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 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.
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.
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
2. Pre-operative Animal Care
3. Positioning in the Operative Suite
4. Sterile Preparation of Operative Field for a Left-sided Injection
NOTE: From this point forward, proceed in strict sterile fashion.
5. Skin Incision and Subperiosteal Dissection
6. Single-level Laminotomy
7. Dissection of DRG
8. Injection of DRG
9. Closure
10. Post-operative Animal Care
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 1B represent a successful injection of one DRG. DAPI staining was evenly dispersed through both the central and peripheral DRG parenchyma. Thus, a successful DRG injection is illustrated by the diffuse spread of DAPI staining throughout the three-dimensional DRG architecture. Sub-optimal injection is illustrated by inconsistent staining. For instance, minimal staining (Figure 1C) or focal staining along the outer rim but not inner aspect of DRG parenchyma (Figure 1D) indicates unsuccessful injection. Also, considered together, Figure 1C (central DRG section) and Figure 1D (peripheral DRG section) illustrate a lack of consistent staining within three dimensions for this single lumbar DRG.
Figure 1: Three-dimensional Assessment of DAPI Distribution in Injected DRG. (A) A central section from an injected lumbar DRG representative of a successful outcome. The staining of the marker dye DAPI is evenly dispersed throughout the entire DRG in two dimensions. (B) A parallel, peripheral section of the same DRG in (A), illustrating the consistency of DAPI spread within a second plane of section, confirming a successful injection in three dimensions. (C) A central section from an injected lumbar DRG representative of a sub-optimal outcome. Minimal to no staining of DAPI is seen except for occasional foci. (D) A parallel, peripheral section of the same DRG in (C), illustrating partial distribution of DAPI along the DRG periphery. Blue: DAPI. Red: autofluorescence. Scale bars = 500 μm (A and C),100 μm (B and D). Please click here to view a larger version of this figure.
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. The results from the above small animal studies are promising and we hope our protocol will pave the way for others to translate these prior findings to swine. Healthy and diseased animals were used in the above studies, supporting the utility of small animals in preclinical research. Inevitably, large animal models will be required to make the best available comparison to human DRG in terms of size and injectate distribution. For instance, the discrepancy in DRG size is clearly evident between rats and humans. L5 DRG in adult, male rats measure approximately 2.6 mm x 1.5 mm 5 compared to 11.6 mm x 6.6 mm in adult, male humans 21. Based on live radiographic measurements in swine, L5 DRG were found to be approximately 8.0 mm x 6.0 mm 22. Therefore, swine are situated as a particularly useful species for preclinical study due to structural similarities with the human nervous system and nearby musculoskeletal anatomy. This is evidenced by the reverse translation used to design this protocol according to that performed in the clinic. Moreover, swine are use animals and are of growing importance in biomedical research. This protocol will support preclinical studies of intraganglionic delivery of injectable solutions to advance prior findings from work in rodents to large animals. Thereby, this protocol may foster novel strategies for treating chronic pain that integrate anatomically selective delivery techniques with novel molecularly selective agents, which, we predict, may have the potential to transform pain medicine 3.
Additional notes for successful DRG injection
The prelaminar periosteum at lumbar vertebral levels in swine continues cephalad in place of the ligamentum flavum. During laminotomy, the periosteum can be easily separated by placement of the Kerrison rongeur and can mimic the appearance of dura mater. It is a critical step to differentiate the ligamentum flavum, periosteum, epidural fat, and dura sac as the dissection is carried out. Furthermore, exposure of the epidural space is more efficient and incurs less bleeding if the periosteum is removed simultaneously with the lamina. If the periosteum is not removed along with bone, it can be incised with a #11 blade and removed to expose the underlying epidural fat.
To do no harm is a top priority and this must be balanced with the goal of DRG access and injection. Thus, care is taken not to advance the evacuation of epidural fat farther anteriorly or anteromedially than is needed to allow positive identification of the dural sac, dural nerve root sleeve, and DRG. Dissection in the anteromedial direction where the dural sac gives rise to the dural nerve root sleeve is particularly dangerous as a longitudinal epidural vein will be encountered. Furthermore, dissection in this direction increases the risk for unintended durotomy, signaled by the outflow of cerebrospinal fluid from the surface of the dural sac.
A final critical point is that of identifying the dural sac, dorsal roots, DRG in its entirety, and spinal nerve. This helps to establish 4 pieces of converging anatomical evidence that ensure complete DRG definition. Defining the DRG in its entirety is required in order to position the needle tip at its three-dimensional center, which permits the CED needle to establish a consistent pressure gradient while maximizing the distance to the surrounding anatomical boundaries. Both factors greatly increase the volumes delivered and range of intraparenchymal spread 19,20. Delivery of injectate at a location that is not the true anatomic center results in sub-optimal injection because inconsistent pressures result when injectate leaks from the nearby site of DRG puncture 20.
One difficulty with using a CED needle for DRG injection is that of compliance. Once the injection has started, the needle tip must be kept as still as possible or else pressure gradients will dissipate due to abrupt changes in compliance 20. Respiratory motion is a source of continuous movement during the injection. However, the risk of needle movement secondary to respiratory excursion is largely removed by anchoring the CED and guide needle within the paraspinal musculature prior to puncture of the DRG as both the needle and DRG move in synchrony with respiration. The injection duration for a volume of 100 μL totals 24 min at the stepped rate described here. Care should be taken to limit external disruption of the entire injection apparatus during this time. Arrangement of the surgical field, personnel, and surrounding obstacles should be modified as needed before the injection is started to ensure an undisturbed interface between the CED needle tip and DRG.
The authors have nothing to disclose.
The study was performed with support by the Schulze Family Foundation (to A.S.B.).
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 | – |
4×4 surgical gauze | Dynarex | 3262 | – |
Weitlaner self-retaining retractor, 8 inch | Miltex | 11-618 | – |
Meyerding self-retaining retractor, 1×2 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, 1/2×4 inch | 3M | R1547 | – |