Pain Hypersensitivity in SLURP1 and SLURP2 Knock-out Mouse Models of Hereditary Palmoplantar Keratoderma.

SLURP1 and SLURP2 are both small secreted members of the Ly6/u-PAR family of proteins and are highly expressed in keratinocytes. Loss-of-function mutations in SLURP1 lead to a rare autosomal recessive palmoplantar keratoderma (PPK), Mal de Meleda (MdM), which is characterized by diffuse, yellowish palmoplantar hyperkeratosis. Some individuals with MdM experience pain in conjunction with the hyperkeratosis that has been attributed to fissures or microbial superinfection within the affected skin. By comparison, other hereditary PPKs such as pachyonychia congenita and Olmsted syndrome show prevalent pain in PPK lesions. Two mouse models of MdM, Slurp1 knock-out and Slurp2X knock-out, exhibit robust PPK in all four paws. However, whether the sensory experience of these animals includes augmented pain sensitivity remains unexplored. In this study, we demonstrate that both models exhibit hypersensitivity to mechanical and thermal stimuli as well as spontaneous pain behaviors in males and females. Anatomical analysis revealed slightly reduced glabrous skin epidermal innervation and substantial alterations in palmoplantar skin immune composition in Slurp2X knock-out mice. Primary sensory neurons innervating hindpaw glabrous skin from Slurp2X knock-out mice exhibit increased incidence of spontaneous activity and mechanical hypersensitivity both in vitro and in vivo. Thus, Slurp knock-out mice exhibit polymodal PPK-associated pain that is associated with both immune alterations and neuronal hyperexcitability and might therefore be useful for the identification of therapeutic targets to treat PPK-associated pain.

The Transcription Factor Tbx5-Dependent Epigenetic Modification Contributes to Neuropathic Allodynia by Activating TRPV1 Expression in the Dorsal Horn.

Nerve injury can induce aberrant changes in the spine; these changes are due to, or at least partly governed by, transcription factors that contribute to the genesis of neuropathic allodynia. Here, we showed that spinal nerve ligation (SNL, a clinical neuropathic allodynia model) increased the expression of the transcription factor Tbx5 in the injured dorsal horn in male Sprague Dawley rats. In contrast, blocking this upregulation alleviated SNL-induced mechanical allodynia, and there was no apparent effect on locomotor function. Moreover, SNL-induced Tbx5 upregulation promoted the recruitment and interaction of GATA4 and Brd4 by enhancing its binding activity to H3K9Ac, which was enriched at the Trpv1 promotor, leading to an increase in TRPV1 transcription and the development of neuropathic allodynia. In addition, nerve injury-induced expression of Fbxo3, which abates Fbxl2-dependent Tbx5 ubiquitination, promoted the subsequent Tbx5-dependent epigenetic modification of TRPV1 expression during SNL-induced neuropathic allodynia. Collectively, our findings indicated that spinal Tbx5-dependent TRPV1 transcription signaling contributes to the development of neuropathic allodynia via Fbxo3-dependent Fbxl2 ubiquitination and degradation. Thus, we propose a potential medical treatment strategy for neuropathic allodynia by targeting Tbx5.

Development of opioid-induced hyperalgesia depends on reactive astrocytes controlled by Wnt5a signaling.

Opioids are the frontline analgesics for managing various types of pain. Paradoxically, repeated use of opioid analgesics may cause an exacerbated pain state known as opioid-induced hyperalgesia (OIH), which significantly contributes to dose escalation and consequently opioid overdose. Neuronal malplasticity in pain circuits has been the predominant proposed mechanism of OIH expression. Although glial cells are known to become reactive in OIH animal models, their biological contribution to OIH remains to be defined and their activation mechanism remains to be elucidated. Here, we show that reactive astrocytes (a.k.a. astrogliosis) are critical for OIH development in both male and female mice. Genetic reduction of astrogliosis inhibited the expression of OIH and morphine-induced neural circuit polarization (NCP) in the spinal dorsal horn (SDH). We found that Wnt5a is a neuron-to-astrocyte signal that is required for morphine-induced astrogliosis. Conditional knock-out of Wnt5a in neurons or its co-receptor ROR2 in astrocytes blocked not only morphine-induced astrogliosis but also OIH and NCP. Furthermore, we showed that the Wnt5a-ROR2 signaling-dependent astrogliosis contributes to OIH via inflammasome-regulated IL-1ß. Our results reveal an important role of morphine-induced astrogliosis in OIH pathogenesis and elucidate a neuron-to-astrocyte intercellular Wnt signaling pathway that controls the astrogliosis.

Genome-wide association studies with experimental validation identify a protective role for B lymphocytes against chronic post-surgical pain.

BACKGROUND: Chronic post-surgical pain (CPSP) significantly impacts patients' recovery and quality of life. Although environmental risk factors are well-established, genetic risk remains less understood. METHODS: A meta-analysis of genome-wide association studies followed by partitioned heritability was performed on 1350 individuals across five surgery types: hysterectomy, mastectomy, abdominal, hernia, and knee. In subsequent animal studies, withdrawal thresholds to evoked mechanical stimulation were measured in Rag1 null mutant and wild-type mice after plantar incision and laparotomy. Cell sorting by flow cytometry tracked recruitment of immune cell types. RESULTS: We discovered 77 genome-wide significant single-nucleotide polymorphism (SNP) hits, distributed among 24 loci and 244 genes. Meta-analysis of all cohorts estimated a SNP-based narrow-sense heritability for CPSP at ∼39%, indicating a substantial genetic contribution. Partitioned heritability analysis across a wide variety of tissues revealed enrichment of heritability in immune system-related genes, particularly those associated with B and T cells. Rag1 null mutant mice lacking both T and B cells exhibited exacerbated and prolonged allodynia up to 42 days after surgery, which was rescued by B-cell transfer. Recruitment patterns of B cells but not T cells differed significantly during the first 7 days after injury in the footpad, lymph nodes, and dorsal root ganglia. CONCLUSIONS: These findings suggest a key protective role for the adaptive immune system in the development of chronic post-surgical pain.

Protein Arginine Methyltransferase 5 Contributes to Paclitaxel-Induced Neuropathic Pain by Activating Transient Receptor Potential Vanilloid 1 Epigenetic Modification in Dorsal Root Ganglion.

BACKGROUND: Paclitaxel (PTX), which is a first-line chemotherapy drug used to treat various types of cancers, exhibits peripheral neuropathy as a common side effect that is difficult to treat. Protein arginine methyltransferase 5 (PRMT 5) is a key regulator of the chemotherapy response, as chemotherapy drugs induce PRMT5 expression. However, little is known about the PRMT5-mediated epigenetic mechanisms involved in PTX-induced neuropathic allodynia. METHODS: Sprague-Dawley rats were intraperitoneally given PTX to induce neuropathic pain. Biochemical analyses were conducted to measure the protein expression levels in the dorsal root ganglion (DRG) of the animals. The von Frey test and hot plate test were used to evaluate nociceptive behaviors. RESULTS: PTX increased the PRMT5 (mean difference [MD]: 0.68, 95% confidence interval [CI], 0.88-0.48; P < .001 for vehicle)-mediated deposition of histone H3R2 dimethyl symmetric (H3R2me2s) at the transient receptor potential vanilloid 1 ( Trpv1 ) promoter in the DRG. PRMT5-induced H3R2me2s recruited WD repeat domain 5 (WDR5) to increase trimethylation of lysine 4 on histone H3 (H3K4me3) at Trpv1 promoters, thus resulting in TRPV1 transcriptional activation (MD: 0.65, 95% CI, 0.82-0.49; P < .001 for vehicle) in DRG in PTX-induced neuropathic pain. Moreover, PTX increased the activity of NADPH oxidase 4 (NOX4) (MD: 0.66, 95% CI, 0.81-0.51; P < .001 for vehicle), PRMT5-induced H3R2me2s, and WDR5-mediated H3K4me3 in the DRG in PTX-induced neuropathic pain. Pharmacological antagonism and the selective knockdown of PRMT5 in DRG neurons completely blocked PRMT5-mediated H3R2me2s, WDR5-mediated H3K4me3, or TRPV1 expression and neuropathic pain development after PTX injection. Remarkably, NOX4 inhibition not only attenuated allodynia behavior and reversed the above-mentioned signaling but also reversed NOX4 upregulation via PTX. CONCLUSIONS: Thus, the NOX4/PRMT5-associated epigenetic mechanism in DRG has a dominant function in the transcriptional activation of TRPV1 in PTX-induced neuropathic pain.

Sirtuin 3 Mediated by Spinal cMyc-Enhancer of Zeste Homology 2 Pathway Plays an Important Role in Human Immunodeficiency Virus-Related Neuropathic Pain Model.

BACKGROUND: Clinical data demonstrate that chronic use of opioid analgesics increases neuropathic pain in people living with human immunodeficiency virus (HIV). Therefore, it is important to elucidate the molecular mechanisms of HIV-related chronic pain. In this study, we investigated the role of the transcription factor cMyc, epigenetic writer enhancer of zeste homology 2 (EZH2), and sirtuin 3 (Sirt3) pathway in HIV glycoprotein gp120 with morphine (gp120M)-induced neuropathic pain in rats. METHODS: Neuropathic pain was induced by intrathecal administration of recombinant gp120 with morphine. Mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. Spinal expression of cMyc, EZH2, and Sirt3 were measured using Western blots. Antinociceptive effects of intrathecal administration of antisense oligodeoxynucleotide against cMyc, a selective inhibitor of EZH2, or recombinant Sirt3 were tested. RESULTS: In the spinal dorsal horn, gp120M upregulated expression of cMyc (ratio of gp120M versus control, 1.68 ± 0.08 vs 1.00 ± 0.14, P = .0132) and EZH2 (ratio of gp120M versus control, 1.76 ± 0.05 vs 1.00 ± 0.16, P = .006), and downregulated Sirt3 (ratio of control versus gp120M, 1.00 ± 0.13 vs 0.43 ± 0.10, P = .0069) compared to control. Treatment with intrathecal antisense oligodeoxynucleotide against cMyc, GSK126 (EZH2 selective inhibitor), or recombinant Sirt3 reduced mechanical allodynia and thermal hyperalgesia in this gp120M pain model. Knockdown of cMyc reduced spinal EZH2 expression in gp120M treated rats. Chromatin immunoprecipitation (ChIP) assay showed that enrichment of cMyc binding to the ezh2 gene promoter region was increased in the gp120M-treated rat spinal dorsal horn, and that intrathecal administration of antisense ODN against cMyc (AS-cMyc) reversed the increased enrichment of cMyc. Enrichment of trimethylation of histone 3 on lysine residue 27 (H3K27me3; an epigenetic mark associated with the downregulation of gene expression) binding to the sirt3 gene promoter region was upregulated in the gp120M-treated rat spinal dorsal horn; that intrathecal GSK126 reversed the increased enrichment of H3K27me3 in the sirt3 gene promoter. Luciferase reporter assay demonstrated that cMyc mediated ezh2 gene transcription at the ezh2 gene promoter region, and that H3K27me3 silenced sirt3 gene transcription at the gene promoter region. CONCLUSION: These results demonstrated that spinal Sirt3 decrease in gp120M-induced neuropathic pain was mediated by cMyc-EZH2/H3K27me3 activity in an epigenetic manner. This study provided new insight into the mechanisms of neuropathic pain in HIV patients with chronic opioids.

αO-Conotoxin GeXIVA[1,2] Reduced Neuropathic Pain and Changed Gene Expression in Chronic Oxaliplatin-Induced Neuropathy Mice Model.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting painful neuropathy that occurs commonly during cancer management, which often leads to the discontinuation of medication. Previous studies suggest that the α9α10 nicotinic acetylcholine receptor (nAChR)-specific antagonist αO-conotoxin GeXIVA[1,2] is effective in CIPN models; however, the related mechanisms remain unclear. Here, we analyzed the preventive effect of GeXIVA[1,2] on neuropathic pain in the long-term oxaliplatin injection-induced CIPN model. At the end of treatment, lumbar (L4-L6) spinal cord was extracted, and RNA sequencing and bioinformatic analysis were performed to investigate the potential genes and pathways related to CIPN and GeXIVA[1,2]. GeXIVA[1,2] inhibited the development of mechanical allodynia induced by chronic oxaliplatin treatment. Repeated injections of GeXIVA[1,2] for 3 weeks had no effect on the mice's normal pain threshold or locomotor activity and anxiety-like behavior, as evaluated in the open field test (OFT) and elevated plus maze (EPM). Our RNA sequencing results identified 209 differentially expressed genes (DEGs) in the CIPN model, and simultaneously injecting GeXIVA[1,2] with oxaliplatin altered 53 of the identified DEGs. These reverted genes were significantly enriched in immune-related pathways represented by the cytokine-cytokine receptor interaction pathway. Our findings suggest that GeXIVA[1,2] could be a potential therapeutic compound for chronic oxaliplatin-induced CIPN management.

A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior.

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aß fibers. However, the mechanism by which Aß fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP+) and show that specific ablation or silencing of AAV-NpyP+ SDH interneurons converted touch-sensing Aß fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP+ neurons received excitatory inputs from Aß fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP+ neurons exhibited deeper resting membrane potentials, and their excitation by Aß fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP+ neurons in nerve-injured rats reversed Aß fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aß fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.

NFATc2-dependent epigenetic downregulation of the TSC2/Beclin-1 pathway is involved in neuropathic pain induced by oxaliplatin.

Neuropathic pain is a common dose-limiting side effect of oxaliplatin, which hampers the effective treatment of tumors. Here, we found that upregulation of transcription factor NFATc2 decreased the expression of Beclin-1, a critical molecule in autophagy, in the spinal dorsal horn, and contributed to neuropathic pain following oxaliplatin treatment. Meanwhile, manipulating autophagy levels by intrathecal injection of rapamycin (RAPA) or 3-methyladenine (3-MA) differentially altered mechanical allodynia in oxaliplatin-treated or naïve rats. Utilizing chromatin immunoprecipitation-sequencing (ChIP-seq) assay combined with bioinformatics analysis, we found that NFATc2 negatively regulated the transcription of tuberous sclerosis complex protein 2 (TSC2), which contributed to the oxaliplatin-induced Beclin-1 downregulation. Further assays revealed that NFATc2 regulated histone H4 acetylation and methylation in the TSC2 promoter site 1 in rats' dorsal horns with oxaliplatin treatment. These results suggested that NFATc2 mediated the epigenetic downregulation of the TSC2/Beclin-1 autophagy pathway and contributed to oxaliplatin-induced mechanical allodynia, which provided a new therapeutic insight for chemotherapy-induced neuropathic pain.

LncRNA NONRATT014888.2 contributes to cancer-induced bone pain through downregulation of natriuretic peptide receptor 3 in rats.

Long noncoding RNA (lncRNA) is implicated in both cancer development and pain process. However, the role of lncRNA in the development of cancer-induced bone pain (CIBP) is unclear. LncRNA NONRATT014888.2 is highly expressed in tibia related dorsal root ganglions (DRGs) in CIBP rats which function is unknown. CIBP was induced by injection of Walker 256 mammary gland tumor cells into the tibia canal of female SD rats. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of rats were measured. Down-regulation of NONRATT014888.2 by siRNA in CIBP rats markedly attenuated hind-paw mechanical pain hypersensitivity. LncRNA-predicted target mRNAs analysis and mRNA sequencing results cued Socs3, Npr3 were related with NONRATT014888.2. Intrathecal injection of NONRATT014888.2-siR206 upregulated Npr3 both in mRNA and protein level. Npr3 was co-expressed in NONRATT014888.2-positive DRGs neurons and mainly located in cytoplasm, but not in Glial fibrillary acidic protein (GFAP)-positive cells. Intrathecal injection of ADV-Npr3 upregulated Npr3 expression and enhanced the PWT of CIBP rats. Our results suggest that upregulated lncRNA NONRATT014888.2 contributed to hyperalgesia in CIBP rats, and the mechanism may through downregulation of Npr3.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

BACKGROUND: The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. METHODS: Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. RESULTS: PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. CONCLUSIONS: pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

Nerve Injury-Induced γH2AX Reduction in Primary Sensory Neurons Is Involved in Neuropathic Pain Processing.

Phosphorylation of the serine 139 of the histone variant H2AX (γH2AX) is a DNA damage marker that regulates DNA damage response and various diseases. However, whether γH2AX is involved in neuropathic pain is still unclear. We found the expression of γH2AX and H2AX decreased in mice dorsal root ganglion (DRG) after spared nerve injury (SNI). Ataxia telangiectasia mutated (ATM), which promotes γH2AX, was also down-regulated in DRG after peripheral nerve injury. ATM inhibitor KU55933 decreased the level of γH2AX in ND7/23 cells. The intrathecal injection of KU55933 down-regulated DRG γH2AX expression and significantly induced mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. The inhibition of ATM by siRNA could also decrease the pain threshold. The inhibition of dephosphorylation of γH2AX by protein phosphatase 2A (PP2A) siRNA partially suppressed the down-regulation of γH2AX after SNI and relieved pain behavior. Further exploration of the mechanism revealed that inhibiting ATM by KU55933 up-regulated extracellular-signal regulated kinase (ERK) phosphorylation and down-regulated potassium ion channel genes, such as potassium voltage-gated channel subfamily Q member 2 (Kcnq2) and potassium voltage-gated channel subfamily D member 2 (Kcnd2) in vivo, and KU559333 enhanced sensory neuron excitability in vitro. These preliminary findings imply that the down-regulation of γH2AX may contribute to neuropathic pain.

RNA-Seq Reveals Sex Differences in Gene Expression during Peripheral Neuropathic Inflammation and in Pain Relief from a COX-2 Inhibiting Theranostic Nanoemulsion.

Given decades of neuroinflammatory pain research focused only on males, there is an urgent need to better understand neuroinflammatory pain in females. This, paired with the fact that currently there is no long-term effective treatment for neuropathic pain furthers the need to evaluate how neuropathic pain develops in both sexes and how it can be relieved. Here we show that chronic constriction injury of the sciatic nerve caused comparable levels of mechanical allodynia in both sexes. Using a COX-2 inhibiting theranostic nanoemulsion with increased drug loading, both sexes achieved similar reduction in mechanical hypersensitivity. Given that both sexes have improved pain behavior, we specifically explored differential gene expression between sexes in the dorsal root ganglia (DRG) during pain and relief. Total RNA from the DRG revealed a sexually dimorphic expression for injury and relief caused by COX-2 inhibition. Of note, both males and females experience increased expression of activating transcription factor 3 (Atf3), however, only the female DRG shows decreased expression following drug treatment. Alternatively, S100A8 and S100A9 expression appear to play a sex specific role in relief in males. The sex differences in RNA expression reveal that comparable behavior does not necessitate the same gene expression.

Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences.

Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sex-dependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line, Cx3CR1-CreERT2-eYFP;TLR4fl/fl (TLR4 cKO) to specifically knock out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time- and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.SIGNIFICANCE STATEMENT The contribution of myeloid cell TLR4 to sex-specific pain progression remains controversial. We used complementary pharmacologic and transgenic approaches to specifically manipulate TLR4 based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We discovered that microglial TLR4 contributes to early pain progression in males, and to a lesser extent in females. We further found that maintenance of chronic pain likely occurs through myeloid TLR4-independent mechanisms in both sexes. Together, we define a more nuanced contribution of this receptor to the acute-to-chronic pain transition in a mouse model of complex regional pain syndrome.

LncRNA NONRATT009773.2 promotes bone cancer pain progression through the miR-708-5p/CXCL13 axis.

Bone cancer pain (BCP) is the most frequently observed chronic cancer pain, and its development remains largely unexplored. Dysregulation of non-coding RNAs greatly contributes to the pathogenesis of BCP. In the present study, we found a new long noncoding RNA (lncRNA), NONRATT009773.2, and investigated its role in the spinal cord of BCP rats. Our results showed that NONRATT009773.2 was significantly up-regulated in BCP model rats, whereas depletion of NONRATT009773.2 attenuated BCP. In contrast, overexpression of NONRATT009773.2 triggered pain-like symptoms in normal animals. Moreover, NONRATT009773.2 functioned as a microRNA (miRNA) sponge to absorb miR-708-5p and up-regulated miRNA downstream target CXCL13, which plays fundamental roles in the initiation and maintenance of neuroinflammation and hyperalgesia. Collectively, our current findings indicated that NONRATT009773.2 could be employed as a new therapeutic target for BCP.

The Role of Epigenomic Regulatory Pathways in the Gut-Brain Axis and Visceral Hyperalgesia.

The gut-brain axis (GBA) is broadly accepted to describe the bidirectional circuit that links the gastrointestinal tract with the central nervous system (CNS). Interest in the GBA has grown dramatically over past two decades along with advances in our understanding of the importance of the axis in the pathophysiology of numerous common clinical disorders including mood disorders, neurodegenerative disease, diabetes mellitus, non-alcohol fatty liver disease (NAFLD) and enhanced abdominal pain (visceral hyperalgesia). Paralleling the growing interest in the GBA, there have been seminal developments in our understanding of how environmental factors such as psychological stress and other extrinsic factors alter gene expression, primarily via epigenomic regulatory mechanisms. This process has been driven by advances in next-generation multi-omics methods and bioinformatics. Recent reviews address various components of GBA, but the role of epigenomic regulatory pathways in chronic stress-associated visceral hyperalgesia in relevant regions of the GBA including the amygdala, spinal cord, primary afferent (nociceptive) neurons, and the intestinal barrier has not been addressed. Rapidly developing evidence suggests that intestinal epithelial barrier dysfunction and microbial dysbiosis play a potentially significant role in chronic stress-associated visceral hyperalgesia in nociceptive neurons innervating the lower intestine via downregulation in intestinal epithelial cell tight junction protein expression and increase in paracellular permeability. These observations support an important role for the regulatory epigenome in the development of future diagnostics and therapeutic interventions in clinical disorders affecting the GBA.

The pathological involvement of spinal cord EphB2 in visceral sensitization in male rats.

Patients with post-traumatic stress disorder (PTSD) are usually at an increased risk for chronic disorders, such as irritable bowel syndrome (IBS), characterized by hyperalgesia and allodynia, but its subsequent effect on visceral hyperalgesia and the mechanism remain unclear. The present study employed single prolonged stress (SPS), a model of PTSD-pain comorbidity, behavioral evaluation, intrathecal drug delivery, immunohistochemistry, Western blotting, and RT-PCR techniques. When detecting visceral sensitivity, the score of the abdominal withdrawal reflex (AWR) induced by graded colorectal distention (CRD) was used. The AWR score was reduced in the SPS day 1 group but increased in the SPS day 7 and SPS day 14 groups at 40 mmHg and 60 mmHg, and the score was increased significantly with EphrinB1-Fc administration. The EphB2+ cell density and EphB2 protein and mRNA levels were downregulated in the SPS day 1 group and then upregulated significantly in the SPS day 7 group; these changes were more noticeable with EphrinB1-Fc administration compared with the SPS-only group. The C-Fos-positive reaction induced by SPS was mainly localized in neurons of the spinal dorsal horn, in which the C-Fos-positive cell density and its protein and mRNA levels were upregulated on SPS days 7 and 14; these changes were statistically significant in the SPS + EphrinB1-Fc group compared with the SPS alone group. The present study confirmed the time window for the AWR value, EphB2 and C-Fos changes, and the effect of EphrinB1-Fc on these changes, which suggests that spinal cord EphB2 activation exacerbates visceral pain after SPS.

TNF-α-Mediated RIPK1 Pathway Participates in the Development of Trigeminal Neuropathic Pain in Rats.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) participates in the regulation of cellular stress and inflammatory responses, but its function in neuropathic pain remains poorly understood. This study evaluated the role of RIPK1 in neuropathic pain following inferior alveolar nerve injury. We developed a model using malpositioned dental implants in male Sprague Dawley rats. This model resulted in significant mechanical allodynia and upregulated RIPK1 expression in the trigeminal subnucleus caudalis (TSC). The intracisternal administration of Necrosatin-1 (Nec-1), an RIPK1 inhibitor, blocked the mechanical allodynia produced by inferior alveolar nerve injury The intracisternal administration of recombinant rat tumor necrosis factor-α (rrTNF-α) protein in naive rats produced mechanical allodynia and upregulated RIPK1 expression in the TSC. Moreover, an intracisternal pretreatment with Nec-1 inhibited the mechanical allodynia produced by rrTNF-α protein. Nerve injury caused elevated TNF-α concentration in the TSC and a TNF-α block had anti-allodynic effects, thereby attenuating RIPK1 expression in the TSC. Finally, double immunofluorescence analyses revealed the colocalization of TNF receptor and RIPK1 with astrocytes. Hence, we have identified that astroglial RIPK1, activated by the TNF-α pathway, is a central driver of neuropathic pain and that the TNF-α-mediated RIPK1 pathway is a potential therapeutic target for reducing neuropathic pain following nerve injury.

Deletion of the Gene Encoding the NMDA Receptor GluN1 Subunit in Schwann Cells Causes Ultrastructural Changes in Remak Bundles and Hypersensitivity in Pain Processing.

Abnormalities in interactions between sensory neurons and Schwann cells (SCs) may result in heightened pain processing and chronic pain states. We previously reported that SCs express the NMDA receptor (NMDA-R), which activates cell signaling in response to glutamate and specific protein ligands, such as tissue-type plasminogen activator. Herein, we genetically targeted grin1 encoding the essential GluN1 NMDA-R subunit, conditionally in SCs, to create a novel mouse model in which SCs are NMDA-R-deficient (GluN1- mice). These mice demonstrated increased sensitivity to light touch, pinprick, and thermal hyperalgesia in the absence of injury, without associated changes in motor function. Ultrastructural analysis of adult sciatic nerve in GluN1- mice revealed increases in the density of Aδ fibers and Remak bundles and a decrease in the density of Aß fibers, without altered g-ratios. Abnormalities in adult Remak bundle ultrastructure were also present including aberrant C-fiber ensheathment, distances between axons, and increased poly-axonal pockets. Developmental and post radial sorting defects contributed to altered nerve fiber densities in adult. Uninjured sciatic nerves in GluN1- mice did not demonstrate an increase in neuroinflammatory infiltrates. Transcriptome profiling of dorsal root ganglia (DRGs) revealed 138 differentially regulated genes in GluN1- mice. One third of the regulated genes are known to be involved in pain processing, including sprr1a, npy, fgf3, atf3, and cckbr, which were significantly increased. The intraepidermal nerve fiber density (IENFD) was significantly decreased in the skin of GluN1- mice. Collectively, these findings demonstrate that SC NMDA-R is essential for normal PNS development and for preventing development of pain states.SIGNIFICANCE STATEMENT Chronic unremitting pain is a prevalent medical condition; however, the molecular mechanisms that underlie heightened pain processing remain incompletely understood. Emerging data suggest that abnormalities in Schwann cells (SCs) may cause neuropathic pain. We established a novel mouse model for small fiber neuropathy (SFN) in which grin1, the gene that encodes the NMDA receptor (NMDA-R) GluN1 subunit, is deleted in SCs. These mice demonstrate hypersensitivity in pain processing in the absence of nerve injury. Changes in the density of intraepidermal small fibers, the ultrastructure of Remak bundles, and the transcriptome of dorsal root ganglia (DRGs) provide possible explanations for the increase in pain processing. Our results support the hypothesis that abnormalities in communication between sensory nerve fibers and SCs may result in pain states.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.