Tomoya Oe

Biogenic amine depletion causes chronic muscular pain and tactile allodynia accompanied by depression: A putative animal model of fibromyalgia.

ABSTRACT Fibromyalgia is a prevalent and burdensome disorder characterized by chronic widespread pain and complex comorbid symptoms. To develop better treatments for pain‐centered fibromyalgia symptoms, there is still a need for animal models which mimic the features of fibromyalgia patients. In the present study, we have established a fibromyalgia animal model by utilizing a never‐before‐published pharmacological effect of reserpine. Repeated administration of reserpine (1 mg/kg s.c., once daily, for three consecutive days) causes a significant decrease in the muscle pressure threshold and tactile allodynia, which are sustained for 1 week or more in both male and female rats. This treatment regimen decreases the amount of biogenic amines (dopamine, norepinephrine, and 5‐hydroxytryptamine) in the spinal cord, thalamus, and prefrontal cortex, which are deeply involved in pain signal processing. It also significantly increases immobility time in the forced swim test, which is indicative of depression, a common comorbid symptom of fibromyalgia. Pregabalin, duloxetine, and pramipexole significantly attenuated the reserpine‐induced decrease in muscle pressure threshold, but diclofenac did not. The validity of the use of this reserpinized animal as a fibromyalgia model is demonstrated from three different aspects, i.e., face validity (manifestation of chronic pain and comorbid symptoms), construct validity (dysfunction of biogenic amine‐mediated central nervous system pain control is involved), and predictive validity (similar responses to treatments used in fibromyalgia patients). This animal model is expected to contribute to the better understanding of fibromyalgia pathophysiology and the evaluation of drugs, especially those which would activate biogenic amine system.

Diclofenac, a non-steroidal anti-inflammatory drug, suppresses apoptosis induced by endoplasmic reticulum stresses by inhibiting caspase signaling.

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used in the treatment of inflammation and pain. In many reports, NSAIDs have induced apoptosis in a variety of cell lines such as colon cancer cells. On the other hand, more recently a few reports have found that NSAIDs protect against apoptosis. Here we investigate endoplasmic reticulum (ER)-stress-induced apoptosis of neuronal cells. The aim of this study is to examine the involvement of NSAIDs, in particular diclofenac, on ER-stress-induced apoptosis of human neuroblastoma SH-SY5Y cells. Diclofenac significantly suppressed SH-SY5Y cell death induced by two types of ER-stress-inducing agents: thapsigargin, an inhibitor of Ca2+-ATPase on the endoplasmic reticulum membrane, and tunicamycin, a glycosylation blocker. Other NSAIDs, such as indomethacin, ibuprofen, aspirin, and ketoprofen, also suppressed ER-stress-induced SH-SY5Y cell death. The dose-dependent anti-apoptotic effect of diclofenac did not correlate with the reduction of prostaglandin release. Administration of prostaglandin E2, which was a primary product of arachidonic metabolism, showed no effects against anti-apoptotic effects produced by diclofenac. Thapsigargin and tunicamycin each significantly activated caspase-3, -9, and -2 in the intrinsic apoptotic pathway in SH-SY5Y cells. Diclofenac suppressed the activation of caspases induced by both ER stresses. Thapsigargin and tunicamycin decreased the mitochondrial membrane potential in SH-SY5Y cells. Diclofenac suppressed the mitochondrial depolarization induced by both ER stresses. Diclofenac inhibited ER-stress-induced apoptosis of SH-SY5Y cells by suppressing the activation of caspases in the intrinsic apoptotic pathway. This is the first report to find that diclofenac has protective effects against ER-stress-induced apoptosis.

A selective peroxisome proliferator-activated receptor γ modulator with distinct fat cell regulation properties

Adipogenesis is an important process for the improvement of insulin resistance by peroxisome proliferator-activated receptor (PPAR) γ agonists, such as rosiglitazone and pioglitazone. FK614 [3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3-Hbenzimidazole-5-carboxamide] is a structurally novel class of PPARγ agonist that improves insulin sensitivity in animal models of type 2 diabetes. Herein, we characterize FK614, a selective PPARγ modulator (SPPARM) with differential properties affecting the regulation of fat cell function. FK614 behaves as a partial agonist in inducing the interaction of PPARγ with both transcriptional coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, but as a full agonist with both PPAR-binding protein and PPAR-interacting protein, which are required for PPARγ-mediated adipogenesis. In the differentiating 3T3-L1 adipocytes, the levels of adipose fatty acid-binding protein (aP2) mRNA expression and triglyceride accumulation induced by FK614 were as efficacious as those of rosiglitazone and pioglitazone. In contrast, the effect of FK614 on aP2 gene expression in mature adipocytes was less than that of the other PPARγ agonists. Furthermore, the long-term treatment of mature adipocytes with rosiglitazone and pioglitazone reduced the expression of phosphodiesterase 3B, the down-regulation of which has an important role in the development of insulin resistance; however, FK614 had no such effect in mature adipocytes. Thus, FK614 behaves as an SPPARM with differential effects on the activation of PPARγ at each stage of adipocyte differentiation. The stage-dependent selectivity of FK614 may contribute to its enhanced insulin sensitization in differentiating adipocytes and to reduced insulin resistance at the stage of adipocyte hypertrophy.

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.

A Simple, Flexible, Nonfluorescent System for the Automated Screening of Neurite Outgrowth:

Measurement of neurite outgrowth is a common assay of neurotrophic activity. However, currently available techniques for measuring neurite outgrowth are either time or resource intensive. The authors established a system in which chronic treatment of a subcloned SH-SY5Y cell line with aphidicolin and various concentrations of nerve growth factor (NGF) induced discernable alterations in proliferation and differentiation. Cells were fixed, labeled with a nonfluorescent dye, and evaluated both manually and with an automated analysis system. NGF increased multiple parameters of differentiation, including neurite length, the proportion of cells extending neurites, and branching, as well as promoting cellular survival/proliferation. Interestingly, although NGF treatment increased the total number of branches, it actually decreased the proportion of branches per neurite length. The authors observed no differences in results obtained using the manual and automated systems, but the automated system was orders of magnitude faster. To demonstrate the flexibility of the system, the authors also show that they could measure changes in differentiation induced by a small-molecule Rho kinase inhibitor, as well as by retinoic acid cotreatment with brain-derived neurotrophic factor. In addition to this flexibility, this system does not require specialized equipment or fluorescent antibodies for analysis and therefore provides a less resource-intensive alternative to fluorescence-based systems.

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF‐induced neurite‐positive (NIN+) subclones that showed high responsiveness to NGF‐induced neurite outgrowth and three NGF‐induced neurite‐negative (NIN–) subclones that abolished NGF‐induced neurite outgrowth from parental SH‐SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506‐mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF‐induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF‐induced increases in mRNAs for the immediate‐early genes (IEGs) c‐fos and NGF inducible (NGFI) genes NGFI‐A, NGFI‐B and NGFI‐C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN– cell line, particularly in terms of axonal vesicle‐related genes and growth cone guidance‐related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN– cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH‐SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.

Different pathophysiology underlying animal models of fibromyalgia and neuropathic pain: Comparison of reserpine-induced myalgia and chronic constriction injury rats

The reserpine-induced myalgia (RIM) rat manifests fibromyalgia-like chronic pain symptoms. The present study explored the pathophysiology underlying the pain symptoms in the RIM rat and the chronic constriction injury (CCI) rat, an animal model of neuropathic pain as a reference. Nerve tissue samples were collected from the nociception-tested animals for pathological examinations. Additionally, the therapeutic efficacy of a sodium channel blocker mexiletine was assessed in both rats. A slight vacuolization in the substantia nigra (SN) occurred in some of the RIM rats without any other histopathological changes in the brain or peripheral neurons. All the RIM rats, with or without vacuolization, showed hypersensitivity to tactile, muscle pressure, and cold stimuli. In the CCI rat, neurodegenerative changes were apparent in the sciatic nerve and the spinal cord only. CCI rats displayed muscle hyperalgesia in addition to tactile and cold allodynia. Pharmacotherapy with mexiletine did not attenuate the pain in the RIM rat, although it was effective in the CCI rat. Taken together, it is not likely that pain symptoms in RIM rats are caused by degenerative changes at the level of primary afferents and spinal cord, as is the case for CCI rats. The significance of the vacuolization in the SN is less clear at present because of the minor extent of the change and the lack of correlation with nociceptive sensitivity. The pain symptoms in RIM rats could be associated with dysfunction of biogenic amines-mediated CNS pain control even without apparent pathologies in the nervous system.

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.

CyclinB2 and BIRC5 genes as surrogate biomarkers for neurite outgrowth in SH-SY5Y subclonal cells

Neurite outgrowth plays a key role in neuronal development and regeneration, and is the hallmark assay for the effects of neurotrophic factors such as nerve growth factor (NGF). However, measuring neurite outgrowth is a slow and resource-intensive process. We therefore wanted to identify surrogate biomarkers for neurite outgrowth activity by gene expression analysis in SH-O10 cells, a subclone of the human SH-SY5Y neuroblastoma cell line but with much higher NGF-induced neurite outgrowth activity. Microarray analysis identified seven genes where mRNA levels were changed. NGF-induced decreases in levels of two genes, CyclinB2 and BIRC5, were confirmed by quantitative real-time RT-PCR. Levels of NGF-induced decreases in CyclinB2 and BIRC5 mRNA in several SH-SY5Y subclones with different neurite outgrowth responses correlated with their neurite outgrowth activities. Decreases in CyclinB2 and BIRC5 mRNA induced by FK506 or retinoic acid, both of which exert potentiation of NGF-induced neurite outgrowth effects but with different mechanisms, also correlated with their neurite outgrowth activities. In conclusion, decreasing levels of CyclinB2 and BIRC5 mRNA strongly correlate with neurite outgrowth activities in terms of NGF-related effect in SH-SY5Y subclonal cells, and have potential to become quantitative surrogate biomarkers for measuring NGF-related neurite outgrowth.