Pain that persists well beyond the healing of the injury, including surgical wounds, is a major clinical problem (1). Multiple mechanisms are likely responsible for persistent postinjury pain; some might be related to the transition of acute pain to chronicity, a concept that is supported by reports of the relationship between the intensity of acute postoperative pain and subsequent development of chronic pain after surgery (2–4). It is possible that a long-lasting imprint of acute pain contributes to its transition to chronic pain.
Long-term potentiation (LTP) is proposed as a fundamental mechanism of memory (5). LTP of synaptic transmission in the spinal dorsal horn after repetitive high-frequency electrical stimulation has been described for both C and A-δ fibers (6,7). This form of LTP was not affected by neonatal capsaicin treatment, which destroys most C fibers (6). There are striking similarities between the synaptic plasticity contributing to memory and that contributing to pain (8,9).
In the rat model, injuries of a paw cause a secondary hyperalgesia that can spread to paws distant from the inflamed plantar region (10–12). Kayser et al. (12) demonstrated that with carrageenan-induced inflammation, hyperalgesia in the injected paw lasts less than a week and not much longer than 24 h in the noninjected paws. In experiments with repeated acute tissue injury, these authors (13) found that distant hyperalgesia in the previously injected hindpaw was exacerbated when the second carrageenan injection (into previously noninjected paw) was given seven days after the first injection (by that time the initially induced hyperalgesia in the injected paw had disappeared). These and similar experiments (12–16) demonstrated that when the index of hyperalgesia after inflammation indicates recovery, the central nervous system (CNS) imprint of secondary hyperalgesia continues, and can be revealed by carrageenan injection into the other (not previously injected) hindpaw. These results indicate that the repeated paw inflammation induced by carrageenan can be used as a model for evaluation of nociceptive memory that might facilitate the establishment of chronic pain.
The aim of the present study was to determine whether selective blockade of C fibers of the sciatic and saphenous nerves with resiniferatoxin (RTX) can prevent the development of distant hyperalgesia induced by repeated paw inflammation. Such an effect would indicate that the long-lasting imprint of secondary hyperalgesia depends on C-fibers.
Experiments were performed on male Sprague-Dawley rats weighing 250–300 g. The rats were housed with a 12-h light:dark cycle, and food and water were available ad libitum. The study for this protocol was approved by the Institutional Panel on Laboratory Animal Care.
Inflammation was induced by subcutaneous injection (26-gauge needle) of 0.1 mL of 2% carrageenan (Sigma Chemical Co, St. Louis, MO) in the plantar surface of the hindpaw under halothane (2%) anesthesia. The response to noxious pressure was determined by detecting a threshold of coordinated struggle to increasing pressure (17) with the use of an Analgesy-Meter (Ugo Basile, Milan, Italy). The threshold was measured by positioning the hindpaws on a Teflon platform and directing the device's 2-mm pressure cone on their dorsal surface. The cutoff pressure was 250 g. Three consecutive measurements were taken, and the last two were averaged. If at the time of the initial measurement the pressure threshold was more than a third less than baseline, the other two measurements were not taken because of ethical considerations. The response to noxious heat was determined by the paw-flick test with the use of radiant infrared heat (18). 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 Basile). The infrared radiation intensity was set to elicit a withdrawal latency of 6–7 s, the cutoff time was 10 s. Three consecutive measurements were averaged. The volume of the rat paw was measured by a Plethysmometer (Paw Volume Meter; Ugo Basile). Two consecutive measurements were averaged.
Blockade of the nociceptive input from the inflamed paw was achieved by percutaneous injections at two nerves: the sciatic and saphenous. The two injections were made under brief halothane anesthesia; the sciatic nerve was injected at the greater trochanter level and the saphenous nerve at the midthigh level. To prevent the initial excitatory effect of RTX, its administration was preceded by bupivacaine (Bup) blockade. Bup (0.5%) was injected in a volume of 0.1 mL at the sciatic nerve and 0.05 mL at the saphenous nerve, providing blockade of 60–90 min. Ten minutes after Bup, the injections at both nerves were repeated with the same volumes of the solutions of RTX. RTX (Sigma) was dissolved in dimethyl sulfoxide (Sigma) to a concentration of 1 μg/μL and stored at –80°C under nitrogen. It was diluted to 0.001% before the administration in 0.9% saline with 0.3% Tween 80 (to avoid precipitation). With the Bup-RTX administrations, there were no signs of behavior associated with pain (flinching or licking of the hindpaw).
Nociceptive memory regarding secondary hyperalgesia was assessed by measuring distant hyperalgesia after repeated carrageenan-induced inflammation, as described by Guilbaud et al. (13). During the first inflammation induced by the injection of carrageenan into the hindpaw, the mechanical hyperalgesia was profound on the side of injection and minimal or absent in the contralateral hindpaw. However, repeated injections of carrageenan into the previously noninjected hindpaw resulted in pronounced hyperalgesia in the contralateral, previously injected hindpaw (Fig. 1). The difference between distant hyperalgesia after the initial and repeated-crossover injection of carrageenan into the hindpaw was used as a measure of the memory of secondary hyperalgesia.
The general procedure for measuring changes in behavioral responses was as follows: For several days after arrival, rats were placed in the testing environment, and during the two days preceding the experiment pressure threshold, heat latency and hindpaw volumes were measured. On the experimental day, basal values were determined twice with a 30-min interval; the average value of two readings was used as a base for evaluating the effect of the agents. The time of the last baseline measurement was noted as time 0. The rats were assigned randomly (blocked randomization) to one of four groups. In Group 1, carrageenan was injected into the right hindpaw and into the left hindpaw 2 wk later. Noxious pressure threshold, noxious heat latency, and paw volumes were measured on both sides 2, 4, and 21 h and 5–6 days after each of the carrageenan injections (5, 7, and 24 h after the last baseline measurement). In Group 2, carrageenan was injected only once into the left hindpaw 2 wk after the last baseline measurement; all the above-indicated measurements were made at the same time points as in Group 1. In Group 3, Bup was injected at the sciatic and saphenous nerves, and the completeness of blockade was confirmed (by first- and fifth-digit pinch). Ten minutes later, Bup was followed by RTX, and behavioral variables were measured 3 h later. Immediately after this, carrageenan was injected into the right hindpaw and into the left hindpaw 2 wk later. Again, noxious pressure threshold, noxious heat latency, and paw volume were measured on both sides 2, 4, and 21 h and 5–6 days after each of the carrageenan injections. The experiments in Group 4 were similar to those in Group 3, with one exception: carrageenan was injected only once, 14 days after the Bup-RTX injection.
Raw data were expressed in grams for the noxious pressure threshold, in seconds for noxious heat latency, and in milliliters for the paw volume. Results are presented as mean ± sd. They were analyzed using a two-way (group and time) analysis of variance, with time treated as a repeated-measures factor. Comparisons among the groups mean ± sd at each time point were performed with one-way analysis of variance. Multiple comparisons among mean ± sd were made using Fisher's protected least significant difference test. The results were declared significant if P < 0.05.
In Group 1, with 2 carrageenan injections separated by a 2-wk interval, the first injection of carrageenan caused a profound decrease in noxious pressure threshold on the side of injection (right hindpaw) but no significant changes on the contralateral side (left hindpaw) (Table 1; first period). The second injection of carrageenan (2 wk later into the previously noninjected left hindpaw) caused a profound decrease in noxious pressure threshold on the side of injection. On the right, previously injected side (which did not receive an injection this time), the noxious pressure threshold decreased from the baseline of 141 ± 23 g to 122 ± 26 g (P < 0.001) at 5 h, 105 ± 16 g (P < 0.0001) at 7 h, and 96 ± 19 g (P < 0.0001) at 24 h (Table 1; second period). The sensitivity to noxious heat in the injected paw was decreased during both periods of inflammation; at the peak of inflammation (7 h), the measurements of the latency were impossible because of excessive sensitivity of the animals. In contrast to the noxious pressure threshold, noxious heat latency in the paw contralateral to the inflamed paw did not change during either period of inflammation (Table 2). Paw volume on the side of carrageenan injection was profoundly increased almost to the same degree during the first and the second periods of inflammation (Table 3).
In Group 2, with the single injection of carrageenan 2 wk after the baseline measurement, the response to inflammation was identical to that in Group 1 after the first injection (Tables 1–3). Most importantly, there was a statistically significant difference between Group 2 and Group 1 regarding noxious pressure threshold in the noninflamed (right) hindpaw during the second period: 140 ± 11 g (Group 2) versus 105 ± 16 g (P < 0.001) at 7 h and 134 ± 15 g (Group 2) versus 96 ± 19 g (P < 0.001) at 24 h.
In Group 3, with the administration of RTX 3 h before the injection of carrageenan, noxious pressure threshold after the first carrageenan injection was uninformative because of the RTX-induced increases of the threshold. At 5 and 7 h, it was more than the cutoff limits (Table 1). Two weeks later, after the second carrageenan injection, the hyperalgesia in the inflamed hindpaw was as profound as in Group 1, but hyperalgesia in the noninflamed, previously injected hindpaw was absent. The differences between Group 3 and Group 1 regarding noxious pressure threshold in the noninflamed (right) hindpaw during the second period were statistically significant: 139 ± 22 g (Group 3) versus 105 ± 16 g (P < 0.001) at 7 h and 140 ± 22 g (Group 3) versus 96 ± 19 g (P < 0.0001) at 24 h (Table 1). Noxious heat latency was immeasurable after RTX injection (Table 2). In 2 wk, the response to heat almost completely recovered; however, the baseline values on the side of RTX injection during the second period were somewhat higher than during the first period (8.09 ± 0.3 s versus 6.86 ± 0.5 s in Group 3 and 8.09 ± 1.0 s versus 6.99 ± 0.4 s in Group 4, a statistically significant difference in both cases). There were significant differences between Group 3 and Group 1 regarding inflamed paw volumes: 1.61 ± 0.2 mL (Group 3) versus 3.23 ± 0.6 mL (P < 0.0001) at 7 h and 1.78 ± 0.3 mL (Group 3) versus 2.32 ± 0.2 mL (P < 0.001) at 24 h. However, the inflamed paw volumes after the second carrageenan injection (2 wk after RTX) were not different from corresponding volumes in Group 1 (Table 3).
In Group 4, when the administration of RTX was not followed by the carrageenan injection (first period), the results obtained during the second period (with the injection of carrageenan) did not significantly differ from those in Group 3 (Tables 1–3).
Our results with mechanical hyperalgesia during repeated carrageenan-induced inflammation confirmed the observation by Guilbaud et al. (13) that the second injection of carrageenan, given when the hyperalgesia caused by the first injection had disappeared, resulted in an enhanced distant hyperalgesia in the previously injected paw. This effect has been reproduced in several other studies (14,15). Fletcher et al. (15) observed that after the second carrageenan injection, distant hyperalgesia in the previously injected paw was increased and that the infiltration of the paw with Bup before the first carrageenan injection can prevent this hyperalgesia reinforcement. It is interesting that the effect of Bup preinfiltration was very insignificant when the authors used a single injection of carrageenan (19). The enhanced distant hyperalgesia after the repeated-crossover injection of carrageenan into the previously noninflamed paw suggests that, despite recovery from the initial injury, there is a trace in the CNS that makes a further injury more painful. In our experiments, as in the work of Kayser and Guilbaud (12), the first injection of carrageenan did not cause significant changes in noxious pressure threshold in the contralateral hindpaw. This might be a result of the concealment of distant hyperalgesia by various central inhibitory controls responding to the injury (for example, diffuse noxious inhibitory controls) (20). Our experiments demonstrated that the repeated-crossover injection of carrageenan caused a decrease in the noxious pressure threshold in the previously inflamed paw, which at 24 hours, reached a maximum of 30% (P < 0.0001). Thus, the decrease was pronounced not only with the one-week interval between carrageenan injections (13–16), but also with a two-week interval. It was also observed in our preliminary experiments with a four-week interval. An example with a 4-week interval is presented in Figure 1. It demonstrates a sustained decrease of pressure threshold by approximately a third in the contralateral hindpaw after the repeated injection of carrageenan (Fig. 1C). At the same time, there was no significant change in pressure threshold in the contralateral hindpaw after the initial injection (Fig. 1B). Thus, the nociceptive imprint of the initial inflammation was revealed as a distant hyperalgesia by the repeated carrageenan injection four weeks later.
The noxious pressure threshold in the previously injected hindpaw completely recovered before the second carrageenan injection into the opposite hindpaw (Table 1; Group 1). That was an indication that hyperalgesia caused by the first carrageenan injection disappeared but that its imprint in the CNS remained and resulted in marked distant hyperalgesia from the repeated injury.
The decrease of pressure threshold observed in the right paw 24 hours after the second carrageenan injection (into the left paw) was more pronounced than that in the left paw (96 ± 19 g versus 126 ± 18 g; Table 1). This difference does not necessarily indicate that the response of the injected paw in general was less profound than that of the contralateral paw. It only indicates that the response of the contralateral paw lasted longer. Because of high sensitivity of the injected paw at five and seven hours after carrageenan, the measurement of pressure threshold was not performed because of the ethical considerations (but it was performed in the contralateral paw). The longer lasting response of the contralateral paw might have been caused by the different time course of the response reflecting central sensitization (related to the contralateral paw) versus the local inflammatory response (related to the injected paw).
For a relatively short time after the RTX administration, noxious pressure thresholds were increased, even in the collateral paw. This effect cannot be explained by the systemic effect of RTX after absorption from the injected leg because in the collateral paw, latency to noxious heat (which with RTX is a more sensitive index than pressure threshold) did not change. The likely explanation for this phenomenon is that Bup did not completely block the excitatory effect of RTX (it could extend beyond the duration of Bup's action −1 to 1.5 hours), and this led to the activation of inhibitory descending projections from the CNS. A similar effect was demonstrated by Gjerstad et al. (21) with capsaicin. The authors observed a profound inhibition of electrically evoked C-fiber responses in the dorsal horn after contralateral IM injection of the vanilloid receptor agonist.
Noxious heat latency did not demonstrate any significant change in the noninjected hindpaw during the first or second period of the experiment, which is in agreement with the notion that hyperalgesia outside the zone of the injury occurs only with mechanical stimuli (22).
The increases in the injected hindpaw volume during the first and the second periods of the experiment in Group 1 were similar. In Group 3, after the first carrageenan injection, paw volume was increased to 1.61 ± 0.2 mL at 7 hours (P < 0.05 versus baseline) and to 1.78 ± 0.3 mL at 24 hours (P < 0.0005). The increase in the paw volume lasted for several days. As it was demonstrated in our previous study (23), the perineural administration of RTX counteracts carrageenan-induced inflammation to a significant degree, by delaying inflammation development. This type of effect was clearly visible in the present experiments. In the RTX-treated rats, the carrageenan-induced increase in paw volume was small at 7 hours but much more extensive at 24 hours (from 1.40 ± 0.1 mL to 1.78 ± 0.3 mL; P < 0.0005) and later. The comparison of Groups 1 and 3 demonstrates that RTX administration attenuated the paw volume changes during the first, but not during the second, period. We previously observed that RTX perineural blockade of the sciatic and saphenous nerves significantly reduced carrageenan-induced paw edema (23). In the present study, the degree of edema reduction was even greater. Lam and Ferrell (24), using the Evans blue content method, demonstrated that inflammation induced by an injection of intraarticular carrageenan was reduced by 44% in the knees of rats that had previously been injected with capsaicin. They also observed that chronic joint denervation produced a 37% reduction in the inflammation. The above results suggest that RTX and other vanilloids counteract neurogenic inflammation. This agrees well with the observation by Jansco et al. (25) 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.
RTX is an ultrapotent vanilloid receptor agonist; and its initial excitatory effects relative to its inactivating effects are far less pronounced compared with those of capsaicin (26). Nevertheless, to prevent the initial excitatory effect of RTX, its administration in our experiments was preceded by Bup (0.5%), which produced a blockade lasting 60–90 minutes. It was demonstrated previously in rat experiments that a local anesthetic nerve block of such duration provides no noticeable effect on carrageenan-induced hyperalgesia (27). In our preliminary experiments for the present study, rats received the Bup (0.5%) nerve blocks without a subsequent RTX injection; then 2 carrageenan injections with the two-week interval followed (as in Group 3). As expected, with Bup alone, there was a profound distant hyperalgesia in the contralateral hindpaw caused by the repeated-crossover injection of carrageenan.
Our results indicate that the distant hyperalgesia caused by repeated-crossover injection of carrageenan two weeks after the initial injection was completely prevented by perineural RTX administered before the initial injection of carrageenan (Fig. 2). This effect may be interpreted as prevention of long-term nociceptive imprint that lasts long after recovery from the hyperalgesia. Woolf and Wall (28) demonstrated that selective blockade of nociceptive fibers with the application of capsaicin to the sciatic nerve prevents the development of central sensitization. In their experiments, capsaicin abolished prolonged facilitation of the flexor reflex caused by electrical stimulation or mustard oil-induced inflammation. These results led to the conclusion that C afferents are primarily responsible for the induction of central sensitization. Central sensitization has several distinct forms, including LTP-like changes (9). It is possible that RTX-induced blockade of central sensitization, caused in our experiments by the first injection of carrageenan, is, in fact, the mechanism responsible for the prevention of distant hyperalgesia after the repeated carrageenan injection.
The other possibility is that the blockade of nociceptive fibers prevented activation of molecular mechanisms specific for memory. LTP of synaptic transmission in the spinal cord after stimulation of C- and A-δ fibers is well known (6,7) and might be a mechanism responsible for the RTX-induced prevention of distant hyperalgesia after the repeated injection of carrageenan. However, Randic et al. (6), who studied LTP in a slice preparation of young rat spinal cord, found that it was not affected by neonatal capsaicin treatment.
Blockade of the C-fibers leads to the inevitable antiinflammatory effect caused by the suppression of neurogenic inflammation. As a result, we observed the lesser degree of hindpaw inflammation after the first injection of carrageenan, administered after perineural RTX. This effect could contribute to the absence of distant hyperalgesia after the second injection of carrageenan.
Postsurgical chronic pain can be a result of injury to major peripheral nerves; it also can be caused by continuing inflammation. The other possibility is that severe and long-lasting acute pain can transit to chronic pain via memory-like alterations in the CNS, and, as a result, chronic pain can persist without any significant peripheral nerve damage or continuing inflammation. Persistent, activity-dependent alterations in synapses between neurons are thought to be mechanisms for memory (29). Similar synaptic alterations contribute to information storage and behavioral plasticity not only in regions of the brain specialized for memory function, but also in other CNS regions. For example, in the spinal cord, they are contributing to nociceptive plasticity (9). Reports of the correlations between the intensity of acute postoperative pain and subsequent development of chronic pain after breast surgery (2), thoracotomy (3), and inguinal hernia repair (4) may indicate that the memory-like synaptic plasticity generated by acute pain can contribute to its transition to chronic pain. It was demonstrated that duration of nociceptive blockade is an important factor in the prevention of persistent hyperalgesia (27). Selective nociceptive blockade with vanilloid receptor agonists might have an advantage over blockade provided by local anesthetics, especially when requirements for mobilization and protective sensations are important.
In conclusion, using the model of repeated carrageenan-induced inflammation, we demonstrated that after recovery of hyperalgesia induced by the initial inflammation, repeated inflammation led to the development of distant hyperalgesia that was absent during the initial inflammation. The development of distant hyperalgesia during repeated inflammation was completely prevented by perineural RTX administered before the initial injection of carrageenan. These results suggest that selective blockade of nociceptive fibers prevents formation of a long-term hyperalgesia-related imprint in the CNS that lasts long after recovery of hyperalgesia induced by the initial injury. This effect could be achieved via several mechanisms, including blockade of central sensitization or prevention of activation of molecular mechanisms specific for memory. Blockade of the C-fibers leads to the inevitable antiinflammatory effect (suppression of neurogenic inflammation), which could also be a significant contributing factor. Thus, pain memory can be preempted by selective and prolonged blockade of C-fibers.
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