Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
TSUDA MAKOTO Last modified date:2023.10.06

Professor / Department of Molecular and System Pharmacology / Department of Pharmaceutical Health Care and Sciences / Faculty of Pharmaceutical Sciences


Papers
1. Teruaki Ono, Tomohiro Yamashita, Ryota Kano, Mariko Inoue, Shota Okada, Koki Kano, Schuichi Koizumi, Kazuhisa Iwabuchi, Yoshio Hirabayashi, Ichiro Matsuo, Yasuharu Nakashima, Hiroyuki Kamiguchi, Yuta Kohro, Makoto Tsuda, GPR55 contributes to neutrophil recruitment and mechanical pain induction after spinal cord compression in mice., Brain, behavior, and immunity, 10.1016/j.bbi.2023.03.008, 110, 276-287, 2023.03, Pain transmission and processing in the nervous system are modulated by various biologically active substances, including lysophospholipids, through direct and indirect actions on the somatosensory pathway. Lysophosphatidylglucoside (LysoPtdGlc) was recently identified as a structurally unique lysophospholipid that exerts biological actions via the G protein-coupled receptor GPR55. Here, we demonstrated that GPR55-knockout (KO) mice show impaired induction of mechanical pain hypersensitivity in a model of spinal cord compression (SCC) without the same change in the models of peripheral tissue inflammation and peripheral nerve injury. Among these models, only SCC recruited peripheral inflammatory cells (neutrophils, monocytes/macrophages, and CD3+ T-cells) in the spinal dorsal horn (SDH), and GPR55-KO blunted these recruitments. Neutrophils were the first cells recruited to the SDH, and their depletion suppressed the induction of SCC-induced mechanical hypersensitivity and inflammatory responses in compressed SDH. Furthermore, we found that PtdGlc was present in the SDH and that intrathecal administration of an inhibitor of secretory phospholipase A2 (an enzyme required for producing LysoPtdGlc from PtdGlc) reduced neutrophil recruitment to compressed SDH and suppressed pain induction. Finally, by screening compounds from a chemical library, we identified auranofin as a clinically used drug with an inhibitory effect on mouse and human GPR55. Systemically administered auranofin to mice with SCC effectively suppressed spinal neutrophil infiltration and pain hypersensitivity. These results suggest that GPR55 signaling contributes to the induction of inflammatory responses and chronic pain after SCC via the recruitment of neutrophils and may provide a new target for reducing pain induction after spinal cord compression, such as spinal canal stenosis..
2. Tadayuki Ishibashi, Daichi Sueto, Yu Yoshikawa, Keisuke Koga, Ken Yamaura, Makoto Tsuda, Identification of Spinal Inhibitory Interneurons Required for Attenuating Effect of Duloxetine on Neuropathic Allodynia-like Signs in Rats., Cells, 10.3390/cells11244051, 11, 24, 2022.12, Neuropathic pain is a chronic pain condition that occurs after nerve damage; allodynia, which refers to pain caused by generally innocuous stimuli, is a hallmark symptom. Although allodynia is often resistant to analgesics, the antidepressant duloxetine has been used as an effective therapeutic option. Duloxetine increases spinal noradrenaline (NA) levels by inhibiting its transporter at NAergic terminals in the spinal dorsal horn (SDH), which has been proposed to contribute to its pain-relieving effect. However, the mechanism through which duloxetine suppresses neuropathic allodynia remains unclear. Here, we identified an SDH inhibitory interneuron subset (captured by adeno-associated viral (AAV) vectors incorporating a rat neuropeptide Y promoter; AAV-NpyP+ neurons) that is mostly depolarized by NA. Furthermore, this excitatory effect was suppressed by pharmacological blockade or genetic knockdown of α1B-adrenoceptors (ARs) in AAV-NpyP+ SDH neurons. We found that duloxetine suppressed Aβ fiber-mediated allodynia-like behavioral responses after nerve injury and that this effect was not observed in AAV-NpyP+ SDH neuron-selective α1B-AR-knockdown. These results indicate that α1B-AR and AAV-NpyP+ neurons are critical targets for spinal NA and are necessary for the therapeutic effect of duloxetine on neuropathic pain, which can support the development of novel analgesics..
3. Ikuko Takeda, Kohei Yoshihara, Dennis L Cheung, Tomoko Kobayashi, Masakazu Agetsuma, Makoto Tsuda, Kei Eto, Schuichi Koizumi, Hiroaki Wake, Andrew J Moorhouse, Junichi Nabekura, Controlled activation of cortical astrocytes modulates neuropathic pain-like behaviour., Nature communications, 10.1038/s41467-022-31773-8, 13, 1, 4100-4100, 2022.07, Chronic pain is a major public health problem that currently lacks effective treatment options. Here, a method that can modulate chronic pain-like behaviour induced by nerve injury in mice is described. By combining a transient nerve block to inhibit noxious afferent input from injured peripheral nerves, with concurrent activation of astrocytes in the somatosensory cortex (S1) by either low intensity transcranial direct current stimulation (tDCS) or via the chemogenetic DREADD system, we could reverse allodynia-like behaviour previously established by partial sciatic nerve ligation (PSL). Such activation of astrocytes initiated spine plasticity to reduce those synapses formed shortly after PSL. This reversal from allodynia-like behaviour persisted well beyond the active treatment period. Thus, our study demonstrates a robust and potentially translational approach for modulating pain, that capitalizes on the interplay between noxious afferents, sensitized central neuronal circuits, and astrocyte-activation induced synaptic plasticity..
4. Kazuki Fujimori, Misuzu Sekine, Moeka Watanabe, Ryoichi Tashima, Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Chemogenetic silencing of spinal cord-projecting cortical neurons attenuates Aβ fiber-derived neuropathic allodynia in mice., Neuroscience research, 10.1016/j.neures.2022.05.001, 2022.05, Mechanical allodynia (pain caused by innocuous mechanical stimulation) is a hallmark symptom of neuropathic pain occurring following peripheral nerve injury (PNI). Using a transgenic mouse line, in which myelinated primary afferents, including Aβ fibers, express channelrhodopsin-2, we found that illumination of the plantar skin of mice following PNI produced an Aβ fiber-mediated pain-like withdrawal behavior and increased c-FOS+ neurons in the superficial spinal dorsal horn (SDH). These two responses were attenuated by chemogenetic silencing of primary sensory cortex (S1) neurons projecting directly to the SDH. These findings indicate that spinally projecting cortical S1 neurons contribute to Aβ fiber-derived neuropathic allodynia..
5. Kensho Kanehisa, Keisuke Koga, Sho Maejima, Yuto Shiraishi, Konatsu Asai, Miho Shiratori-Hayashi, Mei-Fang Xiao, Hirotaka Sakamoto, Paul F Worley, Makoto Tsuda, Neuronal pentraxin 2 is required for facilitating excitatory synaptic inputs onto spinal neurons involved in pruriceptive transmission in a model of chronic itch., Nature communications, 10.1038/s41467-022-30089-x, 13, 1, 2367-2367, 2022.05, An excitatory neuron subset in the spinal dorsal horn (SDH) that expresses gastrin-releasing peptide receptors (GRPR) is critical for pruriceptive transmission. Here, we show that glutamatergic excitatory inputs onto GRPR+ neurons are facilitated in mouse models of chronic itch. In these models, neuronal pentraxin 2 (NPTX2), an activity-dependent immediate early gene product, is upregulated in the dorsal root ganglion (DRG) neurons. Electron microscopy reveals that NPTX2 is present at presynaptic terminals connected onto postsynaptic GRPR+ neurons. NPTX2-knockout prevents the facilitation of synaptic inputs to GRPR+ neurons, and repetitive scratching behavior. DRG-specific NPTX2 expression rescues the impaired behavioral phenotype in NPTX2-knockout mice. Moreover, ectopic expression of a dominant-negative form of NPTX2 in DRG neurons reduces chronic itch-like behavior in mice. Our findings indicate that the upregulation of NPTX2 expression in DRG neurons contributes to the facilitation of glutamatergic inputs onto GRPR+ neurons under chronic itch-like conditions, providing a potential therapeutic target..
6. Keita Kohno, Ryoji Shirasaka, Kohei Yoshihara, Satsuki Mikuriya, Kaori Tanaka, Keiko Takanami, Kazuhide Inoue, Hirotaka Sakamoto, Yasuyuki Ohkawa, Takahiro Masuda, Makoto Tsuda, A spinal microglia population involved in remitting and relapsing neuropathic pain., Science (New York, N.Y.), 10.1126/science.abf6805, 376, 6588, 86-90, 2022.04, Neuropathic pain is often caused by injury and diseases that affect the somatosensory system. Although pain development has been well studied, pain recovery mechanisms remain largely unknown. Here, we found that CD11c-expressing spinal microglia appear after the development of behavioral pain hypersensitivity following nerve injury. Nerve-injured mice with spinal CD11c+ microglial depletion failed to recover spontaneously from this hypersensitivity. CD11c+ microglia expressed insulin-like growth factor-1 (IGF1), and interference with IGF1 signaling recapitulated the impairment in pain recovery. In pain-recovered mice, the depletion of CD11c+ microglia or the interruption of IGF1 signaling resulted in a relapse in pain hypersensitivity. Our findings reveal a mechanism for the remission and recurrence of neuropathic pain, providing potential targets for therapeutic strategies..
7. Takahiro Masuda, Lukas Amann, Gianni Monaco, Roman Sankowski, Ori Staszewski, Martin Krueger, Francesca Del Gaudio, Liqun He, Neil Paterson, Elisa Nent, Francisco Fernández-Klett, Ayato Yamasaki, Maximilian Frosch, Maximilian Fliegauf, Lance Fredrick Pahutan Bosch, Hatice Ulupinar, Nora Hagemeyer, Dietmar Schreiner, Cayce Dorrier, Makoto Tsuda, Claudia Grothe, Anne Joutel, Richard Daneman, Christer Betsholtz, Urban Lendahl, Klaus-Peter Knobeloch, Tim Lämmermann, Josef Priller, Katrin Kierdorf, Marco Prinz, Specification of CNS macrophage subsets occurs postnatally in defined niches., Nature, 10.1038/s41586-022-04596-2, 604, 7907, 740-748, 2022.04, All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS..
8. Ruqayya Afridi, Makoto Tsuda, Hoon Ryu, Kyoungho Suk, The Function of Glial Cells in the Neuroinflammatory and Neuroimmunological Responses., Cells, 10.3390/cells11040659, 11, 4, 2022.02, The historical concept of glia just as the glue of brain tissue has been challenged by the accumulation of concrete evidence showing the multifunctional role of these cells during development and in the adult brain [...]..
9. Chinatsu Shinozaki, Keita Kohno, Mitsunori Shiroishi, Daisuke Takahashi, Yu Yoshikawa, Yoshito Abe, Kenji Hamase, Makoto Nakakido, Kohei Tsumoto, Kazuhide Inoue, Makoto Tsuda, Tadashi Ueda, Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions., Scientific reports, 10.1038/s41598-021-03784-w, 12, 1, 131-131, 2022.01, We have recently developed a mouse monoclonal antibody (12-10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12-10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the "hot spot" in the 12-10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12-10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the "hot spot" in the paratope..
10. Sawako Uchiyama, Kohei Yoshihara, Riku Kawanabe, Izuho Hatada, Keisuke Koga, Makoto Tsuda, Stress-induced antinociception to noxious heat requires α1A-adrenaline receptors of spinal inhibitory neurons in mice., Molecular brain, 10.1186/s13041-021-00895-3, 15, 1, 6-6, 2022.01, It is well known that acute exposure to physical stress produces a transient antinociceptive effect (called stress-induced analgesia [SIA]). One proposed mechanism for SIA involves noradrenaline (NA) in the central nervous system. NA has been reported to activate inhibitory neurons in the spinal dorsal horn (SDH), but its in vivo role in SIA remains unknown. In this study, we found that an antinociceptive effect on noxious heat after acute exposure to restraint stress was impaired in mice with a conditional knockout of α1A-adrenaline receptors (α1A-ARs) in inhibitory neurons (Vgat-Cre;Adra1aflox/flox mice). A similar reduction was also observed in mice treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a selective neurotoxin for NAergic neurons in the locus coeruleus (LC). Furthermore, whole-cell patch-clamp recordings using spinal cord slices revealed that NA-induced increase in the frequency of spontaneous inhibitory postsynaptic currents in the substantia gelatinosa neurons was suppressed by silodosin, an α1A-AR antagonist, and by conditional knockout of α1A-ARs in inhibitory neurons. Moreover, under unstressed conditions, the antinociceptive effects of intrathecal NA and phenylephrine on noxious heat were lost in Vgat-Cre;Adra1aflox/flox mice. Our findings suggest that activation of α1A-ARs in SDH inhibitory neurons, presumably via LC-NAergic neurons, is necessary for SIA to noxious heat..
11. Riku Kawanabe, Kohei Yoshihara, Izuho Hatada, Makoto Tsuda, Activation of spinal dorsal horn astrocytes by noxious stimuli involves descending noradrenergic signaling., Molecular brain, 10.1186/s13041-021-00788-5, 14, 1, 79-79, 2021.05, Astrocytes are critical regulators of neuronal function in the central nervous system (CNS). We have previously shown that astrocytes in the spinal dorsal horn (SDH) have increased intracellular Ca2+ levels following intraplantar injection of the noxious irritant, formalin. However, the underlying mechanisms remain unknown. We investigated these mechanisms by focusing on the role of descending noradrenergic (NAergic) signaling because our recent study revealed the essential role of the astrocytic Ca2+ responses evoked by intraplantar capsaicin. Using in vivo SDH imaging, we found that the Ca2+ level increase in SDH astrocytes induced by intraplantar formalin injection was suppressed by ablation of SDH-projecting locus coeruleus (LC)-NAergic neurons. Furthermore, the formalin-induced Ca2+ response was dramatically decreased by the loss of α1A-adrenaline receptors (ARs) in astrocytes located in the superficial laminae of the SDH. Moreover, similar inhibition was observed in mice pretreated intrathecally with an α1A-AR-specific antagonist. Therefore, activation of α1A-ARs via descending LC-NAergic signals may be a common mechanism underlying astrocytic Ca2+ responses in the SDH evoked by noxious stimuli, including chemical irritants..
12. Yuto Shiraishi, Keisuke Koga, Ryo Yamagata, Izuho Hatada, Miho Shiratori-Hayashi, Makoto Tsuda, α1A-adrenaline receptors in dorsal horn inhibitory neurons have an inhibitory role in the regulation of chloroquine-induced itch in mice., Molecular brain, 10.1186/s13041-021-00768-9, 14, 1, 55-55, 2021.03, Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR-Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch..
13. Shuntaro Uchida, Shingo Soya, Yuki C Saito, Arisa Hirano, Keisuke Koga, Makoto Tsuda, Manabu Abe, Kenji Sakimura, Takeshi Sakurai, A Discrete Glycinergic Neuronal Population in the Ventromedial Medulla That Induces Muscle Atonia during REM Sleep and Cataplexy in Mice., The Journal of neuroscience : the official journal of the Society for Neuroscience, 10.1523/JNEUROSCI.0688-20.2020, 41, 7, 1582-1596, 2021.02, During rapid eye movement (REM) sleep, anti-gravity muscle tone and bodily movements are mostly absent, because somatic motoneurons are inhibited by descending inhibitory pathways. Recent studies showed that glycine/GABA neurons in the ventromedial medulla (VMM; GlyVMM neurons) play an important role in generating muscle atonia during REM sleep (REM-atonia). However, how these REM-atonia-inducing neurons interconnect with other neuronal populations has been unknown. In the present study, we first identified a specific subpopulation of GlyVMM neurons that play an important role in induction of REM-atonia by virus vector-mediated tracing in male mice in which glycinergic neurons expressed Cre recombinase. We found these neurons receive direct synaptic input from neurons in several brain stem regions, including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD; GluSLD neurons). Silencing this circuit by specifically expressing tetanus toxin light chain (TeTNLC) resulted in REM sleep without atonia. This manipulation also caused a marked decrease in time spent in cataplexy-like episodes (CLEs) when applied to narcoleptic orexin-ataxin-3 mice. We also showed that GlyVMM neurons play an important role in maintenance of sleep. This present study identified a population of glycinergic neurons in the VMM that are commonly involved in REM-atonia and cataplexy.SIGNIFICANCE STATEMENT We identified a population of glycinergic neurons in the ventral medulla that plays an important role in inducing muscle atonia during rapid eye movement (REM) sleep. It sends axonal projections almost exclusively to motoneurons in the spinal cord and brain stem except to those that innervate extraocular muscles, while other glycinergic neurons in the same region also send projections to other regions including monoaminergic nuclei. Furthermore, these neurons receive direct inputs from several brainstem regions including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD). Genetic silencing of this pathway resulted in REM sleep without atonia and a decrease of cataplexy when applied to narcoleptic mice. This work identified a neural population involved in generating muscle atonia during REM sleep and cataplexy..
14. Tomohiro Yamashita, Sawako Kamikaseda, Aya Tanaka, Hidetoshi Tozaki-Saitoh, Jose M M Caaveiro, Kazuhide Inoue, Makoto Tsuda, New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1β Release: An Involvement in its Alleviating Effect on Neuropathic Pain., Cells, 10.3390/cells10020434, 10, 2, 2021.02, P2X7 receptors (P2X7Rs) belong to a family of ATP-gated non-selective cation channels. Microglia represent a major cell type expressing P2X7Rs. The activation of microglial P2X7Rs causes the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β). This response has been implicated in neuroinflammatory states in the central nervous system and in various diseases, including neuropathic pain. Thus, P2X7R may represent a potential therapeutic target. In the present study, we screened a chemical library of clinically approved drugs (1979 compounds) by high-throughput screening and showed that the Ca2+ channel blocker cilnidipine has an inhibitory effect on rodent and human P2X7R. In primary cultured rat microglial cells, cilnidipine inhibited P2X7R-mediated Ca2+ responses and IL-1β release. Moreover, in a rat model of neuropathic pain, the intrathecal administration of cilnidipine produced a reversal of nerve injury-induced mechanical hypersensitivity, a cardinal symptom of neuropathic pain. These results point to a new inhibitory effect of cilnidipine on microglial P2X7R-mediated inflammatory responses and neuropathic pain, proposing its therapeutic potential..
15. Teruaki Ono, Makoto Tsuda, Reply to Letter to the Editor "Regarding letter on mechanical pain of the lower extremity after compression of the upper spinal cord involves signal transducer and activator of transcription 3-dependent reactive astrocytes and interleukin-6"., Brain, behavior, and immunity, 10.1016/j.bbi.2020.11.014, 91, 796-796, 2021.01.
16. Ryoichi Tashima, Keisuke Koga, Yu Yoshikawa, Misuzu Sekine, Moeka Watanabe, Hidetoshi Tozaki-Saitoh, Hidemasa Furue, Toshiharu Yasaka, Makoto Tsuda, A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior., Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.2021220118, 118, 3, 2021.01, A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aβ fibers. However, the mechanism by which Aβ fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP+) and show that specific ablation or silencing of AAV-NpyP+ SDH interneurons converted touch-sensing Aβ fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP+ neurons received excitatory inputs from Aβ fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP+ neurons exhibited deeper resting membrane potentials, and their excitation by Aβ fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP+ neurons in nerve-injured rats reversed Aβ fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aβ fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia..
17. Tatsuhiro Igawa, Shuhei Kishikawa, Yoshito Abe, Makoto Tsuda, Kazuhide Inoue, Tadashi Ueda, Analysis of binding residues in monoclonal antibody with high affinity for the head domain of the rat P2X4 receptor., Journal of biochemistry, 10.1093/jb/mvaa124, 2020.11, P2X4 receptor is known to be involved in neuropathic pain. In order to detect the expression of P2X4 receptor on microglia at the time of onset of neuropathic pain, one approach consist on the preparation of the monoclonal antibodies with both selective binding and high affinity. We have recently established a monoclonal antibody (named 12-10H) which had high affinity to rat P2X4 receptor expressed in 1321N1 cells. The dissociation constants of the complex between the monoclonal antibodies obtained so far and the head domain in the rat P2X4 receptor were in the nanomolar range. To improve the affinity by rational mutations, we need to know the precious location of the binding site in these monoclonal antibodies. Here, we have analyzed and identified the binding residues in the monoclonal antibody (12-10H) with high affinity for the head domain of the rat P2X4 receptor by site-directed mutagenesis..
18. Yuta Kohro, Tsuyoshi Matsuda, Kohei Yoshihara, Keita Kohno, Keisuke Koga, Ryuichi Katsuragi, Takaaki Oka, Ryoichi Tashima, Sho Muneta, Takuya Yamane, Shota Okada, Kazuya Momokino, Aogu Furusho, Kenji Hamase, Takumi Oti, Hirotaka Sakamoto, Kenichiro Hayashida, Ryosuke Kobayashi, Takuro Horii, Izuho Hatada, Hidetoshi Tozaki-Saitoh, Katsuhiko Mikoshiba, Verdon Taylor, Kazuhide Inoue, Makoto Tsuda, Spinal astrocytes in superficial laminae gate brainstem descending control of mechanosensory hypersensitivity., Nature neuroscience, 10.1038/s41593-020-00713-4, 23, 11, 1376-1387, 2020.11, Astrocytes are critical regulators of CNS function and are proposed to be heterogeneous in the developing brain and spinal cord. Here we identify a population of astrocytes located in the superficial laminae of the spinal dorsal horn (SDH) in adults that is genetically defined by Hes5. In vivo imaging revealed that noxious stimulation by intraplantar capsaicin injection activated Hes5+ SDH astrocytes via α1A-adrenoceptors (α1A-ARs) through descending noradrenergic signaling from the locus coeruleus. Intrathecal norepinephrine induced mechanical pain hypersensitivity via α1A-ARs in Hes5+ astrocytes, and chemogenetic stimulation of Hes5+ SDH astrocytes was sufficient to produce the hypersensitivity. Furthermore, capsaicin-induced mechanical hypersensitivity was prevented by the inhibition of descending locus coeruleus-noradrenergic signaling onto Hes5+ astrocytes. Moreover, in a model of chronic pain, α1A-ARs in Hes5+ astrocytes were critical regulators for determining an analgesic effect of duloxetine. Our findings identify a superficial SDH-selective astrocyte population that gates descending noradrenergic control of mechanosensory behavior..
19. Miho Shiratori-Hayashi, Makoto Tsuda, Role of reactive astrocytes in the spinal dorsal horn under chronic itch conditions., Journal of pharmacological sciences, 10.1016/j.jphs.2020.07.010, 144, 3, 147-150, 2020.11, Astrocytes are the most abundant glial cells in the central nervous system (CNS), including the spinal cord. Neuronal damage induces astrocytes to become reactive and contribute to various CNS pathologies. Recent studies have demonstrated that astrocytes in the spinal dorsal horn (SDH) become reactive in a transcription factor signal transducer and activator of transcription 3-dependent manner without neuronal damage under chronic itch conditions, causing release of the factor lipocalin-2, leading to induction of sensitization of gastrin releasing peptide-induced chemical itch signaling in the SDH. In this review, we describe recent advances in our understanding of SDH neuronal pathways for itch transmission, the mechanisms of SDH astrocytic activation and its contribution to abnormal itch processing and discuss the role of reactive astrocytes in the SDH in abnormal sensory processing under chronic itch conditions..
20. Keisuke Koga, Yuto Shiraishi, Ryo Yamagata, Hidetoshi Tozaki-Saitoh, Miho Shiratori-Hayashi, Makoto Tsuda, Intrinsic braking role of descending locus coeruleus noradrenergic neurons in acute and chronic itch in mice., Molecular brain, 10.1186/s13041-020-00688-0, 13, 1, 144-144, 2020.10, Itch is defined as an unpleasant sensation that provokes a desire to scratch. Our understanding of neuronal circuits for itch information transmission and processing in the spinal dorsal horn (SDH) has progressively advanced following the identification of SDH neuron subsets that are crucial for scratching behavior in models of itch. However, little is known about the control of acute and chronic itch by descending signals from the brain to the SDH. In this study, using genetic approaches that enable cell-type and circuit-specific functional manipulation, we reveal an intrinsic potential of locus coeruleus (LC)-noradrenergic (NAergic) neurons that project to the SDH to control acute and chronic itch. Activation and silencing of SDH-projecting LC-NAergic neurons reduced and enhanced scratching behavior, respectively, in models of histamine-dependent and -independent acute itch. Furthermore, in a model of chronic itch associated with contact dermatitis, repetitive scratching behavior was suppressed by the activation of the descending LC-NAergic pathway and by knocking out NA transporters specific to descending LC-NAergic neurons using a CRISPR-Cas9 system. Moreover, patch-clamp recording using spinal slices showed that noradrenaline facilitated inhibitory synaptic inputs onto gastrin-releasing peptide receptor-expressing SDH neurons, a neuronal subset known to be essential for itch transmission. Our findings suggest that descending LC-NAergic signaling intrinsically controls acute and chronic itch and provide potential therapeutic strategies for the treatment of acute and chronic itch..
21. Kazuhide Inoue, Makoto Tsuda, Nociceptive signaling mediated by P2X3, P2X4 and P2X7 receptors., Biochemical pharmacology, 10.1016/j.bcp.2020.114309, 114309-114309, 2020.10, Chronic pain is a debilitating condition that often occurs following peripheral tissue inflammation and nerve injury. This pain, especially neuropathic pain, is a significant clinical problem because of the ineffectiveness of clinically available drugs. Since Burnstock proposed new roles of nucleotides as neurotransmitters, the roles of extracellular ATP and P2 receptors (P2Rs) in pain signaling have been extensively studied, and ATP-P2R signaling has subsequently received much attention as it can provide clues toward elucidating the mechanisms underlying chronic pain and serve as a potential therapeutic target. This review summarizes the literature regarding the role of ATP signaling via P2X3Rs (as well as P2X2/3Rs) in primary afferent neurons and via P2X4Rs and P2X7Rs in spinal cord microglia in chronic pain, and discusses their respective therapeutic potentials..
22. Hiroyuki Konishi, Takayuki Okamoto, Yuichiro Hara, Okiru Komine, Hiromi Tamada, Mitsuyo Maeda, Fumika Osako, Masaaki Kobayashi, Akira Nishiyama, Yosky Kataoka, Toshiyuki Takai, Nobuyuki Udagawa, Steffen Jung, Keiko Ozato, Tomohiko Tamura, Makoto Tsuda, Koji Yamanaka, Tomoo Ogi, Katsuaki Sato, Hiroshi Kiyama, Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction., The EMBO journal, 10.15252/embj.2020104464, e104464, 2020.09, Microglia are the principal phagocytes that clear cell debris in the central nervous system (CNS). This raises the question, which cells remove cell debris when microglial phagocytic activity is impaired. We addressed this question using Siglechdtr mice, which enable highly specific ablation of microglia. Non-microglial mononuclear phagocytes, such as CNS-associated macrophages and circulating inflammatory monocytes, did not clear microglial debris. Instead, astrocytes were activated, exhibited a pro-inflammatory gene expression profile, and extended their processes to engulf microglial debris. This astrocytic phagocytosis was also observed in Irf8-deficient mice, in which microglia were present but dysfunctional. RNA-seq demonstrated that even in a healthy CNS, astrocytes express TAM phagocytic receptors, which were the main astrocytic phagocytic receptors for cell debris in the above experiments, indicating that astrocytes stand by in case of microglial impairment. This compensatory mechanism may be important for the maintenance or prolongation of a healthy CNS..
23. Toshiaki Teratani, Yohei Mikami, Nobuhiro Nakamoto, Takahiro Suzuki, Yosuke Harada, Koji Okabayashi, Yuya Hagihara, Nobuhito Taniki, Keita Kohno, Shinsuke Shibata, Kentaro Miyamoto, Harumichi Ishigame, Po-Sung Chu, Tomohisa Sujino, Wataru Suda, Masahira Hattori, Minoru Matsui, Takaharu Okada, Hideyuki Okano, Masayuki Inoue, Toshihiko Yada, Yuko Kitagawa, Akihiko Yoshimura, Mamoru Tanida, Makoto Tsuda, Yusaku Iwasaki, Takanori Kanai, The liver-brain-gut neural arc maintains the Treg cell niche in the gut., Nature, 10.1038/s41586-020-2425-3, 585, 7826, 591-596, 2020.09, Recent clinical and experimental evidence has evoked the concept of the gut-brain axis to explain mutual interactions between the central nervous system and gut microbiota that are closely associated with the bidirectional effects of inflammatory bowel disease and central nervous system disorders1-4. Despite recent advances in our understanding of neuroimmune interactions, it remains unclear how the gut and brain communicate to maintain gut immune homeostasis, including in the induction and maintenance of peripheral regulatory T cells (pTreg cells), and what environmental cues prompt the host to protect itself from development of inflammatory bowel diseases. Here we report a liver-brain-gut neural arc that ensures the proper differentiation and maintenance of pTreg cells in the gut. The hepatic vagal sensory afferent nerves are responsible for indirectly sensing the gut microenvironment and relaying the sensory inputs to the nucleus tractus solitarius of the brainstem, and ultimately to the vagal parasympathetic nerves and enteric neurons. Surgical and chemical perturbation of the vagal sensory afferents at the hepatic afferent level reduced the abundance of colonic pTreg cells; this was attributed to decreased aldehyde dehydrogenase (ALDH) expression and retinoic acid synthesis by intestinal antigen-presenting cells. Activation of muscarinic acetylcholine receptors directly induced ALDH gene expression in both human and mouse colonic antigen-presenting cells, whereas genetic ablation of these receptors abolished the stimulation of antigen-presenting cells in vitro. Disruption of left vagal sensory afferents from the liver to the brainstem in mouse models of colitis reduced the colonic pTreg cell pool, resulting in increased susceptibility to colitis. These results demonstrate that the novel vago-vagal liver-brain-gut reflex arc controls the number of pTreg cells and maintains gut homeostasis. Intervention in this autonomic feedback feedforward system could help in the development of therapeutic strategies to treat or prevent immunological disorders of the gut..
24. Miho Shiratori-Hayashi, Chiharu Yamaguchi, Kazushi Eguchi, Yuto Shiraishi, Keita Kohno, Katsuhiko Mikoshiba, Kazuhide Inoue, Motohiro Nishida, Makoto Tsuda, Astrocytic STAT3 activation and chronic itch require IP3R1/TRPC-dependent Ca2+ signals in mice., The Journal of allergy and clinical immunology, 10.1016/j.jaci.2020.06.039, 2020.08, BACKGROUND: Chronic itch is a debilitating symptom of inflammatory skin diseases, but the underlying mechanism is poorly understood. We have recently demonstrated that astrocytes in the spinal dorsal horn become reactive in models of atopic and contact dermatitis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and critically contribute to chronic itch. In general, STAT3 is transiently activated; however, STAT3 activation in reactive astrocytes of chronic itch model mice persistently occurs via an unknown mechanism. OBJECTIVE: We aimed to determine the mechanisms of persistent activation of astrocytic STAT3 in chronic itch conditions. METHODS: To determine the factors that are required for persistent activation of astrocytic STAT3, Western blotting and calcium imaging with cultured astrocytes or spinal cord slices were performed. Thereafter, chronic itch model mice were used for genetic and behavioral experiments to confirm the role of the factors determined to mediate persistent STAT3 activation from in vitro and ex vivo experiments in chronic itch. RESULTS: IP3 receptor type 1 (IP3R1) knockdown in astrocytes suppressed IL-6-induced persistent STAT3 activation and expression of lipocalin-2 (LCN2), an astrocytic STAT3-dependent inflammatory factor that is required for chronic itch. IP3R1-dependent astrocytic Ca2+ responses involved Ca2+ influx through the cation channel transient receptor potential canonical (TRPC), which was required for persistent STAT3 activation evoked by IL-6. IL-6 expression was upregulated in dorsal root ganglion neurons in a mouse model of chronic itch. Dorsal root ganglion neuron-specific IL-6 knockdown, spinal astrocyte-specific IP3R1 knockdown, and pharmacologic spinal TRPC inhibition attenuated LCN2 expression and chronic itch. CONCLUSION: Our findings suggest that IP3R1/TRPC channel-mediated Ca2+ signals elicited by IL-6 in astrocytes are necessary for persistent STAT3 activation, LCN2 expression, and chronic itch, and they may also provide new targets for therapeutic intervention..
25. Kakeru Shimoda, Akiyuki Nishimura, Caroline Sunggip, Tomoya Ito, Kazuhiro Nishiyama, Yuri Kato, Tomohiro Tanaka, Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Motohiro Nishida, Modulation of P2Y6R expression exacerbates pressure overload-induced cardiac remodeling in mice., Scientific reports, 10.1038/s41598-020-70956-5, 10, 1, 13926-13926, 2020.08, Cardiac tissue remodeling caused by hemodynamic overload is a major clinical outcome of heart failure. Uridine-responsive purinergic P2Y6 receptor (P2Y6R) contributes to the progression of cardiovascular remodeling in rodents, but it is not known whether inhibition of P2Y6R prevents or promotes heart failure. We demonstrate that inhibition of P2Y6R promotes pressure overload-induced sudden death and heart failure in mice. In neonatal cardiomyocytes, knockdown of P2Y6R significantly attenuated hypertrophic growth and cell death caused by hypotonic stimulation, indicating the involvement of P2Y6R in mechanical stress-induced myocardial dysfunction. Unexpectedly, compared with wild-type mice, deletion of P2Y6R promoted pressure overload-induced sudden death, as well as cardiac remodeling and dysfunction. Mice with cardiomyocyte-specific overexpression of P2Y6R also exhibited cardiac dysfunction and severe fibrosis. In contrast, P2Y6R deletion had little impact on oxidative stress-mediated cardiac dysfunction induced by doxorubicin treatment. These findings provide overwhelming evidence that systemic inhibition of P2Y6R exacerbates pressure overload-induced heart failure in mice, although P2Y6R in cardiomyocytes contributes to the progression of cardiac fibrosis..
26. Yusuke Ohmichi, Mika Ohmichi, Ryoichi Tashima, Koji Osuka, Kaori Fukushige, Dominika Kanikowska, Yugo Fukazawa, Hiromu Yawo, Makoto Tsuda, Munekazu Naito, Takashi Nakano, Physical disuse contributes to widespread chronic mechanical hyperalgesia, tactile allodynia, and cold allodynia through neurogenic inflammation and spino-parabrachio-amygdaloid pathway activation., Pain, 10.1097/j.pain.0000000000001867, 161, 8, 1808-1823, 2020.08, Physical disuse could lead to a state of chronic pain typified by complex regional pain syndrome type I due to fear of pain through movement (kinesiophobia) or inappropriate resting procedures. However, the mechanisms by which physical disuse is associated with acute/chronic pain and other pathological signs remain unresolved. We have previously reported that inflammatory signs, contractures, disuse muscle atrophy, spontaneous pain-like behaviors, and chronic widespread mechanical hyperalgesia based on central plasticity occurred after 2 weeks of cast immobilization in chronic post-cast pain (CPCP) rat model. In this study, we also demonstrated dystrophy-like changes, both peripheral nociceptive signals and activation of the central pain pathway in CPCP rats. This was done by the following methods: (1) vascular permeability (Evans blue dye) and inflammatory- and oxidative stress-related messenger RNA changes (real-time quantitative polymerase chain reaction); (2) immunofluorescence of pERK and/or c-Fos expression in the spino-parabrachio-amygdaloid pathway; and (3) blockade of nociceptive-related signals using sciatic nerve block. Furthermore, we demonstrated tactile allodynia using an optogenetic method in a transgenic rat line (W-TChR2V4), cold allodynia using the acetone test, and activation of dorsal horn neurons in the chronic phase associated with chronic mechanical hyperalgesia using c-Fos immunofluorescence. In addition, we showed that nociceptive signals in the acute phase are involved in chronic pathological pain-like behaviors by studying the effects of sciatic nerve block. Thus, we conclude that physical disuse contributes to dystrophy-like changes, spontaneous pain-like behavior, and chronic widespread pathological pain-like behaviors in CPCP rats after 2 weeks of cast immobilization..
27. Teruaki Ono, Yuta Kohro, Keita Kohno, Hidetoshi Tozaki-Saitoh, Yasuharu Nakashima, Makoto Tsuda, Mechanical pain of the lower extremity after compression of the upper spinal cord involves signal transducer and activator of transcription 3-dependent reactive astrocytes and interleukin-6., Brain, behavior, and immunity, 10.1016/j.bbi.2020.07.025, 2020.07, Chronic pain is one of the main symptoms of spinal disorders such as spinal canal stenosis. A major cause of this pain is related to compression of the spinal cord, and chronic pain can develop at the level of the compressed spinal segment. However, in many patients chronic pain arises in an area that does not correspond to the compressed segment, and the underlying mechanism involved remains unknown. This was investigated in the present study using a mouse model of spinal cord compression in which mechanical pain of the hindpaws develops after compression of the first lumbar segment (L1) of the spinal cord. Compression induced the activation of astrocytes in the L1 spinal dorsal horn (SDH)-but not the L4 SDH that corresponds to the hindpaws-and activated signal transducer and activator of transcription 3 (STAT3). Suppressing reactive astrocytes by expressing a dominant negative form of STAT3 (dnSTAT3) in the compressed SDH prevented mechanical pain. Expression of interleukin (IL)-6 was also upregulated in the compressed SDH, and it was inhibited by astrocytic expression of dnSTAT3. Intrathecal administration of a neutralizing anti-IL-6 antibody reversed the compression-induced mechanical pain. These results suggest that astrocytic STAT3 and IL-6 in the compressed SDH are involved in remote mechanical pain observed in the lower extremity, and may provide a target for treating chronic pain associated with spinal cord compression such as spinal canal stenosis..
28. Keisuke Koga, Ryo Yamagata, Keita Kohno, Takuya Yamane, Miho Shiratori-Hayashi, Yuta Kohro, Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Sensitization of spinal itch transmission neurons in a mouse model of chronic itch requires an astrocytic factor, Journal of Allergy and Clinical Immunology, 10.1016/j.jaci.2019.09.034, 145, 1, 183-191.e10, 2020.01, Background: Chronic itch is a highly debilitating symptom among patients with inflammatory skin diseases. Recent studies have revealed that gastrin-releasing peptide (GRP) and its receptor (gastrin-releasing peptide receptor [GRPR]) in the spinal dorsal horn (SDH) play a central role in itch transmission. Objective: We aimed to investigate whether GRP-GRPR signaling is altered in SDH neurons in a mouse model of chronic itch and to determine the potential mechanisms underlying these alterations. Methods: Patch-clamp recordings from enhanced green fluorescent protein (EGFP)–expressing (GRPR+) SDH neurons were used to examine GRP-GRPR signaling in spinal cord slices obtained from Grpr-EGFP mice. Immunohistochemical, genetic (gene expression and editing through adeno-associated virus vectors), and behavioral approaches were also used for in vivo experiments. Results: We observed potentiation of GRP-evoked excitation in the GRPR+ SDH neurons of mice with contact dermatitis, without concomitant changes in GRPR expression. Interestingly, increases in excitation were attenuated by suppressing the reactive state of SDH astrocytes, which are known to be reactive in patients with chronic itch conditions. Furthermore, CRISPR-Cas9–mediated astrocyte-selective in vivo editing of a gene encoding lipocalin-2 (LCN2), an astrocytic factor implicated in chronic itch, suppressed increases in GRP-induced excitation of GRPR+ neurons, repetitive scratching, and skin damage in mice with contact dermatitis. Moreover, LCN2 potentiated GRP-induced excitation of GRPR+ neurons in normal mice. Conclusion: Our findings indicate that, under chronic itch conditions, the GRP-induced excitability of GRPR+ SDH neurons is enhanced through a non–cell-autonomous mechanism involving LCN2 derived from reactive astrocytes..
29. Keisuke Koga, Ryo Yamagata, Keita Kohno, Takuya Yamane, Miho Shiratori-Hayashi, Yuta Kohro, Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Sensitization of spinal itch transmission neurons in a mouse model of chronic itch requires an astrocytic factor., The Journal of allergy and clinical immunology, 10.1016/j.jaci.2019.09.034, 145, 1, 183-191, 2020.01, BACKGROUND: Chronic itch is a highly debilitating symptom among patients with inflammatory skin diseases. Recent studies have revealed that gastrin-releasing peptide (GRP) and its receptor (gastrin-releasing peptide receptor [GRPR]) in the spinal dorsal horn (SDH) play a central role in itch transmission. OBJECTIVE: We aimed to investigate whether GRP-GRPR signaling is altered in SDH neurons in a mouse model of chronic itch and to determine the potential mechanisms underlying these alterations. METHODS: Patch-clamp recordings from enhanced green fluorescent protein (EGFP)-expressing (GRPR+) SDH neurons were used to examine GRP-GRPR signaling in spinal cord slices obtained from Grpr-EGFP mice. Immunohistochemical, genetic (gene expression and editing through adeno-associated virus vectors), and behavioral approaches were also used for in vivo experiments. RESULTS: We observed potentiation of GRP-evoked excitation in the GRPR+ SDH neurons of mice with contact dermatitis, without concomitant changes in GRPR expression. Interestingly, increases in excitation were attenuated by suppressing the reactive state of SDH astrocytes, which are known to be reactive in patients with chronic itch conditions. Furthermore, CRISPR-Cas9-mediated astrocyte-selective in vivo editing of a gene encoding lipocalin-2 (LCN2), an astrocytic factor implicated in chronic itch, suppressed increases in GRP-induced excitation of GRPR+ neurons, repetitive scratching, and skin damage in mice with contact dermatitis. Moreover, LCN2 potentiated GRP-induced excitation of GRPR+ neurons in normal mice. CONCLUSION: Our findings indicate that, under chronic itch conditions, the GRP-induced excitability of GRPR+ SDH neurons is enhanced through a non-cell-autonomous mechanism involving LCN2 derived from reactive astrocytes..
30. Shota Yamamoto, Tomohiro Yamashita, Mayu Ito, Jose M.M. Caaveiro, Nobuaki Egashira, Hidetoshi Tozaki-Saitoh, Makoto Tsuda, New pharmacological effect of fulvestrant to prevent oxaliplatin-induced neurodegeneration and mechanical allodynia in rats, International Journal of Cancer, 10.1002/ijc.32043, 145, 8, 2107-2113, 2019.10, Oxaliplatin, which is widely used as chemotherapy for certain solid cancers, frequently causes peripheral neuropathy. Commonly described neuropathic symptoms include aberrant sensations such as mechanical allodynia (hypersensitivity to normally innocuous stimuli). Although oxaliplatin neuropathy is a dose-limiting toxicity, there are no established preventive strategies available at present. By screening several sets of small-molecule chemical libraries (more than 3,000 compounds in total) using a newly established in vitro high-throughput phenotypic assay, we identified fulvestrant, a clinically approved drug for the treatment of breast cancer in postmenopausal women, as having a protective effect on oxaliplatin-induced neuronal damage. Furthermore, histological and behavioural analyses using a rat model of oxaliplatin neuropathy demonstrated the in vivo efficacy of fulvestrant to prevent oxaliplatin-induced axonal degeneration of the sciatic nerve and mechanical allodynia. Furthermore, fulvestrant did not interfere with oxaliplatin-induced cytotoxicity against cancer cells. Thus, our findings reveal a previously unrecognised pharmacological effect of fulvestrant to prevent oxaliplatin-induced painful peripheral neuropathy without impairing its cytotoxicity against cancer cells and may represent a novel prophylactic option for patients receiving oxaliplatin chemotherapy..
31. Kazu Kobayakawa, Yasuyuki Ohkawa, Shingo Yoshizaki, Tetsuya Tamaru, Takeyuki Saito, Ken Kijima, Kazuya Yokota, Masamitsu Hara, Kensuke Kubota, Yoshihiro Matsumoto, Katsumi Harimaya, Keiko Ozato, Takahiro Masuda, Makoto Tsuda, Tomohiko Tamura, Kazuhide Inoue, V. Reggie Edgerton, Yukihide Iwamoto, Yasuharu Nakashima, Seiji Okada, Macrophage centripetal migration drives spontaneous healing process after spinal cord injury, Science Advances, 10.1126/sciadv.aav5086, 5, 5, 2019.05, Traumatic spinal cord injury (SCI) brings numerous inflammatory cells, including macrophages, from the circulating blood to lesions, but pathophysiological impact resulting from spatiotemporal dynamics of macrophages is unknown. Here, we show that macrophages centripetally migrate toward the lesion epicenter after infiltrating into the wide range of spinal cord, depending on the gradient of chemoattractant C5a. However, macrophages lacking interferon regulatory factor 8 (IRF8) cannot migrate toward the epicenter and remain widely scattered in the injured cord with profound axonal loss and little remyelination, resulting in a poor functional outcome after SCI. Time-lapse imaging and P2X/YRs blockade revealed that macrophage migration via IRF8 was caused by purinergic receptors involved in the C5a-directed migration. Conversely, pharmacological promotion of IRF8 activation facilitated macrophage centripetal movement, thereby improving the SCI recovery. Our findings reveal the importance of macrophage centripetal migration via IRF8, providing a novel therapeutic target for central nervous system injury..
32. Hidetoshi Saitoh, Junya Masuda, Ryu Kawada, Chinami Kojima, Sosuke Yoneda, Takahiro Masuda, Kazuhide Inoue, Tsuda Makoto, Transcription factor MafB contributes to the activation of spinal microglia underlying neuropathic pain development, GLIA, 10.1002/glia.23570, 67, 4, 729-740, 2019.04, Microglia, which are pathological effectors and amplifiers in the central nervous system, undergo various forms of activation. A well-studied microglial-induced pathological paradigm, spinal microglial activation following peripheral nerve injury (PNI), is a key event for the development of neuropathic pain but the transcription factors contributing to microglial activation are less understood. Herein, we demonstrate that MafB, a dominant transcriptional regulator of mature microglia, is involved in the pathology of a mouse model of neuropathic pain. PNI caused a rapid and marked increase of MafB expression selectively in spinal microglia but not in neurons. We also found that the microRNA mir-152 in the spinal cord which targets MafB expression decreased after PNI, and intrathecal administration of mir-152 mimic suppressed the development of neuropathic pain. Reduced MafB expression using heterozygous Mafb deficient mice and by intrathecal administration of siRNA alleviated the development of PNI-induced mechanical hypersensitivity. Furthermore, we found that intrathecal transfer of Mafb deficient microglia did not induce mechanical hypersensitivity and that conditional Mafb knockout mice did not develop neuropathic pain after PNI. We propose that MafB is a key mediator of the PNI-induced phenotypic alteration of spinal microglia and neuropathic pain development..
33. Masaya Yasui, Yuki Menjyo, Kyohei Tokizane, Akiko Shiozawa, Tsuda Makoto, Kazuhide Inoue, Hiroshi Kiyama, Hyperactivation of proprioceptors induces microglia-mediated long-lasting pain in a rat model of chronic fatigue syndrome, Journal of Neuroinflammation, 10.1186/s12974-019-1456-x, 16, 1, 2019.03, Background: Patients diagnosed with chronic fatigue syndrome (CFS) or fibromyalgia experience chronic pain. Concomitantly, the rat model of CFS exhibits microglial activation in the lumbar spinal cord and pain behavior without peripheral tissue damage and/or inflammation. The present study addressed the mechanism underlying the association between pain and chronic stress using this rat model. Methods: Chronic or continuous stress-loading (CS) model rats, housed in a cage with a thin level of water (1.5 cm in depth), were used. The von Frey test and pressure pain test were employed to measure pain behavior. The neuronal and microglial activations were immunohistochemically demonstrated with antibodies against ATF3 and Iba1. Electromyography was used to evaluate muscle activity. Results: The expression of ATF3, a marker of neuronal hyperactivity or injury, was first observed in the lumbar dorsal root ganglion (DRG) neurons 2 days after CS initiation. More than 50% of ATF3-positive neurons simultaneously expressed the proprioceptor markers TrkC or VGluT1, whereas the co-expression rates for TrkA, TrkB, IB4, and CGRP were lower than 20%. Retrograde labeling using fluorogold showed that ATF3-positive proprioceptive DRG neurons mainly projected to the soleus. Substantial microglial accumulation was observed in the medial part of the dorsal horn on the fifth CS day. Microglial accumulation was observed around a subset of motor neurons in the dorsal part of the ventral horn on the sixth CS day. The motor neurons surrounded by microglia were ATF3-positive and mainly projected to the soleus. Electromyographic activity in the soleus was two to three times higher in the CS group than in the control group. These results suggest that chronic proprioceptor activation induces the sequential activation of neurons along the spinal reflex arc, and the neuronal activation further activates microglia along the arc. Proprioceptor suppression by ankle joint immobilization significantly suppressed the accumulation of microglia in the spinal cord, as well as the pain behavior. Conclusion: Our results indicate that proprioceptor-induced microglial activation may be a key player in the initiation and maintenance of abnormal pain in patients with CFS..
34. Tatsuhiro Igawa, Shuhei Kishikawa, Yoshito Abe, Tomohiro Yamashita, Saki Nagai, Mitsunori Shiroishi, Chinatsu Shinozaki, Hiroyuki Tanaka, Hidetoshi Saitoh, Tsuda Makoto, Kazuhide Inoue, Tadashi Ueda, Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure, Purinergic Signalling, 10.1007/s11302-019-09646-5, 2019.03, P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4’s head domain (rHD, Gln111–Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells..
35. Shota Yamamoto, Nobuaki Egashira, Tsuda Makoto, Satohiro Masuda, Riluzole prevents oxaliplatin-induced cold allodynia via inhibition of overexpression of transient receptor potential melastatin 8 in rats, Journal of Pharmacological Sciences, 10.1016/j.jphs.2018.10.006, 138, 3, 214-217, 2018.11, Oxaliplatin causes acute cold hypersensitivity in most patients. We previously reported oxalate derived from oxaliplatin induced cold allodynia via overexpression of transient receptor potential melastatin 8 (TRPM8) in the dorsal root ganglion (DRG) in rats. In this study, we examined the effect of riluzole on oxaliplatin-induced cold allodynia. In cultured DRG neurons, riluzole suppressed oxalate-induced increase of the number of menthol (TRPM8 agonist)-sensitive cells. Moreover, riluzole prevented cold allodynia and increase in levels of TRPM8 mRNA in oxaliplatin-treated rats. These results suggest that riluzole prevents oxaliplatin-induced cold allodynia via inhibition of TRPM8 overexpression in the DRG..
36. Tao Chen, Wataru Taniguchi, Qi Yu Chen, Hidetoshi Saitoh, Qian Song, Ren Hao Liu, Kohei Koga, Tsuyoshi Matsuda, Yae Kaito-Sugimura, Jian Wang, Zhi Hua Li, Ya Cheng Lu, Kazuhide Inoue, Tsuda Makoto, Yun Qing Li, Terumasa Nakatsuka, Min Zhuo, Top-down descending facilitation of spinal sensory excitatory transmission from the anterior cingulate cortex, Nature Communications, 10.1038/s41467-018-04309-2, 9, 1, 2018.05, Spinal sensory transmission is under descending biphasic modulation, and descending facilitation is believed to contribute to chronic pain. Descending modulation from the brainstem rostral ventromedial medulla (RVM) has been the most studied, whereas little is known about direct corticospinal modulation. Here, we found that stimulation in the anterior cingulate cortex (ACC) potentiated spinal excitatory synaptic transmission and this modulation is independent of the RVM. Peripheral nerve injury enhanced the spinal synaptic transmission and occluded the ACC-spinal cord facilitation. Inhibition of ACC reduced the enhanced spinal synaptic transmission caused by nerve injury. Finally, using optogenetics, we showed that selective activation of ACC-spinal cord projecting neurons caused behavioral pain sensitization, while inhibiting the projection induced analgesic effects. Our results provide strong evidence that ACC stimulation facilitates spinal sensory excitatory transmission by a RVM-independent manner, and that such top-down facilitation may contribute to the process of chronic neuropathic pain..
37. Tashima R, Koga K, Sekine M, Kanehisa K, Kohro Y, Tominaga K, Matsushita K, Tozaki-Saitoh H, Fukazawa Y, Inoue K, Yawo H, Furue H, Tsuda M, Optogenetic activation of non-nociceptive Aβ fibers induces neuropathic pain-like sensory and emotional behaviors after nerve injury in rats., eNeuro, 10.1523/ENEURO.0450-17.2018, 5, ENEURO.0450-17.2018, 2018.02.
38. Kohei Yoshihara, Tsuyoshi Matsuda, Yuta Koro, Hidetoshi Saitoh, Kazuhide Inoue, Tsuda Makoto, Astrocytic Ca2+ responses in the spinal dorsal horn by noxious stimuli to the skin, Journal of Pharmacological Sciences, 10.1016/j.jphs.2018.04.007, 2018.01, The role of astrocytes in the spinal dorsal horn (SDH) for sensory information processing under normal conditions is poorly understood. In this study, we investigated whether SDH astrocytes respond to noxious and innocuous stimuli to the skin of normal mice using in vivo two-photon Ca2+ imaging under anesthesia. We found that noxious stimulation evoked by intraplantar formalin injection provoked an elevation in intracellular Ca2+ levels in SDH astrocytes. By contrast, neither instantaneous noxious pinching nor innocuous stimuli (cooling or brushing) to the hindpaw elicited astrocytic Ca2+ responses. Thus, SDH astrocytes could respond preferentially to a strong and/or sustained noxious stimulus..
39. Kensho Kanehisa, Miho Shiratori, Keisuke Koga, Hidetoshi Saitoh, Yuta Koro, Kenji Takamori, Tsuda Makoto, Specific activation of inhibitory interneurons in the spinal dorsal horn suppresses repetitive scratching in mouse models of chronic itch, Journal of Dermatological Science, 10.1016/j.jdermsci.2017.05.017, 88, 2, 251-254, 2017.11.
40. Ohgidani M, Kato TA, Hosoi M, Tsuda M, Hayakawa K, Hayaki C, Iwaki R, Sagata N, Hashimoto R, Inoue K, Sudo N, Kanba S, Fibromyalgia and microglial TNF-α: Translational research using human blood induced microglia-like cells., Sci Rep, 10.1038/s41598-017-11506-4, 7, 11882, 2017.09.
41. Koga K, Kanehisa K, Kohro Y, Shiratori-Hayashi M, Tozaki-Saitoh H, Inoue K, Furue H, Tsuda M, Chemogenetic silencing of GABAergic dorsal horn interneurons induces morphine-resistant spontaneous nocifensive behaviours., Sci Rep, 10.1038/s41598-017-04972-3, 7, 4739, 2017.07.
42. Hidetoshi Saitoh et al., P2Y12 receptors in primary microglia activate nuclear factor of activated T cell signaling to induce C-C chemokine 3 expression, J Neurochem, 141, 100-110, 2017.04.
43. Nakaya Michio et al., Cardiac myofibroblast engulfment of dead cells facilitates recovery after myocardial infarction, J Clin Invest, 127, 383-401 , 2017.01.
44. Matsunaga Naoya et al., Inhibition of G0/G1 Switch 2 Ameliorates Renal Inflammation in Chronic Kidney Disease, EBioMedicine , S2352-3964, 30463-30467 , 2016.11.
45. Koyanagi S et al., Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia, Nat Commun, 7, 13102 , 2016.11.
46. Tomohiro Yamashita et al., Duloxetine inhibits microglial P2X4 receptor function and alleviates neuropathic pain after peripheral nerve injury, PLoS One, 11, e0165189 , 2016.10.
47. Matsumura Y et al., A novel P2X4 receptor-selective antagonist produces anti-allodynic effect in a mouse model of herpetic pain, Sci Rep, 6, 32461, 2016.08.
48. Masuda T et al., Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain, Nat Commun, 7, 12529, 2016.08.
49. Tashima R et al., Bone marrow-derived cells in the population of spinal microglia after peripheral nerve injury, Sci Rep, 6, 23701, 2016.03.
50. Nishimura et al., Purinergic P2Y6 receptors heterodimerize with angiotensin AT1 receptors to promote angiotensin II-induced hypertension, Sci Signal, 9, ra7, 2016.01.
51. Yuta Kohro et al., A new minimally-invasive method for microinjection into the mouse spinal dorsal horn, Sci Rep, 5, 14306, 2015.09.
52. Miho Shiatori-Hayashi et al., STAT3-dependent reactive astrogliosis in the spinal dorsal horn underlies chronic itch, NATURE MEDICINE, 10.1038/nm.3912, 21, 8, 927-931, 2015.08, Chronic itch is an intractable symptom of inflammatory skin diseases, such as atopic and contact dermatitis. Recent studies have revealed neuronal pathways selective for itch, but the mechanisms by which itch turns into a pathological chronic state are poorly understood. Using mouse models of atopic and contact dermatitis, we demonstrate a long-term reactive state of astrocytes in the dorsal horn of the spinal segments that corresponds to lesioned, itchy skin. We found that reactive astrogliosis depended on the activation of signal transducer and activator of transcription 3 (STAT3). Conditional disruption of astrocytic STAT3 suppressed chronic itch, and pharmacological inhibition of spinal STAT3 ameliorated the fully developed chronic itch. Mice with atopic dermatitis exhibited an increase in scratching elicited by intrathecal administration of the itch-inducer gastrin-releasing peptide (GRP), and this enhancement was normalized by suppressing STAT3-mediated reactive astrogliosis. Moreover, we identified lipocalin-2 (LCN2) as an astrocytic STAT3-dependent upregulated factor that was crucial for chronic itch, and we demonstrated that intrathecal administration of LCN2 to normal mice increased spinal GRP-evoked scratching. Our findings indicate that STAT3-dependent reactive astrocytes act as critical amplifiers of itching through a mechanism involving the enhancement of spinal itch signals by LCN2, thereby providing a previously unrecognized target for treating chronic itch..
53. Takahiro Masuda et al., Transcription factor IRF1 is responsible for IRF8-mediated IL-1β expression in reactive microglia, J Pharmacol Sci, 128, 4, 216-220, 2015.08.
54. Igawa T et al., Solution structure of the rat P2X4 receptor head domain involved in inhibitory metal binding.
, FEBS Lett, 589, 680-686, 2015.03.
55. Akagi et al.,, Interferon regulatory factor 8 expressed in microglia contributes to tactile allodynia induced by repeated cold stress in rodents., J Pharmacol Sci, 121, 172-176, 2014.11.
56. Masuda Takahiro et al., IRF8 is a transcriptional determinant for microglial motility., Purinergic Signal, 10, 3, 515-521, 2014.09.
57. Yasui et al.,, A chronic fatigue syndrome model demonstrates mechanical allodynia and muscular hyperalgesia via spinal microglial activation., Glia, 62, 9, 1407-1417, 2014.09.
58. Ohi-ishi et al.,, Involvement of the chemokine CCL3 and the purinoceptor P2X7 in the spinal cord in paclitaxel-induced mechanical allodynia., Mol Pain, 10, 53, 2014.08.
59. Matsushita K et al., Chemokine (C-C motif) receptor 5 is an important pathological regulator in the development and maintenance of neuropathic pain., Anesthesiology, 120, 6, 1491-1503, 2014.06.
60. Masuda Takahiro et al., Transcription factor IRF5 drives P2X4R+-reactive microglia gating neuropathic pain., Nat Commun, 5, 3771, 2014.05, 神経のダメージで発症する慢性的な痛み(神経障害性疼痛)の原因タンパク質として「IRF5」を突き止めました。IRF5は、神経の損傷後に脳・脊髄の免疫細胞であるミクログリアで増え、IRF5欠損マウスでは痛みが弱くなっていました。さらに、2003年にP2X4受容体タンパク質のミクログリアでの増加が神経障害性疼痛に重要であることを英国科学誌Natureで発表していますが、今回見つかったIRF5がP2X4受容体を増やす実行役であることも明らかにしました。この研究成果は、慢性疼痛メカニズムの解明へ向けた大きな前進となり、痛みを緩和する治療薬の開発に応用できることが期待されます(Nature Communications誌掲載)。.
61. Igawa T, Higashi S, yoshito abe, Takatoshi Ohkuri, Hiroyuki Tanaka, satoshi morimoto, 山下 智大, TSUDA MAKOTO, Kazuhide Inoue, Tadashi Ueda, Preparation and characterization of a monoclonal antibody against the refolded and functional extracellular domain of rat P2X4 receptor, JOURNAL OF BIOCHEMISTRY, 10.1093/jb/mvs143, 153, 3, 275-282, 2013.03.
62. Nakaya Michio, Tajima M, Kosako H, Nakaya T, Hashimoto A, Watari K, Nishihara H, Ohba M, Komiya S, Tani N, Motohiro Nishida, Taniguchi H, Sato Y, Matsumoto M, TSUDA MAKOTO, Kuroda M, Kazuhide Inoue, Hitoshi Kurose, GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance, NATURE COMMUNICATIONS, 10.1038/ncomms2540, 4, 1532, 2013.02.
63. Muratake H, Ito A, Toda T, Suzuki H, Fukasawa H, TSUDA MAKOTO, Kazuhide Inoue, Sugiyama K, Shudo K, (R)- and (S)-4-Amino-3-(trimethylsilyl)methylbutanoic acids ameliorate neuropathic pain without central nervous system-related side effects, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 10.1016/j.bmcl.2012.10.001, 22, 24, 7602-7604, 2012.12.
64. Sun L, Wu Z, Yoshinori Hayashi, Peters C, TSUDA MAKOTO, Kazuhide Inoue, Hiroshi Nakanishi, Microglial Cathepsin B Contributes to the Initiation of Peripheral Inflammation-Induced Chronic Pain, JOURNAL OF NEUROSCIENCE, 10.1523/JNEUROSCI.0677-12.2012, 32, 33, 11330-11342, 2012.08.
65. Uesugi A, Kataoka A, Tozaki-Saitoh H, Koga Y, Tsuda M, Robaye B, Boeynaems JM, Inoue K., Involvement of protein kinase D in uridine diphosphate-induced microglial macropinocytosis and phagocytosis., Glia, 60, 7, 1094-1105, 2012.07.
66. Toyomitsu E, Tsuda M, Yamashita T, Tozaki-Saitoh H, Tanaka Y, Inoue K., CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia., Purinergic Signal., 8, 2, 301-310, 2012.06.
67. Masuda Takahiro, TSUDA MAKOTO, Ryohei Yoshinaga, Hidetoshi Saitoh, Keiko Ozato, Tomohiko Tamura, Kazuhide Inoue, IRF8 Is a Critical Transcription Factor for Transforming Microglia into a Reactive Phenotype, CELL REPORTS, 10.1016/j.celrep.2012.02.014, 1, 4, 334-340, 2012.04, 神経のダメージで発症する慢性的な激しい痛み(神経障害性疼痛)の原因タンパク質として「IRF8」を突き止めました。神経の損傷後,IRF8は脳・脊髄の免疫細胞と呼ばれる「ミクログリア」だけで劇的に増えており,同細胞の過度な活性化状態をつくりだして激しい痛みを引き起こすことを明らかにしました。また,IRF8は中枢神経においてミクログリア特異的に発現する転写因子として世界初の例であり,他の神経疾患においてもミクログリアの活性化が報告されていることから,他の領域にも本成果が波及することが期待できる。この研究成果は,慢性疼痛メカニズムの解明へ向けた大きな前進となり,痛みを緩和する治療薬の開発に応用できることが期待されます(Cell Reports誌掲載)。.
68. Kataoka A, Koga Y, Uesugi A, Tozaki-Saitoh H, Tsuda M, Inoue K., Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways., Purinergic Signal., 7, 4, 403-411, 2011.12.
69. Kuboyama K, Harada H, Tozaki-Saitoh H, Tsuda M, Ushijima K, Inoue K, Astrocytic P2Y1 receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia., J Cereb Blood Flow Metab, 10.1038/jcbfm.2011.49., 31, 9, 1930-1941, 2011.09.
70. Biber K*, Tsuda M*, Tozaki-Saitoh H*, Tsukamoto K, Toyomitsu E, Masuda T, Boddeke H, Inoue K. (*equal contributors), Neuronal CCL21 up-regulates microglia P2X4 expression and initiates neuropathic pain development., EMBO J, 30, 9, 1864-1873, 2011.05.
71. Tsuda M, Kohro Y, Yano T, Tsujikawa T, Kitano J, Tozaki-Saitoh H, Koyanagi S, Ohdo S, Ji RR, Salter MW, Inoue K, JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats., Brain, 134, 4, 1127-1139, 2011.04.
72. Maeda M, Tsuda M, Tozaki-Saitoh H, Inoue K, Kiyama H, Nerve injury-activated microglia engulf myelinated axons in a P2Y12 signaling-dependent manner in the dorsal horn., Glia, 58, 15, 1838-1846, 2010.11.
73. Kusunose N, Koyanagi S, Hamamura K, Matsunaga N, Yoshida M, Uchida T, Tsuda M, Inoue K, Ohdo S, Molecular basis for the dosing time-dependency of anti-allodynic effects of gabapentin in a mouse model of neuropathic pain. , Mol Pain, 6, 83, 2010.11.
74. Shiratori M, Tozaki-Saitoh H, Yoshitake M, Tsuda M, Inoue K, P2X7 receptor activation induces CXCL2 production in microglia through NFAT and PKC/MAPK pathways., J Neurochem, 114, 3, 810-819, 2010.08.
75. Hasegawa S, Kohro Y, Shiratori M, Ishii S, Shimizu T, Tsuda M, Inoue K, Role of PAF receptor in proinflammatory cytokine expression in the dorsal root ganglion and tactile allodynia in a rodent model of neuropathic pain., PLoS ONE 5: e10467 (2010), 2010.05.
76. Tsuda M, Toyomitsu E, Kometani M, Tozaki-Saitoh H, Inoue K, Mechanisms underlying fibronectin-induced up-regulation of P2X4R expression in microglia: distinct roles of PI3K-Akt and MEK-ERK signalling pathways, J Cell Mol Med, 13, 9b, 3251-9, 2009.09.
77. Tsuda M, Kuboyama K, Inoue T, Nagata K, Tozaki-Saitoh H, Inoue K, Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays, Mol Pain, 5, 28, 2009.06.
78. Tsuda M, Masuda T, Kitano J, Shimoyama H, Tozaki-Saitoh H, Inoue K, IFN-gamma receptor signaling mediates spinal microglia activation driving neuropathic pain, Proc Natl Acad Sci U S A, 106, 19, 8032-8037, 2009.05.
79. Hasegawa S, Kohro Y, Tsuda M, Inoue K, Activation of cytosolic phospholipase A2 in dorsal root ganglion neurons by Ca2+/calmodulin-dependent protein kinase II after peripheral nerve injury, Mol Pain, 5, 22, 2009.05.
80. Shinozaki Y, Sumitomo K, Tsuda M, Koizumi S, Inoue K, Torimitsu K, Direct Observation of ATP-Induced Conformational Changes in Single P2X4 Receptors, PLoS Biol, 7, 5, e103, 2009.05.
81. Masuda J, Tsuda M, Tozaki-Saitoh H, Inoue K, Intrathecal delivery of PDGF produces tactile allodynia through its receptors in spinal microglia, Mol Pain, 5, 23, 2009.05.
82. Nagata K, Imai T, Yamashita T, Tsuda M, Tozaki-Saitoh H, Inoue K., Antidepressants inhibit P2X4 receptor function: a possible involvement in neuropathic pain relief, Mol Pain, 5, 20, 2009.04.
83. Kataoka A, Tozaki-Saitoh H, Koga Y, Tsuda M, Inoue K, Activation of P2X7 receptors induces CCL3 production in microglial cells through transcription factor NFAT. , J Neurochem 108(1): 115-125 (2009), 2009.01.
84. Tozaki-Saitoh H, Tsuda M, Miyata H, Ueda K, Kohsaka S, Inoue K, P2Y12 receptors in spinal microglia are required for neuropathic pain after peripheral nerve injury, J Neurosci 28: 4949-4956 (2008), 28: 4949-4956, 2008.05.
85. Tsuda M, Toyomitsu E, Komatsu T, Masuda T, Kunifusa E, Nasu-Tada K, Koizumi S, Yamamoto K, Ando J, Inoue K, Fibronectin/integrin system is involved in P2X(4) receptor upregulation in the spinal cord and neuropathic pain after nerve injury, Glia 56: 579-585 (2008), 2008.04.
86. Tsuda M, Ueno H, Kataoka A, Tozaki-Saitoh H, Inoue K, Activation of dorsal horn microglia contributes to diabetes-induced tactile allodynia via extracellular signal-regulated protein kinase signaling, Glia 56: 378-386 (2008), 2008.03.
87. Lyn tyrosine kinase is required for P2X4 receptor upregulation and neuropathic pain after peripheral nerve injury, Tsuda M, Tozaki-Saitoh H, Masuda T, Toyomitsu E, Tezuka T, Yamamoto T, Inoue K, Glia 56: 50-58 (2008), 2008.01.
88. Tsuda M, Hasegawa S, Inoue K, P2X receptors-mediated cytosolic phospholipase A2 activation in primary afferent sensory neurons contributes to neuropathic pain, J Neurochem 103: 1408-1416 (2007), 2007.11.
89. Tsuda M, Ishii S, Masuda T, Hasegawa S, Nakamura K, Nagata K, Yamashita T, Furue H, Tozaki-Saito H, Yoshimura M, Koizumi S, Shimizu T and Inoue K, Reduced pain behaviors and ERK activation in primary sensory neurons by peripheral tissue injury in mice lacking platelet-activating factor receptor, J Neurochem 102: 1658–1668 (2007), 2007.09.
90. Koizumi S, Shigemoto-Mogami Y, Nasu-Tada K, Shinozaki Y, Ohsawa K, Tsuda M, Joshi BV, Jacobson KA, Kohsaka S, Inoue K., UDP acting at P2Y(6) receptors is a mediator of microglial phagocytosis., Nature. 446: 1091-1095 (2007), 2007.04.
91. Ikeda H, Tsuda M, Inoue K, Murase K., Long-term potentiation of neuronal excitation by neuron-glia interactions in the rat spinal dorsal horn., Eur J Neurosci. 2007 Mar;25(5):1297-306., 2007.03.
92. Tozaki-Saitoh H, Koizumi S, Sato Y, Tsuda M, Nagao T, Inoue K., Retinoic acids increase P2X2 receptor expression through the 5'-flanking region of P2rx2 gene in rat phaeochromocytoma PC-12 cells., Mol Pharmacol. 2006 Jul;70(1):319-28. , 2006.07.
93. Shiokawa H, Nakatsuka T, Furue H, Tsuda M, Katafuchi T, Inoue K, Yoshimura M., Direct excitation of deep dorsal horn neurones in the rat spinal cord by the activation of postsynaptic P2X receptors., J Physiol. 2006 Jun 15;573(Pt 3):753-63., 2006.06.
94. Nasu-Tada K, Koizumi S, Tsuda M, Kunifusa E, Inoue K., Possible involvement of increase in spinal fibronectin following peripheral nerve injury in upregulation of microglial P2X4, a key molecule for mechanical allodynia., Glia. 2006 May;53(7):769-75., 2006.05.
95. Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y., BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain., Nature. 2005 Dec 15;438(7070):1017-21. , 10.1038/nature04223, 438, 7070, 1017-1021, 2005.12.
96. Narita M, Miyatake M, Shibasaki M, Tsuda M, Koizumi S, Narita M, Yajima Y, Inoue K, Suzuki T., Long-lasting change in brain dynamics induced by methamphetamine: enhancement of protein kinase C-dependent astrocytic response and behavioral sensitization., J Neurochem. 2005 Jun;93(6):1383-92., 2005.06.
97. Koizumi S, Fujishita K, Inoue K, Shigemoto-Mogami Y, Tsuda M, Inoue K., Ca2+ waves in keratinocytes are transmitted to sensory neurons: the involvement of extracellular ATP and P2Y2 receptor activation., Biochem J. 2004 Jun 1;380(Pt 2):329-38., 10.1042/BJ20031089, 380, 329-338, 2004.06.
98. Tsuda M, Mizokoshi A, Shigemoto-Mogami Y, Koizumi S, Inoue K., Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury., Glia. 2004 Jan 1;45(1):89-95., 10.1002/glia.10308, 45, 1, 89-95, 2004.01.
99. Inoue K, Koizumi S, Tsuda M, Shigemoto-Mogami Y., Signaling of ATP receptors in glia-neuron interaction and pain., Life Sci. 2003 Dec 5;74(2-3):189-97., 10.1016/j.lfs.2003.09.006, 74, 2-3, 189-197, 2003.12.
100. Koizumi S, Fujishita K, Tsuda M, Shigemoto-Mogami Y, Inoue K., Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures., Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):11023-8., 10.1073/pnas.1834448100, 100, 19, 11023-11028, 2003.09.
101. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K. Related Articles, Links , P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury., Nature. 2003 Aug 14;424(6950):778-83., 10.1038/nature01786, 424, 6950, 778-783, 2003.08.
102. Tsuda M, Shigemoto-Mogami Y, Ueno S, Koizumi S, Ueda H, Iwanaga T, Inoue K., Downregulation of P2X3 receptor-dependent sensory functions in A/J inbred mouse strain., Eur J Neurosci. 2002 May;15(9):1444-50., 10.1046/j.1460-9568.2002.01982.x, 15, 9, 1444-1450, 2002.05.
103. Shigemoto-Mogami Y, Koizumi S, Tsuda M, Ohsawa K, Kohsaka S, Inoue K., Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5., J Neurochem. 2001 Sep;78(6):1339-49., 10.1046/j.1471-4159.2001.00514.x, 78, 6, 1339-1349, 2001.09.
104. Tsuda M, Koizumi S, Inoue K., Role of endogenous ATP at the incision area in a rat model of postoperative pain.
, Neuroreport. 2001 Jun 13;12(8):1701-4., 10.1097/00001756-200106130-00036, 12, 8, 1701-1704, 2001.06.
105. Yajima Y, Narita M, Tsuda M, Imai S, Kamei J, Nagase H, Suzuki T., Modulation of NMDA- and (+)TAN-67-induced nociception by GABA(B) receptors in the mouse spinal cord., Life Sci. 2000 Dec 29;68(6):719-25., 2000.12.
106. Tsuda M, Koizumi S, Kita A, Shigemoto Y, Ueno S, Inoue K., Mechanical allodynia caused by intraplantar injection of P2X receptor agonist in rats: involvement of heteromeric P2X2/3 receptor signaling in capsaicin-insensitive primary afferent neurons., J Neurosci. 2000 Aug 1;20(15):RC90., 20, 15, 2000.08.
107. Suzuki T, Kato H, Aoki T, Tsuda M, Narita M, Misawa M., Effects of the non-competitive NMDA receptor antagonist ketamine on morphine-induced place preference in mice., Life Sci. 2000 Jun 16;67(4):383-9., 2000.06.
108. Tsuda M, Ueno S, Inoue K., Evidence for the involvement of spinal endogenous ATP and P2X receptors in nociceptive responses caused by formalin and capsaicin in mice.
, Br J Pharmacol. 1999 Dec;128(7):1497-504. , 1999.12.
109. Tsuda M, Ueno S, Inoue K., In vivo pathway of thermal hyperalgesia by intrathecal administration of alpha,beta-methylene ATP in mouse spinal cord: involvement of the glutamate-NMDA receptor system., Br J Pharmacol. 1999 May;127(2):449-56., 1999.05.
110. Suzuki T, Shimizu N, Tsuda M, Soma M, Misawa M., Role of metabotropic glutamate receptors in the hypersusceptibility to pentylenetetrazole-induced seizure during diazepam withdrawal.
, Eur J Pharmacol. 1999 Mar 19;369(2):163-8., 1999.03.
111. Suzuki T, Kato H, Tsuda M, Suzuki H, Misawa M., Effects of the non-competitive NMDA receptor antagonist ifenprodil on the morphine-induced place preference in mice.
, Life Sci. 1999;64(12):PL151-6., 1999.02.
112. Ueno S, Tsuda M, Iwanaga T, Inoue K., Cell type-specific ATP-activated responses in rat dorsal root ganglion neurons., Br J Pharmacol. 1999 Jan;126(2):429-36., 1999.01.
113. Tsuda M, Suzuki T, Misawa M., NMDA receptor antagonists potently suppress the spontaneous withdrawal signs induced by discontinuation of long-term diazepam treatment in Fischer 344 rats.
, Brain Res. 1998 Apr 20;790(1-2):82-90., 1998.04.
114. Tsuda M, Shimizu N, Yajima Y, Suzuki T, Misawa M., Hypersusceptibility to DMCM-induced seizures during diazepam withdrawal in mice: evidence for upregulation of NMDA receptors.
, Naunyn Schmiedebergs Arch Pharmacol. 1998 Mar;357(3):309-15., 1998.03.
115. Tsuda M, Suzuki T, Misawa M., Subsensitivity to mitochondrial diazepam binding inhibitor receptor agonist FGIN-1-27-induced antiseizure effect in diazepam-withdrawn mice., Life Sci. 1998;62(14):PL213-7., 1998.02.
116. Tsuda M, Shimizu N, Yajima Y, Suzuki T, Misawa M., Role of nitric oxide in the hypersusceptibility to pentylenetetrazole-induced seizure in diazepam-withdrawn mice.
, Eur J Pharmacol. 1998 Feb 26;344(1):27-30., 1998.02.
117. Tsuda M, Suzuki T, Misawa M., Region-specific changes in [3H]dizocilpine binding in diazepam-withdrawn rats.
, Neurosci Lett. 1998 Jan 9;240(2):113-5., 1998.01.
118. Tsuda M, Chiba Y, Suzuki T, Misawa M., Upregulation of NMDA receptor subunit proteins in the cerebral cortex during diazepam withdrawal.
, Eur J Pharmacol. 1998 Jan 12;341(2-3):R1-2. , 1998.01.
119. Tsuda M, Suzuki T, Misawa M., Age-related decrease in the antiseizure effect of ifenprodil against pentylenetetrazole in mice.
, Brain Res Dev Brain Res. 1997 Dec 19;104(1-2):201-4., 1997.12.
120. Tsuda M, Suzuki T, Misawa M., Aggravation of DMCM-induced seizure by nitric oxide synthase inhibitors in mice., Life Sci. 1997;60(23):PL339-43., 1997.05.
121. Kamei J, Sodeyama M, Tsuda M, Suzuki T, Nagase H., Antinociceptive effect of buprenorphine in mu1-opioid receptor deficient CXBK mice., Life Sci. 1997;60(22):PL 333-7., 1997.04.
122. Tsuda M, Suzuki T, Misawa M., Recovery of decreased seizure threshold for pentylenetetrazole during diazepam withdrawal by NMDA receptor antagonists.
, Eur J Pharmacol. 1997 Apr 11;324(1):63-6., 1997.04.
123. Tsuda M, Suzuki T, Misawa M., Role of the NMDA receptor complex in DMCM-induced seizure in mice., Neuroreport. 1997 Feb 10;8(3):603-6., 1997.02.
124. Suzuki T, Ise Y, Tsuda M, Maeda J, Misawa M., Mecamylamine-precipitated nicotine-withdrawal aversion in rats., Eur J Pharmacol. 1996 Oct 31;314(3):281-4., 1996.10.
125. Tsuda M, Suzuki T, Misawa M, Nagase H., Involvement of the opioid system in the anxiolytic effect of diazepam in mice., Eur J Pharmacol. 1996 Jun 20;307(1):7-14., 1996.06.
126. Suzuki T, Tsuda M, Narita M, Funada M, Mizoguchi H, Misawa M., Diazepam pretreatment suppresses morphine withdrawal signs in the mouse., Life Sci. 1996;58(4):349-57., 1995.12.
127. Mizoguchi H, Shirayama N, Tsuda M, Yoshiike M, Suzuki T, Misawa M., Potentiation of physical dependence on diazepam by ondansetron in rats., Life Sci. 1994;54(9):PL131-6., 1994.07.
128. Suzuki T, Tsuda M, Funada M, Misawa M., Blockade of morphine-induced place preference by diazepam in mice., Eur J Pharmacol. 1995 Jul 14;280(3):327-30., 1995.07.