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Nerve injury-induced alternations of gene expression in primary sensory neurons of the dorsal root ganglion (DRG) are molecular basis of neuropathic pain genesis. Transcription factors regulate gene expression. In this study, we examined whether early B cell factor 1 (EBF1), a transcription factor, in the DRG, participated in neuropathic pain caused by chronic constriction injury (CCI) of the sciatic nerve. EBF1 was distributed exclusively in the neuronal nucleus and coexpressed with cytoplasmic/membrane Kv1.2 in individual DRG neurons. The expression of Ebf1 mRNA and protein was time-dependently downregulated in the ipsilateral lumbar (L) 3/4 DRGs after unilateral CCI. Rescuing this downregulation through microinjection of the adeno-associated virus 5 expressing full-length Ebf1 mRNA into the ipsilateral L3/4 DRGs reversed the CCI-induced decrease of DRG Kv1.2 expression and alleviated the development and maintenance of mechanical, heat and cold hypersensitivities. Conversely, mimicking the downregulation of DRG EBF1 through microinjection of AAV5-expressing Ebf1 shRNA into unilateral L3/4 DRGs produced a reduction of Kv1.2 expression in the ipsilateral L3/4 DRGs, spontaneous pain, and the enhanced responses to mechanical, heat and cold stimuli in naive mice. Mechanistically, EBF1 not only bound to the Kcna2 gene (encoding Kv1.2) promoter but also directly activated its activity. CCI decreased the EBF1 binding to the Kcna2 promoter in the ipsilateral L3/4 DRGs. Our findings suggest that DRG EBF1 downregulation contributes to neuropathic pain likely by losing its binding to Kcna2 promoter and subsequently silencing Kv1.2 expression in primary sensory neurons. Exogenous EBF1 administration may mitigate neuropathic pain by rescuing DRG Kv1.2 expression. [ABSTRACT FROM AUTHOR]

Metastatic bone pain and chemotherapy-induced peripheral neuropathic pain are the most common clinical symptoms in cancer patients. The current clinical management of these two disorders is ineffective and/or produces severe side effects. The present study employed a dual-target compound named as ZL006-05 and examined the effect of systemic administration of ZL006-05 on RM-1–induced bone cancer pain and paclitaxel-induced neuropathic pain. Intravenous injection of ZL006-05 dose-dependently alleviated RM-1–induced mechanical allodynia, heat hyperalgesia, cold hyperalgesia, and spontaneously ongoing nociceptive responses during both induction and maintenance periods, without analgesic tolerance, affecting basal/acute pain and locomotor function. Similar behavioral results were observed in paclitaxel-induced neuropathic pain. This injection also decreased neuronal and astrocyte hyperactivities in the lumbar dorsal horn after RM-1 tibial inoculation or paclitaxel intraperitoneal injection. Mechanistically, intravenous injection of ZL006-05 potentiated the GABAA receptor agonist–evoked currents in the neurons of the dorsal horn and anterior cingulate cortex and also blocked the paclitaxel-induced increase in postsynaptic density-95–neuronal nitric oxide synthase interaction in dorsal horn. Our findings strongly suggest that ZL006-05 may be a new candidate for the management of cancer pain and chemotherapy-induced peripheral neuropathic pain. [ABSTRACT FROM AUTHOR]

Genetic knockout or knockdown of fat-mass and obesity-associated protein (FTO), a demethylase that participates in RNA N6-methyladenosine modification in injured dorsal root ganglion (DRG), has been demonstrated to alleviate nerve trauma-induced nociceptive hypersensitivities. However, these genetic strategies are still impractical in clinical neuropathic pain management. The present study sought to examine the effect of intrathecal administration of two specific FTO inhibitors, meclofenamic acid (MA) and N-CDPCB, on the development and maintenance of nociceptive hypersensitivities caused by unilateral L5 spinal nerve ligation (SNL) in rats. Intrathecal injection of either MA or N-CDPCB diminished dose-dependently the SNL-induced mechanical allodynia, heat hyperalgesia, cold hyperalgesia, and spontaneous ongoing nociceptive responses in both development and maintenance periods, without altering acute/basal pain and locomotor function. Intrathecal MA also reduced the SNL-induced neuronal and astrocyte hyperactivities in the ipsilateral L5 dorsal horn. Mechanistically, intrathecal injection of these two inhibitors blocked the SNL-induced increase in the histone methyltransferase G9a expression and rescued the G9a-controlled downregulation of mu opioid receptor and Kv1.2 proteins in the ipsilateral L5 DRG. These findings further indicate the role of DRG FTO in neuropathic pain and suggest potential clinical application of the FTO inhibitors for management of this disorder. [ABSTRACT FROM AUTHOR]

Chemotherapy-induced peripheral neuropathic pain (CIPNP) often occurs in cancer patients treated with antineoplastic drugs. Therapeutic management of CIPNP is very limited, at least in part due to the largely unknown mechanisms that underlie CIPNP genesis. Here, we showed that systemic administration of the chemotherapeutic drug paclitaxel significantly and time-dependently increased the levels of cyclic AMP response element-binding protein (CREB) in dorsal root ganglion (DRG) neurons. Blocking this increase through DRG microinjection of Creb siRNA attenuated paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities. Mimicking this increase through DRG microinjection of the adeno-associated virus 5 expressing full-length Creb mRNA led to enhanced responses to basal mechanical, heat, and cold stimuli in mice in absence of paclitaxel treatment. Mechanically, paclitaxel-induced increase of DRG CREB protein augmented Dnmt3a promoter activity and participated in the paclitaxel-induced upregulation of DNMT3a protein in the DRG. CREB overexpression also elevated the expression of DNMT3a in in vivo and in vitro DRG neurons of naïve mice. Given that DNMT3a is an endogenous instigator of CIPNP and that CREB co-expresses with DNMT3a in DRG neurons, CREB may be a key player in CIPNP through transcriptional activation of the Dnmt3a gene in primary sensory neurons. CREB is thus a likely potential target for the therapeutic management of this disorder. [ABSTRACT FROM AUTHOR]

Analgesic tolerance and hyperalgesia hinder the long-term utility of opioids. We examined whether spinal high mobility group box 1 (HMGB1) is involved in morphine tolerance and its underlying mechanisms by using a model of repeated intrathecal (i.t.) injections of morphine. The results showed that chronic i.t. morphine exposure led to increased expression of HMGB1, Toll-like receptor 4 (TLR4), and receptor for advanced glycation end products (RAGE) and their mRNAs in the dorsal horn. Morphine challenge also promoted HMGB1 expression and release in cultured spinal neurons, but these effects were inhibited by TAK-242, naloxone (antagonists of TLR4), and TLR4 siRNA. Intrathecal coadministration of morphine with TAK-242 or PDTC (inhibitor of NF-κB activation) also reduced HMGB1 expression in the spinal cord. Repeated i.t. coinjections of morphine with glycyrrhizin (GL, an HMGB1 inhibitor) or HMGB1 siRNA prevented reduction of the maximal possible analgesic effect (MPAE) of morphine and alleviated morphine withdrawal-induced hyperalgesia. The established morphine tolerance and hyperalgesia were partially reversed when i.t. injections of GL or HMGB1 antibody started at day 7 of morphine injection. Repeated i.t. injections of morphine with HMGB1 siRNA inhibited the activation of NF-κB, but not that of JNK and p38. A single i.t. injection of HMGB1 in naïve rats caused pain-related hypersensitivity and reduction in MPAE. Moreover, phosphorylated NF-κB p65, TNF-α, and IL-1β levels in the dorsal horn were upregulated following this treatment, but this upregulation was prevented by coinjection with TAK-242. Together, these results suggest that morphine-mediated upregulation of spinal HMGB1 contributes to analgesic tolerance and hyperalgesia via activation of TLR4/NF-κB signaling, and the HMGB1 inhibitor might be a promising adjuvant to morphine in the treatment of intractable pain in the clinic. [ABSTRACT FROM AUTHOR]

Peripheral nerve injury downregulates the expression of the μ-opioid receptor (MOR) and voltage-gated potassium channel subunit Kv1.2 by increasing their DNA methylation in the dorsal root ganglion (DRG). Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) causes DNA demethylation. Given that DRG MOR and Kv1.2 downregulation contribute to neuropathic pain genesis, this study investigated the effect of DRG TET1 overexpression on neuropathic pain. Overexpression of TET1 in the DRG through microinjection of herpes simplex virus expressing full-length TET1 mRNA into the injured rat DRG significantly alleviated the fifth lumbar spinal nerve ligation (SNL)-induced pain hypersensitivities during the development and maintenance periods, without altering acute pain or locomotor function. This microinjection also restored morphine analgesia and attenuated morphine analgesic tolerance development after SNL. Mechanistically, TET1 microinjection rescued the expression of MOR and Kv1.2 by reducing the level of 5-methylcytosine and increasing the level of 5-hydroxymethylcytosine in the promoter and 5' untranslated regions of the Oprml1 gene (encoding MOR) and in the promoter region of the Kcna2 gene (encoding Kv1.2) in the DRG ipsilateral to SNL. These findings suggest that DRG TET1 overexpression mitigated neuropathic pain likely through rescue of MOR and Kv1.2 expression in the ipsilateral DRG. Virus-mediated DRG delivery of TET1 may open a new avenue for neuropathic pain management. [ABSTRACT FROM AUTHOR]

Abstract Aims Histone acetylation and deacetylation are two histone posttranslational modifications that are usually controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although HATs or HDACs Inhibitors could relieve pain hypersensitivities in chronic pain animal models, it is not clear on the expression of global histone acetylation in the dorsal root ganglion (DRG) or spinal dorsal horn in chronic pain conditions. Main methods A spinal nerve ligation (SNL)-induced neuropathic pain model and a complete Freund's adjuvant (CFA)-induced inflammatory pain model in rats were used to examine the expression of total acetyl-histone H3 (AcH3) and total acetyl-histone H4 (AcH4) by immunofluorescence or western blot. Key findings AcH3 and AcH4 not only localized in neuronal nuclei, but also in nuclei of glial cells in the DRG. Unilateral SNL induced the increase of AcH3 and AcH4 expression in the injured lumbar 5 (L5) DRG, but not in the uninjured L5 DRG or the spinal dorsal horn, while unilateral intraplantar injection of CFA increased AcH3 and AcH4 expression in the ipsilateral L4/5 spinal dorsal horn, but not in the L4/5 DRG. Significance These results provide morphological evidence for global histone acetylation expression in the DRG and spinal cord and indicate the differential expression in the DRG and spinal dorsal horn in different chronic pain models. More precise epigenetic mechanisms of histone acetylation on the target genes need to be revealed. [ABSTRACT FROM AUTHOR]

Unlabelled: Chronic sleep disturbance-induced stress is known to increase basal pain sensitivity. However, most surgical patients frequently report short-term sleep disturbance/deprivation during the pre- and postoperation periods and have normal pain perception presurgery. Whether this short-term sleep disturbance affects postsurgical pain is elusive. Here, we report that pre- or postexposure to rapid eye movement sleep disturbance (REMSD) for 6 hours daily for 3 consecutive days did not alter basal responses to mechanical, heat, and cold stimuli, but did delay recovery in incision-induced reductions in paw withdrawal threshold to mechanical stimulation and paw withdrawal latencies to heat and cold stimuli on the ipsilateral side of male or female rats. This short-term REMSD led to stress shown by an increase in swim immobility time, a decrease in sucrose consumption, and an increase in the level of corticosterone in serum. Blocking this stress via intrathecal RU38486 or bilateral adrenalectomy abolished REMSD-caused delay in recovery of incision-induced reductions in behavioral responses to mechanical, heat, and cold stimuli. Moreover, this short-term REMSD produced significant reductions in the levels of mu opioid receptor and kappa opioid receptor, but not Kv1.2, in the ipsilateral L4/5 spinal cord and dorsal root ganglia on day 9 after incision (but not after sham surgery).Perspective: Our findings show that short-term sleep disturbance either pre- or postsurgery does not alter basal pain perception, but does exacerbate postsurgical pain hypersensitivity. The latter may be related to the reductions of mu and kappa opioid receptors in the spinal cord and dorsal root ganglia caused by REMSD plus incision. Prevention of short-term sleep disturbance may help recovery from postsurgical pain in patients. [ABSTRACT FROM AUTHOR]

• Orai1 deficiency drastically decreases lipopolysaccharides (LPS)-induced TNF-α and IL-6 production in astrocytes. • Inhibition of CRACs or deficiency of Orai1 does not alter LPS-induced MAPK activation. • LPS exposure increases Orai1 protein expression vis NF-κB. • LPS exposure enhances basal Ca2+ level and SOCE. • Orai1 deficiency almost eliminates Ca2+ entry induced by elevating extracellular Ca2+ concentration, suggesting that Orai1 functions as a leak Ca2+ channel maintaining astrocytic Ca2+ homeostasis. Toll-like receptor 4 (TLR4) has been implicated in pathological conditions including chronic pain. Activation of astrocytic TLRs leads to the synthesis of pro-inflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor-ɑ (TNF-α), which can cause pathological inflammation and tissue damage in the central nervous system. However, the mechanisms of TLR4-mediated cytokine releases from astrocytes are incomplete understood. Our previous study has shown that Orai1, a key component of calcium release activated calcium channels (CRACs), mediates Ca2+ entry in astrocytes. How Orai1 contributes to TLR4 signaling remains unclear. Here we show that Orai1 deficiency drastically attenuated lipopolysaccharides (LPS)-induced TNF-α and IL-6 production in astrocytes. Acute LPS treatment did not induce Ca2+ response and had no effect on thapsigargin (Ca2+-ATPase inhibitor)-induced store-dependent Ca2+ entry. Inhibition or knockdown of Orai1 showed no reduction in LPS-induced p-ERK1/2, p-c-Jun N-terminal kinase, or p-p38 MAPK activation. Interestingly, Orai1 protein level was significantly increased after LPS exposure, which was blocked by inhibition of NF-κB activity. LPS significantly increased basal Ca2+ level and SOCE after exposure to astrocytes. Moreover, elevating extracellular Ca2+ concentration increased cytosolic Ca2+ level, which was almost eliminated in Orai1 KO astrocytes. Our study reports novel findings that Orai1 acts as a Ca2+ leak channel regulating the basal Ca2+ level and enhancing cytokine production in astrocytes under the inflammatory condition. These findings highlight an important role of Orai1 in astrocytic TRL4 function and may suggest that Orai1 could be a potential therapeutic target for neuroinflammatory disorders including chronic pain. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: Voltage-gated sodium channels (Nav) are essential for the generation and conduction of action potentials. Peripheral inflammation increases the expression of Nav1.7 and Nav1.8 in dorsal root ganglion (DRG) neurons, suggesting that they participate in the induction and maintenance of chronic inflammatory pain. However, how Nav1.7 and Nav1.8 are regulated in the DRG under inflammatory pain conditions remains unclear. Using a complete Freund's adjuvant (CFA)-induced chronic inflammatory pain model and Western blot analysis, we found that phosphorylated Akt (p-Akt) was significantly increased in the ipsilateral L4/5 DRGs of rats on days 3 and 7 after intraplantar CFA injection. Immunohistochemistry showed that the percentage of p-Akt-positive neurons in the DRG was also significantly increased in the ipsilateral L4/5 DRGs at these time points. Moreover, CFA injection increased the colocalization of p-Akt with Nav1.7 and Nav1.8 in L4/5 DRG neurons. Pretreatment of rats with an intrathecal injection of Akt inhibitor IV blocked CFA-induced thermal hyperalgesia and CFA-induced increases in Nav1.7 and Nav1.8 in the L4/5 DRGs on day 7 after CFA injection. Our findings suggest that the Akt pathway participates in inflammation-induced upregulation of Nav1.7 and Nav1.8 expression in DRG neurons. This participation might contribute to the maintenance of chronic inflammatory pain. Perspective: This article presents that inhibition of Akt blocks CFA-induced thermal hyperalgesia and CFA-induced increases in dorsal root ganglion Nav1.7 and Nav1.8. These findings have potential implications for use of Akt inhibitors to prevent and/or treat persistent inflammatory pain. [Copyright &y& Elsevier]

Unlabelled: Persistent inflammation promotes internalization of synaptic GluR2-containing, Ca(2+)-impermeable AMPA receptors (AMPARs) and insertion of GluR1-containing, Ca(2+)-permeable AMPARs at extrasynaptic sites in dorsal horn neurons. Previously we have shown that internalization of synaptic GluR2-containing AMPARs requires activation of spinal cord protein kinase C alpha (PKCα), but molecular mechanisms that underlie altered trafficking of extrasynaptic AMPARs are unclear. Here, using antisense (AS) oligodeoxynucleotides (ODN) that specifically knock down PKCα, we found that a decrease in dorsal horn PKCα expression prevents complete Freund's adjuvant (CFA)-induced increase in functional expression of extrasynaptic Ca(2+)-permeable AMPARs in substantia gelatinosa (SG) neurons of the rat spinal cord. Augmented AMPA-induced currents and associated [Ca(2+)](i) transients were abolished, and the current rectification 1 day post-CFA was reversed. These changes were observed specifically in SG neurons characterized by intrinsic tonic firing properties, but not in those that exhibited strong adaptation. Finally, dorsal horn PKCα knockdown produced an antinociceptive effect on CFA-induced thermal and mechanical hypersensitivity during the maintenance period of inflammatory pain, indicating a role for PKCα in persistent inflammatory pain maintenance. Our results indicate that inflammation-induced trafficking of extrasynaptic Ca(2+)-permeable AMPARs in tonically firing SG neurons depends on PKCα, and that this PKCα-dependent trafficking may contribute to persistent inflammatory pain maintenance.Perspective: This study shows that PKCα knockdown blocks inflammation-induced upregulation of extrasynaptic Ca(2+)-permeable AMPARs in dorsal horn neurons and produces an antinociceptive effect during the maintenance period of inflammatory pain. These findings have potential implications for use of PKCα gene-silencing therapy to prevent and/or treat persistent inflammatory pain. [ABSTRACT FROM AUTHOR]

Abstract: Mammalian target of rapamycin (mTOR) controls mRNA translation and is critical for neuronal plasticity. However, how it participates in central sensitization underlying chronic pain is unclear. Here, we show that NMDA receptors are required for the functional role of spinal cord mTOR in bone cancer pain induced by injecting prostate cancer cells (PCCs) into the tibia. Intrathecal rapamycin, a specific mTOR inhibitor, dose dependently attenuated the development and maintenance of PCC-induced mechanical allodynia and thermal hyperalgesia. Rapamycin alone did not affect locomotor activity and acute responses to thermal or mechanical stimuli. Phosphorylation of mTOR and p70S6K (a downstream effector) was increased time dependently in L4-5 dorsal horn and transiently in L4-5 dorsal root ganglions on the ipsilateral side after PCC injection, although total expression of mTOR or p70S6K was not changed in these regions. The increases in dorsal horn were abolished by intrathecal infusion of DL-AP5, an NMDA receptor antagonist. Moreover, NMDA receptor subunit NR1 colocalized with mTOR and p70S6K in dorsal horn neurons. These findings suggest that PCC-induced dorsal horn activation of the mTOR pathway participates in NMDA receptor-triggered dorsal central sensitization under cancer pain conditions. Perspective: The present study shows that inhibition of spinal mTOR blocks cancer-related pain without affecting acute pain and locomotor function. Given that mTOR inhibitors are FDA-approved drugs, mTOR in spinal cord may represent a potential new target for preventing and/or treating cancer-related pain. [Copyright &y& Elsevier]

Abstract: In certain patients with neuropathic pain, the pain is dependent on activity in the sympathetic nervous system. To investigate whether the spared nerve injury model (SNI) produced by injury to the tibial and common peroneal nerves and leaving the sural nerve intact is a model for sympathetically maintained pain, we measured the effects of surgical sympathectomy on the resulting mechanical allodynia, mechanical hyperalgesia, and cold allodynia. Decreases of paw withdrawal thresholds to von Frey filament stimuli and increases in duration of paw withdrawal to pinprick or acetone stimuli were observed in the ipsilateral paw after SNI, compared with their pre-SNI baselines. Compared with sham surgery, surgical lumbar sympathectomy had no effect on the mechanical allodynia and mechanical hyperalgesia induced by SNI. However, the sympathectomy significantly attenuated the cold allodynia induced by SNI. These results suggest that the allodynia and hyperalgesia to mechanical stimuli in the SNI model is not sympathetically maintained. However, the sympathetic nervous system may be involved, in part, in the mechanisms of cold allodynia in the SNI model. Perspective: The results of our study suggest that the SNI model is not an appropriate model of sympathetically maintained mechanical allodynia and hyperalgesia but may be useful to study the mechanisms of cold allodynia associated with sympathetically maintained pain states. [Copyright &y& Elsevier]

Chronic pain, particularly neuropathic pain, is notoriously difficult to treat. NMDA receptor antagonists are effective in reducing pain hypersensitivity in animal models and clinical settings but are associated with an unacceptable level of side-effects. Recent studies of the role of a family of synaptic membrane-associated guanylate kinase proteins in chronic pain provide new insights into central mechanisms of chronic pain that could result in new biochemical targets for its treatment. [Copyright &y& Elsevier]

Rodents are the main model systems for pain research, but determining their pain state is challenging. To develop an objective method to assess pain sensation in mice, we adopt high-speed videography to capture sub-second behavioral features following hind paw stimulation with both noxious and innocuous stimuli and identify several differentiating parameters indicating the affective and reflexive aspects of nociception. Using statistical modeling and machine learning, we integrate these parameters into a single index and create a "mouse pain scale," which allows us to assess pain sensation in a graded manner for each withdrawal. We demonstrate the utility of this method by determining sensations triggered by three different von Frey hairs and optogenetic activation of two different nociceptor populations. Our behavior-based "pain scale" approach will help improve the rigor and reproducibility of using withdrawal reflex assays to assess pain sensation in mice. • High-speed videography identifies sub-second pain-related behavioral features • Statistical modeling converts behavioral features to a single index (mouse pain scale) • Mouse pain scale classifies sensation induced by Von Frey hair stimulation • Mouse pain scale classifies sensation triggered by optogenetic activation Abdus-Saboor et al. develop a behavior-centered "mouse pain scale" using high-speed videography, statistical modeling, and machine learning. With this method, they assess the sensation induced by noxious, innocuous, and optogenetic stimuli. This method will improve the reliability of using the mouse hind paw withdrawal to measure pain. [ABSTRACT FROM AUTHOR]

Background: Blocking increased expression of nerve injury-specific long non-coding RNA (NIS-lncRNA) in injured dorsal root ganglia (DRG) through DRG microinjection of NIS-lncRNA small hairpin interfering RNA or generation of NIS-lncRNA knockdown mice mitigates neuropathic pain. However, these strategies are impractical in the clinic. This study employed a Food and Drug Administration (FDA)-approved antisense oligonucleotides strategy to examine the effect of NIS-lncRNA ASOs on neuropathic pain.Methods: Effects of intrathecal injection of NIS-lncRNA antisense oligonucleotides on day 7 or 14 after chronic constriction injury (CCI) of the sciatic nerve, fourth lumbar (L4) spinal nerve ligation, or intraperitoneal injection of paclitaxel or streptozotocin on the expression of DRG NIS-lncRNA and C-C chemokine ligand 2 (CCL2, an NIS-lncRNA downstream target) and nociceptive hypersensitivity were examined. We also assessed whether NIS-lncRNA antisense oligonucleotides produced cellular toxicity.Results: Intrathecal NIS-lncRNA antisense oligonucleotides attenuated CCI-induced mechanical allodynia, heat hyperalgesia, cold hyperalgesia, and ongoing nociceptive responses, without changing basal or acute nociceptive responses and locomotor function. Intrathecal NIS-lncRNA antisense oligonucleotides also blocked CCI-induced increases in NIS-lncRNA and CCL2 in the ipsilateral L3 and L4 DRG and hyperactivities of neurones and astrocytes in the ipsilateral L3 and L4 spinal cord dorsal horn. Similar results were found in antisense oligonucleotides-treated mice after spinal nerve ligation or intraperitoneal injection of paclitaxel or streptozotocin. Normal morphologic structure and no cell loss were observed in the DRG and spinal cord of antisense oligonucleotides-treated mice.Conclusion: These findings further validate the role of NIS-lncRNA in trauma-, chemotherapy-, or diabetes-induced neuropathic pain and demonstrate potential clinical application of NIS-lncRNA antisense oligonucleotides for neuropathic pain management. [ABSTRACT FROM AUTHOR]

Background: Nerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to neuropathic pain genesis. Eukaryotic initiation factor 4 gamma 2 (eIF4G2) is a general repressor of cap-dependent mRNA translation. Whether DRG eIF4G2 participates in nerve injury-induced alternations in gene expression and nociceptive hypersensitivity is unknown.Methods: The expression and distribution of eIF4G2 mRNA and protein in mouse DRG after spinal nerve ligation (SNL) were assessed. Effects of eIF4G2 siRNA microinjected through a glass micropipette into the injured DRG on the SNL-induced DRG mu opioid receptor (MOR) and Kv1.2 downregulation and nociceptive hypersensitivity were examined. In addition, effects of DRG microinjection of adeno-associated virus 5-expressing eIF4G2 (AAV5-eIF4G2) on basal DRG MOR and Kv1.2 expression and nociceptive thresholds were analysed.Results: eIF4G2 protein co-expressed with Kv1.2 and MOR in DRG neurones. Levels of eIF4G2 mRNA (1.7 [0.24] to 2.3 [0.14]-fold of sham, P<0.01) and protein (1.6 [0.14] to 2.5 [0.22]-fold of sham, P<0.01) in injured DRG were time-dependently increased on days 3-14 after SNL. Blocking increased eIF4G2 through microinjection of eIF4G2 siRNA into the injured DRG attenuated SNL-induced downregulation of DRG MOR and Kv1.2 and development and maintenance of nociceptive hypersensitivities. DRG microinjection of AAV5-eIF4G2 reduced DRG MOR and Kv1.2 expression and elicited hypersensitivities to mechanical, heat and cold stimuli in naïve mice.Conclusions: eIF4G2 contributes to neuropathic pain through participation in downregulation of Kv1.2 and MOR in injured DRG and is a potential target for treatment of this disorder. [ABSTRACT FROM AUTHOR]

Nerve injury-induced mechanical hypersensitivity is one of major clinical symptoms in neuropathic pain patients. Understanding molecular mechanisms underlying this symptom is crucial for developing effective therapies. The present study was to investigate whether sensory neuron-specific long noncoding RNA (SS-lncRNA) predominantly expressed in small non-peptidergic dorsal root ganglion (DRG) neurons repaired nerve injury-induced mechanical hypersensitivity. SS-lncRNA downregulation in the mas-related G protein-coupled receptor member D (Mrgprd)-expressed DRG neurons was rescued and mimicked by crossbreeding MrgprdCreERT2/+ lines with Rosa26SS-lncRNA knock-in mice and SS-lncRNA fl/fl mice, respectively, followed by tamoxifen injection. Rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons significantly reversed the spinal nerve ligation (SNL)-induced reduction of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in these DRG neurons and alleviated the SNL-induced mechanical hypersensitivity, without affecting the SNL-induced heat and cold nociceptive hypersensitivities, on the ipsilateral side. Conversely, mimicking SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons reduced basal KCNN1 expression in these DRG neurons and produced the enhanced response to mechanical stimulation, but not thermal and cold stimuli, on bilateral sides. Mechanistically, SS-lncRNA downregulation caused a reduction in its binding to lysine-specific demethylase 6B (KDM6B) and consequent recruitment of less KDM6B to Kcnn1 promoter and an increase of H3K27me3 enrichment in this promoter in injured DRG. Our findings suggest that SS-lncRNA downregulation in small non-peptidergic sensory neurons is required specifically for nerve injury-induced mechanical hypersensitivity likely through silencing KCNN1 expression caused by KDM6B-gated increase of H3K27me3 enrichment in Kcnn1 promoter in these neurons. [ABSTRACT FROM AUTHOR]

• Transient receptor potential vanilloid 1 is activated and sensitized in the dorsal root ganglion neurons through multiple mechanisms under sickle cell disease-associated pain conditions. • Endothelin-1 and endothelin type A receptor are upregulated in the dorsal root ganglion neurons from the sickle cell disease mice. • Both transient receptor potential vanilloid 1 and endothelin-1/endothelin type A receptor are required for the development of sickle cell disease-associated pain. • Cannabinoids might be used as a therapeutical approach, but remains to be confirmed in the management of sickle cell disease-associated pain. Sickle cell disease (SCD) is one of the most common severe genetic diseases around the world. A majority of SCD patients experience intense pain, leading to hospitalization, and poor quality of life. Opioids form the bedrock of pain management, but their long-term use is associated with severe side effects including hyperalgesia, tolerance and addiction. Recently, excellent research has shown some new potential mechanisms that underlie SCD-associated pain. This review focused on how transient receptor potential vanilloid 1, endothelin-1/endothelin type A receptor, and cannabinoid receptors contributed to the pathophysiology of SCD-associated pain. Understanding these mechanisms may open a new avenue in managing SCD-associated pain and improving quality of life for SCD patients. [ABSTRACT FROM AUTHOR]

Abstract High mobility group box 1 protein (HMGB1) is potentially triggered by Aβ oligomers and other sterile injuries, and is a non-histone DNA binding nuclear protein with roles in neural development and neurodegeneration, which contribute to memory impairment and chronic neuroinflammation in the brain. However, the exact molecular mechanisms of HMGB1 activation in Alzheimer's disease (AD) were previously unknown. The present study aimed to elucidate the effects of HMGB1 in Aβ 25–35 -induced neuroinflammation in hippocampal neuron cultures. RNA interference (RNAi) HMGB1 treatment significantly reduced Aβ 25–35 -induced HMGB1 expression by almost 70% in primary hippocampal neurons. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assay (ELISA) demonstrated that short hairpin RNA (shRNA) for HMGB1 ameliorated Aβ 25–35 -treated neuroinflammation, including activation of advanced glycosylation end product-specific receptor (RAGE), toll-like receptor 4 (TLR4), and nuclear factor-kappa B (NF-κB)-p65, as well as induced the release of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), IL-6, and HMGB1 in primary hippocampal neurons and the culture supernatant. In addition, pretreatment with HMGB1-shRNA dramatically reduced both the degree of nuclear-cytoplasmic HMGB1 translocation of HMGB1 and NF-κB DNA binding. Together, the data indicate that HMGB1 mediates the pathogenesis of AD by activating RAGE/TLR4 signaling and that shRNA targeting HMGB1 may be a promising therapeutic strategy for treating AD. Highlights • Molecular mechanisms of HMGB1 activation in Alzheimer's disease were investigated. • We evaluated HMGB1 in Aβ 25–35 -induced hippocampal neuron neuroinflammation. • HMGB1 mediates Alzheimer's disease pathogenesis via RAGE/TLR4 signaling. • Targeting HMGB1 with shRNA may be a promising therapeutic strategy for treating AD. [ABSTRACT FROM AUTHOR]

Abstract Hemorrhages occurring within the thalamus lead to a pain syndrome. Clinical treatment of thalamic pain is ineffective, at least in part, due to the elusive mechanisms that underlie the induction and maintenance of thalamic pain. The present study investigated the possible contribution of a protein-protein interaction between postsynaptic density protein 95 (PSD-95) and neuronal nitric oxide synthase (nNOS) to thalamic pain in mice. Thalamic hemorrhage was induced by microinjection of type IV collagenase into unilateral ventral posterior medial/lateral nuclei of the thalamus. Pain hypersensitivities, including mechanical allodynia, heat hyperalgesia, and cold allodynia, appeared at day 1 post-microinjection, reached a peak 5–7 days post-microinjection, and persisted for at least 28 days post-microinjection on the contralateral side. Systemic pre-treatment (but not post-treatment) of ZL006, a small molecule that disrupts PSD-95-nNOS interaction, alleviated these pain hypersensitivities. This effect is dose-dependent. Mechanistically, ZL006 blocked the hemorrhage-induced increase of binding of PSD-95 with nNOS and membrane translocation of nNOS in thalamic neurons. Our findings suggest that the protein-protein interaction between PSD-95 and nNOS in the thalamus plays a significant role in the induction of thalamic pain. This interaction may be a promising therapeutic target in the clinical management of hemorrhage-induced thalamic pain. Highlights • The thalamus hemorrhagic mice exhibit hyperalgesia phenomena. • Pre-treatment of ZL006 attenuates hemorrhage-induced thalamic pain. • ZL006 takes effect by disrupting the PSD-95-nNOS protein-protein interaction in thalamic neurons. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that Loperamide, originally known as an anti-diarrheal drug, is a promising analgesic agent primarily targeting mu-opioid receptors. However some evidences suggested that non-opioid mechanisms may be contributing to its analgesic effect. In the present study, Loperamide was identified as a Nav1.7 blocker in a pilot screen. In HEK293 cells expressing Nav1.7 sodium channels, Loperamide blocked the resting state of Nav1.7 channels (IC 50 = 1.86 ± 0.11 μM) dose-dependently and reversibly. Loperamide produced a 10.4 mV of hyperpolarizing shift for the steady-state inactivation of Nav1.7 channels without apparent effect on the voltage-dependent activation. The drug displayed a mild use- and state-dependent inhibition on Nav1.7 channels, which was removed by the local anesthetic-insensitive construct Nav1.7-F1737A. Inhibition of Nav1.7 at resting state was not altered significantly by the F1737A mutation. Compared to its effects on Nav1.7, Loperamide exhibited higher potency on recombinant Nav1.8 channels in ND7/23 cells (IC 50 = 0.60 ± 0.10 μM) and weaker potency on Nav1.9 channels (3.48 ± 0.33 μM). Notably more pronounced inhibition was observed in the native Nav1.8 channels (0.11 ± 0.08 μM) in DRG neurons. Once mu-opioid receptor was antagonized by Naloxone in DRG neurons, potency of Loperamide on Nav1.8 was identical to that of recombinant Nav1.8 channels. The inhibition on Nav channels may be the main mechanism of Loperamide for pain relief beyond mu-opioid receptor. In the meanwhile, the opioid receptor pathway may also influence the blocking effect of Loperamide on sodium channels, implying a cross-talk between sodium channels and opioid receptors in pain processing. [ABSTRACT FROM AUTHOR]

Blockade of lysophosphatidic acid receptor 5 (LPA5) by a recently reported antagonist AS2717638 (2) attenuated inflammatory and neuropathic pains, although it showed moderate in vivo efficacy and its structure-activity relationships and the ADME properties are little studied. We therefore designed and synthesized a series of isoquinolone derivatives and evaluated their potency in LPA5 calcium mobilization and cAMP assays. Our results show that substituted phenyl groups or bicyclic aromatic rings such as benzothiophenes or benzofurans are tolerated at the 2-position, 4-substituted piperidines are favored at the 4-position, and methoxy groups at the 6- and 7-positions are essential for activity. Compounds 65 and 66 showed comparable in vitro potency, excellent selectivity against LPA1-LPA4 and >50 other GPCRs, moderate metabolic stability, and high aqueous solubility and brain permeability. Both 65 and 66 significantly attenuated nociceptive hypersensitivity at lower doses than 2 and had longer-lasting effects in an inflammatory pain model, and 66 also dose-dependently reduced mechanical allodynia in the chronic constriction injury model and opioid-induced hyperalgesia at doses that had no effect on the locomotion in rats. These results suggest that these isoquinolone derivatives as LPA5 antagonists are of promise as potential analgesics. [Display omitted] • Structure-activity relationships at multiple positions of the isoquinolone were explored and a total of 63 isoquinolone derivatives were obtained. • A novel strategy using Chan-Lam coupling, conducted open to the air, was established to prepare 2-substituted isoquinolones with good yields and reproducibility. • Representative compounds (65 , 66) showed good potency and selectivity against LPA1-LPA4 and >50 other GPCRs, moderate metabolic stability, and high aqueous solubility and brain permeability. • Both 65 and 66 significantly attenuated nociceptive hypersensitivity at lower doses than previously reported AS2717638 and had longer-lasting effects in an inflammatory pain model. • 66 also dose-dependently reduced mechanical allodynia in the chronic constriction injury model and opioid-induced hyperalgesia. [ABSTRACT FROM AUTHOR]

Paclitaxel-induced downregulation of two-pore domain K+ channel 1.1 (K 2p 1.1) caused by increasing DNA methylation within its gene promoter in the dorsal root ganglion (DRG) contributes to neuropathic pain. Given that ten-eleven translocation methylcytosine dioxygenase 1 (TET1) promotes DNA demethylation and gene transcription, the present study investigated whether DRG overexpression of TET1 produces an antinociceptive effect on the paclitaxel-induced nociceptive hypersensitivity. TET1 was overexpressed in the DRG through unilateral microinjection of the herpes simplex virus expressing full-length Tet1 mRNA into the fourth and fifth lumbar DRGs of male rats. Behavioral tests were carried out to examine the effect of this overexpression on the paclitaxel-induced nociceptive hypersensitivity. Western blot analysis, chromatin immunoprecipitation assay and 5-hydroxymethylcytosine detection assay were performed to assess the levels of TET1/K 2p 1.1, 5-methylcytosine and 5-hydroxymethylcytosine, respectively. DRG overexpression of TET1 mitigated the paclitaxel-induced mechanical allodynia, heat hyperalgesia and cold hyperalgesia on the ipsilateral side during the development and maintenance periods. Locomotor function or basal (acute) responses to mechanical, heat or cold stimuli were not affected. Mechanistically, DRG overexpression of TET1 rescued the expression of K 2p 1.1 by blocking the paclitaxel-induced increase in the level of 5-methylcytosine and correspondingly reversing the paclitaxel-induced decreases in the amount of 5-hydroxymethylcytosine within the K 2p 1.1 promoter region in the microinjected DRGs of male rats. Our findings suggest that DRG overexpression of TET1 alleviated chemotherapy-induced neuropathic pain likely through rescuing DRG K 2p 1.1 expression. Our findings may provide a potential avenue for the management of this disorder. [ABSTRACT FROM AUTHOR]

Abstract: Protein translation controlled through activation of mammalian target of rapamycin (mTOR) participates in many physiological and pathological processes. However, whether such activation is required for chronic pain is still unknown. Here, we examined activation of the mTOR signaling pathway during complete Freund's adjuvant (CFA)-induced chronic inflammatory pain and L5 spinal nerve ligation (SNL)-induced neuropathic pain in rats. Western blot analysis showed significantly increased levels of phosphorylated mTOR (p-mTOR) and phosphorylated p70S6 kinase 1 (p-S6K1, a downstream effector of mTOR) in the ipsilateral L4/5 spinal cord 2h, 1 day, 3 days, and 7 days after intraplantar CFA injection and in the ipsilateral L4/5 dorsal root ganglions (DRGs) 1 and 3 days after CFA injection. Immunohistochemistry also demonstrated increases in number of p-mTOR-labeled neurons in the ipsilateral L4/5 DRGs and in density of p-mTOR-labeled immunoreactivity in the ipsilateral L4/5 superficial dorsal horn 1 day after CFA injection. Moreover, intrathecal administration of rapamycin, a selective inhibitor of mTOR, significantly blocked CFA-induced mechanical allodynia and thermal hyperalgesia 1 day post-CFA injection. Interestingly, expression of neither p-mTOR nor p-S6K1 was markedly altered on days 3, 7, or 14 after L5 SNL in L5 spinal cord or DRG. These findings indicate that in DRG and spinal cord, mTOR and S6K1 are activated during chronic inflammatory pain, but not during neuropathic pain. Our results strongly suggest that mTOR and its downstream pathway contribute to the development of chronic inflammatory pain. [Copyright &y& Elsevier]

Abstract: Mammalian target of rapamycin (mTOR) controls protein translation and has an important role in the mechanism of pain hypersensitivity under persistent pain conditions. However, its expression and localization in pain-related regions of the nervous system is not completely understood. Here, we examined the expression and distribution of mTOR, eukaryotic initiation factor 4E-binding protein1/2 (4E-BP1/2), p70 ribosomal S6 protein kinase (p70S6K), and their phosphorylated (active) counterparts in two major pain-related regions, the dorsal root ganglion (DRG) and spinal cord dorsal horn. Reverse transcriptase-polymerase chain reaction showed that mTOR, 4E-BP1, and p70S6K mRNA are expressed in the DRG and dorsal horn. Western blot analysis further confirmed the existence of their protein products in these two regions, but expression of their phosphorylated counterparts was very low in dorsal horn and was not detected in the DRG. Immunohistochemistry revealed mTOR and p70S6K in the DRG neurons. Quantitative analysis showed that approximately 26.1% (± 3.2%) of DRG neurons were positive for mTOR and 19.1% (± 1.9%) were positive for p70S6K. Most of these neurons were small—less than 600µm2 in cross-sectional area—and some co-labeled with substance P or isolectin B4. Surprisingly, 4E-BP1 was observed only in the DRG satellite glial cells. In the dorsal horn, mTOR, p70S6K, and 4E-BP1 were detected in neurons, but not in astrocytes or microglia. They were distributed in the whole dorsal horn, especially in the superficial dorsal horn. Immunostaining for their phosphorylated counterparts was very low or undetectable in DRG and dorsal horn. Behavioral study showed that intrathecal mTOR inhibitor, rapamycin, did not affect acute nocicepetive transmission. The results indicate that although mTOR, p70S6K, and 4E-BP1 are highly expressed in the DRG and dorsal horn, their activate forms are very low in both regions under normal conditions. Our findings support the view that mTOR and its downstream effectors do not play a key role in acute pain. [Copyright &y& Elsevier]