The study was performed with support by the Schulze Family Foundation (to A.S.B.).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic.
1. Prerequisites of Rigor and Reproducibility
<ol>
	<li>To ensure rigor of design, follow national standards of good laboratory practices are at all times and obtain internal approval by IACUC (or similar committee) prior to any animal involvement in experiments. 
	NOTE: This protocol was designed to maintain a clinically faithful approach. Thus, the materials, instru...

<ol>
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None; the authors have no conflicts of interest related to this study.

We sought to describe a method for surgical exposure of DRG via laminotomy and intraganglionic injection in a healthy large animal species, specifically, swine. In rodents, a similar method has been detailed 12 and used to deliver conventional pharmacologic agents 8,10 and viral vectors 6,7,9,12 to DRG. ...

Dorsal root ganglia (DRG) are anatomically discrete, neuronal collections located along the vertebral column. Each DRG contains the primary sensory neurons that encode and relay peripheral stimuli to the central nervous system (CNS) from specific body regions. For instance, the pain of osteoarthritis begins when pain receptors located about a joint perceive noxious stimuli. This process is termed nociception. Long-term awareness of noxious stimuli leads to chronic pain 1.
Chronic pain is a frequent subject of preclinical study 2 where one goal is to develop useful methods for targeted delivery of analgesics to DRG, such as intraganglionic injection 3. However, DRG are difficult to access because they reside within the boney confines of intervertebral foramina 4. Several groups have successfully overcome this obstacle through the use of spine surgery in rodents 5,6,7,8,9,10.
In the clinic, laminectomy is a common spine operation and refers to surgical removal of the vertebral lamina, thereby unroofing the vertebral canal 11. Incorporation of surgical techniques to afford direct DRG access has been successful in rodents 5,12, however, translation may be unrealistic considering differences in size of relevant structures and how that influences pharmacokinetics or technical feasibility 13,14. For example, one study determined the transverse spinal cord diameter at T10 to be 3.0, 7.0, and 8.2 mm for rat, pig, and human, respectively 15. Thus, large animal models better approximate human dimensions of nervous structures.
In swine, Raore et al. used multi-level laminectomy to gain access to the cervical spinal cord for multiple intraspinal injections 16. The procedure was well tolerated and led to a phase I clinical trial where comparable surgical outcomes were documented 17. These results encourage continued use of preclinical large animal models as predictors of technical feasibility and safety in humans.
To date, no detailed methodology exists for surgical access and injection of DRG in a large animal species. To narrow this translational gap, we report a protocol for DRG exposure and injection via laminotomy in swine. Standard neurosurgical techniques, instruments, and materials were used and the method was designed to mimic modern surgical practice. We demonstrate intraganglionic injection using an aqueous solution for lumbar DRG and confirm successful delivery via histology after post-operative day 21.

<table border="0" cellpadding="0" cellspacing="0" width="1311">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Large humane animal sling</td>
			<td style="width:167px;">Britz &#38; Company</td>
			<td style="width:119px;">002539</td>
			<td style="width:484px;">Modified to include abdominal aperture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Adhesive patient return electrode - 9 inch</td>
			<td style="width:167px;">Medtronic</td>
			<td style="width:119px;">E7506</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Ranger blood &#38; fluid warming system</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">24500</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Lactated Ringer&#39;s fluid</td>
			<td style="width:167px;">Hospira</td>
			<td style="width:119px;">0409-7953-09</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Force air warming device</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">77500</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:543px;">Duraprep solution with applicator, 26 ml (0.7% iodine povacrylex, 74% isopropyl alcohol</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">8630</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Sterile disposable surgical towels</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDT2168286</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Ioban 2 incise drape</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">6651EZSB</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Disposable suction canister and tubing</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">DYND44703H</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Button switch electrosurgical monopolar pencil</td>
			<td style="width:167px;">Medtronic</td>
			<td style="width:119px;">E2450H</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fine smooth straight bipolar electrosurgical forceps, 4 1/2 inch</td>
			<td style="width:167px;">Bovie</td>
			<td style="width:119px;">A826</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">#15 blade</td>
			<td style="width:167px;">Miltex</td>
			<td style="width:119px;">4-315</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">#11 blade</td>
			<td style="width:167px;">Miltex</td>
			<td style="width:119px;">4-311</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">4x4 surgical gauze</td>
			<td style="width:167px;">Dynarex</td>
			<td style="width:119px;">3262</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Weitlaner self-retaining retractor, 8 inch</td>
			<td style="width:167px;">Miltex</td>
			<td style="width:119px;">11-618</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Meyerding self-retaining retractor, 1x2 3/8 inch</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">42-2078</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Gelpi self-retaining retractor, 7 inch</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">60-6570</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Freer elevator, 5 mm</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDS4641518F</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Bone wax</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:119px;">W31G</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Spurling intervertebral disc rongeur, 3 mm</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">42-2852</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Spurling 45-degree, up-biting Kerrison rongeur, 2 mm</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDS4052802</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Leksell angled rongeur, 2 mm</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">40-4097</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Gelfoam, size 50</td>
			<td style="width:167px;">Pfizer</td>
			<td style="width:119px;">AZL32301</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Cottonoid patty</td>
			<td style="width:167px;">Medtronic</td>
			<td style="width:119px;">8004007</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Frazier suction tip, 6 Fr</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">50-2006</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Frazier suction tip, 10 Fr</td>
			<td style="width:167px;">Sklar</td>
			<td style="width:119px;">50-2010</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Dandy blunt right angle nerve hook</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDS4005220</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Nylon suture, 6-0</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:119px;">697G</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Castroviejo smooth micro needle holder</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDG2428614</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">22 gauge Quinke point spinal needle</td>
			<td style="width:167px;">Halyard Health</td>
			<td style="width:119px;">18397</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:543px;">32 gauge CED needle with locking Luer hub</td>
			<td style="width:167px;">See comments</td>
			<td style="width:119px;">n/a</td>
			<td style="width:484px;">As in: Pleticha, J., Maus, T.P., Christner, J.A., Marsh, M.P., Lee, K.H., Hooten, W.M., Beutler, A.S. Minimally invasive convection-enhanced delivery of biologics into dorsal root ganglia: validation in the pig model and prospective modeling in humans. Technical note. J Neurosurg. 121(4), 851-8 (2014).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Polyethylene tubing, 5 feet</td>
			<td style="width:167px;">Scientific Commodities</td>
			<td style="width:119px;">BB31695-PE/05</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Monoject syringe, 3 ml</td>
			<td style="width:167px;">Kendall</td>
			<td style="width:119px;">SY15352</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">NanoJet syringe pump</td>
			<td style="width:167px;">Chemyx</td>
			<td style="width:119px;">10050</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">DAPI</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fast Green FCF</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7252</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Bulb irrigation syringe</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">DYND20125</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fine-toothed Adson forceps</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">MDS1000212</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Vicryl suture, 0</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:119px;">J603H</td>
			<td style="width:484px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Vicryl suture, 2-0</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:119px;">J317H</td>
			<td style="width:484px;">-</td>
		</tr>
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			<td height="21" style="height:21px;width:543px;">Needle counter</td>
			<td style="width:167px;">Medline</td>
			<td style="width:119px;">NC20FBRGS</td>
			<td style="width:484px;">-</td>
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			<td height="21" style="height:21px;width:543px;">Steri-strip skin closure, 1/2x4 inch</td>
			<td style="width:167px;">3M</td>
			<td style="width:119px;">R1547</td>
			<td style="width:484px;">-</td>
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laminotomy for lumbar dorsal root ganglion access and injection in swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

Histologic assessment of injectate spread 
Successful delivery of injectate to DRG is determined by histologic assessment of DAPI spread. The technique involves positioning the needle tip in the three-dimensional center of the DRG. Therefore, successful delivery is determined by evaluating the extent of DAPI staining from histologic sections both near (central DRG sections) and distant (peripheral DRG sections) to the needle tip. Figure 1A and Figur...

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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.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

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.

laminotomy-for-lumbar-dorsal-root-ganglion-access-injection

Research

JoVE Journal

Neuroscience

12.5K Views.  Mayo Clinic, Translational Science Track, Mayo Graduate School. 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 ...