Mina Tsukamoto

Pharmacological characterization and gene expression profiling of an L5/L6 spinal nerve ligation model for neuropathic pain in mice

L5/L6 spinal nerve ligation (SNL) in rodents induces behavioral signs similar to the symptoms of neuropathic pain in humans. L5/L6 SNL in rats has been well characterized so far, but there have been few studies using mice. In this study, we established an L5/L6 SNL model in mice and examined the effects of known antinociceptive drugs in the model. We also analyzed the changes in gene expression in dorsal root ganglions with special reference to those which are known to change in a neuropathic pain state to validate the model. Mechanical allodynia in the ipsilateral side paw was observed beginning on day 1 and lasted for at least 2 months following surgery. Diclofenac showed no significant effect on the mechanical allodynia. Gabapentin and pregabalin completely reversed allodynia, but they also caused a decrease in locomotor activity. Duloxetine caused a partial recovery of the threshold. Mexiletine completely reversed allodynia, but it also caused sedation or motor impairment. Morphine caused a partial recovery of the threshold and hyper-locomotion. This mouse L5/L6 SNL model represents a robust mechanical allodynia, which shows a similar pharmacological response to that reported in rats and human patients with neuropathic pain. The pattern changes in gene expression also resembled those reported in rats. This model will therefore be useful for investigation of the effects of novel antinociceptive compounds and the mechanisms of neuropathic pain.

Reserpine causes biphasic nociceptive sensitivity alteration in conjunction with brain biogenic amine tones in rats.

The present study investigated the precise relationship between brain biogenic amine (dopamine, noradrenaline, and serotonin) tones and nociception. Nociceptive sensitivities to multimodal (muscle pressure, tactile, cold, and heat) stimuli were assessed in acute phase (up to 24 h after reserpine or tetrabenazine injection) and chronic phase (on day 2 or later) in rats. A single injection of reserpine (3 mg/kg s.c.) significantly decreased biogenic amines in the spinal cord (SC), thalamus (THA), and prefrontal cortex (PFC) in both acute and chronic phases, but significantly increased a dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the SC and a serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the SC and THA in acute phase. The content of all biogenic amine metabolites was at low level in chronic phase. Animals exhibited hypersensitivities to tactile and heat stimuli and hyposensitivity to muscle pressure stimulus in acute phase. In chronic phase, they manifested hypersensitivities to all modes of stimuli. Tetrabenazine (20 mg/kg i.p.) significantly decreased brain biogenic amines for a short time, although it did not significantly affect the nociceptive sensitivities. In conclusion, a single injection of reserpine causes a biphasic alteration of nociceptive sensitivities, which is in conjunction with the dynamic change of brain biogenic amine tones, in rats. Cold and heat hypersensitivities in addition to mechanical ones are induced by the reserpine treatment. Sustained modification of brain biogenic amine tones would be critical to induce a robust change in nociceptive sensitivities based on the different effects between reserpine and tetrabenazine.

Spinal mechanism of standard analgesics: Evaluation using mouse models of allodynia

Spinal neurotransmission plays an important role in the perception of pain signaling. In the present study, we investigated the spinal anti-nociceptive mechanism of current standard analgesics in mouse models of tactile allodynia induced by intrathecal administration of N-methyl-D-aspartic acid (NMDA), prostaglandin E2 (PGE2), and bicuculline. NMDA-induced allodynia is induced by postsynaptic NMDA receptor activation, while PGE2-induced allodynia is triggered by the enhancement of presynaptic glutamate release via EP1 receptor activation. In contrast, bicuculline induces allodynia by the blockade of gamma-aminobutyric acid (GABA)A receptor-mediated inhibitory system. As the clinically available analgesics, pregabalin (alpha2delta-subunit calcium channel ligand), ziconotide (N-type calcium channel blocker), mexiletine (sodium channel blocker), and duloxetine (serotonin and norepinephrine reuptake inhibitors) were evaluated in these neurochemically-induced allodynia models. Pregabalin almost completely alleviated NMDA-, PGE2-, and bicuculline-induced allodynia. Despite being classified as an agent with a similar molecular target mechanism, ziconotide could only alleviate PGE2-induced allodynia, but not NMDA- or bicuculline-induced allodynia, as did mexiletine and duloxetine. These results taken together suggest that ziconotide, mexiletine, and duloxetine suppress spinal hyperactivity via the presynaptic site mechanism. In contrast, pregabalin could suppress via the downstream step during spinal hyperactivation such as postsynaptic NMDA activation or dysfunction of GABAergic control in addition to presynaptic mechanism. In conclusion, present findings provide implication that the spinal anti-nociceptive mechanistic site of pregabalin is different from that of ziconotide, mexiletine, and duloxetine, and pregabalin could have a broader anti-nociceptive mechanism other than N-type calcium channel blockade.

AS1069562, the (+)-Isomer of Indeloxazine, Exerts Analgesic Effects in a Rat Model of Neuropathic Pain with Unique Characteristics in Spinal Monoamine Turnover

AS1069562 [(R)-2-[(1H-inden-7-yloxy)methyl]morpholine monobenzenesulfonate] is the (+)-isomer of indeloxazine, which had been used clinically for the treatment of cerebrovascular diseases with multiple pharmacological actions, including serotonin (5-HT) and norepinephrine (NE) reuptake inhibition. Here we investigated the analgesic effects of AS1069562 in a rat model of chronic constriction injury (CCI)–induced neuropathic pain and the spinal monoamine turnover. These effects were compared with those of the antidepressants duloxetine and amitriptyline. AS1069562 significantly elevated extracellular 5-HT and NE levels in the rat spinal dorsal horn, although its 5-HT and NE reuptake inhibition was much weaker than that of duloxetine in vitro. In addition, AS1069562 increased the ratio of the contents of both 5-HT and NE to their metabolites in rat spinal cord, whereas duloxetine slightly increased only the ratio of the content of 5-HT to its metabolite. In CCI rats, AS1069562 and duloxetine significantly ameliorated mechanical allodynia, whereas amitriptyline did not. AS1069562 and amitriptyline significantly ameliorated thermal hyperalgesia, and duloxetine tended to ameliorate it. Furthermore, AS1069562, duloxetine, and amitriptyline significantly improved spontaneous pain–associated behavior. In a gastric emptying study, AS1069562 affected gastric emptying at the same dose that exerted analgesia in CCI rats. On the other hand, duloxetine and amitriptyline significantly reduced gastric emptying at lower doses than those that exerted analgesic effects. These results indicate that AS1069562 broadly improved various types of neuropathic pain–related behavior in CCI rats with unique characteristics in spinal monoamine turnover, suggesting that AS1069562 may have potential as a treatment option for patients with neuropathic pain, with a different profile from currently available antidepressants.

Systemic administration of 5-HT2C receptor agonists attenuates muscular hyperalgesia in reserpine-induced myalgia model

Fibromyalgia is a prevalent musculoskeletal disorder characterized by chronic widespread pain that significantly reduces quality of life in patients. Due to the lack of consistently effective treatment, the development of improved therapies for treating fibromyalgia is necessary. As dysfunction of serotonergic analgesic control appears to be involved in the pathophysiology of fibromyalgia, the present study explored the potential of 5-HT(2C) receptor agonists as novel therapies for treating this disease. Three 5-HT(2C) receptor agonists (lorcaserin, vabicaserin and YM348) that have been suggested to be useful in the treatment of several central nervous system diseases, including obesity and schizophrenia, were used. The effect of systemic administration of these agents on the muscular hyperalgesia that develops in the reserpine-induced myalgia (RIM) rat, a putative animal model of fibromyalgia, was investigated. RIM rats exhibited decreased muscle pressure thresholds. Microdialysis experiments showed that the concentration of serotonin (5-HT) in the spinal cord of RIM rats was significantly lower than that of controls. Lorcaserin (0.3-3 mg/kg p.o.), vabicaserin (0.3-3 mg/kg s.c.) and YM348 (0.03-0.3 mg/kg p.o.) recovered the muscle pressure threshold. The effect of lorcaserin was reversed by the pretreatment with SB242084, a 5-HT(2C) receptor antagonist. Our findings demonstrate that 5-HT(2C) receptors play a critical role in muscular hyperalgesia in RIM rats and suggest that 5-HT(2C) receptor agonists have therapeutic potential for treating chronic pain in patients with fibromyalgia although clinical extrapolation remains to be a future challenge.

Antinociceptive effects of AS1069562, the (+)-isomer of indeloxazine, on spinal hypersensitivity induced by intrathecal injection of prostaglandin in mice: comparison with duloxetine and amitriptyline.

The (+)-isomer of indeloxazine AS1069562 exerts multiple pharmacological actions including the inhibition of serotonin (5-HT) and norepinephrine reuptake and analgesia in experimental animal pain models. Here, we evaluated the antinociceptive effects of AS1069562 and the antidepressants duloxetine and amitriptyline in mouse models of prostaglandin-induced spinal hypersensitivity. Prostaglandin E2 (PGE2) and F2α (PGF2α) were intrathecally administered to induce spinal hypersensitivity, causing tactile allodynia in mice. Allodynia induced by PGF2α but not by PGE2 was suppressed by desensitization of C-fibers with systemic pretreatment with resiniferatoxin. C-fiber hyperexcitability might therefore play a role in allodynia induced by PGF2α but not PGE2. In the PGE2-induced allodynia model, AS1069562 and duloxetine significantly suppressed allodynia, whereas amitriptyline did not. In the PGF2α-induced allodynia model, AS1069562 and amitriptyline significantly ameliorated allodynia, whereas duloxetine did not. To demonstrate the broad effects of AS1069562 compared to duloxetine, additional studies were conducted to elucidate other target mechanisms of AS1069562 beyond 5-HT and norepinephrine reuptake inhibition. AS1069562 exhibited affinity for both 5-HT1A and 5-HT3 receptors, and the analgesic effect of AS1069562 on PGF2α-induced allodynia was significantly blocked by the 5-HT1A receptor antagonist (S)-WAY100135 and the 5-HT3 receptor agonist SR57227. Taken together, these results indicate that AS1069562 inhibits both C-fiber- and non-C-fiber-dependent prostaglandin-induced allodynia, while duloxetine inhibits only non-C-fiber-triggered allodynia, and amitriptyline inhibits only C-fiber-triggered allodynia. These broad antinociceptive effects of AS1069562 may be due not only to 5-HT and norepinephrine reuptake inhibition but also to its effects on 5-HT receptors such as 5-HT1A and 5-HT3 receptors.

New index of pain triggered by spinal activation of voltage-dependent sodium channels

Voltage-dependent sodium channels (VDSCs) are crucial for pain generation. Here, to develop a new behavioral index of pain induced by spinal VDSC activation, we examined whether intrathecal veratridine injection produced nociceptive behavior. Intrathecal injection of the VDSC opener veratridine in mice dose-dependently induced nociceptive responses, with response times subsequently reduced by administration of morphine or pregabalin. Systemic administration of lidocaine and mexiletine, but not amitriptyline, also decreased this response time. Taken together, these results demonstrated that response time of nociceptive behavior induced by intrathecal veratridine injection is a quantitative index of pain triggered by spinal VDSC activation.

Analgesic effects of ASP3662, a novel 11β‐hydroxysteroid dehydrogenase 1 inhibitor, in rat models of neuropathic and dysfunctional pain

Glucocorticoids are a major class of stress hormones known to participate in stress‐induced hyperalgesia. Although 11β‐hydroxysteroid dehydrogenase 1 (11β‐HSD1) is a key enzyme in the intracellular regeneration of glucocorticoids in the CNS, its role in pain perception has not been assessed. Here, we examined the effects of ASP3662, a novel 11β‐HSD1 inhibitor, on neuropathic and dysfunctional pain.

211 ESTABLISHMENT OF L5/L6 SPINAL NERVE LIGATION MODEL OF NEUROPATHIC PAIN IN MICE AND PHARMACOLOGICAL CHARACTERIZATION

of protein gene product 9.5 (PGP 9.5), a marker of nerve tissue, was utilized to detect the nerve growth in scar tissue under electron microscopy. Results: Results illustrate that the whirl body and the detachment of plasmalemma could be found in Schwann cells of large myelinated fibers. Neurotubule disruption and neurofilament disorganization also developed in small myelinated or unmyelinated fibers. In addition, numerous nerve fibers labeled by PGP 9.5 were observed in scar tissue on light or electron microscopy after 3 months lumbar laminectomy. These small fibers frequently accompany vessels and spread from periphery to central connective tissue of scar. Conclusions: We conclude that the dysmyelination and axonopathy in roots, and the nerve ingrowth in scar tissue could induce neuropathic pain, which might be the main causes of pain syndrome after spine operation.