Microglia in Neuropathic Pain.

Neuropathic pain (NP) is pain resulting from lesions or disease of the somatosensory system. A cardinal feature of NP is tactile allodynia (a painful response to normally innocuous stimulation). In 2003, a breakthrough strategy for inducing NP was proposed in which microglia of the spinal dorsal horn (SDH) are activated after peripheral nerve injury (PNI) to overexpress P2X4 receptor (P2X4R) and play an important role in inducing tactile allodynia. In 2005, it was reported that stimulation of microglial P2X4Rs evokes the release of brain-derived neurotrophic factor (BDNF), which causes a depolarizing shift of the anion reversal potential (Eanion) of secondary sensory neurons. These findings and other facts suggest the mechanism by which innocuous touch stimuli cause severe pain and the important role of microglia in the mechanism.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism through which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in the injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3-/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8-/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8-/- bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism through which migrating macrophages attract astrocytes and affect the pathophysiology and outcome after SCI.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism by which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3 -/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8 -/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8 -/ bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism in which migrating macrophages attracted astrocytes and affected the pathophysiology and outcome after SCI.

Overview for the study of P2 receptors: From P2 receptor history to neuropathic pain studies.

Since new roles of nucleotides as neurotransmitters were proposed by Geoffrey Burnstock, the roles of ATP and P2 receptors (P2Rs) have been extensively studied in pain signaling. This review primarily focuses on the history and roles of P2X2Rs and P2X2/3Rs in acute and chronic pain, and P2X4Rs in neuropathic pain after peripheral nerve injury (PNI). Spinal microglial activity mediated by P2X4Rs shows a very important contribution to evoking neuropathic pain, and P2X4Rs might be targets for the treatment of neuropathic pain. The advantage of P2X4Rs of microglia as therapeutic targets is that P2X4Rs are predominantly enhanced in activated microglia after PNI, and P2X4R blockers do not affect normal pain signaling. Currently, many excellent P2R-related drug candidates are being developed, and it seems that the day when we will use them in clinical practice is not too far away.

The Role of Microglial Purinergic Receptors in Pain Signaling.

Pain is an essential modality of sensation in the body. Purinergic signaling plays an important role in nociceptive pain transmission, under both physiological and pathophysiological conditions, and is important for communication between both neuronal and non-neuronal cells. Microglia and astrocytes express a variety of purinergic effectors, and a variety of receptors play critical roles in the pathogenesis of neuropathic pain. In this review, we discuss our current knowledge of purinergic signaling and of the compounds that modulate purinergic transmission, with the aim of highlighting the importance of purinergic pathways as targets for the treatment of persistent pain.

A spinal microglia population involved in remitting and relapsing neuropathic pain.

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.

The Role of ATP Receptors in Pain Signaling.

Since new roles of nucleotides as neurotransmitters were proposed by Geoffrey Burnstock, the roles of ATP and P2 receptors (P2Rs) have been extensively studied in pain signaling. Chronic pain is a debilitating condition that often occurs following peripheral tissue inflammation and nerve injury. Especially neuropathic pain is a significant clinical problem because there are few clinically effective drugs. This review summarizes the findings for the role of ATP signaling through P2X3Rs and P2X2/3Rs in primary afferent neurons and through P2X4Rs, P2X7Rs, and P2Y12R in spinal microglia in chronic pain to discuss the therapeutic potentials with considering active situation of drug development of P2R compounds.

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions.

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.

New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1ß Release: An Involvement in its Alleviating Effect on Neuropathic Pain.

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-1ß (IL-1ß). 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-1ß 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.

Nociceptive signaling of P2X receptors in chronic pain states.

P2X3 monomeric receptors (P2X3Rs) and P2X2/3 heteromeric receptors (P2X2/3Rs) in primary sensory neurons and microglial P2X4 monomeric receptors (P2X4Rs) in the spinal dorsal horn (SDH) play important roles in neuropathic pain. In particular, P2X4R in the spinal microglia during peripheral nerve injury (PNI), experimental autoimmune neuritis, and herpes models are useful to explore the potential strategies for developing new drugs to treat neuropathic pain. Recently, novel P2X4 antagonists, NP-1815-PX and NC-2600, were developed, which demonstrated potent and specific inhibition against rodent and human P2X4Rs. The phase I study of NC-2600 has been completed, and no serious side effects were reported. The roles played by purinergic receptors in evoking neuropathic pain provide crucial insights into the pathogenesis of neuropathic pain.

Astrocytic STAT3 activation and chronic itch require IP3R1/TRPC-dependent Ca2+ signals in mice.

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.

Nociceptive signaling mediated by P2X3, P2X4 and P2X7 receptors.

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.

Analysis of binding residues in monoclonal antibody with high affinity for the head domain of the rat P2X4 receptor.

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 consists 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 (HD) 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 analysed and identified the binding residues in the monoclonal antibody (12-10H) with high affinity for the HD of the rat P2X4 receptor by site-directed mutagenesis.

Spinal astrocytes in superficial laminae gate brainstem descending control of mechanosensory hypersensitivity.

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.

Macrophage centripetal migration drives spontaneous healing process after spinal cord injury.

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.

Hyperactivation of proprioceptors induces microglia-mediated long-lasting pain in a rat model of chronic fatigue syndrome.

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.

Role of the P2X4 receptor in neuropathic pain.

Neuropathic pain is the most important type of chronic pain because it is refractory to available medications. Neuropathic pain occurs after peripheral nerve injury (PNI) or nerve damage by various reasons. In recent decades, a growing body of evidence shows that spinal microglia and P2X4 receptor (P2X4R), a subtype of ionotropic ATP receptors, play a principal role in evoking this pain. Understanding the functions of microglial P2X4R in evoking this pain provides potential strategies for developing new treatments for neuropathic pain.

Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure.

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.

Transcription factor MafB contributes to the activation of spinal microglia underlying neuropathic pain development.

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.