Maintenance Of Neuropathic Pain Research Articles

Astrocytic NDRG2 is critical in the maintenance of neuropathic pain

Activation of astrocytes and abnormal synaptic glutamate metabolism are closely associated with the induction and maintenance of neuropathic pain (NP), but the exact mechanism underlying this association remains unclear. N-myc downstream-regulated gene 2 (NDRG2), a novel tumor-suppressor protein and stress-response gene, is involved in the pathogenesis of several neurodegenerative diseases. However, its role in nociceptive transduction has rarely been investigated. Here, we found that NDRG2, which was mainly expressed in the astrocytes in the central nervous system (CNS), was increased in the spinal cord of a spinal nerve ligation (SNL) rat model for NP. Suppression of NDRG2 by intrathecal injection of an NDRG2-RNAi-adenovirus significantly alleviated SNL-induced mechanical and thermal hypersensitivity, as well as elevated astrocytic glutamate transporter 1 (GLT-1) expression and downregulated pro-inflammatory cytokine levels, in the spinal dorsal horn of rats on Day 10 after SNL. Furthermore, in lipopolysaccharide (LPS)-stimulated primary astrocytic cultures derived from neonatal rats, inhibition of NDRG2 significantly reversed both the LPS-induced activation of astrocytes and decreased expression of GLT-1. By contrast, overexpression of NDRG2 by an adenoviral vector carrying NDRG2 resulted in astrocytic activation, aberrant glutamatergic neurotransmission, and spontaneous nociceptive responses in rats. Intrathecal injection of AG490, which is an inhibitor of the Janus tyrosine kinase and signal transducer and activator of the transcription 3 (JAK/STAT3) signaling pathway, significantly attenuated both mechanical and thermal hyperalgesia, as well as inhibited reactive astrocytes and restored normal expression levels of astrocytic GLT-1, in the spinal dorsal horn of NDRG2-overexpression rats. In conclusion, spinal astrocytic NDRG2 is critical in the maintenance of NP. Moreover, NDRG2 modulates astrocytic activation and inflammatory responses via regulating GLT-1 expression through the JAK/STAT3 signaling pathway. Our findings suggested that NDRG2 could be a novel therapeutic target for the treatment of NP.

Quercetin attenuates diabetic neuropathic pain by inhibiting mTOR/p70S6K pathway-mediated changes of synaptic morphology and synaptic protein levels in spinal dorsal horn of db/db mice

Numerous studies indicate that the changes of synaptic morphology and synaptic protein levels in spinal dorsal horn neurons contributes to the development and maintenance of neuropathic pain. Quercetin, a bioflavonoid compound, has been shown to have analgesic effect in several pain models. However, the underlying mechanism for quercetin to allieviate pain is unclear. Therefore, in this study, we observed the effect of quercetin on diabetic neuropathic pain in db/db mice and explored the underlying mechanisms. Our results showed that chronic quercetin treatment alleviated thermal hyperalgesia in db/db mice. Moreover, quercetin administration significantly reduced the total dendritic length, the number of dendritic branches, and the dendritic spine density in the spinal dorsal horn neurons of db/db mice. Meanwhile, the up-regulated expressions of synaptic plasticity-associated proteins postsynaptic density protein 95 (PSD-95) and synaptophysin in spinal dorsal horn of db/db mice were decreased by quercetin treatment. In addition, quercetin treatment reduced the phosphorylated levels of mammalian target of rapamycin (mTOR) and p70 ribosomal S6 kinase (p70S6K) in spinal dorsal horn of db/db mice. These results demonstrate that quercetin may alleviate diabetic neuropathic pain by inhibiting mTOR/p70S6K pathway-mediated changes of synaptic morphology and synaptic protein levels in spinal dorsal horn neurons of db/db mice. These findings suggest that quercetin may be a promising therapeutic drug in neuropathic pain.

Microglia: sculptors of neuropathic pain?

Neuropathic pain presents a huge societal and individual burden. The limited efficacy of current analgesics, diagnostic markers and clinical trial outcome measures arises from an incomplete understanding of the underlying mechanisms. A large and growing body of evidence has established the important role of microglia in the onset and possible maintenance of neuropathic pain, and these cells may represent an important target for future therapy. Microglial research has further revealed their important role in structural remodelling of the nervous system. In this review, we aim to explore the evidence for microglia in sculpting nervous system structure and function, as well as their important role in neuropathic pain, and finally integrate these studies to synthesize a new model for microglia in somatosensory circuit remodelling, composed of six key and inter-related mechanisms. Summarizing the mechanisms through which microglia modulate nervous system structure and function helps to frame a better understanding of neuropathic pain, and provide a clear roadmap for future research.

Anti-inflammatory protein TSG-6 secreted by bone marrow mesenchymal stem cells attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-\u03baB signaling pathway in spinal microglia

BackgroundNeuroinflammation plays a vital role in the development and maintenance of neuropathic pain. Recent evidence has proved that bone marrow mesenchymal stem cells (BMSCs) can inhibit neuropathic pain and possess potent immunomodulatory and immunosuppressive properties via secreting a variety of bioactive molecules, such as TNF-α-stimulated gene 6 protein (TSG-6). However, it is unknown whether BMSCs exert their analgesic effect against neuropathic pain by secreting TSG-6. Therefore, the present study aimed to evaluate the analgesic effects of TSG-6 released from BMSCs on neuropathic pain induced by chronic constriction injury (CCI) in rats and explored the possible underlying mechanisms in vitro and in vivo.MethodsBMSCs were isolated from rat bone marrow and characterized by flow cytometry and functional differentiation. One day after CCI surgery, about 5 × 106 BMSCs were intrathecally injected into spinal cerebrospinal fluid. Behavioral tests, including mechanical allodynia, thermal hyperalgesia, and motor function, were carried out at 1, 3, 5, 7, 14 days after CCI surgery. Spinal cords were processed for immunohistochemical analysis of the microglial marker Iba-1. The mRNA and protein levels of pro-inflammatory cytokines (IL-1β, TNFα, IL-6) were detected by real-time RT-PCR and ELISA. The activation of the TLR2/MyD88/NF-κB signaling pathway was evaluated by Western blot and immunofluorescence staining. The analgesic effect of exogenous recombinant TSG-6 on CCI-induced mechanical allodynia and heat hyperalgesia was observed by behavioral tests. In the in vitro experiments, primary cultured microglia were stimulated with the TLR2 agonist Pam3CSK4, and then co-cultured with BMSCs or recombinant TSG-6. The protein expression of TLR2, MyD88, p-p65 was evaluated by Western blot. The mRNA and protein levels of IL-1β, TNFα, IL-6 were detected by real-time RT-PCR and ELISA. BMSCs were transfected with the TSG-6-specific shRNA and then intrathecally injected into spinal cerebrospinal fluid in vivo or co-cultured with Pam3CSK4-treated primary microglia in vitro to investigate whether TSG-6 participated in the therapeutic effect of BMSCs on CCI-induced neuropathic pain and neuroinflammation.ResultsWe found that CCI-induced mechanical allodynia and heat hyperalgesia were ameliorated by intrathecal injection of BMSCs. Moreover, intrathecal administration of BMSCs inhibited CCI-induced neuroinflammation in spinal cord tissues. The analgesic effect and anti-inflammatory property of BMSCs were attenuated when TSG-6 expression was silenced. We also found that BMSCs inhibited the activation of the TLR2/MyD88/NF-κB pathway in the ipsilateral spinal cord dorsal horn by secreting TSG-6. Meanwhile, we proved that intrathecal injection of exogenous recombinant TSG-6 effectively attenuated CCI-induced neuropathic pain. Furthermore, in vitro experiments showed that BMSCs and TSG-6 downregulated the TLR2/MyD88/NF-κB signaling and reduced the production of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, in primary microglia treated with the specific TLR2 agonist Pam3CSK4.ConclusionsThe present study demonstrated a paracrine mechanism by which intrathecal injection of BMSCs targets the TLR2/MyD88/NF-κB pathway in spinal cord dorsal horn microglia to elicit neuroprotection and sustained neuropathic pain relief via TSG-6 secretion.

The AMPK pathway triggers autophagy during CSF1-induced microglial activation and may be implicated in inducing neuropathic pain

The development and maintenance of neuropathic pain is now given far more attention in the clinic work. Increasing evidence has shown that colony-stimulating factor 1 (CSF1) is involved in microglial activation and may further induce pain. Here, we observed the signaling events that link the CSF1-induced microglial activated and consequences for pain processing. For the in vitro study, flow cytometry showed the microglial activity was markedly increased after CSF1 stimulation. Western blot showed the increased expression of p-PRKAA1/PRKAA1, p-AMPK/AMPK, p-ULK1/ULK1, p-S6k/S6k and LC3-II/LC3-I. QRT-PCR showed the IL-1, TNF-α and BDNF were simultaneously upregulated in the activated microglia cells, whereas the specific AMPK inhibitor compound C exhibited reverse effects in microglia. Using immunofluorescence staining and electron microscopy, we found CSF1 decreased microglial p62 expression and induced the number of autophagosomes, whereas compound C significantly exhibited the reverse effects. For the in vivo study, compared with the control and AMPK-siRNA transfection, the mice under CSF1 intrathecal injection increased CSF1 receptor and LC3 expressed in the activated spinal microglia. More importantly, qRT-PCR showed CSF1 intrathecal injection substantially upregulated BDNF and c-Fos mRNA expression as well as the ensuing neuropathic pain. Our findings demonstrated that CSF1 induced a significant upregulation of microglial activation via the AMPK signaling pathway and resulted in an increasing microglial autophagic level. An increasing CSF1 level in the central nervous system can mimic and cause pain syndromes by up-regulation of AMPK-depended autophagy, thus offering a new target for the therapy of neuropathic pain.

PARP-1-regulated TNF-α expression in the dorsal root ganglia and spinal dorsal horn contributes to the pathogenesis of neuropathic pain in rats

Emerging evidence has implicated poly-(ADP-ribose) polymerase 1 (PARP-1), a transcriptional coregulator, in a variety of inflammatory diseases. In the current study, the role of PARP-1 in neuropathic pain and the underlying mechanisms were investigated. Neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rates. Western blotting, qRT-PCR, immunohistochemistry, chromatin immunoprecipitation (ChIP), and Co-IP assays were performed to elucidate the mechanisms. The results showed that SNL resulted in a significant increase in the expression and activation of PARP-1 in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn, which occurred on day one, reached peak on day 7, and persisted more than 2 weeks after surgery. Double immunofluorescence staining revealed that PARP-1 was expressed exclusively in DRG A-type and C-type neurons. In the spinal cord, PARP-1 mainly colocalized with the neuronal marker NeuN and the astrocytic marker GFAP specifically in the superficial lamina. Prior intrathecal (i.t.) injection of PJ-34, a PARPs inhibitor, or Tiq-A, a specific PARP-1 inhibitor, dose-dependently prevented the reductions in PWT and PWL following SNL. Established neuropathic pain-like hypersensitivity was also attenuated with i.t. injection of PJ-34 and Tiq-A starting on day 7 following SNL, a timepoint at which neuropathic pain was fully established. SNL-induced mechanical allodynia and thermal hyperalgesia were also alleviated by i.t. injection of PARP-1 siRNA following a reduction in PARP-1 expression in the dorsal horn. Moreover, the SNL-induced increases in TNF-α protein and mRNA in the dorsal horn and DRG were dramatically suppressed by i.t. injection of Tiq-A or PARP-1 siRNA. The i.t. lipopolysaccharide (LPS)-induced increase in the production of TNF-α in the dorsal horn was also inhibited by prior to i.t. injection of PARP-1 siRNA. Results of ChIP assay showed that SNL-induced PARP-1 activation promoted the binding of NF-κB p65 with the TNF-α promoter in the dorsal horn and that PARP-1 inhibition reduced this binding and suppressed TNF-α expression. Co-IP assay revealed that SNL caused a significant increase in the level of histone H1 poly(ADP)–ribosylation. Together, these results indicate that PARP-1-regulated TNF-α expression in the DRG and spinal dorsal horn following SNL contributes to the development and maintenance of neuropathic pain. Targeting PARP-1 might be a promising therapeutic strategy for the treatment of the chronic pain.

Development of GPR183 Antagonists to Treat Neuropathic Pain

Chronic neuropathic pain affects millions of people in the United States today. Opioids are currently the frontline agent for the treatment of neuropathic pain; however, as their use continues to grow, serious side effects can arise including addiction and death. The need for a safer alternative to opioids is becoming increasingly more urgent. GPR183 (Ebstein‐Barr induced gene 2, EBI2) is a G‐protein coupled receptor (GPCR) that binds to 7a,25‐dihydroxycholesterol (7a,25‐OHC). A homology model for GPR183 was constructed and a pharmacophore hypothesis was built around the binding of other known ligands to the receptor. Over 5 million compounds were screened in silico for their similarity to our pharmacophore hypothesis and the top 20k hits were funneled into a high‐throughput docking protocol. Several compounds showed strong inhibition of 7a,25‐OHC calcium mobilization with nanomolar potency. Intrathecal injections of these GPR183 antagonists reversed CCI‐induced mechano‐allodynia in mice when given at peak neuropathic pain. These results identify a prominent and functional role for GPR183 in the spinal cord in the maintenance of neuropathic pain. A preliminary structure‐activity relationship study of one of these compounds is being performed to study the pharmacology and medicinal properties of this new class of compounds.Support or Funding InformationWe would like to acknowledge Dr. Dale Dorsett and the Saint Louis University Genomics Core Facility for their assistance with the RNA‐Sequencing. This research was supported by funds from Dr. Salvemini and Dr. Arnatt and T32 GM008306‐01 training grant (KB). Dr. Salvemini and Dr. Arnatt have filed a patent describing the intellectual property generated at SLU. All other authors claim no conflict of interest.

Upregulation of Myeloid Zinc Finger 1 in Dorsal Root Ganglion via Regulating Matrix Metalloproteinase-2/9 and Voltage-gated Potassium 1.2 Expression Contributes to Complete Freund’s Adjuvant-induced Inflammatory Pain

Myeloid zinc finger 1 (MZF1) belongs to the Kruppel family of zinc-finger transcription factors. Recent studies have demonstrated that in dorsal root ganglion (DRG) neurons, MZF1 is involved in the development and maintenance of neuropathic pain. However, the role of MZF1 in inflammatory pain still remains unknown. In the present study, the mechanism of MZF1 in chronic inflammatory pain was investigated in rats received an intraplantar injection of complete Freund’s adjuvant (CFA). Subsequently, a series of assays including Western blotting, qRT-PCR, immunohistochemistry, and chromatin immunoprecipitation (ChIP) were performed. We found that CFA led to MZF1 upregulation in ipsilateral L4/5 DRGs. Pre- and post-microinjection of MZF1 siRNA into the ipsi-L5 DRG blocked the development of CFA-induced chronic inflammatory pain and alleviated the mechanical allodynia and thermal hyperalgesia in the maintenance phase. CFA also increased MMP-2/9 and Nav1.8 expression but reduced voltage-gated potassium 1.2 (Kv1.2) and Cav1.2 expression in L4/L5 DRGs. Microinjection of MZF1 siRNA into DRG diminished the CFA-induced changes in MMP-2/9 and Kv1.2 expression. However, the expressions of Nav1.8 and Cav1.2 were not changed by the treatment. Double immunofluorescence staining showed that MMP-2/9 and Kv1.2 were co-localized with MZF1 in DRGs. The ChIP-PCR results revealed that MZF1 binds directly to the promoter region of MMP-2/9 gene. Together, the above results imply that upregulation of MZF1 in DRGs might contribute to the development and maintenance of CFA-induced chronic inflammatory pain by regulating MMP-2/9 and Kv1.2 expression. Targeting DRG-localized MZF1 might be a promising therapeutic strategy for the treatment of chronic inflammatory pain in the clinic.

Bilateral activation of glial cells and cellular distribution of the chemokine CCL2 and its receptor CCR2 in the trigeminal subnucleus caudalis of trigeminal neuropathic pain model.

Glial cells activated by peripheral nerve injury contribute to the induction and maintenance of neuropathic pain by releasing neuromodulating cytokines and chemokines. We investigated the activation of microglia and astrocytes as well as the cellular distribution of the chemokine CCL2 and its receptor CCR2 in the trigeminal subnucleus caudalis (TSC) ipsilateral and contralateral to infraorbital nerve ligature (IONL). The left infraorbital nerve was ligated under aseptic conditions, and sham controls were operated without nerve ligature. Tactile hypersensitivity was significantly increased bilaterally in vibrissal pads of both sham- and IONL-operated animals from day 1 to 7 and tended to normalize in sham controls surviving for 14days. Activated microglial cells significantly increased bilaterally in the TSC of both sham- and IONL-operated animals with a marked but gradual increase in the ipsilateral TSC from 1 to 7days followed by a decrease by day 14. In contrast, robust activation of astrocytes was found bilaterally in the TSC of IONL-operated rats from 3 to 14days with a transient activation in the ipsilateral TSC of sham-operated animals. Cellular distribution of CCL2 varied with survival time. CCL2 immunofluorescence was detected in neurons within 3days and in astrocytes at later time points. In contrast, CCR2 was found only in astrocytes at all time points with CCR2 intensity being dominant in the ipsilateral TSC. In summary, our results reveal bilateral activation of microglial cells and astrocytes as well as changes in the cellular distribution of CCL2 and its receptor CCR2 in the TSC during the development and maintenance of orofacial neuropathic pain.

Multiple Morphometric Assessment of Microglial Cells in Deafferented Spinal Trigeminal Nucleus.

Microglia (MG) are the first cells to react to the abnormal incoming signals that follow an injury of sensory nerves and play a critical role in the development and maintenance of neuropathic pain, a common sequel of nerve injuries. Here we present population data on cell number, soma size, and length of processes of MG in the caudal division of the spinal trigeminal nucleus (Sp5C) in control mice and at the peak of microgliosis (7 days) following unilateral transection of the infraorbital nerve (IoN). The study is performed combining several bias- and assumption-free imaging and stereological approaches with different immunolabeling procedures, with the objective of tackling some hard problems that often hinder proper execution of MG morphometric studies. Our approach may easily be applied to low-density MG populations, but also works, with limited biases, in territories where MG cell bodies and processes form dense meshworks. In controls, and contralaterally to the deafferented side, MG cell body size and shape and branching pattern matched well the descriptions of “resting” or “surveillant” MG described elsewhere, with only moderate intersubject variability. On the superficial laminae of the deafferented side, however, MG displayed on average larger somata and remarkable diversity in shape. The number of cells and the length of MG processes per mm3 increased 5 and 2.5 times, respectively, indicating a net 50% decrease in the mean length of processes per cell. By using specific immunolabeling and cell sorting of vascular macrophages, we found only a negligible fraction of these cells in Sp5C, with no differences between controls and deafferented animals, suggesting that blood-borne monocytes play at most a very limited role in the microgliosis occurring following sensory nerve deafferentation. In sum, here we present reliable morphometric data on MG in control and deafferented trigeminal nuclei using efficient methods that we propose may equally be applied to any morphometric population analysis of these cells under different physiological or pathological conditions.

Long Noncoding RNA H19 in the Injured Dorsal Root Ganglion Contributes to Peripheral Nerve Injury-Induced Pain Hypersensitivity

Peripheral nerve injury-induced changes in gene transcription and translation in the dorsal root ganglion (DRG) play a critical role in the development and maintenance of neuropathic pain. Long noncoding RNAs (lncRNAs) regulate gene expression. Here, we report that peripheral nerve injury caused by ligation of the fourth spinal nerve (SNL) led to a time-dependent increase in the expression in H19, an lncRNA, in the injured DRG. Microinjection of a specific H19 siRNA, but not negative control scrambled siRNA, into the injured DRG 4 days before SNL alleviated mechanical allodynia and thermal hyperalgesia on days 3 and 5 post-SNL. Additionally, DRG microinjection of the H19 siRNA on day 7 after SNL reduced mechanical allodynia and thermal hyperalgesia on days 10 and 12 post-SNL. DRG microinjection of neither siRNA affected locomotor activity and acute basal responses to mechanical and thermal stimuli. Our findings suggest that H19 participates in the peripheral mechanism underlying the development and maintenance of neuropathic pain. H19 may be a potential target for treatment of this disorder.

Indomethacin attenuates mechanical allodynia during the organization but not the maintenance of the peripheral neuropathic pain induced by nervus ischiadicus chronic constriction injury.

The neurochemical mechanisms underlying neuropathic pain (NP) are related to peripheral and central sensitization caused by the release of inflammatory mediators in the peripheral damaged tissue and ectopic discharges from the injured nerve, leading to a hyperexcitable state of spinal dorsal horn neurons. The aim of this work was to clarify the role played by cyclooxygenase (COX) in the lesioned peripheral nerve in the development and maintenance of NP by evaluating at which moment the non-steroidal anti-inflammatory drug indomethacin, a non-selective COX inhibitor, attenuated mechanical allodynia after placing one loose ligature around the nervus ischiadicus, an adaptation of Bennett and Xie's model in rodents. NP was induced in male Wistar rats by subjecting them to chronic constriction injury (CCI) of the nervus ischiadicus, placing one loose ligature around the peripheral nerve, and a sham surgery (without CCI) was used as control. Indomethacin (2 mg/kg) or vehicle was intraperitoneally and acutely administered in each group of rats and at different time windows (1, 2, 4, 7, 14, 21, and 28 days) after the CCI or sham surgical procedures, followed by von Frey's test for 30 min. The data showed that indomethacin decreased the mechanical allodynia threshold of rats on the first, second, and fourth days after CCI (P<0.05). These findings suggested that inflammatory mechanisms are involved in the induction of NP and that COX-1 and COX-2 are involved in the induction but not in the maintenance of NP.

MicroRNA-547-5p-mediated interleukin-33/suppressor of tumorigenicity 2 signaling underlies the genesis and maintenance of neuropathic pain and is targeted by the therapy with bone marrow stromal cells.

Interleukin-33 (IL-33)/suppressor of tumorigenicity 2 (ST2) signaling is known to promote inflammation and the genesis and maintenance of neuropathic pain. However, it remained mostly unknown how IL-33/ST2 signaling can be enhanced by neuropathic stimulations. Here, we report that the chronic constriction nerve injury (CCI)-induced increases in the expression of IL-33 and ST2 and a decrease in microRNA (miRNA)-547-5p not only in the dorsal root ganglia (DRG) but also in spinal dorsal horn (SDH) ipsilateral to the CCI. We found that increasing endogenous miRNA-547-5p by the intrathecal (i.t.) infusion of agomir-miR-547-5p did not produce any effect in naive rats but blocked the CCI-induced increases in the IL-33 and ST2, and pain sensitivity. The reducing endogenous miRNA-547-5p by the i.t. delivering antagomir-miR-547-5p into naive rats caused significant changes in IL-33 and ST2 expressions in both the DRG and SDH, and pain sensitivity, which were similar to those induced by the CCI. Since increasing IL-33 by the i.t. infusion of recombinant IL-33 produced no change in the expression of miR-547-5p, and the CCI still reduced miR-547-5p expression in rats with the IL-33 knockdown, we conclude that the reduction of miR-547-5p can be an upstream event leading to the enhancement of IL-33/ST2 signaling induced by the CCI. The intravenous application of bone marrow stromal cells (BMSCs) reduced the depression of miR-547-5p in both the DRG and SDH, and pain hypersensitivity produced by the CCI or antagomir-miR547-5p application. However, the BMSC effect was significantly occluded by the pretreatment with miR-547-5p agomir or the IL-33 knockdown, demonstrating a novel mechanism underlying the BMSC therapy.

Dorsal Root Ganglia Homeobox downregulation in primary sensory neurons contributes to neuropathic pain in rats.

Transcriptional changes in primary sensory neurons are involved in initiation and maintenance of neuropathic pain. However, the transcription factors in primary sensory neurons responsible for neuropathic pain are not fully understood. Dorsal Root Ganglia Homeobox (DRGX) is a paired-like homeodomain transcription factor necessary for the development of nociceptive primary sensory neurons during the early postnatal period. However, roles for DRGX after development are largely unknown. Here, we report that DRGX downregulation in primary sensory neurons as a result of post-developmental nerve injury contributes to neuropathic pain in rats. DRGX expression was decreased in nuclei of small and medium primary sensory neurons after spinal nerve ligation. DRGX downregulation by transduction of a short hairpin RNA with an adeno-associated viral vector induced mechanical allodynia and thermal hyperalgesia. In contrast, DRGX overexpression in primary sensory neurons suppressed neuropathic pain. DRGX regulated matrix metalloproteinase-9 (MMP-9) and prostaglandin E receptor 2 mRNA expression in the DRG. MMP-9 inhibitor attenuated DRGX downregulation-induced pain. These results suggest that DRGX downregulation after development contributes to neuropathic pain through transcriptional modulation of pain-related genes in primary sensory neurons.

DRG神経におけるホメオボックス遺伝子DRGXの神経障害性疼痛への関与

Neuropathic pain is caused by lesions or diseases of the somatosensory system and is less responsive to pain medications. Transcriptomic changes in dorsal root ganglion (DRG) neurons are involved in initiation and maintenance of neuropathic pain. Dorsal Root Ganglia Homeobox (DRGX) is a paired-like homeodomain transcription factor crucial for the development of nociceptive DRG neurons. However, roles for DRGX after development are almost unclarified. Here, we report that DRGX downregulation in DRG neurons due to nerve injury in the post-developmental stage is involved in neuropathic pain in rats. DRGX expression was persistently decreased in DRG neurons in neuropathic pain model rats produced by spinal nerve ligation. DRGX protein was mainly downregulated in nuclei of small-to-medium DRG neurons after the nerve injury. Additionally, DRGX downregulation using an adeno-associated viral vector expressing short hairpin RNA induced mechanical allodynia and thermal hyperalgesia in intact rats, while DRGX overexpression suppressed neuropathic pain. DRGX regulated mRNA expression of matrix metalloproteinase-9 and prostaglandin E receptor 2 in the DRG. These results suggest that DRGX downregulation after development contributes to neuropathic pain through transcriptional modulation of pain-related genes in DRG neurons.

Beneficial effects of fish oil enriched in omega-3 fatty acids on the development and maintenance of neuropathic pain.

The aim of this work was to assess the preventive effect of an eicosapentaenoic acid/docosahexaenoic acid-concentrate fish oil on neuropathic pain development and regenerative features of sciatic nerve in rats. In the present study, rats with chronic constriction injury (CCI) of the sciatic nerve and sham-operated ones received fish oil enriched in omega-3 fatty acids (0.36 or 0.72g/kg per day, oral) or saline solution for 21days, with thermal hyperalgesia and mechanical allodynia being assessed before and 3, 7, 14 and 21days after injury. Fish oil enriched in omega-3 fatty acids (0.72g/kg) reversed thermal hyperalgesia and significantly reduced mechanical allodynia. In addition, ω-3 treatment (0.72g/kg) promoted the recovery of the Sciatic Functional Index as well as restored axonal density and morphology, without the formation of neuroma in the injured sciatic nerves after 21days. We conclude that the fish oil enriched in omega-3 fatty acids administration relieves thermal hyperalgesia and mechanical allodynia effectively and also enhances the recovery process in rats with CCI of the sciatic nerve. These findings might contribute to new therapeutic approaches including omega-3 fatty acids in neuropathic pain treatment.

LncRNAs linc00311 and AK141205 are identified as new regulators in STAT3-mediated neuropathic pain in bCCI rats

Neuropathic pain is a severe disease caused by lesions or diseases in the somatosensory system. Long non-coding RNAs (lncRNAs) are important in the development and maintenance of neuropathic pain. However, the precise role of lncRNAs in regulating neuropathic pain remains largely unknown. In this study, a rat model of bilateral chronic constriction injury (bCCI) was established, and microarray was applied to analyze differentially expressed lncRNAs among sham group, bCCI group and the experimental group (bCCI rats administrated with a specific STAT3 inhibitor WP1066). Linc00311 and lncRNA-AK141205 were uncharacterized lncRNAs both upregulated by > 2 folds in bCCI model compared with Sham group, and they were downregulated by > 2 folds following WP1066 administration compared with bCCI group. Downregulation of linc00311 and lncRNA-AK141205 by specific siRNAs significantly attenuated mechanical allodynia, thermal and cold hyperalgesia in bCCI rats. In addition, inhibition of linc00311 and lncRNA-AK141205 inactivated the signal transducer and activator of transcription 3 (STAT3) signaling in spinal microglia in vivo and in vitro. Inhibition of linc00311 and lncRNA-AK141205 could reduce activation of STAT3 and production of proinflammatory cytokines. Moreover, activating STAT3 with SD19 could antagonize the effect of the suppressive effect of siRNAs on production of proinflammatory cytokines. Hence, it is likely that silencing linc00311 and lncRNA-AK141205 may be a promising and novel treatment for neuropathic pain.

Mechanisms for Reducing Neuropathic Pain.

Injury typically results in the development of neuropathic pain, but the pain normally decreases and disappears in paralleled with wound healing. The pain results from cells resident at, and recruited to, the injury site releasing pro-inflammatory cytokines and other mediators leading to the development of pro-inflammatory environment and causing nociceptive neurons to develop chronic ectopic electrical activity, which underlies neuropathic pain. The pain decreases as some of the cells that induce pro-inflammation, changing their phenotype leading to the blocking the release of pro-inflammatory mediators while releasing anti-inflammatory mediators, and blocking nociceptive neuron chronic spontaneous electrical activity. Often, despite apparent wound healing, the neuropathic pain becomes chronic. This raises the question of how chronic pain can be eliminated. While many of the cells and mediators contributing to the development and maintenance of neuropathic pain are known, a better understanding is required of how the injury site environment can be controlled to permanently eliminate the pro-inflammatory environment and silence the chronically electrically active nociceptive neurons. This paper examines how methods that can promote the transition of the pro-inflammatory injury site to an anti-inflammatory state, by changing the composition of local cell types, modifying the activity of pro- and anti-inflammatory receptors, inducing the release of anti-inflammatory mediators, and silencing the chronically electrically active nociceptive neurons. It also examines the hypothesis that factors released from platelet-rich plasma applied to chronic pain sites can permanently eliminate chronic inflammation and its associated chronic pain.

Upregulation of Cdh1 in the trigeminal spinal subnucleus caudalis attenuates trigeminal neuropathic pain via inhibiting GABAergic neuronal apoptosis

Trigeminal neuropathic pain (TNP) remains a tremendous clinical challenge due to its elusive mechanisms. Previous studies showed that peripheral nerve injury facilitated a selective GABAergic neuronal apoptosis in the superficial dorsal horn and contributed to the development and maintenance of neuropathic pain. It has also demonstrated that downregulation of the anaphase-promoting complex/cyclosome(APC/C) and its coactivator Cdh1 contribute to neuronal apoptosis in diverse neurodegenerative diseases. However, whether APC/C-Cdh1 downregulation could induce GABAergic neuronal apoptosis in trigeminal caudalis nucleus (Vc), and then contribute to the development and maintenance of TNP remains unknown. In this study, we aimed to investigate the role of APC/C-Cdh1 in a TNP rat model and its underlying mechanisms. Our results showed that Cdh1 was primarily distributed in superficial laminae of Vc and significantly downregulated in Vc at day 14 post trigeminal nerve injury. Furthermore, trigerminal nerve injury leads to neuronal apoptosis, especially GABAergic interneurons in the superficial of Vc. Upregulating Cdh1 in Vc ameliorated mechanical allodynia and inhibited GABAergic neuronal apoptosis induced by chronic constriction injury of trigeminal infraorbital nerve (CCI-ION).

Systems Pathology of Neuropathic Pain and Fibromyalgia.

Currently, only a few medicines have been approved for use in the clinical treatment of chronic pain, but they are not fully satisfying due to their side effects. From the view that radical treatment, rather than simply treating symptoms, is more important in addressing life-long chronic pain, we have been investigating translational research for a mechanism-based medicine to treat pain. Through the characterization of various types of peripheral and central neuropathic pain in mice, we discovered that lysophosphatidic acid (LPA) plays roles in definitive mechanisms of the development and maintenance of neuropathic pain. We found LPA1 receptor- and LPA3 receptor-mediated amplification of LPA production could be a key mechanism underlying the initiation and maintenance of this pain. We have developed stress-induced fibromyalgia models, and have revealed that LPA1 receptor-signaling also plays key roles in the mechanism. Throughout these studies, we found that LPA plays a key role in pain memory, and that LPA1 receptor- and LPA3 receptor-antagonists could reverse the established pain, and thereby cure the disease source of pain.