Peripheral nerve injury produces a sustained shift in the balance between glutamate release and uptake in the dorsal horn of the spinal cord

Peripheral nerve injury provokes heightened excitability of primary sensory afferents including nociceptors, and elicits ectopic activity in lesioned and neighboring intact nerve fibers. The major transmitter released by sensory afferents in the superficial dorsal horn of the spinal cord is glutamate. Glutamate is critically involved in nociceptive signaling and the development of neuropathic pain. We recorded miniature excitatory postsynaptic currents (mEPSCs) from neurons in lamina II of the rat dorsal horn to assess spontaneous synaptic activity after spared nerve injury (SNI), a model of chronic neuropathic pain. Following SNI, the frequency of mEPSCs doubled, indicating heightened glutamate release from primary afferents or spinal interneurons. Consistent with this finding, glutamate concentrations in the cerebrospinal fluid were elevated at 1 and 4 weeks after SNI. Transmitter uptake was insufficient to prevent the rise in extracellular glutamate as the expression of glutamate transporters remained unchanged or decreased. 2-Methyl-6-(phenylethynyl)pyridine hydrochloride, an antagonist of metabotropic glutamate receptor 5 (mGluR5), reduced the frequency of mEPSCs to its preinjury level, suggesting a positive feedback mechanism that involves facilitation of transmitter release by mGluR5 activation in the presence of high extracellular glutamate. Treatment with the β-lactam antibiotic ceftriaxone increased the expression of glutamate transporter 1 (Glt1) in the dorsal horn after SNI, raised transmitter uptake, and lowered extracellular glutamate. Improving glutamate clearance prevented the facilitation of transmitter release by mGluR5 and attenuated neuropathic pain-like behavior. Balancing glutamate release and uptake after nerve injury should be an important target in the management of chronic neuropathic pain.     

Unilateral Focal Burn Injury Is Followed by Long-Lasting Bilateral Allodynia and Neuronal Hyperexcitability in Spinal Cord Dorsal Horn

Pain after burn injury can be intense and long lasting. Treatment is often ineffective, and there is a need for increased knowledge of the underlying pain mechanisms. In the present study, we established a unilateral partial-thickness burn injury model, which produces ipsilateral mechanical allodynia soon after injury, followed by contralateral allodynia. Chronic bilateral allodynia lasts up to 8 weeks postinjury in this model. In addition to the change in pain behavior, electrophysiological analyses showed that dorsal horn neurons become hyperexcitable and display significantly increased evoked activity with enlarged receptive fields, initially on the side ipsilateral to the injury, and subsequently on both sides of the spinal cord. It is known that, following nerve injury, activation of p38 mitogen-activated protein kinase (MAPK) pathways within spinal microglia contributes to the pathogenesis of pain. In our burn injury model, rapid and prolonged activation of phospho-p38-expressing microglia occurs bilaterally in the spinal cord dorsal horn. Taken together, these data demonstrate that a unilateral peripheral burn injury can produce long-lasting allodynia that can spread to the contralateral limb, together with dorsal horn neuronal hyperexcitability and microglial activation on both ipsilateral and contralateral sides of the spinal cord. Our results suggest that central neuropathic mechanisms can contribute to pain after burn injury.PerspectiveMechanisms contributing to pain following burn injury are incompletely understood. In a novel animal model of burn injury, we have demonstrated hyperexcitability of second-order sensory neurons, activation of microglia, and chronic bilateral pain following the burn injury. This work identifies potential therapeutic targets to alleviate pain after burn injury.     

Spinal microglia and neuropathic pain in young rats

Neuropathic pain behaviour is not observed in neonatal rats and tactile allodynia does not develop in the spared nerve injury (SNI) model until rats are 4 weeks of age at the time of surgery. Since activated spinal microglia are known to play a key role in neuropathic pain, we have investigated whether the microglial response to nerve injury in young rats differs from that in adults. Here we show that dorsal horn microglial activation, visualised with IBA-1 immunostaining, is significantly less in postnatal day (P) 10 rat pups than in adults, 7 days after SNI. This was confirmed by qPCR analysis of IBA-1 mRNA and mRNA of other microglial markers, integrin-alpha M, MHC-II DMalpha and MHC-II DMbeta. Dorsal horn IBA-1+ve microglia could be activated, however, by intraspinal injections of lipopolysaccharide (LPS) or N-methyl-d-aspartate (NMDA) at P10, although the increase in the levels of mRNA for all microglial markers was less than in the adult rat. In addition, P10 rats developed a small but significant mechanical allodynia in response to intrathecal LPS. Intrathecal injection of cultured ATP-activated microglia, known to cause mechanical allodynia in adult rats, had no behavioural effect at P10 and only began to cause allodynia if injections were performed at P16. The results clearly demonstrate immaturity of the microglial response triggered by nerve injury in the first postnatal weeks which may explain the absence of tactile allodynia following peripheral nerve injury in young rats.     

Peripheral axonal injury results in reduced mu opioid receptor pre- and post-synaptic action in the spinal cord

In both the spared nerve injury (SNI) and spinal nerve ligation (SNL) rat peripheral neuropathic pain models the presynaptic inhibitory effect of the mu opioid receptor (MOR) agonist (DAMGO) on primary afferent-evoked excitatory postsynaptic currents (EPSCs) and miniature EPSCs in superficial dorsal horn neurons is substantially reduced, but only in those spinal cord segments innervated by injured primary afferents. The two nerve injury models also reduce the postsynaptic potassium channel opening action of DAMGO on lamina II spinal cord neurons, but again only in segments receiving injured afferent input. The inhibitory action of DAMGO on ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons is also reduced in affected segments following nerve injury. MOR expression decreases substantially in injured dorsal root ganglion neurons (DRG), while intact neighboring DRGs are unaffected. Decreased activation of MOR on injured primary afferent central terminals and the second order neurons they innervate may minimize any reduction by opioids of the spontaneous pain mediated by ectopic input from axotomized small diameter afferents. Retention of MOR expression and activity in nearby non-injured afferents will enable, however, an opioid-mediated reduction of stimulus-evoked and spontaneous pain carried by intact nociceptor afferents and we find that intrathecal DAMGO (1000 ng) reduces mechanical hypersensitivity in rats with SNL. Axotomy-induced changes in MOR may contribute to opioid- insensitive components of neuropathic pain while the absence of these changes in intact afferents may contribute to the opioid sensitive components.     

Interleukin‐6 induces microglial CX3CR1 expression in the spinal cord after peripheral nerve injury through the activation of p38 MAPK

Peripheral nerve injury leading to neuropathic pain induces the upregulation of interleukin (IL)‐6 and microglial CX3CR1 expression, and activation of p38 mitogen‐activated protein kinase (MAPK) in the spinal cord. Here, we investigated whether IL‐6 regulates CX3CR1 expression through p38 MAPK activation in the spinal cord in rats with chronic constriction injury (CCI) of the sciatic nerve. Similar temporal changes in the expression of IL‐6, phosphorylated p38 MAPK and CX3CR1 were observed following CCI. The increases in CX3CR1 expression, p38 MAPK activation and pain behavior after CCI were suppressed by blocking IL‐6 action with a neutralizing antibody, while they were enhanced by supplying exogenous recombinant rat IL‐6 (rrIL‐6). rrIL‐6 also induced increases in spinal CX3CR1 expression, p38 MAPK activation and pain behavior in naïve rats without nerve injury. Furthermore, treatment with the p38 MAPK‐specific inhibitor, SB203580, suppressed the increase in CX3CR1 expression induced by CCI or rrIL‐6 treatment. Finally, blocking CX3CR1 or p38 MAPK activation prevented the development of mechanical allodynia and thermal hyperalgesia induced by CCI or rrIL‐6 treatment. These results suggest a new mechanism of neuropathic pain, in which IL‐6 induces microglial CX3CR1 expression in the spinal cord through p38 MAPK activation, enhancing the responsiveness of microglia to fractalkine in the spinal cord, thus playing an important role in neuropathic pain after peripheral nerve injury.     

Genetic deletion of synapsin II reduces neuropathic pain due to reduced glutamate but increased GABA in the spinal cord dorsal horn

The synaptic vesicle protein synapsin II is specifically expressed in synaptic terminals of primary afferent nociceptive neurons and regulates transmitter release in the spinal cord dorsal horn. Here, we assessed its role in nerve injury-evoked molecular and behavioral adaptations in models of peripheral neuropathic pain using mice genetically lacking synapsin II. Deficiency of synapsin II resulted in reduced mechanical and cold allodynia in two models of peripheral neuropathic pain. This was associated with decreased glutamate release in the dorsal horn of the spinal cord upon sciatic nerve injury or capsaicin application onto the sciatic nerve and reduced calcium signals in spinal cord slices upon persistent activation of primary afferents. In addition, the expression of the vesicular glutamate transporters, VGLUT1 and VGLUT2, was strongly reduced in synapsin II knockout mice in the spinal cord. Conversely, synapsin II knockout mice showed a stronger and longer-lasting increase of GABA in lamina II of the dorsal horn after nerve injury than wild type mice. These results suggest that synapsin II is involved in the regulation of glutamate and GABA release in the spinal cord after nerve injury, and that a imbalance between glutamatergic and GABAergic synaptic transmission contributes to the manifestation of neuropathic pain.     

Characterization of cell proliferation in rat spinal cord following peripheral nerve injury and the relationship with neuropathic pain

Glial activation is a typical response of the central nervous system to nerve injury. In the current investigation, we characterized the temporal and spatial pattern of glial proliferation, one of the most conspicuous features of glial activation, in relation to nerve injury-induced neuropathic pain. Using bromodeoxyuridine (BrdU) as a mitotic marker, we analyzed cell proliferation in the spinal cord, identified the phenotype of dividing cells, traced their fate, and correlated these phenomena with behavioural assays of the neuropathic pain syndrome. Our results demonstrated that peripheral nerve injury induced an early and transient cell proliferation, on the spinal cord ipsilateral to the nerve lesion which peaked at day 3 post-surgery. The majority of the proliferating cells were Iba-1(+) microglia, together with some NG2(+) oligodendrocyte progenitors, and GFAP(+) astrocytes. These newly generated cells continued to divide over time with the response peaking at day 14 post-injury. Microglia were always the predominant phenotype which made up over 60% of activated microglia derived from this newly generated cell population. There was a close temporal correlation between microglial proliferation in the spinal cord dorsal horn and the abnormal pain responses, suggesting a contribution of the new microglia to the genesis of the neuropathic pain symptoms.     

Intrathecal neuropeptide Y reduces behavioral and molecular markers of inflammatory or neuropathic pain

Our previous work indicates that the intrathecal administration of neuropeptide Y (NPY) acts at its cognate receptors to reduce behavioral signs of nociception in several models of inflammatory pain, including the formalin test. The present study extends these findings to a rat model of peripheral neuropathic pain, and then evaluates the hypothesis that NPY inhibits inflammation- and nerve injury-induced activation of spinal nociceptive transmission. Here we show that NPY dose-dependently reduced behavioral signs of mechanical and cold hypersensitivity in the spared nerve injury (SNI) model. Intrathecal administration of either a Y1 (BIBO3304) or a Y2 (BIIE0246) receptor antagonist dose-dependently reversed the anti-allodynic actions of NPY. To monitor the effects of NPY on the stimulus-induced activation of spinal nociresponsive neurons, we quantified protein expression of the immediate-early gene c-fos in lamina I-VI of the L4-L5 dorsal horn, with special attention to the mediolateral pattern of Fos immunohistochemical staining after SNI. Either tactile stimulation of the hindpaw ipsilateral to nerve injury, or intraplantar injection of noxious formalin, increased the number of Fos-like immunoreactive profiles. Tactile stimulation evoked a mediolateral pattern of Fos expression corresponding to the innervation territory of the uninjured (sural) nerve. We found that intrathecal NPY reduced both formalin- and SNI-induced Fos expression. NPY inhibition of SNI-induced Fos expression was localized to the sural (uninjured) innervation territory, and could be blocked by intrathecal BIBO3304 and BIIE0246. We conclude that NPY acts at spinal Y1 and Y2 receptors to reduce spinal neuron activity and behavioral signs of inflammatory or neuropathic pain.     

Brain derived nerve growth factor induces spinal noradrenergic fiber sprouting and enhances clonidine analgesia following nerve injury in rats

Many treatments for neuropathic pain activate or augment norepinephrine release in the spinal cord, yet these treatments are less effective against acute nociceptive stimuli. We previously showed in mice that peripheral nerve injury results in sprouting of spinal noradrenergic fibers, possibly reflecting the substrate for this shift in drug efficacy. Here, we tested whether such sprouting also occurs in rats after nerve injury and examined one signal for such sprouting. Ligation of L5 and L6 spinal nerves unilaterally in rats resulted in hypersensitivity to tactile stimulation of the ipsilateral paw, and sprouting of noradrenergic fibers in the dorsal horn of the lumbar spinal cord. Brain derived nerve growth factor (BDNF) content increased in L4-L6 dorsal root ganglia ipsilateral to injury and in lumbar spinal cord following nerve injury, and intrathecal infusion of BDNF antiserum prevented spinal noradrenergic sprouting. This treatment also prevented the increased analgesic efficacy of intrathecal clonidine observed after nerve injury. Intraspinal injection of BDNF in non-injured rats mimicked the sprouting of spinal noradrenergic fibers seen after nerve injury. These results suggest that increased BDNF synthesis and release drives spinal noradrenergic sprouting following nerve injury, and that this sprouting may paradoxically increase the capacity for analgesia in the setting of neuropathic pain from drugs which utilize or mimic the noradrenergic pathway.     

Depletion of endogenous spinal 5-HT attenuates the behavioural hypersensitivity to mechanical and cooling stimuli induced by spinal nerve ligation

There is compelling evidence for a strong facilitatory drive modulating spinal nociceptive transmission. This is in part via serotonergic pathways and originates from the rostroventral medulla. We previously demonstrated that neuropathic pain is associated with an enhanced descending facilitatory drive onto the mechanical evoked responses of dorsal horn neurones, mediated by 5-HT acting at spinal 5-HT3 receptors. Furthermore, depletion of spinal 5-HT has been shown to reduce the at-level mechanical allodynia that follows spinal cord injury. To further clarify the role and direction of effect of endogenous 5-HT, we investigated the effects of depleting spinal 5-HT, via intrathecal injection of 5,7di-hydroxytryptamine (5,7DHT), on pain behaviours after spinal nerve ligation (SNL). Depletion of spinal 5-HT in normal animals leads to reductions in mechanical and thermal evoked responses of deep dorsal horn neurones implying that spinal 5-HT has a predominant facilitatory function. After nerve injury, the frequency of paw withdrawals to low intensity mechanical and cooling stimulation of the ipsilateral hindpaw in the SNL-5,7DHT group was significantly attenuated when compared with the SNL-saline group from day seven post-nerve injury. Sham-5,7DHT and sham-saline animals showed very little response sensitivity on either hindpaw. This 5-HT-mediated difference in behaviour was independent of both the up-regulation of the NK1 receptor and spinal microglial activation produced by nerve injury. These data suggest that supraspinal serotonergic influences under these conditions are facilitatory and are implicated in the maintenance of spinal cord neuronal events leading to the behavioural hypersensitivity manifested after peripheral nerve damage.     

Depletion of endogenous noradrenaline does not prevent spinal cord plasticity following peripheral nerve injury

The present study examined the role of endogenous noradrenaline on glial and neuronal plasticity in the spinal cord in rats after peripheral nerve injury. An intrathecal injection of dopamine-β-hydroxylase antibody conjugated to saporin (DβH-saporin) completely depleted noradrenergic axons in the spinal cord and also reduced noradrenergic neurons in the locus coeruleus (A6) and A5 noradrenergic nucleus in the brainstem and noradrenergic axons in the paraventricular nucleus of the hypothalamus. DβH-saporin treatment itself did not alter mechanical withdrawal threshold, but enhanced mechanical hypersensitivity and intrathecal clonidine analgesia after L5-L6 spinal nerve ligation. In the spinal dorsal horn of spinal nerve ligation rats, DβH-saporin treatment increased choline acetyltransferase immunoreactivity as well as immunoreactivity in microglia of ionized calcium binding adaptor molecule 1[IBA1] and in astrocytes of glial fibrillary acidic protein, and brain-derived nerve growth factor content. DβH-saporin treatment did not, however, alter the fractional release of acetylcholine from terminals by dexmedetomidine after nerve injury. These results suggest that endogenous tone of noradrenergic fibers is not necessary for the plasticity of α2-adrenoceptor analgesia and glial activation after nerve injury, but might play an inhibitory role on glial activation. PERSPECTIVE: This study demonstrates that endogenous noradrenaline modulates plasticity of glia and cholinergic neurons in the spinal cord after peripheral nerve injury and hence influences the pathophysiology of spinal cord changes associated with neuropathic pain.     

Minocycline Attenuates Mechanical Allodynia and Central Sensitization Following Peripheral Second-Degree Burn Injury

Burn injury induces severe pain that can be refractory to existing pharmacotherapies. The underlying mechanism of burn pain remains unclear. We previously established an animal model and reported that unilateral burn injury induces chronic and bilateral mechanical allodynia, which is associated with central sensitization and microglial activation in the spinal cord dorsal horn. Modulation of the activity of microglia and p38 mitogen-activated protein kinase (MAPK) has been shown to ameliorate neuropathic pain in several nerve-injury pain models. In the present study, we show in this rat model that daily treatment with the microglial inhibitor minocycline (10 mg/kg), administered at the time of burn injury and for 7 days thereafter, significantly attenuates ipsilateral and contralateral allodynia as assessed up to 1 month following burn injury. These sensory changes are paralleled by significant suppression of evoked hyperexcitability of dorsal-horn neurons and of the expression of phosphorylated p38 (phospho-p38) in OX42+ microglial cells within the dorsal horn. Our results suggest that modulation of inflammation at early times after burn injury may have long-lasting effects, attenuating central neuropathic mechanisms which contribute to pain after burn injury.     

Injury discharges regulate calcium channel alpha-2-delta-1 subunit upregulation in the dorsal horn that contributes to initiation of neuropathic pain

Previous studies have shown that peripheral nerve injury in rats induces increased expression of the voltage gated calcium channel (VGCC) alpha-2-delta-1 subunit (Ca v alpha2 delta1) in spinal dorsal horn and sensory neurons in dorsal root ganglia (DRG) that correlates to established neuropathic pain states. To determine if injury discharges trigger Ca v alpha2 delta1 induction that contributes to neuropathic pain initiation, we examined allodynia onset and Ca v alpha2 delta1 levels in DRG and spinal dorsal horn of spinal nerve ligated rats after blocking injury induced neural activity with a local brief application of lidocaine on spinal nerves before the ligation. The lidocaine pretreatment blocked ligation-induced discharges in a dose-dependent manner. Similar pretreatment with the effective concentration of lidocaine diminished injury-induced increases of the Ca v alpha2 delta1 in DRG and abolished that in spinal dorsal horn specifically, and resulted in a delayed onset of tactile allodynia post-injury. Both dorsal horn Ca v alpha2 delta1 upregulation and tactile allodynia in the lidocaine pretreated rats returned to levels similar to that in saline pretreated controls 2 weeks post the ligation injury. In addition, preemptive intrathecal Ca v alpha2 delta1 antisense treatments blocked concurrently injury-induced allodynia onset and Ca v alpha2 delta1 upregulation in dorsal spinal cord. These findings indicate that injury induced discharges regulate Ca v alpha2 delta1 expression in the spinal dorsal horn that is critical for neuropathic allodynia initiation. Thus, preemptive blockade of injury-induced neural activity or Ca v alpha2 delta1 upregulation may be a beneficial option in neuropathic pain management.     

Intrathecal AAV Serotype 9-mediated Delivery of shRNA Against TRPV1 Attenuates Thermal Hyperalgesia in a Mouse Model of Peripheral Nerve Injury

Gene therapy for neuropathic pain requires efficient gene delivery to both central and peripheral nervous systems. We previously showed that an adenoassociated virus serotype 9 (AAV9) vector expressing short-hairpin RNA (shRNA) could suppress target molecule expression in the dorsal root ganglia (DRG) and spinal cord upon intrathecal injection. To evaluate the therapeutic potential of this approach, we constructed an AAV9 vector encoding shRNA against vanilloid receptor 1 (TRPV1), which is an important target gene for acute pain, but its role in chronic neuropathic pain remains unclear. We intrathecally injected it into the subarachnoid space at the upper lumbar spine of mice 3 weeks after spared nerve injury (SNI). Delivered shTRPV1 effectively suppressed mRNA and protein expression of TRPV1 in the DRG and spinal cord, and it attenuated nerve injury-induced thermal allodynia 10–28 days after treatment. Our study provides important evidence for the contribution of TRPV1 to thermal hypersensitivity in neuropathic pain and thus establishes intrathecal AAV9-mediated gene delivery as an investigative and potentially therapeutic platform for the nervous system.     

低频电针对选择性神经损伤大鼠神经痛维持期脊髓背角PKA-TRPV1通路及痛敏递质的干预作用

目的 探讨低频电针干预神经病理痛维持期脊髓背角（SCDH）蛋白激酶A（PKA）、辣椒素受体（TRPV1）通路的调控机制。 方法 将大鼠随机分为空白对照组、假手术组、模型对照组、电针干预组4组。采用坐骨神经分支选择性神经损伤（SNI）方法建立神经病理痛模型。电针干预取术侧足三里、昆仑穴,频率2Hz,每日1次,连续干预14d。检测大鼠术侧后足缩足阈值（PWT）、SCDH PKA和TRPV1以及降钙素基因相关肽（CGRP）和P物质（SP）水平。 结果 SNI模型大鼠术侧PWT下降（P<0.01）,术侧SCDH PKA、TRPV1、CGRP、SP水平均上调（P<0.05）;2Hz电针可提高SNI模型大鼠PWT（P<0.01）,降低术侧SCDH PKA、TRPV1、CGRP、SP水平（P<0.05）。 结论 低频电针能改善神经病理痛,可能与其下调SCDH PKA-TRPV1通路以及CGRP、SP痛敏递质水平有关。     

Central glucocorticoid receptors regulate the upregulation of spinal cannabinoid-1 receptors after peripheral nerve injury in rats

Previous studies have shown that peripheral nerve injury upregulated both glucocorticoid receptors (GR) and cannabinoid-1 receptors (CB1R) within the spinal cord dorsal horn in rats. However, the relationship between the expression of spinal GR and CB1R after nerve injury remains unclear. Here, we examined the hypothesis that the upregulation of spinal CB1R induced by chronic constriction nerve injury (CCI) in rats would be regulated by spinal GR. CCI induced the upregulation of spinal CB1R primarily within the ipsilateral spinal cord dorsal horn as revealed by Western blot and immunohistochemistry. The expression of CB1R in CCI rats was substantially attenuated by intrathecal treatment with either the GR antagonist RU38486 or a GR antisense oligonucleotide given twice daily for postoperative day 1-6, whereas the expression of spinal CB1R was enhanced following intrathecal administration of a GR sense oligonucleotide twice daily for postoperative day 1-6. Furthermore, the upregulation of spinal CB1R after nerve injury was prevented in adrenalectomized rats, which was at least partially restored with the intrathecal administration of an exogenous GR agonist dexamethasone, indicating that corticosteroids (endogenous GR agonists) were critical to spinal GR actions. Since the development of neuropathic pain behaviors in CCI rats was attenuated by either RU38486 or a GR antisense oligonucleotide, these results suggest that CB1R is a downstream target for spinal GR actions contributory to the mechanisms of neuropathic pain.     

Role of SIP30 in the development and maintenance of peripheral nerve injury-induced neuropathic pain

Using the chronic constriction injury (CCI) model of neuropathic pain, we profiled gene expression in the rat spinal cord, and identified SIP30 as a gene whose expression was elevated after CCI. SIP30 was previously shown to interact with SNAP25, but whose function was otherwise unknown. We now show that in the spinal cord, SIP30 was present in the dorsal horn laminae where the peripheral nociceptive inputs first synapse, co-localizing with nociception-related neuropeptides CGRP and substance P. With the onset of neuropathic pain after CCI surgery, SIP30 mRNA and protein levels increased in the ipsilateral side of the spinal cord, suggesting a potential association between SIP30 and neuropathic pain. When CCI-upregulated SIP30 was inhibited by intrathecal antisense oligonucleotide administration, neuropathic pain was attenuated. This neuropathic pain-reducing effect was observed both during neuropathic pain onset following CCI, and after neuropathic pain was fully established, implicating SIP30 involvement in the development and maintenance phases of neuropathic pain. Using a secretion assay in PC12 cells, anti-SIP30 siRNA decreased the total pool of synaptic vesicles available for exocytosis, pointing to a potential function for SIP30. These results suggest a role of SIP30 in the development and maintenance of peripheral nerve injury-induced neuropathic pain.     

Downregulation of spinal glutamate transporter EAAC1 following nerve injury is regulated by central glucocorticoid receptors in rats

Previous studies have shown that glucocorticoid receptors (GR) were upregulated, whereas glutamate transporters were downregulated, within the spinal cord dorsal horn after peripheral nerve injury. However, the relationship between the expression of spinal GR and glutamate transporter after nerve injury remains unknown. In the present study, we examined the hypothesis that central GR would regulate the expression of spinal glutamate transporter EAAC1 following chronic constriction nerve injury (CCI) in rats. CCI induced a significant downregulation of EAAC1 expression primarily within the ipsilateral spinal cord dorsal horn when examined on postoperative day 7 using both Western blot and immunohistochemistry. The downregulation of EAAC1 was significantly diminished after either the GR antagonist RU38486 (4 > 2 = 0.5 microg = vehicle) or a GR antisense oligonucleotide was administered intrathecally twice daily for postoperative day 1-6. Moreover, CCI induced a significant downregulation of nuclear factor kappaB (NF-kappaB) within the ipsilateral spinal cord dorsal horn, which also was attenuated by either RU38486 (4 > 2 = 0.5 microg = vehicle) or a GR antisense oligonucleotide. The immunohistochemical data indicated a pattern of colocalization between GR and EAAC1 as well as GR and NF-kappaB within the spinal cord dorsal horn. Since, NF-kappaB has been shown to regulate the expression of those cellular elements linked to inflammation and tissue injury and its activity can be negatively regulated by GR activation, these results suggest that spinal GR through NF-kappaB may play a significant role in the regulation of EAAC1 expression after peripheral nerve injury, a cellular pathway that may contribute to the development of neuropathic pain behaviors in rats.     

MrgC agonism at central terminals of primary sensory neurons inhibits neuropathic pain

Chronic neuropathic pain is often refractory to current pharmacotherapies. The rodent Mas-related G-protein-coupled receptor subtype C (MrgC) shares substantial homogeneity with its human homologue, MrgX1, and is located specifically in small-diameter dorsal root ganglion neurons. However, evidence regarding the role of MrgC in chronic pain conditions has been disparate and inconsistent. Accordingly, the therapeutic value of MrgX1 as a target for pain treatment in humans remains uncertain. Here, we found that intrathecal injection of BAM8-22 (a 15-amino acid peptide MrgC agonist) and JHU58 (a novel dipeptide MrgC agonist) inhibited both mechanical and heat hypersensitivity in rats after an L5 spinal nerve ligation (SNL). Intrathecal JHU58-induced pain inhibition was dose dependent in SNL rats. Importantly, drug efficacy was lost in Mrg-cluster gene knockout (Mrg KO) mice and was blocked by gene silencing with intrathecal MrgC siRNA and by a selective MrgC receptor antagonist in SNL rats, suggesting that the drug action is MrgC dependent. Further, in a mouse model of trigeminal neuropathic pain, microinjection of JHU58 into ipsilateral subnucleus caudalis inhibited mechanical hypersensitivity in wild-type but not Mrg KO mice. Finally, JHU58 attenuated the miniature excitatory postsynaptic currents frequency both in medullary dorsal horn neurons of mice after trigeminal nerve injury and in lumbar spinal dorsal horn neurons of mice after SNL. We provide multiple lines of evidence that MrgC agonism at spinal but not peripheral sites may constitute a novel pain inhibitory mechanism that involves inhibition of peripheral excitatory inputs onto postsynaptic dorsal horn neurons in different rodent models of neuropathic pain.