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

Stroke is the leading cause of mortality and disability worldwide. Although thrombolysis and surgical removal of cerebral vascular occlusion can be performed in select patients, there are still an urgent need for effective therapies against stroke. Here, we demonstrate a catheter-based, intra-arterial delivery of stem cells to the ischemic hemisphere as a potential regimen for stroke therapy.

First, we introduce how to prepare the injection catheter and surgical hooks. We designed two types of injection catheters for intra-arterial injection. Design one is used for injection of a solution or suspension that does not require flushing of the tube.

Design two allows injection of neural stem cells, followed by a flush of the dead volume to deliver the full volume. To build a set of injection catheters, you will need 26 gauge and 20 gauge needles, a 10 to 15 centimeter length of MRE 25 catheter and three centimeter length of MRE 50 and MRE 10 catheters. Cut off both needle tips and polish the metal end on sandpaper to reopen the tube and smooth the edges and then flush with distilled water to clean out the bore and wash off debris.

The figure shows a closed-end of a 20 gauge needle after clipping off the needle tip, which is reopened with a smooth bore after polishing. Under a microscope, insert the 20 gauge needle into the end of the MRE 50 tubing. Bend the 26 gauge needle and then penetrate the tubing below the end of the 20 gauge needle tip.

Secure the 26 gauge needle with super glue. Then embed the two needle hubs and the tubing and epoxy to build a solid block to enhance strength. You will also need three surgical hooks to hold the tissue in position for better exposure of the surgical sites.

Cut a one and a half to two centimeter long needle shaft from a 27 gauge needle and polish both ends on sandpaper until dull. Use a small hemostatic clamp to bend the shaft into a hook at one end and a ring-shaped at the other end. Insert a 10 to 15 centimeter long MRE 25 catheter through the ring and secure with clear surgical tape.

Make two more hooks using the same method. The hooks and catheter system should be soaked overnight in seventy percent ethanol for sterilization. All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee at the University of Kentucky.

We use ENT embryonic mouse brain to culture GFP, positive neural stem cells. GFP positive embryos can be readily visualized in the FITC channel on a fluorescent microscope. While wild-type embryos show no fluorescence using the same exposure.

The autofluorescence of wild-type embryos is visible only when the exposure time is increased 100 times. Embryonic cortex is collected and used to establish neural stem cell cultures following the manufacturer's protocol. These neural stem cells can form neurospheres and can be passage over 10 generations and they can differentiate into glia are neurons.

Panel of embryonic stem cell markers such as nanog, SSEA1, Oct4 and Sox3 can be used to identify the stem cell properties while in neurospheres. Neurospheres were cultured in media, following the manufacturer's protocol and use between passages three and five for neural stem cell delivery. Ischemic stroke was induced in the left hemisphere for both mice and rats, using a filament based, middle cerebral artery occlusion, MCAO with some modifications depending on the species.

In mice, the filament is inserted through the common carotid artery, CCA and advanced into the internal carotid artery, ICA. While in rats, the filament is introduced through the external carotid artery, ECA into the ICA because of the larger working space. Here we demonstrate the mouse surgery.

After making a midline incision along the neck, separate the underlying tissues using blunt dissection. Use the three microsurgical hooks made from 27-gauge needle shafts to achieve a better visual field. The black square indicates the surgical area for the next step.

The muscle layer above the left CCA is carefully open to expose the left CCA and vagus nerve. Follow the CCA until you reach and identify the bifurcation. Caution should be exercised not to stretch, displace or squeeze this CCA or vagus nerve.

Carefully open the clear connective tissue, covering the CCA and vagus nerve. Free the space underneath this CCA with a tip of the forceps and isolate the CCA stem as much as possible. Thread a double-stranded six O braided nylon surgical suture underneath the CCA.

Cut the middle point to leave to sutures underlying the CCA. Place another nylon suture under the CCA. Tie a slipknot on the upper suture, keeping it loose.

Separate the two lower sutures. Tie a slip knot with the middle suture, keeping it loose for now. Tie a surgeon's knot with a lower suture and move it to the proximal end as far as possible.

Trim down the suture ends. Further expose the CCA beyond the bifurcation. Push the upper loose slipknot to the bifurcation and tighten it to prevent backflow of blood.

Lay the silicon rubber coated 70 nylon MCAO suture next to the CCA. Using micro scissors, cut a small incision on the CCA, next to the lower knot. Insert the tip of the MCAO suture into the CCA through this incision and advance it to the upper knot.

Adjust the position of the middle knot and tighten it until it can hold the MCAO suture in position. Do not excessively tighten the knot. Release the upper knot, but make another loose slip knot at the same location.

Advance the MCAO suture into the ICA. Confirm the MCAO suture passes the bifurcation into the ICA. The path of the suture should be relatively straight, entering deeper into the tissue as it advances.

Check the silver marker on the suture end. Make sure it reaches the bifurcation. Check the direction of the MCAO suture again.

Adjust upper and middle knots to ensure no blood leakage. Further advance the MCAO suture, tighten the two upper knots to secure the MCAO suture in place. Start recording time for one hour of ischemia.

After one hour of ischemia, we anesthetize the animal and reopen the skin wound. Expose the surgical area. Locate upper and middle slipknots.

Gently release the upper knot. Gently pull out the MCAO suture until the white silicone rubber part reaches the middle knot. Tighten the upper knot to hold the MCAO suture in place to avoid bleeding.

Gently release the middle slipknot. Pull out the MCAO suture until its end passes the upper knot. Tighten the upper knot.

Place the middle slipknot right above the incision on the CCA. Tighten the middle slipknot. Remove the upper knot.

Trim suture ends of the middle knot. Close the skin wound and allow the animal to recover. Based on our experience, neural stem cells safely injected through this arterial route between one and three days after stroke.

Again, there are minor variations between mouse and rat surgeries for neural stem cell injection. And here we demonstrate the mouse surgery. On day three post-stroke, anesthetize the animal, reopen the wound and re-expose the CCA.

Lay a double-stranded surgical suture under the CCA and cut it at the midpoint to get two sutures. Place the two sutures properly. Make a slipknot with the upper suture.

Place it right below the bifurcation. Tighten the upper slipknot. Make another slipknot with the middle suture.

Cut a small incision right above the trimmed knot remaining from the stroke surgery. Trim the tip of the MRE 10 catheter with an angle of approximately 45 degrees. Use this sharp end to enter the incision on the CCA.

Be careful to properly adjust the entry angle and the force used to insert the catheter. Otherwise, the catheter may not enter the CCA or may puncture it. Insert the catheter and advance until the tip reaches the upper knot.

Tighten the middle slipknot to hold the catheter in place and ensure no blood linkage. Release the upper knot. Make a slip knot with the upper suture.

Adjust the angle of the catheter within the CCA and the positions of both knots until a quick backflow of blood is visible, indicating successful communication between the catheter and CCA flow. Start the syringe pump. The blood in the catheter will be flushed back into the CCA.

Terminate the injection after five minutes. Ligate the upper knot. Release the middle knot and withdraw the catheter.

Ligate the middle knot. Trim all knots. Close the wound and allow the animal to recover.

Following intra-arterial injection, GFP labeled neural stem cells are mostly found in the ipsilateral hemisphere. Here, we show the distribution in the hippocampal dentate gyrus at one day after injection. In the ipsilateral hemisphere, some of the GFP positive neural stem cells localized in the cortex and striatum, express double cort, a marker of immature neurons.

These are representative images from sham and stroke animals with or without neural stem cell injection at seven days after injection. At 30 days after delivery, only minor auto fluorescence can be detected through FITC channel in the animals that receive vehicle injection. While in stem cells injected animals, GFP labeled neural stem cells can be found in the injured brain with expression of various cell markers like the gleo marker GFP or the neuronal marker tujuan.

The white arrows in each image indicate neural stem cells expressing GFP or tujuan markers respectively. In conclusion, intra-arterial injection is a feasible method for delivery of neural stem cells for stroke therapy. Neural stem cells can survive and differentiate into different cell types within the injured brain.

And finally, the intra-arterial injection method introduced here can also be used for delivery of solutions and suspensions.