Several studies have demonstrated that perineural administration of the naturally occurring vanilloids, capsaicin, and resiniferatoxin (RTX) produces selective nociceptive blockade (1–5). These agents cause an initial activation of the TRPV1 receptors that leads to their desensitization, and to the inactivation of nociceptive nerve fibers lasting for a very long period. In initial studies with perineural administration of vanilloids, only capsaicin was used and almost exclusively in high concentrations—-1%–1.5% (1,6). With these concentrations, the effect of capsaicin was still present at the end of the study period (4–6 wk). This was the basis for the suggestion that the local treatment of peripheral nerves with capsaicin results in a permanent impairment of the function of capsaicin-sensitive afferent nerve fibers and possibly in their degeneration.
Initial studies conducted with the use of electron microscopy found no axonal degeneration after topical (cornea) (7) or perineural (sciatic nerve) (6) application of capsaicin even in high concentrations (up to 1.5%). Two subsequent electron microscopy studies on the effect of perineural capsaicin on C-fibers, performed by the same group of authors, resulted in conflicting outcomes. In rats, treatment of the saphenous nerve with 1% capsaicin caused the C:A fiber ratio to decrease by approximately 35% (3); at the same time, in rabbits (also saphenous nerve), 1% capsaicin did not change the C:A fiber ratio (8). The authors suggested that a concentration of capsaicin lower than 1% may suppress nociception in the rat without C-fiber degeneration. This suggestion was based on the finding that even 0.01% capsaicin applied to rat nerves causes long-lasting suppression of neurogenic inflammation, axonal transport, and nociceptive reactions. Another study in rats (9), using quantitative electron microscopy of the saphenous nerve treated with capsaicin (1%), described a 32% reduction in the number of unmyelinated axons.
In relatively recent studies on the neurotoxic effects of capsaicin applied to the skin (10,11), administered either perineurally (12), or into the urinary bladder (13) to provide nerve fiber visualization, the authors used immunohistochemical methods based on determination of substance P, calcitonin gene-related peptide, and/or the pan-neuronal marker protein gene product (PGP 9.5). These studies demonstrated a profound loss of nerve fiber staining in epidermis (or urinary bladder wall) that led many of the authors to conclude that capsaicin induced axonal degeneration. However, none of these studies used electron microscopy to confirm this conclusion. When Avelino and Cruz (14) re-examined the problem of capsaicin-induced axonal degeneration in the rat bladder with the parallel use of immunohistochemical and electron microscopic methods they found that both capsaicin and RTX caused desensitization and a profound reduction in substance P and calcitonin gene-related peptide immunoreactive fibers without causing significant nerve changes. The authors concluded that vanilloids, applied intravesically (urinary bladder) in full desensitizing concentrations, exert an effect on the bladder (including depletion of substance P and calcitonin gene-related peptide) that lasts for 8–12 wk without nerve fiber degeneration. They suggested that PGP immunoreactivity, which was used in several capsaicin studies as an index of axon destruction, could be lost because vanilloids cause axonal transport blockade that slows the arrival of PGP to peripheral axons, a process that occurs without nerve degeneration.
Perineural RTX inhibits responses to noxious stimuli in a dose-dependent manner (15). Depending on the concentration (0.00003%–0.003% in 0.1 mL) the effect of a single administration to the sciatic nerve can last from several hours to several weeks. Only at the concentration of 0.003% did the blockade have a chance of lasting longer than 3 wk. The relatively rapid recovery indicates that this effect of RTX on C-fibers is reversible. The controversy regarding nerve fiber degeneration versus long-lasting loss of function without degeneration is relevant to the high concentrations of vanilloid agonists, probably far in excess of the concentrations that produce nociceptive blockade lasting only several days. All published studies on vanilloid toxicity after perineural administration was performed with capsaicin. At the same time, RTX differs from capsaicin in the spectrum of its actions. Capsaicin acts on vanilloid receptors and other targets in overlapping concentration ranges, whereas RTX has a profound separation between its various effects (16). Therefore, even if very high concentrations of RTX could cause nerve fiber degeneration, these concentrations may be well above RTX concentrations producing analgesia, perhaps a situation similar to that with local anesthetics. Peripheral nerve blocks induced by local anesthetics are associated with some degree of nerve fiber injury (17). Even at clinical concentrations, they are capable of producing dose-dependent injury to the myelinated and unmyelinated nerve fibers (18).
The aim of the present study was to determine whether RTX-induced reversible sciatic nerve block results in degeneration of unmyelinated fibers.
The protocol for this study was approved by the Institutional Panel on Laboratory Animal Care. Male Sprague-Dawley rats weighing 275–325 g were used for the experiments. The rats were housed with a 12-h light/dark cycle, and food and water were available ad libitum. RTX was injected in various concentrations percutaneously at the sciatic nerve. The injections were made at the greater trochanter level in a volume of 0.1 mL with animals under brief halothane anesthesia (2%). India ink was added to the RTX solution to identify the site of injection. RTX was purchased from Sigma (St. Louis, MO), dissolved in dimethyl sulfoxide (Sigma) to a concentration of 1 μg/μL, and stored at −80°C under nitrogen. It was diluted to the required concentration before the experiment in 0.9% Sal with 0.3% Tween 80. The effect of RTX was monitored by measuring the rat’s response to noxious heat stimulation. For this aim, animals were placed in a clear acrylic box on a glass platform, a beam of radiant heat was applied through the platform to the plantar surface of the hind paw, and withdrawal latency was determined by a photocell (Plantar Test device, Ugo Basil, Milan, Italy). The infrared radiation intensity was set to elicit a withdrawal latency of 6–7 s, with a cut-off time 10.9 s. Three consecutive measurements were averaged.
Forty-eight hours after the RTX injection, animals were anesthetized with pentobarbital sodium (55 mg/kg, IP) and perfused via a cannular in the aorta (access through the puncture at the apex of the left ventricle) with chilled modified Karnovsky’s fixative containing 2.5% paraformaldehyde and 2.0% glutaraldehyde in 0.1 M cacodylate buffer. The fixative was delivered with an infusion pump (Harvard Apparatus, model 55-2222, Natick, MA) into the aorta. The speed of the fixation was accelerated by making several incisions in the liver. Perfusion continued until both the upper and lower limbs of the rat became stiff (usually it required 100–150 mL of the fixative). The sciatic nerve was exposed and a 1 cm segment at the site of injection was removed and placed in the same fixative. It was then postfixed in 2% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated, and embedded in epoxy resin. Two transverse sections of each nerve were cut, one at the proximal end of the segment, the other at the distal end. The sections were mounted on grids and stained with uranyl acetate and lead citrate. They underwent light and ultrastructural examination. The specimen preparation was performed in the Department of Pathology Electron Microscope Facility of the Children’s Hospital Boston by Howard Mulhern. The evaluation was performed by Umberto DeGirolami.
The ultrastructural examination was performed with a fei Phillips EM208S electron microscope equipped with an AMT digital camera. A series of micrographs were taken of each cross-section according to a scheme that covered the entire cross-section, avoiding major vessels. The total area photographed corresponded to approximately 5% of the total cross-sectional area of the sciatic nerve. The micrographs (20 per cross-sectional area) were made at magnifications up to 15000×. The images were used for counting unmyelinated fibers. Fiber diameters were measured with the AMT Capture Engine calibrated with a standard grating replica. Unmyelinated fibers were inspected for evidence of injury, including swelling, disintegration, dystrophy with dark-staining axoplasm, accumulation of abnormal deposits, and destruction or degeneration of organelles. The approximate number of observed unmyelinated fibers was 700 per nerve (two cross-sections).
Two concentrations of RTX were selected for the study: 0.0001% (0.1 μg) and 0.001% (1 μg). There were three groups of animals with five rats per group (one group for each of the concentrations and a group of control rats that received an injection of the RTX vehicle). The RTX concentrations were selected to have a reversible sciatic nerve block lasting approximately 2 days (one group) or 2 wk (another group) (15). The time interval between the RTX administration and the removal of the nerve segment (48 h) was selected on the basis of preliminary experiments.
Preliminary experiments with injections of RTX (0.01%, 0.001%, 0.1 mL for both) and an RTX vehicle (0.1 mL) were performed without in vivo fixation (the removed segment of the sciatic nerve was immersed in Karnovsky’s fixative). The segments were taken 8 days after the RTX (or RTX vehicle) injection at the sciatic nerve. There were five animals per each of three groups. Because the preliminary experiments showed no abnormalities in unmyelinated and myelinated fibers, the earlier (48 h) timing after RTX administration and the method providing better quality of nerve fixation (in vivo fixative administration) were chosen for the final experiments.
The rats were assigned to the group randomly, and the investigators responsible for the evaluation of the micrographs were blinded as to type of treatment.
Comparisons among groups were performed with one-way analysis of variance. The results were declared significant if P value was <0.05. Data are reported as the mean ± sd.
Cross-sections of the sciatic nerve 48 h after the perineural administration of RTX at 0.001% (0.1 μg) or 0.0001% (1 μg) appeared essentially normal, as shown in Figure 1. This was true of both myelinated and unmyelinated fibers. Since the main purpose of the study was the search for ultrastructural evidence of injury, it was directed toward finding the breakdown of unmyelinated axons, degeneration of their organelles, or accumulation of abnormal deposits. One of our rarely observed findings was the irregularly compacted membranous deposits in the unmyelinated axons (Fig. 2). Fibers filled with these deposits were regarded as degenerating axons.
Table 1 demonstrates that the frequency of finding degenerating axons in the nerves following the RTX administration was approximately one per thousand with both concentrations of RTX. Degenerating axons were not found in the control group; however, the difference between the control and RTX groups was not statistically significant.
The number of unmyelinating axons and their diameters are shown in Table 2, which demonstrates no statistically significant decline in the total number of unmyelinated axons in the RTX groups compared with the control group. The only statistically significant change was related to the size of axons. In the group with RTX 0.0001%, the axon diameter was 1.03 ± 0.08 μm vs 1.18 ± 0.08 μm in control (a 13% reduction, P < 0.001). In this group of animals, recovery of response to noxious heat (before removal of the nerve segment) was observed in four of five rats. With much higher RTX concentration (0.001%), when there was no functional recovery after RTX administration at 48 h in any of the five rats, the decline in axon diameter was not statistically significant.
Our initial experiments with the segments of the sciatic nerve removed on the 8th day after the initiation of RTX-induced blockade did not reveal any signs of degenerating fibers. Figure 3 illustrates findings typical for five rats on the 8th day after the RTX administration: the unmyleinated fibers look normal. This was observed with 1 μg of RTX (0.001%, 0.1 mL) when the response to noxious heat stimulation recovered in all five rats. Similar morphologic outcome was reported by Ainsworth et al. (6) 14 days after treatment of the sciatic nerve with capsaicin. Possibly, the damage to the unmyelinated fibers occurred early after RTX administration and by the 8th day the signs of damage disappeared. Studies on the reactions of unmyelinated fibers to various injuries indicate such possibility (19,20). They describe early (24–48 h) occurrence of unmyelinated axon swelling and the presence of electron dense bodies, but most of degenerative changes disappeared 1 or 2 wk after the injury. To detect signs of axonal degeneration at its maximum, we concentrated our attention on the status of unmyelinated fibers 48 h after the RTX administration.
No significant degeneration of unmyelinated fibers attributable to the RTX-induced blockade was found in our study. We did not observe any axon swelling. On the contrary, there was a very modest decline in the size of unmyelinated fibers. However, infrequent signs of fiber degeneration were observed, consisting of irregularly compacted membranous deposits in the unmyelinated axons. The deposits probably represent degenerating mitochondria. Similar inclusions were described by the Powell et al. (21) in rats with alloxan diabetic neuropathy. It is interesting that Szolcsanyi et al. (7) reported that, 46 h after local administration of capsaicin (1%) to the cornea of rats, swollen mitochondria with disorganized crystae can be found. But these authors also concluded that electron microscopy showed no signs of axon degeneration.
In several studies, capsaicin (1%) was directly applied to the saphenous nerve, and electron microscopy was used to determine its effect on unmyelinated fibers several months after its administration (3,8,9). In one of these studies, performed in rats, the authors concluded that cross-sections of treated saphenous nerves had essentially normal appearance (3). However, quantitative measurements in this study demonstrated that C-fibers had declined by approximately one-third. When the same group of authors used perineural capsaicin (1%) in rabbits, they found a profound decline in the substance P content of the skin innervated by the saphenous nerve, but no changes in C-fiber numbers (8). A decline in the number of C-fibers in the rat saphenous nerve several months after capsaicin administration was reported by Jancso and Lawson (9).
In the present study, we did not observe any significant decrease in the number of unmyelinated axons, although there was a tendency (statistically insignificant) towards a decline in the number of counted axons (Table 2). Since there was also a tendency towards an increase in the total area of the nerve cross-sections (probably, due to tissue irritation caused by RTX) decline in the axon number could also be explained by this factor. When comparing our study with previous electron microscopic studies, one should consider that different vanilloids were used for the blockade and that there were differences in blockade duration: reversible blockade lasted for only several days (with 0.0001% RTX in our experiments), whereas in some studies it lasted for several months (reversibility in question). In addition, we studied early (48 h) changes after blockade, and other studies examined nerve segments taken 14 days (6) or 2–12 mo (3,9) after the blockade initiation. Some early signs of fiber degeneration may disappear in several weeks or months.
Signs of degeneration in our study were rarely noted, especially compared with the effect of local anesthetics. For example, quantitative electron microscopic evaluation of the effects of different local anesthetics (including lidocaine up to 3%) on rat sciatic nerves removed 48 h after extraneural injections demonstrated degeneration of approximately 5% of unmyelinated axons (18). This frequency of degeneration is more than an order of magnitude higher than the frequency of degeneration observed in the rats with reversible RTX blockade.
The assessment of the size of unmyelinated fibers in our study demonstrated a statistically significant difference between the control group and the RTX 0.0001% group. The degree of decline in size in the RTX group (13%) was similar to that reported for capsaicin (3). The decrease in fiber size may be explained by RTX-induced blockade of the intra-axonal transport of macromolecules and depletion of various sensory neuropeptides (16).
Capsaicin is able to kill adult sensory neurons in culture. This action is mimicked by RTX and is most likely mediated by calcium influx (22). Given to newborn rats, capsaicin kills most small- to medium-sized dorsal root ganglion neurons (23). In adult rats, a significant loss of dorsal root ganglion neurons induced by systemic capsaicin was reported (24); however, the dose of the agent was extremely high—-100 mg/kg, s.c. Systemic administration of high doses of capsaicin demonstrated that different parts of peripheral sensory axons have different sensitivity to the agent, with the subepidermal part of the axon being the most vulnerable. Such high doses can cause severe depression of respiration. Comparison of the therapeutic dose ranges (limited by respiratory depression) of systemic capsaicin and RTX in rats demonstrated that they include doses that differ by two orders of magnitude with capsaicin and three orders with RTX (16).
In microscopic studies with perineural administration of vanilloids, only capsaicin was used and almost exclusively in high concentrations—1%–1.5% to the sciatic nerve (1,6,12). The effect of capsaicin was still present at the end of the study period (4–6 wk). The long duration of this effect was the basis for the suggestion that the local treatment of peripheral nerves with capsaicin results in permanent impairment of the function of capsaicin-sensitive afferent nerve fibers. Whether it is a result of the degeneration of afferent fibers or a long-lasting but eventually reversible loss of fiber functions is a matter of controversy.
The possibility of nerve fiber degeneration may be relevant only to the effects of RTX lasting more than 3 wk. This conclusion is based on the results of studies in rats on the recovery of nociceptive functions of sciatic nerve fibers damaged by different methods. Whether it was a neurolytic block by a local anesthetic agent (25) or mechanical nerve injury (26), regeneration of sciatic nerve fibers began to contribute nociception no earlier than 3 wk after destruction of the fibers. Thus, restoration of nociceptive responses beyond a 3-wk period is not necessarily due to the reversible nature of the effect of vanilloids on the fiber, but may result from nerve regeneration with the growth of new fibers. However, the restoration of nociceptive responses before this time speaks against fiber degeneration. Rather it indicates that the restoration of fiber function following RTX-induced blockade (lasting 3 wk or less) is in agreement with the absence of any significant fiber degeneration demonstrated by electron microscopy. Both concentrations of RTX used in this study provide nociceptive blockade lasting <3 wk (2 days with RTX 0.0001% and 2 wk with RTX 0.001%) (15).
The Idarola group recently suggested the use of RTX for selective nociceptive neuronal deletion as an approach to controlling nociceptive processes (27,28). In principle, both reversible and irreversible blockade of C-fibers can result from RTX administration. The type of blockade probably depends on the agent’s concentration (dose) and the sensitivity of the target nerve chosen for selective antinociceptive action. As with local anesthetics, this response may depend on the concentration at which it can produce anesthetic or neurolytic blockade.
One can argue that unmyelinated fibers do not leave much signs of degeneration behind when they die. Therefore, the very little overt axon damage found in this study does not indicate the absence of degeneration. However, the number of unmyelinated axons also did not significantly change in our experiments. In addition, there is other evidence that these fibers are not irreversibly damaged by RTX: the recovery of the thermal nociception by the 8th day after RTX (0.001%, 0.1 mL) administration.
Our results suggest that a selective and long-lasting sciatic nerve block can be provided by RTX without any significant damage to the unmyelinated nerve fibers.
We thank Professor Edwin L. Bradley, Jr., for statistical analysis.
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