Nerve Injury-induced Changes Research Articles

ZNF382 controls mouse neuropathic pain via silencer-based epigenetic inhibition of Cxcl13 in DRG neurons.

Nerve injury-induced changes of gene expression in dorsal root ganglion (DRG) are critical for neuropathic pain genesis. However, how these changes occur remains elusive. Here we report the down-regulation of zinc finger protein 382 (ZNF382) in injured DRG neurons after nerve injury. Rescuing this down-regulation attenuates nociceptive hypersensitivity. Conversely, mimicking this down-regulation produces neuropathic pain symptoms, which are alleviated by C-X-C motif chemokine 13 (CXCL13) knockdown or its receptor CXCR5 knockout. Mechanistically, an identified cis-acting silencer at distal upstream of the Cxcl13 promoter suppresses Cxcl13 transcription via binding to ZNF382. Blocking this binding or genetically deleting this silencer abolishes the ZNF382 suppression on Cxcl13 transcription and impairs ZNF382-induced antinociception. Moreover, ZNF382 down-regulation disrupts the repressive epigenetic complex containing histone deacetylase 1 and SET domain bifurcated 1 at the silencer-promoter loop, resulting in Cxcl13 transcriptional activation. Thus, ZNF382 down-regulation is required for neuropathic pain likely through silencer-based epigenetic disinhibition of CXCL13, a key neuropathic pain player, in DRG neurons.

Neurotherapeutic Potential of Cervus elaphus Sibericus on Axon Regeneration and Growth Cone Reformation after H2O2-Induced Injury in Rat Primary Cortical Neurons.

Simple SummaryOxidative stress plays a crucial role in nerve injury-induced changes, including the cellular redox state, greater disease severity, and reduced function. Most studies have demonstrated that the modulation of oxidative damage to neurons leads to improved survival and functional recovery. Deer antlers are considered a potent natural source of antioxidants. Thus, we examined the neurotherapeutic effect of Cervus elaphus sibericus in the axon regeneration and growth cone reformation of cortical neurons. The effect of three doses of C. elaphus sibericus on cortical neurons was determined in a hydrogen peroxide-induced injury model using immunocytochemistry, flow-cytometry, and real-time PCR. We found that C. elaphus sibericus effectively inhibited neuronal death following hydrogen peroxide-induced injury by significantly inhibiting oxidative stress and consequently promoting neuron repair and axon regeneration in cortical neurons.Cervus elaphus sibericus (CES), commonly known as deer antler, has been used as a medicinal herb because of its various pharmacological activities, including its anti-infective, anti-arthritic, anti-allergic, and anti-oxidative properties. However, the precise mechanisms by which CES functions as a potent anti-oxidative agent remain unknown; particularly, the effects of CES on cortical neurons and its neurobiological mechanism have not been examined. We used primary cortical neurons from the embryonic rat cerebral cortex and hydrogen peroxide to induce oxidative stress and damage in neurons. After post-treatment of CES at three concentrations (10, 50, and 200 µg/mL), the influence of CES on the neurobiological mechanism was assessed by immunocytochemistry, flow cytometry, and real-time PCR. CES effectively prevented neuronal death caused by hydrogen peroxide-induced damage by regulating oxidative signaling. In addition, CES significantly induced the expression of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated genes. We also observed newly processing elongated axons after CES treatment under oxidative conditions. In addition, filopodia tips generally do not form a retraction bulb, called swollen endings. Thus, CES shows therapeutic potential for treating neurological diseases by stimulating neuron repair and regeneration.

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.

Pathophysiological Changes of Physical Barriers of Peripheral Nerves After Injury.

Peripheral nerves are composed of complex layered anatomical structures, including epineurium, perineurium, and endoneurium. Perineurium and endoneurium contain many physical barriers, including the blood-nerve barrier at endoneurial vessels and the perineurial barrier. These physical barriers help to eliminate flux penetration and thus contribute to the establishment of a stable microenvironment. In the current review, we introduce the anatomical compartments and physical barriers of peripheral nerves and then describe the cellular and molecular basis of peripheral physical barriers. We also specifically explore peripheral nerve injury-induced changes of peripheral physical barriers, including elevated endoneurial fluid pressure, increased leakage of tracer, decreased barrier-type endothelial cell ratio, and altered distributions and expressions of cellular junctional proteins. The understanding of the pathophysiological changes of physical barriers following peripheral nerve injury may provide a clue for the treatment of peripheral nerve injury.

Dorsal root ganglion myeloid zinc finger protein 1 contributes to neuropathic pain after peripheral nerve trauma.

Peripheral nerve injury-induced changes in gene transcription and translation in primary sensory neurons of the dorsal root ganglion (DRG) are considered to contribute to neuropathic pain genesis. Transcription factors control gene expression. Peripheral nerve injury increases the expression of myeloid zinc finger protein 1 (MZF1), a transcription factor, and promotes its binding to the voltage-gated potassium 1.2 (Kv1.2) antisense (AS) RNA gene in the injured DRG. However, whether DRG MZF1 participates in neuropathic pain is still unknown. Here, we report that blocking the nerve injury-induced increase of DRG MZF1 through microinjection of MZF1 siRNA into the injured DRG attenuated the initiation and maintenance of mechanical, cold, and thermal pain hypersensitivities in rats with chronic constriction injury (CCI) of the sciatic nerve, without affecting locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking the nerve injury-induced increase of DRG MZF1 through microinjection of recombinant adeno-associated virus 5 expressing full-length MZF1 into the DRG produced significant mechanical, cold, and thermal pain hypersensitivities in naive rats. Mechanistically, MZF1 participated in CCI-induced reductions in Kv1.2 mRNA and protein and total Kv current and the CCI-induced increase in neuronal excitability through MZF1-triggered Kv1.2 AS RNA expression in the injured DRG neurons. MZF1 is likely an endogenous trigger of neuropathic pain and might serve as a potential target for preventing and treating this disorder.

Epigenetic regulation of chronic pain.

Chronic pain arising from peripheral inflammation and tissue or nerve injury is a common clinical symptom. Although intensive research on the neurobiological mechanisms of chronic pain has been carried out during previous decades, this disorder is still poorly managed by current drugs such as opioids and nonsteroidal anti-inflammatory drugs. Inflammation, tissue injury and/or nerve injury-induced changes in gene expression in sensory neurons of the dorsal root ganglion, spinal cord dorsal horn and pain-associated brain regions are thought to participate in chronic pain genesis; however, how these changes occur is still elusive. Epigenetic modifications including DNA methylation and covalent histone modifications control gene expression. Recent studies have shown that peripheral noxious stimulation changes DNA methylation and histone modifications and that these changes may be related to the induction of pain hypersensitivity under chronic pain conditions. This review summarizes the current knowledge and progress in epigenetic research in chronic pain and discusses the potential role of epigenetic modifications as therapeutic antinociceptive targets in this disorder.

P.1.g.055 5-HT2A/2C serotonergic receptors of lateral septal area modulate fear-induced responses elicited by activation of dorsolateral periaqueductal grey matter

While group 1 metabotropic glutamate receptors (mGluRs) and ionotropic N-methyl-d-aspartate (NMDA) receptors regulate nociception, the precise molecular mechanism(s) contributing to glutamate signaling in chronic pain remain unclear. Here we not only confirmed the key involvement of Homer proteins in neuropathic pain, but also distinguished between the functional roles for different Homer family members and isoforms. Chronic constriction injury (CCI) of the sciatic nerve induced long-lasting, time-dependent increases in the postsynaptic density expression of the constitutively expressed (CC) isoforms Homer1b/c and/or Homer2a/b in the spinal dorsal horn and supraspinal structures involved in nociception (prefrontal cortex, thalamus), that co-occurred with increases in their associated mGluRs, NR2 subunits of the NMDA receptor, and the activation of downstream kinases. Virus-mediated overexpression of Homer1c and Homer2b after spinal (intrathecal) virus injection exacerbated CCI-induced mechanical and cold hypersensitivity, however, Homer1 and Homer2 gene knockout (KO) mice displayed no changes in their neuropathic phenotype. In contrast, overexpression of the immediate early gene (IEG) Homer1a isoform reduced, while KO of Homer1a gene potentiated neuropathic pain hypersensitivity. Thus, nerve injury-induced increases in CC-Homers expression promote pain in pathological states, but IEG-Homer induction protects against both the development and maintenance of neuropathy. Additionally, exacerbated pain hypersensitivity in transgenic mice with reduced Homer binding to mGluR5 supports also an inhibitory role for Homer interactions with mGluR5 in mediating neuropathy. Such data indicate that nerve injury-induced changes in glutamate receptor/Homer signaling contribute in dynamic but distinct ways to neuropathic pain processing, which has relevance for the etiology of chronic pain symptoms and its treatment.

Nerve injury-induced changes in Homer/glutamate receptor signaling contribute to the development and maintenance of neuropathic pain

While group 1 metabotropic glutamate receptors (mGluRs) and ionotropic N-methyl-d-aspartate (NMDA) receptors regulate nociception, the precise molecular mechanism(s) contributing to glutamate signaling in chronic pain remain unclear. Here we not only confirmed the key involvement of Homer proteins in neuropathic pain, but also distinguished between the functional roles for different Homer family members and isoforms. Chronic constriction injury (CCI) of the sciatic nerve induced long-lasting, time-dependent increases in the postsynaptic density expression of the constitutively expressed (CC) isoforms Homer1b/c and/or Homer2a/b in the spinal dorsal horn and supraspinal structures involved in nociception (prefrontal cortex, thalamus), that co-occurred with increases in their associated mGluRs, NR2 subunits of the NMDA receptor, and the activation of downstream kinases. Virus-mediated overexpression of Homer1c and Homer2b after spinal (intrathecal) virus injection exacerbated CCI-induced mechanical and cold hypersensitivity, however, Homer1 and Homer2 gene knockout (KO) mice displayed no changes in their neuropathic phenotype. In contrast, overexpression of the immediate early gene (IEG) Homer1a isoform reduced, while KO of Homer1a gene potentiated neuropathic pain hypersensitivity. Thus, nerve injury-induced increases in CC-Homers expression promote pain in pathological states, but IEG-Homer induction protects against both the development and maintenance of neuropathy. Additionally, exacerbated pain hypersensitivity in transgenic mice with reduced Homer binding to mGluR5 supports also an inhibitory role for Homer interactions with mGluR5 in mediating neuropathy. Such data indicate that nerve injury-induced changes in glutamate receptor/Homer signaling contribute in dynamic but distinct ways to neuropathic pain processing, which has relevance for the etiology of chronic pain symptoms and its treatment.

Nerve injury-induced changes in GABAA and GABAB sub-unit expression in area 3b and cuneate nucleus of adult squirrel monkeys: Further evidence of developmental recapitulation

The primate somatosensory system provides an excellent model system with which to investigate adult neural plasticity. Here, we report immunohistochemical staining data for the GABAA α1, GABABR1a, and GABABR1b receptor subunits in somatosensory area 3b, and cuneate nucleus one week after median nerve compression in adult squirrel monkeys. We find a significant decrease in GABAA α1 subunit staining across all cortical layers and within both soma and neuropil of the deprived cortical and brainstem regions. The GABAB staining showed an opposing shift in deprived regions, with a significant increase in presynaptic GABABR1a staining, and a significant decrease in postsynaptic GABABR1b staining in deprived regions of the cortex and brainstem. These changes in receptor subunit expression generate patterns that are very similar to those reported in the neonate. Furthermore, the similarities between brainstem and cortical expression suggest conserved forms of adult plasticity in these two regions. Taken together these results, along with the results from our previous paper investigating AMPA subunit expression in these same animals, support the hypothesis that deprived neurons enter a previously hidden state of developmental recapitulation that serves to prime the brain for NMDA receptor mediated receptive field reorganization.

(104) Peripheral nerve injury insult alters proteomic profile of the spinal dorsal horn synaptosome-associated proteins

Peripheral nerve injury leads to the changes in protein expression in the spinal cord that are thought to be one of central mechanisms underlying the development and maintenance of nerve injury-induced neuropathic pain. However, the changes in synaptic protein profiles in the spinal dorsal horn after nerve injury are unknown. Using two-dimensional gel electrophoresis in combination with mass spectrometry, we, here, reported the global changes in synaptosome-associated proteins in spinal dorsal horn after peripheral nerve injury. Neuropathic pain was induced by unilateral the fifth spinal nerve injury (SNI) in rats and evaluated by measurement of paw withdrawal thresheds in response to mechanical stimuli on day 14 post-surgery. Sham surgery was performed as a control. Followed by behavioral examination, the fifth lumbar dorsal horns ipsilateral to spinal nerve injury or sham surgery were harvested. The synaptosome was fractioned and proteins were resolved in 2-DE. Among 30 identified differentially expressed proteins, 28 proteins were significantly up-regulated and 2 proteins down-regulated on day 14 post-nerve injury compared to sham surgery. Some of these proteins were further validated by Western blot analysis. These proteins were involved in transmission and modulation of central pain signaling, amino acid metabolism, oxidative stress, apoptosis, or energy metabolism. Nerve injury-induced changes in expression and/or redistribution of the proteins, especially involved in transmission and modulation of central pain signaling, in dorsal horn synaptosome may play a role in the central mechanism that underlies the development and maintenance of nerve injury-induced chronic neuropathic pain. It is possible that these identified proteins may serve as potential therapeutic targets for treatment of chronic pain.

NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice

Previous research has shown that peripheral inflammation and peripheral nerve injury alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices. Peripheral nerve injury-induced changes in the pharmacological and electrophysiological properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 μM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation.

Artemin has potent neurotrophic actions on injured C‐fibres

In this study, we have investigated the effects of artemin (ARTN), one of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors, on C-fibres following nerve injury in the adult rat. GDNF family receptor alpha (GFRalpha) 3, the ligand binding domain of the ARTN receptor, is expressed in 34% of dorsal root ganglion (DRG) cells, predominantly in the peptidergic population of C-fibres and in a proportion of the isolectin B4 (IB4)-binding population. Interestingly, only 30% of GFRalpha3-expressing DRG cells co-expressed RET (the signal transducing domain). In agreement with previous studies, treatment with ARTN prevented many of the nerve injury-induced changes in the histochemistry of both the peptidergic and the IB4-binding populations of small, but not large, diameter DRG cells. In addition, ARTN treatment maintained C-fibre conduction velocity, and C-fibre evoked substance P release within the dorsal horn following nerve injury. ARTN was also protective following capsaicin treatment, which produces selective C-fibre injury. Given the potent neurotrophic actions of ARTN on C-fibres, it may therefore provide potential for the treatment of nerve injury, particularly in the maintenance of small fibre function.

New Approaches for the Treatment of Pain: The GDNF Family of Neurotrophic Growth Factors

This article focuses on the GDNF family of neurotrophic factors as a potential new class of therapeutics for neuropathic pain, with a particular emphasis on the ligands, artemin and GDNF. In vivo activity of the ligands, expression of ligands and receptors after peripheral nerve injury, and modulation of nerve injury-induced changes by the ligands are reviewed in detail. Structural considerations, particularly with regard to implications for binding interactions and biological activity are discussed.

Coupling gene chip analyses and rat genetic variances in identifying potential target genes that may contribute to neuropathic allodynia development.

Genetic factors and nerve injury-induced changes of gene expression in sensory neurons are potential contributors to tactile allodynia, a neuropathic pain state manifested as hypersensitivity to innocuous mechanical stimulation. To uncover genes relevant to neuropathic allodynia, we analyzed gene expression profiles in dorsal root ganglia (DRG) of spinal nerve-ligated Harlan and Holtzman Sprague Dawley rats, strains with different susceptibilities to neuropathic allodynia. Using Affymetrix gene chips, we identified genes showing differential basal-level expression in these strains without injury-induced regulation. Of more than 8000 genes analyzed, less than 180 genes in each strain were regulated after injury, and 19-22% of that was regulated in a strain-specific manner. Importantly, we identified functionally related genes that were co-regulated post injury in one or both strains. In situ hybridization and real-time PCR analyses of a subset of identified genes confirmed the patterns of the microarray data, and the former also demonstrated that injury-induced changes occurred, not only in neurons, but also in non-neuronal cells. Together, our studies provide a global view of injury plasticity in DRG of these rat stains and support a plasticity-based mechanism mediating variations in allodynia susceptibility, thus providing a source for further characterization of neuropathic pain-relevant genes and potential pathways.

Peripheral nerve injury alters excitatory synaptic transmission in lamina II of the rat dorsal horn.

Using the blind whole cell patch-clamp recording technique, we investigated peripheral nerve injury-induced changes in excitatory synaptic transmission to neurones in lamina II of the dorsal horn. Partial (i.e. chronic constriction injury (CCI) and spared nerve injury (SNI)) and complete (i.e. sciatic nerve transection (SNT)) peripheral nerve injury altered the mean threshold intensity for eliciting A fibre-mediated EPSCs in lamina II neurones. Following SNT and CCI, EPSC threshold was significantly decreased, but following SNI, EPSC threshold was increased (naive: 32 +/- 2 mu A, SNT: 22 +/- 2 mu A, CCI: 23 +/- 2 mu A, SNI: 49 +/- 4 mu A; P < 0.01, Student's unpaired t test). Despite this disparity between models, dorsal root compound action potential recordings revealed no significant difference in the conduction velocity or activation threshold of A beta and A delta fibres in naive, SNT, CCI and SNI rats. In addition to the changes in EPSC threshold, we also observed a shift in the distribution of EPSCs. In spinal cord slices from naive rats, polysynaptic A beta fibre-evoked EPSCs were observed in 24 % of lamina II neurones, monosynaptic A delta fibre EPSCs were observed in 34 % and polysynaptic A delta fibre EPSCs were observed in 7 %. Following SNT and CCI, the percentage of neurones with polysynaptic A beta fibre EPSCs increased to > or = 65 % of the sampled population, while the percentage of neurones with monosynaptic A delta fibre EPSCs decreased to < 10 %. The percentage of neurones with polysynaptic A delta fibre EPSCs was unchanged. In contrast, following SNI, A beta fibre EPSCs decreased in incidence while the percentage of neurones with polysynaptic A delta fibre EPSCs increased to 44 %. Similar to the other injury models, however, monosynaptic A delta fibre EPSCs decreased in frequency following SNI. Thus, excitatory synaptic transmission is subject to divergent plasticity in different peripheral nerve injury models, reflecting the complexity of responses to different forms of deafferentation.

Nerve injury-induced changes in opioid modulation of wide dynamic range dorsal column nuclei neurones

In the present study we investigated the effects of spinal morphine on the electrically and naturally evoked responses of gracile nuclei neurones in a rat model of neuropathy, induced by the tight ligation of lumbar L5/6 spinal nerves. Two weeks after surgery, animals were prepared for electrophysiological recordings and neuronal responses were characterised to a range of controlled natural (brush, low- and high-intensity von Frey filaments, heat 45°C) and peripheral electrical stimuli. Morphine (0.1, 0.25, 1 and 5 μg) was applied spinally and its effect was compared to that in sham-operated or naive animals. Following surgery, all neuropathic rats exhibited signs of mechanical allodynia. Nerve injury induced a significant increase in the receptive field size of gracile nuclei neurones, and also produced a non-significant increase in the proportion and level of spontaneous activity in these neurones. The baseline electrical and natural evoked responses remained unaltered. Spinal morphine reduced both the Aδ-fibre- and C-fibre-evoked responses of gracile nuclei neurones, and similarly inhibited the heat-evoked responses of neuropathic, sham-operated and naive rats. Morphine, however, produced only minor reductions (<30% inhibition of pre-drug control responses) of the Aβ-fibre- and brush-evoked responses of gracile nuclei neurones. These drug effects were similar in all animal groups. In complete contrast, morphine produced a marked inhibition of the low-intensity punctate mechanical evoked responses (von Freys 2 and 9 g) after nerve injury, an effect that was totally lacking in the sham-operated or naive animal groups. This dramatic shift was selective for the low-intensity punctate mechanical stimuli and such an effect was not seen with the noxious mechanical punctate stimulus (von Frey 75 g) where there was a modest inhibition in all groups. Our results suggest that there is plasticity in the opioid modulation of dorsal column projection pathways following spinal nerve ligation and these alterations appear to interact with sensory pathways conveying low-threshold punctate stimuli.

Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates

The generation of knock-out and transgenic mice offers a promising approach to the identification of novel biochemical factors that contribute to persistent pain conditions. To take advantage of these mice, however, it is important to demonstrate that the traditional models of persistent pain, which were largely developed for studies in the rat, can be used in the mouse. Here, we combined behavioral and anatomical methods to characterize the pathophysiology of a partial nerve injury-evoked pain condition in the `normal' mouse. In male C57BL6 mice we tied a tight ligature around 1/3 to 1/2 of the diameter of the sciatic nerve and evaluated the time-course and magnitude of the ensuing mechanical and thermal allodynia. We also used immunocytochemistry to analyze nerve injury-induced changes in substance P (SP) and NK-1 (SP) receptor expression in the spinal cord. As in the rat, partial nerve injury markedly decreased paw withdrawal thresholds to both mechanical and thermal stimuli on the injured side. We detected threshold changes one day after the injury. The thermal allodynia resolved by 49 days, but the mechanical allodynia persisted for the duration of the study (70 days). We found no changes contralateral to the nerve injury. Sympatholytic treatment with guanethidine significantly reduced both the thermal and mechanical allodynia. We observed a reduction of SP immunoreactivity in the superficial dorsal horn on the injured side at 7 and 14, but not at 3 or 70 days after the nerve injury, and we observed an increase of NK-1 receptor expression at 3, 7, 14 and 42, but not at 70 days after the injury. We conclude that partial injury to the sciatic nerve produces a comparable allodynia and neurochemical plasticity in the rat and mouse. These results establish a valuable model for future studies of the biochemical basis of neuropathic pain in mice with specific gene modifications.