Modeling Sensory Axon Regeneration in a Rat Using Dorsal Root Ganglion Injection and Dorsal Root Crush Injury

Begin with an anesthetized rat.

Make a midline skin incision between the cervical C2 and thoracic T2 vertebrae.

Next, incise along the white fibrous tissue midline and retract the first muscle layer.

Dissect the thin membranous tissue at the midline of the second muscle layer, then retract the striated longitudinal muscles.

Finally, incise the third muscle layer to expose the vertebral column.

Remove the lamina and pedicle overlying the left C4 to T1 vertebrae to reveal the C5 to C8 dorsal root ganglia or DRGs.

Inject a recombinant adeno-associated viral suspension into the DRGs.

The virus encodes a growth-promoting protein that aids in axonal regeneration.

Using forceps, crush the C5 to C8 dorsal roots to sever axonal connections from the DRG to the spinal cord, creating a controlled axonal injury.

The model is ready to study virus-mediated axonal regeneration. 

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 jelly-like texture.

Make a similar sized incision on the first layer of muscle along the white midline. Do not go beyond the 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 muzzle 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 DRGs, use a pair of fine rongeurs to perform a left hemilaminectomy 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 injected solution throughout the DRG.

Once the needle is inserted, use the infusion syringe pump to inject 1 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 the 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.