Dorsal Horn Of Rat Spinal Cord Research Articles

(+)-Catechin attenuates CCI-induced neuropathic pain in male rats by promoting the Nrf2 antioxidant pathway to inhibit ROS/TLR4/NF-κB-mediated activation of the NLRP3 inflammasome.

The objective of this study was to investigate the potential mechanisms by which (+)-catechin alleviates neuropathic pain. Thirty-two male Sprague-Dawley rats were divided into four groups: the sham group, the chronic constriction injury (CCI)group, the CCI+ ibuprofen group, and the CCI+ (+)-catechin group. CCI surgery induces thermal hyperalgesia in rats and (+)-catechin ameliorated CCI-induced thermal hyperalgesia and repaired damaged sciatic nerve in rats. CCI decreased SOD levels in male rat spinal cord dorsal horn and promoted MDA production, induced oxidative stress by increasing NOX4 levels and decreasing antioxidant enzyme HO-1 levels, and also increased protein levels of TLR4, p-NF-κB, NLRP3 inflammasome components, and IL-1β. In contrast, (+)-catechin reversed the above results. In i vitro experiments, (+)-catechin reduced the generation of reactive oxygen species (ROS) in GMI-R1 cells after LPS stimulation and attenuated the co-expression of IBA-1 and NLRP3. It also showed significant inhibition of the NF-κB and NLRP3 inflammatory pathways and activation of the Nrf2-mediated antioxidant system. Overall, these findings suggest that (+)-catechin inhibits the activation of the NLRP3 inflammasome through the triggering of the Nrf2-induced antioxidant system, the inhibition of the TLR4/NF-κB pathway, and the production of ROS to alleviate CCI-induced neuropathic pain in male rats.

Exploring the effect of Tuina on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation based on NR2B/PSD-95 pathway

ObjectiveTo investigate the analgesic mechanism of Tuina (Chinese therapeutic massage) by observing the effect of the N-methyl-D-aspartate receptor subunit 2B (NR2B)/postsynaptic density-95 (PSD-95) pathway on the dendritic structure of spinal cord dorsal horn in rats with lumbar disc herniation.MethodsFifty Sprague-Dawley rats were randomly divided into a blank group, a model group, a Tuina group, a blocker agent group, and a blocker agent + Tuina group. The sciatic nerve chronic constriction injury (CCI) model was prepared by the sciatic nerve ligation method. From the 4th day after modeling, rats in the Tuina group and the blocker agent + Tuina group were subject to daily Tuina intervention, and those in the blocker agent group and the blocker agent + Tuina group were daily intrathecally injected with NR2B blocker agent (MK-801). The spontaneous pain score was used to observe the pain behavior of all rats. The expression levels of NR2B and downstream PSD-95 were measured by immunohistochemistry, and the dendritic structure changes were observed by Golgi staining for rat spinal cord dorsal horn after 14 d of continuous intervention.ResultsCompared with the blank group, the degree of rat spontaneous pain after CCI was elevated in both the model and the Tuina groups (P<0.01) and was reduced in the Tuina group after the Tuina intervention compared with the model group (P<0.05). Compared with the model group, the rat spontaneous pain level after blocking NR2B was reduced in both the blocker agent group and the blocker agent + Tuina group (P<0.05). The NR2B and PSD-95 protein levels were significantly higher in the model group compared with the blank group (P<0.01); the total number of dendritic branches was increased (P<0.01), and the total dendritic length became longer (P<0.01) in the spinal cord dorsal horn. The rat NR2B and PSD-95 protein levels were significantly decreased in the Tuina group compared with the model group (P<0.01); the total dendritic branch number was reduced (P<0.01) and the total length was shortened (P<0.01) in the spinal cord dorsal horn. After blocking NR2B, the expression levels of NR2B and downstream PSD-95 protein were significantly lower in both the blocker agent group and the blocker agent + Tuina group compared to the model group (P<0.01). The total branch number was significantly reduced (P<0.01), and the total length was significantly shortened (P<0.01) of the dendrites in the spinal cord dorsal horn.ConclusionTuina may exert an analgesic effect by remodeling the dendritic structure in the spinal cord dorsal horn in rats with lumbar disc herniation, and its mechanism may be related to the inhibition of NR2B/PSD-95 signaling pathway.

Electroacupuncture Alleviates Diabetic Neuropathic Pain and Downregulates p-PKC and TRPV1 in Dorsal Root Ganglions and Spinal Cord Dorsal Horn.

Diabetic neuropathic pain (DNP) is a common complication of diabetes. Streptozotocin (STZ)-induced changes of protein in dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) are critical for DNP genesis. However, which proteins change remains elusive. Here, the DNP model was established by a single intraperitoneal injection of STZ, accompanied by increased fasting blood glucose (FBG), decreased body weight (BW), and decreased paw withdrawal latency (PWL). Proteins change in L4-L6 DRGs and SCDH of rats were detected. Western blot and immunofluorescence results showed that expression levels of phosphorylated protein kinase C (p-PKC), transient receptor potential vanilloid-1 (TRPV1), Substance P (SP) and calcitonin gene-related peptide (CGRP) in the DRG and the SCDH of rats were increased after STZ injection. A preliminary study from our previous study showed that 2 Hz electroacupuncture (EA) effectively alleviates DNP. However, the analgesic mechanism of EA needs further elucidation. Here, EA at the bilateral Zusanli (ST36) and KunLun (BL60) acupoints was applied for one week, and to investigate the effect on DNP. EA reversed thermal hyperalgesia in DNP rats and downregulated the expression of p-PKC, TRPV1, SP, and CGRP in DRG and SCDH.

Sonic Hedgehog Signaling Contributes to Chronic Post-Thoracotomy Pain via Activating BDNF/TrkB Pathway in Rats.

PurposeSome patients undergoing thoracotomy may suffer from chronic post-thoracotomy pain (CPTP). Treatment of CPTP has been a clinical challenge and the underlying mechanisms of CPTP remain elusive. Recently, sonic hedgehog (Shh) signaling has been shown to be associated with various pain states but its role in the pathogenesis of CPTP is still unclear.MethodsCPTP was induced in rats by thoracotomy. Rats were divided into CPTP group and non-CPTP group based on the mechanical withdrawal threshold (MWT). Rats were administered with Shh signaling inhibitor cyclopamine and activator smoothened agonist (SAG), and then evaluated by MWT and cold allodynia testing. The expressions of Shh signaling (Shh ligand, patched and smoothened receptor, Gli transcription factors), brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase receptor B (Trk-B), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) in rat T4-5 spinal cord dorsal horn (SDH) were detected by Western blotting and immunohistochemistry.ResultsThe expression of Shh signaling significantly increased and the BDNF/TrkB pathway was activated in T4-5 SDH of CPTP rats. Cyclopamine attenuated hyperalgesia and down-regulated the expressions of Gil1, BDNF, p-TrkB, p-PI3K and p-Akt in CPTP rats. SAG induced hyperalgesia in non-CPTP rats and elevated the expressions of Gil1, BDNF, p-TrkB, p-PI3K and p-Akt.ConclusionShh signaling may contribute to CPTP via activating BDNF/TrkB signaling pathway, and inhibition of Shh signaling may effectively alleviate CPTP.

Effects of sevoflurane and desflurane on the nociceptive responses of substantia gelatinosa neurons in the rat spinal cord dorsal horn: An in vivo patch-clamp analysis.

BackgroundVolatile anesthetics suppress noxiously evoked activity in the spinal dorsal horn, which could contribute in part to analgesia, immobility. Modulation of excitatory and inhibitory synaptic transmission in substantia gelatinosa neurons could lead to the suppression of dorsal horn activity; however, this phenomenon has not yet been investigated fully.MethodsIn urethane-anesthetized rats, extracellular activity of dorsal horn neurons (action potentials) and excitatory/inhibitory postsynaptic currents in substantia gelatinosa neurons were recorded using extracellular and in vivo patch-clamp techniques, respectively, to assess the spontaneous and the noxious-evoked activity. Sevoflurane or desflurane at concentrations ranging from 0.1 to 2 minimum alveolar concentration was administered by inhalation. Hot- and cold-plate tests were performed to assess nociceptive responses during the inhalation of volatile anesthetics at lower anesthetic doses (0.1–0.5 minimum alveolar concentration).ResultsAt anesthetic doses (1 and 2 minimum alveolar concentration), both sevoflurane and desflurane decreased the frequency of action potentials in the dorsal horn and the activities of excitatory postsynaptic currents in substantia gelatinosa neurons during pinch stimulation and decreased the activities of spontaneous and miniature excitatory postsynaptic currents. Inhibition of the frequencies was more prominent than that of amplitudes in spontaneous and miniature excitatory postsynaptic currents at these anesthetic doses. However, at subanesthetic doses (0.1 and 0.2 minimum alveolar concentration), desflurane facilitated action potentials and excitatory postsynaptic currents. Inhibitory postsynaptic currents were inhibited by both anesthetics at anesthetic doses (1 and 2 minimum alveolar concentration). Hot- or cold-plate tests showed hyperalgesic effects of desflurane at subanesthetic doses (0.1 and 0.2 minimum alveolar concentration) and a dose-dependent analgesic effect of sevoflurane.ConclusionsSevoflurane and desflurane at anesthetic doses suppressed dorsal horn activity mainly via inhibition of excitatory postsynaptic currents in substantia gelatinosa neurons, which would contribute to their analgesic properties. Presynaptic mechanisms were likely in excitatory postsynaptic currents inhibition. Desflurane but not sevoflurane may have a hyperalgesic effect at subanesthetic doses.

The mitochondrial calcium uniporter contributes to morphine tolerance through pCREB and CPEB1 in rat spinal cord dorsal horn

BackgroundThe long-term use of opioid analgesics is limited by the development of unwanted side-effects, such as tolerance. The molecular mechanisms of morphine anti-nociceptive tolerance are still unclear. The mitochondrial calcium uniporter (MCU) is involved in painful hyperalgesia, but the role of MCU in morphine tolerance has not been uncharacterised. MethodsRats received intrathecal injection of morphine for 7 days to induce morphine tolerance. The mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. The effects of an MCU inhibitor, antisense oligodeoxynucleotide against cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) or cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in morphine tolerance were examined. ResultsSpinal morphine tolerance was associated with an increased expression of neuronal MCU, phospho-CREB (pCREB), and CPEB1 in the spinal cord dorsal horn. MCU inhibition increased the mechanical threshold and thermal latency, and reduced the accumulation of mitochondrial calcium in morphine tolerance. Intrathecal antisense oligodeoxynucleotide against CREB or CPEB1 restored the anti-nociceptive effects of morphine compared with mismatch oligodeoxynucleotide in von Frey test and hotplate test. Chromatin immunoprecipitation with quantitative PCR assay showed that CREB knockdown reduced the interaction of pCREB with the ccdc109a gene (encoding MCU expression) promoter and decreased the MCU mRNA transcription. RNA immunoprecipitation assay suggested that CPEB1 binds to the MCU mRNA 3′ untranslated region. CPEB1 knockdown decreased the expression of MCU protein. ConclusionsThese findings suggest that spinal MCU is regulated by pCREB and CPEB1 in morphine tolerance, and that inhibition of MCU, pCREB, or CPEB1 may be useful in preventing the development of opioid tolerance.

Effects of 1,8-cineole on neuropathic pain mediated by P2X2 receptor in the spinal cord dorsal horn

As an intractable health threat, neuropathic pain is now a key problem in clinical therapy, which can be caused by lesions affecting the peripheral nervous systems. 1,8-cineole is a natural monoterpene cyclic ether present in eucalyptus and has been reported to exhibit anti-inflammatory and antioxidant effects. Research has shown that 1,8-cineole inhibits P2X3 receptor-mediated neuropathic pains in dorsal root ganglion. The P2X2 and P2X3 receptors participate in the transmission of algesia and nociception information by primary sensory neurons. In the present study, We thus investigated in the spinal cord dorsal horn whether 1,8-cineole inhibits the expression of P2X2 receptor-mediated neuropathic pain. This study used rats in five random groups: group of chronic constriction injury(CCI) with dimethysulfoxide control (CCI + DMSO); group of CCI; sham group(Sham); group of CCI treated with a low dose 1,8-cineole (CCI + 50 mg/kg); group of CCI with a high dose (CCI + 100 mg/kg). We observed the effects of 1,8-cineole on thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT). We examined P2X2 receptors mRNA change in rat spinal cord dorsal horn by In situ nucleic acid hybridization(ISH) and Quantitative realtime polymerase chain reaction (qRT-PCR) methods. Western Blotting and Immunohistochemical staining methods were used to observe P2X2 receptor protein expressions in the rat spinal cord dorsal horn. It demonstrated that oral administration of 1,8-cineole inhibits over-expression of P2X2 receptor protein and mRNA in the spinal cord and dorsal horn in the CCI rats. And the study explored new methods for the prevention and treatment of neuropathic pain.

Effect of the spinal apelin‑APJ system on the pathogenesis of chronic constriction injury‑induced neuropathic pain in rats.

Apelin is hypothesized to serve a dual function in pain processing. Spinal administration of apelin induces hyperalgesia, while opioid receptors are implicated in the antinociceptive effects of apelin in acute nociceptive models. However, whether the apelin-apelin receptor (APJ) system is involved in neuropathic pain remains to be elucidated. The present study aimed to evaluate the impact and mechanism of the spinal apelin-APJ system in neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve produced sustained spinal apelin and APJ upregulation, which was associated with mechanical allodynia and heat hyperalgesia development in the hind-paw plantar surface. Immunofluorescence demonstrated that apelin and APJ were localized to the superficial dorsal horns. In order to further clarify the function of the apelin-APJ system, a single intrathecal administration of ML221, an APJ antagonist, was used; this transiently reduced CCI-induced pain hypersensitivity. However, apelin-13 (the isoform which binds most strongly to APJ) exhibited no effect on the nociceptive response, suggesting an essential role for the spinal apelin-APJ system in neuropathic pain sensitization. The present study demonstrated that a single application of ML221 alleviated mechanical allodynia and heat hyperalgesia 7 days following CCI, in a dose-dependent manner. Intraspinal delivery of ML221, at the onset of and in fully-established neuropathic pain, persistently attenuated CCI-induced pain hypersensitivity, indicating that the apelin-APJ system was involved in initiating and maintaining pain. It was demonstrated, using immunoblotting, that intrathecal ML221 downregulated phosphorylated extracellular signal-related kinase (ERK) in the rat spinal cord dorsal horn, suggesting that the effect of apelin on neuropathic pain may be mediated via ERK signaling. The results of the present study suggested that the spinal apelin-APJ system may drive neuropathic pain. Inhibition of APJ may provide novel pharmacological interventions for neuropathic pain.

Gabapentin Effects on PKC-ERK1/2 Signaling in the Spinal Cord of Rats with Formalin-Induced Visceral Inflammatory Pain.

Currently, the clinical management of visceral pain remains unsatisfactory for many patients suffering from this disease. While preliminary animal studies have suggested the effectiveness of gabapentin in successfully treating visceral pain, the mechanism underlying its analgesic effect remains unclear. Evidence from other studies has demonstrated the involvement of protein kinase C (PKC) and extracellular signal-regulated kinase1/2 (ERK1/2) in the pathogenesis of visceral inflammatory pain. In this study, we tested the hypothesis that gabapentin produces analgesia for visceral inflammatory pain through its inhibitory effect on the PKC-ERK1/2 signaling pathway. Intracolonic injections of formalin were performed in rats to produce colitis pain. Our results showed that visceral pain behaviors in these rats decreased after intraperitoneal injection of gabapentin. These behaviors were also reduced by intrathecal injections of the PKC inhibitor, H-7, and the ERK1/2 inhibitor, PD98059. Neuronal firing of wide dynamic range neurons in L6–S1 of the rat spinal cord dorsal horn were significantly increased after intracolonic injection of formalin. This increased firing rate was inhibited by intraperitoneal injection of gabapentin and both the individual and combined intrathecal application of H-7 and PD98059. Western blot analysis also revealed that PKC membrane translocation and ERK1/2 phosphorylation increased significantly following formalin injection, confirming the recruitment of PKC and ERK1/2 during visceral inflammatory pain. These effects were also significantly reduced by intraperitoneal injection of gabapentin. Therefore, we concluded that the analgesic effect of gabapentin on visceral inflammatory pain is mediated through suppression of PKC and ERK1/2 signaling pathways. Furthermore, we found that the PKC inhibitor, H-7, significantly diminished ERK1/2 phosphorylation levels, implicating the involvement of PKC and ERK1/2 in the same signaling pathway. Thus, our results suggest a novel mechanism of gabapentin-mediated analgesia for visceral inflammatory pain through a PKC-ERK1/2 signaling pathway that may be a future therapeutic target for the treatment of visceral inflammatory pain.

Pronociceptive and Antinociceptive Effects of Buprenorphine in the Spinal Cord Dorsal Horn Cover a Dose Range of Four Orders of Magnitude.

Due to its distinct pharmacological profile and lower incidence of adverse events compared with other opioids, buprenorphine is considered a safe option for pain and substitution therapy. However, despite its wide clinical use, little is known about the synaptic effects of buprenorphine in nociceptive pathways. Here, we demonstrate dose-dependent, bimodal effects of buprenorphine on transmission at C-fiber synapses in rat spinal cord dorsal horn in vivo. At an analgesically active dose of 1500 μg·kg(-1), buprenorphine reduced the strength of spinal C-fiber synapses. This depression required activation of spinal opioid receptors, putatively μ1-opioid receptors, as indicated by its sensitivity to spinal naloxone and to the selective μ1-opioid receptor antagonist naloxonazine. In contrast, a 15,000-fold lower dose of buprenorphine (0.1 μg·kg(-1)), which caused thermal and mechanical hyperalgesia in behaving animals, induced an enhancement of transmission at spinal C-fiber synapses. The ultra-low-dose buprenorphine-induced synaptic facilitation was mediated by supraspinal naloxonazine-insensitive, but CTOP-sensitive μ-opioid receptors, descending serotonergic pathways, and activation of spinal glial cells. Selective inhibition of spinal 5-hydroxytryptamine-2 receptors (5-HT2Rs), putatively located on spinal astrocytes, abolished both the induction of synaptic facilitation and the hyperalgesia elicited by ultra-low-dose buprenorphine. Our study revealed that buprenorphine mediates its modulatory effects on transmission at spinal C-fiber synapses by dose dependently acting on distinct μ-opioid receptor subtypes located at different levels of the neuraxis.

Effects of remifentanil and fentanyl on N-methyl-D-aspartate receptor currents in rat spinal cord dorsal horn neurons

Objective To investigate the effect of remifentanil and fentanyl on N-methyl-D-aspartate (NMDA) receptor currents in rat spinal cord dorsal horn neurons.Methods Whole-cell patch-clamp technique was used to record the NMDA receptor currents.The primary cultured E14SD rat spinal cord dorsal horn neurons (DH cells) were randomly divided into 3 groups (n =10 each):remifentanil group (group R),fentanyl group (group F) and control group (group C).DH cells were perfused with 4 nmol/L remifentanil (group R) or 10 μmol/L fentanyl (group F) for 60 min followed by washout.NMDA receptor currents were recorded immediately after administration (T0),at 15,30,45 and 60 min of action of drugs (T1-4),and at 15 and 30 min (T5-6) after washout.Results Compared with group C,no significant change in the peak NMDA receptor current was found at each time point in group F and at T0 and T1 in group R (P ＞ 0.05),and the peak NMDA receptor current was significantly increased at T2-6 in group R (P ＜ 0.01).The peak NMDA receptor current was significantly higher at T2-6 than at T0,while lower at T2-4 and T6 than at T5 in group R (P ＜ 0.01).Conclusion Remifentanil can increase NMDA receptor function in rat spinal cord horn neurons,and the peak effect is reached after washout,but fentanyl dose not have the effect. Key words: Piperidines; Receptors, N-methyl-D-aspartate; Drug tolerance

Effects of propofol on phosphorylation of AMPA receptor GluR1 subunits in a rat model of visceral pain

Objective To investigate the effects of propofol on the phosphoryhtion of α-amino-3-hydroxy5-methyl-4-isoxazolepropionate(AMPA)receptor GluR1 subunits at Serine-831 and Serine-845 sites in the spinal cord dorsal horn in a rat model of visceral pain.Methods Thirty male SD rars,aged 6-8 weeks,weighing 200300 g,in which intrathecal catheters were successfully placed without complications,were randomly divided into 3 groups(n-=10 each):sham operation group(group Ⅰ),visceral pain group(group Ⅱ)and propofol group (group Ⅲ).Visceral pain was induced by injection of 10％ capsaicin 50μl via the rectum in groups Ⅱ and Ⅲ.While the equal volume of normal saline was given instead in group Ⅰ.Group Ⅲ received intrathecal injection of propofol 20 tg at 10 min before injection of capsaicin.While the equal volume of dimethyl sulfoxide was given instead of propofol in groups Ⅰ and Ⅱ.The cumulative pain score was recorded during 30 min after capsaicin injection.The rats were then sacrificed,and the lumbar segment(L3-6)of the spinal cord was removed for determination of the expression of GluR1 subunits and phosphorylation of GluR1 subunits at Serine-831 and Serine-845 sites in the spinal cord dorsal horn.Results Compared with group Ⅰ,the cumulative pain score and phosphorylation of GluR1 subunits at Serine-831 sites and Serine-845 in the spinal cord dorsal horn were significantly increased(P ＜0.05 or 0.01),but there were no significant differences in the expression of GluR1 subunits in groups Ⅱ and Ⅲ (P ＞ 0.05).Compared with group Ⅱ,the cumulative pain score and phosphorylation of GluRl subunits at Serine831 and Serine-845 sites in the spinal cord dorsal horn were significantly decreased in group Ⅲ(P ＜ 0.05 or 0.01).Conclusion Propofol can attenuate the visceral pain through the inhibition of the phosphorylation of AMPA receptor GluR1 subunits at Serine-831 and Serine-845 sites in tte rat spinal cord dorsal horn. Key words: Propofol; Receptors,AMPA; Spinal cord; Pain

Amitriptyline attenuates astrocyte activation and morphine tolerance in rats: Role of the PSD-95/NR1/nNOS/PKCγ signaling pathway

The tricyclic antidepressant amitriptyline binds with high affinity to N-methyl-d-aspartate receptors (NMDARs) and inhibits NMDAR-mediated events. Activation of the postsynaptic density protein-95 (PSD-95)/NMDAR-mediated downstream signaling cascade, including neuronal nitric oxide synthase (nNOS) and protein kinase gamma (PKCγ), has been shown to be involved in morphine tolerance. The present study examined the potential effect of amitriptyline on chronic morphine infusion-induced spinal PSD-95/NMDAR/nNOS/PKCγ signaling in morphine tolerance. Male Wistar rats were implanted with an intrathecal catheter and received an intrathecal infusion of saline or amitriptyline (15μg/h), morphine+saline (tolerance induction, 15μg/h), or morphine+amitriptyline for 5 days. Co-administration of amitriptyline with morphine not only preserved the antinociceptive effect of morphine, but also attenuated astrocyte activation in the rat spinal cord dorsal horn. On day 5 after drug infusion, increased expression and phosphorylation of spinal membrane NMDAR NR1 subunit and expression of PSD-95 were observed following chronic morphine infusion and these effects were attenuated by amitriptyline co-infusion. Upregulation of NMDAR-induced intracellular nNOS expression was also inhibited by amitriptyline co-infusion in chronic morphine-infused rats. Furthermore, amitriptyline co-infusion significantly inhibited morphine-induced PKCγ expression in both the cytosol and membrane of spinal neurons. These findings suggest that the attenuation of morphine tolerance caused by amitriptyline is due to downregulation of NMDAR NR1 subunit expression in the synaptosomal membrane accompanied by decreased expression of the scaffolding protein PSD-95. The effects of amitriptyline in attenuating astrocyte activation and reversing tolerance to morphine may be due, at least in part, to inhibition of the PSD-95/NMDAR NR1/nNOS/PKCγ signaling cascade.

Neurotensin inhibits background K+ channels and facilitates glutamatergic transmission in rat spinal cord dorsal horn

Neurotensin (NT) is a neuropeptide involved in the modulation of nociception. We have investigated the actions of NT on cultured postnatal rat spinal cord dorsal horn (DH) neurons. NT induced an inward current associated with a decrease in membrane conductance in 46% of the neurons and increased the frequency of glutamatergic miniature excitatory synaptic currents in 37% of the neurons. Similar effects were observed in acute slices. Both effects of NT were reproduced by the selective NTS1 agonist JMV449 and blocked by the NTS1 antagonist SR48692 and the NTS1/NTS2 antagonist SR142948A. The NTS2 agonist levocabastine had no effect. The actions of NT persisted after inactivation of G(i/o) proteins by pertussis toxin but were absent after inactivation of protein kinase C (PKC) by chelerythrine or inhibition of the MAPK (ERK1/2) pathway by PD98059. Pre- and postsynaptic effects of NT were insensitive to classical voltage- and Ca(2+) -dependent K(+) channel blockers. The K(+) conductance inhibited by NT was blocked by Ba(2+) and displayed no or little inward rectification, despite the presence of strongly rectifying Ba(2+) -sensitive K(+) conductance in these neurons. This suggested that NT blocked two-pore domain (K2P) background K(+) -channels rather than inwardly rectifying K(+) channels. Zn(2+) ions, which inhibit TRESK and TASK-3 K2P channels, decreased NT-induced current. Our results indicate that in DH neurons NT activates NTS1 receptors which, via the PKC-dependent activation of the MAPK (ERK1/2) pathway, depolarize the postsynaptic neuron and increase the synaptic release of glutamate. These actions of NT might modulate the transfer and the integration of somatosensory information in the DH.

P.1.c.058 The involvement of adenosine A1 receptors in the antihyperalgesic effect of levetiracetam

We have reported previously that the dipeptidyl peptidase IV inhibitor Ile-Pro-Ile had an antihyperalgesic action in rats when given intrathecally in the carrageenan-induced hyperalgesia, as detected by the Randall–Selitto test. Vildagliptin, a non-peptide inhibitor of the same enzyme, which is already on the market as an “euglycemic” agent in diabetics, has a slightly more potent and more sustained antihyperalgesic effect in the same test when given by the same route. The action of 3 nmol/rat vildagliptin could be antagonized by subcutaneous naltrexone (0.5 mg/kg) pretreatment, or by intrathecally co-administered specific antiserum to endomorphin-2. Thus, the antihyperalgesia by vildagliptin, similarly to Ile-Pro-Ile, was opioid receptor-mediated and could be attributed to the promotion of endomorphin-2 generation in rat spinal cord dorsal horn. Furthermore, vildagliptin (1 mg/kg) is a potent antihyperalgesic also when given subcutaneously.

The dipeptidyl peptidase IV (CD26, EC 3.4.14.5) inhibitor vildagliptin is a potent antihyperalgesic in rats by promoting endomorphin-2 generation in the spinal cord

We have reported previously that the dipeptidyl peptidase IV inhibitor Ile-Pro-Ile had an antihyperalgesic action in rats when given intrathecally in the carrageenan-induced hyperalgesia, as detected by the Randall–Selitto test. Vildagliptin, a non-peptide inhibitor of the same enzyme, which is already on the market as an “euglycemic” agent in diabetics, has a slightly more potent and more sustained antihyperalgesic effect in the same test when given by the same route. The action of 3 nmol/rat vildagliptin could be antagonized by subcutaneous naltrexone (0.5 mg/kg) pretreatment, or by intrathecally co-administered specific antiserum to endomorphin-2. Thus, the antihyperalgesia by vildagliptin, similarly to Ile-Pro-Ile, was opioid receptor-mediated and could be attributed to the promotion of endomorphin-2 generation in rat spinal cord dorsal horn. Furthermore, vildagliptin (1 mg/kg) is a potent antihyperalgesic also when given subcutaneously.

Cocaine- and amphetamine-regulated transcript (CART) peptide activates ERK pathways via NMDA receptors in rat spinal cord dorsal horn in an age-dependent manner

Activation of extracellular signal-regulated kinase (ERK) cascade in the spinal cord dorsal horn may contribute to pain hypersensitivity. Our recent study showed that cocaine- and amphetamine-regulated transcript peptide fragment 55–102 (CARTp) increased the levels of phosphoserine 896 and phosphoserine 897 on the N-methyl- d-aspartate (NMDA) receptor NR1 subunit (pNR1-ser896 and pNR1-ser897) via protein kinase A (PKA) and protein kinase C (PKC) signaling pathways leading to increases in NMDA receptor function in spinal cord dorsal horn neurons. Because NMDA receptor, PKC, and PKA signaling pathways may participate in ERK activation, we examined the effects of CARTp on ERK activation in spinal cord dorsal horn neurons in vitro. Western blot analysis showed a significant increase in the level of phosphorylated (activated) ERK (pERK) in the dorsal part of the spinal cord slices after incubation of the slices with CARTp (300 nM). Co-administration of CARTp with an NMDA receptor antagonist, MK801 or AP5, or an ERK inhibitor PD98059 blocked the increase in the level of pERK. Interestingly, the increase in the level of pERK by CARTp was observed in postnatal week 3 (W3) and postnatal week 4 (W4), but not in postnatal week 2 (W2) rats. The age-related responses were also noted by CARTp-induced increases in the levels of pNR1-ser896 and pNR1-ser897. In the in vitro electrophysiological study, CARTp increased the amplitude of NMDA-mediated depolarizations in spinal substantia gelatinosa neurons of W3 and W4 rats, but not W2 rats. The results suggest that CARTp activation of ERK signals via the NMDA receptor in the spinal cord dorsal horn was age-dependent.

Expression and distribution of mTOR, p70S6K, 4E-BP1, and their phosphorylated counterparts in rat dorsal root ganglion and spinal cord dorsal horn

Mammalian target of rapamycin (mTOR) controls protein translation and has an important role in the mechanism of pain hypersensitivity under persistent pain conditions. However, its expression and localization in pain-related regions of the nervous system is not completely understood. Here, we examined the expression and distribution of mTOR, eukaryotic initiation factor 4E-binding protein1/2 (4E-BP1/2), p70 ribosomal S6 protein kinase (p70S6K), and their phosphorylated (active) counterparts in two major pain-related regions, the dorsal root ganglion (DRG) and spinal cord dorsal horn. Reverse transcriptase-polymerase chain reaction showed that mTOR, 4E-BP1, and p70S6K mRNA are expressed in the DRG and dorsal horn. Western blot analysis further confirmed the existence of their protein products in these two regions, but expression of their phosphorylated counterparts was very low in dorsal horn and was not detected in the DRG. Immunohistochemistry revealed mTOR and p70S6K in the DRG neurons. Quantitative analysis showed that approximately 26.1% (± 3.2%) of DRG neurons were positive for mTOR and 19.1% (± 1.9%) were positive for p70S6K. Most of these neurons were small—less than 600 µm 2 in cross-sectional area—and some co-labeled with substance P or isolectin B4. Surprisingly, 4E-BP1 was observed only in the DRG satellite glial cells. In the dorsal horn, mTOR, p70S6K, and 4E-BP1 were detected in neurons, but not in astrocytes or microglia. They were distributed in the whole dorsal horn, especially in the superficial dorsal horn. Immunostaining for their phosphorylated counterparts was very low or undetectable in DRG and dorsal horn. Behavioral study showed that intrathecal mTOR inhibitor, rapamycin, did not affect acute nocicepetive transmission. The results indicate that although mTOR, p70S6K, and 4E-BP1 are highly expressed in the DRG and dorsal horn, their activate forms are very low in both regions under normal conditions. Our findings support the view that mTOR and its downstream effectors do not play a key role in acute pain.

Expression and distribution of mTOR, p70S6K, and 4E-BP1 in rat dorsal root ganglion and spinal cord dorsal horn

Mammalian target of rapamycin (mTOR) controls protein translation and has an important role in the mechanism of pain hypersensitivity under persistent pain conditions. However, a complete understanding of its localization in pain-related regions of the nervous system is still lacking. Here, we examined the expression and distribution of mTOR and its two downstream effectors, eukaryotic initiation factor 4E-binding protein1/2 (4E-BP1/2) and p70 ribosomal S6 protein kinase (p70S6K), in two major pain-related regions, the dorsal root ganglion (DRG) and spinal cord dorsal horn.

Exogenous tumor necrosis factor‐α rapidly alters synaptic and sensory transmission in the adult rat spinal cord dorsal horn

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is involved in the generation of inflammatory and neuropathic pain. This study investigated if TNF-alpha has any effect on spinal synaptic and/or sensory transmission by using whole-cell recordings of substantia gelatinosa (SG) neurons in transverse lumbar spinal cord slices of adult rats and by using behavioral tests. After intrathecal administration of TNF-alpha in adult rats, spontaneous hind paw withdrawal behavior and thermal hyperalgesia were rapidly induced (approximately 30 min), while mechanical allodynia slowly developed. Bath application of TNF-alpha (0.1-1 nM, 8 min) depressed peak amplitude of monosynaptic Adelta and C fiber-evoked excitatory postsynaptic currents (EPSCs) without changing in holding currents and input resistances, whereas this application generally potentiated polysynaptic Adelta fiber-evoked EPSCs. Moreover, the frequencies, but not the amplitudes, of spontaneous and miniature EPSCs and spontaneous inhibitory postsynaptic currents were significantly increased by bath-applied TNF-alpha in most of the SG neurons. The effects of TNF-alpha on Adelta/C fiber-evoked monosynaptic and polysynaptic or spontaneous EPSCs were significantly blocked by 5 microM TNF-alpha antagonist that inhibits TNF-alpha binding to its type 1 receptor (TNFR1). Because this study also found high protein expression of TNFR1 in the adult dorsal root ganglion and no change of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) induced whole-cell currents by TNF-alpha, we conclude that presynaptic TNFR1 at Adelta/C primary afferent terminals contributes to the rapid alteration of synaptic transmission in the spinal SG, and the development of abnormal pain hypersensitivity by exogenous TNF-alpha.