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Gentle touch such as stroking of the skin produces a pleasant feeling, which is detected by a rare subset of sensory neurons that express Mas-related G protein-coupled receptor B4 (MrgprB4) in mice. We examined small populations of MrgprB4-positive neurons in the trigeminal ganglion and the dorsal root ganglion, and most of these were sensitive to transient receptor potential ankyrin 1 (TRPA1) agonist but not TRPV1, TRPM8, or TRPV4 agonists. Deficiency of MrgprB4 did not affect noxious pain or itch behaviors in the hairless plantar and hairy cheek. Although behavior related to acetone-induced cold sensing in the hind paw was not changed, unpleasant sensory behaviors in response to acetone application or sucrose splash to the cheek were significantly enhanced in Mrgprb4-knockout mice as well as in TRPA1-knockout mice. These results suggest that MrgprB4 in the trigeminal neurons produces pleasant sensations in cooperation with TRPA1, rather than noxious or cold sensations. Pleasant sensations may modulate unpleasant sensations on the cheek via MrgprB4.

Microglia are immune cells in the central nervous system (CNS) and essential for homeostasis that are important for both neuroprotection and neurotoxicity, and are activated in a variety of CNS diseases. Microglia aggravate cognitive impairment induced by chronic cerebral hypoperfusion, but their precise roles under these conditions remain unknown. Here, we used PLX3397, a colony-stimulating factor 1 receptor inhibitor, to deplete microglia in mice with chronic cerebral hypoperfusion induced by bilateral common carotid artery stenosis (BCAS). Cognitive impairment induced 28 days after BCAS was significantly improved in mice fed a diet containing PLX3397. In PLX3397-fed mice, microglia were depleted and white matter injury induced by BCAS was suppressed. In addition, the expression of proinflammatory cytokines, interleukin 6 and tumor necrosis factor alpha, was suppressed in PLX3397-fed mice. Taken together, these findings suggest that microglia play destructive roles in the development of cognitive impairment and white matter injury induced by chronic cerebral hypoperfusion. Thus, microglia represent a potential therapeutic target for chronic cerebral hypoperfusion-related diseases.

Aging causes various functional changes, including cognitive impairment and inflammatory responses in the brain. Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable channel expressed abundantly in immune cells, exacerbates inflammatory responses. Previously, we reported that TRPM2 on resident microglia plays a critical role in exacerbating inflammation, white matter injury, and cognitive impairment during chronic cerebral hypoperfusion; however, the physiological or pathophysiological role of TRPM2 during age-associated inflammatory responses remains unclear. Therefore, we examined the effects of TRPM2 deletion in young (2–3 months) and older (12–24 months) mice. Compared with young wild-type (WT) mice, middle-aged (12–16 months) WT mice showed working and cognitive memory dysfunction and aged (20–24 months) WT mice exhibited impaired spatial memory. However, these characteristics were not seen in TRPM2 knockout (TRPM2-KO) mice. Consistent with the finding of cognitive impairment, aged WT mice exhibited white matter injury and hippocampal damage and an increase in the number of Iba1-positive cells and amounts of pro-inflammatory cytokines in the brain; these characteristics were not seen in TRPM2-KO mice. These findings suggest that TRPM2 plays a critical role in exacerbating inflammatory responses and cognitive dysfunction during aging.

Antipsychotics often cause tardive dyskinesia, an adverse symptom of involuntary hyperkinetic movements. Analysis of the US Food and Drug Administration Adverse Event Reporting System and JMDC insurance claims revealed that acetaminophen prevented the dyskinesia induced by dopamine D2 receptor antagonists. In vivo experiments further showed that a 21-day treatment with haloperidol increased the number of vacuous chewing movements (VCMs) in rats, an effect that was inhibited by oral acetaminophen treatment or intracerebroventricular injection of N-(4-hydroxyphenyl)-arachidonylamide (AM404), an acetaminophen metabolite that acts as an activator of the transient receptor potential vanilloid 1 (TRPV1). In mice, haloperidol-induced VCMs were also mitigated by treatment with AM404 applied to the dorsal striatum, an effect not seen in TRPV1-deficient mice. Acetaminophen prevented the haloperidol-induced decrease in the number of c-Fos+preproenkephalin+ striatal neurons in wild-type mice but not in TRPV1-deficient mice. Finally, chemogenetic stimulation of indirect pathway medium spiny neurons in the dorsal striatum decreased haloperidol-induced VCMs. These results suggest that acetaminophen activates the indirect pathway neurons by activating TRPV1 channels via AM404.

Intracerebral hemorrhage (ICH) is one of the most severe subtypes of stroke with high morbidity and mortality. Although a lot of drug discovery studies have been conducted, the drugs with satisfactory therapeutic effects for motor paralysis after ICH have yet to reach clinical application. Transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable cation channel and activated by hypoosmolarity and warm temperature, is expressed in various cell types. The present study investigated whether TRPV4 would participate in the brain damage in a mouse model of ICH. ICH was induced by intrastriatal treatment of collagenase. Administration of GSK1016790A, a selective TRPV4 agonist, attenuated neurological and motor deficits. The inhibitory effects of the TRPV4 agonist in collagenase-injected WT mice were completely disappeared in TRPV4-KO mice. The TRPV4 agonist did not alter brain injury volume and brain edema at 1 and 3 days after ICH induction. The TRPV4 agonist did not show any differences with respect to the increased number of Iba1-positive microglia/macrophages, GFAP-positive astrocytes, and Gr1-positive neutrophils at 1 and 3 days after ICH induction. Quantitative RT-PCR experiments revealed that the TRPV4 agonist significantly upregulated the expression level of c-fos, a marker of neuronal activity, while the agonist gave no effects on the expression level of cytokines/chemokines at 1 day after ICH induction, These results suggest that stimulation of TRPV4 would ameliorate ICH-induced brain injury, presumably by increased neuronal activity and TRPV4 provides a novel therapeutic target for the treatment for ICH.

Central nervous system (CNS) inflammation is a potential therapeutic target for neurodegenerative diseases. In recent years, a number of studies have focused on the links between neurodegenerative diseases and CNS glial cells, especially microglia. Microglia are the main resident immune cells in the CNS and represent approximately 10-15% of all CNS cells. Microglia play an important role in maintaining brain homeostasis at rest by surveying the environment, and engulfing apoptotic cells and debris in the healthy brain. However, under certain pathological conditions, microglia can generate neurotoxic factors, such as pro-inflammatory cytokines and molecules like nitric oxide (NO), which lead to CNS inflammatory diseases. In this review, we discuss the evidence that regulation of microglial ion channels may modulate CNS inflammation and subsequent tissue damage in neurological disorders. In particular, we discuss the role of transient receptor potential (TRP) channels in microglia in both acute and chronic inflammatory conditions, and describe the physiological and pathophysiological roles of TRP channels in CNS inflammatory pathways. Additionally, we describe the benefits of stimulation/inhibition of TRP channels in animal models of microglia-related CNS inflammatory diseases.

Chronic cerebral hypoperfusion is a characteristic seen in widespread CNS diseases, including neurodegenerative and mental disorders, and is commonly accompanied by cognitive impairment. Recently, several studies demonstrated that chronic cerebral hypoperfusion can induce the excessive inflammatory responses that precede neuronal dysfunction; however, the precise mechanism of cognitive impairment due to chronic cerebral hypoperfusion remains unknown. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable channel that is abundantly expressed in immune cells and is involved in aggravation of inflammatory responses. Therefore, we investigated the pathophysiological role of TRPM2 in a mouse chronic cerebral hypoperfusion model with bilateral common carotid artery stenosis (BCAS). When male mice were subjected to BCAS, cognitive dysfunction and white matter injury at day 28 were significantly improved in TRPM2-knockout (TRPM2-KO) mice compared with wild-type (WT) mice, whereas hippocampal damage was not observed. There were no differences in blood-brain barrier breakdown and H2O2 production between the two genotypes at 14 and 28 days after BCAS. Cytokine production was significantly suppressed in BCAS-operated TRPM2-KO mice compared with WT mice at day 28. In addition, the number of Iba1-positive cells gradually decreased from day 14. Moreover, daily treatment with minocycline significantly improved cognitive perturbation. Surgical techniques using bone marrow chimeric mice revealed that activated Iba1-positive cells in white matter could be brain-resident microglia, not peripheral macrophages. Together, these findings suggest that microglia contribute to the aggravation of cognitive impairment by chronic cerebral hypoperfusion, and that TRPM2 may be a potential target for chronic cerebral hypoperfusion-related disorders., 脳の血流低下が認知機能障害を引き起こす --脳の免疫細胞「ミクログリア」による脳内炎症と白質傷害が原因か--. 京都大学プレスリリース. 2018-03-09.

Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder characterized by the repeated rise of concerns (obsessions) and repetitive unwanted behavior (compulsions). Although selective serotonin reuptake inhibitors (SSRIs) is the first-choice drug, response rates to SSRI treatment vary between symptom dimensions. In this study, to find a therapeutic target for SSRI-resilient OCD symptoms, we evaluated treatment responses of quinpirole (QNP) sensitization-induced OCD-related behaviors in mice. SSRI administration rescued the cognitive inflexibility, as well as hyperactivity in the lateral orbitofrontal cortex (lOFC), while no improvement was observed for the repetitive behavior. D2receptor signaling in the central striatum (CS) was involved in SSRI-resistant repetitive behavior. An adenosine A2Aantagonist, istradefylline, which rescued abnormal excitatory synaptic function in the CS indirect pathway medium spiny neurons (MSNs) of sensitized mice, alleviated both of the QNP-induced abnormal behaviors with only short-term administration. These results provide a new insight into therapeutic strategies for SSRI-resistant OCD symptoms and indicate the potential of A2Aantagonists as a rapid-acting anti-OCD drug.

Multiple sclerosis (MS) is the common demyelinating disease of human central nervous system. Among mouse models available to study MS, including the cuprizone application and lysolecithin-injection models, experimental autoimmune encephalomyelitis (EAE) model is widely used so that chronic EAE model of C57BL/6J can reflect the autoimmune pathogenesis of MS well. Here we introduce the EAE model based on C57BL/6J mice, which is generated by injection of myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) as an antigen. After immunization with complete Freund's adjuvant, clinical signs and changes in body weight are observed one or two weeks later. The EAE model will continue to be useful for development of therapeutics for MS.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS) characterized by demyelination and axonal injury. Current therapies that mainly target lymphocytes do not fully meet clinical need due to the risk of severe side effects and lack of efficacy against progressive MS. Evidence suggests that MS is associated with CNS inflammation, although the underlying molecular mechanism is poorly understood. Transient receptor potential melastatin 2 (TRPM2), a Ca²⁺-permeable nonselective cation channel, is expressed at high levels in the brain and by immune cells, including monocyte lineage cells. Here, we show that TRPM2 plays a pathological role in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Knockout or pharmacological inhibition of TRPM2 inhibited progression of EAE, and TRPM2-knockout (TRPM2-KO) mice showed lower activation of Iba1-immunopositive monocyte lineage cells and neutrophil infiltration of the CNS than wild-type (WT) mice. Moreover, CXCL2 production in TRPM2-KO mice was significantly reduced at Day 14 although the severity of EAE was the same as that in WT mice at that time point. In addition, we used bone marrow chimeric mice to show that TRPM2 expressed by CNS-infiltrating macrophages contributes to progression of EAE. Since CXCL2 induces migration of neutrophils, these results indicate that reduced expression of CXCL2 in the CNS suppresses neutrophil infiltration and slows progression of EAE in TRPM2-KO mice. Together, the results suggest that TRPM2 plays an important role in progression of EAE pathology and shed light on its putative role as a therapeutic target for MS. SIGNIFICANCE STATEMENT: Current therapies for multiple sclerosis (MS), which mainly target lymphocytes, carry the risk of severe side effects and lack efficacy against the progressive form of the disease. Here, we found that the transient receptor potential melastatin 2 (TRPM2) channel, abundantly expressed in CNS-infiltrating macrophages, plays a crucial role in development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE progression was suppressed by knockout or pharmacological inhibition of TRPM2; this was attributed to a reduction in CXCL2 chemokine production by CNS-infiltrating macrophages in TRPM2-knockout mice, resulting in suppression of neutrophil infiltration into the CNS. These results reveal an important role of TRPM2 in the pathogenesis of EAE and shed light on its potential as a therapeutic target., 多発性硬化症の新たな病態増悪機構を解明 --TRPM2を介したケモカイン産生が神経炎症の増悪に至る好中球の浸潤を引き起こす--. 京都大学プレスリリース. 2018-09-13.

We previously established a long-lasting cystitis model by an intravesical injection of hydrogen peroxide (H2O2) into mice. In this study, we assessed the pain-related behaviors in the cystitis model. An intravesical injection of 1.5% H2O2 transiently decreased spontaneous locomotor activity at 3 h after injection, indicative of acute spontaneous pain. In contrast, licking response to a bladder distention was slowly observed as licks to the lower abdomen at 7 and 14 days after injection, which was attenuated by amitriptyline and morphine, but not by oxybutynin. These results suggest that H2O2-induced chronic cystitis model shows delayed and long-lasting painful pathological condition.

Chronic inflammatory bladder disorders, such as interstitial cystitis/bladder pain syndrome, are associated with poor quality of life. The exact pathological processes remain unclear, but accumulating evidence suggests that reactive oxidative species (ROS) are involved in urinary bladder disorders. Transient receptor potential ankyrin 1 (TRPA1), the most sensitive TRP channel to ROS, was shown to be responsible for urinary bladder abnormalities and hyperalgesia in an acute cystitis model. However, the roles of TRPA1 in chronic inflammatory bladder are not fully understood. We previously established a novel mouse cystitis model induced by intravesical injection of hydrogen peroxide (H2O2), resulting in long-lasting frequent urination, bladder inflammation, pain-related behavior, and histopathological changes. In the present study, we investigated the pathophysiological role of TRPA1 in the H2O2-induced long-lasting cystitis mouse model. Under anesthesia, 1.5% H2O2 solution was introduced transurethrally into the bladder of female wild-type (WT) and TRPA1-knockout mice and maintained for 30 min. This increased the number of voids in WT mice at 1 and 7 days after injection, but reduced the number in TRPA1-knockout mice at 1 day but not 7 days after injection. Spontaneous locomotor activities (increase in freezing time and decrease in distance moved) were reduced at 3 h after injection in WT mice, whereas the spontaneous visceral pain-related behaviors were attenuated in TRPA1-knockout mice. Furthermore, upregulation of c-fos mRNA in the spinal cord at 1 day after injection was observed in WT but not TRPA1-knockout mice. However, there was no difference in histopathological changes in the urinary bladder, such as edematous thickening in the submucosa, between WT and TRPA1-knockout mice at 1 or 7 days after injection. Finally, Trpa1 mRNA levels in the L5-S1 dorsal root ganglion were not altered, but levels in the urinary bladder were drastically increased at 1 and 7 days after injection. Taken together, these results suggest that TRPA1 contributes to acute bladder hyperactivity such as frequent urination and bladder pain, but does not appear to play a major role in the pathological processes of long-lasting cystitis.

Positron emission tomography with 15O-labeled gases (15O-PET) is important for in vivo measurement of cerebral oxygen metabolism both in clinical and basic settings. However, there are currently no reports concerning 15O-PET in mice. Here, we developed an 15O-PET method applicable to mice with spontaneous respiration of 15O-gas without a tracheotomy catheter. Sequential 15O-PET was also performed in a mouse model of chronic cerebral hypoperfusion with bilateral common carotid artery stenosis (BCAS) induced by placement of microcoils. 15O-gas with isoflurane was supplied to the nose of mouse with evacuation of excess 15O-gas surrounding the body. 15O-PET was performed on days 3, 7, 14, 21, and 28 after surgery. Cerebral blood flow (CBF), cerebral blood volume, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO2) were calculated in whole brains. A significant decrease in CBF and compensatory increase in OEF in the BCAS group produced CMRO2 values comparable to that of the sham group at three days post-operation. Although CBF and OEF in the BCAS group gradually recovered over the first 28 days, the CMRO2 showed a gradual decrease to 68% of sham values at 28 days post-operation. In conclusion, we successfully developed a noninvasive 15O-PET method for mice.

Novel longer lasting inflammatory bladder animal models are needed to better understand the pathophysiology of chronic cystitis. We previously developed a relatively long‐lasting mouse cystitis model by intravesical injection of hydrogen peroxide (H2O2). To further evaluate its pathophysiology, in this study, we established and analyzed a rat cystitis model. Under anesthesia, 1.5% H2O2 solution was introduced transurethrally into the bladder of female rats, and kept for 30 min. The H2O2 injection significantly increased the number of micturition events up to day 14 and decreased urine volume per micturition, with the smallest volumes on day 3, compared with the vehicle‐treated group. Cystometric analysis on day 7 revealed that intercontraction intervals were significantly shortened without affecting the baseline, threshold, or maximum pressures. Intravesical resiniferatoxin‐evoked nociceptive behaviors, such as freezing, were significantly enhanced on days 7 and 14. Furthermore, histopathology revealed hemorrhage, edema, infiltration of neutrophils into the lamina propria, and urothelial denudation in the early phase (day 1). These damages were gradually repaired, while hyperplasia of the urothelium, vascularization, increases in fibroblast counts, and infiltration of mast cells and eosinophils were observed through the later phase (days 7 and 14). These results suggest that intravesical H2O2 injection induces relatively long‐lasting cystitis with enhanced bladder activity and pain sensation in rats. This approach thus provides a novel rat long‐lasting cystitis model that allows us to analyze detailed symptoms and pathophysiology of H2O2‐induced cystitis model than the mouse model and may be used to investigate the pathophysiology and treatment of chronic bladder hypersensitive disorders, such as bladder pain syndrome/interstitial cystitis.

Oxaliplatin, a platinum-based chemotherapeutic agent, causes an acute peripheral neuropathy triggered by cold in almost all patients during or within hours after its infusion. We recently reported that a single administration of oxaliplatin induced cold hypersensitivity 2 h after the administration in mice. In this study, we examined whether standard analgesics relieve the oxaliplatin-induced acute cold hypersensitivity. Gabapentin, tramadol, mexiletine, and calcium gluconate significantly inhibited and morphine and milnacipran decreased the acute cold hypersensitivity, while diclofenac and amitriptyline had no effects. These results suggest that gabapentin, tramadol, mexiletine, and calcium gluconate are effective against oxaliplatin-induced acute peripheral neuropathy. Keywords:: oxaliplatin, acute peripheral neuropathy, analgesic

Background and Purpose— Intracerebral hemorrhage (ICH) stems from the rupture of blood vessels in the brain, with the subsequent accumulation of blood in the parenchyma. Increasing evidence suggests that blood-derived factors induce excessive inflammatory responses that are involved in the progression of ICH-induced brain injury. Thrombin, a major blood-derived factor, leaks into the brain parenchyma on blood–brain barrier disruption and induces brain injury and astrogliosis. Furthermore, thrombin dynamically upregulates transient receptor potential canonical 3 channel, which contributes to pathological astrogliosis through a feed-forward upregulation of its own expression. The present study investigated whether Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), a specific transient receptor potential canonical 3 inhibitor, can improve functional outcomes and attenuate astrogliosis after ICH in mice. Methods— Male C57BL6 mice received an intracerebral infusion of collagenase or autologous blood to induce ICH. Pyr3 was given both intracerebroventricularly and intraperitoneally after ICH induction. ICH-induced brain injury was evaluated by quantitative assessment of neurological deficits, brain swelling, and injury volume after ICH. Astrocyte activation was evaluated by immunohistochemical assessment of changes in S100 protein expression. Results— Neurological deficits, neuronal injury, brain edema, and astrocyte activation were all significantly improved by administration of Pyr3. Moreover, delayed administration of Pyr3 at 6 hours or 1 day after blood or collagenase infusion, respectively, also improved the symptoms. Conclusions— Pyr3, a specific inhibitor of transient receptor potential canonical 3, reduced the perihematomal accumulation of astrocytes and ameliorated ICH–induced brain injury. Therefore, transient receptor potential canonical 3 provides a new therapeutic target for the treatment of hemorrhagic brain injury.

There is so far no generally accepted animal model of chronic cystitis by which potential therapies can be evaluated. In this study, we aimed to establish a new mouse model of cystitis based on the proinflammatory effects of reactive oxygen species. A single intravesical injection of 1.5% hydrogen peroxide (H2O2) significantly increased the numbers of voids by 1 day after injection in female mice, which lasted up to 7 days. The H2O2 injection rapidly increased the bladder weight by 3 h in parallel with the histological damage and hyperpermeability of urothelial barrier. Although the urothelial dysfunction was recovered to normal by 7 days, increase in bladder weight, edematous thickening of the submucosa, and vascular hyperpermeability were apparent even 7 days after injection. During the time course, massive infiltration of neutrophils and increased expression of inflammatory cytokines were observed in the bladder. An intraperitoneal administration of oxybutynin, amitriptyline, indomethacin, or morphine attenuated the H2O2-induced frequent urination. These findings suggest that an intravesical injection of H2O2 induces relatively long-lasting inflammatory and overactive bladder, compared with existing cystitis models. The intravesical H2O2 injection model may be a simple and useful tool in the pathological study and drug discovery for chronic cystitis.[Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.12265FP] Keywords:: chronic cystitis, hydrogen peroxide, interstitial cystitis, overactive bladder, long-lasting

Atypical antipsychotics are associated with an increased risk of hyperglycaemia, thus limiting their clinical use. This study focused on finding the molecular mechanism underlying antipsychotic-induced hyperglycaemia. First, we searched for drug combinations in the FDA Adverse Event Reporting System (FAERS) database wherein a coexisting drug reduced the hyperglycaemia risk of atypical antipsychotics, and found that a combination with vitamin D analogues significantly decreased the occurrence of quetiapine-induced adverse events relating diabetes mellitus in FAERS. Experimental validation using mice revealed that quetiapine acutely caused insulin resistance, which was mitigated by dietary supplementation with cholecalciferol. Further database analysis of the relevant signalling pathway and gene expression predicted quetiapine-induced downregulation of Pik3r1, a critical gene acting downstream of insulin receptor. Focusing on the phosphatidylinositol 3-kinase (PI3K) signalling pathway, we found that the reduced expression of Pik3r1 mRNA was reversed by cholecalciferol supplementation in skeletal muscle, and that insulin-stimulated glucose uptake into C2C12 myotube was inhibited in the presence of quetiapine, which was reversed by concomitant calcitriol in a PI3K-dependent manner. Taken together, these results suggest that vitamin D coadministration prevents antipsychotic-induced hyperglycaemia and insulin resistance by upregulation of PI3K function., 有害事象ビッグデータから副作用の分子メカニズムを解明－統合失調症治療薬が起こす高血糖はビタミンDで防止できる. 京都大学プレスリリース. 2016-05-23.

Dysesthesia is an unpleasant abnormal sensation, which is often accompanied by peripheral neuropathy or vascular impairment. Here, we examined the roles of transient receptor potential ankyrin 1 (TRPA1) in dysesthesia-like behaviours elicited by transient hindlimb ischemia (15–60 min) by tightly compressing the hindlimb, and reperfusion by releasing the ligature. The paw-withdrawal responses to tactile stimulation were reduced during ischemia and lasted for a while after reperfusion. Hindlimb ischemia/reperfusion elicited spontaneous licking of the ischemic hindpaw that peaked within 10 min. The licking was inhibited by reactive oxygen species (ROS) scavengers, a TRPA1 antagonist, or TRPA1 deficiency, but not by TRPV1 deficiency. In human TRPA1-expressing cells as well as cultured mouse dorsal root ganglion neurons, the H[2]O[2]-evoked TRPA1 response was significantly increased by pretreatment with hypoxia (80 mmHg) for 30 min. This hypoxia-induced TRPA1 sensitisation to H[2]O[2] was inhibited by overexpressing a catalytically-inactive mutant of prolyl hydroxylase (PHD) 2 or in a TRPA1 proline mutant resistant to PHDs. Consistent with these results, a PHD inhibitor increased H[2]O[2]-evoked nocifensive behaviours through TRPA1 activation. Our results suggest that transient hindlimb ischemia/reperfusion-evoked spontaneous licking, i.e. painful dysesthesia, is caused by ROS-evoked activation of TRPA1 sensitised by hypoxia through inhibiting PHD-mediated hydroxylation of a proline residue in TRPA1., 「しびれ」による痛みのメカニズムを解明 -糖尿病や血流障害によるしびれ治療薬の開発に期待-. 京都大学プレスリリース. 2016-03-18.

[Background] Diabetic peripheral neuropathy is a common long-term complication of diabetes. Accumulating evidence suggests that vascular impairment plays important roles in the pathogenesis of diabetic peripheral neuropathy, while the mechanism remains unclear. We recently reported that transient receptor potential ankyrin 1 (TRPA1) is sensitized by hypoxia, which can contribute to cold hypersensitivity. In this study, we investigated the involvement of TRPA1 and vascular impairment in painful diabetic peripheral neuropathy using streptozotocin-induced diabetic model mice. [Results] Streptozotocin-induced diabetic model mice showed mechanical and cold hypersensitivity with a peak at two weeks after the streptozotocin administration, which were likely to be paralleled with the decrease in the skin blood flow of the hindpaw. Streptozotocin-induced cold hypersensitivity was significantly inhibited by an antagonist HC-030031 (100 mg/kg) or deficiency for TRPA1, whereas mechanical hypersensitivity was unaltered. Consistent with these results, the nocifensive behaviors evoked by an intraplantar injection of the TRPA1 agonist allyl isothiocyanate (AITC) were enhanced two weeks after the streptozotocin administration. Both streptozotocin-induced cold hypersensitivity and the enhanced AITC-evoked nocifensive behaviors were significantly inhibited by a vasodilator, tadalafil (10 mg/kg), with recovery of the decreased skin blood flow. Similarly, in a mouse model of hindlimb ischemia induced by the ligation of the external iliac artery, AITC-evoked nocifensive behaviors were significantly enhanced three and seven days after the ischemic operation, whereas mechanical hypersensitivity was unaltered in TRPA1-knockout mice. However, no difference was observed between wild-type and TRPA1-knockout mice in the hyposensitivity for current or mechanical stimulation or the deceased density of intraepidermal nerve fibers eight weeks after the streptozotocin administration. [Conclusion] These results suggest that TRPA1 sensitization during diabetic vascular impairment causes cold, but not mechanical, hypersensitivity in the early painful phase of diabetic peripheral neuropathy. However, TRPA1 may play little or no role in the progression of diabetic peripheral neuropathy.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel with high Ca(2+) permeability, which functions as a polymodal nociceptor activated by heat, protons and several vanilloids, including capsaicin and anandamide. Although TRPV1 channels are widely distributed in the mammalian brain, their pathophysiological roles in the brain remain to be elucidated. In this study, we investigated whether TRPV1 is involved in cerebral ischemic injury using a middle cerebral artery (MCA) occlusion model in wild-type (WT) and TRPV1-knockout (KO) mice. For transient ischemia, the left MCA of C57BL/6 mice was occluded for 60 min and reperfused at 1 and 2 days after ischemia. We found that neurological and motor deficits, and infarct volumes in TRPV1-KO mice were lower than those of WT mice. Consistent with these results, intracerebroventricular injection of a TRPV1 antagonist, capsazepine (20 nmol), 30 min before the onset of ischemia attenuated neurological and motor deficits and improved infarct size without influencing cerebral blood flow in the occluded MCA territory. The protective effect of capsazepine on ischemic brain damage was not observed in TRPV1-KO mice. WT and TRPV1-KO mice did not show any differences with respect to the increased number of Iba1-positive microglia/macrophages, GFAP-positive astrocytes, and Gr1-positive neutrophils at 1 and 2 days after cerebral ischemia. Taken together, we conclude that brain TRPV1 channels are activated by ischemic stroke and cause neurological and motor deficits and infarction after brain ischemia.

脳虚血再灌流後に神経細胞が変性する過程では，グリア細胞が異常活性化し，血液脳関門が破綻することで末梢血由来免疫細胞が浸潤し，炎症性サイトカインや細胞傷害性因子の産生を介した過剰な炎症応答が惹起され，脳組織傷害が増悪されることが示されつつあるが，その治療標的に関する知見は非常に乏しいのが現状である．Transient receptor potential melastatin 2（TRPM2）は脳や免疫系細胞に広く分布する活性酸素感受性Ca2＋透過型陽イオンチャネルであり，免疫系細胞におけるTRPM2 を介したCa2＋流入が特定の免疫／炎症応答に重要な役割を果たすことが近年明らかとなってきた．我々は以前，炎症性疼痛や神経障害性疼痛モデルマウスにおいて，ミクログリアやマクロファージに発現するTRPM2 が特定のサイトカイン産生に関与することを明らかとしてきた．そこでTRPM2 遺伝子欠損マウスを用いて脳虚血傷害の形成におけるTRPM2 の関与について研究を行った結果，TRPM2 遺伝子欠損により神経症状や梗塞巣体積は脳虚血24 時間後の急性期では変化がないのに対して， 48 時間および72 時間後では顕著に改善されることを見出した．ミクログリア／マクロファージの活性化抑制薬ミノサイクリンやiNOS選択的阻害薬1400Wを用いた薬理学的および組織学的検討より，ミクログリア／マクロファージに発現するTRPM2 を介したiNOS の過剰発現によるNO の過剰産生が，脳虚血傷害の病態進展に関与することを明らかとした．本研究の成果は，TRPM2 がミクログリア／マクロファージによる過剰な炎症応答に重要な役割を果たすことを示唆するものであり，脳虚血傷害に伴う遅発性神経傷害の新たな治療薬の開発のみならず，過剰な脳内の炎症応答が関与する他の中枢性疾患創薬研究への足がかりになる ことも期待される．

Recent evidence suggests a role of transient receptor potential melastatin 2 (TRPM2) in immune and inflammatory responses. We previously reported that TRPM2 deficiency attenuated inflammatory and neuropathic pain in some pain mouse models, including formalin- or carrageenan-induced inflammatory pain, and peripheral nerve injury-induced neuropathic pain models, while it had no effect on the basal mechanical and thermal nociceptive sensitivities. In this study, we further explored the involvement of TRPM2 in various pain models using TRPM2-knockout mice. There were no differences in the chemonociceptive behaviors evoked by intraplantar injection of capsaicin or hydrogen peroxide between wildtype and TRPM2-knockout mice, while acetic acid-induced writhing behavior was significantly attenuated in TRPM2-knockout mice. In the postoperative incisional pain model, no difference in mechanical allodynia was observed between the two genotypes. By contrast, mechanical allodynia in the monosodium iodoacetate-induced osteoarthritis pain model and the experimental autoimmune encephalomyelitis model were significantly attenuated in TRPM2-knockout mice. Furthermore, mechanical allodynia in paclitaxel-induced peripheral neuropathy and streptozotocin-induced painful diabetic neuropathy models were significantly attenuated in TRPM2-knockout mice. Taken together, these results suggest that TRPM2 plays roles in a wide range of pathological pain models based on peripheral and central neuroinflammation, rather than physiological nociceptive pain.

Neuropathic pain is a pathological pain condition that often results from peripheral nerve injury. Several lines of evidence suggest that neuroinflammation mediated by the interaction between immune cells and neurons plays an important role in the pathogenesis of neuropathic pain. Transient receptor potential melastatin 2 (TRPM2) is a nonselective Ca(2+)-permeable cation channel that acts as a sensor for reactive oxygen species. Recent evidence suggests that TRPM2 expressed on immune cells plays an important role in immune and inflammatory responses. In this study, we examined the roles of TRPM2 expressed on immune and glial cells in neuropathic pain. TRPM2 deficiency attenuated pain behaviors (mechanical allodynia, thermal hyperalgesia and spontaneous pain behaviors) in various kinds of inflammatory and neuropathic pain, but not in nociceptive pain models. In peripheral nerve injury-induced neuropathic pain models, TRPM2 deficiency diminished infiltration of neutrophils mediated through CXCL2 production from macrophages around the injured peripheral nerve and activation of spinal microglia, suggesting that TRPM2 expressed on macrophages and microglia aggravates peripheral and spinal pronociceptive inflammatory responses. Furthermore, we examined the infiltration of peripheral immune cells into the injured nerve and spinal cord using bone marrow chimeric mice by crossing wildtype and TRPM2-knockout mice. The results suggest that TRPM2 plays an important role in the infiltration of peripheral immune cells, particularly macrophages, into the spinal cord, rather than into the injured nerves. The spinal infiltration of macrophages mediated by TRPM2 may contribute to the pathogenesis of neuropathic pain.

Most clinically-used antidepressants acutely increase monoamine levels in synaptic clefts, while their therapeutic effects often require several weeks of administration. Slow neuroadaptive changes in serotonergic neurons are considered to underlie this delayed onset of beneficial actions. Recently, we reported that sustained exposure of rat organotypic raphe slice cultures containing abundant serotonergic neurons to selective serotonin (5-HT) reuptake inhibitors (citalopram, fluoxetine and paroxetine) caused the augmentation of exocytotic serotonin release. However, the ability of other classes of antidepressants to evoke a similar outcome has not been clarified. In this study, we investigated the sustained actions of two tricyclic antidepressants (imipramine and desipramine), one tetracyclic antidepressant (mianserin), three 5-HT and noradrenaline reuptake inhibitors (milnacipran, duloxetine and venlafaxine) and one noradrenergic and specific serotonergic antidepressant (mirtazapine) on serotonin release in the slice cultures. For seven of nine antidepressants, sustained exposure to the agents at concentrations of 0.1–100 µm augmented the level of increase in extracellular serotonin. The rank order of their potency was as follows: milnacipran>duloxetine>citalopram>venlafaxine>imipramine>fluoxetine>desipramine. Neither mirtazapine nor mianserin caused any augmentation. The highest augmentation by sustained exposure to milnacipran was partially attenuated by an α1-adrenoceptor antagonist, benoxathian, while the duloxetine-, venlafaxine- and citalopram-mediated increases were not affected. These results suggest that inhibition of the 5-HT transporter is required for the enhancement of serotonin release. Furthermore, the potent augmentation by milnacipran is apparently due to the accompanied activation of the α1-adrenoceptor.

Recent evidence suggests that transient receptor potential melastatin 2 (TRPM2) expressed in immune cells plays an important role in immune and inflammatory responses. We recently reported that TRPM2 expressed in macrophages and spinal microglia contributes to the pathogenesis of inflammatory and neuropathic pain aggravating peripheral and central pronociceptive inflammatory responses in mice. To further elucidate the contribution of TRPM2 expressed by peripheral immune cells to neuropathic pain, we examined the development of peripheral nerve injury-induced neuropathic pain and the infiltration of immune cells (particularly macrophages) into the injured nerve and spinal cord by using bone marrow (BM) chimeric mice by crossing wildtype (WT) and TRPM2-knockout (TRPM2-KO) mice. Four types of BM chimeric mice were prepared, in which irradiated WT or TRPM2-KO recipient mice were transplanted with either WT-or TRPM2-KO donor mouse-derived green fluorescence protein-positive (GFP(+)) BM cells (TRPM2(BM+/Rec+), TRPM2(BM-/Rec+), TRPM2(BM+/Rec-), and TRPM2(BM-/Rec-) mice). Mechanical allodynia induced by partial sciatic nerve ligation observed in TRPM2(BM+/Rec+) mice was attenuated in TRPM2(BM-/Rec+), TRPM2(BM+/Rec-), and TRPM2(BM-/Rec-) mice. The numbers of GFP(+) BM-derived cells and Iba1/GFP double-positive macrophages in the injured sciatic nerve did not differ among chimeric mice 14 days after the nerve injury. In the spinal cord, the number of GFP(+) BM-derived cells, particularly GFP/Iba1 double-positive macrophages, was significantly decreased in the three TRPM2-KO chimeric mouse groups compared with TRPM2(BM+/Rec+) mice. However, the numbers of GFP(-)/Iba1(+) resident microglia did not differ among chimeric mice. These results suggest that TRPM2 plays an important role in the infiltration of peripheral immune cells, particularly macrophages, into the spinal cord, rather than the infiltration of peripheral immune cells into the injured nerves and activation of spinal-resident microglia. The spinal infiltration of macrophages mediated by TRPM2 may contribute to the pathogenesis of neuropathic pain.

Accumulating evidence suggests that neuroimmune interactions contribute to pathological pain. Transient receptor potential melastatin 2 (TRPM2) is a nonselective Ca(2+)-permeable cation channel that acts as a sensor for reactive oxygen species. TRPM2 is expressed abundantly in immune cells and is important in inflammatory processes. The results of the present study show that TRPM2 plays a crucial role in inflammatory and neuropathic pain. While wild-type and TRPM2 knock-out mice showed no difference in their basal sensitivity to mechanical and thermal stimulation, nocifensive behaviors in the formalin test were reduced in TRPM2 knock-out mice. In carrageenan-induced inflammatory pain and sciatic nerve injury-induced neuropathic pain models, mechanical allodynia and thermal hyperalgesia were attenuated in TRPM2 knock-out mice. Carrageenan-induced inflammation and sciatic nerve injury increased the expression of TRPM2 mRNA in the inflamed paw and around the injured sciatic nerve, respectively. TRPM2 deficiency diminished the infiltration of neutrophils and the production of chemokine (C-X-C motif) ligand-2 (CXCL2), a major chemokine that recruits neutrophils, but did not alter the recruitment of F4/80-positive macrophages in the inflamed paw or around the injured sciatic nerve. Microglial activation after nerve injury was suppressed in the spinal cord of TRPM2 knock-out mice. Furthermore, CXCL2 production and inducible nitric oxide synthase induction were diminished in cultured macrophages and microglia derived from TRPM2 knock-out mice. Together, these results suggest that TRPM2 expressed in macrophages and microglia aggravates peripheral and spinal pronociceptive inflammatory responses and contributes to the pathogenesis of inflammatory and neuropathic pain., 慢性痛の原因となる神経炎症応答の増悪機構を解明－新しい鎮痛薬開発の可能性－. 京都大学プレスリリース. 2012-03-15.

Whether a high plasma aldosterone concentration induced by strict salt restriction promotes cardiac remodeling remains controversial. Male Sprague-Dawley rats at 10weeks of age were given normal salt (NS) (1.5% NaCl) or low salt (LS) (0.05% NaCl) diets. Each animal underwent aortocaval fistula creation for volume-overloaded heart failure or sham surgery. All rats with a fistula received either vehicle or a non-hypotensive dose of spironolactone (200mg/kg/day) by gavage. Two weeks later, the LS diet significantly increased the plasma aldosterone level in the sham-operated and fistula-created rats (2677+/-662pg/ml and 2406+/-422pg/ml) compared with that in rats given the NS diet (518+/-18pg/ml and 362+/-45pg/ml, respectively). In sham-operated rats, the difference in plasma aldosterone level did not affect the extent of myocardial fibrosis (1.8+/-0.1% with LS diet vs. 1.5+/-0.3% with NS diet). However, the increase in myocardial fibrosis in fistula-created rats was more prominent with the LS diet than with the NS diet (4.7+/-0.3% vs. 3.4+/-0.1%). In addition, the fistula-created rats on the LS diet expressed significantly increased oxidative stress and transforming growth factor-beta compared with those on the NS diets (P0.05). These increases in the fistula-created rats on the LS diet were significantly suppressed by the non-hypotensive dose of spironolactone (P0.05). These results suggest that increased plasma aldosterone level with strict salt restriction activated the mineralocorticoid receptor signaling in volume-overloaded condition, resulting in increased myocardial fibrosis.

Several lines of evidence suggest that activation of spinal mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) and p38 MAPK, contributes to the induction and maintenance of chronic pain. We recently reported that an intrathecal (i.t.) administration of ATP evoked tactile allodynia, which lasted more than 1 week in rats. The long-lasting allodynia was induced by activation of spinal P2X 2/3-receptors, and the induction and early phase of maintenance, but not the late phase, was mediated, at least in part, by the activation of spinal glial cells. In this study, we examined the involvement of spinal ERK and p38 MAPK in each phase of i.t. ATP-evoked long-lasting allodynia. I.t. administration of ATP (100 nmol) markedly increased phosphorylated ERK, which peaked at 1-8 h before gradually decreasing to a level that was sustained until 7 d after administration. In contrast, only a slight increase in phosphorylated p38 MAPK was observed. Consistent with the increased phosphorylation of MAPKs, the ERK kinase MEK inhibitor, U0126 (3 nmol), attenuated the induction phase (co-administration with ATP) and early maintenance phase (1-d post-ATP administration) of the i.t. ATP-evoked allodynia, but not the late maintenance phase (7-d post-ATP administration), while the p38 MAPK inhibitor, SB203580 (10 microg), had little effect. These results suggest that the induction phase and early maintenance phase, but not the late maintenance phase of long-lasting allodynia is mediated by the activation of ERK, rather than by the activation of p38 MAPK, in the spinal cord. These findings are informative for elucidating the mechanisms underlying the pathogenesis of chronic pain.

Background: Oxaliplatin, a platinum-based chemotherapeutic agent, causes an unusual acute peripheral neuropathy. Oxaliplatin-induced acute peripheral neuropathy appears in almost all patients rapidly after infusion, and is triggered or exacerbated by cold, while its mechanisms are poorly understood. In this study, the involvement of thermosensitive transient receptor potential channels (TRPA1, TRPM8 and TRPV1) in oxaliplatin-induced acute hypersensitivity was investigated in mice. Results: A single intraperitoneal administration of oxaliplatin (1–10 mg/kg) induced cold but not mechanical hypersensitivity within 2 h in a dose-dependent manner. Infusion of the oxaliplatin metabolite, oxalate (1.7 mg/kg), also induced acute cold hypersensitivity, while another platinum-based chemotherapeutic agent, cisplatin (5 mg/kg), or the non-platinum-containing chemotherapeutic agent, paclitaxel (6 mg/kg) failed to induce mechanical or cold hypersensitivity. The oxaliplatin-induced acute cold hypersensitivity was abolished by the TRPA1 antagonist HC-030031 (100 mg/kg) and by TRPA1 deficiency. The nocifensive behaviors evoked by intraplantar injections of allyl-isothiocyanate (AITC; TRPA1 agonist) were significantly enhanced in mice treated for 2 h with oxaliplatin (1–10 mg/kg) in a dose-dependent manner, while capsaicin (TRPV1 agonist)-evoked nocifensive behaviors were not affected. Menthol (TRPM8/TRPA1 agonist)-evoked nocifensive-like behaviors were also enhanced by oxaliplatin pretreatment, which were inhibited by TRPA1 deficiency. Similarly, oxalate enhanced, but neither cisplatin nor paclitaxel affected AITC-evoked nocifensive behaviors. Pretreatment of cultured mouse dorsal root ganglia (DRG) neurons with oxaliplatin (30–300 μM) for 1, 2, or 4 h significantly increased the number of AITC-sensitive neurons in a concentration-dependent manner whereas there was no change in the number of menthol- or capsaicin-sensitive neurons. Conclusions: Taken together, these results suggest that a brief treatment with oxaliplatin or its metabolite oxalate is sufficient to enhance the responsiveness of TRPA1 but not that of TRPM8 and TRPV1 expressed by DRG neurons, which may contribute to the characteristic acute peripheral neuropathy induced by oxaliplatin.

Background: The glial glutamate transporter GLT-1 is abundantly expressed in astrocytes and is crucial for glutamate removal from the synaptic cleft. Decreases in glutamate uptake activity and expression of spinal glutamate transporters are reported in animal models of pathological pain. However, the lack of available specific inhibitors and/or activators for GLT-1 makes it difficult to determine the roles of spinal GLT-1 in inflammatory and neuropathic pain. In this study, we examined the effect of gene transfer of GLT-1 into the spinal cord with recombinant adenoviruses on the inflammatory and neuropathic pain in rats. Results: Intraspinal infusion of adenoviral vectors expressing the GLT-1 gene increased GLT-1 expression in the spinal cord 2–21 days after the infusion. Transgene expression was primarily localized to astrocytes. The spinal GLT-1 gene transfer had no effect on acute mechanical and thermal nociceptive responses in naive rats, whereas it significantly reduced the inflammatory mechanical hyperalgesia induced by hindlimb intraplantar injection of carrageenan/kaolin. Spinal GLT-1 gene transfer 7 days before partial sciatic nerve ligation recovered the extent of the spinal GLT-1 expression in the membrane fraction that was decreased following the nerve ligation, and prevented the induction of tactile allodynia. However, the partial sciatic nerve ligation-induced allodynia was not reversed when the adenoviruses were infused 7 or 14 days after the nerve ligation. Conclusion: These results suggest that overexpression of GLT-1 on astrocytes in the spinal cord by recombinant adenoviruses attenuates the induction, but not maintenance, of inflammatory and neuropathic pain, probably by preventing the induction of central sensitization, without affecting acute pain sensation. Upregulation or functional enhancement of spinal GLT-1 could be a novel strategy for the prevention of pathological pain.