Minimally invasive convection-enhanced delivery of biologics into dorsal root ganglia: validation in the pig model and prospective modeling in humans. Technical note

Abstract

Dorsal root ganglia (DRG) are critical anatomical structures involved in nociception. Intraganglionic (IG) drug delivery is therefore an important route of administration for novel analgesic therapies. Although IG injection in large animal models is highly desirable for preclinical biodistribution and toxicology studies of new drugs, no method to deliver pharmaceutical agents into the DRG has been reported in any large species. The present study describes a minimally invasive technique of IG agent delivery in domestic swine, one of the most common large animal models. The technique utilizes CT guidance for DRG targeting and a custom-made injection assembly for convection enhanced delivery (CED) of therapeutic agents directly into DRG parenchyma. The DRG were initially visualized by CT myelography to determine the optimal access route to the DRG. The subsequent IG injection consisted of 3 steps. First, a commercially available guide needle was advanced to a position dorsolateral to the DRG, and the dural root sleeve was punctured, leaving the guide needle contiguous with, but not penetrating, the DRG. Second, the custom-made stepped stylet was inserted through the guide needle into the DRG parenchyma. Third, the stepped stylet was replaced by the custom-made stepped needle, which was used for the IG CED. Initial dye injections performed in pig cadavers confirmed the accuracy of DRG targeting under CT guidance. Intraganglionic administration of adeno-associated virus in vivo resulted in a unilateral transduction of the injected DRG, with 33.5% DRG neurons transduced. Transgene expression was also found in the dorsal root entry zones at the corresponding spinal levels. The results thereby confirm the efficacy of CED by the stepped needle and a selectivity of DRG targeting. Imaging-based modeling of the procedure in humans suggests that IG CED may be translatable to the clinical setting.

Keywords: AAV = adeno-associated virus; AAV1 = AAV serotype 1; CED = convection-enhanced delivery; CTF = CT fluoroscopy; DRG = dorsal root ganglia; EGFP = enhanced green fluorescent protein; IG = intraganglionic; PBS = phosphate-buffered saline; computed tomography; convection-enhanced delivery; dorsal root ganglia; gene therapy; intraganglionic injection; pain; peripheral nerve.

Figure 1. Injection assembly used for DRG targeting and intraganglionic (IG) convection enhanced delivery (CED)

The 22 G 6’’ guide needle and three insets (proprietary stylet of the guide needle, custom made stepped stylet, and custom made stepped needle) were used throughout the 3-step procedure detailed in the main text. A. Overview of the guide needle (with its proprietary stylet in place), the stepped stylet, and the stepped needle. The term “step” (arrowhead) refers to the sharp transition between the wide needle/stylet shaft and their narrow tips. B. Longitudinal section of the guide needle with its three insets in place: proprietary stylet (top), custom made stepped stylet (middle); or custom made stepped needle (bottom). C. Cross-section of the stepped needle.

Figure 2. Targeting the DRG under computed tomography (CT) guidance in the pig model

CT imaging was used to visualize the pertinent spinal anatomy, determine the optimal needle path, and monitor the advancement of the guide needle to the DRG. A. DRG visualization and planning of the injection route. A CT myelogram (left) opacified the thecal sac (arrowhead) and visualized the DRG (arrows). The myelogram was used to determine the optimal trajectory of the needle (dotted line), here shown for the left L6 DRG. CT image of the same spinal level without IT contrast is shown for comparison (right). B. Placement of the guide needle under CTF guidance. The skin entry point determined by the myelogram was first matched with the corresponding point on the body surface of the animal by placing a radiopaque lead marker (arrowhead) on the skin. The needle was then advanced in increments along its predetermined path (middle) until its tip was located directly adjacent to the dorsal surface of the DRG (right). C. Confirmation of the guide needle placement. The additional contrast delivered to the left lateral IT sleeve was visualized by CTF as a crescent-shaped hyperdense area (arrowhead), further outlining the targeted DRG. The guide needle on an adjacent slice is not shown to allow better comparison with the myelogram alone presented in Panel A. D. Bilateral DRG targeting. Once the first needle reached the lateral sleeve of the IT space, a second needle could be advanced to the contralateral DRG using the same technique. The DRG could be safely targeted bilaterally at up to 3 spinal levels during one session with no adverse effects. E. Needle path and neighboring skeletal structures. Volume-rendered reconstruction (left) provides an overview of the trajectory of the guide needle (solid arrow). The lumbar puncture needle, used for obtaining the myelogram, is also shown (empty arrow). Coronal view (center) shows the cauda equina and L6 DRG (arrows) bilaterally. The tip of the guide needle was passed between the articular processes (asterisks) and into the L5-L6 intervertebral foramen. Oblique axial view (right), parallel with the long axis of intervertebral foramen, details the position of the tip of the guide needle immediately dorsal to the DRG (arrow) and ventral to the facet joint (arrowhead). Scale bars: 2.5 cm.

Figure 3. Validation of the IG injectate delivery

Efficacy of CED and accuracy of DRG targeting was first verified by dye injection both in vitro and post mortem, and then confirmed in vivo by IG administration of adeno-associated virus (AAV). A. Performance of the stepped needle used for CED (left) was compared to a conventional needle (22G spinal needle with Quincke tip; right) in vitro by administration of Evans blue dye into agarose gel, here shown for the flow rate of 10 μL/min. B. Accurate radiographic visualization of the DRG and the needle was verified in a pig cadaver. Unilateral administration of Chicago Blue dye at two spinal levels confirmed correct targeting of the DRG and the adjacent spinal root. C. CED of AAV1 via the stepped needle transduced 33.5% of DRG sensory neurons (right), as evidenced by EGFP expression (top left). The transduced cells demonstrated morphology characteristic of primary sensory neurons. 7.5×10¹⁰ genome copies of the vector suspended in 50 μl PBS were delivered into the left L6 DRG at the flow rate of 10 μL/min. Transduction was detected 4 weeks later by laser scanning microscopy. No transduction was found in the DRG that were not injected (bottom left), suggesting that no spillage of the vector to the cerebrospinal fluid had occurred. Scale bars: 100 μm. D. Transduction of the spinal cord was found in the dorsal root entry zones (DREZ), corresponding to the centripetal axons of the DRG sensory neurons. The transduction was thereby restricted to the cells whose cell bodies or axons came into a direct contact with the transducing agent within the DRG, indicating that no trans-synaptic spread of the vector had occurred. DH, dorsal horn; arrow, posterior median sulcus; arrowhead; postero-lateral sulcus. Scale bars: 200 μm. All microscopic images were acquired in the lambda stack mode, and linear unmixing was used to distinguish the specific EGFP signal (green) from non-specific autofluorescence (red). Original magnification:×200 for the DRG and the spinal cord inset;×50 for the spinal cord overview.

Figure 4. Modeling the IG injection in humans

Analysis of human spinal anatomy by MRI and CT-myelography sustains the feasibility of DRG targeting from a postero-lateral approach under CTF guidance in human patients. A. MRI images (T1 weighted, fat saturated) of the human lumbar spine, here shown for L3, L4, and L5 levels, identified the DRG by gadolinium enhancement (arrows). B. CT-myelography of the corresponding segments showed unobstructed access to the DRG. The normodense contours of the DRG (arrows) stood out from the hypodense background of the epidural fat; the IT contrast did not spread into the root sleeves and therefore did not further outline the DRG. The optimal trajectory for accessing the DRG is indicated by a dotted line. Scale bars: 5 cm.