Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

The goal of this surgical approach is to directly apply treatments at the level of the neuronal cell body, and assess axon regeneration following therapeutic intervention, without having to perform a more damaging lesion within the central nervous system. This method can help answer key questions in the field of axon regeneration, such as, can therapeutically treated axons regenerate in the spinal cord, and possibly reach the brain stem targets? The main advantage of this technique is that researchers can examine CNS axon regeneration without having to directly injure the spinal cord.

This reduces post-surgical complication, and improves animal welfare. Begin by properly anesthetizing the animal. Confirming a surgical plane of anesthesia by the absence of a reaction to a toe pinch.

Then weigh the animal and record the preoperative weight. For a surgical DRG injection, shave the fur on the neck from between the ears to just below to the scapuli. Disinfect the shaved area with an antiseptic product.

Inject two milliliters of saline subcutaneously on the flank of the animal, along with an appropriate dose of analgesic and antibiotic. Apply eye ointment to both eyes to prevent corneal drying during the procedure. Place the animal in a spinal stereotaxic frame, and place a heating pad at 37 degrees Celsius underneath the animal.

Begin the surgery by locating the prominent C2 and T2 spinous processes over the skin. Then use a number 10 scalpel to make a skin incision between the C2 and T2 spinous processes. Once the skin is opened, a white fibrous tissue midline should be visible on the first layer of muscle, which has a jellylike texture.

Make a similar sized incision on the first layer of muscle along the white midline. Do not go beyond the first prominent T2 spinous process, as there is a major blood vessel located near there. Retract the first layer of muscle using two retractors, one placed rostrally, and one caudally.

The second layer of muscle with a striated appearance should now be visible. Locate the midline of the second layer of muscle, where two longitudinal muscles can be observed, connected by a thin, membranous tissue. Then use a pair of micro scissors to dissect the membranous tissue and separate the two longitudinal muscles.

Adjust the retractors to expose the third layer of thin muscle covering the spine. The spinous processes can be felt by lightly touching with a pair of forceps over the third layer of muscle. Use micro scissors to make a small incision on the third layer of muscle, and gently scrape off the muscle from the bone, using a curette or scalpel in a sideways manner, to clearly expose the vertebrae.

To expose the left C5 to C8 DRG's, use a pair of fine bone shears to perform left hemi-laminectomy on the C4 to T1 vertebrae, by carefully removing part of the lamina and pedicle. Once enough of the DRG has been exposed for injection, prepare the syringe by placing the virus-filled microliter syringe fitted with a custom-made 33 gauge blunt needle onto the stereotaxic syringe holder. Next, use a 30 gauge beveled needle to make a small, superficial opening on each of the targeted DRG to assist with the insertion of the injection needle.

Gently adjust the stereotaxic coordinates to insert the 33 gauge needle into the center of the DRG. Ideally, the needle should be placed at the very center of the DRG to ensure uniform diffusion of the injector solution throughout the DRG. Once the needle is inserted, use the infusion syringe pump to inject one microliter of the virus into each DRG.

During the injection, the DRG will slowly change color if the virus solution contains a colored dye. Do not over-insert the needle, as this may cause fluid to leak out from the ventral side of the DRG. Should leakage occur during injection, adjust the position of the needle immediately.

Three minutes after the end of the injection, withdraw the injection needle. To perform a concurrent C5 to C8 dorsal root crush injury, crush each root three times for 10 seconds, using a pair of fine-tipped forceps. Completely oppose the ends of the forceps.

A white line in the tissue should appear at the crush site. Following the surgeries, ensure that there is no bleeding or small pieces of bone fragments left at the incision site before closing up. If preferred, place a small piece of surgical absorbable sponge on top of the exposed spinal cord and DRG.

Allow the third layer of the muscle to retract back naturally onto the spine, without suturing. Loosely suture the two longitudinal muscles on the second layer with absorbable 6-0 suture material. Suture the first layer of muscle with absorbable 6-0 suture material.

Suture the skin with absorbable 5-0 suture material. If heavy bleeding occurred during the surgery, subcutaneously inject one to two milliliters of saline to replenish the loss of fluid from the animal, as permitted under local regulations. Provide edible hydrating gel, and allow the animal to recover fully from anesthesia.

Frequently monitor the animal for at least one hour before returning to the holding area. The following images show recovery of post-surgical animals at day one, day seven, after skin suture removal, and at day 14. One week prior to tissue collection, properly anesthetize the animal as before, and then stabilize the left forepaw by taping the limb to the table.

Before injection of CTB, use a 30 gauge beveled needle to make a small, superficial opening on the skin to assist with the insertion of the injection needle. Then use a microliter syringe fitted with a custom-made 33 gauge short needle, to slowly inject one microliter of 1%CTB subcutaneously into the center of the glabrous footpad and four digits. Allow the animal to recover fully from anesthesia, before returning to the holding area.

This image shows the result of the DRG injection without dorsal root crush injury. GFP positive cell bodies and axons in the spinal cord are clearly visible four weeks after the injection of AAV5GFP. This higher magnification image shows the axons in the dorsal column and dorsal horn.

The GFP labeled cell bodies in the DRG are seen here. This image shows CTB positive axon terminals in the Cuneate nucleus one week following CTB injection. The following images show results of a complete dorsal root crush injury.

As seen here, GFP labeled axotomized sensory axons can regenerate up to the dorsal root entry zone, but not into the spinal cord after injury. This is an image of the Cuneate nucleus in the same animal. CTB positive axon terminals are absent following a complete dorsal root crush injury.

The presence of labeled axons in the spinal cord after injury represents either incomplete injury or regeneration. The presence of CTB positive terminals in the Cuneate nucleus suggests incomplete injury, while their absence suggest complete injury and potentially partial regeneration into the spinal cord. While attending this procedure, it is important to remove just the right amount of vertebrae bones to expose the DRG and dorsal root, without inducing any unwanted damage, especially to the spinal cord.

Otherwise, this may result in unexpected post-surgical complications. After watching this video, you should have a good understanding of how to perform a DRG injection. You can incorporate in other techniques such as central spinal injury, electrophysiology, behavior testing, and anatomical analysis to examine axon regenerations.