Organic anion transporter 1 is an HDAC4-regulated mediator of nociceptive hypersensitivity in mice.

Persistent pain is sustained by maladaptive changes in gene transcription resulting in altered function of the relevant circuits; therapies are still unsatisfactory. The epigenetic mechanisms and affected genes linking nociceptive activity to transcriptional changes and pathological sensitivity are unclear. Here, we found that, among several histone deacetylases (HDACs), synaptic activity specifically affects HDAC4 in murine spinal cord dorsal horn neurons. Noxious stimuli that induce long-lasting inflammatory hypersensitivity cause nuclear export and inactivation of HDAC4. The development of inflammation-associated mechanical hypersensitivity, but neither acute nor basal sensitivity, is impaired by the expression of a constitutively nuclear localized HDAC4 mutant. Next generation RNA-sequencing revealed an HDAC4-regulated gene program comprising mediators of sensitization including the organic anion transporter OAT1, known for its renal transport function. Using pharmacological and molecular tools to modulate OAT1 activity or expression, we causally link OAT1 to persistent inflammatory hypersensitivity in mice. Thus, HDAC4 is a key epigenetic regulator that translates nociceptive activity into sensitization by regulating OAT1, which is a potential target for pain-relieving therapies.

NADPH-Oxidase 2 Promotes Autophagy in Spinal Neurons During the Development of Morphine Tolerance.

Repeated morphine administration results in analgesic tolerance. However, the underlying mechanism of morphine analgesic tolerance remains unclear. NADPH-oxidase 2 (NOX2) is the first discovered NADPH oxidase, which mainly functions to produce reactive oxygen species. Its specific role in morphine tolerance has not been fully investigated. In this work, we found that chronic morphine administration significantly increased the expression of NOX2 in spinal cord. Pretreatment of NOX2 inhibitor blocked the upregulation of NOX2 and autophagy markers, including LC3B and P62, and consequently the development of morphine tolerance. NOX2 and LC3B were both colocalized with NeuN in spinal dorsal horn in morphine-tolerant rats. Our results suggest that the increased autophagy activity in spinal neurons promoted by NOX2 activation contributes to the development of morphine tolerance. NOX2 may be considered as a new therapeutic target for morphine tolerance.

BDNF modulated KCC2 ubiquitylation in spinal cord dorsal horn of mice.

The K+-Cl- co-transporter 2 (KCC2) is a neuron-specific Cl- extruder in the dorsal horn of spinal cord. The low intracellular Cl- concentration established by KCC2 is critical for GABAergic and glycinergic systems to generate synaptic inhibition. Peripheral nerve lesions have been shown to cause KCC2 dysfunction in adult spinal cord through brain-derived neurotrophic factor (BDNF) signaling, which switches the hyperpolarizing inhibitory transmission to be depolarizing and excitatory. However, the mechanisms by which BDNF impairs KCC2 function remain to be elucidated. Here we found that BDNF treatment enhanced KCC2 ubiquitination in the dorsal horn of adult mice, a post-translational modification that leads to KCC2 degradation. Our data showed that spinal BDNF application promoted KCC2 interaction with Casitas B-lineage lymphoma b (Cbl-b), one of the E3 ubiquitin ligases that are involved in the spinal processing of nociceptive information. Knockdown of Cbl-b expression decreased KCC2 ubiquitination level and attenuated the pain hypersensitivity induced by BDNF. Spared nerve injury significantly increased KCC2 ubiquitination, which could be reversed by inhibition of TrkB receptor. Our data implicated that KCC2 was one of the important pain-related substrates of Cbl-b and that ubiquitin modification contributed to BDNF-induced KCC2 hypofunction in the spinal cord.

Intrathecal Injection of GRIP-siRNA Reduces Postoperative Synaptic Abundance of Kainate Receptor GluK2 Subunits in Rat Dorsal Horns and Pain Hypersensitivity.

The mechanisms underlying postoperative pain differ from the inflammatory or neuropathic pain. Previous studies have demonstrated that intrathecal α-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) -kainate (KA) receptor antagonist inhibits the guarding pain behavior and mechanical hyperalgesia, indicating a critical role of spinal KA receptors in postoperative pain hypersensitivity. However, how the functional regulations of spinal KA receptor subunits are involved in the postoperative pain hypersensitivity remains elusive. Therefore, in the current study, we investigated the synaptic delivery of spinal KA receptor subunits and the interaction between KA receptor subunits and glutamate receptor-interacting protein (GRIP) during the postoperative pain. Our data indicated that plantar incision induced the synaptic delivery of GluK2, but not GluK1 or GluK3 in ipsilateral spinal cord dorsal horns. The co-immunoprecipitation showed an increased GluK2 -GRIP interaction in ipsilateral dorsal horn neurons at 6 h post-incision. Interestingly, Intrathecal pretreatment of GRIP siRNA increased the paw withdrawal thresholds to mechanical stimuli and decreased the cumulative pain scores in the paws ipsilateral to the incision at 6 h post-incision. Additionally, Intrathecal pretreatment of GRIP siRNA reduced the synaptic abundance of GluK2 in ipsilateral spinal dorsal horn at 6 h after plantar incision. In general, our data have demonstrated that the GluK2- GRIP interaction-mediated synaptic abundance of GluK2 in dorsal horn neurons plays an important role in the postoperative pain hypersensitivity. Disrupting the GluK2- GRIP interaction may provide a new approach for relieving postoperative pain.

Manifestation of lipopolysaccharide-induced tolerance in neuro-glial primary cultures of the rat afferent somatosensory system.

OBJECTIVE: Bacterial lipopolysaccharide (LPS) may contribute to the manifestation of inflammatory pain within structures of the afferent somatosensory system. LPS can induce a state of refractoriness to its own effects termed LPS tolerance. We employed primary neuro-glial cultures from rat dorsal root ganglia (DRG) and the superficial dorsal horn (SDH) of the spinal cord, mainly including the substantia gelatinosa to establish and characterize a model of LPS tolerance within these structures. METHODS: Tolerance was induced by pre-treatment of both cultures with 1 µg/ml LPS for 18 h, followed by a short-term stimulation with a higher LPS dose (10 µg/ml for 2 h). Cultures treated with solvent were used as controls. Cells from DRG or SDH were investigated by means of RT-PCR (expression of inflammatory genes) and immunocytochemistry (translocation of inflammatory transcription factors into nuclei of cells from both cultures). Supernatants from both cultures were assayed for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by highly sensitive bioassays. RESULTS: At the mRNA-level, pre-treatment with 1 µg/ml LPS caused reduced expression of TNF-α and enhanced IL-10/TNF-α expression ratios in both cultures upon subsequent stimulation with 10 µg/ml LPS, i.e. LPS tolerance. SDH cultures further showed reduced release of TNF-α into the supernatants and attenuated TNF-α immunoreactivity in microglial cells. In the state of LPS tolerance macrophages from DRG and microglial cells from SDH showed reduced LPS-induced nuclear translocation of the inflammatory transcription factors NFκB and NF-IL6. Nuclear immunoreactivity of the IL-6-activated transcription factor STAT3 was further reduced in neurons from DRG and astrocytes from SDH in LPS tolerant cultures. CONCLUSION: A state of LPS tolerance can be induced in primary cultures from the afferent somatosensory system, which is characterized by a down-regulation of pro-inflammatory mediators. Thus, this model can be applied to study the effects of LPS tolerance at the cellular level, for example possible modifications of neuronal reactivity patterns upon inflammatory stimulation.

Primary culture of the rat spinal dorsal horn: a tool to investigate the effects of inflammatory stimulation on the afferent somatosensory system.

One maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E2 (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca2+]i. Short-term stimulation of SDH primary cultures with LPS (10 µg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E2 synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca2+-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 µg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca2+ transients in SDH neurons, while substance P-evoked Ca2+ signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses.

Role of reactive astrocytes in the spinal dorsal horn under chronic itch conditions.

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.

Neonatal vincristine administration modulates intrinsic neuronal excitability in the rat dorsal root ganglion and spinal dorsal horn during adolescence.

Our recent work has shown that the early-life administration of vincristine (VNC), commonly used to treat pediatric cancers, evokes mechanical pain hypersensitivity in rats that emerges during adolescence and persists into adulthood. However, the underlying mechanisms remain unclear, as nothing is known about how neonatal VNC treatment influences peripheral and central nociceptive processing at the cellular level. Here, we used in vitro intracellular microelectrode and whole-cell patch-clamp recordings to evaluate the consequences of early-life VNC administration on the intrinsic membrane properties of adolescent dorsal root ganglion and spinal superficial dorsal horn neurons. The results demonstrate that VNC treatment increased the prevalence and rate of repetitive firing in both large- and medium-diameter sensory neurons, while reducing repetitive firing in small-diameter neurons, in comparison with vehicle-treated littermate controls. By contrast, passive membrane properties and peripheral conduction velocities were similar between experimental groups across all classes of primary afferents. Within the adolescent superficial dorsal horn, neonatal VNC exposure significantly enhanced the intrinsic membrane excitability of lamina I spinoparabrachial neurons, as evidenced by a decrease in rheobase and elevation of repetitive firing frequency compared with controls. Meanwhile, putative interneurons within lamina I exhibited a reduction in repetitive action potential discharge after early-life chemotherapy. Collectively, these findings suggest that neonatal VNC treatment evokes cell type-specific changes in intrinsic excitability at multiple levels of the ascending pain pathway. Overall, this work lays an essential foundation for the future exploration of the ionic mechanisms that drive chemotherapy-induced chronic pain in children and adolescents.

[Roles of CX3CR1 in mediation of post-incision induced mechanical pain hypersensitivity: Effects of acupuncture-combined anesthesia].

Post-incision pain often occurs after surgery and is emergent to be treated in clinic. It hinders the rehabilitation of patients and easily leads to various types of postoperative complications. Acupuncture-combined anesthesia (ACA) is the combination of traditional acupuncture and modern anesthesia, which means acupuncture is applied at acupoints with general anesthesia. It was testified that ACA strengthened the analgesic effect and reduced the occurrence of postoperative pain, but its mechanism was not clear. Numerous reports have shown that chemokine receptor CX3CR1 is involved in the development and progression of many pathological pains. The present study was aimed to reveal whether ACA played the analgesic roles in the post-incision pain by affecting CX3CR1. A model of toe incision pain was established in C57BL/6J mice. The pain threshold was detected by behavioral test, and the expression of CX3CR1 protein was detected by immunohistochemical method and Western blot. The results showed that the significant mechanical allodynia and thermal hyperalgesia were induced by paw incision in the mice. Mechanical allodynia was significantly suppressed by ACA, but thermal hyperalgesia was not changed. CX3CR1 was mainly expressed in microglia in the spinal cord dorsal horn, and its protein level was significantly increased at 3 d after incision compared with that of naïve C57BL/6J mice. ACA did not affect CX3CR1 protein expression at 3 d after incision in the toe incision model mice. Paw withdrawal threshold was significantly increased at 3 d after incision in CX3CR1 knockout (KO) mice compared with that in the C57BL/6J mice. But the analgesic effect of ACA was disappeared in CX3CR1 KO mice. Accordingly, it was also blocked when neutralizing antibody of CX3CR1 was intrathecally injected (i.t.) 1 h before ACA in the C57BL/6J mice. These results suggest that CX3CR1 in microglia is involved in post-incision pain and analgesia of ACA.

Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

Electroacupuncture Attenuates CFA-induced Inflammatory Pain by suppressing Nav1.8 through S100B, TRPV1, Opioid, and Adenosine Pathways in Mice.

Pain is associated with several conditions, such as inflammation, that result from altered peripheral nerve properties. Electroacupuncture (EA) is a common Chinese clinical medical technology used for pain management. Using an inflammatory pain mouse model, we investigated the effects of EA on the regulation of neurons, microglia, and related molecules. Complete Freund's adjuvant (CFA) injections produced a significant mechanical and thermal hyperalgesia that was reversed by EA or a transient receptor potential V1 (TRPV1) gene deletion. The expression of the astrocytic marker glial fibrillary acidic protein (GFAP), the microglial marker Iba-1, S100B, receptor for advanced glycation end-products (RAGE), TRPV1, and other related molecules was dramatically increased in the dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) of CFA-treated mice. This effect was reversed by EA and TRPV1 gene deletion. In addition, endomorphin (EM) and N6-cyclopentyladenosine (CPA) administration reliably reduced mechanical and thermal hyperalgesia, thereby suggesting the involvement of opioid and adenosine receptors. Furthermore, blocking of opioid and adenosine A1 receptors reversed the analgesic effects of EA. Our study illustrates the substantial therapeutic effects of EA against inflammatory pain and provides a novel and detailed mechanism underlying EA-mediated analgesia via neuronal and non-neuronal pathways.

Preprotachykinin A is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli.

The superficial dorsal horn, which is the main target for nociceptive and pruritoceptive primary afferents, contains a high density of excitatory interneurons. Our understanding of their roles in somatosensory processing has been restricted by the difficulty of distinguishing functional populations among these cells. We recently defined 3 nonoverlapping populations among the excitatory neurons, based on the expression of neurotensin, neurokinin B, and gastrin-releasing peptide. Here we identify and characterise another population: neurons that express the tachykinin peptide substance P. We show with immunocytochemistry that its precursor protein (preprotachykinin A, PPTA) can be detected in ∼14% of lamina I-II neurons, and these are concentrated in the outer part of lamina II. Over 80% of the PPTA-positive cells lack the transcription factor Pax2 (which determines an inhibitory phenotype), and these account for ∼15% of the excitatory neurons in this region. They are different from the neurotensin, neurokinin B, or gastrin-releasing peptide neurons, although many of them contain somatostatin, which is widely expressed among superficial dorsal horn excitatory interneurons. We show that many of these cells respond to noxious thermal and mechanical stimuli and to intradermal injection of pruritogens. Finally, we demonstrate that these cells can also be identified in a knock-in Cre mouse line (Tac1), although our findings suggest that there is an additional population of neurons that transiently express PPTA. This population of substance P-expressing excitatory neurons is likely to play an important role in the transmission of signals that are perceived as pain and itch.

Downregulation of the spinal dorsal horn clock gene Per1 expression leads to mechanical hypersensitivity via c-jun N-terminal kinase and CCL2 production in mice.

Disturbances of circadian rhythm and dysregulation of clock gene expression are involved in the induction of various neurological disorder states, including chronic pain. However, the relationship between the CNS circadian-clock gene system and nociception remains poorly defined. Significant circadian oscillations of Period (Per1, Per2), Bmal1 and Cryptochrome 1 (Cry1) mRNA expression have been observed in the lumbar spinal dorsal horn of naïve mice. The current study examined the expression of clock genes in the lumbar spinal dorsal horn of mice with neuropathic pain due to a partial sciatic nerve ligation (PSNL). Seven days after PSNL, the mice displayed a robust unilateral hind paw mechanical hypersensitivity. The normal circadian oscillations of Per1, Per2 and Cry1, but not Bmal1, mRNA expression were significantly suppressed in the ipsilateral lumbar spinal dorsal horn of PSNL mice 7days following surgery. The circadian expression of PER1 protein, in particular, was also significantly suppressed in the ipsilateral spinal dorsal horn of PSNL mice. Double-labeling immunohistochemistry revealed downregulation of PER1 in neurons and astrocytes, but not microglia. Knockdown of Per1 expression by intrathecal treatment with Per1 siRNA also induced mechanical hypersensitivity, phosphorylation of c-jun N-terminal kinase (JNK) and the upregulation of chemokine (C-C motif) ligand 2 (CCL2) production in the lumbar spinal dorsal horn. Per1 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of either the JNK inhibitor SP600125 or the selective CCL2 receptor (CCR2) antagonist RS504393, indicating that these intracellular messengers are crucial in mediating the mechanical hypersensitivity following the downregulation of PER1 expression. These results suggest that the downregulation of the spinal dorsal horn clock genes such as Per1 expressed could be crucial in the induction of neuropathic pain following peripheral nerve injury. Modulating clock gene Per1 expression could be a novel therapeutic strategy in alleviating neuropathic pain.

Recombinant neural progenitor transplants in the spinal dorsal horn alleviate chronic central neuropathic pain.

Neuropathic pain induced by spinal cord injury (SCI) is clinically challenging with inadequate long-term treatment options. Partial pain relief offered by pharmacologic treatment is often counterbalanced by adverse effects after prolonged use in chronic pain patients. Cell-based therapy for neuropathic pain using GABAergic neuronal progenitor cells (NPCs) has the potential to overcome untoward effects of systemic pharmacotherapy while enhancing analgesic potency due to local activation of GABAergic signaling in the spinal cord. However, multifactorial anomalies underlying chronic pain will likely require simultaneous targeting of multiple mechanisms. Here, we explore the analgesic potential of genetically modified rat embryonic GABAergic NPCs releasing a peptidergic NMDA receptor antagonist, Serine-histogranin (SHG), thus targeting both spinal hyperexcitability and reduced inhibitory processes. Recombinant NPCs were designed using either lentiviral or adeno-associated viral vectors (AAV2/8) encoding single and multimeric (6 copies of SHG) cDNA. Intraspinal injection of recombinant cells elicited enhanced analgesic effects compared with nonrecombinant NPCs in SCI-induced pain in rats. Moreover, potent and sustained antinociception was achieved, even after a 5-week postinjury delay, using recombinant multimeric NPCs. Intrathecal injection of SHG antibody attenuated analgesic effects of the recombinant grafts suggesting active participation of SHG in these antinociceptive effects. Immunoblots and immunocytochemical assays indicated ongoing recombinant peptide production and secretion in the grafted host spinal cords. These results support the potential for engineered NPCs grafted into the spinal dorsal horn to alleviate chronic neuropathic pain.

Activation of toll like receptor 4 attenuates GABA synthesis and postsynaptic GABA receptor activities in the spinal dorsal horn via releasing interleukin-1 beta.

Toll like receptor 4 (TLR4) is an innate immune pattern recognition receptor, expressed predominantly on microglia in the CNS. Activation of spinal TLR4 plays a critical role in the genesis of pathological pain induced by nerve injury, bone cancer, and tissue inflammation. Currently, it remains unknown how synaptic activities in the spinal dorsal horn are regulated by TLR4 receptors. Through recording GABAergic currents in neurons and glial glutamate transporter currents in astrocytes in rodent spinal slices, we determined whether and how TLR4 modulates GABAergic synaptic activities in the superficial spinal dorsal horn. We found that activation of TLR4 by lipopolysaccharide (LPS) reduces GABAergic synaptic activities through both presynaptic and postsynaptic mechanisms. Specifically, LPS causes the release of IL-1ß from microglia. IL-1ß in turn suppresses GABA receptor activities at the postsynaptic site through activating protein kinase C (PKC) in neurons. GABA synthesis at the presynaptic site is reduced upon activation of TLR4. Glial glutamate transporter activities are suppressed by IL-1ß and PKC activation induced by LPS. The suppression of glial glutamate transporter activities leads to a deficiency of glutamine supply, which results in an attenuation of the glutamate-glutamine cycle-dependent GABA synthesis. These findings shed light on understanding synaptic plasticity induced by activation of TLR4 under neuroinflammation and identify GABA receptors, glial glutamate transporters, IL-1ß and PKC as therapeutic targets to abrogate abnormal neuronal activities following activation of TLR4 in pathological pain conditions.

Connections between EM2- and SP-containing terminals and GABAergic neurons in the mouse spinal dorsal horn.

Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect in pain modulation. To investigate the potential interactions of EM2- and substance P (SP)-containing primary afferents and γ-amino butyric acid (GABA)-containing interneurons in lamina II in nociceptive transmission, connections between EM2- and SP-containing terminals and GABAergic neurons in the spinal dorsal horn were studied. Double-immunofluorescent labeling showed that approximately 62.3 % of EM2-immunoreactive neurons exhibited SP-immunostaining, and 76.9 % of SP-immunoreactive neurons demonstrated EM2-immunoreactivities in the dorsal root ganglion (DRG). Dense double-labeled EM2- and SP-immunoreactivities were mainly observed in lamina II of the lumbar dorsal horn. Furthermore, triple-immunofluorescent labeling results revealed that EM2 and SP double-labeled terminals overlapped with GABAergic neurons. Immuno-electron microscopy confirmed that the EM2- or SP-immunoreactive terminals formed synapses with GABA-immunoreactive dendrites in lamina II of the lumbar dorsal horn. During noxious information transmission induced by formalin plantar injection, GABAergic neurons expressing FOS in their nuclei were contacted with EM2- or SP-immunoreactive terminals. These results suggest that the interactions between EM2- and SP-containing terminals and GABAergic interneurons in the lamina II influence pain transmission and modulation in the spinal dorsal horn.