Hisashi Shirakawa

TRPM2 Contributes to Inflammatory and Neuropathic Pain through the Aggravation of Pronociceptive Inflammatory Responses in Mice

Accumulating evidence suggests that neuroimmune interactions contribute to pathological pain. Transient receptor potential melastatin 2 (TRPM2) is a nonselective Ca2+-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.

Acute cold hypersensitivity characteristically induced by oxaliplatin is caused by the enhanced responsiveness of TRPA1 in mice.

BackgroundOxaliplatin, 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.ResultsA 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.ConclusionsTaken 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.

Gene transfer of GLT-1, a glial glutamate transporter, into the spinal cord by recombinant adenovirus attenuates inflammatory and neuropathic pain in rats.

BackgroundThe 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.ResultsIntraspinal 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.ConclusionThese 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.

Transient Receptor Potential Canonical 3 (TRPC3) Mediates Thrombin-Induced Astrocyte Activation and Upregulates Its Own Expression in Cortical Astrocytes

Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood–brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca2+-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca2+ influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca2+ influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH2-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca2+ signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca2+ signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca2+-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH2, a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own expression.

TRPV1 stimulation triggers apoptotic cell death of rat cortical neurons

Transient receptor potential vanilloid 1 (TRPV1) functions as a polymodal nociceptor and is activated by several vanilloids, including capsaicin, protons and heat. Although TRPV1 channels are widely distributed in the brain, their roles remain unclear. Here, we investigated the roles of TRPV1 in cytotoxic processes using TRPV1-expressing cultured rat cortical neurons. Capsaicin induced severe neuronal death with apoptotic features, which was completely inhibited by the TRPV1 antagonist capsazepine and was dependent on extracellular Ca(2+) influx. Interestingly, nifedipine, a specific L-type Ca(2+) channel blocker, attenuated capsaicin cytotoxicity, even when applied 2-4 h after the capsaicin. ERK inhibitor PD98059 and several antioxidants, but not the JNK and p38 inhibitors, attenuated capsaicin cytotoxicity. Together, these data indicate that TRPV1 activation triggers apoptotic cell death of rat cortical cultures via L-type Ca(2+) channel opening, Ca(2+) influx, ERK phosphorylation, and reactive oxygen species production.

Isolation of a diterpenoid substance with potent neuroprotective activity from fetal calf serum

Excess activation of glutamate receptors and production of free radicals including nitric oxide may result in severe and irreversible damage to the mammalian central nervous system (CNS), but endogenous defense systems that protect neurons from these insults are poorly understood. Here, we purified and isolated a neuroprotective substance, which has been named “serofendic acid,” from a lipophilic fraction of FCS based on the ability to protect rat primary cortical neurons against nitric oxide cytotoxicity. Mass spectrometry and NMR spectroscopy revealed the chemical structure of serofendic acid (15-hydroxy-17-methylsulfinylatisan-19-oic acid) as a sulfur-containing atisane-type diterpenoid, which is unique among known endogenous substances. Synthetic serofendic acid exhibited potent protective actions on cortical neurons against cytotoxicity of a nitric oxide donor as well as of glutamate, although it did not show appreciable influences on glutamate receptor-mediated responses in these neurons. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on nitric oxide radicals but was capable of inhibiting the generation of hydroxyl radical, a presumed “executor” radical in the nitric oxide-mediated neurotoxic cascade. These findings suggest that serofendic acid is a low-molecular-weight bioactive factor that promotes survival of CNS neurons, probably through the attenuation of free radical-mediated insults.

Stimulation of transient receptor potential vanilloid 4 channel suppresses abnormal activation of microglia induced by lipopolysaccharide.

Microglia are intrinsic immune cells in the brain. In response to neurodegenerative events, excessively activated microglia change their shapes and release various cytokines leading to the pathogenesis of central nervous system (CNS) disease. Because the intracellular mechanisms of this process are still unclear, we have evaluated the functional roles of transient receptor potential vanilloid 4 (TRPV4) channel expressed in the microglia. Robust microglial activation after an injection of lipopolysaccharide (LPS) into the mouse cerebral ventricle was suppressed by concurrent administration of a selective TRPV4 agonist, 4α‐phorbol 12,13‐didecanoate (4α‐PDD). When the mechanism was further investigated using cultured rat microglia intrinsically expressing functional TRPV4, release of tumor necrosis factor‐α (TNF‐α) and expression of galectin‐3 were both increased by LPS. These increases were significantly suppressed by cotreatment with 4α‐PDD, and the inhibitory effects of 4α‐PDD were abolished by knockdown of TRPV4 or TRPV4 antagonists. The amplitude of voltage‐dependent K+ current, which is augmented during microglial activation, was also suppressed by 4α‐PDD treatment. Opening of TRPV4 channels with 4α‐PDD induced membrane depolarization mainly by increasing Na+ influx. In addition, mimicking depolarization with a high‐K+ solution suppressed LPS‐induced TNF‐α release and galectin‐3 upregulation. Both depolarizing treatments with 4α‐PDD and high‐K+ solution decreased store‐operated Ca2+ influx caused by thapsigargin. These results suggest that depolarization in response to opening of the TRPV4 channel attenuates the driving force for extracellular Ca2+ and suppresses microglial activation.

Pregnenolone sulphate attenuates AMPA cytotoxicity on rat cortical neurons

Neuroactive steroids can modulate brain excitability by interaction with several neurotransmitter receptor‐associated channels. These compounds may thus exert profound influences on excitotoxic injury, i.e. neuronal cell death triggered by over‐activation of glutamate receptors. It has been reported that pregnenolone sulphate (PS) and pregnenolone hemisuccinate (PHS) augment N‐methyl‐d‐aspartate (NMDA) neurotoxicity in rat cultured neurons. Here we show that the effects of neuroactive steroids on AMPA cytotoxicity display features distinct from those on NMDA cytotoxicity. Concomitant application of PS (30–300 µm) attenuated, rather than augmented, AMPA neurotoxicity in cortical slice cultures in a concentration‐dependent manner, whereas various other steroids including pregnenolone and PHS had no effect. Inhibition of steroid sulphatase by estrone‐3‐O‐sulphamate led to a shift of the minimum effective concentration of PS against AMPA cytotoxicity from 30 to 10 µm. The protective action of PS was not affected by inhibition of protein synthesis or by blockade of glucocorticoid receptors, GABAA receptors or σ‐receptors. In dissociated cortical neurons, PS attenuated AMPA‐induced inward currents whereas pregnenolone and PHS exhibited no significant effect. Thus, with strict structural specificity, PS but not pregnenolone or PHS attenuates AMPA cytotoxicity, probably by inhibiting activities of AMPA receptor‐associated channels.

Transient Receptor Potential Canonical 3 Inhibitor Pyr3 Improves Outcomes and Attenuates Astrogliosis After Intracerebral Hemorrhage in Mice

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.

Involvement of TRPM2 in Peripheral Nerve Injury-Induced Infiltration of Peripheral Immune Cells into the Spinal Cord in Mouse Neuropathic Pain Model

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 (TRPM2BM+/Rec+, TRPM2BM–/Rec+, TRPM2BM+/Rec–, and TRPM2BM–/Rec– mice). Mechanical allodynia induced by partial sciatic nerve ligation observed in TRPM2BM+/Rec+ mice was attenuated in TRPM2BM–/Rec+, TRPM2BM+/Rec–, and TRPM2BM–/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 TRPM2BM+/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.