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From the *Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; and Department of Biostatistics, University of Alabama, Birmingham, Alabama.
Address correspondence and reprint requests to Igor Kissin, MD, PhD, Pain Research Center, Brigham and Women's Hospital, 75 Francis Street, MRB 611, Boston, MA 02115. Address e-mail to kissin{at}zeus.bwh.harvard.edu.
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Abstract |
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METHODS: Male Sprague–Dawley rats received a single percutaneous injection of RTX (0.0005%, 0.1 mL) or vehicle at the sciatic nerve, and underwent surgery 3 h later to place four loose ligatures around the nerve on the side of drug administration. Responses to noxious heat (withdrawal latency, paw-lift duration), repetitive stimulation with von Frey filaments, and changes in hindpaw posture (toe spread, ventroflexion, and foot exorotation) were assessed.
RESULTS: Perineural RTX administered before surgery completely prevented ligation-induced reduction in withdrawal latency, increase in paw lift duration and increase in withdrawal frequency to von Frey filaments. The preventive effect of RTX on the development of deficits in hindpaw posture was pronounced but not complete, e.g., on day 7 after surgery, the cumulative paw-posture score (0–6) was 1.69 ± 0.92 with RTX and 4.06 ± 1.68 with vehicle (P < 0.005). The effect of RTX used against the background of already developed neuropathy was limited to thermal hypoalgesia lasting for a relatively short period.
CONCLUSION: Perineural RTX prevents the development of neuropathy caused by placing loosely constrictive ligatures on the sciatic nerve. Perioperative use of drugs acting via the TRPV1 receptors may hold the promise for preventing neuropathic pain after surgery on peripheral nerves.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONSREFERENCES |
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The TRPV1 receptors are found in peripheral nerve fibers (4,6,7). Electron microscopy demonstrated that TRPV1 immunostaining is associated with the axonal plasma membrane of unmyelinated fibers (8). Stimulation of the peripheral nerve by capsaicin leads to calcitonin gene-related peptide (CGRP) exocytosis along unmyelinated axons (8). The release of CGRP and other neuropeptides causes vasodilatation and plasma extravasation, both important elements of neuroinflammation. On the other hand, various inflammatory mediators, released after nerve damage, can activate or sensitize the TRPV1 receptors. Taken together, these findings suggest a possible protective effect of desensitization of the TRPV1 receptors in neuropathic pain. The review of pertinent literature reveals a number of studies that may confirm this suggestion. For example, Meller et al. (9) reported that neonatal (48 h after birth) capsaicin treatment (that destroys most C- and some A
-fibers) prevented the development of thermal hyperalgesia produced by placing loosely constrictive ligatures around the sciatic nerve 18 wk later. However, the authors did not observe any change of thermal withdrawal latency in capsaicin-treated animals compared with vehicle-treated animals. Neonatal capsaicin had a similar preventive effect on hyperalgesia produced by partial sciatic nerve ligation (10). Ossipov et al. (11) observed that the systemic administration of large doses of RTX (producing desensitization) resulted in a significant and long-lasting decrease in sensitivity to thermal noxious stimuli and abolished thermal hyperalgesia, but did not affect tactile allodynia caused by spinal nerve ligation. Gaus et al. (12) also demonstrated that systemic capsaicin administered before the placement of loose ligatures around the sciatic nerve prevented the development of heat hyperalgesia and the injury-induced decline in bone mineral density. Gaus et al. (12) also observed these effects when the drug was administered after the development of neuropathy.
If desensitization of the TRPV1 receptors has a protective effect in neuropathic pain, the presence of the effect could be the most pronounced in the model of placing ligatures around the sciatic nerve (13) because the neuroinflammation in this model plays a major role in the ensuing hyperalgesia (14,15). Our aim in the present study was to test the hypothesis that perineural RTX can prevent the development of hyperalgesia caused by placing loosely constrictive ligatures on the sciatic nerve. The perineural administration of RTX can provide a local long-lasting action without major systemic effects (16).
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METHODS |
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Nerve injury was induced by placing loosely constrictive ligatures around the common sciatic nerve as described by Bennett and Xie (13). Animals were anesthetized with a combination of ketamine (60 mg/kg) and xylazine (12 mg/kg) administered intraperitoneally. The common sciatic nerve was exposed at the middle of the thigh by blunt dissection through biceps femoris. Proximal to the sciatic trifurcation, 
7 mm of nerve was freed of adhering tissue and four ligatures (4.0 chromic gut) were tied loosely around the nerve 1 mm apart. The ligatures were tied such that there was no observable indentation of the sciatic nerve. The incision was closed in layers. Immediately after surgery, the animals were kept in a soft-bed cage with some food inside so that they could feed themselves without standing on their hind legs. In our experiments, the procedure was modified by the introduction of subsequent release of the nerve from the constriction. On Day 7 after placement of the ligatures, the rats were anesthetized (ketamine–xylazine combination) and after the access to the sciatic nerve similar to that of the initial surgery, all ligatures around the nerve were removed and the incision closed.
The development of neuropathy was assessed by the following tests. Heat hyperalgesia was determined by the paw-flick test with the use of radiant infrared heat (17). 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 hindpaw, 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, the cut-off time was 10.9 s. Three consecutive measurements were averaged. A difference score was calculated by subtracting the average latency of the operated side from that of the control side. A negative difference score indicated a lower threshold at the operated side, i.e., hyperalgesia to noxious heat (13). Paw-lift duration was measured as the time in seconds during which the animal kept its hindpaw elevated off the glass surface of the Planter Test device in response to the radiant heat. Three consecutive measurements were averaged. The measurements were taken for each hindpaw.
Mechanical hyperalgesia was determined by measuring the paw withdrawal in response to repetitive stimulation with von Frey filament with a bending force of 12 g [according to Flatters and Bennett (18), the responses to 8 g to 15 g filaments are best described as hyperalgesia, not allodynia]. The rats were placed in individual boxes with a mesh floor and allowed to acclimatize. The von Frey testing was performed as described previously (19). The 12 g filament was applied 10 times, at 5-s intervals, to each hindpaw. The number of withdrawals in response to 10 filament applications was determined for both paws and expressed as a right–left difference score.
Three variables were used to estimate changes in hindpaw posture: toe spread, ventroflexion, and exorotation of the foot. After the placement of loose ligatures, the toes, which are normally spread apart while the animal is walking or standing, are together and ventroflexed; in addition, the paw is everted (exorotated) with the animal walking on the medial edge of the paw touching the floor (13). Each of these three variables was scored separately after the animal was observed walking on an open bench. A score of 0 was assigned if the rat displayed no evidence of change; a score of 1, if the rat displayed a slight disturbance in the walking pattern; and a score of 2 if the disturbance was severe. The sum of the three variables (0–2 each) was recorded as a cumulative paw-posture score (0–6 total).
RTX (Sigma) was injected percutaneously at the sciatic nerve (single injection of 0.1 mL of 0.0005% RTX at the greater trochanter level 3 h before the surgery). To prevent the initial excitatory effect of RTX, its administration was preceded (10 min) by bupivacaine blockade. Bupivacaine (0.5%) was injected in a volume of 0.1 mL providing blockade of 60–90 min. Both injections were made with animals under brief anesthesia with halothane (2%). RTX was dissolved in dimethyl sulfoxide (Sigma) to a concentration of 1 µg/µL and stored at –80°C under nitrogen. It was diluted to 0.0005% before administration in 0.9% saline with 0.3% Tween 80 (to avoid precipitation).
Two series of experiments were performed. In the first series, the general procedure was as follows. For several days after arrival, rats were placed in the testing environment, and during the two days preceding the experiment, withdrawal latency to noxious heat, paw-lift duration, and frequency of foot withdrawal to von Frey filaments were determined. On the day of experiment, basal values were measured twice at a 20-min interval; the average value of two readings was used as a base for evaluating the effect of the agents. The rats were randomly assigned to 1 of 4 groups (n = 8, per group). In Group 1 (B/V + L, bupivacaine/vehicle + ligature) after baseline measurements, bupivacaine 0.5% was injected (0.1 mL) to the sciatic nerve, and the completeness of the nerve blockade was determined (by pinch of the first digit). Vehicle for RTX (dimethyl sulfoxide in saline with Tween 80) was injected 10 min after the bupivacaine injection, and behavioral variables were measured 3 h later. Surgery for placement of loose ligatures was then performed as described above. On Day 7 after the surgery, the behavioral variables, including the paw posture, were measured, after which the ligatures around the nerve were removed. On days 10 and 14 all the variables were measured again. In Group 2 (B/R + L, bupivacaine/RTX + ligature), the rats received RTX. All other conditions were identical to those in the Group 1. In Group 3 (B/R, bupivacaine/RTX), the animals did not have ligatures placed around the sciatic nerve, all other conditions were identical to those in Group 2. In Group 4 (N), the animals underwent neither the placement of ligatures nor percutaneous injections at the sciatic nerve.
The second series of experiments evaluated the effects of RTX after neuropathy caused by loosely constrictive ligatures had already developed. After the measurement of basal values (same as in the first series), the rats underwent surgery for placement of four ligatures on the sciatic nerve. On Day 7 after surgery, the behavioral variables, including paw posture, were measured, and the animals were randomly assigned to 1 of 2 groups (n = 8, per group). The rats in Group 1 (L + B/R, ligature + bupivacaine/RTX) received perineural RTX (0.0005%, 0.1 mL) after (10 min) bupivacaine injection (0.5%, 0.1 mL). The rats in Group 2 (L+B/V, ligature + bupivacaine/vehicle) received the injection of vehicle for RTX (also 10 min after bupivacaine injection). In both groups, all variables were measured 4 h and 24 h after the injection (on Days 7 and 8 after surgery, respectively) and on Days 9, 10, and 14 after surgery. Unlike in the first series of experiments, the ligatures were not removed.
The rats were assigned to the groups randomly (blocked randomization) and the examiner who measured the effects was blinded to the type of treatment (B/R or B/V). Each animal was housed individually and identified only with a sequential number assigned before the beginning of the experiment. Because the control solution and the RTX solution were in identical vials and were both clear, they were indistinguishable to the examiner.
Results are presented as mean ± sd. They were analyzed by a two-way (group and time) analysis of variance, with time treated as a repeated-measures factor. Comparisons between group mean ± sd at each time point were performed with one-way analysis of variance. Multiple comparisons among mean ± sd were made with Fisher's protected least significant difference test. The results were declared significant if P < 0.05.
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RESULTS |
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The right-left difference score in withdrawal latency is presented in Figure 1A. It demonstrates that the negative latency difference score caused by the placement of ligatures was absent in the RTX group. The right-left difference in paw-lift duration (Fig. 1B) was also eliminated by the preoperative administration of RTX. Mechanical hyperalgesia revealed by the response to a von Frey filament with a bending force of 12 g was also absent with RTX administration. The increase in the right–left difference score in response to the filament was completely prevented on Day 7 and also on Day 10 (3 days after the removal of ligatures) (Fig. 1C).
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The placement of ligatures caused significant deficits in hindpaw posture. The changes were related to all three assessed variables: toe spread, ventroflexion, and exorotation. Cumulative paw-posture score (0–6) in Group 1 (B/V + L) was 4.06 ± 1.68 on Day 7, 2.06 ± 1.15 on Day 10 (3 days after the removal of ligatures), and 1.47 ± 1.92 on Day 14 (7 days after the removal of ligatures). With the preoperative administration of RTX (Group 2, B/R + L), the changes induced by ligation were less prominent: 1.69 ± 0.92 (Day 7), 1.66 ± 0.87 (Day 10), and 1.25 ± 0.81 (Day 14). The difference between Groups 1 and 2 was statistically significant (P < 0.005) only on Day 7. When these three paw-posture variables were assessed separately, the effect of RTX was the most pronounced for exorotation (Fig. 1D). On Day 7, the exorotation score (0–2) in Group 1 (B/V + L) was 1.00 ± 0.53, and in Group 2 (B/R + L) was 0.12 ± 0.23 (P < 0.001).
The results on the withdrawal latency to noxious heat in the second series of experiments are shown in Figure 2A. The placement of ligatures caused a significant (P < 0.001) decline in withdrawal latency. In Group 1 (L + B/R), 4 h and 24 h after injection of RTX, withdrawal latency was increased above the preinjection level (P < 0.0001). In Group 2 (L + B/V), withdrawal latency was at approximately the same level during the 7- to 14-day period. As a result, the difference between groups was profound at both 4 h (8.89 ± 2.01 s vs 4.62 ± 1.01 s, P < 0.0001) and 24 h (7.11 ± 2.43 s vs 4.22 ± 0.67 s, P < 0.01) after the injection. However, on Day 9 (48 h after injection), the difference between groups decreased and was not significant. The difference between the groups (L + B/R and L + B/V) for paw-lift duration (Fig. 2B) was significant (P < 0.05) only at 4 h and 24 h after drug administration. The group differences in the response to von Frey filament or in paw-posture deficits were not significant. The cumulative paw-posture score on Day 8 was (L + B/R vs L + B/V) 2.63 ± 0.27 vs 3.34 ± 1.28 and on Day 9, 2.75 ± 0.30 vs 3.22 ± 1.00 (P > 0.05 for both days).
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DISCUSSIONS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONSREFERENCES |
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In our study, perineural RTX prevented the development of both heat and mechanical hyperalgesia. In addition, the deficits in paw posture after the nerve injury were also diminished. The deficits in paw posture after the loose ligation of the sciatic nerve is most likely due to the injury of the motor neuron projections (A
axons) that innervate the muscles below the knee (22). Capsaicin and RTX act on unmyelinated and, to a lesser degree, on myelinated, (predominantly small and medium size) afferent neurons, whereas they do not directly affect other afferent or non-sensory nerve fibers (23). Ma (24) studied the expression of the TRPV1 receptors by primary afferent neurons in rats and confirmed that these receptors are most likely expressed by C- and A-fiber neurons. Thus, the deficits in paw posture we observed were probably due to an indirect effect of RTX on motor fibers. Most likely these deficits were a consequence of the preventive effect of RTX on the development of neuroinflammation caused by the placement of ligatures around the sciatic nerve. The TRPV1 receptors integrate multiple noxious stimuli generated by inflammatory mediators (1,2). They are present in peripheral nerve fibers, to a significant degree in axonal plasma membranes (6–8,24). Stimulation of the TRPV1 receptors in the peripheral nerves leads to CGRP exocytosis along unmyelinated axons (8). The release of CGRP and other neuropeptides causes vasodilatation and plasma extravasation, both important elements of neuroinflammation. In addition, in inflammation, the TRPV1 receptors are sensitized and upregulated (3–5). Menendez et al. (25) reported that the analgesic effect induced by capsaicin is enhanced both in carrageenan-induced and Freund adjuvant-induced inflammation. The RTX-induced blockade of C-fibers results in the suppression of antidromic vasodilation (26). This confirms an earlier observation that local application of capsaicin to the sciatic nerve prevents neurogenic inflammation in the lateral part of the dorsal skin of the rat's paw (27). The suppression of antidromic vasodilation can contribute to preventing the development of neuropathy caused by the placement of ligatures. If a long-lasting desensitization of the TRVP1 receptors produced by RTX has a preventive effect with regard to the inflammatory response to the placement of loose ligatures around the sciatic nerve, the alleviation of all signs of neuropathy, including the deficits in paw posture, is not an unexpected outcome.
In contrast to the preoperative RTX administration, the effect of administration against the background of already developed neuropathy was limited to thermal hypoalgesia lasting a relatively short period, not much longer than 24 h Duration of this effect does not exceed duration of the effect of a similar dose of perineural RTX on the response to noxious heat in rats without neuropathy. For example, duration of the marked effect of perineural RTX (0.0003–0.0005%) on the response to noxious heat usually does not exceed 24 h (16). The difference between the effects of RTX administered before or after the development of neuropathy may indicate that the TRPV1 receptors are more important for the induction of hyperalgesia than for its maintenance.
The analgesic interventions with the use of capsaicin or RTX are based on an initial activation of the TRPV1 receptors that leads to desensitization of the receptors and defunctionalization of nociceptive nerve fibers lasting for a very long period of time. The other strategy of providing analgesia via the TRVP1 receptors is based on the use of TRVP1 receptor antagonists. For example, systemic BCTC, a TRVP1 antagonist has been reported to significantly reduce both mechanical and thermal hyperalgesia in inflammatory (Freund's adjuvant) and neuropathic (partial sciatic nerve injury) pain (28). Another TRVP1 receptor antagonist, iodo-resiniferatoxin applied intraplantarly inhibited the heat threshold-decreasing effect of intraplantar RTX (29). Similarly, intraplantar injection of capsazepine prevented capsaicin-induced hyperalgesia (21).
Pain that persists well beyond the healing of the injury, including surgical wounds, is a major clinical problem. Multiple mechanisms are likely responsible for chronic postinjury pain. Nerve damage is probably the most important factor in the development of persistent postsurgical pain (30), especially with surgeries on the peripheral nerves. It was recently demonstrated that perioperatively administered corticosteroid diminished damage to the C-fibers in microscopic lumbar disk surgery (31). Perioperative use of drugs acting via the TRPV1 receptors may hold promise for the prevention of neuropathic pain after surgery on the peripheral nerves.
In conclusion, a single preoperative injection of RTX at the sciatic nerve prevented the development of neuropathic changes caused by temporary placement of loose ligatures around the nerve on the side of the injection. Thermal and mechanical hyperalgesia were prevented completely, and the prevention of deficits in paw posture was significant. In contrast to the preoperative nerve treatment, the effect of RTX used against the background of already developed neuropathy was relatively short and limited to thermal hypoalgesia.
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Footnotes |
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Supported by the National Institutes of Health Grant GM065834.
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REFERENCES |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONSREFERENCES |
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