Rat Dorsal Horn Research Articles

Down-regulation of Stargazin inhibits the enhanced surface delivery of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor GluR1 subunit in rat dorsal horn and ameliorates postoperative pain.

Stargazin is the first transmembrane protein known to regulate synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. However, it is unclear whether regulation of the surface delivery of spinal AMPA receptor subunits by stargazin contributes to postoperative pain development. Western blot analysis was used to examine changes in the surface delivery of AMPA receptor subunits, GluR1 and GluR2, in rat dorsal horn. The interaction between stargazin and GluR1 and GluR2 was examined by coimmunoprecipitation. Expression of stargazin was suppressed by intrathecal administration of small interfering RNA311. Membrane-bound GluR1, but not GluR2, in ipsilateral dorsal horn was increased at 3 h (1.49 ± 0.15-fold of β-tubulin, mean ± SEM) and 1 day (1.03 ± 0.25) after incision, as compared with that in control rats (naive, 0.63 ± 0.23, P < 0.05, n = 6 per group). The amount of GluR1 coimmunoprecipitated with stargazin was greater at 3 h after incision (1.48 ± 0.31-fold of input) than that in control animals (0.45 ± 0.24, P < 0.05, n = 6 per group). Importantly, the increase in membrane GluR1 at 3 h after incision was normalized to near control level (0.72 ± 0.20-fold of β-tubulin) by pretreatment with intrathecal stargazin small interfering RNA311 (0.87 ± 0.09), but not scrambled small interfering RNA (1.48 ± 0.24) or vehicle (1.25 ± 0.13, P < 0.05, n = 6 per group). Stargazin small interfering RNA311 pretreatment prevented the increase in stargazin-GluR1 interaction and decreased postoperative pain after incision. This study suggests a critical role of stargazin-mediated surface delivery of GluR1 subunit in the development of postoperative pain. A better therapeutic strategy for postoperative pain may involve selectively down-regulating spinal stargazin to inhibit synaptic targeting of GluR1 subunit.

The expression of calcium/calmodulin-dependent protein kinase II in the dorsal horns of rats with type 1 and type 2 diabetes

The activation of calcium/calmodulin-dependent protein kinase II (CaMKII) has been proposed as a key factor in chronic pain development. This study therefore aimed to investigate the expression of CaMKII in the dorsal horn in a rat model of early phase diabetes mellitus (DM) types 1 and 2. Sprague–Dawley rats were used. DM1 was induced using streptozotocin (STZ) (55mg/kg injected intraperitoneally (i.p.)). DM2 was induced using a combination of a high fat diet (HFD) and STZ (35mg/kg i.p.). Controls received an i.p. injection of pure citrate buffer solution. DM2 animals and their controls also received HFD 2 weeks prior to the i.p. injection. Rats were sacrificed 2 weeks and 2 months after diabetes induction. The expression of tCaMKII, pCaMKIIα and IB4 in the dorsal horns was quantified using immunohistochemistry. Increased expression of tCaMKII and pCaMKIIα was seen in the dorsal horns of DM1 animals 2 weeks and 2 months after diabetes induction. In DM2 animals, similar changes in the expression of tCaMKII and pCaMKIIα were observed after 2 weeks, but not after 2 months. The expression of pCaMKIIα was most pronounced in laminae I–III. No difference in IB4 expression was observed between the groups. These results suggest a potential role for CaMKII in diabetic neuropathy development. Inhibition of CaMKII signaling pathways should be further explored as a potential treatment target in painful diabetic neuropathy.

Role of glycogen synthase kinase-3β in expression and trafficking of GluR1-and GluR2-containing AMPA receptors in spinal dorsal horns of rats with remifentanil-induced hyperalgesia

Objective To evaluate the role of glycogen synthase kinase-3β (GSK-3β) in the expression and trafficking of GluR1-and GluR2-containing AMPA receptors in the spinal dorsal horns of rats with remifentanil-induced hyperalgesia.Methods Twenty-four male Sprague-Dawley rats,weighing 240-260 g,aged 2-3 months,were randomly divided into 3 groups (n =8 each) using a random number table:control gourp (group C),remifentanil group (group R),and remifentanil + TDZD-8 (GSK-3β inhibitor) group (group R + T).In group R,remifentanil was infused intravenously at 1.0 μg· kg-1 ·min-1 for 1 h.In group R + T,TDZD-8 1 mg/kg was injected intravenously and then remifentanil was infused intravenously at 1.0 μg· kg-1 · min-1 for 1 h.In group C,normal saline was infused intravenously at 0.1 ml· kg-1 · min-1 for 1 h.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before remifentanil infusion and 2,6,24 and 48 h after remifentanil infusion.The rats were sacrificed after the last behavioral test,and L4.6 segments of the spinal cord were removed to determine the expression of AMPA receptor GluR1 and GluR2 in membrane and total protein in the spinal dorsal horn by Western blot.The ratio of GluR1 expression to GluR2 expression (GluR1/GluR2) in membrane protein was calculated.Results Compared with group C,MWT was significantly decreased,TWL was shortened,the expression of GluR1 in membrane and total protein was up-regualted,the expression of GluR2 in membrane and total protein was down-regulated,and GluR1/GluR2 in membrane protein was increased in group R.Compared with group R,MWT was significantly increased,TWL was prolonged,the expression of GluR1 in membrane and total protein was down-regulated,the expression of GluR2 in membrane and total protein was upregulated,and GluR1/GluR2 in membrane protein was decreased in group R + T.Conclusion GSK-3β mediates up-regulated expression and insertion of GluR1-containing AMPA receptors and down-regulated expression and internalization of GluR2-containing AMPA receptors in the spinal dorsal horns of rats with remifentanil-induced hyperalgesia. Key words: Glycogen synthesis kinase 3 ; Receptors, AMPA ; Piperidines ; Hyperalgesia ; Spinal cord

Intrathecal baclofen, a GABAB receptor agonist, inhibits the expression of p-CREB and NR2B in the spinal dorsal horn in rats with diabetic neuropathic pain.

This study aimed to investigate the effect of baclofen, a γ-aminobutyric acid B (GABAB) receptor agonist, on the expression of p-CREB and NR2B in the spinal dorsal horn of rats with diabetic neuropathic pain (DNP). The DNP rats, which were successfully induced with streptozocin, were distributed among 3 groups that were treated with saline (D1 group), baclofen (D2 group), or CGP55845 + baclofen (D3 group) continuously for 4 days. The rats induced with saline and subsequently treated with saline were used as controls (C group). The times for the paw withdrawal threshold and thermal withdrawal latency of the D1 group were lower than those for the C group, and were significantly increased after baclofen treatment, but not when GABA receptor was pre-blocked with CGP55845 (D3 group). Increased protein expression levels of NR2B and p-CREB and mRNA levels of NR2B were found in the D1 group when compared with the controls. Baclofen treatment significantly suppressed their expression, bringing it close to the levels of controls. However, in the D3 group, the expression of p-CREB and NR2B were still significantly higher than that of the controls. Activation of GABAB receptor by baclofen attenuates diabetic neuropathic pain, which may partly be accomplished via down-regulating the expression of p-CREB and NR2B.

Different effects of local anesthetics on extracellular signal-regulated kinase phosphorylation in rat dorsal horn neurons

Local anesthetics, which are widely known to be neuronal voltage-gated Na+ channel blockers, also affect a variety of other ion channels, N-methyl-d-asparate (NMDA) receptors and α-amino-3-hydroxy-5-methyl-4-izoxazolepropionic acid (AMPA) receptors. Glutamate, which is released from presynaptic fibers, activates extracellular signal-regulated kinase (ERK) through NMDA and AMPA receptors in spinal dorsal horn neurons. ERK plays a key role in central sensitization, which contributes to the chronicity of pain. We investigated the effects of four representative local anesthetics, lidocaine, tetracaine, levobupivacaine, and ropivacaine on ERK phosphorylation induced by capsaicin, which releases glutamate from presynaptic neurons, NMDA, AMPA, or ionomycin, a calcium ionophore, in dorsal neurons. We observed capsaicin-induced phosphorylation of ERK, which was suppressed by lidocaine, tetracaine, or ropivacaine, but not by levobupivacaine. NMDA-induced phosphorylation of ERK was suppressed by lidocaine, tetracaine, or levobupivacaine, but not by ropivacaine. AMPA-induced phosphorylation of ERK was suppressed by lidocaine or tetracaine, but not by levobupivacaine or ropivacaine. Finally, ionomycin-induced ERK phosphorylation was suppressed by lidocaine, tetracaine, or ropivacaine, but not by levobupivacaine. Our results suggest that local anesthetics contribute to the prevention of the incidence of persistent postsurgical pain with varying intensities and through different mechanisms of action.

Effects of dezocine on diabetic neuropathic pain and expression of NMDA receptor subunit 2B in spinal dorsal horns of rats

Objective To evaluate the effects of dezocine on diabetic neuropathic pain （DNP ） and expression of NMDA receptor subunit 2B （NR2B） in the spinal dorsal horns of rats .Methods Forty-eight male Sprague-Dawley rats , aged 4 weeks , weighing 150-170 g , with DNP induced by intraperitoneal injection of streptozocin （STZ） 50 mg/kg （successful induction of diabetes was defined as blood glucose 〉16.7 mmol/L） , were randomly divided into 2 groups （ n=24 each） using a random number table：DNP group and dezocine group （group D） .Twenty-four normal rats were chosen and served as normal control group （group C） .In group D , dezocine 2.52 mg/kg was injected intramuscularly once a day for 7 consecutive days starting from 2nd week after STZ injection ,while the rats in DNP and C groups received the equal volume of normal saline .Paw withdrawl threshold （PWT） to mechanical stimulation was measured before dezocine injection （T0 ） ,and on 1st ,3rd ,5th and 7th days after dezocine injection （T1-4 ） and on 7th day after the end of dezocine injection （T5 ） .Twelve rats in each group were sacrificed after measurement of PWT at T4 ,and T5 .The lumbar segments of the spinal cord were removed for determination of NR2B protein expression （by immuno-histochemistry and Western blot ） and NR2B＆nbsp;mRNA expression （by RT-PCR ） in the spinal dorsal horns .Results Compared with group C ,the PWT at T0-5 in group DNP and at T0 and T5 in group D was significantly decreased , and the expression of NR2B protein and mRNA at T4 ,5 in DNP group and at T5 in D group was up-regulated （ P0.05） . The PWT was significantly lower at T0 and T5 ,and the expression of NR2B protein and mRNA was higher at T5 than at T4 in group D （ P〈0.05 ） .Conclusion Dezocine can effectively relieve DNP in rats and inhibition of NR2B expression in the spinal dorsal horns is involved in the mechanism . Key words: Analgesics,opioid; Neuralgia; Diabetes mellitus; Spinal cord; Receptors,N-methyl-D-aspartate

Relationship between VEGF and ERK signaling pathway in spinal dorsal horns of rats with neuropathic pain

Objective To evaluate the relationship between vascular endothelial growth factor (VEGF) and extracellular signal-regulated kinase (ERK) signaling pathway in spinal dorsal horns of rats with neuropathic pain.Methods Twenty male Sprague-Dawley rats,aged 2 months,weighing 160-320 g,were randomly divided into 4 groups (n =5 each):sham operation group (group S); chronic constrictive injury (CCI) group; normal saline group (NS group); VEGF antibody group.Neuropathic pain was induced by CCI.The animals were anesthetized with intraperitoneal 10％ chloral hydrate 350 mg/kg.The left sciatic nerve was exposed and 4 ligatures were placed on the sciatic nerve at 1 mm intervals.In group S,the left sciatic nerve was only exposed but not ligated.In VEGF antibody group,VEGF antibody 0.3μg/15 μl was injected intrathecally every 2 days for 4 times starting from 2 h after CCI,while the equal volume of normal saline was injected in NS group.Paw withdrawal threshold to von Frey filament stimulation (PWMT) and paw withdrawal latency to nociceptive thermal stimulation (PWTL) were measured at 1 day before CCI (T1),and 1,3,5 and 7 days after CCI (T2-5).The rats were sacrificed after PWMT and PWTL were measured and the L4-6 segments of the spinal cord were removed for determination of phosphorylated ERK (p-ERK) expression in spinal dorsal horns by immunohistochemistry and Western blot.Results Compared with group S,PWMT and PWTL were significantly decreased at T2-5,and the p-ERK expression in spinal dorsal horns was up-regulated in CCI and VEGF antibody groups (P ＜ 0.05).Compared with CCI group,PWMT and PWTL were significantly increased at T3-5,and the p-ERK expression in spinal dorsal horns was down-regualted in VEGF antibody group (P ＜ 0.05).Conclusion VEGF in the spinal dorsal horn is involved in the development and maintenance of neuropathic pain in rats through activating ERK signaling pathway. Key words: Vascular endothelial growth factors; Extracellular signal-regulated MAP kinases; Neuralgia ; Spinal cord

Effects of sinomenine on neuronal apoptosis in spinal dorsal horns in a rat model of neuropathic pain

Objective To evaluate the effects of sinomenine on neuronal apoptosis in the spinal dorsal horns in a rat model of neuropathic pain (NP).Methods One hundred and eight male Wistar rats,weighing 180-220 g,were randomly divided into 3 groups with 36 animals in each group:sham operation group (group S),NP group and sinomenine group (group SIN).The animals were anesthetized with intraperitoneal chloral hydrate.In groups NP and SIN,NP was induced by chronic constrictive injury.The sciatic nerve was exposed and four ligatures were placed on the right sciatic nerve at 1 mm intervals with 4-0 chromic catgut.In group S,the right sciatic nerves were exposed,but not ligated.In group SIN,sinomenine 40 mg/kg was injected intraperitoneally once a day starting from the end of operation until one day before the rats were sacrificed.In groups NP and S,the rats received the equal volume of normal saline instead of sinomenine.Twelve animals in each group were randomly chosen at 1 day before operation (T0) and 3,7 and 14 days after operation (T1-33) to measure mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL).The rats in each group were sacrificed at T1-3 after measurement of pain threshold and the lumbar segments (L4-6) of the spinal cord were removed for determination of caspase-3 mRNA and p38MAPK mRNA expression (by real-time fluorescent quantitative PCR) and cell apoptosis (by TUNEL) in the spinal dorsal horn.Apoptosis rate (AR =the number of apoptotic cells/the total number of cells examined) was calculated.Results Compared with group S,MWT and TWL were significantly decreased,the expression of caspase-3 mRNA and p38MAPK mRNA was up-regulated,and AR was increased at T1-3 in NP and SIN groups (P ＜ 0.05).Compared with group NP,MWT and TWL were significantly increased,the expression of caspase-3 mRNA and p38MAPK mRNA was down-regulated,and AR was decreased at T1-3 in group SIN (P ＜ 0.05).MWT and TWL were significantly lower,and the expression of caspase-3 mRNA and p38MAPK mRNA and AR were higher at T2,3 than at T1 in groups NP and SIN (P ＜ 0.05).Conclusion Sinomenine can attenuate NP through inhibiting the expression of p38MAPK signal transduction pathway and reducing neuronal apoptosis in the spinal dorsal horn of rats. Key words: Sinomenine; Neuralgia; Apoptosis; Spinal cord

Down-regulation of Stargazin Inhibits the Enhanced Surface Delivery of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate Receptor GluR1 Subunit in Rat Dorsal Horn and Ameliorates Postoperative Pain

Down-regulation of Stargazin Inhibits the Enhanced Surface Delivery of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate Receptor GluR1 Subunit in Rat Dorsal Horn and Ameliorates Postoperative Pain

A Putative Relay Circuit Providing Low-Threshold Mechanoreceptive Input to Lamina I Projection Neurons via Vertical Cells in Lamina II of the Rat Dorsal Horn

BackgroundLamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents.ResultsWe initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin.ConclusionsThese results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia.

Expressions of neuropathic pain-related proteins in the spinal cord dorsal horn in rats with bilateral chronic constriction injury

To evaluate the pain-related behavioral changes in rats with bilateral chronic constriction injury(bCCI)and identify the expressions of neuropathic pain-related proteins. The bCCI models were established by ligating the sciatic nerves in female Sprague Dawley rats. Both mechanical hyperalgesia and cold hyperalgesia were evaluated through electronic von Frey and acetone method. Liquid chromatography-mass spectrometry/mass spectrometry was applied to characterize the differentially expressed proteins. Both mechanical withdrawal threshold and cold hyperalgesia threshold decreased significantly on the postoperative day 7 and 14, when compared with na ve or sham rats(P <0.05). Twenty five differentially expressed proteins associated with bilateral CCI were discovered, with eighteen of them were upregulated and seven of them downregulated. The bCCT rats have remarkably decreased mechanical and cold hyperalgesia thresholds. Twenty five neuropathic pain-related proteins are found in the spinal cord dorsal horn.

Glycogen synthase kinase 3 beta regulates glial glutamate transporter protein expression in the spinal dorsal horn in rats with neuropathic pain

Dysfunctional glial glutamate transporters and over production of pro-inflammatory cytokines (including interleukin-1β, IL-1β) are two prominent mechanisms by which glial cells enhance neuronal activities in the spinal dorsal horn in neuropathic pain conditions. Endogenous molecules regulating production of IL-1β and glial glutamate functions remain poorly understood. In this study, we revealed a dynamic alteration of GSK3β activities and its role in regulating glial glutamate transporter 1 (GLT-1) protein expression in the spinal dorsal horn and nociceptive behaviors following the nerve injury. Specifically, GSK3β was expressed in both neurons and astrocytes in the spinal dorsal horn. GSK3β activities were suppressed on day 3 but increased on day 10 following the nerve injury. In parallel, protein expression of GLT-1 in the spinal dorsal horn was enhanced on day 3 but reduced on day 10. In contrast to these time-dependent changes, the activation of astrocytes and over-production of IL-1β were found on both day 3 and day 10. Meanwhile, thermal hyperalgesia was observed from day 2 through day 10 and mechanical allodynia from day 4 through day 10. Pre-emptive pharmacological inhibition of GSK3β activities significantly ameliorated thermal hyperalgesia and mechanical allodynia at the late stage but did not have effects at the early stage. These were accompanied with the suppression of GSK3β activities, prevention of decreased GLT-1 protein expression, inhibition of astrocytic activation, and reduction of IL-1β in the spinal dorsal horn on day 10. These data indicate that the increased GSK3β activity in the spinal dorsal horn is attributable to the downregulation of GLT-1 protein expression in neuropathic rats at the late stage. Further, we also demonstrated that the nerve-injury-induced thermal hyperalgesia on day 10 was transiently suppressed by pharmacological inhibition of GSK3β. Our study suggests that GSK3β may be a potential target for the development of analgesics for chronic neuropathic pain.

Design and synthesis of potent and selective P2X3 receptor antagonists derived from PPADS as potential pain modulators

Pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate (7a, PPADS), a nonselective P2X receptor antagonist, was extensively modified to develop more stable, potent, and selective P2X3 receptor antagonists as potential antinociceptive agents. Based on the results of our previous report, all strong anionic groups in PPADS including phosphate and sulfonate groups were changed to carboxylic acids or deleted. The unstable azo (–NN–) linkage of 7a was transformed to more stable carbon-carbon, ether or amide linkages through the synthesis of the 5-hydroxyl-pyridine moieties with substituents at 2 position via a Diels-Alder reaction. This resulted in the retention of antagonistic activity (IC50 = 400 ∼ 700 nM) at the hP2X3 receptor in the two-electrode voltage clamp (TEVC) assay system on the Xenopus oocytes. Introduction of bulky aromatic groups at the carbon linker, as in compounds 13h–n, dramatically improved the selectivity profiles of hP2X3 when compared with mP2X1 and hP2X7 receptors. Among the substituents tested at the 2-position, the m-phenoxybenzyl group showed optimum selectivity and potency at the hP2X3 receptor. In searching for effective substituents at the 4- and 3-positions, we found that compound 36j, with 4-carboxaldehyde, 3-propenoic acid and 2-(m-phenoxy)benzyl groups, was the most potent and selective hP2X3 receptor antagonist with an IC50 of 60 nM at hP2X3 and marginal antagonistic activities of 10 μM at mP2X1 and hP2X7. Furthermore, using an ex-vivo assay system, we found that compound 36j potently inhibited pain signaling in the rat dorsal horn with 20 μM 36j displaying 65% inhibition while 20 μM pregabalin, a clinically available drug, showed only 31% inhibition.

Mapping of the fluoride resistant acid phosphatase (FRAP) activity in cervico-thoracic-lumbar spinal dorsal horn in rats

The fluoride resistant acid phosphatase (FRAP) is considered a biomarker enzyme of presence and function in nociceptive fibers. Its well described spinal dorsal horn (DH) superficial lamina location contrasts to its less known cervico-lumbar DH distribution. Thus, 15 male Sprague Dawley rats (300–400 g) were anaesthetized (thiopental 60 mg kg−1 i.p.) and laminectomized (C 1-L 5) to extract and fix (formaline 4% + NaF 1%) spinal cords. Cervical, thoracic and lumbar segments were coronally cut (1 mm slices) and micropunched (1 mmO) at the dorsal horn (both left and right). Samples were diluted in NaF 1% in saline (1000 μL: fragment) and homogenized. Protein concentration of the homogenate was determined by Bradford method to correct variation on tissue sampling and used to compensate enzymatic dilution. FRAP activity was determined by the Gomorri method and expressed as U gr prot−1 L−1. No differences were found between left and right DH FRAP activities (Mann-Whitney U Test, P > 0.05) in any metamera. DH segment values were: cervical 2.53 (2.67–2.40 U gr prot−1 L−1; mean ± 95% CI), thoracic 3.59 (3.96–3.21) and lumbar 2.42 (2.75–2.09). Thoracic FRAP activity was statistically higher (+45.3%) than those from cervical and lumbar DH segments (ANOVA; Ω2 = 0.29; F = 17.78; P = 0.000001; Levene test P = 0.000006). No significant correlation was found among cervicotoraco-lumbar FRAP activity. The increased FRAP activity for thoracic DH could be explained by the thoracic overlapped primary afferents and collateral fibers with cervical or lumbar input added to the basal thoracic FRAP expression.

Endogenous Interleukin-1β in Neuropathic Rats Enhances Glutamate Release from the Primary Afferents in the Spinal Dorsal Horn through Coupling with Presynaptic N-Methyl-d-aspartic Acid Receptors*♦

Excessive activation of glutamate receptors and overproduction of proinflammatory cytokines, including interleukin-1β (IL-1β) in the spinal dorsal horn, are key mechanisms underlying the development and maintenance of neuropathic pain. In this study, we investigated the mechanisms by which endogenous IL-1β alters glutamatergic synaptic transmission in the spinal dorsal horn in rats with neuropathic pain induced by ligation of the L5 spinal nerve. We demonstrated that endogenous IL-1β in neuropathic rats enhances glutamate release from the primary afferent terminals and non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Myeloid differentiation primary response protein 88 (MyD88) is a mediator used by IL-1β to enhance non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Presynaptic NMDA receptors are effector receptors used by the endogenous IL-1β to enhance glutamate release from the primary afferents in neuropathic rats. This is further supported by the fact that NMDA currents recorded from small neurons in the dorsal root ganglion of normal rats are potentiated by exogenous IL-1β. Furthermore, we provided evidence that functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is mediated by the neutral sphingomyelinase/ceramide signaling pathway. Hence, functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is a crucial mechanism leading to enhanced glutamate release and activation of non-NMDA receptors in the spinal dorsal horn neurons in neuropathic pain conditions. Interruption of such functional coupling could be an effective approach for the treatment of neuropathic pain.

Role of Stargazin in trafficking of GluRl-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn in rats with incisional pain

Objective To evaluate the role of Stargazin in trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in the spinal dorsal horn in rats with incisional pain.Methods Forty-five adult male Sprague-Dawley rats,aged 6-8 yr,weighing 280-300 g,were randomly divided into 5 groups (n =9 each):control group (group C),sham operation group (group S),incisional pain + normal saline group (group P),incisional pain + Stargazin small interference RNA (siRNA) group (group Ⅰ),and incisional pain + non-sense siRNA group (group N).In P,I and N groups,normal saline 10 μl,20 μmol/L siRNA and 20 μmol/L non-siRNA 10 μl were injected intrathecally,respectively,twice a day for 3 consecutive days.A 1 cm long incision was made in the plantar surface of right hindpaw.Cumulative pain score (CPS) and paw-withdrawal threshold to yon Frey stimuli (PWT) were measured at 3 h after incision.The animals were sacrificed after pain behavior assessment.Their lumbar segments of the spinal cord (L3-6) were removed.The expression of GluR1 and GluR2 in cell membrane and cytoplasm in spinal cord dorsal horn was determined by Western blot analysis.The co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was examined by co-immuno-precipitation.Results Compared with group C,the CPS was significantly increased,the PWT was decreased,the expression of GluR1 in cytoplasm was down-regulated and the expression of GluR1 in cell membrane was up-regulated in N and P groups,and the CPS was increasedin group Ⅰ (P ＜ 0.05 or 0.01).Compared with group P,the CPS was significantly decreased,the PWT was increased,the expression of GluR1 in cytoplasm was up-regulated,the expression of GluR1 in cell membrane was down-regulated,and the expression of Stargazin and co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was down-regulated in group Ⅰ (P ＜ 0.05 or 0.01).Conclusion Stargazin mediates trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn and is involved in the development of hyperalgesia in rats with incisional pain. Key words: Receptors, AMPA; Protein transport; Pain; Spinal cord; Stargazin

Scratching inhibits serotonin-evoked responses of rat dorsal horn neurons in a site- and state-dependent manner

Scratching inhibits pruritogen-evoked responses of neurons in the superficial dorsal horn, implicating a spinal site for scratch inhibition of itch. We investigated if scratching differentially affects neurons depending on whether they are activated by itchy vs. painful stimuli, and if the degree of inhibition depends on the relative location of scratching. We recorded from rat lumbar dorsal horn neurons responsive to intradermal (id) microinjection of serotonin (5-hydroxytryptamine, 5-HT). During the response to 5-HT, scratch stimuli (3mm, 300mN, 2Hz, 20s) were delivered at the injection site within the mechanosensitive receptive field (on-site), or 4–30mm away, outside of the receptive field (off-site). During off-site scratching, 5-HT-evoked firing was significantly attenuated followed by recovery. On-site scratching excited neurons, followed by a significant post-scratch decrease in 5-HT-evoked firing. Most neurons additionally responded to mustard oil (allyl isothiocyanate). Off-site scratching had no effect, while on-site scratching excited the neurons. These results indicate that scratching exerts a state-dependent inhibitory effect on responses of spinal neurons to pruritic but not algesic stimuli. Moreover, on-site scratching first excited neurons followed by inhibition, while off-site scratching immediately evoked the inhibition of pruritogen-evoked activity. This accounts for the suppression of itch by scratching at a distance from the site of the itchy stimulus.

Injection of nerve growth factor into a low back muscle induces long-lasting latent hypersensitivity in rat dorsal horn neurons

Little is known about the central mechanisms underlying the transition from local or regional to widespread pain in low back pain patients. The aim of the study was to find out if muscle input induced by injection of nerve growth factor (NGF) can be used as an animal model for studying spinal mechanisms involved in widespread myofascial low back pain. Electrophysiological recordings from rat dorsal horn neurons were made in vivo to study alterations in their responsiveness caused by 2 injections of NGF into the multifidus muscle at an interval of 5days. NGF is known to be closely associated with many painful muscle disorders. The results demonstrate that the 2 NGF injections—but not a single one—caused a significant hyperexcitability of spinal neurons. Five days after the first NGF injection, the neurons were not significantly sensitized but were easier to sensitize by a second injection. The state of the neurons resembles nociceptive priming. Important findings were that the proportion of neurons having multiple receptive fields (RFs) in various tissues was significantly higher after 2 NGF injections, and new RFs appeared on the distal hind limb. The new RFs were located not in the skin but in deep tissues (muscles, thoracolumbar fascia). If similar changes occur in patients, the data might explain the diffuse nature and spread of myofascial low back pain.

Neural coding of nociceptive stimuli—from rat spinal neurones to human perception

Translational studies are key to furthering our understanding of nociceptive signalling and bridging the gaps between molecules and pathways to the patients. This requires use of appropriate preclinical models that accurately depict outcome measures used in humans. Whereas behavioural animal studies classically involve reports related to nociceptive thresholds of, for example, withdrawal, electrophysiological recordings of spinal neurones that receive convergent input from primary afferents permits investigation of suprathreshold events and exploration of the full-range coding of different stimuli. We explored the central processing of nociceptive inputs in a novel parallel investigation between rats and humans. Using radiant laser pulses, we first compared the electrophysiological responses of deep wide dynamic range and superficial nociceptive-specific neurones in the rat dorsal horn with human psychophysics and cortical responses. Secondly, we explored the effects of spatial summation using laser pulses of identical energy and different size. We observed 3 main findings. Firstly, both rodent and human data confirmed that neodymium–yttrium aluminium perovskite laser stimulation is a nociceptive-selective stimulus that never activates Aβ afferents. Secondly, graded laser stimulation elicited similarly graded electrophysiological and behavioural responses in both species. Thirdly, there was a significant degree of spatial summation of laser nociceptive input. The remarkable similarity in rodent and human coding indicates that responses of rat dorsal horn neurones can translate to human nociceptive processing. These findings suggest that recordings of spinal neuronal activity elicited by laser stimuli could be a valuable predictive measure of human pain perception.

Effect of glial cell line-derived neurotrophic factor on Fos protein expression in the spinal dorsal horn of neuropathic pain rat model

Objective To study the effect of glial cell line-derived neurotrophic factor (GDNF)on Fos protein expression in the spinal dorsal horn of neuropathic pain rat model.Methods Adult Sprague-Dawley male rats were randomly divided into normal control group,sham operation group,spinal nerve ligation (SNL) group and SNL combined with treatment of GDNF group.Every group was divided into three sub-groups:SNL 3,7 and 14 days (n =10 in each group).Fos protein expression in the spinal dorsal horn was examined by using inununohistochemistry and Western blotting.Results Compared with normal control and sham operation groups,the Fos protein expression in the spinal dorsal horn of the rats in SNL group was increased significantly (1.00 ± 0.00,1.08 ± 0.12,and 4.62 ± 0.52 respectively).As compared with the rats in SNL group,there was an apparent decrease in GDNF treatment group three days later (1.82 ± 0.51).Conclusion Fos protein in the spinal dorsal horn of rats was involved in the mechanism of neuropathic pain.Intrathecal administration of GDNF could inhibit Fos protein expression in the spinal dorsal horn. Key words: Glial cell line-derived neurotrophic factor; Neuropathic pain; Fos protein