Rat Spinal Dorsal Horn Research Articles

Small-Conductance Ca2+-Activated K+ Channel 2 in the Dorsal Horn of Spinal Cord Participates in Visceral Hypersensitivity in Rats.

Visceral hypersensitivity is a highly complex and subjective phenomenon associated with multiple levels of the nervous system and a wide range of neurotransmission. The dorsal horn (DH) in spinal cord relays the peripheral sensory information into the brain. Small conductance Ca2+-activated K+ (SK) channels regulate neuronal excitability and firing by allowing K+ to efflux in response to increase in the intracellular Ca2+ level. In this study, we examined the influence of SK2 channels in the spinal DH on the pathogenesis of visceral hypersensitivity induced by colorectal distension (CRD) in rats. Electrophysiological results showed that rats with visceral hypersensitivity presented a decrease in the SK channel-mediated afterhyperpolarization current (IAHP), and an increase in neuronal firing rates and c-Fos positive staining in the spinal DH. Western blot data revealed a decrease in the SK2 channel protein in the membrane fraction. Moreover, intrathecal administration of the SK2 channel activator 1-EBIO or CyPPA alleviated visceral hypersensitivity, reversed the decrease in IAHP and the increase in neuronal firing rates in spinal DH in rats that experienced CRD. 1-EBIO or CyPPA effect could be prevented by SK2 channel blocker apamin. CRD induced an increase in c-Fos protein expression in the spinal DH, which was prevented by 1-EBIO. Together, these data suggest that visceral hypersensitivity and pain is associated with a decrease in the number and function of membrane SK2 channels in the spinal DH. Pharmacological manipulation of SK2 channels may open a new avenue for the treatment of visceral hypersensitivity and pain.Highlights: -Neonatal colorectal distension induced visceral hypersensitivity in rats.-Visceral hypersensitivity rats presented a decrease in afterhyperpolarization current (IAHP) and membrane SK2 channel protein in the spinal dorsal horn.-Visceral hypersensitivity rats presented an increase in neuronal firing rate in the spinal dorsal horn.-Intrathecal administration of SK2 channel activator 1-EBIO or CyPPA prevented visceral hypersensitivity and decrease in IAHP.

Dexmedetomidine attenuates persistent postsurgical pain by upregulating K+-Cl- cotransporter-2 in the spinal dorsal horn in rats.

BackgroundDexmedetomidine (DEX) could have an analgesic effect on pain transmission through the modulation of brain-derived neurotrophic factor (BDNF). In addition, KCC2-induced shift in neuronal Cl− homeostasis is crucial for postsynaptic inhibition mediated by GABAA receptors. Accumulating evidence shows that nerve injury, peripheral inflammation and stress activate the spinal BDNF/TrkB signal, which results in the downregulation of KCC2 transport and expression, eventually leads to GAGAergic disinhibition and hyperalgesia. The aim of this experiment was to explore the interaction between DEX and KCC2 at a molecular level in rats in the persistent postsurgical pain (PPSP).MethodsPPSP in rats was evoked by the skin/muscle incision and retraction (SMIR). Mechanical hypersensitivity was assessed with the Dynamic Plantar Aesthesiometer. Western blot and immunofluorescence assay were used to assess the expressions of related proteins.ResultsIn the first part of our experiment, the results revealed that the BDNF/TrkB-KCC2 signal plays a critical role in the development of SMIR-evoked PPSP; the second part showed that intraperitoneal administrations of 40 µg/kg DEX at 15 min presurgery and 1 to 3 days post-surgery significantly attenuated SMIR-evoked PPSP. Simultaneously, SMIR-induced KCC2 downregulation was partly reversed, which coincided with the inhibition of the BDNF/TrkB signal in the spinal dorsal horn. Moreover, intrathecal administrations of KCC2 inhibitor VU0240551 significantly reduced the analgesic effect of DEX on SMIR-evoked PPSP.ConclusionThe results of our study indicated that DEX attenuated PPSP by restoring KCC2 function through reducing BDNF/TrkB signal in the spinal dorsal horn in rats, which provides a new insight into the treatment of chronic pain in clinical postsurgical pain management.

VEGFR2 promotes central endothelial activation and the spread of pain in inflammatory arthritis

Chronic pain can develop in response to conditions such as inflammatory arthritis. The central mechanisms underlying the development and maintenance of chronic pain in humans are not well elucidated although there is evidence for a role of microglia and astrocytes. However in pre-clinical models of pain, including models of inflammatory arthritis, there is a wealth of evidence indicating roles for pathological glial reactivity within the CNS. In the spinal dorsal horn of rats with painful inflammatory arthritis we found both a significant increase in CD11b+ microglia-like cells and GFAP+ astrocytes associated with blood vessels, and the number of activated blood vessels expressing the adhesion molecule ICAM-1, indicating potential glio-vascular activation. Using pharmacological interventions targeting VEGFR2 in arthritic rats, to inhibit endothelial cell activation, the number of dorsal horn ICAM-1+ blood vessels, CD11b+ microglia and the development of secondary mechanical allodynia, an indicator of central sensitization, were all prevented. Targeting endothelial VEGFR2 by inducible Tie2-specific VEGFR2 knock-out also prevented secondary allodynia in mice and glio-vascular activation in the dorsal horn in response to inflammatory arthritis. Inhibition of VEGFR2 in vitro significantly blocked ICAM-1-dependent monocyte adhesion to brain microvascular endothelial cells, when stimulated with inflammatory mediators TNF-α and VEGF-A165a. Taken together our findings suggest that a novel VEGFR2-mediated spinal cord glio-vascular mechanism may promote peripheral CD11b+ circulating cell transmigration into the CNS parenchyma and contribute to the development of chronic pain in inflammatory arthritis. We hypothesise that preventing this glio-vascular activation and circulating cell translocation into the spinal cord could be a new therapeutic strategy for pain caused by rheumatoid arthritis.

Regulation of Nociceptive Glutamatergic Signaling by Presynaptic Kv3.4 Channels in the Rat Spinal Dorsal Horn.

Presynaptic voltage-gated K+ (Kv) channels in dorsal root ganglion (DRG) neurons are thought to regulate nociceptive synaptic transmission in the spinal dorsal horn. However, the Kv channel subtypes responsible for this critical role have not been identified. The Kv3.4 channel is particularly important because it is robustly expressed in DRG nociceptors, where it regulates action potential (AP) duration. Furthermore, Kv3.4 dysfunction is implicated in the pathophysiology of neuropathic pain in multiple pain models. We hypothesized that, through their ability to modulate AP repolarization, Kv3.4 channels in DRG nociceptors help to regulate nociceptive synaptic transmission. To test this hypothesis, we investigated Kv3.4 immunoreactivity (IR) in the rat cervical superficial dorsal horn (sDH) in both sexes and implemented an intact spinal cord preparation to investigate glutamatergic synaptic currents from second order neurons in the sDH under conditions that selectively inhibit the Kv3.4 current. We found presynaptic Kv3.4 IR in peptidergic and nonpeptidergic nociceptive fibers of the sDH. The Kv3.4 channel is hypersensitive to 4-aminopyridine and tetraethylammonium (TEA). Accordingly, 50 μm 4-aminopyridine and 500 μm TEA significantly prolong the AP, slow the maximum rate of repolarization in small-diameter DRG neurons, and potentiate monosynaptic excitatory postsynaptic currents (EPSCs) in dorsal horn laminae I and II through a presynaptic mechanism. In contrast, highly specific inhibitors of BK, Kv7, and Kv1 channels are less effective modulators of the AP and have little to no effect on EPSCs. The results strongly suggest that presynaptic Kv3.4 channels are major regulators of nociceptive synaptic transmission in the spinal cord.SIGNIFICANCE STATEMENT Intractable neuropathic pain can result from disease or traumatic injury and many studies have been conducted to determine the underlying pathophysiological changes. Voltage-gated ion channels, including the K+ channel Kv3.4, are dysregulated in multiple pain models. Kv3.4 channels are ubiquitously expressed in the dorsal root ganglion (DRG), where they are major regulators of DRG excitability. However, little is known about the ionic mechanisms that regulate nociceptive synaptic transmission at the level of the first synapse in the spinal cord, which is critical to pain transmission in both intact and pathological states. Here, we show that Kv3.4 channels have a significant impact on glutamatergic synaptic transmission in the dorsal horn, further illuminating its potential as a molecular pain therapeutic target.

Endoplasmic Reticulum Stress in Spinal Cord Contributes to the Development of Morphine Tolerance.

Morphine tolerance remains an intractable problem, which hinders its prolonged use in clinical practice. Endoplasmic reticulum (ER) stress has been proved to play a fundamental role in the pathogenesis of Alzheimer’s disease, diabetes, atherosclerosis, cancer, etc. In this study, we provide the first direct evidence that ER stress may be a significant driver of morphine tolerance. Binding immunoglobulin protein (BiP), the ER stress marker, was significantly upregulated in neurons in spinal dorsal horn in rats being treated with morphine for 7 days. Additionally, chronic morphine treatment resulted in the activation of three arms of unfolded protein response (UPR): inositol-requiring enzyme 1/X-box binding protein 1 (IRE1/XBP1), protein kinase RNA-like ER kinase/eukaryotic initiation factor 2 subunit alpha (PERK/eIF2α), and activating transcription factor 6 (ATF6). More importantly, inhibiting either one of the three cascades could attenuate the development of morphine tolerance. Taken together, our results suggest that ER stress in spinal cord might contribute to the development of morphine tolerance. These findings implicate a potential clinical strategy for preventing morphine tolerance and may contribute to expanding the morphine usage in clinic.

Glial cell type-specific changes in spinal dipeptidyl peptidase 4 expression and effects of its inhibitors in inflammatory and neuropatic pain

Altered pain sensations such as hyperalgesia and allodynia are characteristic features of various pain states, and remain difficult to treat. We have shown previously that spinal application of dipeptidyl peptidase 4 (DPP4) inhibitors induces strong antihyperalgesic effect during inflammatory pain. In this study we observed low level of DPP4 mRNA in the rat spinal dorsal horn in physiological conditions, which did not change significantly either in carrageenan-induced inflammatory or partial nerve ligation-generated neuropathic states. In naïve animals, microglia and astrocytes expressed DPP4 protein with one and two orders of magnitude higher than neurons, respectively. DPP4 significantly increased in astrocytes during inflammation and in microglia in neuropathy. Intrathecal application of two DPP4 inhibitors tripeptide isoleucin-prolin-isoleucin (IPI) and the antidiabetic drug vildagliptin resulted in robust opioid-dependent antihyperalgesic effect during inflammation, and milder but significant opioid-independent antihyperalgesic action in the neuropathic model. The opioid-mediated antihyperalgesic effect of IPI was exclusively related to mu-opioid receptors, while vildagliptin affected mainly delta-receptor activity, although mu- and kappa-receptors were also involved. None of the inhibitors influenced allodynia. Our results suggest pathology and glia-type specific changes of DPP4 activity in the spinal cord, which contribute to the development and maintenance of hyperalgesia and interact with endogenous opioid systems.

Effects of metformin on the expression of AMPK and STAT3 in the spinal dorsal horn of rats with neuropathic pain.

Neuropathic pain (NP) is a frustrating and burdensome problem. Current treatments for NP have unendurable side effects and/or questionable efficacy, and once these therapies are stopped, the symptoms often return. Thus, novel drugs are needed to enhance the effectiveness of treatments for NP. One novel target for pain treatments is adenosine monophosphate-activated protein kinase (AMPK), which regulates a variety of cellular processes, including protein translation, which is considered to be affected in NP. Metformin is a widely available drug that possesses the ability to activate AMPK. The signal transducer and activator of transcription 3 (STAT3) pathway plays an important role in neuroinflammation. The present study investigated the analgesic effect of metformin on NP induced by chronic constriction injury (CCI), and the influence of metformin on the expression of AMPK and STAT3 in the spinal dorsal horn (SDH). In CCI rats, paw withdrawal latencies in response to thermal hyperalgesia were significantly shorter, while phosphorylated (p)-AMPK was expressed at lower levels and p-STAT3 was expressed at higher levels in the SDH. Administering intraperitoneal injections of metformin (200 mg/kg) for 6 successive days activated AMPK and suppressed the expression of p-STAT3, in addition to reversing hyperalgesia. Finally, metformin inhibited the activation of microglia and astrocytes in the SDH, which may explain how it alleviates NP.

Endomorphin-2 Inhibits the Activity of the Spinoparabrachial Projection Neuron through Presynaptic Mechanisms in the Spinal Dorsal Horn in Rats

Background/Aims: Spinal dorsal horn (SDH) is one of the most important regions for analgesia produced by endomorphin-2 (EM2), which has a higher affinity and specificity for the µ-opioid receptor (MOR) than morphine. Many studies have focused on substantia gelatinosa (SG, lamina II) neurons to elucidate the cellular basis for its antinociceptive effects. However, the complicated types and local circuits of interneurons in the SG make it difficult to understand the real effects of EM2. Therefore, in the present study, we examined the effects of EM2 on projection neurons (PNs) in lamina I. Methods: Tracing, immunofluoresence, and immunoelectron methods were used to examine the morphological connections between EM2-immunoreactive (-ir) terminals and PNs. By using in vitro whole cell patch clamp recording technique, we investigated the functional effects of EM2 on PNs. Results: EM2-ir afferent terminals directly contacted PNs projecting to the parabrachial nucleus in lamina I. Their synaptic connections were further confirmed by immunoelectron microscopy, most of which were asymmetric synapses. It was found that EM2 had a strong inhibitory effect on the frequency, but not amplitude, of the spontaneous excitatory postsynaptic current (sEPSC) of the spinoparabrachial PNs in lamina I, which could be reversed by MOR antagonist CTOP. However, their spontaneous inhibitory postsynaptic current (sIPSC) and intrinsic properties were not changed after EM2 application. Conclusion: Applying EM2 to the SDH could produce analgesia through inhibiting the activities of the spinoparabrachial PNs in lamina I by reducing presynaptic neurotransmitters release from the primary afferent terminals.

Relationship between neuropeptide S in amygdala and 5-hydroxytryptamine and GABA in spinal dorsal horns of rats with neuropathic pain

Objective To evaluate the relationship between neuropeptide S (NPS) in the amygdala and 5-hydroxytryptamine (5-HT) and GABA in spinal dorsal horns of rats with neuropathic pain. Methods Eighty pathogen-free healthy male Sprague-Dawley rats, weighing 200-260 g, aged 2 months, were divided into 4 groups (n=20 each) using a random number table: sham operation group (group Sham), neuropathic pain group (group NP), low dose NPS group (group L-NPS) and high dose NPS group (group H-NPS). The neuropathic pain model was established by left L5, 6 spinal nerve ligation (SNL) in anesthetized rats.NPS was injected into the bilateral amygdala at 3, 6, 9, 12, 15 and 18 days after SNL in L-NPS group (10 pmol per side) and H-NPS group (100 pmol per side). The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 2 days before SNL and 1, 4, 7, 11, 14, 17 and 21 days after SNL.Five rats were selected at 7, 14 and 21 days after SNL and sacrificed, and the lumbar segment (L5) of the spinal cord was removed for detection of the expression of 5-HT and GABA in spinal dorsal horns by immunofluorescence histochemistry. Results Compared with group Sham, the MWT was significantly decreased, the TWL was shortened, and the expression of 5-HT and GABA in spinal dorsal horns was down-regulated in NP, L-NPS and H-NPS groups (P<0.05). Compared with group NP, the MWT was significantly increased, the TWL was prolonged, and the expression of 5-HT and GABA in spinal dorsal horns was up-regulated in L-NPS and H-NPS groups (P<0.05). Compared with group L-NPS, the MWT was significantly increased, the TWL was prolonged, and the expression of 5-HT and GABA in spinal dorsal horns was up-regulated in group H-NPS (P<0.05). Conclusion The spinal mechanism of endogenous analgesia induced by NPS in the amygdala may be related to up-regulation of the expression of 5-HT and GABA in spinal dorsal horns of rats with neuropathic pain. Key words: Amygdala; Neuralgia; Spinal cord; Serotonin; Gamma-aminobutyric acid; Neuropeptides

Changes in expression of neuroligin1 in excitatory postsynaptic membrane of spinal dorsal horn in a rat model of incisional pain

Objective To evaluate the changes in the expression of neuroligin1 in excitatory postsynaptic membrane of the spinal dorsal horn in a rat model of incisional pain. Methods Forty-eight pathogen-free healthy adult male Sprague-Dawley rats, aged 6-8 weeks, weighing 280-320 g, were divided into control group(group C, n=12)and incisional pain group(group I, n=36)using a random number table.A 1 cm long incision was made in the plantar surface of the right hindpaw in group I. Cumulative pain score(CPS)was assessed and mechanical paw withdrawal threshold to von Frey stimuli was measured at 3 h and 1 and 3 days after operation(T1, 2, 3). The animals were then sacrificed and their lumbar segments(L3-6)of the spinal cord were removed for detection of the expression of neuroligin1, postsynaptic density-95 protein(PSD-95), glutamate receptor 1(GluR1)and GluR2 in the postsynaptic membrane of spinal dorsal horn(by Western blot)and co-expression of neuroligin1 with PSD-95 in spinal dorsal horn(by co-immuno-precipitation). Results Compared with group C, CPS was significantly increased and mechanical paw withdrawal threshold was decreased at T1-3, and the expression of neuroligin1 and GluR1 in the postsynaptic membrane of spinal dorsal horn at T1, 2 and co-expression of neuroligin1 with PSD-95 at T1 were up-regulated in group I(P<0.01 or 0.05). Conclusion The development and maintenance of incisional pain is related to the signaling pathway regulated by neuroligin1 in excitatory postsynaptic membrane of the spinal dorsal horn of rats. Key words: Cell Adhesion Molecules; Pain; Spinal cord; Receptors, AMPA

CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.

Neonatal Colonic Inflammation Increases Spinal Transmission and Cystathionine β-Synthetase Expression in Spinal Dorsal Horn of Rats with Visceral Hypersensitivity.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by chronic abdominal pain and alteration of bowel movements. The pathogenesis of visceral hypersensitivity in IBS patients remains largely unknown. Hydrogen sulfide (H2S) is reported to play an important role in development of visceral hyperalgesia. However, the role of H2S at spinal dorsal horn level remains elusive in visceral hypersensitivity. The aim of this study is designed to investigate how H2S takes part in visceral hypersensitivity of adult rats with neonatal colonic inflammation (NCI). Visceral hypersensitivity was induced by neonatal colonic injection of diluted acetic acid. Expression of an endogenous H2S synthesizing enzyme cystathionine β-synthetase (CBS) was determined by Western blot. Excitability and synaptic transmission of neurons in the substantia gelatinosa (SG) of spinal cord was recorded by patch clamping. Here, we showed that expression of CBS in the spinal dorsal horn was significantly upregulated in NCI rats. The frequency of glutamatergic synaptic activities in SG was markedly enhanced in NCI rats when compared with control rats. Application of NaHS increased the frequency of both spontaneous and miniature excitatory post-synaptic currents of SG neurons in control rats through a presynaptic mechanism. In contrast, application of AOAA, an inhibitor of CBS, dramatically suppressed the frequency of glutamatergic synaptic activities of SG neurons of NCI rats. Importantly, intrathecal injection of AOAA remarkably attenuated visceral hypersensitivity of NCI rats. These results suggest that H2S modulates pain signaling likely through a presynaptic mechanism in SG of spinal dorsal horn, thus providing a potential therapeutic strategy for treatment for chronic visceral pain in patients with IBS.

Effects of naftopidil on inhibitory transmission in substantia gelatinosa neurons of the rat spinal dorsal horn in vitro

ObjectiveNaftopidil is used clinically for the treatment of voiding disorders in benign prostatic hyperplasia. Previous in vivo experiments in which naftopidil was applied intrathecally abolished rhythmic bladder contraction, suggesting that naftopidil might inhibit a voiding reflex through interaction with spinal dorsal horn neurons. Here we aimed to clarify the mechanism of action of naftopidil on dorsal horn neurons. MethodsWhole-cell patch-clamp recordings were performed using substantia gelatinosa neurons of adult rat spinal cord slices. Miniature or evoked inhibitor and excitatory postsynaptic currents (IPSCs and EPSCs, respectively) were analyzed. ResultsBath-applied naftopidil increased the frequency but not the amplitude of miniature IPSCs (mIPSCs) in 38% of neurons tested; in contrast, the effect of naftopidil on miniature EPSCs (mEPSCs) were mild and observed in only 2 out of 19 neurons. Naftopidil enhanced the amplitude of both GABAergic and glycinergic evoked-IPSCs (eIPSCs) that were elicited by focal stimuli in the presence of either the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV). ConclusionsAlthough naftopidil was developed as an alpha-1 adrenoceptor antagonist, our previous spinal cord slice experiments showed that the activation of an alpha-1 adrenoceptor in substantia gelatinosa increases the frequency of mIPSCs. This result suggested that, under our conditions, naftopidil may interact with a receptor(s) other than an alpha-1 adrenoceptor in the spinal dorsal horn. The present results suggested that naftopidil enhances the release of GABA and glycine by activating inhibitory interneuron terminals in the spinal dorsal horn via a receptor other than an alpha-1 adrenoceptor, thereby modulating sensory transmission in the substantia gelatinosa.

Activation of RAGE/STAT3 pathway by methylglyoxal contributes to spinal central sensitization and persistent pain induced by bortezomib

Bortezomib is a first-line chemotherapeutic drug widely used for multiple myeloma and other nonsolid malignancies. Although bortezomib-induced persistent pain is easily diagnosed in clinic, the pathogenic mechanism remains unclear. Here, we studied this issue with use of a rat model of systemic intraperitoneal administration of bortezomib for consecutive 5days. Consisted with our previous study, we found that bortezomib treatment markedly induced mechanical allodynia in rats. Furthermore, we first found that bortezomib treatment significantly induced the upregulation of methylglyoxal in spinal dorsal horn of rats. Spinal local application of methylglyoxal also induced mechanical allodynia and central sensitization in normal rats. Moreover, administration of bortezomib upregulated the expression of receptors for advanced glycation end products (RAGE) and phosphorylated STAT3 (p-STAT3) in dorsal horn. Importantly, intrathecal injection of metformin, a known scavenger of methylglyoxal, significantly attenuated the upregulation of methylglyoxal and RAGE in dorsal horn, central sensitization and mechanical allodynia induced by bortezomib treatment, and blockage of RAGE also prevented the upregulation of p-STAT3, central sensitization and mechanical allodynia induced by bortezomib treatment. In addition, inhibition of STAT3 activity by S3I-201 attenuated bortezomib-induced mechanical allodynia and central sensitization. Local knockdown of STAT3 also ameliorated the mechanical allodynia induced by bortezomib administration. Our results suggest that accumulation of methylglyoxal may activate the RAGE/STAT3 signaling pathway in dorsal horn, and contributes to the spinal central sensitization and persistent pain induced by bortezomib treatment.

Activated leukocyte cell adhesion molecule is involved in excitatory synaptic transmission and plasticity in the rat spinal dorsal horn.

Activated leukocyte cell adhesion molecule (ALCAM), a member of type I transmembrane immunoglobulin superfamily of cell adhesion molecule, is expressed in the surface membrane of various cell types including neurons. In the spinal cord dorsal horn (DH), the first gate for the sensory and pain transmission to the brain, the expression and function of ALCAM have not been known yet. Therefore, we here investigate the synaptic function of ALCAM in the substantia gelatinosa (lamina II) of the spinal DH, as well as its expression in the DH. Bath-application of ALCAM/Fc or CD6/Fc, the recombinant human IgG1-Fc chimeric proteins, specifically potentiated C-fiber-mediated excitatory synaptic transmission and predominantly increased spontaneous release of glutamate. In addition, the development of long-term potentiation, a form of synaptic plasticity, at excitatory synapses was significantly inhibited in the presence of the recombinant proteins. The functional roles of ALCAM in the spinal DH were further supported by immunohistochemical analysis; it showed that ALCAM intensely expressed through laminae I/II with the exception of lateral portion of the dorsal part of inner lamina II and distinctly co-localized with molecular markers of C-fibers, such as peptidergic calcitonin gene-related protein and transient receptor potential vanilloid type 1 and non-peptidergic isolectin B4. This study, for the first time, suggests the modulatory roles of ALCAM in the excitatory synaptic transmission and plasticity in the rat spinal DH.

Chronic constriction injury of sciatic nerve changes circular RNA expression in rat spinal dorsal horn.

BackgroundMechanisms of neuropathic pain are still largely unknown. Molecular changes in spinal dorsal horn may contribute to the initiation and development of neuropathic pain. Circular RNAs (circRNAs) have been identified as microRNA sponges and involved in various biological processes, but whether their expression profile changes in neuropathic pain condition is not reported.MethodsTo test whether neuropathic pain influences circRNA expression, we developed a sciatic chronic constriction injury (CCI) model in rats. The CCI ipsilateral spinal dorsal horns of lumbar enlargement segments (L3–L5) were collected, and the total RNA was extracted and subjected to Arraystar Rat circRNA Microarray. Quantitative real-time polymerase chain reaction (qPCR) was used to confirm the circRNA expression profile. To estimate functions of differential circRNAs, bioinformatics analyses including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes Pathway analyses were performed for the top 100 circRNAs and circRNA–microRNA networks were constructed for the top 10 circRNAs.ResultscircRNA microarrays showed that 469 circRNAs were differentially expressed between CCI and sham-operated rats (fold change ≥2). In all, 363 of them were significantly upregulated, and the other 106 were downregulated in the CCI group. Three of them (circRNA_013779, circRNA_008008, and circRNA_003724) overexpressed >10 times after CCI insult. Expression levels of eight circRNAs were verified using qPCR. GO analysis revealed that thousands of predicted target genes were involved in the biological processes, cellular component, and molecular function; in addition, dozens of these genes were enriched in the Hippo signaling pathway, MAPK signaling pathway, and so on. Competing endogenous RNAs analysis showed that circRNA_008008 and circRNA_013779 are the two largest nodes in the circRNA–microRNA interaction network of the top 10 circRNAs.ConclusionCCI resulted in a comprehensive expression profile of circRNAs in the spinal dorsal horn in rats. CircRNAs in the dorsal horn could be helpful to reveal molecular mechanisms of neuropathic pain.

The developmental emergence of differential brainstem serotonergic control of the sensory spinal cord

Descending connections from brainstem nuclei are known to exert powerful control of spinal nociception and pain behaviours in adult mammals. Here we present evidence that descending serotonergic fibres not only inhibit nociceptive activity, but also facilitate non-noxious tactile activity in the healthy adult rat spinal dorsal horn via activation of spinal 5-HT3 receptors (5-HT3Rs). We further show that this differential serotonergic control in the adult emerges from a non-modality selective system in young rats. Serotonergic fibres exert background 5-HT3R mediated facilitation of both tactile and nociceptive spinal activity in the first three postnatal weeks. Thus, differential descending serotonergic control of spinal touch and pain processing emerges in late postnatal life to allow flexible and context-dependent brain control of somatosensation.

Stereological study on the number of synapses in the rat spinal dorsal horn with painful diabetic neuropathy induced by streptozotocin.

Our previous studies showed that direct injury to the sciatic nerve (chronic constriction injury or axotomy) is associated with a numerical increase in synaptic number in the rat spinal dorsal horn. The aim of this study was to determine whether painful diabetic neuropathy (PDN) was also associated with numerical changes in the synaptic or neuronal numbers in the spinal dorsal horn. Overall, 17 adult SD rats were allocated randomly into the control group (n=5) and the streptozotocin (STZ) group (n=12). STZ was injected intraperitoneally to induce diabetes. In the STZ group, seven rats (STZ-H) showed hyperglycemia (fasting blood glucose >11.1 mM) and the rest of the five rats (STZ-N) did not. Rats were fed and observed for 28 days after hyperglycemia. Two of the seven STZ-H rats died of infection during the observation period. Body weight and paw withdraw threshold (PWT) decreased in the rest of the five STZ-H rats. Twenty-eight days after hyperglycemia, the L5 segment of the spinal cord was removed; paraffin-embedded sections were prepared and stained with Nissl's method and synaptophysin immunohistochemistry, respectively. The optical dissector (a stereological technique) was used to estimate the numbers of neurons and synapses in the spinal dorsal horn. Compared with the control group, the synaptic number and ratio between the numbers of synapses and neurons in the L5 segment of the spinal dorsal horn were increased significantly in the STZ-H rats (P<0.05), whereas the neuronal number did not change significantly (P>0.05). Parameters of STZ-N rats showed no significant changes. In conclusion, PDN, a form of neuropathic pain, is also associated with a synaptic plasticity (numerical increase) in the spinal dorsal horn. This numerical change might be the reason for central sensitization resulting in reduced pain threshold, enhanced responsiveness, and expanded receptive fields associated with PDN. Therefore, our studies indicate that neuropathic pain conditions with different etiologies might show the same synaptic numerical plasticity changes.

EZH2 regulates spinal neuroinflammation in rats with neuropathic pain

Alteration in gene expression along the pain signaling pathway is a key mechanism contributing to the genesis of neuropathic pain. Accumulating studies have shown that epigenetic regulation plays a crucial role in nociceptive process in the spinal dorsal horn. In this present study, we investigated the role of enhancer of zeste homolog-2 (EZH2), a subunit of the polycomb repressive complex 2, in the spinal dorsal horn in the genesis of neuropathic pain in rats induced by partial sciatic nerve ligation. EZH2 is a histone methyltransferase, which catalyzes the methylation of histone H3 on K27 (H3K27), resulting in gene silencing. We found that levels of EZH2 and tri-methylated H3K27 (H3K27TM) in the spinal dorsal horn were increased in rats with neuropathic pain on day 3 and day 10 post nerve injuries. EZH2 was predominantly expressed in neurons in the spinal dorsal horn under normal conditions. The number of neurons with EZH2 expression was increased after nerve injury. More strikingly, nerve injury drastically increased the number of microglia with EZH2 expression by more than sevenfold. Intrathecal injection of the EZH2 inhibitor attenuated the development and maintenance of mechanical and thermal hyperalgesia in rats with nerve injury. Such analgesic effects were concurrently associated with the reduced levels of EZH2, H3K27TM, Iba1, GFAP, TNF-α, IL-1β, and MCP-1 in the spinal dorsal horn in rats with nerve injury. Our results highly suggest that targeting the EZH2 signaling pathway could be an effective approach for the management of neuropathic pain.

Mechanism of spinal monocyte chemoattractant protein-1-mediated maintenance of chronic pathological pain in rats: the relationship with synaptic transmission in spinal dorsal horns

Objective To evaluate the relationship between the mechanism of spinal monocyte chemoattractant protein-1 (MCP-1)-mediated maintenance of chronic pathological pain and synaptic transmission in spinal dorsal horns of rats. Methods Female Sprague-Dawley rats, aged 2-3 weeks after birth, weighing 150-210 g, were studied.The experiment was performed in 2 parts.Experiment Ⅰ Eighteen Sprague-Dawley rats were randomly divided into 2 groups (n=9 each) on 7 days after intrathecal catheters were inserted: phosphate buffer solution(PBS)group and MCP-1 group.PBS 10 μl was intrathecally injected in group PBS, and PBS 10 μl containing 100 ng MCP-1 was intrathecally injected in group MCP-1.The mechanical pain threshold was measured at 30 and 60 min before intrathecal injection, and 30, 60, 90, 120, 150 and 180 min and 1, 2 and 3 days after intrathecal injection.Experiment Ⅱ The transverse spinal cord slices were prepared, and substantia gelatinosa neurons were selected for whole-cell patch-clamp recording.Electrophysiological recording was performed at 1 h of incubation with artificial cerebrospinal fluid (ACSF) and immediately after adding MCP-1: for excitatory synaptic transmission recording, MCP-1 (final concentration 100 nmol/L), N-methyl-D-aspartate (NMDA, final concentration 100 μmol/L) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA, final concentration 20 μmol/L) were added to ACSF, and spontaneous excitatory postsynaptic currents (sEPSCs), AMPA receptors-mediated currents and NMDA receptors-mediated currents were recorded; for inhibitory synaptic transmission recording, MCP-1 (final concentration 100 nmol/L) and γ-aminobutyric acid (GABA, final concentration 1 mmol/L) were added to ACSF, and spontaneous inhibitory postsynaptic currents (sIPSCs) and GABA receptors-mediated currents were recorded. Results Compared with group PBS, the mechanical pain threshold was significantly decreased at 30 min-2 days after intrathecal injection in group MCP-1 (P<0.01). Compared with those at 1 h of incubation with ACSF, the frequency and amplitude of sEPSCs were significantly increased, the amplitude of NMDA receptors- and AMPA receptors-mediated currents were increased, the frequency and amplitude of sIPSCs were decreased, and the amplitude of GABA receptors-mediated currents was decreased immediately after adding MCP-1 (P<0.05). Conclusion MCP-1 enhances excitatory synaptic transmission through enhancing the function of NMDA and AMPA receptors in the posterior substantia gelatinosa neurons of the spinal cord; MCP-1 weakens inhibitory synaptic transmission through inhibiting GABA receptor function, which may be involved in MCP-1-mediated maintenance of chronic pathological pain in rats. Key words: Chemokine CCL2; Pain; Synaptic transmission; Spinal cord