Uninjured Dorsal Root Ganglion Research Articles

Satellite glial cells bridge sensory neuron crosstalk in visceral pain and cross-organ sensitization.

Following colonic inflammation, the uninjured bladder afferent neurons are also activated. The mechanisms and pathways underlying this sensory neuron cross-activation (from injured neurons to uninjured neurons) are not fully understood. Colonic and bladder afferent neurons reside in the same spinal segments and are separated by satellite glial cells (SGCs) and extracellular matrix in dorsal root ganglia (DRG). SGCs communicate with sensory neurons in a bidirectional fashion. In this review, I summarize the differentially regulated genes/proteins in the injured and uninjured DRG neurons and explore the role of SGCs in regulation of sensory neuron crosstalk in visceral cross-organ sensitization. I also highlight the paracrine pathways in mediating neuron-SGC and SGC-neuron coupling with an emphasis on the neurotrophins and purinergic systems. Finally, I discuss the results from recent RNAseq profiling of SGCs to reveal useful molecular markers for characterization, functional study, and therapeutic targets of SGCs. Significance Statement Satellite glial cells (SGCs) are the largest glial subtypes in sensory ganglia and play a critical role in mediating sensory neuron crosstalk, an underlying mechanism in colon-bladder cross-sensitization. Identification of novel and unique molecular markers of SGCs can advance the discovery of therapeutics targets in treatment of chronic pain including visceral pain comorbidity.

Evaluation of motor and sensory neuron populations in a mouse median nerve injury model

BackgroundPeripheral nerves can regenerate and restore function after injury but this process is hindered by many factors including chronic denervation, motor end-plate resorption and Schwann cell senescence. Forelimb injury models in rodents are becoming increasingly popular as they more accurately reflect the physiology and biomechanics of upper extremity nerve injuries. However several aspects of this surgical model remain poorly characterized. New MethodC57Bl/6 mice underwent enumeration of median nerve motor and sensory neuron pools using retrograde labeling with or without nerve transection. Distal histomorphometry of uninjured mouse median nerves was also examined. Baseline reference values of volitional forelimb grip strength measurements were determined and the rate of neural elongation was also estimated. ResultsWe identified 1363 ± 165 sensory and 216 ± 16 motor neurons within the uninjured dorsal root ganglia (DRG) and ventral spinal cord, respectively. Eight days following injury, approximately 34% of motoneurons had elongated a distance of 5 mm beyond the repair site 8 days following injury. Volitional grip strength decreased 50% with unilateral median nerve transection and was negligible with contralateral flexor tendon tenotomy. Comparison with existing methodOur spinal cord and DRG harvesting technique presented here was technically straightforward and reliable. Estimates of motor and sensory neuron numbers for the mouse median nerve compared favourably with studies using intramuscular injection of retrograde neurotracer. Histomorphometry data was consistent with and reinforced reference values in the literature. ConclusionsThis study provides data that further develops an increasingly popular surgical model for studying peripheral nerve injury and repair.

Bestrophin-1 Participates in Neuropathic Pain Induced by Spinal Nerve Transection but not Spinal Nerve Ligation

Previous studies have reported that L5/L6 spinal nerve ligation (SNL), but not L5 spinal nerve transection (SNT), enhances anoctamin-1 in injured and uninjured dorsal root ganglia (DRG) of rats suggesting some differences in function of the type of nerve injury. The role of bestrophin-1 in these conditions is unknown. The aim of this study was to investigate the role of bestrophin-1 in rats subjected to L5 SNT and L5/L6 SNL. SNT up-regulated bestrophin-1 protein expression in injured L5 and uninjured L4 DRG at day 7, whereas it enhanced GAP43 mainly in injured, but also in uninjured DRG. In contrast, SNL enhanced GAP43 at day 1 and 7, while bestrophin-1 expression increased only at day 1 after nerve injury. Accordingly, intrathecal injection of the bestrophin-1 blocker CaCCinh-A01 (1-10 µg) reverted SNT- or SNL-induced tactile allodynia in a concentration-dependent manner. Intrathecal injection of CaCCinh-A01 (10 µg) prevented SNT-induced upregulation of bestrophin-1 and GAP43 at day 7. In contrast, CaCCinh-A01 did not affect SNL-induced up-regulation of GAP43 nor bestrophin-1. Bestrophin-1 was mainly expressed in small- and medium-size neurons in naïve rats, while SNT increased bestrophin-1 immunoreactivity in CGRP+, but not in IB4+ neuronal cells in DRG. Intrathecal injection of bestrophin-1 plasmid (pCMVBest) induced tactile allodynia and increased bestrophin-1 expression in DRG and spinal cord in naïve rats. CaCCinh-A01 reversed bestrophin-1 overexpression-induced tactile allodynia and restored bestrophin-1 expression. Our data suggest that bestrophin-1 plays a relevant role in neuropathic pain induced by SNT, but not by SNL. PerspectiveSNT, but not SNL, up-regulates bestrophin-1 and GAP43 protein expression in injured L5 and uninjured L4 DRG. SNT increases bestrophin-1 immunoreactivity in CGRP+ neurons in DRG. Bestrophin-1 overexpression induces allodynia. CaCCinh-A01 reduces allodynia and restores bestrophin-1 expression. Our data suggest bestrophin-1 is differentially regulated depending on the neuropathic pain model.

Adrenomedullin is an Important Pathological Mediator in Progression of Chronic Neuropathic Pain.

The characterization of neuropathic pain is maladaptive plasticity within the nociceptive system. Multiple alterations contribute to complex pain phenotypes. Adrenomedullin (AM) has been documented to be a pain mediator. However, its involvement in pathological pain is poorly understood. We studied the contribution of AM to chronic neuropathic pain in the spinal nerve ligation (SNL) model. Daily injection of the AM receptor antagonist AM22-52 (10 nmol) via an intrathecal (i.t.) route after SNL inhibited mechanical allodynia starting on day 6. Single administration of AM22-52 produced an immediate attenuation on pain hypersensitivity on day 2 or 10 post-SNL. Protein and mRNA levels were assayed by immunofluorescent staining and qRT-PCR, respectively, on days 1, 3, 7 and 15 post-SNL. The results showed that AM at both protein and mRNA levels was increased in both injured (L5) and adjacent uninjured (L4) nerves starting on day 3 post-SNL. In dorsal root ganglion (DRG) at L5, AM was increase on days 1-7 at mRNA level but only on day 7 at protein level. However, AM was increase at mRNA level on days 1-7 and at protein level on days 3-15 in L4 DRG. AM mRNA expression was upregulated on days 1-7 in the spinal cord. Expression of receptor activity-modifying protein 2 (RAMP2), an essential AM1 receptor component, was upregulated in small and medium-diameter neurons on days 1-15 in both L5 and L4 DRG. Furthermore, single administration of AM22-52 suppressed the increase of nNOS in DRG induced by SNL and daily injection of AM22-52 for 7 days inhibited SNL-induced increase of CGRP mRNA in the spinal dorsal horn. This study indicates that the increased AM bioactivity in injured and uninjured peripheral nerves, uninjured adjacent DRG and the spinal dorsal horn play a critical role mainly in the late-phase development of neuropathic pain. The mechanism may involve the recruitment of nNOS and CGRP.

FOXO3a as a sensor of unilateral nerve injury in sensory neurons ipsilateral, contralateral and remote to injury.

Emerging evidence supports that the stress response to peripheral nerve injury extends beyond the injured neuron, with alterations in associated transcription factors detected both locally and remote to the lesion. Stress-induced nuclear translocation of the transcription factor forkhead class box O3a (FOXO3a) was initially linked to activation of apoptotic genes in many neuronal subtypes. However, a more complex role of FOXO3a has been suggested in the injury response of sensory neurons, with the injured neuron expressing less FOXO3a. To elucidate this response and test whether non-injured sensory neurons also alter FOXO3a expression, the temporal impact of chronic unilateral L4–6 spinal nerve transection on FOXO3a expression and nuclear localization in adult rat dorsal root ganglion neurons ipsilateral, contralateral or remote to injury relative to naïve controls was examined. In naïve neurons, high cytoplasmic and nuclear levels of FOXO3a colocalized with calcitonin gene related peptide, a marker of the nociceptive subpopulation. One hour post-injury, an acute increase in nuclear FOXO3a in small size injured neurons occurred followed by a significant decrease after 1, 2 and 4 days, with levels increasing toward pre-injury levels by 1 week post-injury. A more robust biphasic response to the injury was observed in uninjured neurons contralateral to and those remote to injury. Nuclear levels of FOXO3a peaked at 1 day, decreased by 4 days, then increased by 1 week post-injury, a response mirrored in C4 dorsal root ganglion neurons remote to injury. This altered expression contralateral and remote to injury supports that spinal nerve damage has broader systemic impacts, a response we recently reported for another stress transcription factor, Luman/CREB3. The early decreased expression and nuclear localization of FOXO3a in the injured neuron implicate these changes in the cell body response to injury that may be protective. Finally, the broader systemic changes support the existence of stress/injury-induced humeral factor(s) influencing transcriptional and potentially behavioral changes in uninjured dorsal root ganglion neurons. Approval to conduct this study was obtained from the University of Saskatchewan Animal Research Ethics Board (protocol #19920164).

Activation of µ-δ opioid receptor heteromers inhibits neuropathic pain behavior in rodents.

Several reports support the idea that µ- and δ-opioid receptors (ORs) may exist as heterodimers in brain regions involved in pain signaling. The unique pharmacology of these heteromers may present a novel analgesic target. However, the role of µ-δ heteromers in sensory neurons involved in pain and opioid analgesia remains unclear, particularly during neuropathic pain. We examined the effects of spinal nerve injury on µ-δ heteromer expression in dorsal root ganglion (DRG) neurons and the effects of a µ-δ heteromer-targeting agonist, CYM51010, on neuropathic pain behavior in rats and mice. An L5 spinal nerve ligation (SNL) in rats significantly decreased µ-δ heteromer expression in L5 DRG but increased heteromer levels in uninjured L4 DRG. Importantly, in SNL rats, subcutaneous injection of CYM51010 inhibited mechanical hypersensitivity in a dose-related manner (EC50: 1.09 mg/kg) and also reversed heat hyperalgesia and attenuated ongoing pain (2 mg/kg, subcutaneously). HEK-293T cell surface-labeled with µ- and δ-ORs internalized both receptors after exposure to CYM51010. By contrast, in cells transfected with µ-OR alone, CYM51010 was significantly less effective at inducing receptor internalization. Electrophysiologic studies showed that CYM51010 inhibited the C-component and windup phenomenon in spinal wide dynamic range neurons of SNL rats. The pain inhibitory effects of CYM51010 persisted in morphine-tolerant rats but was markedly attenuated in µ-OR knockout mice. Our studies show that spinal nerve injury may increase µ-δ heterodimerization in uninjured DRG neurons, and that µ-δ heteromers may be a potential therapeutic target for relieving neuropathic pain, even under conditions of morphine tolerance.

Voltage-gated sodium channel 1.7 expression decreases in dorsal root ganglia in a spinal nerve ligation neuropathic pain model.

The role of the voltage-gated sodium channel 1.7 (Nav1.7) is unclear in models of neuropathic pain induced by nerve injury. In the present study, we measured expression levels of Nav1.7 in two distinct neuropathic pain models: spinal nerve ligation (SNL) and chronic constriction injury (CCI). In the SNL model, both mRNA and protein levels of Nav1.7 were markedly lower in the L5 dorsal root ganglia (DRG) but were significantly higher in the L4 DRG. Nav1.7 protein levels were notably higher in both L4 and L5 DRGs under CCI conditions. We found that excessive damage of L5 nerves such as SNL reduced expression levels of Nav1.7 in the injured L5 DRG and activated the adjacent uninjured DRG, resulting in Nav1.7 level increases in the adjacent L4 DRG. We confirmed again that Nav1.7 was closely related to neuropathic pain induced by nerve injury. More importantly, our results suggest that tracing the molecular changes exclusively in the L5 DRG in SNL model may not completely explain the pain mechanism; it is necessary to study the adjacent uninjured L4 DRG.

Axotomy Induces Phasic Alterations in Luman/CREB3 Expression and Nuclear Localization in Injured and Contralateral Uninjured Sensory Neurons: Correlation With Intrinsic Axon Growth Capacity.

Luman/CREB3 is an important early retrograde axotomy signal regulating acute axon outgrowth in sensory neurons through the adaptive unfolded protein response. As the injury response is transcriptionally multiphasic, a spatiotemporal analysis of Luman/CREB3 localization in rat dorsal root ganglion (DRG) with unilateral L4-L6 spinal nerve injury was conducted to determine if Luman/CREB3 expression was similarly regulated. Biphasic alterations in Luman/CREB3 immunofluorescence and nuclear localization occurred in neurons ipsilateral to 1-hour, 1-day, 2-day, 4-day, and 1-week injury, with a largely parallel, but less avid response contralaterally. This biphasic response was not observed at the transcript level. To assess whether changes in neuronal Luman expression corresponded with an altered intrinsic capacity to grow an axon/neurite in vitro, injury-conditioned and contralateral uninjured DRG neurons underwent a 24-hour axon growth assay. Two-day injury-conditioned neurons exhibited maximal outgrowth capacity relative to naïve, declining at later injury-conditioned timepoints. Only neurons contralateral to 1-week injury exhibited significantly higher axon growth capacity than naïve. In conclusion, alterations in neuronal injury-associated Luman/CREB3 expression support that a multiphasic cell body response occurs and reveal a novel contralateral plasticity in axon growth capacity at 1-week post-injury. These adaptive responses have the potential to inform when repair or therapeutic intervention may be most effective.

Expression of acetyl-histone H3 and acetyl-histone H4 in dorsal root ganglion and spinal dorsal horn in rat chronic pain models

AimsHistone acetylation and deacetylation are two histone posttranslational modifications that are usually controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although HATs or HDACs Inhibitors could relieve pain hypersensitivities in chronic pain animal models, it is not clear on the expression of global histone acetylation in the dorsal root ganglion (DRG) or spinal dorsal horn in chronic pain conditions. Main methodsA spinal nerve ligation (SNL)-induced neuropathic pain model and a complete Freund's adjuvant (CFA)-induced inflammatory pain model in rats were used to examine the expression of total acetyl-histone H3 (AcH3) and total acetyl-histone H4 (AcH4) by immunofluorescence or western blot. Key findingsAcH3 and AcH4 not only localized in neuronal nuclei, but also in nuclei of glial cells in the DRG. Unilateral SNL induced the increase of AcH3 and AcH4 expression in the injured lumbar 5 (L5) DRG, but not in the uninjured L5 DRG or the spinal dorsal horn, while unilateral intraplantar injection of CFA increased AcH3 and AcH4 expression in the ipsilateral L4/5 spinal dorsal horn, but not in the L4/5 DRG. SignificanceThese results provide morphological evidence for global histone acetylation expression in the DRG and spinal cord and indicate the differential expression in the DRG and spinal dorsal horn in different chronic pain models. More precise epigenetic mechanisms of histone acetylation on the target genes need to be revealed.

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.

Upregulation of N-type calcium channels in the soma of uninjured dorsal root ganglion neurons contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury

N-type voltage-gated calcium (Cav2.2) channels are expressed in the central terminals of dorsal root ganglion (DRG) neurons, and are critical for neurotransmitter release. Cav2.2 channels are also expressed in the soma of DRG neurons, where their function remains largely unknown. Here, we showed that Cav2.2 was upregulated in the soma of uninjured L4 DRG neurons, but downregulated in those of injured L5 DRG neurons following L5 spinal nerve ligation (L5-SNL). Local application of specific Cav2.2 blockers (ω-conotoxin GVIA, 1–100 μM or ZC88, 10–1000 μM) onto L4 and 6 DRGs on the operated side, but not the contralateral side, dose-dependently reversed mechanical allodynia induced by L5-SNL. Patch clamp recordings revealed that both ω-conotoxin GVIA (1 μM) and ZC88 (10 μM) depressed hyperexcitability in L4 but not in L5 DRG neurons of L5-SNL rats. Consistent with this, knockdown of Cav2.2 in L4 DRG neurons with AAV-Cav2.2 shRNA substantially prevented L5-SNL-induced mechanical allodynia and hyperexcitability of L4 DRG neurons. Furthermore, in L5-SNL rats, interleukin-1 beta (IL-1β) and IL-10 were upregulated in L4 DRGs and L5 DRGs, respectively. Intrathecal injection of IL-1β induced mechanical allodynia and Cav2.2 upregulation in bilateral L4–6 DRGs of naïve rats, whereas injection of IL-10 substantially prevented mechanical allodynia and Cav2.2 upregulation in L4 DRGs in L5-SNL rats. Finally, in cultured DRG neurons, Cav2.2 was dose-dependently upregulated by IL-1β and downregulated by IL-10. These data indicate that the upregulation of Cav2.2 in uninjured DRG neurons via IL-1β over-production contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

Hyperpolarization-activated cyclic nucleotide-gated channels contribute to spontaneous activity in L4 C-fiber nociceptors, but not Aβ-non-nociceptors, after axotomy of L5-spinal nerve in the rat in vivo.

Peripheral neuropathic pain associated with partial nerve injury is believed to be driven partly by aberrant spontaneous activity (SA) in both injured and uninjured dorsal root ganglion (DRG) neurons. The underlying ionic mechanisms are not fully understood, but hyperpolarization-activated cyclic nucleotide-gated (HCN) channels which underlie the excitatory Ih current have been implicated in SA generation in axotomized A-fiber neurons after L5-spinal nerve ligation/axotomy (SNL/SNA). Here, using a modified model of SNA (mSNA) which involves, in addition to L5-SNA, loose ligation of the L4-spinal nerve with neuroinflammation-inducing chromic gut, we examined whether HCN channels also contribute to SA in the adjacent L4-neurons. Intracellular recordings from L4-DRG neurons in control rats, and L4-DRG neurons in mSNA rats were made using in vivo voltage- and current-clamp techniques. Compared with control, L4 C-nociceptors and Aβ-low-threshold mechanoreceptors (LTMs) exhibited SA 7 days after mSNA. This was accompanied, in C-nociceptors, by a significant increase in Ih amplitude, the percentage of Ih-expressing neurons, and Ih activation rate. Hyperpolarization-activated cyclic nucleotide-gated channel blockade with ZD7288 (10 mg/kg, intravenously) suppressed SA in C-nociceptors, but not Aβ-LTMs, and caused in C-nociceptors, membrane hyperpolarization and a decrease in Ih activation rate. Furthermore, intraplantar injection of ZD7288 (100 μM) was found to be as effective as gabapentin (positive control) in attenuating cold hypersensitivity in mSNA rats. These findings suggest that HCN channels contribute to nerve injury-induced SA in L4 C-nociceptors, but not Aβ-LTMs, and that ZD7288 exerts its analgesic effects by altering Ih activation properties and/or causing membrane hyperpolarization in L4 C-nociceptors.

Identification of key genes and pathways associated with neuropathic pain in uninjured dorsal root ganglion by using bioinformatic analysis.

PurposeNeuropathic pain is a complex chronic condition occurring post-nervous system damage. The transcriptional reprogramming of injured dorsal root ganglia (DRGs) drives neuropathic pain. However, few comparative analyses using high-throughput platforms have investigated uninjured DRG in neuropathic pain, and potential interactions among differentially expressed genes (DEGs) and pathways were not taken into consideration. The aim of this study was to identify changes in genes and pathways associated with neuropathic pain in uninjured L4 DRG after L5 spinal nerve ligation (SNL) by using bioinformatic analysis.Materials and methodsThe microarray profile GSE24982 was downloaded from the Gene Expression Omnibus database to identify DEGs between DRGs in SNL and sham rats. The prioritization for these DEGs was performed using the Toppgene database followed by gene ontology and pathway enrichment analyses. The relationships among DEGs from the protein interactive perspective were analyzed using protein–protein interaction (PPI) network and module analysis. Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of DEGs in the rodent neuropathic pain model.ResultsA total of 206 DEGs that might play a role in neuropathic pain were identified in L4 DRG, of which 75 were upregulated and 131 were downregulated. The upregulated DEGs were enriched in biological processes related to transcription regulation and molecular functions such as DNA binding, cell cycle, and the FoxO signaling pathway. Ctnnb1 protein had the highest connectivity degrees in the PPI network. The in vivo studies also validated that mRNA and protein levels of Ctnnb1 were upregulated in both L4 and L5 DRGs.ConclusionThis study provides insight into the functional gene sets and pathways associated with neuropathic pain in L4 uninjured DRG after L5 SNL, which might promote our understanding of the molecular mechanisms underlying the development of neuropathic pain.

Bulleyaconitine A preferably reduces tetrodotoxin-sensitive sodium current in uninjured dorsal root ganglion neurons of neuropathic rats probably via inhibition of protein kinase C.

Oral Bulleyaconitine A (BLA) is effective for treating neuropathic pain in human patients, but the underlying mechanism is poorly understood. Here, we tested whether BLA blocked voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons. Compelling evidence shows that voltage-gated sodium channels are upregulated in uninjured DRG neurons but downregulated in injured ones following peripheral nerve injury. We found that BLA preferably inhibited Na currents in uninjured DRG neurons in neuropathic rats. Compared to sham rats, IC50 values for resting and inactivated Na currents were 113 and 74 times lower in injured and uninjured neurons of L4-6 DRGs in spared nerve injury (SNI) rats (4.55 and 0.56 nM) and were 688 and 518 times lower in the uninjured L4 and L6 DRG neurons of L5 spinal nerve ligation (L5-SNL) rats. The use-dependent blockage of BLA on Na currents was more potent in neuropathic rats compared to sham rats. Bulleyaconitine A facilitated the inactivation of Na channels in each group. IC50 values for resting and inactivated tetrodotoxin-sensitive (TTX-S) channels were 1855 and 1843 times lower than those for TTX-resistant channels in the uninjured neurons of L5 spinal nerve ligation rats. The upregulation of protein kinase C was associated with the preferable effect of BLA on TTX-S Na channels in the uninjured DRG neurons. Local application of BLA onto L4-6 DRGs at 0.1 to 10 nM dose-dependently alleviated the mechanical allodynia and thermal hyperalgesia in L5 spinal nerve ligation model. Thus, preferable blockage of TTX-S Na channels in uninjured DRG neurons may contribute to BLA's antineuropathic pain effect.

Mas-related gene (Mrg) C receptors inhibit mechanical allodynia and spinal microglia activation in the early phase of neuropathic pain in rats

Mas-related gene (Mrg) C receptors are exclusively expressed in the trigeminal and dorsal root ganglia (DRG). However, their functional roles are poorly understood. This study was aimed to determine the effect of MrgC receptors on pain hypersensitivity in the early phase of neuropathic pain and its underlying mechanisms. Intrathecal (i.t.) administration of the selective MrgC receptor agonist bovine adrenal medulla 8–22 (BAM8-22) at 1 or 10nmol attenuated mechanical allodynia one day after L5 spinal nerve ligation (SNL) surgery. I.t. BAM8-22 (10 nmol) inhibited SNL-induced microglia activation in the spinal dorsal horn on day 2 post-SNL. The BAM8-22 treatment also abolished SNL-induced upregulation of neuronal nitric oxide synthesis (nNOS) in the dorsal root ganglia (DRG). On the other hand, SNL, but not sham, surgery reduced the expression of MrgC receptor mRNA in the injured L5 DRG without changing thier levels in the adjacent uninjured L4 or L6 DRG on day 2 following the surgery. These results suggest that the activation of MrgC receptors can relieve pain hypersensitivity by the inhibition of nNOS increase in DRG neurons and microglia activation in the spinal dorsal horn in the early time following peripheral nerve injury. This study provides evidence that MrgC receptors could be targeted as a novel therapy for neuropathic pain with limited unwanted effects.

Effects of peripheral nerve injury on parvalbumin expression in adult rat dorsal root ganglion neurons.

BackgroundParvalbumin (PV) is a calcium binding protein that identifies a subpopulation of proprioceptive dorsal root ganglion (DRG) neurons. Calcitonin gene-related peptide (CGRP) is also expressed in a high proportion of muscle afferents but its relationship to PV is unclear. Little is known of the phenotypic responses of muscle afferents to nerve injury. Sciatic nerve axotomy or L5 spinal nerve ligation and section (SNL) lesions were used to explore these issues in adult rats using immunocytochemistry.ResultsIn naive animals, the mean PV expression was 25 % of L4 or L5 dorsal root ganglion (DRG) neurons, and this was unchanged 2 weeks after sciatic nerve axotomy. Colocalization studies with the injury marker activating transcription factor 3 (ATF3) showed that approximately 24 % of PV neurons expressed ATF3 after sciatic nerve axotomy suggesting that PV may show a phenotypic switch from injured to uninjured neurons. This possibility was further assessed using the spinal nerve ligation (SNL) injury model where injured and uninjured neurons are located in different DRGs. Two weeks after L5 SNL there was no change in total PV staining and essentially all L5 PV neurons expressed ATF3. Additionally, there was no increase in PV-ir in the adjacent uninjured L4 DRG cells. Co-labelling of DRG neurons revealed that less than 2 % of PV neurons normally expressed CGRP and no colocalization was seen after injury.ConclusionThese experiments clearly show that axotomy does not produce down regulation of PV protein in the DRG. Moreover, this lack of change is not due to a phenotypic switch in PV immunoreactive (ir) neurons, or de novo expression of PV-ir in uninjured neurons after nerve injury. These results further illustrate differences that occur when muscle afferents are injured as compared to cutaneous afferents.

Voltage-gated sodium channel function and expression in injured and uninjured rat dorsal root ganglia neurons

The nine members of the voltage-gated sodium channel (Nav) family mediate inward sodium currents that depolarize neurons and lead to action potential firing. Increased Nav expression and function in sensory ganglia may drive ectopic action potentials and result in neuropathic pain. Using patch-clamp electrophysiology and molecular biology techniques, experiments were performed to elucidate the contribution of Nav channels to sodium currents in rat dorsal root ganglion (DRG) neurons following the L5/L6 spinal nerve ligation (SNL) model of neuropathic pain. The abundance of DRG neurons with fast, tetrodotoxin sensitive (TTX-S) currents was seven-fold higher whereas the abundance of DRG neurons with slow, tetrodotoxin resistant (TTX-R) currents was nearly thirty-fold lower when comparing ipsilateral (injured) to contralateral (uninjured) neurons. TTX-S currents were elevated in larger neurons while TTX-R currents were reduced in both small and large neurons. Among Nav transcripts encoding TTX-R channels, Scn10a (Nav1.8) and Scn11a (Nav1.9) expression was twenty- to thirty-fold lower, while among Nav transcripts encoding TTX-S channels, Scn3a (Nav1.3) expression was four-fold higher in injured compared to uninjured DRG by qRT-PCR analysis. In summary, the SNL model of neuropathic pain induced a phenotypic switch in Nav expression from TTX-R to TTX-S channels in injured DRG neurons. Transcriptional reprogramming of Nav genes may drive ectopic action potential firing and contribute to neuropathic pain.

Axotomy of tributaries of the pelvic and pudendal nerves induces changes in the neurochemistry of mouse dorsal root ganglion neurons and the spinal cord

Using immunohistochemical techniques, we characterized changes in the expression of several neurochemical markers in lumbar 4-sacral 2 (L4-S2) dorsal root ganglion (DRG) neuron profiles (NPs) and the spinal cord of BALB/c mice after axotomy of the L6 and S1 spinal nerves, major tributaries of the pelvic (targeting pelvic visceral organs) and pudendal (targeting perineum and genitalia) nerves. Sham animals were included. Expression of cyclic AMP-dependent transcription factor 3 (ATF3), calcitonin gene-related peptide (CGRP), transient receptor potential cation channel subfamily V, member 1 (TRPV1), tyrosine hydroxylase (TH) and vesicular glutamate transporters (VGLUT) types 1 and -2 was analysed seven days after injury. L6-S1 axotomy induced dramatic de novo expression of ATF3 in many L6-S1 DRG NPs, and parallel significant downregulations in the percentage of CGRP-, TRPV1-, TH- and VGLUT2-immunoreactive (IR) DRG NPs, as compared to their expression in uninjured DRGs (contralateral L6-S1-AXO; sham mice); VGLUT1 expression remained unaltered. Sham L6-S1 DRGs only showed a small ipsilateral increase in ATF3-IR NPs (other markers were unchanged). L6-S1-AXO induced de novo expression of ATF3 in several lumbosacral spinal cord motoneurons and parasympathetic preganglionic neurons; in sham mice the effect was limited to a few motoneurons. Finally, a moderate decrease in CGRP- and TRPV1-like-immunoreactivities was observed in the ipsilateral superficial dorsal horn neuropil. In conclusion, injury of a mixed visceral/non-visceral nerve leads to considerable neurochemical alterations in DRGs matched, to some extent, in the spinal cord. Changes in these and potentially other nociception-related molecules could contribute to pain due to injury of nerves in the abdominopelvic cavity.

Temporal changes in MrgC expression after spinal nerve injury

Mas-related G-protein-coupled receptor subtype C (MrgC) may play an important role in pain sensation. However, the distribution of MrgC receptors in different subpopulations of rodent dorsal root ganglion (DRG) neurons has not been clearly demonstrated owing to a lack of MrgC-selective antibody. It is also unclear whether peripheral nerve injury induces different time-dependent changes in MrgC expression in injured and uninjured DRG neurons. Here we showed that MrgC immunoreactivity is distributed in both IB4-positive (non-peptidergic) and calcitonin gene-related peptide-positive (peptidergic) DRG neurons in mice and rats. Importantly, the MrgC mRNA level and MrgC immunoreactivity were both decreased in the injured L5 DRG compared to corresponding levels in the contralateral (uninjured) DRG in rats on days 14 and 30 after an L5 spinal nerve ligation. In contrast, mRNA and protein levels of MrgC were increased in the adjacent uninjured L4 DRG. Thus, nerve injury may induce temporal changes in MrgC expression that differ between injured and uninjured DRG neurons. In animal behavior tests, chronic constriction injury of the sciatic nerve induced mechanical pain hypersensitivity in wild-type mice and Mrg-clusterΔ−/− mice (Mrg KO). However, the duration of mechanical hypersensitivity was longer in the Mrg KO mice than in their wild-type littermates, indicating that activation of Mrgs may constitute an endogenous mechanism that inhibits the maintenance of neuropathic pain in mice. These findings extend our knowledge about the distribution of MrgC in rodent DRG neurons and the regulation of its expression by nerve injury.

Nerve injury-induced upregulation of miR-21 in the primary sensory neurons contributes to neuropathic pain in rats

Neuropathic pain is intractable chronic pain caused by damage to the somatosensory system. Peripheral nerve injury of the primary sensory neurons changes expressions of multiple microRNAs that affect many aspects of cellular functions by regulating specific gene expressions. miR-21, a well-characterized oncogenic miRNA, is consistently upregulated after peripheral nerve injury in the dorsal root ganglion (DRG), where cell bodies of primary sensory neurons exist. However, their causal relationship to the pain is fully unknown. In this study, we therefore investigated the miR-21 expression in the DRGs along with the time course of neuropathic pain and its involvement in the neuropathic pain. Neuropathic pain was induced in rats by specific ligation of the left fifth lumbar spinal nerve. After the injury, miR-21 expression in the injured DRG neurons, but not in the neighboring uninjured DRG neurons, was persistently upregulated following the pain development. Intrathecal administration of interleukin-1β also increased the miR-21 expression in the DRG. Both mechanical allodynia and thermal hyperalgesia in the neuropathic pain were attenuated by intrathecal administration of miR-21 inhibitor. miR-21 is specifically upregulated in the injured DRG neurons and causally involved in the late phase of neuropathic pain. Therefore, miR-21 and its modulatory system may be a therapeutic target for intractable chronic neuropathic pain.