CT-guided injection of a TRPV1 agonist around dorsal root ganglia decreases pain transmission in swine

Abstract

One approach to analgesia is to block pain at the site of origin or along the peripheral pathway by selectively ablating pain-transmitting neurons or nerve terminals directly. The heat/capsaicin receptor (TRPV1) expressed by nociceptive neurons is a compelling target for selective interventional analgesia because it leaves somatosensory and proprioceptive neurons intact. Resiniferatoxin (RTX), like capsaicin, is a TRPV1 agonist but has greater potency. We combine RTX-mediated inactivation with the precision of computed tomography (CT)-guided delivery to ablate peripheral pain fibers in swine. Under CT guidance, RTX was delivered unilaterally around the lumbar dorsal root ganglia (DRG), and vehicle only was administered to the contralateral side. During a 4-week observation period, animals demonstrated delayed or absent withdrawal responses to infrared laser heat stimuli delivered to sensory dermatomes corresponding to DRG receiving RTX treatment. Motor function was unimpaired as assessed by disability scoring and gait analysis. In treated DRG, TRPV1 mRNA expression was reduced, as were nociceptive neuronal perikarya in ganglia and their nerve terminals in the ipsilateral dorsal horn. CT guidance to precisely deliver RTX to sites of peripheral pain transmission in swine may be an approach that could be tailored to block an array of clinical pain conditions in patients.

Conflict of interest statement

Competing interests: M.J.I. is a co-inventor on a U.S. patent describing the use of intrathecal RTX for pain treatment. Patent #8,338,457, issued December 2012: “Selective ablation of pain-sensing neurons by administration of a vanilloid agonist.” The other authors declare that they have no competing interests.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig. 1. CT-guided injections in a pig model

(A) Three-dimensional (3D) surface rendering of posterior skin surface with animal in prone position on CT table. Bilateral spinal needles are in place at the L2/3, L3/4, L4/5, and L5/6 levels (blue arrowheads). (B) In the same animal, 3D volume rendering of pig lumbosacral spine CT with spinal needles in place immediately adjacent to DRGs at the neuroforamina. (C) Axial CT image with pig in prone position. Scan obtained after injection of 2000 ng of RTX at the L4/L5 level. Periganglionic distribution of drug/contrast mixture is seen (black arrowheads) surrounding the exiting L4 DRG (black arrow) on the right (needle tip is out of plane of scan). Needle is in place to deliver vehicle solution (not yet injected) to left DRG. Black appearance distal to needle tip is a common CT artifact due to beam hardening along the needle path.

Fig. 2. Withdrawal latency to C-fiber activation with laser stimulus

(A) Diagram depicts the injected levels in pigs at L2/3, L3/4, L4/5, and L5/6, and the sensory dermatomes innervated by these ganglia (orange shading). C-fiber stimulations were confined to this shaded area. (B) Withdrawal latency at week 2 after injection with a 10.5-s cutoff threshold shows symmetric baseline withdrawal latency in uninjected animals. n = 2 (P = 0.257; left, 6.2 ± 0.4 s; right, 6.5 ± 0.4 s; 68 total stimuli). Significantly delayed or absent response in 500 ng (9.2 ± 0.3 s, n = 2) and 2000 ng (9.5 ± 0.3, n = 3) RTX groups relative to contralateral dermatomes injected with vehicle only (contralateral to 500 ng, 6.8 ± 0.4; contralateral to 2000 ng, 7.8 ± 0.3). Data points reaching 10.5-s cutoff (orange shading) have been staggered slightly to minimize overlap. Individual stimuli, circle or square; mean response time, gray box. (C) Withdrawal latency at week 4 after injection with a 15.5-s cutoff. There is a significantly delayed or absent response in RTX-treated hindlimb (500 ng, 13.7 ± 0.5, n = 3; 2000 ng, 12.1 ± 0.6, n = 4) compared to the contralateral limb injected with vehicle only (500 ng, 9.3 ± 0.6; 2000 ng, 8.5 ± 0.4). (D) RTX-treated dermatomes have an increase in number of trials reaching cutoff (combined 2- and 4-week data): uninjected, 4.4% (3 of 68); 500 ng, 60.7% (37 of 61) versus vehicle control 15.7% (11 of 70); and 2000 ng, 59.6% (53 of 89) versus vehicle control 17.4% (19 of 109). Nonparametric Kruskal-Wallis test with Dunn’s posttest to correct for multiple comparisons.

Fig. 3. Relative TRPV1 mRNA expression in treated DRG compared with control

Relative TRPV1 expression compared to control at the same lumbar level injected with 500 ng of RTX: 63.9 ± 7.3%, n = 7 DRG (P = 0.02), and with 2000 ng of RTX: 33.7 ± 4.1%, n = 12 DRG (P = 0.0005). Wilcoxon matched pairs test.

Fig. 4. Immunofluorescence in L4/5 DRG and nerve terminals in the spinal cord (L5)

(A to C) TRPV1 IR cell bodies from vehicle- (A) and RTX-injected (B) ganglia. (C) Bar graph depicts a 66% relative decrease in TRPV1 IR cells in RTX-treated (11.1 ± 1.6%, 140 of 1259) versus contralateral ganglia (32.7 ± 3.4%, 476 of 1457; P = 0.002). (D to F) NF200-IR cell bodies were not significantly affected by RTX injection (69.3 ± 3.9%, 567 of 818) (D); vehicle-injected DRG (71.6 ± 4.7%, 985 of 1375; P = 0.27) (E). (G to I) SP-IR cell bodies in RTX-treated ganglia (10.7 ± 2.8%, 99 of 923) (G) compared with contralateral ganglia (25.8 ± 2.7%, 418 of 1621) (H) were decreased by 59% (P < 0.0001). (J to L) CGRP-IR cell bodies from vehicle-injected (27 ± 1.4%, 290 of 1055) (J) and RTX-injected (42 ± 0.7%, 440 of 1044; P = 0.005) (K) ganglia. (L) Bar graph depicts a 35.6% relative decrease. (M to O) CGRP-IR nerve terminals in the dorsal horn (DH) from vehicle- (M) and RTX-injected (N) sides of the same animal with a 40.8 ± 8.5% reduction (O) in CGRP nociceptive terminals. Cell counts are a summation of DRG sections from three animals and are expressed as % IR cells per total cells. Scale bars, 100 μm (A to K) and 300 μm (M and N).

Fig. 5. Comparative histology of DRG injected with either vehicle or RTX

Hematoxylin and eosin (H&E) preparation of DRG parenchyma from lumbar DRG at ×40 magnification. (A and C) DRG injected with vehicle only demonstrating multiple large-, medium-, and small-diameter cell bodies with a generally thinly dispersed and uniform distribution of satellite cells, typically arrayed linearly along axons and in single cell layers around cell bodies. (B and D) In contrast, DRG injected with RTX demonstrate an overall reduction in the number of ganglion cells and an increase in the number of intervening, multilayered satellite cells (black arrows). Additionally, in some foci, the satellite cells are seen forming small aggregates resembling the structures that have been previously described as nodules of Nageotte (black arrowheads). Scale bar, 50 μm.