Name: laminotomy for lumbar dorsal root ganglion access and injection in swine
Uploaded: 2017-10-10T15:00:00
Description: Watch this Scientific Journal Video about Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine at JoVE.com

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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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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine at JoVE.com

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

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

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

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

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.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...