Deletion of vanilloid receptor 1_expressing primary afferent neurons for pain control (original) (raw)
Single intraganglionic injection in rat experimental models. Unilateral intraganglionic microinjection of RTX into the TG (Figure 1, A and B) attenuated or eliminated both the afferent (nociceptive) and efferent (neurogenic inflammation) functions supported by C-fiber neurons (Figure 1, C–E). Intraganglionic RTX attenuated plasma extravasation; this is an indicator of neurogenic inflammation and efferent function mediated by C fibers (17, 18). Intravenous Evans Blue dye, which binds to plasma proteins, delineated the areas with intact innervation (Figure 1C), staining them deep blue. The skin remained largely white on the RTX-microinjected side, where trigeminal neurons expressing TRPV1 were deleted. Analgesic actions were observable as soon as 24 hours after injection using a sensitive test for C-fiber function, the eye-wipe response to corneal application of capsaicin (CAP). An intraganglionic dose of 20 ng of RTX nearly eliminated the wiping behavior, and complete suppression was obtained with 200 ng (Figure 1D). The antinociceptive effects from a single RTX injection were maintained for at least 1 year (Figure 1E). The long-duration behavioral effect and loss of TRPV1-immunoreactive (TRPV1-IR) neurons (Figure 1, F and G) suggest that RTX analgesia is the result of permanent cell removal and not the result of prolonged receptor desensitization; the latter phenomenon is observed when CAP is applied to neurons in acute in vitro electrophysiological experiments (19–21). The loss of CAP chemosensitivity did not affect the mechanosensitive aspects of the corneal reflex to the liquid droplet itself. Furthermore, there were no observable alterations in grooming that might indicate the presence of a sensory dysesthesia.
Single intraganglionic treatment. RTX was microinjected unilaterally into the TG using a transcranial stereotaxic approach. (A) Coomassie blue dye depicts trigeminal injection site at the base of the skull and (B) excised, stained TG. (C) RTX-induced blockade of neurogenic inflammation was evaluated by extravasation of Evans blue_stained plasma proteins. Blue areas of skin identify regions with intact C-fiber innervation. Extravasation was blocked on the RTX-injected side, which remains white. (D) Dose-related blockade of nociceptive afferent transmission from the cornea after unilateral intraganglionic RTX administration. CAP eye-wipe response was assessed 1_3 days after injection. *Pairwise t test; P < 0.01; n = 30, 6, and 18 for administration of 200 ng, 20 ng, and vehicle, respectively. (E) Block of CAP-induced eye-wipe response is evident as up to about 1 year in unilaterally intraganglionically treated rats, consistent with the permanent deletion of TRPV1-IR neurons, as shown in F and G (*repeated pairwise t test; P < 0.01; n = 7). On the RTX-treated side (F), there is a reduction (∼80%, similar to animals examined within 1 to 3 days after RTX treatment; see Figure 2) of TRPV1-IR neurons compared with the contralateral nontreated side (G). Bars: 100 μm.
That intraganglionic RTX administration produced extensive deletion of TRPV1-expressing neurons could be seen by immunohistochemistry as early as 1–3 days after RTX treatment (Figure 2, A–D). To establish that another class of sensory ganglion neurons remains intact, we stained for a neurofilament protein (N52) expressed in neurons with large-diameter myelinated axons (22). Both single- and double-stained (N52 and TRPV1) sections were examined. Injected TGs contain many N52+ neurons (684 ± 32, brown in Figure 2, C and D) but very few TRPV1+ neurons (123 ± 36, purple), while contralateral TGs show both N52+ (709 ± 23) and TRPV1+ (604 ± 68) neurons (Figure 2, E and F). The loss of TRPV1+ neurons was significant (P < 0.01). The loss was also evident using RT-PCR; however, a small amount of RNATRPV1 was detectable in the RTX-injected ganglia (Figure 2G), consistent with cell counts showing substantial loss of TRPV1-IR neurons and retention of the N52 neurons not expressing TRPV1 (Figure 2, C and D).
Selective loss of TRPV1-IR sensory ganglion neurons after RTX (200 ng) microinjection. (A) Immunostaining for TRPV1 shows extensive loss of IR neuronal perikarya after RTX injection compared with contralateral noninjected TG (B). (C_F) Double labeling shows that large myelinated N52+ sensory neurons (brown) are retained on the RTX-injected side, whereas TRPV1+ neurons (purple) are deleted (C). On the contralateral, noninjected side, both N52 and TRPV1 neurons are intact (D). Quantification shows no significant difference in the number of N52-IR perikarya after RTX, whereas an 80% reduction in TRPV1+ neurons occurs (E and F). Bars in graph represent the average neuron counts in three sections of TG from three to five different rats assessed between 1 and 3 days after injection (*P < 0.01). (G) RT-PCR shows reduction of mRNATRPV1 in two different rats. Mr, markers; NO, no primer; SC, spinal cord; T, RTX treated; C, contralateral TG. Bars: 0.5 mm (A and B) and 50 ∝m (C and D).
Multiganglionic administration of RTX in rats. Severe pain secondary to advanced metastatic disease is generally more diffuse and not localized to one or two dermatomes. In these cases, multiple ganglia can be treated bilaterally via the cerebrospinal fluid. To test this, RTX was administered by lumbar puncture to target ganglia that innervate the tail and lower limbs. Treatment effectiveness and specificity were assessed by measuring (a) selective attenuation of noxious thermal and inflammatory responses, (b) the degree of spread of the drug by behavioral comparison to the forelimbs, corresponding to the spatial extent of the cell deletion, and (c) retention of locomotor activity and mechanosensation. RTX administered intrathecally (10, 50 ng) produced minor increases in withdrawal latency to a radiant noxious thermal stimulus (23, 24), whereas at doses of 100 and 200 ng, many animals reached the 14-second cutoff for hindpaw and tail stimulation without affecting forepaw latencies (Figure 3A). Intrathecal RTX also blocked carrageenan-induced thermal hyperalgesia, consistent with a substantial role of TRPV1+ neurons in experimental inflammatory conditions (Figure 3B), as shown by targeted disruption of the TRPV1 gene (25, 26). Behavioral effects were reflected at the cellular level by decreases in TRPV1 and calcitonin gene-related peptide (CGRP) immunoreactivities in the lumbar ganglia and dorsal horn, respectively, consistent with deletion of TRPV1-expressing neurons (Figure 3, D–G). In contrast, TRPV1+ neurons in cervical ganglia, which are remote from the level of lumbar RTX application, were unaffected (Figure 3C), corresponding to retention of normal forepaw-withdrawal latencies (Figure 3A). Thus, by targeting one or many ganglia, using the intraganglionic or intrathecal routes, respectively, the spatial extent of the therapeutic action may be adjusted to match varied clinical presentations.
Intrathecal (multiganglionic) or peripheral RTX attenuates thermal nociception and inflammatory hyperalgesia. (A) Dose_response to lumbar intrathecal administration of RTX (n = 50, 10 per group; *,#P < 0.05). Doses lower than 50 ng were without effect. Robust analgesia was obtained for the tail and hindpaws at doses of 100 and 200 ng. (B) Carrageenan inflammatory hyperalgesia (n = 4 per group; ** P < 0.05) is reversed after 200 ng intrathecal RTX (*P < 0.05). No analgesic effect was seen in forepaws (A), correlating with retention of TRPV1-IR neurons in the cervical ganglia (C) and their loss in lumbosacral ganglia (D). Dose-related reduction of CGRP-IR, a neuropeptide expressed by nociceptive afferent terminals, in the lumbar spinal cord at 3 days after administration of RTX (E_G). No significant effect on locomotor performance occurs as examined using an accelerating Rota-Rod (H). Peripheral left hindpaw (LHP) administration of 100 ng RTX yields unilateral and reversible (about 20 days) thermal analgesia (*ANOVA with Scheffe’s post hoc test; P < 0.005) (n = 5 per group). RHP, right hindpaw; FP, forepaw. (I) Retention of TRPV1-IR neurons (arrows) in L5 DRGs from RTX-injected (J) and noninjected (K) hindpaws. Cell counts of TRPV1-IR perikarya showed no significant alteration between left and right lumbar ganglia (see Results). Bars: 50 ∝m (C and D); 100 ∝m (J and K); 300 ∝m (E_G).
Retention of other somatosensory functions and locomotion. Importantly, the response to mechanical stimuli remained intact. Orientation to and withdrawal from a pinch with toothed forceps on the tail, forepaws, and hindpaws were present in all RTX-treated animals, and responses were qualitatively similar to controls. Quantitative assessment of mechanosensation using von Frey hairs also showed no alteration in RTX-treated (3 and 10 days and 1 year) animals in comparison to control animals. No significant difference was observed in the threshold for light touch or for paw withdrawal between untreated controls (10.9 ± 1.8 g for light touch, 20.3 ± 5.7 g for paw withdrawal), treated animals after 3 days (11 ± 1.8 g, 46 ± 30.6 g), treated animals after 10 days (10.1 ± 1.1 g, 22 ± 4.6 g) and treated animals after 1 year (11.7 ± 0.0 g, 33.7 ± 16 g). There were no indications of mechanoallodynia such as prolonged withdrawal to touch with the von Frey hairs, or mechanohyperalgesia as assessed by withdrawal from a pinprick (all animals had a withdrawal duration of 0.5 second or less) (27).
No alteration occurred in locomotor function as evaluated with an accelerating Rota-Rod, indicating the presence of intact motor axons and sensory proprioceptive neurons in ganglia exposed to RTX (Figure 3, D and H). Normal locomotion was also evident in the canine subjects (see later). As with the trigeminal microinjections, no indications of sensory neglect or denervation-induced hyperesthesia syndromes (e.g., autotomy behavior) were observed (28, 29). These features reinforce the clinical applicability of RTX, since the pathological, inflammation-associated aspects of pain are eliminated, while mechanonociception, motor function, and other sensory modalities remain intact.
Peripheral RTX administration. The same concept of ligand-activated calcium cytotoxicity was applied to the peripheral nerve terminals to obtain a transient reduction of nociceptive transmission. Injection of 100 ng RTX subcutaneously into the hindpaw significantly attenuated thermal nociception for approximately 20 days. The analgesic activity was confined to the injected hindpaw, and no alteration in latency was detected for the contralateral paw or the forepaws (Figure 3I). Since the injection was remote from the cell body, it was predicted that the effect would be transient, and that TRPV1+ cells would remain intact; both of these predicted results were observed (Figure 3, I–K). Counts of TRPV1-IR neurons from eight sections of L5 ganglia showed 119 ± 23 neurons on the injected side and 113 ± 10 on the contralateral side. Thus, the dose and anatomical site of RTX application critically determine whether the analgesic action will be reversible, as would be desired for postoperative pain, or irreversible (e.g., terminal cancer pain) (30, 31).
Veterinary clinical application. The canine model was established to assess the efficacy of primary afferent neuronal deletion in a higher order mammal with clinical problems more closely approximating the human situation. On the basis of attenuation of thermal response determined in an initial cohort, a single intrathecal dose of RTX (1 ∝g/kg) was administered under general anesthesia via cisternal puncture. Visual analog scale (VAS) ratings of nociceptive status before the injection averaged 62 ± 7.6 (mean ± SEM); these ratings sharply decreased (11 ± 3.0) at the 2-week follow-up and were maintained at 6 and 10 weeks (9.6 ± 5.3 and 7.5 ± 4.2, respectively) (Figure 4A). The animals initially presented with limb guarding as they walked, but this improved over time and daily activity increased, as was evident from video recordings. In fact, the entire demeanor of the dogs appeared improved following intrathecal RTX treatment. The efficacy of RTX action was further demonstrated by (a) the discontinuation or greatly reduced use of supplementary analgesics (opioids and NSAIDs in all eight dogs) and (b) the fact that neoplastic advancement did not diminish the RTX-induced analgesia.
RTX analgesia in naturally occurring neoplasms or osteoarthritis in the dog and morphological aspects of cell deletion in dog DRG and rat TG over time. (A) Intrathecal RTX reduced pain reports by the owners using a VAS in neoplastic (N), arthritic (A), and neoplastic plus arthritic (N + A) dogs (n = 8). Bars represent individual animals; inset represents summed data (*ANOVA with Scheffe’s post hoc test; P < 0.05). (B) High-power (∞40) image of an adult rat TG, 24 hours after 200 ng intraganglionic RTX administration, shows grossly normal appearance. (C) The enlargement reveals swollen eosinophilic cytoplasm and dislocated nuclei with partial wavy pattern of the nuclear membrane in many small- to medium-sized neurons (black arrows), characteristic of calcium cytotoxicity. Large neurons (red arrowheads) are normal. (D) DRG from an adult dog 21 days after intrathecal RTX injection shows patchy proliferation of satellite cells. (E) At higher magnification, neuronophagia is evident (black arrows). A necrotic neuron in which the nuclear membrane is faintly visible around a spot of condensed nuclear material is also visible (red arrowhead). By 30 days in dog ganglia, damaged and dead neurons are replaced by proliferating satellite cell colonies called nodules of Nageotte, which can be seen as a ball of proliferating cells (not shown). (F) One year after RTX treatment, the rat TG shows extended areas of acellular eosinophilic fields surrounded by rosetting satellite cells, indicated by red asterisks at higher magnification (G). There is no evidence of excessive, distorting glial proliferation. Bars: 100 ∝m (B, D, and F); 25 ∝m (C, E, and G).
Comparative histology of rat and canine sensory ganglia demonstrated that RTX eliminated many small-diameter neuronal cell bodies in both species, leaving no visible damage to surrounding neuropil (Figure 4, B–G). The early morphological signs of specific cell deletion were studied in rats sacrificed at 6, 12, and 24 hours. During this period, nuclear envelope ruffling and displacement of the nucleus in specific ganglion neurons were noted, indicating early stages of neuronal damage (Figure 4, B and C). In dogs, satellite cell activation and neuronophagia occurred around the dying neurons 3 weeks after intrathecal RTX treatment, progressing into characteristic nodules of Nageotte, which have been described in human diseases with pathological sensory aspects (Figure 4, D and E) (32). After 1 year, retention of the large-size neurons and deposition of fine ECM inside satellite cell rosettes were observed in rat ganglia without abnormal scar formation (Figure 4, F and G).
Effect of RTX on human DRG neurons. The ligand-activated cell deletion approach for pain control is based on the enriched expression of TRPV1 in a subset of sensory ganglion neurons. Immunocytochemistry and live-cell imaging were performed to determine expression of TRPV1 in adult human sensory ganglia and demonstrate whether selectivity can be expected upon administration into humans (Figure 5). While the cloned human TRPV1 has been studied in heterologous expression systems (33) and in an immortalized human neuronal cell line (34), there are no data on the physiological response and selectivity of primary human DRG neurons to stimulation by vanilloids. Figure 5 depicts the structure of a normal adult human DRG, and immunofluorescence (green) reveals human TRPV1-expressing neurons. There are “empty” lacunae surrounded by Hoechst 33342+ satellite cells (blue) that are occupied by neurons, which are negative for human TRPV1 expression.
Human DRG neurons show selective sensitivity to RTX treatment. (A) H&E section, adult human DRG (*large-sized neurons). (B_D) Green immunofluorescence human TRPV1-IR neurons (arrows), contrasted with satellite cell nuclei (blue). (C) TRPV1-IR (green) is adsorbed by the peptide antigen (D). Selective response of human embryonic DRG neurons to RTX assessed by increases in [Ca2+]i using Fluo-4 AM imaging (E and F). Arrows indicate responding neurons, and arrowheads indicate nonresponding neurons. E shows the baseline fluorescence depicted as near 0 for the first 30 seconds of the normalized data in the graph (G). In F, the transmembrane calcium flux leads to increases in intracellular calcium in specific neurons, which become brightly fluorescent. Magnification ∞200. (G) Traces of individual cells showing a substantial and abrupt response in vanilloid-sensitive sensory neurons (traces 1_4). Elevation of [Ca2+]i for a prolonged period of time is suggestive of imminent cell death in cells responding to vanilloid treatment. The RTX effect is clearly selective; nonresponding cells (traces 5_7) maintain normal calcium levels. Increase in normalized fluorescence intensity (numbered ØF/F0 traces refer to cells in E and F). Bars: 100 ∝m.
RTX opens the human TRPV1 ion channel and increases the [Ca2+]i inside human TRPV1+ DRG neurons. This change of [Ca2+]i can be detected with Fluo-4 AM, which changes its fluorescence emission intensity according to the [Ca2+]i (Figure 5, E–G). Fluo-4 AM imaging clearly distinguishes two populations of neurons: vanilloid sensitive and vanilloid insensitive. The [Ca2+]i in responding human neurons (Figure 5, E–G; cells 1–4) remains high for a prolonged period of time, suggesting a damaged, incapacitated state and imminent cell death. The images and line traces from nonresponding cells (Figure 5, E–G; cells 5–7) are also important, since they demonstrate the selectivity of the RTX treatment and the lack of toxicity to the nonresponding neurons or satellite cells.