Neurotropin® (NTP) is a nonprotein extract from inflamed skin of rabbits inoculated with vaccinia virus and is widely used in Japan to treat neuropathic pain such as post-herpetic neuralgia (PHN) and other painful conditions. A double-blind placebo-controlled cross-over study investigating its effectiveness in the treatment of acute pain after tooth extraction and complex regional pain syndrome (CRPS) is currently underway at the National Institute of Dental and Craniofacial Research in the United States (US).
The biological activity of NTP is expressed as neurotropin units (NU), and is standardized by an analgesic test in animals loaded with the specific alternation of rhythm in environmental temperature (SART) stress. This test of repeated cold stress creates a hypersensitivity to noxious stimuli (1,2). The antinociceptive effect of NTP has been investigated using SART-stressed animals, and the activation of the descending monoaminergic pain inhibitory systems was implicated in the mechanism of action of NTP (3,4). The noradrenergic neurons of the descending pain inhibitory systems arise mainly from the locus ceruleus in the pons, whereas those of the serotonergic system arise from the nucleus raphe magnus in the medulla oblongata (5). Both of these pathways terminate in the spinal dorsal horn and inhibit pain transmission from the primary afferent neurons. The antinociceptive effect of systemically administered NTP is antagonized by intrathecal (i.t.) administration of a 5-hydroxytryptamine (5-HT)3 receptor antagonist and a noradrenergic α2 antagonist (3). These results indicate the involvement of activation of descending monoaminergic pain inhibitory systems in the antinociceptive effect of NTP. The inhibitory effects of NTP on mechanical (6) and thermal hyperalgesia (7) have been demonstrated in the rat chronic constriction injury (CCI) model. However, the mechanism of action of NTP was not investigated in this model.
In the present study, to elucidate the mechanism of antiallodynia and antihyperalgesia of NTP in neuropathic pain and the involvement in descending monoaminergic pain inhibitory systems, we studied the effect of NTP in mice subjected to spinal nerve ligation (SNL) after chemical denervation of spinal serotonergic or noradrenergic neurons. In addition, we compared the effect of NTP administered via various routes.
Male C57Bl/6 mice (8–12 wk old; Japan SLC Inc., Hamamatsu, Shizuoka, Japan), weighing 25–33 g at the time of testing, were used. They were housed in groups of 6–8 and maintained under standard laboratory conditions with chow and water available ad libitum. Lighting was on a regular light/dark cycle, with lights on from 6:00 am to 8:00 pm. All procedures were performed in accordance with the study protocol approved by the Animal Research Committee of Osaka University Medical School.
NTP was provided by Nippon Zoki Pharmaceutical Co. (Osaka, Japan) and was diluted with physiological saline. Doses of NTP were expressed as NU. Desipramine hydrochloride (Wako Pure Chemical Industries Ltd., Osaka, Japan), 6-hydroxydopamine (6-OHDA; Sigma Chemical Co., St. Louis, MO), 5,7-dihydroxytryptamine (5,7-DHT; Fluka Chemie, Buchs, SG, Switzerland) were purchased. NTP was administered in a volume of 10 mL/kg for intraperitoneal (i.p.) injection. The intracerebroventricular (i.c.v.) injection was performed into the left lateral ventricle of mice. Injection was performed using a Hamilton microsyringe fitted with a 27-gauge needle in a volume of 5 μL, according to the method of Haley and McCormick (8). The site of injection was 2 mm lateral from the midline on a line drawn through the anterior base of the ears, and 3 mm in depth from the skull surface. I.t. injection was performed freehand between L5 and L6 lumber space according to the method of Hylden and Wilcox (9). The injection was performed using a 30-gauge needle attached to a Hamilton glass microsyringe in a volume of 5 μL. For local injection into the nerve-injured paw, the mice were briefly anesthetized with sevoflurane, and the subcutaneous injection was performed with a syringe with a 30-gauge needle into the dorsal skin of the left hindpaw. Behavioral tests were started 30 min after i.p. and local injection or 15 min after i.c.v. and i.t. injection.
The left fifth lumbar nerve of mice was tightly ligated with 6-0 silk under sevoflurane anesthesia as previously described (10). Control animals were sham-operated by exposing, but not ligating, the left fifth lumbar nerve.
Behavioral tests were performed at 7 or 10 days after nerve ligation.
To measure mechanical sensitivity of the hindpaw, mice were placed in individual plastic boxes on a mesh floor and allowed to acclimate for 30 min. A series of calibrated von Frey filaments (Stoelting, Wood Dale, IL) were applied perpendicularly to the plantar surface of the hindpaw with sufficient force to bend the filaments for 6 s. Brisk withdrawal or paw flinching were considered as positive responses. In the absence of a response, the filament of next greater force was applied. In the presence of a response, the filament of next lower force was applied. In the presence of a response, the filament of next greater force was applied. The tactile stimulus producing a 50% likelihood of withdrawal was determined using the “up-down” calculating method.
An automated von Frey test was used to assess sensitivity to nociceptive mechanical stimulation. After habituation, a mechanical stimulus [0.8- to 0.9-mm-diameter filament connected to an automatic Transducer Indicator (model 2290; IITC, Woodland Hills, CA)] was then applied to the middle of the plantar surface of the left hindpaw. Withdrawal threshold (in grams) was measured. A 20-s cut-off time was used to avoid tissue damage.
Thermal sensitivity was assessed by the method of Hargreaves et al. (11). A radiant heat beam (Paw Stimulator Analgesia Meter Model 390; IITC) was focused on the left hind limb footpads of mice placed on a glass surface. The withdrawal-response latency (in seconds) was measured with a 20-s cut-off time. In all cases, the experimenter was blinded to the type of operation (sham or ligated) and drugs administered.
The neurotoxins, 6-OHDA and 5,7-DHT, dissolved in saline containing 1 mg/mL ascorbic acid, were used according to the method of Nakazawa et al. (12) with some modifications. For selective denervation of spinal noradrenergic terminals, i.t. injection of 6-OHDA (20 μg/5 μL/mouse) was performed 3 days before behavioral testing. Selective denervation of spinal serotonergic neurons was induced by i.t. injection of 5,7-DHT (20 μg/5 μL/mouse) 3 days before testing. To block the uptake of 5,7-DHT into noradrenergic terminals, desipramine (25 mg/kg) was administered i.p. 30 min before i.t. injection of 5,7-DHT. Saline (5 μL) was injected i.t. as a control.
To determine the contents of norepinephrine (NE), serotonin (5-HT), and dopamine (DA) in spinal cord, the lumber enlargement (approximately 1 cm) was isolated from normal (non-operated) mice on the same schedule as the antinociceptive test after treatment with the neurotoxins. Tissue was homogenized with 0.28% perchloric acid including 0.2% EDTA-2Na and 0.002% ascorbic acid, and centrifuged (2000g × 30 min) at 4°C. The supernatant was stored at −80°C until the analysis. The content of monoamines was measured using high-performance liquid chromatography.
All data were expressed as mean ± sem. Data on the time course of analgesic effects were analyzed using analysis of variance (ANOVA) followed by Dunnett’s post hoc comparison test. Dose-dependent effect was assessed by t-test and ANOVA followed by Tukey-Kramer’s post hoc comparison test. The effect of neurotoxins was assessed by t-test. Significance levels were set at P < 0.05.
Seven days after SNL, clear hypersensitivity to mechanical and thermal stimuli was observed compared with the preoperative state and the contralateral paw (Figs. 1 and 2). NTP (100 NU/kg) significantly increased both the withdrawal threshold and withdrawal latency in SNL mice between 20 and 60 min after i.p. injection (Fig. 1) compared with the preinjection baseline (0 min). The maximal effect was observed at between 30 and 40 min. Saline had no effect on either withdrawal threshold or withdrawal latency of SNL mice. NTP had no effect on either withdrawal threshold or withdrawal latency of the contralateral paws of SNL mice (Fig. 1) or sham-operated mice (Fig. 2).
NTP increased both withdrawal threshold and withdrawal latency at the doses between 50 and 200 NU/kg and the effect was dose-dependent although the difference between 100 and 200 NU/kg was not significant (Fig. 2). The maximal effect of NTP on both withdrawal threshold and withdrawal latency was only partial and did not completely restore the threshold to the sham-operated control levels.
To examine the involvement of spinal monoaminergic systems in the analgesic effect of NTP, spinal noradrenergic or serotonergic neurons were selectively denervated. The effects of chemical denervation on the contents of spinal NE, 5-HT, and DA are shown in Table 1. I.t. 6-OHDA depleted the content of NE by >97% with only a slight (18%) but significant reduction of DA. 5,7-DHT reduced the content of 5-HT by >96% with no effect on NE or DA. Spinal noradrenergic denervation by 6-OHDA inhibited the increase of both withdrawal threshold and latency induced by 100 NU/kg of NTP (Fig. 3). However, spinal serotonergic denervation by 5,7-DHT did not modify the withdrawal of either the increased threshold or latency induced by 100 NU/kg NTP.
The effect of NTP via various routes was examined by i.c.v., i.t., and local injection (Fig. 4). Although i.c.v. administration of NTP increased withdrawal threshold and withdrawal latency at 100 mNU/mouse, i.t. and local NTP did not show any effects.
NTP had no analgesic effect in contralateral paws or in sham-operated mice. Both antiallodynic and antihyperalgesic effects were observed in SNL mice. This result is in agreement with the report that NTP has antinociceptive effects in SART-stressed animals but little or no analgesic effect in normal animals (4). These properties are different from other analgesics such as opioids and tricyclic antidepressants, for which an antinociceptive effect has been reported in normal animals (13,14). This suggests that the mechanism of action of NTP may be different from other analgesics.
In this study, NTP showed both antiallodynic and antihyperalgesic effects in the mouse SNL model. In another study using the rat CCI model (7), however, NTP showed antihyperalgesia but not antiallodynia. The reasons for this difference are unclear but probably relate to differences between the species (rat or mouse) and/or the two models (CCI or SNL).
A single i.p. injection of 100 NU/kg NTP significantly inhibited allodynia and hyperalgesia, and the effect lasted for 60 minutes with the peak effect at 30–40 minutes after injection (Fig. 1). This result is similar to that seen in a previous study (6) wherein the analgesic effect was observed at the dose of 100 NU/kg in the CCI model. Comparing the peak effect observed at 30 minutes after injection at doses of 50, 100, and 200 NU/kg, NTP dose-dependently inhibited allodynia and hyperalgesia, but the analgesic effect of 200 NU/kg NTP was not significantly more than that of 100 NU/kg, suggesting that the inhibition reached a plateau at <100% inhibition. This is different from the effect seen in the SART-stress model, in which NTP completely inhibited the hyperalgesia induced by SART stress. The different effects of NTP in these two models are probably attributable to the different underlying mechanisms. SART stress has been reported to induce hypersensitivity to pain by decreasing the activity of descending pain inhibitory systems (3). Chronic cold stress was reported to increase the firing rate and the expression of tyrosine hydroxylase in neurons of the locus ceruleus (15). However, CCI was reported to increase the expression of tyrosine hydroxylase both in the locus ceruleus and the dorsal horn (16). Hyperexcitability of locus ceruleus or dorsal horn neurons results in a decreased efficiency of descending pain inhibitory systems.
In the experiments with neurotoxins, 6-OHDA for depletion of NE and 5,7-DHT for depletion of 5-HT, were applied i.t. because these neurotoxins do not pass the blood-brain barrier (12). I.t. 20 μg 6-OHDA depleted the content of spinal NE with no effect on the 5-HT content. Similarly, i.t. 20 μg 5,7-DHT after i.p. desipramine (25 mg/kg) effectively depleted the content of spinal 5-HT with no effect on the content of NE or DA. Spinal monoamine depletion did not increase the allodynia and hyperalgesia induced by SNL (data not shown). The antiallodynic and antihyperalgesic effect of NTP was attenuated by spinal depletion of NE but not by depletion of 5-HT. This suggests that NTP-induced antiallodynia and antihyperalgesia are mediated principally through the spinal noradrenergic neurons but not serotonergic neurons in SNL mice. In contrast, the study using the SART-stressed model has shown that the analgesic effects of NTP are antagonized by i.t. application of a 5-HT3 antagonist and an α2 noradrenergic antagonist, suggesting the implication of both 5-HT and NE in the analgesic effect of NTP in the SART-stressed model. The involvement of spinal NE in antiallodynic and antihyperalgesic effect of NTP is similar between the two models but the spinal 5-HT seems less involved in the effect of NTP in SNL mice. A more limited contribution of 5-HT than NE in the SNL model is suggested by the fact that the selective NE uptake inhibitors exhibit analgesic properties in neuropathic pain models, whereas selective 5-HT uptake inhibitors do not (17,18). Furthermore, α2 agonists have been shown to inhibit hyperalgesia in the mouse SNL model (19,20). Clinical studies have also shown the analgesic and antiallodynic effect of antidepressants with a major noradrenergic component (21–24). Although the exact mechanism of action of these antidepressants is still unknown, these results are consistent with our findings, suggesting the importance of noradrenergic descending pain inhibitory systems.
Although there was no analgesic effect when NTP was administered via either i.t. or local injection, i.c.v. NTP showed antiallodynic and antihyperalgesic effects. These results suggest that NTP may act at a supraspinal site, but not act directly at the spinal or peripheral level. However, we do not know the supraspinal site at which NTP may act. Indirect activation of the spinal dorsal horn by stimulation of the supraspinal site has been suggested, where the firing of dorsal horn neurons in response to noxious skin heating can be inhibited by stimulation in the periaqueductal gray and the lateral reticular formation in the midbrain (25). In addition, the spinal cord neurons can also be inhibited by electrical stimulation in other regions of the brain, such as the raphe nuclei, the locus ceruleus, and various regions of the medullary reticular formation (26). These regions might be candidates for supraspinal sites in which NTP may act.
Antidepressants show the supraspinal, spinal, and peripheral antinociceptive and antihyperalgesic effect. The supraspinal site was suggested to be more important for the antinociceptive effect of imipramine and amitriptyline (27). However, spinally injected tricyclic antidepressants inhibited biting and licking behaviors in a rat formalin test (28) and spinal amitriptyline showed an antihyperalgesic effect in a rat SNL model (29). Amitriptyline and desipramine show the peripheral antinociceptive and antiallodynic effect possibly by an antiinflammatory effect (30), by blocking sodium channels at peripheral sites (31,32), and by the action of adenosine (33,34). These reports suggest the analgesic mechanism of NTP which acts at only the supraspinal site is different from those of antidepressants which act at various sites.
NTP has been widely used in Japan and China to treat chronic pain including lumbago, neck-shoulder-arm syndrome, symptomatic neuralgia, and neuropathic pain such as subacute myelo-opticoneuropathy and PHN. Open clinical trials conducted in Japan have also shown NTP to be useful in patients with CRPS. The results of a double-blind study of NTP in CRPS conducted at the National Institutes of Health in the US are expected shortly.
In the present study, NTP inhibited both allodynia and hyperalgesia induced by SNL. Its effect was dose-dependent and probably involved the supraspinal site of action and the sequential activation of spinal NE neurons as one of its mechanisms of action. Although further preclinical and clinical studies are clearly required, the present study further reinforces the probable usefulness of NTP in the treatment of human neuropathic pain.
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