Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Transcript

The overall goal of this procedure is to provide surgical access of a lumbar dorsal root ganglion in swine for intraganglionic injection of a solution using a convection-enhanced delivery needle. This method can help answer key questions in the basic and translational neuroscience fields regarding the effect of a new drug on DRG cells when delivered directly to DRG parenchyma. The main advantage of this technique is that it provides the necessary technical data to confirm that a solution was successfully delivered to DRG and to compare results between biological replicates.

This technique will be the reference standard for intraganglionic delivery. The ultimate goal is image-guided delivery. With this reference standard, we can uniquely characterize the therapeutic efficacy of the agents and the delivery efficiency.

Anesthetize a swine for surgery and then position it in a modified large humane animal sling with a padded abdominal aperture. Ensure that the metal parts are padded to avoid creating short circuits. To prepare the skin overlying the lumbar spine, scrub the region according to the manufacturer's instructions using a liberal amount of solution.

Continue the surgical scrub towards the midline coronal plane past the marked transverse processes using the same technique. Next, place surgical towels neatly around the incision site over the midline along the marked lumbar spinous processes. Then, drape the entire region well beyond the periphery of the operative site.

Once fully prepared for the surgery, start by using a number-15 scalpel to open an eight to 12 centimeter midline sagittal incision through the incise drape directly posterior to the spinous processes. Maintain hemostasis using gauze tamponades and monopolar electrosurgery. Next, use monopolar electrosurgery to dissect the subcutaneous tissue and fat overlying the thoracal lumbar fascia.

Next, palpate the spinous processes deep to the thoracal lumbar fascia and cut the fascia along the midline to expose the supraspinous ligament spanned between the spinous processes. At this point, the incision may be lengthened in either direction to make three spinous processes are fully visible. Now, make a two-millimeter-deep parasagittal incision through the supraspinous ligament posterior to each spinous process using a number-15 blade.

Make the incisions along the left third of the posterior surface of the spinous processes. Then, use a five-millimeter Freer elevator to gently release the supraspinous ligament at each level along each incision. Now, identify the subperiosteal plane and dissect within that plane along the lateral surface of each spinous process.

Then, perform three subperiosteal dissections in a parallel fashion to make a gentle and even dissection. Next, identify the lamina at each level and perform a subperiosteal dissection laterally. Extend the dissection to the lateral border of the two zygapophyseal joints that connect the three exposed vertebrate into the lateral edge of the lamina between the joints.

This lamina is the pars interarticularis. It demarks the posterior border of the intervertebral foramen where the DRG reside. With care, use a five-millimeter Freer elevator or curette to palpate the transition between the caudal-most edge of the lamina and central canal.

Do not force the elevator anterior. Next, using rongeurs, extract bone in a piecewise fashion. Remove bone along the base of the spinous process superiorly to a level just caudal to the caudal surface of the pedicle and out laterally to its full extent.

Expose the smooth shiny periosteum in full. Cut with a number-11 blade and expose epidural fat. Leave the inferior articular process that was connected to the lamina in place for most of the laminotomy.

Then, remove the inferior articular process in a piecewise fashion, but leave the adjoining superior articular process intact. Now, dissect the DRG with the aid of loop magnification or a dissecting microscope. First, evacuate the epidural fat in a piecewise fashion beginning medially and proceeding laterally.

Make a gentle dissection with bipolar forceps and suction via six to 10 French Frazier suction tips. Next, identify the dural sac along the midline running in a superior inferior direction parallel to the axis of the skin incision. Then, remove epidural fat along the dural sac until the dural sac can be seen to give rise to the dural nerve root sleeve.

Now, continuing the epidural fat evacuation, trace the dural sleeve laterally and inferiorly until it is seen to enlarge around the DRG. The DRG is oval and yellow-orange about four to six millimeters wide. Proceed laterally with the fat removal past the DRG until the adjoining spinal nerve is seen.

Use a 22-gauge spinal needle to guide the trajectory of a 32-gauge CED needle. Puncture the guide needle through the skin and paraspinal muscles. Very carefully, aim the guide needle along a trajectory that approximates the longitudinal access of the DRG.

Gradually advance the guide needle until the tip emerges from the lateral paraspinal wall of the dissection field. Then, advance the guide needle along its long axis to approximate the CED needle tip to the DRG. Now, puncture the DRG with the CED needle tip and submerse the tip into the three-dimensional center of the DRG.

It is critical to again consider the unique size and shape of the exposed DRG. The goal is to achieve a smooth puncture and submerge the needle tip to reach the DRG's three-dimensional center without overshooting or missing your mark. Now, deliver 100 microliters of injectate by CED at a graduated rate over the next 24 minutes.

After the last step, a three-minute rest, withdraw the injection apparatus along its long axis in a smooth, gentle motion. Finally, close the surgical site as described in the text protocol. After closure, clean the skin and apply adhesive bandage strips perpendicular to the incision followed by gauze, and then an adhesive antimicrobial drape.

Successful delivery of the injectate into the DRG can be assessed histologically by examination. Ideally, the DAPI seen in blue will eventually distribute outwards in all directions. When an injection is successful, DAPI is evenly dispersed through both the central and peripheral DRG parenchyma seen in red.

When the injection is suboptimal, the staining will be inconsistent. For instance, there will be minimal staining or focal staining along the outer rim, but none in the inner aspect of the DRG parenchyma. While attempting this procedure, it's important to remember, start small and go slowly.

You can always widen the laminotomy later on in dissection but you can never put bone back.

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Journal (Neuroscience)

Journal (Neuroscience)

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

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