Spinal microglial β‐endorphin signaling mediates IL‐10 and exenatide‐induced inhibition of synaptic plasticity in neuropathic pain

AimThis study aimed to investigate the regulation of pain hypersensitivity induced by the spinal synaptic transmission mechanisms underlying interleukin (IL)‐10 and glucagon‐like peptide 1 receptor (GLP‐1R) agonist exenatide‐induced pain anti‐hypersensitivity in neuropathic rats through spinal nerve ligations.MethodsNeuropathic pain model was established by spinal nerve ligation of L5/L6 and verified by electrophysiological recording and immunofluorescence staining. Microglial expression of β‐endorphin through autocrine IL‐10‐ and exenatide‐induced inhibition of glutamatergic transmission were performed by behavioral tests coupled with whole‐cell recording of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) through application of endogenous and exogenous IL‐10 and β‐endorphin.ResultsIntrathecal injections of IL‐10, exenatide, and the μ‐opioid receptor (MOR) agonists β‐endorphin and DAMGO inhibited thermal hyperalgesia and mechanical allodynia in neuropathic rats. Whole‐cell recordings of bath application of exenatide, IL‐10, and β‐endorphin showed similarly suppressed enhanced frequency and amplitude of the mEPSCs in the spinal dorsal horn neurons of laminae II, but did not reduce the frequency and amplitude of mIPSCs in neuropathic rats. The inhibitory effects of IL‐10 and exenatide on pain hypersensitive behaviors and spinal synaptic plasticity were totally blocked by pretreatment of IL‐10 antibody, β‐endorphin antiserum, and MOR antagonist CTAP. In addition, the microglial metabolic inhibitor minocycline blocked the inhibitory effects of IL‐10 and exenatide but not β‐endorphin on spinal synaptic plasticity.ConclusionThis suggests that spinal microglial expression of β‐endorphin mediates IL‐10‐ and exenatide‐induced inhibition of glutamatergic transmission and pain hypersensitivity via presynaptic and postsynaptic MORs in spinal dorsal horn.     

Supraspinal nociceptive networks in neuropathic pain after spinal cord injury

Neuropathic pain following spinal cord injury involves plastic changes along the whole neuroaxis. Current neuroimaging studies have identified grey matter volume (GMV) and resting‐state functional connectivity changes of pain processing regions related to neuropathic pain intensity in spinal cord injury subjects. However, the relationship between the underlying neural processes and pain extent, a complementary characteristic of neuropathic pain, is unknown. We therefore aimed to reveal the neural markers of widespread neuropathic pain in spinal cord injury subjects and hypothesized that those with greater pain extent will show higher GMV and stronger connectivity within pain related regions. Thus, 29 chronic paraplegic subjects and 25 healthy controls underwent clinical and electrophysiological examinations combined with neuroimaging. Paraplegics were demarcated based on neuropathic pain and were thoroughly matched demographically. Our findings indicate that (a) spinal cord injury subjects with neuropathic pain display stronger connectivity between prefrontal cortices and regions involved with sensory integration and multimodal processing, (b) greater neuropathic pain extent, is associated with stronger connectivity between the posterior insular cortex and thalamic sub‐regions which partake in the lateral pain system and (c) greater intensity of neuropathic pain is related to stronger connectivity of regions involved with multimodal integration and the affective‐motivational component of pain. Overall, this study provides neuroimaging evidence that the pain phenotype of spinal cord injury subjects is related to the underlying function of their resting brain.     

LRP1 deficiency in microglia blocks neuro-inflammation in the spinal dorsal horn and neuropathic pain processing

Following injury to the peripheral nervous system (PNS), microglia in the spinal dorsal horn (SDH) become activated and contribute to the development of local neuro-inflammation, which may regulate neuropathic pain processing. The molecular mechanisms that control microglial activation and its effects on neuropathic pain remain incompletely understood. We deleted the gene encoding the plasma membrane receptor, LDL Receptor-related Protein-1 (LRP1), conditionally in microglia using two distinct promoter-Cre recombinase systems in mice. LRP1 deletion in microglia blocked development of tactile allodynia, a neuropathic pain-related behavior, after partial sciatic nerve ligation (PNL). LRP1 deletion also substantially attenuated microglial activation and pro-inflammatory cytokine expression in the SDH following PNL. Because LRP1 shedding from microglial plasma membranes generates a highly pro-inflammatory soluble product, we demonstrated that factors which activate spinal cord microglia, including lipopolysaccharide (LPS) and colony-stimulating factor-1, promote LRP1 shedding. Proteinases known to mediate LRP1 shedding, including ADAM10 and ADAM17, were expressed at increased levels in the SDH after PNL. Furthermore, LRP1-deficient microglia in cell culture expressed significantly decreased levels of interleukin-1β and interleukin-6 when treated with LPS. We conclude that in the SDH, microglial LRP1 plays an important role in establishing and/or amplifying local neuro-inflammation and neuropathic pain following PNS injury. The responsible mechanism most likely involves proteolytic release of LRP1 from the plasma membrane to generate a soluble product that functions similarly to pro-inflammatory cytokines in mediating crosstalk between cells in the SDH and in regulating neuropathic pain.     

Downregulation of connexin36 in mouse spinal dorsal horn neurons leads to mechanical allodynia

Connexin36 (Cx36), a component of neuronal gap junctions, is crucial for interneuronal communication and regulation. Gap junction dysfunction underlies neurological disorders, including chronic pain. Following a peripheral nerve injury, Cx36 expression in the ipsilateral spinal dorsal horn was markedly decreased over time, which paralleled the time course of hind paw tactile allodynia. Intrathecal (i.t.) injection of Cx36 siRNA (1 and 5 pg) significantly reduced the expression of Cx36 protein in the lumbar spinal cord, peaking 3 days after the injection, which corresponded with the onset of hind paw tactile allodynia. It is possible that some of the tactile allodynia resulting from Cx36 downregulation could be mediated through excitatory neuromodulators, such as glutamate and substance P. The Cx36 knockdown‐evoked tactile allodynia was significantly attenuated by i.t. treatment with the N‐methyl‐D‐aspartate glutamate receptor antagonist MK‐801 but not the substance P receptor antagonist CP96345. Immunohistochemistry showed that Cx36 was colocalized with glycine transporter‐2, a marker for inhibitory glycinergic spinal interneurons, but not with glutamate decarboxylase 67, a marker for inhibitory GABAergic spinal interneurons. The results indicate that spinal inhibition through glycinergic interneurons is reduced, leading to increased glutamatergic neurotransmission, as a result of Cx36 downregulation. The current data suggest that gap junction dysfunction underlies neuropathic pain and further suggest a novel target for the development of analgesics. © 2014 Wiley Periodicals, Inc.     

Downregulation of ciRNA-Kat6b in dorsal spinal horn is required for neuropathic pain by regulating Kcnk1 in miRNA-26a-dependent manner

Aims

Nerve injury-induced maladaptive changes in gene expression in the spinal neurons are essential for neuropathic pain genesis. Circular RNAs (ciRNA) are emerging as key regulators of gene expression. Here, we identified a nervous-system-tissues-specific ciRNA-Kat6 with conservation in humans and mice. We aimed to investigate whether and how spinal dorsal horn ciRNA-Kat6b participates in neuropathic pain.

Methods

Unilateral sciatic nerve chronic constrictive injury (CCI) surgery was used to prepare the neuropathic pain model. The differentially expressed ciRNAs were obtained by RNA-Sequencing. The identification of nervous-system-tissues specificity of ciRNA-Kat6b and the measurement of ciRNA-Kat6b and microRNA-26a (miRNA-26a) expression level were carried out by quantitative RT-PCR. The ciRNA-Kat6b that targets miRNA-26a and miRNA-26a that targets Kcnk1 were predicted by bioinformatics analysis and verified by in vitro luciferase reports test and in vivo experiments including Western-blot, immunofluorescence, and RNA–RNA immunoprecipitation. The correlation between neuropathic pain and ciRNA-Kat6b, miRNA-26a, or Kcnk1 was examined by the hypersensitivity response to heat and mechanical stimulus.

Results

Peripheral nerve injury downregulated ciRNA-Kat6b in the dorsal spinal horn of male mice. Rescuing this downregulation blocked nerve injury-induced increase of miRNA-26a, reversed the miRNA-26a-triggered decrease of potassium channel Kcnk1, a key neuropathic pain player, in the dorsal horn, and alleviates CCI-induced pain hypersensitivities. On the contrary, mimicking this downregulation increased the miRNA-26a level and decreased Kcnk1 in the spinal cord, resulting in neuropathic pain-like syndrome in naïve mice. Mechanistically, the downregulation of ciRNA-Kat6b reduced the accounts of miRNA-26a binding to ciRNA-Kat6b, and elevated the binding accounts of miRNA-26a to the 3′ untranslated region of Kcnk1 mRNA and degeneration of Kcnk1 mRNA, triggering in the reduction of KCNK1 protein in the dorsal horn of neuropathic pain mice.

Conclusion

The ciRNA-Kat6b/miRNA-26a/Kcnk1 pathway in dorsal horn neurons regulates the development and maintenance of neuropathic pain, ciRNA-Kat6b may be a potential new target for analgesic and treatment strategies.     

Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury

Chronic neuropathic pain is a frequent consequence of spinal cord injury (SCI). Yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. Previous studies have demonstrated that SCI results in excessive ATP release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. We sought to assess the role of connexin 43 (Cx43) as a mediator of CNS inflammation and chronic pain. To determine the extent of Cx43 involvement in chronic pain, a weight‐drop SCI was performed on transgenic mice with Cx43/Cx30 deletions. SCI induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild‐type control mice, which developed after 4 weeks and was maintained after 8 weeks. Notably, SCI‐induced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with Cx43/Cx30 deletions, but fully developed in transgenic mice with only Cx30 deletion. SCI‐induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with Cx43/Cx30 deletions, when compared with littermate controls. In comparison, a standard regimen of post‐SCI treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than Cx43 deletion. These findings suggest Cx43 is critically linked to the development of central neuropathic pain following acute SCI. Since Cx43/Cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain. © 2012 Wiley Periodicals, Inc.     

Zinc improves neurological recovery by promoting angiogenesis via the astrocyte‐mediated HIF‐1α/VEGF signaling pathway in experimental stroke

BackgroundIschemic stroke is a serious cerebrovascular disease with high morbidity and disability. Zinc accumulation has been shown to play a vital role in neuronal death and blood–brain barrier damage following ischemia in acute stage. However, almost nothing is known about whether zinc is involved in neurological recovery in ischemic prolonged period. This study investigates whether zinc promotes neurological recovery through astrocytes‐induced angiogenesis during ischemic repair phase.MethodsSprague–Dawley rats were subjected to 2 h ischemia/14, 21, and 28 days reperfusion by middle cerebral artery occlusion, then administered ZnCl₂ (10 mg/kg) via intraperitoneally daily from 7 days to tissue collection to observe brain tissue morphology, neurological function recovery by cortical width index, Adhesive removal test, and Forelimb placing test. Angiogenesis, astrocyte activation, and HIF‐1α/VEGF pathway were assessed via Western blot, immunofluorescence, and BrdU method in vivo and in vitro.ResultsThe results showed that zinc significantly alleviated brain atrophy and improved neurological function recovery during the cerebral ischemia repair stage. Zinc significantly increased the protein levels of HIF‐1α, VEGF‐A, and VEGF‐R2 in astrocytes, and promoted angiogenesis during cerebral ischemia repair. In vitro and in vivo studies confirmed that zinc promoted angiogenesis via the astrocyte‐mediated HIF‐1α/VEGF signaling pathway.ConclusionsZinc significantly improves neurological function recovery during the cerebral ischemia repair stage, providing new evidence supporting zinc as a potential therapeutic target for ischemic stroke by promoting astrocyte induced angiogenesis.     

Netrin‐1 upregulates GPX4 and prevents ferroptosis after traumatic brain injury via the UNC5B/Nrf2 signaling pathway

AimWe aimed to investigate the regulatory role of Netrin‐1 (NTN1) in ferroptosis after traumatic brain injury (TBI) in mice.MethodsWe assessed the expression pattern of NTN1 by RT–PCR, western blot, and immunofluorescence after establishing the TBI model in mice. After treatment with NTN1 shRNA or recombinant NTN1, we determined the biochemical and morphological changes associated with ferroptosis and netrin‐1‐related pathways. We used Nissl staining to assess lesion volume and Morris water maze and beam‐walking test to evaluate ethological manifestation.ResultsThe mRNA and protein levels of NTN1 were upregulated after TBI. The application of NTN1 shRNA increased the number of FJB positive cells, malondialdehyde (MDA), and reactive oxygen species (ROSs) levels. However, the application of NTN1 recombinant had the opposite effect. Furthermore, knockdown or inhibition of GPX4, Nrf2, and UNC5B counteracted the effects of NTN1 recombinant. Intravenous injection of NTN1 recombinant reduced neuronal loss after CCI and improved motor and cognitive function.ConclusionNTN1 had a neuroprotective effect after TBI and inhibited ferroptosis via activating the UNC5B/Nrf2 pathway. These findings may provide potential therapeutic strategies for TBI.     

Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes

Activation of the neuronal-glial network in the spinal cord dorsal horn (SCDH) mediates various chronic painful conditions. We studied spinal neuronal-astrocyte signaling interactions involved in the maintenance of painful diabetic neuropathy (PDN) in type 2 diabetes. We used the db/db mouse, an animal model for PDN of type 2 diabetes, which develops mechanical allodynia from 6 to 12 wk of age. In this study, enhanced substance P expression was detected in the presynaptic sensory fibers innervating lamina I-III in the lumbar SCDH (LSCDH) of the db/db mouse at 10 wk of age. This phenomenon is associated with enhanced spinal ERK1/2 phosphorylation in projection sensory neurons and regional astrocyte activation. In addition, peak phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptor (NMDAR), along with upregulation of neuronal and inducible nitric oxide synthase (nNOS and iNOS) expression were detected in diabetic mice. Expression of nNOS and iNOS was detected in both interneurons and astrocytes in lamina I-III of the LSCDH. Treatment with MK801, an NMDAR inhibitor, inhibited mechanical allodynia, ERK1/2 phosphorylation, and nNOS and iNOS upregulation in diabetic mice. MK801 also reduced astrocytosis and glial acidic fibrillary protein upregulation in db/db mice. In addition, N(G)-nitro-L-arginine methyl ester (L-NAME), a nonspecific NOS inhibitor, had similar effects on NMDAR signaling and NOS expression. These results suggest that nitric oxide from surrounding interneurons and astrocytes interacts with NMDAR-dependent signaling in the projection neurons of the SCDH during the maintenance of PDN.     

Intrathecal injection of dexmedetomidine ameliorates chronic neuropathic pain via the modulation of MPK3/ERK1/2 in a mouse model of chronic neuropathic pain

ABSTRACT

Objective: Despite the application of dexmedetomidine (DEX) as a perioperative adjuvant in local analgesia, the exact analgesic mechanism underpinning chronic neuropathic pain (CNP) awaits our elucidation.

Methods: We investigated the molecular mechanisms of the anti-nociceptive effect of DEX on neuropathic pain in a mouse model induced by chronic constriction injury (CCI).

Results: DEX administration significantly increased the paw withdrawal latency (PWL) values 0.5 to 2 h post-injection in CCI-induced CNP mice at day 5 to 21 versus dimethyl sulfoxide (DMSO)-treated mice, confirming its analgesic effect. The c-Fos expression was significantly elevated in CCI mice versus the sham-operated group, whereas the elevation was mitigated by DEX injection. Subsequently, the involvement of MKP1 and MKP3 in the pathogenesis of chronic neuropathic pain was evaluated. Western blotting analyses revealed significant decrease in both MKP1 and MKP3 in the spinal cord in CCI group versus the sham group. DEX markedly elevated the MKP3 expression and modestly reduced the MKP1 expression, with insignificant difference in the latter. Co-injection of BCI (an MKP3 inhibitor) and DEX evidently reduced the PWL values in CCI mice. Furthermore, DEX significantly downregulated the phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2, down-stream effector of MKP3 in CCI mice, whereas the downregulation was reversed by BCI.

Conclusion: We confirmed that DEX exerts the analgesic effect on chronic neuropathic pain via the regulation of MKP3/ERK1/2 signaling pathway, which may contribute to clarification of the molecular mechanism and novel therapy for chronic neuropathic pain.     

Hypoxic glioma‐derived extracellular vesicles harboring MicroRNA‐10b‐5p enhance M2 polarization of macrophages to promote the development of glioma

IntroductionThe delivery of biomolecules by tumor cell‐secreted extracellular vesicles (EVs) is linked to the development of glioma. Here, the present study was implemented to explore the functional significance of hypoxic glioma cell‐derived EVs carrying microRNA‐10b‐5 (miR‐10b‐5p) on glioma with the involvement of polarization of M2 macrophages.MethodsEVs were isolated from hypoxia‐stimulated glioma cells, and their role in polarization of M2 macrophages was studied by co‐culturing with macrophages. miR‐10b‐5p expression in glioma tissues, glioma‐derived EVs, and macrophages co‐cultured with EVs was characterized. Interaction among miR‐10b‐5p, NEDD4L, and PIK3CA was analyzed. The macrophages or glioma cells were transfected with overexpressing plasmid or shRNA to study the effects of miR‐10b‐5p/NEDD4L/PIK3CA on M2 macrophage polarization, and glioma cell proliferation, migration, and invasion in vitro and in vivo. ResultsPromotive role of hypoxia‐stimulated glioma‐derived EVs in macrophage M2 polarization was confirmed. Elevation of miR‐10b‐5p occurred in glioma tissues, glioma‐derived EVs and macrophages co‐cultured with EVs, and stimulated M2 polarization of macrophages. NEDD4L was a target gene of miR‐10b‐5p. Overexpression of NEDD4L could inhibit PI3K/AKT pathway through increase in ubiquitination and degradation of PIK3CA. Hypoxic glioma‐derived EVs harboring upregulated miR‐10b‐5p triggered an M2 phenotype in macrophages as well as enhanced aggressive tumor biology of glioma cells via inhibition of PIK3CA/PI3K/AKT pathway by targeting NEDD4L.ConclusionsIn summary, miR‐10b‐5p delivered by hypoxic glioma‐derived EVs accelerated macrophages M2 polarization to promote the progression of glioma via NEDD4L/PIK3CA/PI3K/AKT axis.     

Automated slice‐specific z‐shimming for functional magnetic resonance imaging of the human spinal cord

Functional magnetic resonance imaging (fMRI) of the human spinal cord faces many challenges, such as signal loss due to local magnetic field inhomogeneities. This issue can be addressed with slice‐specific z‐shimming, which compensates for the dephasing effect of the inhomogeneities using a slice‐specific gradient pulse. Here, we aim to address outstanding issues regarding this technique by evaluating its effects on several aspects that are directly relevant for spinal fMRI and by developing two automated procedures in order to improve upon the time‐consuming and subjective nature of manual selection of z‐shims: one procedure finds the z‐shim that maximizes signal intensity in each slice of an EPI reference‐scan and the other finds the through‐slice field inhomogeneity for each EPI‐slice in field map data and calculates the required compensation gradient moment. We demonstrate that the beneficial effects of z‐shimming are apparent across different echo times, hold true for both the dorsal and ventral horn, and are also apparent in the temporal signal‐to‐noise ratio (tSNR) of EPI time‐series data. Both of our automated approaches were faster than the manual approach, lead to significant improvements in gray matter tSNR compared to no z‐shimming and resulted in beneficial effects that were stable across time. While the field‐map‐based approach performed slightly worse than the manual approach, the EPI‐based approach performed as well as the manual one and was furthermore validated on an external corticospinal data‐set (N > 100). Together, automated z‐shimming may improve the data quality of future spinal fMRI studies and lead to increased reproducibility in longitudinal studies.     

Peripheral inflammation activated focal adhesion kinase signaling in spinal dorsal horn of mice

Focal adhesion kinase (FAK) is one of the nonreceptor protein tyrosine kinases critical for the dynamic regulation of cell adhesion structures. Recent studies have demonstrated that FAK is also localized at excitatory glutamatergic synapses and is involved in long‐term modification of synaptic strength. However, whether FAK is engaged in nociceptive processing in the spinal dorsal horn remains unresolved. The current study shows that intraplantar injection of complete Freund's adjuvant (CFA) in mice significantly increases FAK autophosphorylation at Tyr397, indicating a close correlation of FAK activation with inflammatory pain. FAK activation depended on the activity of N‐methyl‐D‐aspartate‐subtype glutamate receptor (NMDAR) and metabotropic glutamate receptor (mGluR) because pharmacological inhibition of NMDAR or group I mGluR totally abolished FAK phosphorylation induced by CFA. The active FAK operated to stimulate extracellular signal‐regulated kinase1/2 (ERK1/2), which boosted the protein expression of GluN2B subunit‐containing NMDAR at the synaptosomal membrane fraction. Inhibition of FAK activity by spinal expression of a kinase‐dead FAK(Y397F) mutant repressed ERK1/2 hyperactivity and reduced the synaptic concentration of NMDAR in CFA‐injected mice. Electrophysiological recording demonstrated that intracellular loading of specific anti‐FAK antibody significantly reduced the amplitudes of NMDAR‐mediated excitatory postsynaptic currents on lamina II neurons from inflamed mice but not from naive mice. Behavioral tests showed that spinal expression of FAK(Y397F) generated a long‐lasting alleviation of CFA‐induced mechanical allodynia and thermal hyperalgesia. These data indicate that FAK might exaggerate NMDAR‐mediated synaptic transmission in the spinal dorsal horn to sensitize nociceptive behaviors. © 2015 Wiley Periodicals, Inc.     

Curzerene suppresses progression of human glioblastoma through inhibition of glutathione S‐transferase A4

AimsGlioblastoma is the central nervous system tumor with the highest mortality rate, and the clinical effectiveness of chemotherapy is low. Curzerene can inhibit the progression of non‐small‐cell lung cancer, but its role in glioma has not been reported. The purpose of this study was to clarify the effect of curzerene on glioma progression and further explore its potential mechanism.MethodsThe expression of glutathione S‐transferase A4 (GSTA4) in glioblastoma and the effect of curzerene on the expression of GSTA4 and matrix metalloproteinase 9 and the activation of the mTOR pathway were detected by Western blotting and RT‐PCR, and the effects of curzerene treatment on glioma malignant character were detected by cell biological assays. The in vivo antitumor effects of curzerene were analyzed in a nude mouse xenograft model.ResultsCurzerene was found to inhibit the expression of GSTA4 mRNA and protein in U251 and U87 glioma cells, and this effect correlated with a downregulation of the proliferation of these cells in a time‐ and dose‐dependent manner. Invasion and migration were also inhibited, and curzerene treatment correlated with induction of apoptosis. Curzerene inhibited the activation of the mTOR pathway and the expression of matrix metalloproteinase 9, and it correlated with increased 4‐hydroxynonenal levels. In vivo, curzerene was found to significantly inhibit tumor growth in nude mice and to prolong the survival time of tumor‐bearing nude mice.ConclusionIn conclusion, inhibition of GSTA4 correlates with positive outcomes in glioma models, and thus, this molecule is a candidate drug for the treatment of glioma.     

Long-term synaptic plasticity in the spinal dorsal horn and its modulation by electroacupuncture in rats with neuropathic pain

Our previous study has reported that electroacupuncture (EA) at low frequency of 2 Hz had greater and more prolonged analgesic effects on mechanical allodynia and thermal hyperalgesia than that EA at high frequency of 100 Hz in rats with neuropathic pain. However, how EA at different frequencies produces distinct analgesic effects on neuropathic pain is unclear. Neuronal plastic changes in spinal cord might contribute to the development and maintenance of neuropathic pain. In the present study, we investigated changes of spinal synaptic plasticity in the development of neuropathic pain and its modulation by EA in rats with neuropathic pain. Field potentials of spinal dorsal horn neurons were recorded extracellularly in sham-operated rats and in rats with spinal nerve ligation (SNL). We found for the first time that the threshold for inducing long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn was significantly lower in SNL rats than that in sham-operated rats. The threshold for evoking the C-fiber-evoked field potentials was also significantly lower, and the amplitude of the field potentials was higher in SNL rats as compared with those in the control rats. EA at low frequency of 2 Hz applied on acupoints ST 36 and SP 6, which was effective in treatment of neuropathic pain, induced long-term depression (LTD) of the C-fiber-evoked potentials in SNL rats. This effect could be blocked by N-methyl-d-aspartic acid (NMDA) receptor antagonist MK-801 and by opioid receptor antagonist naloxone. In contrast, EA at high frequency of 100 Hz, which was not effective in treatment of neuropathic pain, induced LTP in SNL rats but LTD in sham-operated rats. Unlike the 2 Hz EA-induced LTD in SNL rats, the 100 Hz EA-induced LTD in sham-operated rats was dependent on the endogenous GABAergic and serotonergic inhibitory system. Results from our present study suggest that (1) hyperexcitability in the spinal nociceptive synaptic transmission may occur after nerve injury, which may contribute to the development of neuropathic pain; (2) EA at low or high frequency has a different effect on modulating spinal synaptic plasticities in rats with neuropathic pain. The different modulation on spinal LTD or LTP by low- or high-frequency EA may be a potential mechanism of different analgesic effects of EA on neuropathic pain. LTD of synaptic strength in the spinal dorsal horn in SNL rats may contribute to the long-lasting analgesic effects of EA at 2 Hz.