><tr><td>27 G needle</td><td>Becton &amp;amp; Dickinson</td><td>BD PrecisionGlide 305136</td><td>preparation of injection catheter</td></tr><tr><td>4-0 NFS-2 suture with needle</td><td>Henry Schein Animal Health</td><td>56905</td><td>surgery</td></tr><tr><td>6-0 nylon suture</td><td>Teleflex/Braintree Scientific</td><td>104-s</td><td>surgery</td></tr><tr><td>Accutase</td><td>STEMCELL Technologies</td><td>7922</td><td>cell detachment solution</td></tr><tr><td>blade</td><td>Bard-Parker</td><td>10</td><td>surgery</td></tr><tr><td>Buprenorphine-SR Lab</td><td>ZooPharm</td><td>Buprenorphine-SR Lab&amp;reg;</td><td>analgesia (0.6-1 mg/kg over 3 d)</td></tr><tr><td>Calcium/magnisum free PBS</td><td>VWR</td><td>02-0119-0500</td><td>NSC dissociation</td></tr><tr><td>DCX antibody</td><td>Millipore</td><td>AB2253</td><td>immunostaining</td></tr><tr><td>GFAP antibody</td><td>Invitrogen</td><td>180063</td><td>immunostaining</td></tr><tr><td>Isoflurane</td><td>Henry Schein Animal Health</td><td>50562-1</td><td>surgery</td></tr><tr><td>MCAO filament for mouse</td><td>Doccol</td><td>702223PK5Re</td><td>surgery</td></tr><tr><td>MCAO filament for rat</td><td>Doccol</td><td>503334PK5Re</td><td>surgery</td></tr><tr><td>MRE010 catheter</td><td>Braintree Scientific</td><td>MRE010</td><td>preparation of injection catheter</td></tr><tr><td>MRE025 catheter</td><td>Braintree Scientific</td><td>MRE025</td><td>preparation of injection catheter</td></tr><tr><td>MRE050 catheter</td><td>Braintree Scientific</td><td>MRE050</td><td>preparation of injection catheter</td></tr><tr><td>Nu-Tears Ointment</td><td>NuLife Pharmaceuticals</td><td>Nu-Tears Ointment</td><td>eye care during surgery</td></tr><tr><td>S&amp;amp;T Forceps - SuperGrip Tips JF-5TC Angled</td><td>Fine Science Tools</td><td>00649-11</td><td>surgery</td></tr><tr><td>S&amp;amp;T Forceps - SuperGrip Tips JF-5TC Straight</td><td>Fine Science Tools</td><td>00632-11</td><td>surgery</td></tr><tr><td>Superglue</td><td>Pacer Technology</td><td>15187</td><td>preparation of injection catheter</td></tr><tr><td>syringe pump</td><td>Kent Scientific</td><td>GenieTouch</td><td>surgery</td></tr><tr><td>Tuj1 antibody</td><td>Millipore</td><td>MAb1637</td><td>immunostaining</td></tr><tr><td>two-component 5 minute epoxy</td><td>Devcon</td><td>20445</td><td>preparation of injection catheter</td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>15000-08</td><td>surgery</td></tr><tr><td>vascular clamps</td><td>Fine Science Tools</td><td>00400-03</td><td>surgery</td></tr><tr><td>Zeiss microscope</td><td>Zeiss</td><td>Axio Imager 2</td><td>microscopy</td></tr></tbody></table>

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

10.3K Views.  Shaanxi University of Chinese Medicine. Beroerte is de belangrijkste oorzaak van sterfte en invaliditeit wereldwijd. Hoewel trombose en chirurgische verwijdering van cerebrale vasculaire occlusie kan worden uitgevoerd bij bepaalde patiënten, zijn er nog steeds een dringende behoefte aan effectieve therapieën tegen een beroerte. Hier demonstreren we een katheter-gebaseerde, intra-arteriële levering van stamcellen aan het ischemische halfrond als een potentieel regime voor beroertetherapie.Eerst introduceren we hoe we de in...

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

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Developmental Biology

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Medicine

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

Common Carotid Artery Repair After Injection in a Mouse Model 

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

Author Spotlight: Advancing Glioblastoma Treatment Through Intraarterial Delivery Strategies for Oncolytic Viruses and MicroRNAs

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

Cancer Research

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Rat "Brain Milking" for Neural Stem and Oligodendrocyte Progenitor Cell Isolation

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Author Spotlight: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

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

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