Rat Spinal Dorsal Horn Research Articles

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

Expression of N-methyl-D-aspartate receptor subunit 2B containing N-methyl-D-aspartate receptors in the spinal dorsal horn of rats with hydrogen sulfide-induced pain

Objective To investigate the role of N-methyl-D-aspartate receptor subunit 2B (NR2B) containing N-methyl-D-aspartate (NMDA) receptors in the spinal dorsal horn of rats with hydrogen sulfide-induced pain.Methods Thirty male SD rats aged 7 weeks were randomly divided into 3 groups (n =10 each):control group (group C),H2S group (group S),and ketamine group (group K).Intraplantar administration of NaHS (H2S donor) 1 nmol (100 μl) for 5 days in group S and group K,and the equal volum (100 μl) of nodal saline (NS) in group C (once each day) was done.Then rats in group K were injected with ketamine 10 mg/kg (0.2 ml) intramuscularly for 7 days,and the equal volum of NS was injected in group C and group S (once each day).The paw withdrawal threshold (PWT) to mechanical stimulation was measured at first day before the first administration of NaHS/NS (Tc),20 min after the first administration of NaHS/NS (T1),20 min after the fifth administration of NaHS/NS (T2),and 20 min after the seventh administration of ketamine/NS (T3).The rats were sacrificed after the last PWT measurement.The lumber segment of the spinal cord was removed for determination of the expressions of NR2B subunit by using RT-PCR and Western blotting.Results The PWT at T1,T2 and T3 was (2.72 ± 1.17),(1.48 ± 0.57) and (2.40 ± 0.89) g respectively in group S,which was significantly lower than in group C,and the expressions of NR2B mRNA and protein in spinal dorsal horn was (0.75 ±0.07) and (1.36 ± 0.08) respectively,which was significantly higher than in group C.The PWT at T3 was (8.89 ±1.09)g in group K,which was higher than in group S,and the expression of NR2B mRNA and protein in spinal dorsal horn was (0.55 ± 0.05) and (1.02 ± 0.06) respectively,which was lower than in group S.Conclusion The upregnlation of NR2B subunit in spinal dorsal horn was one of meachanisms in hydrogen sulfide-induced pain and inhibition of NR2B expression may contribute to the antinociceptive effects of ketamine. Key words: Hydrogen sulfide; Ketamine; N-methyl-D-aspartate receptor subunit 2B; Spinal dorsal horn; Pain

Properties of Quantum Release of Glutamate and Glycine in Synapses between Co-Cultured Primary Afferent and Spinal Dorsal Horn Neurons

Using the patch-clamp technique, we recorded transmembrane currents and potentials in pairs of synaptically connected co-cultured rat primary afferent neurons (dorsal root ganglion, DRG, cells) and spinal dorsal horn (DH) units; the peculiarities of release of glutamate and glycine in the corresponding synapses were determined. By analyzing distributions of the amplitudes of postsynaptic currents recorded in DH neurons, we estimated the quantum parameters of release of these neurotransmitters. We found that the release of neurotransmitter quanta due to the entry of a presynaptic action potential into glutamate- and glycinergic DRG–DH synapses is realized independently. The probability of transmitter release can be described by binomial statistics. There are reasons to believe that, under co-culturing conditions, a pre-synaptic DRG neuron forms a few synaptic connections with a post-synaptic DH cell. As is concluded, presynaptic terminals in such synapses can have more than one release zone for the transmitter; the possibility of multiquantum release of glycine or glutamate in several zones of release during neurotransmission cannot be ruled out.

The Upregulation of Translocator Protein (18 kDa) Promotes Recovery from Neuropathic Pain in Rats

At present, effective drug for treatment of neuropathic pain is still lacking. Recent studies have shown that the ligands of translocator protein (TSPO, 18 kDa), a peripheral receptor for benzodiazepine, modulate inflammatory pain. Here, we report that TSPO was upregulated in astrocytes and microglia in the ipsilateral spinal dorsal horn of rats following L5 spinal nerve ligation (L5 SNL), lasting until the vanishing of the behavioral signs of neuropathic pain (∼50 d). Importantly, a single intrathecal injection of specific TSPO agonists Ro5-4864 or FGIN-1-27 at 7 and 21 d after L5 SNL depressed the established mechanical allodynia and thermal hyperalgesia dramatically, and the effect was abolished by pretreatment with AMG, a neurosteroid synthesis inhibitor. Mechanically, Ro5-4864 substantially inhibited spinal astrocytes but not microglia, and reduced the production of tumor necrosis factor-α (TNF-α) in vivo and in vitro. The anti-neuroinflammatory effect was also prevented by AMG. Interestingly, TSPO expression returned to control levels or decreased substantially, when neuropathic pain healed naturally or was reversed by Ro5-4864, suggesting that the role of TSPO upregulation might be to promote recovery from the neurological disorder. Finally, the neuropathic pain and the upregulation of TSPO by L5 SNL were prevented by pharmacological blockage of Toll-like receptor 4 (TLR4). These data suggested that TSPO might be a novel therapeutic target for the treatment of neuropathic pain.

Enhancement by Interleukin-1β of AMPA and NMDA Receptor-Mediated Currents in Adult Rat Spinal Superficial Dorsal Horn Neurons

BackgroundProinflammatory cytokine interleukin-1β (IL-1β) released from spinal microglia plays an important role in the maintenance of acute and chronic pain states. However, the cellular basis of this action remains poorly understood. Using whole-cell patch-clamp recordings, we examined the action of IL-1β on AMPA- and NMDA-receptor-mediated currents recorded from substantia gelatinosa (SG) neurons of adult rat spinal cord slices which are key sites for regulating nociceptive transmission from the periphery.ResultsAMPA- and NMDA-induced currents were increased in peak amplitude by IL-1β in a manner different from each other in SG neurons. These facilitatory actions of IL-1β were abolished by IL-1 receptor (IL-1R) antagonist (IL-1ra), which by itself had no detectable effects on AMPA- and NMDA-induced currents. The AMPA- but not NMDA-induced current facilitated by IL-1β was recovered to control level 30 min after IL-1β washout and largely depressed in Na+-channel blocker tetrodotoxin-containing or nominally Ca2+-free Krebs solution. Minocycline, a microglia inhibitor, blocked the facilitatory effect of IL-1β on AMPA- but not NMDA-induced currents, where minocycline itself depressed NMDA- but had not any effects on AMPA-induced currents.ConclusionsIL-1β enhances AMPA and NMDA responses in SG neurons through IL-1R activation; the former but not latter action is reversible and due to an increase in neuronal activity in a manner dependent on extracellular Ca2+ and minocycline. It is suggested that AMPA and NMDA receptors are positively modulated by IL-1β in a manner different from each other; the former but not latter is mediated by a neurotransmitter released as a result of an increase in neuronal activity. Since IL-1β contributes to nociceptive behavior induced by peripheral nerve or tissue injury, the present findings also reveal an important cellular link between neuronal and glial cells in the spinal dorsal horn.

Effects of electroacupuncture on expression of c-fos protein in the spinal dorsal horn of rats with chronic visceral hyperalgesia

Acupuncture is widely used in clinics to suppress chronic visceral pain in patients with irritable bowel syndrome (IBS); however, the exact neurobiological mechanisms for its therapeutic effects need further exploration. The aim of this study was to investigate the possible involvement of spinal neurons in the effects of electroacupuncture (EA) in relieving chronic visceral hyperalgesia in a rat model of IBS. Colon mechanical irritation was applied to male neonatal Sprague-Dawley rats to establish the IBS model. Behavioral test of the abdominal withdraw reflex (AWR) response to colorectal distention stimuli was conducted to judge the degree of colorectal sensitivity. EA at acupoints Zusanli (ST36) and Shangjuxu (ST37) was applied bilaterally in a total of four times every other day, while sham-EA at similar acupoints was done by inserting needles without electrical stimulation. Immunohistochemical methods were used to display the expression of proto-oncogene protein c-fos in the spinal dorsal horn. It was found that AWR scores were significantly increased in the IBS model rats (P<0.01), accompanied with significant increase in the expression of c-fos protein in the superficial laminae (SDH, laminae I and II) and nucleus proprius (NP, laminae III and IV), the neck of the dorsal horn (NECK, laminae V and VI) at lumbosacral (L6-S2) spinal level, and in NECK at thoracolumbar (T13-L2) spinal level, when compared with normal rats (P<0.05). After EA treatment, AWR scores and the expression of c-fos protein in SDH, NP and NECK at similar spinal levels were significantly decreased in the IBS model rats (P<0.05). No such effects on either AWR scores or the expression of c-fos protein were observed in IBS model rats after sham-EA treatment. The abnormally high neuronal excitability in the spinal dorsal horn may be an important reason underlying the visceral hyperalgesia in IBS model rats. EA treatment can relieve the chronic visceral hyperalgesia in IBS rats by suppressing the abnormal neuronal excitability in the spinal dorsal horn.

Temporal distribution of p300/CBP immunoreactivity in the adult rat spinal dorsal horn following chronic constriction injury (CCI).

p300 and its homolog cyclic AMP response element binding protein (CBP) are coactivators that were identified to participate in many biological processes including neural development and cognition. Their roles within the rodent spinal cord have not been reported systematically; in this study, their spatiotemporal distribution in the spinal cord of adult rat following chronic constriction injury (CCI) was studied. p300 and CBP expressed predominantly in nuclei in the gray matter of rat spinal cord. Rats undergoing CCI surgery showed increased p300/CBP immunoreactivity (IR) compared with normal control and sham-operated rats. The number of IR cells reached the peak at day 14 following CCI compared with those on day 3, 7, and 21, accompanied with significant behavioral changes of neuropathic pain. Cell-type determination by immunofluorescence at day 14 following CCI revealed that p300 and CBP expressed in neurons, but not in astrocytes or microglial cells. These results suggest that p300 and CBP are probably involved in the maintenance of neuropathic pain on spinal cord level. Furthermore, p300 and CBP may serve as a sensor only in neurons but not in astrocytes or microglia cells in the adult rat spinal cord.

Automatic System for Identification and Analysis of Burst

Although there are complex patterns of discharges exist in neurons, which are often clustered in bursts, the pattern of the burst discharge can substantially change with the alternation of the living environment of the neurons. Furthermore, there is a big difference in the physiological and pharmacological characteristics of various types of burst discharges. So it is necessary to analyze the information contained in various types of burst discharges. Here, an automatic system for identification and analysis of the neuron discharge based on plotting histograms of the logarithm of the interspike interval was designed. The system consists of neuron discharge collecting unit, neuron discharge processing unit, battery monitoring, real-time charging unit and bursts processing software. The system can identify the burst discharge from the neuron discharge without any omission and make statistical analysis. By using this device, the electrophysiological experiment that the spontaneous and evoked discharges of wide dynamic range neurons in spinal dorsal horn of rats were recorded was smoothly completed. The result of statistical analysis indicated that the device can give the corresponding interspike interval aimed at various types of burst discharges and respectively identify the burst discharges in the different amplitude spikes, which provide a tool for further research on the biosensor and the neural communication.

Increased synaptophysin is involved in inflammation-induced heat hyperalgesia mediated by cyclin-dependent kinase 5 in rats.

Mechanisms associated with cyclin-dependent kinase 5 (Cdk5)-mediated heat hyperalgesia induced by inflammation remain undefined. This study was designed to examine whether Cdk5 mediates heat hyperalgesia resulting from peripheral injection of complete Freund's adjuvant (CFA) in the spinal dorsal horns of rats by interacting with synaptophysin, a well known membrane protein mediating the endocytosis-exocytosis cycle of synaptic vesicles as a molecular marker associated with presynaptic vesicle membranes. The role of Cdk5 in mediating synaptophysin was examined through the combined use of behavioral approaches, imaging studies, and immunoprecipitation following CFA-induced inflammatory pain. Results showed that Cdk5 colocalized with both synaptophysin and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) consisting of VAMP-2, SNAP-25, and syntaxin 1A in spinal dorsal horn of rats. Increased synaptophysin expression of spinal cord horn neurons post intraplantar injection of CFA coincided with increased duration of heat hyperalgesia lasting from 6 h to 3 d. Intrathecal administration of roscovitine, a Cdk5 specific inhibitor, significantly depressed synaptophysin expression during peak heat hyperalgesia and heat hyperalgesia induced by peripheral injection of CFA. Data presented in this report indicated that calpain activity was transiently upregulated 6 h post CFA-treatment despite previous reports suggesting that calpain was capable of cleaving p35 into p25. Results from previous studies obtained by other laboratories demonstrated that significant changes in p35 expression levels within spinal cord horn neurons were not observed in the CFA-treated inflammatory pain model although significant upregulation of Cdk5 kinase was observed between 2 h to 7 d. Therefore, generation of p25 occurred in a calpain-independent fashion in a CFA-treated inflammatory pain model. Our results demonstrated that increased synaptophysin levels were involved in heat hyperalgesia mediated by Cdk5 in spinal cord dorsal horns of CFA-treated rats, suggesting that inhibiting abnormal activation of Cdk5-synaptophysin may present a novel target for diminishing inflammatory pain.

Electrical Stimulation of Large Myelinated Afferents Inhibits Responses of Rat Spinal Dorsal Horn Neurons to Noxious and Innocuous Cutaneous Stimulation

Electrical Stimulation of Large Myelinated Afferents Inhibits Responses of Rat Spinal Dorsal Horn Neurons to Noxious and Innocuous Cutaneous Stimulation

Neurotoxicity of intrathecal injections of dexmedetomidine into the rat spinal dorsal horn.

To investigate the neurotoxicity of intrathecal injections of dexmedetomidine, Sprague-Dawley rats were intrathecally injected with dexmedetomidine at doses of 0.75, 1.50 and 3.00 μg/kg into the spinal dorsal horn. We found that c-Fos expression in the rat spinal dorsal horn peaked at 7 hours following the 3.00 μg/kg dexmedetomidine injection, while the levels of c-Fos expression following 0.75 and 1.50 μg/kg dexmedetomidine were similar to those in the spinal dorsal horn of normal rats. At 48 hours following administration, the level of c-Fos expression was similar to normal levels. In addition, the intrathecal injections of dexmedetomidine increased paw withdrawal mechanical thresholds and prolonged thermal tail flick latencies. These results indicate that dexmedetomidine has pronounced antinociceptive effects. However, dexmedetomidine appears to have neurotoxic effects in the spinal cord because it increased c-Fos expression in the spinal dorsal horn within 7 hours following administration.

Subunit-Specific Inhibition of Glycine Receptors by Curcumol

Emerging evidence has suggested that inhibitory glycine receptors (GlyRs) are an important molecular target in the treatment of numerous neurological disorders. Rhizoma curcumae is a medicinal plant with positive neurological effects. In this study, we showed that curcumol, a major bioactive component of R. curcumae, reversibly and concentration-dependently inhibited the glycine-activated current (IGly) in cultured rat hippocampal neurons. The inhibitory effect was neither voltage- nor agonist concentration-dependent. Moreover, curcumol selectively inhibited homomeric α2-containing, but not α1- or α3-containing, GlyRs. The addition of β subunit conferred the curcumol sensitivity of α3-containing, but not α1-containing, GlyRs. Site-directed mutagenesis analysis revealed that a threonine at position 59 of the α2 subunit is critical for the susceptibility of GlyRs to curcumol-mediated inhibition. Furthermore, paralleling a decline of α2 subunit expression during spinal cord development, the degree of IGly inhibition by curcumol decreased with prolonged culture of rat spinal dorsal horn neurons. Taken together, our results suggest that the GlyRs are novel molecular targets of curcumol, which may underlie its pharmaceutical effects in the central nervous system.

Differences in Neural-Immune Gene Expression Response in Rat Spinal Dorsal Horn Correlates with Variations in Electroacupuncture Analgesia

BackgroundElectroacupuncture (EA) has been widely used to alleviate diverse pains. Accumulated clinical experiences and experimental observations indicated that significant differences exist in sensitivity to EA analgesia for individuals of patients and model animals. However, the molecular mechanism accounting for this difference remains obscure.Methodology/Principal FindingsWe classified model male rats into high-responder (HR; TFL changes >150) and non-responder (NR; TFL changes ≤0) groups based on changes of their pain threshold detected by tail-flick latency (TFL) before and after 2 Hz or 100 Hz EA treatment. Gene expression analysis of spinal dorsal horn (DH) revealed divergent expression in HR and NR after 2 Hz/100 Hz EA. The expression of the neurotransmitter system related genes was significantly highly regulated in the HR animals while the proinflammation cytokines related genes were up-regulated more significantly in NR than that in HR after 2 Hz and 100 Hz EA stimulation, especially in the case of 2 Hz stimulation.Conclusions/SignificanceOur results suggested that differential regulation and coordination of neural-immune related genes might play an important role for individual variations in analgesic effects responding to EA in DH. It also provided new candidate genes related to EA responsiveness for future investigation.

Effect of intrathecal sufentanil and protein kinase C inhibitor on pain threshold and the expression of NMDA receptor/ CGRP in spinal dorsal horn in rats with neuropathic pain

To investigate the effect of intrathecal sufentanil and protein kinase C inhibitor on pain threshold and the expression of N-methyl-D-aspartate receaptors (NMDAR)/calcitonin generelated peptide (CGRP) in spinal dorsal horn in rats with neuropathic pain. Fifty-four healthy male Sprague-Dawley rats were randomly divided into 6 groups (9 in each group). The rats in the sham group(Group S) + spared nerve injury (SNI), SP+SNI, and P+SNI were intrathecally injected sufentanil (1 μg), sufentanil (1 μg) and chelerythrine chloride (11 μg), chelerythrine chloride (11 μg) followed by 10 μL normal saline once every day for 14 days postoperatively, respectively. Similarly, rats in the control group (Group C), the sham group (Group S), and SNI model group (Group SNI) were intrathecally injected 20 μL normal saline in the uniform interval. Pain behaviours were measured on Day 1 pre-surgery and on Day 1, 2, 7, and 14 after the intrathecal injection. The expressions of NMDAR and CGRP in the spinal dorsal horn of L5 segment were determined by immunohistochemistry on Day 2, 7, and 14 after the intrathecal injection. Compared with Group C and Group S, mechanical allodynia threshold in group SNI was decreased after the surgery (P<0.01), and expressions of NMDAR and CGRP immunoreactive soma in the spinal dorsal horn was significantly increased (P<0.01). Mechanical stimulation pain threshold was elevated in Group S+SNI, Group P+SNI, and Group SP+SNI compared with Group SNI (P<0.01), while expressions of NMDAR and CGRP immunoreactive soma in Group S+SNI, Group P +SNI, and Group SP+SNI were significantly decreased (P<0.05 or 0.01). Intrathecal administration of sulfentanil and protein kinase C inhibitor can provide significant antinociception in rats with neuropathic pain and obviously inhibit the upregulation of NMDAR and CGRP expressions in the spinal dorsal horn of SNI rat models.

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND 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

Differential distribution of diacylglycerol lipase‐alpha and N‐acylphosphatidylethanolamine‐specific phospholipase d immunoreactivity in the superficial spinal dorsal horn of rats

It is generally accepted that the endocannabinoid system plays important roles in spinal pain processing. Although it is documented that cannabinoid-1 receptors are strongly expressed in the superficial spinal dorsal horn, the cellular distribution of enzymes that can synthesize endocannabinoid ligands is less well studied. Thus, using immunocytochemical methods at the light and electron microscopic levels, we investigated the distribution of diacylglycerol lipase-alpha (DGL-α) and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), enzymes synthesizing the endocannabinoid ligands, 2-arachidonoylglycerol (2-AG) and anandamide, respectively. Positive labeling was revealed only occasionally in axon terminals, but dendrites displayed strong immunoreactivity for both enzymes. However, the dendritic localization of DGL-α and NAPE-PLD showed a remarkably different distribution. DGL-α immunolabeling in dentrites was always revealed at membrane compartments in close vicinity to synapses. In contrast to this, dendritic NAPE-PLD labeling was never observed in association with synaptic contacts. In addition to dendrites, a substantial proportion of astrocytic (immunoreactive for GFAP) and microglial (immunoreactive for CD11b) profiles were also immunolabeled for both DGL-α and NAPE-PLD. Glial processes immunostained for DGL-α were frequently found near to synapses in which the postsynaptic dendrite was immunoreactive for DGL-α, whereas NAPE-PLD immunoreactivity on glial profiles at the vicinity of synapses was only occasionally observed. Our results suggest that both neurons and glial cells can synthesize and release 2-AG and anandamide in the superficial spinal dorsal horn. The 2-AG can primarily be released by postsynaptic dendrites and glial processes adjacent to synapses, whereas anandamide can predominantly be released from nonsynaptic dendritic and glial compartments.

PI3-kinase/Akt Pathway-Regulated Membrane Insertion of Acid-Sensing Ion Channel 1a Underlies BDNF-Induced Pain Hypersensitivity

Central neural plasticity plays a key role in pain hypersensitivity. This process is modulated by brain-derived neurotrophic factor (BDNF) and also involves the type 1a acid-sensing ion channel (ASIC1a). However, the interactions between the BDNF receptor, tropomyosin-related kinase B (TrkB), and ASIC1a are unclear. Here, we show that deletion of ASIC1 gene suppressed the sustained mechanical hyperalgesia induced by intrathecal BDNF application in mice. In both rat spinal dorsal horn neurons and heterologous cell cultures, the BDNF/TrkB pathway enhanced ASIC1a currents via phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt) cascade and phosphorylation of cytoplasmic residue Ser-25 of ASIC1a, resulting in enhanced forward trafficking and increased surface expression. Moreover, in both rats and mice, this enhanced ASIC1a activity was required for BDNF-mediated hypersensitivity of spinal dorsal horn nociceptive neurons and central mechanical hyperalgesia, a process that was abolished by intrathecal application of a peptide representing the N-terminal region of ASIC1a encompassing Ser-25. Thus, our results reveal a novel mechanism underlying central sensitization and pain hypersensitivity, and reinforce the critical role of ASIC1a channels in these processes.

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Effects of Glial Cell Line-Derived Neurotrophic Factor Intrathecal Injection on Spinal Dorsal Horn Glial Fibrillary Acidic Protein Expression in a Rat Model of Neuropathic Pain

ABSTRACTGlial fibrillary acidic protein (GFAP) is a specific astrocytic marker in the central nervous system. Few studies on the effects of glial cell line-derived neurotrophic factor (GDNF) intrathecal injection on GFAP expression exist in the literature. The present study determined GFAP expression in rat spinal dorsal horn following a spinal nerve ligation (SNL). The effects of GDNF intrathecal injection on GFAP expression were examined to gather experimental evidence on the mechanisms underlying neuropathic pain. Following L5–6 SNL, male Sprague-Dawley rats were randomly divided into four groups: normal control, sham-operated, SNL, and GDNF. Each group was further divided into three subgroups (n = 10) according to the times of sacrifice: 3, 7, and 14 days after surgery. Compared with the normal control and the sham-operated groups, GFAP expression in the SNL group increased at day 3 after surgery and lasted until 14 days after. GFAP expression was significantly less in the GDNF group compared with the SNL group which lasted until 14 days after surgery, suggesting that rat spinal dorsal horn GFAP expression contributes to SNL-induced neuropathic pain. The mechanisms underlying GDNF alleviation of neuropathic pain were shown to be related to the GDNF inhibition of GFAP expression in the spinal dorsal horn.