Injured Dorsal Root Ganglion Research Articles

TET1 Overexpression Mitigates Neuropathic Pain Through Rescuing the Expression of μ-Opioid Receptor and Kv1.2 in the Primary Sensory Neurons.

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.

Subnetwork identification and chemical modulation for neural regeneration: A study combining network guided forest and heat diffusion model

BackgroundThe induction of neural regeneration is vital to the repair of spinal cord injury (SCI). While compared with peripheral nervous system (PNS), the regenerative capacity of the central nervous system (CNS) is extremely limited. This indicates that modulating the molecular pathways underlying PNS repair may lead to the discovery of potential treatment for CNS injury.MethodsBased on the gene expression profiles of dorsal root ganglion (DRG) after a sciatic nerve injury, we utilized network guided forest (NGF) to rank genes in terms of their capacity of distinguishing injured DRG from sham‐operated controls. Gene importance scores deriving from NGF were used as initial heat in a heat diffusion model (HotNet2) to infer the subnetworks underlying neural regeneration in the DRG. After potential regulators of the subnetworks were found through Connectivity Map (cMap), candidate compounds were experimentally evaluated for their capacity to regenerate the damaged neurons.ResultsGene ontology analysis of the subnetworks revealed ubiquinone biosynthetic process is crucial for neural regeneration. Moreover, almost half of the genes in these subnetworks are found to be related to neural regeneration via text mining. After screening compounds that are likely to modulate gene expressions of the subnetworks, three compounds were selected for the experiment. Of them, trichostatin A, a histone deacetylase inhibitor, was validated to enhance neurite outgrowth in vivo via an optic nerve crush mouse model.ConclusionsOur study identified subnetworks underlying neural regeneration, and validated a compound can promote neurite outgrowth by modulating these subnetworks. This work also suggests an alternative approach for drug repositioning that can be easily extended to other disease phenotypes.

Endoplasmic Reticulum Stress in the Dorsal Root Ganglion Contributes to the Development of Pain Hypersensitivity after Nerve Injury

Little is known about the functional relationship between endoplasmic reticulum (ER) stress and development of pain hypersensitivity after nerve injury. The aim of this study was to investigate the role of ER stress in the development of pain hypersensitivity in the dorsal root ganglion (DRG) after spinal nerve ligation (SNL).SNL was performed in male Sprague–Dawley rats. Real-time PCR and immunohistochemistry were performed to investigate ER stress markers including glucose-regulated protein (GRP) 78, C/EBP homologous protein (CHOP), and spliced form of the X-box binding protein 1 (sXBP1) in L4 and L5 DRG. Behavioral assessment with von Frey filaments, radiant heat, and acetone stimulation was performed to investigate pain hypersensitivity. The ER stress inhibitor salubrinal was administered prior to and 1, 3, and 5 days after SNL treatment. Separately, the ER stress inducer tunicamycin was applied to L5 DRG.GRP78, CHOP, and sXBP1 mRNA and protein expression in L5 DRG was increased 1 and 3 days after SNL but returned to baseline 7 days after SNL. In L4 DRG, ER stress markers showed no remarkable change. Immunohistochemistry demonstrated that GRP78 expression was detected in the majority of DRG neurons and in satellite glial cells. Treatment with salubrinal inhibited CHOP expression in L5 DRG and alleviated pain hypersensitivity for 5 days after SNL. Tunicamycin induced ER stress in the DRG and pain hypersensitivity 2 h after treatment.These results demonstrated that ER stress is induced in the injured DRG and contributes to the development of pain hypersensitivity after nerve injury.

Dorsal Root Ganglia Coactivator-associated Arginine Methyltransferase 1 Contributes to Peripheral Nerve Injury-induced Pain Hypersensitivities

Neuropathic pain is associated with gene expression changes within the dorsal root ganglion (DRG) after peripheral nerve injury, which involves epigenetic mechanisms. Coactivator-associated arginine methyltransferase 1 (CARM1), an epigenetic activator, regulates gene transcriptional activity by protein posttranslational modifications. However, whether CARM1 plays an essential role in the development and maintenance of neuropathic pain is unknown. We report here that peripheral nerve injury induced the upregulation of the mRNA and protein expression of CARM1 in the injured DRG, and blocking its expression through small interfering RNA (siRNA) in the injured DRG attenuated the development and maintenance of neuropathic pain. Furthermore, pharmacological inhibition of CARM1 mitigated peripheral nerve injury-induced mechanical allodynia and thermal hyperalgesia. Given that CARM1 inhibition or knockdown attenuated the induction and maintenance of neuropathic pain after peripheral nerve injury, our findings suggest that CARM1 may serve as a promising therapeutic target for neuropathic pain treatment in clinical applications.

Comprehensive analysis of long noncoding RNA expression in dorsal root ganglion reveals cell-type specificity and dysregulation after nerve injury.

Dorsal root ganglion (DRG) neurons provide connectivity between peripheral tissues and the spinal cord. Transcriptional plasticity within DRG sensory neurons after peripheral nerve injury contributes to nerve repair but also leads to maladaptive plasticity, including the development of neuropathic pain. This study presents tissue and neuron-specific expression profiling of both known and novel long noncoding RNAs (LncRNAs) in the rodent DRG after nerve injury. We have identified a large number of novel LncRNAs expressed within the rodent DRG, a minority of which were syntenically conserved between the mouse, rat, and human, and including, both intergenic and antisense LncRNAs. We have also identified neuron type-specific LncRNAs in the mouse DRG and LncRNAs that are expressed in human IPS cell-derived sensory neurons. We show significant plasticity in LncRNA expression after nerve injury, which in mice is strain and gender dependent. This resource is publicly available and will aid future studies of DRG neuron identity and the transcriptional landscape in both the naive and injured DRG. We present our work regarding novel antisense and intergenic LncRNAs as an online searchable database, accessible from PainNetworks (http://www.painnetworks.org/). We have also integrated all annotated gene expression data in PainNetworks, so they can be examined in the context of their protein interactions.

Contribution of dorsal root ganglion octamer transcription factor 1 to neuropathic pain after peripheral nerve injury.

Neuropathic pain genesis is related to gene alterations in the dorsal root ganglion (DRG) after peripheral nerve injury. Transcription factors control gene expression. In this study, we investigated whether octamer transcription factor 1 (OCT1), a transcription factor, contributed to neuropathic pain caused by chronic constriction injury (CCI) of the sciatic nerve. Chronic constriction injury produced a time-dependent increase in the level of OCT1 protein in the ipsilateral L4/5 DRG, but not in the spinal cord. Blocking this increase through microinjection of OCT1 siRNA into the ipsilateral L4/5 DRG attenuated the initiation and maintenance of CCI-induced mechanical allodynia, heat hyperalgesia, and cold allodynia and improved morphine analgesia after CCI, without affecting basal responses to acute mechanical, heat, and cold stimuli as well as locomotor functions. Mimicking this increase through microinjection of recombinant adeno-associated virus 5 harboring full-length OCT1 into the unilateral L4/5 DRG led to marked mechanical allodynia, heat hyperalgesia, and cold allodynia in naive rats. Mechanistically, OCT1 participated in CCI-induced increases in Dnmt3a mRNA and its protein and DNMT3a-mediated decreases in Oprm1 and Kcna2 mRNAs and their proteins in the injured DRG. These findings indicate that OCT1 may participate in neuropathic pain at least in part by transcriptionally activating Dnmt3a and subsequently epigenetic silencing of Oprm1 and Kcan2 in the DRG. OCT1 may serve as a potential target for therapeutic treatments against neuropathic pain.

Expression and Manipulation of the APC-\u03b2-Catenin Pathway During Peripheral Neuron Regeneration

Molecules and pathways that suppress growth are expressed in postmitotic neurons, a potential advantage in mature neural networks, but a liability during regeneration. In this work, we probed the APC (adenomatous polyposis coli)-β-catenin partner pathway in adult peripheral sensory neurons during regeneration. APC had robust expression in the cytoplasm and perinuclear region of adult DRG sensory neurons both before and after axotomy injury. β-catenin was expressed in neuronal nuclei, neuronal cytoplasm and also in perineuronal satellite cells. In injured dorsal root ganglia (DRG) sensory neurons and their axons, we observed paradoxical APC upregulation, despite its role as an inhibitor of growth whereas β-catenin was downregulated. Inhibition of APC in adult sensory neurons and activation of β-catenin, LEF/TCF transcriptional factors were associated with increased neuronal plasticity in vitro. Local knockdown of APC, at the site of sciatic nerve crush injury enhanced evidence for electrophysiological, behavioural and structural regeneration in vivo. This was accompanied by upregulation of β-catenin. Collectively, the APC-β-catenin-LEF/TCF transcriptional pathway impacts intrinsic mechanisms of axonal regeneration and neuronal plasticity after injury, offering new options for addressing axon regeneration.

NGF and BDNF expression in mouse DRG after spared nerve injury

Neuropathic pain is initiated by a primary lesion in the peripheral nervous system and spoils quality of life. Neurotrophins play important roles in the development and transmission of neuropathic pain. There are conflicting reports that the dorsal root ganglion (DRG) in an injured nerve contribute to neuropathic pain, whereas several studies have highlighted the important contribution of the DRG in a non-injured nerve. Clarifying the role of neurotrophins in neuropathic pain is problematic because we cannot distinguish injured and intact neurons in most peripheral nerve injury models. In the present study, to elicit neuropathic pain, we used the spared nerve injury (SNI) model, in which injured DRG neurons are distinguishable from intact ones, and mechanical allodynia develops in the intact sural nerve skin territory. We examined nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in the DRGs of SNI model mice. NGF and BDNF levels increased in the injured L3 DRG, while NGF decreased in the intact L5 DRG. These data offer a new point of view on the role of these neurotrophins in neuropathic pain induced by peripheral nerve injury.

Membrane potential oscillations are not essential for spontaneous firing generation in L4 Aβ-afferent neurons after L5 spinal nerve axotomy and are not mediated by HCN channels.

What is the central question of this study? Is spontaneous activity (SA) in L4 dorsal root ganglion (DRG) neurons induced by L5 spinal nerve axotomy associated with membrane potential oscillations in these neurons, and if so, are these membrane oscillations mediated by HCN channels? What is the main finding and its importance? Unlike injured L5 DRG neurons, which have been shown to be incapable of firing spontaneously without membrane potential oscillations, membrane potential oscillations are not essential for SA generation in conducting 'uninjured' L4 neurons, and they are not mediated by HCN channels. These findings suggest that the underlying cellular mechanisms of SA in injured and 'uninjured' DRG neurons induced by spinal nerve injury are distinct. The underlying cellular and molecular mechanisms of peripheral neuropathic pain are not fully understood. However, preclinical studies using animal models suggest that this debilitating condition is driven partly by aberrant spontaneous activity (SA) in injured and uninjured dorsal root ganglion (DRG) neurons, and that SA in injured DRG neurons is triggered by subthreshold membrane potential oscillations (SMPOs). Here, using in vivo intracellular recording from control L4-DRG neurons, and ipsilateral L4-DRG neurons in female Wistar rats that had previously undergone L5 spinal nerve axotomy (SNA), we examined whether conducting 'uninjured' L4-DRG neurons in SNA rats exhibit SMPOs, and if so, whether such SMPOs are associated with SA in those L4 neurons, and whether they are mediated by hyperpolarization-activated cyclic nucleotide gated (HCN) channels. We found that 7days after SNA: (a) none of the control A- or C-fibre DRG neurons showed SMPOs or SA, but 50%, 43% and 0% of spontaneously active cutaneous L4 Aβ-low threshold mechanoreceptors, Aβ-nociceptors and C-nociceptors exhibited SMPOs, respectively, in SNA rats with established neuropathic pain behaviors; (b) neither SMPOs nor SA in L4 Aβ-neurons was suppressed by blocking HCN channels with ZD7288 (10mgkg-1 , i.v.); and (c) there is a tendency for female rats to show greater pain hypersensitivity than male rats. These results suggest that SMPOs are linked to SA only in some of the conducting L4 Aβ-neurons, that such oscillations are not a prerequisite for SA generation in those L4 A- or C-fibre neurons, and that HCN channels are not involved in their electrogenesis.

Inhibition of sympathetic sprouting in CCD rats by lacosamide.

Early hyperexcitability activity of injured nerve/neuron is critical for developing sympathetic nerve sprouting within dorsal root ganglia (DRG) since lacosamide (LCM), an anticonvulsant, inhibits Na+ channel. The present study tried to test the potential effect of LCM on inhibiting sympathetic sprouting invivo. Lacosamide (50mg/kg) was daily injected intraperitoneally into rats subjected to chronic compression DRG (CCD), an animal model of neuropathic pain that exhibits sympathetic nerve sprouting, for the 1st 7days after injury. Mechanical sensitivity was tested from day 3 to day 18 after injury, and then DRGs were removed off. Immunohistochemical staining for tyrosine hydroxylase (TH) was examined to observe sympathetic sprouting, and patch-clamp recording was performed to test the excitability and Na+ current of DRG neurons. Early systemic LCM treatment significantly reduced TH immunoreactivity density in injured DRG, lowered the excitability level of injured DRG neurons and increased paw withdrawal threshold. These effects on reducing sympathetic sprouting, inhibiting excitability and suppressing pain behaviour were observed 10days after the end of early LCM injection. In vitro 100μmol/L LCM instantly reduced the excitability of CCD neurons via inhibiting Na+ current and reducing the amplitude of AP. All the findings suggest, for the first time, that early administration of LCM inhibited sympathetic sprouting and then alleviated neuropathic pain. Early LCM administration inhibited sympathetic sprouting within DRG in CCD rats via reducing hyperexcitability of neurons. Early LCM administration suppressed neuropathic pain in CCD rats.

TLR4/NF-κB signaling activation in plantar tissue and dorsal root ganglion involves in the development of postoperative pain

BackgroundSevere postoperative pain remains a clinical problem that impacts patient’s rehabilitation. The present work aims to investigate the role of Toll-like receptor-4 (TLR4) activation in wounded plantar tissue and dorsal root ganglion (DRG) in the genesis of postoperative pain and its underlying mechanisms.ResultsPostoperative pain was induced by plantar incision in rat hind paw. Plantar incision led to increased expression of TLR4 in ipsilateral lumbar 4–5 (L4/L5) DRGs, which occurred at 2 h and was persistent to the third day after surgery. Similar to the change in TLR4 expression, there was also significant increase in phosphorylated nuclear factor-kappa B p65 (p-p65) in DRGs after surgery. Immunofluorescence staining revealed that the increased expressions of TLR4 and p-p65 not only in neuronal cells but also in satellite glial cells in DRG. Furthermore, the enhanced expressions of TLR4 and p-p65 were also detected in plantar tissues around the incision, which was observed starting at 2 h and lasting until the third day after surgery. Prior intrathecal (i.t.) injections of TAK-242 (a TLR4-specific antagonist) or 4',6-diamidino-2-phenylindole-dihydrochloride (PDTC, a nuclear factor-kappa B activation inhibitor) dose dependently alleviated plantar incision-induced mechanical allodynia and thermal hyperalgesia and inhibited the increased expressions of p-p65, tumor necrosis factor-alpha, and interleukin-1 beta in DRG. Prior subcutaneous (s.c.) plantar injection of TAK-242 or PDTC also ameliorated pain-related hypersensitivity following plantar incision. Moreover, the plantar s.c. injection of TAK-242 or PDTC inhibited the increased expressions of p-p65, tumor necrosis factor-alpha, and interleukin-1 beta not only in local wounded plantar tissue but also dramatically in ipsilateral lumbar 4–5 DRGs.ConclusionTLR4/ nuclear factor-kappa B signaling activation in local injured tissue and DRG contribute to the development of postoperative pain via regulating pro-inflammatory cytokines release. Targeting TLR4/ nuclear factor-kappa B signaling in local tissue at early stage of surgery may be an effective strategy for the treatment of postoperative pain.

Role of MicroRNA-143 in Nerve Injury-Induced Upregulation of Dnmt3a Expression in Primary Sensory Neurons.

Peripheral nerve injury increased the expression of the DNA methyltransferase 3A (Dnmt3a) mRNA and its encoding Dnmt3a protein in injured dorsal root ganglia (DRG). This increase is considered as an endogenous instigator in neuropathic pain genesis through epigenetic silencing of pain-associated genes (such as Oprm1) in injured DRG. However, how DRG DNMT3a is increased following peripheral nerve injury is still elusive. We reported here that peripheral nerve injury caused by the fifth spinal nerve ligation (SNL) downregulated microRNA (miR)-143 expression in injured DRG. This downregulation was required for SNL-induced DRG Dnmt3a increase as rescuing miR-143 downregulation through microinjection of miR-143 mimics into injured DRG blocked the SNL-induced increase in Dnmt3a and restored the SNL-induced decreases in Oprm1 mRNA and its encoding mu opioid receptor (MOR) in injured DRG, impaired spinal cord central sensitization and neuropathic pain, and improved morphine analgesic effects following SNL. Mimicking SNL-induced DRG miR-143 downregulation through DRG microinjection of miR143 inhibitors in naive rats increased the expression of Dnmt3a and reduced the expression of Oprm1 mRNA and MOR in injected DRG and produced neuropathic pain-like symptoms. These findings suggest that miR-143 is a negative regulator in Dnmt3a expression in the DRG under neuropathic pain conditions and may be a potential target for therapeutic management of neuropathic pain.

Nerve Injury Increases the Expression of Alpha-2/Delta-1 Subunit of L-Type Calcium Channel in Sensory Neurons of Rat Spinal and Trigeminal Nerves

By immunohistochemistry, an effect of nerve injury on distribution of alpha-2/delta-1 subunit of L-type calcium channel was investigated in rat's 4th and 5th lumbar dorsal root ganglia (DRGs), trigeminal ganglion (TG), and mesencephalic trigeminal nucleus (Mes5). The immunoreactivity was expressed by 52.2% of DRG neurons and 31.4% of TG neurons in intact animals. These neurons mostly had small-to-medium-sized cell bodies. In the DRG and TG, alpha-2/delta-1 subunit-positive neurons were lightly or moderately stained. However, the number of alpha-2/delta-1 subunit-immunoreactive (-IR) neurons dramatically increased in the ipsilateral DRG at 3-28 days after sciatic nerve transection (75.3-79.5%) and in the ipsilateral TG at 7 days after infraorbital nerve transection (66.3%). The IR density of alpha-2/delta-1 subunit in DRG and TG neurons was also elevated by the transection. In the injured DRG and TG, many sensory neurons with small-to-medium-sized cell bodies were strongly stained. Some large DRG and TG neurons showing strong staining intensity also appeared after the treatment. In the intact Mes5, sensory neurons were mostly devoid of alpha-2/delta-1 subunit-immunoreactivity (0.4%). However, alpha-2/delta-1-IR sensory neurons on the ipsilateral side of the Mes5 dramatically increased at 7 days after masseteric nerve transection (31.3%). A double immunofluorescence method also demonstrated that c-Jun activating transcription factor 3 (ATF3)-positive DRG (98.3-99.9%) and Mes5 (81.8%) neurons mostly co-expressed alpha-2/delta-1 subunit after the nerve injuries. However, alpha-2/delta-1 subunit immunoreactivity was relatively infrequent among ATF3-immunonegative DRG neurons (51.6-74.1%) and Mes5 neurons (<1%). The present study indicates that the nerve injury increases the protein level of alpha-2/delta-1 subunit among several types of axotomized sensory neurons in the spinal and trigeminal nervous systems.

DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons

Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG.

Nerve injury-induced epigenetic silencing of opioid receptors controlled by DNMT3a in primary afferent neurons.

Opioids are the gold standard for pharmacological treatment of neuropathic pain, but their analgesic effects are unsatisfactory in part due to nerve injury-induced downregulation of opioid receptors in dorsal root ganglia (DRG) neurons. How nerve injury drives such downregulation remains elusive. DNA methyltransferase (DNMT)-triggered DNA methylation represses gene expression. We show here that blocking the nerve injury-induced increase in DRG DNMT3a (a de novo DNMT) rescued the expression of Oprm1 and Oprk1 mRNAs and their respective encoding mu-opioid receptor (MOR) and kappa-opioid receptor (KOR) proteins in the injured DRG. Blocking this increase also prevented the nerve injury-induced increase in DNA methylation in the promoter and 5'-untranslated region of the Oprm1 gene in the injured DRG, restored morphine or loperamide (a peripheral acting MOR preferring agonist) analgesic effects, and attenuated the development of their analgesic tolerance under neuropathic pain conditions. Mimicking this increase reduced the expression of Oprm1 and Oprk1 mRNAs and their coding MOR and KOR in DRG and augmented MOR-gated neurotransmitter release from the primary afferents. Mechanistically, DNMT3a regulation of Oprm1 gene expression required the methyl-CpG-binding protein 1, MBD1, as MBD1 knockout resulted in the decreased binding of DNMT3a to the Oprm1 gene promoter and blocked the DNMT3a-triggered repression of Oprm1 gene expression in DRG neurons. These data suggest that DNMT3a is required for nerve injury-induced and MBD1-mediated epigenetic silencing of the MOR and KOR in the injured DRG. DNMT3a inhibition may serve as a promising adjuvant therapy for opioid use in neuropathic pain management.

HMG-CoA synthase isoenzymes 1 and 2 localize to satellite glial cells in dorsal root ganglia and are differentially regulated by peripheral nerve injury

In dorsal root ganglia (DRG), satellite glial cells (SGCs) tightly ensheathe the somata of primary sensory neurons to form functional sensory units. SGCs are identified by their flattened and irregular morphology and expression of a variety of specific marker proteins. In this report, we present evidence that the 3-hydroxy-3-methylglutaryl coenzyme A synthase isoenzymes 1 and 2 (HMGCS1 and HMGCS2) are abundantly expressed in SGCs. Immunolabeling with the validated antibodies revealed that both HMGCS1 and HMGCS2 are highly colabeled with a selection of SGC markers, including GS, GFAP, Kir4.1, GLAST1, GDNF, and S100 but not with microglial cell marker Iba1, myelin sheath marker MBP, and neuronal marker β3-tubulin or phosphorylated CaMKII. HMGCS1 but not HMGCS2 immunoreactivity in SGCs is reduced in the fifth lumbar (L5) DRGs that contain axotomized neurons following L5 spinal nerve ligation (SNL) in rats. Western blot showed that HMGCS1 protein level in axotomized L5 DRGs is reduced after SNL to 66±8% at 3 days (p<0.01, n=4 animals in each group) and 58±13% at 28 days (p<0.001, n=9 animals in each group) of its level in control samples, whereas HMGCS2 protein was comparable between injured and control DRGs. These results identify HMGCSs as the alternative markers for SGCs in DRGs. Downregulated HMGCS1 expression in DRGs after spinal nerve injury may reflect a potential role of abnormal sterol metabolism of SGCs in the nerve injured-induced neuropathic pain.

Contribution of the Suppressor of Variegation 3-9 Homolog 1 in Dorsal Root Ganglia and Spinal Cord Dorsal Horn to Nerve Injury–induced Nociceptive Hypersensitivity

Peripheral nerve injury-induced gene alterations in the dorsal root ganglion (DRG) and spinal cord likely participate in neuropathic pain genesis. Histone methylation gates gene expression. Whether the suppressor of variegation 3-9 homolog 1 (SUV39H1), a histone methyltransferase, contributes to nerve injury-induced nociceptive hypersensitivity is unknown. Quantitative real-time reverse transcription polymerase chain reaction analysis, Western blot analysis, or immunohistochemistry were carried out to examine the expression of SUV39H1 mRNA and protein in rat DRG and dorsal horn and its colocalization with DRG μ-opioid receptor (MOR). The effects of a SUV39H1 inhibitor (chaetocin) or SUV39H1 siRNA on fifth lumbar spinal nerve ligation (SNL)-induced DRG MOR down-regulation and nociceptive hypersensitivity were examined. SUV39H1 was detected in neuronal nuclei of the DRG and dorsal horn. It was distributed predominantly in small DRG neurons, in which it coexpressed with MOR. The level of SUV39H1 protein in both injured DRG and ipsilateral fifth lumbar dorsal horn was time dependently increased after SNL. SNL also produced an increase in the amount of SUV39H1 mRNA in the injured DRG (n = 6/time point). Intrathecal chaetocin or SUV39H1 siRNA as well as DRG or intraspinal microinjection of SUV39H1 siRNA impaired SNL-induced allodynia and hyperalgesia (n = 5/group/treatment). DRG microinjection of SUV39H1 siRNA also restored SNL-induced DRG MOR down-regulation (n = 6/group). The findings of this study suggest that SUV39H1 contributes to nerve injury-induced allodynia and hyperalgesia through gating MOR expression in the injured DRG. SUV39H1 may be a potential target for the therapeutic treatment of nerve injury-induced nociceptive hypersensitivity.

MiR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair.

Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers including inflammation. microRNA (miR)-155-5p is a small, noncoding RNA that negatively regulates mRNA translation. In macrophages, miR-155-5p is induced by inflammatory stimuli and elicits a response that could be toxic after SCI. miR-155 may also independently alter expression of genes that regulate axon growth in neurons. Here, we hypothesized that miR-155 deletion would simultaneously improve axon growth and reduce neuroinflammation after SCI by acting on both neurons and macrophages. New data show that miR-155 deletion attenuates inflammatory signaling in macrophages, reduces macrophage-mediated neuron toxicity, and increases macrophage-elicited axon growth by ∼40% relative to control conditions. In addition, miR-155 deletion increases spontaneous axon growth from neurons; adult miR-155 KO dorsal root ganglion (DRG) neurons extend 44% longer neurites than WT neurons. In vivo, miR-155 deletion augments conditioning lesion-induced intraneuronal expression of SPRR1A, a regeneration-associated gene; ∼50% more injured KO DRG neurons expressed SPRR1A versus WT neurons. After dorsal column SCI, miR-155 KO mouse spinal cord has reduced neuroinflammation and increased peripheral conditioning-lesion-enhanced axon regeneration beyond the epicenter. Finally, in a model of spinal contusion injury, miR-155 deletion improves locomotor function at postinjury times corresponding with the arrival and maximal appearance of activated intraspinal macrophages. In miR-155 KO mice, improved locomotor function is associated with smaller contusion lesions and decreased accumulation of inflammatory macrophages. Collectively, these data indicate that miR-155 is a novel therapeutic target capable of simultaneously overcoming neuron-intrinsic and neuron-extrinsic barriers to repair after SCI. Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers, including inflammation. Here, new data show that deleting microRNA-155 (miR-155) affects both mechanisms and improves repair and functional recovery after SCI. Macrophages lacking miR-155 have altered inflammatory capacity, which enhances neuron survival and axon growth of cocultured neurons. In addition, independent of macrophages, adult miR-155 KO neurons show enhanced spontaneous axon growth. Using either spinal cord dorsal column crush or contusion injury models, miR-155 deletion improves indices of repair and recovery. Therefore, miR-155 has a dual role in regulating spinal cord repair and may be a novel therapeutic target for SCI and other CNS pathologies.

Thrombospondin-4 divergently regulates voltage-gated Ca2+ channel subtypes in sensory neurons after nerve injury

Loss of high-voltage-activated (HVA) calcium current (ICa) and gain of low-voltage-activated (LVA) ICa after painful peripheral nerve injury cause elevated excitability in sensory neurons. Nerve injury is also accompanied by increased expression of the extracellular matrix glycoprotein thrombospondin-4 (TSP4), and interruption of TSP4 function can reverse or prevent behavioral hypersensitivity after injury. We therefore investigated TSP4 regulation of ICa in dorsal root ganglion (DRG) neurons. During depolarization adequate to activate HVA ICa, TSP4 decreases both N- and L-type ICa and the associated intracellular calcium transient. In contrast, TSP4 increases ICa and the intracellular calcium signal after low-voltage depolarization, which we confirmed is due to ICa through T-type channels. These effects are blocked by gabapentin, which ameliorates neuropathic pain by targeting the α2δ1 calcium subunit. Injury-induced changes of HVA and LVA ICa are attenuated in TSP4 knockout mice. In the neuropathic pain model of spinal nerve ligation, TSP4 application did not further regulate ICa of injured DRG neurons. Taken together, these findings suggest that elevated TSP4 after peripheral nerve injury may contribute to hypersensitivity of peripheral sensory systems by decreasing HVA and increasing LVA in DRG neurons by targeting the α2δ1 calcium subunit. Controlling TSP4 overexpression in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.

Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-mediated Transcriptional Repression of μ-Opioid Receptors in Primary Sensory Neurons

The μ-opioid receptor (MOR, encoded by Oprm1) agonists are the mainstay analgesics for treating moderate to severe pain. Nerve injury causes down-regulation of MORs in the dorsal root ganglion (DRG) and diminishes the opioid effect on neuropathic pain. However, the epigenetic mechanisms underlying the diminished MOR expression caused by nerve injury are not clear. G9a (encoded by Ehmt2), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined the role of G9a in diminished MOR expression and opioid analgesic effects in animal models of neuropathic pain. We found that nerve injury in rats induced a long-lasting reduction in the expression level of MORs in the DRG but not in the spinal cord. Nerve injury consistently increased the enrichment of the G9a product histone 3 at lysine 9 dimethylation in the promoter of Oprm1 in the DRG. G9a inhibition or siRNA knockdown fully reversed MOR expression in the injured DRG and potentiated the morphine effect on pain hypersensitivity induced by nerve injury. In mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce the expression level of MORs and the morphine effect. In addition, G9a inhibition or Ehmt2 knockout in DRG neurons normalized nerve injury-induced reduction in the inhibitory effect of the opioid on synaptic glutamate release from primary afferent nerves. Our findings indicate that G9a contributes critically to transcriptional repression of MORs in primary sensory neurons in neuropathic pain. G9a inhibitors may be used to enhance the opioid analgesic effect in the treatment of chronic neuropathic pain.