Subpial Gene Delivery in the Mouse Spinal Cord to Evaluate Gene Expression

Take an anesthetized mouse, shave its back, and disinfect the skin.

Incise the skin, detach the muscles from the vertebrae, and retract them using clamps.

Drill into the vertebral laminae to create cracks, then remove the bone fragments to expose the dura mater, the outer spinal cord membrane.

Using a needle, create a dural opening and puncture the underlying pia mater to enter the subpial space for direct access to the spinal tissue.

Take a delivery needle containing a suspension of recombinant adeno-associated virus carrying a fluorescent protein-expressing gene and inject the suspension.

Withdraw the needle, suture the muscles and skin, and allow the mouse to recover.

The virus binds to receptors on neurons and glial cells and gets endocytosed.

Once inside the nucleus, the virus triggers gene expression to produce fluorescent proteins.

The subpial gene delivery model is now ready to assess target gene expression in the spinal cord.

Begin by properly anesthetizing the animal for surgery. Isoflurane is used here. Ensure the absence of a paw pinch response before proceeding. Then cover the eyes with ophthalmic ointment. Next, shave the back of the animal with clippers, and clean the skin with 2% chlorhexidine.

If lumbar subpial injections are to be performed, cut the skin overlying the T8 to L1 vertebrae with a scalpel. And then use scissors to detach the paravertebral muscle from the T10 to T12 spinal vertebrae. Mount the animal into a standard stereotaxic frame using mouse spinal clamps. Next, use a dental drill to shave both sides of the lamina of the T10 to T12 vertebrae until cracks appear.

Then, use forceps to remove cracked bone fragments and expose the dorsal surface of the lumbar spinal cord. The dura is now visible. Use a 30 gauge stainless steel needle to make a 1 to 2-millimeter incision into the dura.

It is important to carefully cut open the dura to avoid injury to the underlying pia membrane.

Now, grasp the edge of the cut dura with forceps and extend the dura opening for up to 5 millimeters. Mount the 34 gauge pia penetrating needle and 36 gauge injection needle onto the Z arms of two separate XYZ manipulators using glass capillary holders.

Next, use a 50-microliter micro syringe connected with PE-10 or PE-20 tubing to load AAV9-UBI-GFP virus. After the virus is loaded, connect the end of the tubing to the injection needle. Next, while looking through a surgical dissecting scope set to 8 to 10x magnification, use the X arm to lower the pia penetrating needle into the pia to a depth of 1 millimeter. Keep the angle of the penetrating needle at 5 to 10 degrees, relative to the tissue surface.

After opening the pia, use the X arm to remove the pia penetrating needle from the subpial space. Note the penetrated site using a landmark, such as a blood vessel. Move the X, Y, and Z arms of the second manipulator to position the tip of the AAV9 virus-loaded injection needle into the pia-penetrated site.

Then, after inserting the tip of the injection needle about 0.5 millimeters into the subpial space, use the Z arm to lift the pia about 0.3 millimeters, then use the X arm to continue advancing the needle horizontally into the subpial space. Advance the needle until it enters the subpial space at a depth of about 2 to 3 millimeters.

Then, use the 50-microliter micro syringe to inject the AAV9-UBI-GFP virus into the subpial space. Remove the injection needle from the subpial space after the AAV9-UBI-GFP injection is complete. Close the muscle and skin using fluoro monofilament suture and surgical clips. Allow the animal to recover on a heating pad before housing in a clean cage.