The overall goal of this subpial AAV9 delivery technique is to achieve a potent spinal parenchymal transgene expression throughout the entire length of spinal chord in adult mice. This method can facilitate studies targeted to manipulate spinal gene expression and help to understand the mechanism of several spinal neurodegenerative disorders including amyotrophic lateral sclerosis, spinal traumatic injury, or chronic pain. The main advantage of this technique is that highly potent transgene expression is achieved through the spinal gray and white matter in multiple spinal segments, which are adjacent to subpial AAV9 injected segment.
Visual demonstration of this method is critical because the dura-opening step and placement of subpial needle are difficult to learn without visual demonstration. Demonstrating the procedure will be Takahiro Tadokoro, a post-doctorate fellow in our laboratory. 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 chord. The dura is now visible, use a 30 gauge stainless steel needle to make a one to two 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 five millimeters. If cervical subpial injections are to be performed, immobilize the head of the mouse by placing the head into a stereotaxic frame using ear bars and use scissors to make a 1.5 to two centimeter incision in the dorsal neck skin to expose the C1 and C2 segments and occipital bone.
Then, shave a portion of exposed occipital bone using a dental drill and remove the bone fragments using forceps. Then, use a 23 gauge stainless steel needle and forceps to remove the atlanto occipital membrane of the cisterna magna. Use cotton swabs to clean the incision site of any tissue and bone debris, then cut open the dura, making a two to three millimeter incision.
Mount the 34 gauge pia-penetrating needle and 36 gauge injection needle on to the Z arms of two separate X, Y, Z manipulators using glass capillary holders. Next, use a 50 microliter microsyringe connected with PE10 or PE20 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 eight to ten X magnification, use the X arm to lower the pia-penetrating needle into the pia to a depth of one millimeter. Keep the angle of the penetrating needle at five 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 in to 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 two to three millimeters, then use the 50 microliter microsyringe 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 foromonofilament suture and surgical clips.
Allow the animal to recover on a heating pad before housing in a clean cage. Two bilateral injections of AAV9-UBI-GFP were delivered into the upper lumbar subpial space and the animals were profusion fixed 14 days after AAV9 delivery. Intense GFP expression is seen in the gray matter located within the dotted area and white matter outside the dotted area.
This extends from the lumbar to the upper thoracic segments in animals injected with three plus three microliters of AAV9. Animals injected with a lower volume of AAV9 have less intense expression of GFP. The following images depict potent retrograde and anterograde AAV9-UBI-GFP mediated GFP expression in brain motor and sensory centers.
This low-power image clearly shows the presence of intense GFP positivity in the cervical spinal cord, medulla oblongata, cerebellum, and motor cortex. This high-power image taken from a sagittal brain section shows the presence of GFP florescence in neurons in the reticular formation, nucleus ruber, and axons of the spinocerebellar tract. This lower-power image taken from a coronal brain section demonstrates the presence of GFP florescence in pyramidal neurons in the motor cortex and in the terminals of the spinothalamic tract in areas of the reticular thalamic nuclei.
Finally, this high-power image demonstrates an intense GFP expression in pyramidal neurons in the motor cortex. Once mastered, this technique can be completed in 30 to 40 minute. The use of this technique by researchers in the field of neuroscience will be an effective tool to study the role of specific genes in the evolution of spinal degenerative disorders.
It will also help to explore potential treatment efficacy by suppressing mutated genes known to be linked with progressive spinal neuronal degeneration as in the familiar form of amyotrophic lateral sclerosis.
