Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Summary

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.

Abstract

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',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.

Introduction

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....

Protocol

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

1. 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, instruments, and techniques involved are described in a fashion identical to the highest clinical standards in humans. For instance, strict sterile techniqu....

Results

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 Figure .......

Discussion

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. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Large humane animal sling	Britz & Company	002539	Modified to include abdominal aperture
Adhesive patient return electrode - 9 inch	Medtronic	E7506	-
Ranger blood & fluid warming system	3M	24500	-
Lactated Ringer's fluid	Hospira	0409-7953-09	-
Force air warming device	3M	77500	-
Duraprep solution with applicator, 26 mL (0.7% iodine povacrylex, 74% isopropyl alcohol)	3M	8630	-
Sterile disposable surgical towels	Medline	MDT2168286	-
Ioban 2 incise drape	3M	6651EZSB	-
Disposable suction canister and tubing	Medline	DYND44703H	-
Button switch electrosurgical monopolar pencil	Medtronic	E2450H	-
Fine smooth straight bipolar electrosurgical forceps, 4 1/2 inch	Bovie	A826	-
#15 blade	Miltex	4-315	-
#11 blade	Miltex	4-311	-
4 x 4 surgical gauze	Dynarex	3262	-
Weitlaner self-retaining retractor, 8 inch	Miltex	11-618	-
Meyerding self-retaining retractor, 1 x 2 3/8 inch	Sklar	42-2078	-
Gelpi self-retaining retractor, 7 inch	Sklar	60-6570	-
Freer elevator, 5 mm	Medline	MDS4641518F	-
Bone wax	Ethicon	W31G	-
Spurling intervertebral disc rongeur, 3 mm	Sklar	42-2852	-
Spurling 45-degree, up-biting Kerrison rongeur, 2 mm	Medline	MDS4052802	-
Leksell angled rongeur, 2 mm	Sklar	40-4097	-
Gelfoam, size 50	Pfizer	AZL32301	-
Cottonoid patty	Medtronic	8004007	-
Frazier suction tip, 6 Fr	Sklar	50-2006	-
Frazier suction tip, 10 Fr	Sklar	50-2010	-
Dandy blunt right angle nerve hook	Medline	MDS4005220	-
Nylon suture, 6-0	Ethicon	697G	-
Castroviejo smooth micro needle holder	Medline	MDG2428614	-
22 gauge Quinke point spinal needle	Halyard Health	18397	-
32 gauge CED needle with locking Luer hub	See comments	n/a	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).
Polyethylene tubing, 5 feet	Scientific Commodities	BB31695-PE/05	-
Monoject syringe, 3 mL	Kendall	SY15352	-
NanoJet syringe pump	Chemyx	10050	-
DAPI	Sigma-Aldrich	D9542	-
Fast Green FCF	Sigma-Aldrich	F7252	-
Bulb irrigation syringe	Medline	DYND20125	-
Fine-toothed Adson forceps	Medline	MDS1000212	-
Vicryl suture, 0	Ethicon	J603H	-
Vicryl suture, 2-0	Ethicon	J317H	-
Needle counter	Medline	NC20FBRGS	-
Steri-strip skin closure, 1/2x4 inch	3M	R1547	-