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

1. 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.
   1. 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.
2. Design 1: Insert a 3-4 cm length MRE010 catheter into a 15 cm length MRE025 catheter and secure with superglue.
   1. 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).
   2. 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).
3. Design 2: Insert a 3-4 cm length MRE010 catheter into a 15 cm length MRE025 catheter and secure with superglue.
   1. 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.
   2. 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.
   3. Use a 20 G needle for NSC injection in order to minimize damage to the NSCs, which could adversely affect viability.
4. After construction, flush the catheters with 10 mL of double distilled water, followed by 70% ethanol, and then soak them in 70% ethanol overnight.
5. 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.
6. Preparation of the surgical hooks
   1. 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.
   2. 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.
   3. Soak all hooks and catheter systems in 70% ethanol until use.

2. Animal preparation: Delivery, housing, environment adaptation

1. 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.
2. House them in an environmentally controlled animal vivarium with food and water ad libitum.
3. 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.

3. Culture of mouse and rat neural stem cells (NSCs)

NOTE: NSCs were isolated and cultured following an established protocol²⁰.

1. Mouse
   1. 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).
2. Rat
   1. Isolate NSCs from the subventricular zone (SVZ) of young adult WT rats. Label them with DiI just prior to injection following manufacturer’s instructions²¹.
3. Culture mouse or rat NSCs until they develop into neurospheres, and passage them when the diameter of sphere reaches around 100 µm (Figure 3B). Use the NSCs for injection between passages 3 and 5.
4. Verify their stem cell properties using an embryonic stem cell marker panel (Figure 3C).
5. 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.

4. Surgical preparation

1. 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.
2. 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.
3. 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’s eyes from drying by applying ophthalmological ointment (e.g., artificial tear ointment) during surgery.
4. Have surgeons thoroughly scrub their hands with a bacteriocidal scrub and wear a mask, sterile gloves, and a clean lab coat.

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’ previous reports^17,18,19.

1. 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.
2. MCAO surgery on mouse (Figure 4A)
   1. 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.
   2. 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).
   3. Cut a small incision (~ ¼ – ⅓ 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.
   4. 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.
   5. 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.
3. MCAO surgery on rat (Figure 4B)
   1. 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).
   2. 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.
   3. 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.
   4. 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 2^nd knot on the ECA (Figure 4B, step 6).
   5. 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.

6. Recovery

1. After stroke surgery, place animals on a heating pad until they fully regain consciousness.
2. Provide analgesia via subcutaneous injection. Return animals to their home cages with access to water and food ad libitum.

7. Intra-arterial injection

1. 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.
2. 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.
   1. Set the syringe pump injection rate at 20 µL/min for mice and 50 µL/min for rats. Excessive speed or duration of the injection can result in systemic volume overload, to which mice are more vulnerable than rats.
   2. In brief, at 3 d after stroke surgery, anesthetize the animals with isoflurane and lay them supine on a heating pad.
   3. 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.
3. Intra-arterial injection through the CCA in mouse
   1. 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).
   2. 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.
   3. Place a vessel clip on the ECA, inject 1 x 10⁶ GFP-NSCs through this catheter at 20 µL/min for 5 min with a syringe pump, followed by a flush with 50-100 µL of PBS at the same speed.
   4. 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.
   5. Close the wound with 4-0 surgical suture.
   6. After providing adequate recovery on a heating pad and subcutaneous analgesic injection, return animals to their home cage.
4. Intra-arterial injection through the ECA in rat
   1. Temporarily occlude the ECA and CCA with vessel clips (Figure 5, step 2).
   2. 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).
   3. Remove both vessel clips, inject 5 x 10⁶ NSCs in 100 µL of PBS at 50 µL/min for 2 min, followed by a flush with 50-100 µL of PBS (Figure 5, step 5) at the same speed, using a motorized syringe pump.
   4. 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).
   5. Remove the two vessel clips (Figure 5, step 7) and close the wound with 4-0 surgical suture.
   6. After providing adequate recovery on a heating pad and subcutaneous analgesic injection, return animals to their home cage.
5. Histological assay
   1. 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.
   2. Fix brains overnight and cryopreserve in 30% sucrose for 3 d.
   3. Embed the brains into OCT, slice at 40 µm 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.