Is functional state of spinal microglia involved in the anti-allodynic and anti-hyperalgesic effects of electroacupuncture in rat model of monoarthritis?

Spinal microglia play a key role for creating exaggerated pain following tissues inflammation or injury. Electroacupuncture (EA) can effectively control the exaggerated pain both in humans with inflammatory disease and animals with experimental inflammatory pain. However, little is known about the relationship between spinal glial activation and EA analgesia. Using immunohistochemistry, RT-PCR analysis, and behavioral testing, the present study demonstrated that (1) Unilateral intra-articular injection of CFA produced a robust microglial activation and the up-regulation of the tumor necrosis factor (TNF)-alpha, interleukin (IL-1beta), and IL-6 mRNA levels in the spinal cord; (2) Repeated intrathecal (i.t.) injection of minocycline (100 microg), a microglial inhibitor, or EA stimulation of ipsilateral "Huantiao"(GB30) and "Yanglingquan" (GB34) acupoints significantly suppressed CFA-induced nociceptive behavioral hypersensitivity and spinal microglial activation; (3) Combination of EA with minocycline significantly enhanced the inhibitory effects of EA on allodynia and hyperalgesia. For the first time, these data provide direct evidence for the involvement of spinal microglial functional state in anti-nociception of EA. Thus, anti-neuroinflammatory effect of EA might be considered as one of the mechanisms of its anti-arthritic pain effects, and thereby a multidisciplinary integrated approach to treating symptoms related to arthritis might be raised.     

Cyclooxygenase inhibition in nerve-injury- and TNF-induced hyperalgesia in the rat

After nerve injury, cyclooxygenase-2 (COX-2) is upregulated in spinal cord and peripheral nerve, the latter being dependent on tumor necrosis factor-alpha (TNF). Here we asked whether COX inhibitors attenuate pain behavior induced by chronic constrictive sciatic nerve injury (CCI) or intraneural injection of TNF (2.5 pg/ml). Rats received either 0.9% saline, the nonselective COX inhibitor ibuprofen (40 mg/kg) or the selective COX-2 inhibitor celecoxib (10 or 30 mg/kg) twice daily by gavage started 2 days before, 12 h or 7 days after surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI was moderately, but consistently attenuated by early (day -2 or 12 h after CCI), but not late (7 days after CCI) ibuprofen and celecoxib treatment. Mechanical allodynia, but not thermal hyperalgesia induced by intraneural TNF, was reduced by ibuprofen, but not by celecoxib treatment 5 and 7 days after injection. Sciatic nerves, lumbar dorsal root ganglia (DRG) and spinal cords from rats with treatment started 12 h after surgery were analyzed for prostaglandin E2 (PGE2) levels 10 days after CCI. In injured nerves and ipsilateral DRG, PGE2 levels were increased. Ibuprofen treatment reversed PGE2 levels in injured nerves and DRG, whereas celecoxib blocked increased PGE2 levels only in nerves. In spinal cord, no change in PGE2 levels was observed. In contrast to the marked inhibition of nerve-injury-induced upregulation of PGE2 by COX inhibitors, the effect on pain behavior was modest. Nerve-injury- and TNF-induced pain-related behavior seem to be only partly dependent on peripheral prostaglandins.     

Effects of pertussis toxin on electroacupuncture-produced anti-hyperalgesia in inflamed rats

Our previous study showed that electroacupuncture (EA) significantly attenuated hyperalgesia in an animal model of persistent inflammatory pain. The present study was designed to show if Gi/o protein is involved in EA-produced anti-hyperalgesia. Spinal Gi/o-protein function was destroyed by intrathecal pretreatment with pertussis toxin (PTX). Seven days after the placement of an intrathecal PE-10 tube, PTX was injected into the intrathecal space of the lumbar spinal cord of rats. Seven days after PTX, complete Freund's adjuvant (CFA) was injected into the plantar surface of one hind paw of the rat to induce hyperalgesia in the injected paw. EA treatment was given at acupoint GB30 immediately post-CFA and then hyperalgesia was assessed by measuring the degree of decreased paw withdrawal latency (PWL) to a noxious thermal stimulus. The results showed that PTX pretreatment prevented EA-produced anti-hyperalgesia in the CFA inflammatory pain model but did not affect either baseline pain threshold or CFA-induced hyperalgesia. The data suggest that EA-produced anti-hyperalgesia is mediated by PTX-sensitive Gi/o proteins and the relevant signaling pathways.     

Severity-dependent expression of pro-inflammatory cytokines in traumatic spinal cord injury in the rat.

The post-traumatic inflammatory response in acute spinal cord contusion injury was studied in the rat. Mild and severe spinal cord injury (SCI) was produced by dropping a 10 g weight from 3 and 12 cm at the T12 vertebral level. Increased immunoreactivity of TNF-alpha in mild and severe SCI was detected in neurons at 1 h post-injury, and in neurons and microglia at 6 h post-injury, with a less significant increase in mild SCI. Expression was short-lived and declined sharply by 1 d post-injury. RT-PCR showed an early significant up-regulation of IL-1 beta, IL-6 and TNF-alpha mRNAs, maximal at 6 h post-injury with return to control levels by 24 h post-injury, the changes being less statistically significantly in mild SCI. Western blot showed early transient increases of IL-1 beta, IL-6 and TNF-alpha proteins in severe SCI but not mild SCI. Immunocytochemical, western blotting and RT-PCR analyses suggest that endogenous cells (neurons and microglia) in the spinal cord, not blood-borne leucocytes, contribute to IL-1 beta, IL-6 and TNF-alpha production in the post-traumatic inflammatory response and that their up-regulation is greater in severe than mild SCI.     

Spinal IL-36γ/IL-36R participates in the maintenance of chronic inflammatory pain through astroglial JNK pathway

Emerging evidence indicates that spinal neuroinflammation contributes to the maintenance of chronic inflammatory pain. IL-36, as a novel member of the interleukin (IL)-1 super-family cytokines, plays an important role in inflammatory responses. The present study aimed to investigate the role of spinal IL-36 and IL-36 receptor (IL-36R) signaling in the pathology of chronic inflammatory pain. IL-36γ and IL-36R, but not IL-36α and IL-36β, were persistently upregulated in the spinal cord of mice with intraplantar injections of complete Freund's adjuvant (CFA). Intrathecal administration of both IL-36R antagonist (IL-36Ra) and IL-36γ siRNA significantly attenuated CFA-induced chronic inflammatory pain behaviors. Furthermore, CFA-induced IL-36γ expression was mainly observed in spinal neurons whereas IL-36R was primarily expressed in spinal astrocytes. Additionally, the intrathecal injection of IL-36γ was sufficient to induce pain hypersensitivity and astrocyte activation in naive mice, and these effects could be inhibited by blocking c-Jun N-terminal kinase (JNK) phosphorylation. In vitro experiments also demonstrated that the IL-36γ could induce astrocytic JNK activation and inflammatory cytokines release, which was mediated by IL-36R. Finally, intrathecal injection of IL-36γ-activated astrocytes in a pJNK-dependent manner induced mechanical allodynia and thermal hyperalgesia in naive mice. Collectively, these findings reveal that the neuronal/astrocytic interaction in the spinal cord by which neuronally produced IL-36γ activates astrocytes via IL-36R-mediated JNK pathway is crucial for the maintenance of chronic inflammatory pain. Thus, IL-36γ/IL-36R-mediated astrocyte signaling may be a suitable therapeutic target for chronic inflammatory pain.     

Intrathecal interleukin-1beta administration induces thermal hyperalgesia by activating inducible nitric oxide synthase expression in the rat spinal cord

The effect of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) on the inducible nitric oxide synthase-nitric oxide (iNOS-NO) cascade in nociceptive signal transduction was examined in the intact rat spinal cord. All rats were implanted with an intrathecal (i.t.) catheter; some were also implanted with an i.t. microdialysis probe. The paw withdrawal latency to radiant heat was used to assess thermal hyperalgesia. The iNOS protein expression in the spinal cord dorsal horn was examined by western blot analysis and NOS activity assay. NO production in the CSF dialysate was also measured. IL-1beta i.t. (100 ng) produced thermal hyperalgesia from 4 to 24 h after i.t. injection. The iNOS protein expression was induced at 4 h after i.t. IL-1beta injection, peaked at the 6th hour, and disappeared at 24 h. The iNOS activity showed a similar time-dependent change as the iNOS protein expression. NO release increased by 1.1- to 1.9-fold between 4 and 12 h, also with a peak at the 6th hour, after i.t. IL-1beta administration. Pretreatment with the iNOS inhibitor 1400W (10 microg, i.t.) 1 h before i.t. IL-1beta injection prevented all the responses of IL-1beta. Neither 1400W nor artificial CSF (aCSF) affected the thermal nociceptive threshold and NO production. These results demonstrate that i.t. administration of IL-1beta induced thermal hyperalgesia by activating the iNOS-NO cascade in the rat spinal cord. On the basis of the present findings, we suggest that i.t. administration of iNOS inhibitors may have potential in the treatment of inflammatory and neuropathic pain syndromes.     

Glucocorticoid Receptor Contributes to Electroacupuncture-Induced Analgesia by Inhibiting Nav1.7 Expression in Rats With Inflammatory Pain Induced by Complete Freund's Adjuvant

BackgroundWhile electroacupuncture (EA) has been used traditionally for the treatment of chronic pain, its analgesic mechanisms have not been fully clarified. We observed in an earlier study that EA could reverse inflammatory pain and suppress high Nav1.7 expression. However, the molecular mechanism underlying Nav1.7 expression regulation is unclear. In this study, we studied the relationship between the glucocorticoid receptor (GR) and Nav1.7 and the role of these molecules in EA analgesia.Materials and MethodsIn this study, we established an inflammatory pain model by intraplantar injection of complete Freund's adjuvant (CFA) in rats. EA stimulation was applied to the ipsilateral “Huantiao” (GB30) and “Zusanli” (ST36) acupoints in the rat model. Western blotting, real-time polymerase chain reaction, immunostaining, intrathecal injection, and chromatin immunoprecipitation (ChIP) assay were performed to determine whether the sodium channel protein Nav1.7 plays a role in CFA-induced pain and whether GR regulates Nav1.7 expression during analgesia following EA stimulation.ResultsEA application significantly decreased the paw withdrawal threshold thresholds and thermal paw withdrawal latency and suppressed GR and Nav1.7 expression in the dorsal root ganglion. Moreover, treatment with a GR sense oligonucleotide (OND) markedly reversed these alterations. In contrast, treatment with a GR antisense OND along with EA application exerted a better analgesic effect, which was accompanied by the suppression of Nav1.7 and GR protein expression. The ChIP assay showed that the binding activity of GR to the Nav1.7 promoter was enhanced in CFA injected rats and suppressed in EA-treated rats.ConclusionsThe present study demonstrated that EA exerted anti-hyperalgesic effects by inhibiting GR expression, which led to Nav1.7 expression modulation in the rat model of CFA-induced inflammatory pain.     

A novel immune-to-CNS communication pathway: cells of the meninges surrounding the spinal cord CSF space produce proinflammatory cytokines in response to an inflammatory stimulus

Pain is enhanced in response to elevations of proinflammatory cytokines in spinal cerebrospinal fluid (CSF), following either intrathecal injection of these cytokines or intrathecal immune challenge with HIV-1 gp120 that induces cytokine release. Spinal cord glia have been assumed to be the source of endogenous proinflammatory cytokines that enhance pain. However, assuming that spinal cord glia are the sole source of CSF cytokines may be an underestimate, as the cellular composition of the meninges surrounding the spinal cord CSF space includes several cell types known to produce proinflammatory cytokines. The present experiments provide the first investigation of the immunocompetent nature of the spinal cord meninges. Here, we explore whether rat meninges are responsive to intrathecal gp120. These studies demonstrate that: (a) intrathecal gp120 upregulates meningeal gene expression of proinflammatory signals, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin 6 (IL-6), and inducible nitric oxide synthase (iNOS), and (b) intrathecal gp120 induces meningeal release of TNF-alpha, IL-1beta, and IL-6. In addition, stimulation of isolated meninges in vitro with gp120 induced the release of TNF-alpha and IL-1beta, indicating that the resident cells of the meninges are able to respond without immune cell recruitment. Taken together, these data document that the meninges are responsive to immunogenic stimuli in the CSF and that the meninges may be a source of immune products detected in CSF. The ability of the meninges to release to proinflammatory signals suggests a potential role in the modulation of pain.     

Role of prostaglandin receptor subtype EP1 in prostaglandin E2-induced nociceptive transmission in the rat spinal dorsal horn

It has been indicated that prostaglandin E2 (PGE2) and the receptor for PGE2 (EP receptor) are key factors contributing to the facilitated generation of nociception. This study was designed to investigate the roles of PGE2 and EP1 receptors in the spinal cord in the nociceptive transmission, using behavioral and intracellular calcium ion concentration ([Ca2+]i) assays and in situ hybridization. Experiments were conducted on Sprague-Dawley rats. In behavioral assays, withdrawal thresholds to mechanical stimuli were evaluated using von Frey filament. The effect of an intrathecally administered selective EP1 antagonist, 6-[(2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo[2.2.2]octan-2-yl]-5Z-hexenoic acid (ONO-8711), on the intrathecal PGE2-induced hyperalgesia was examined. In [Ca2+]i assays, we measured [Ca2+]i in the dorsal horn of spinal cord slices and examined the effects of PGE2 and ONO-8711 perfusion on the [Ca2+]i changes. In situ hybridization using EP1 digoxigenin probe was performed on the slice sections of the lumbar spinal cord and bilateral L4 and L5 dorsal root ganglions (DRGs). Mechanical hyperalgesia was observed after intrathecal PGE2 administration. Intrathecal administration of ONO-8711 attenuated the PGE2-induced mechanical hyperalgesia in a dose- and time-dependent manner. Perfusion of ONO-8711 markedly suppressed PGE2-induced [Ca2+]i increment in laminae II-VI in dorsal horn of the spinal cord slice. Moreover, in situ hybridization revealed EP1 hybridization signals in the DRG neurons, but not in the spinal cord. The results of this study suggested that spinal PGE2 activates the EP1 receptors existing on the central terminals of primary afferents, subsequently increasing in [Ca2+]i in the spinal dorsal horn, which are involved in the mechanisms of spinal PGE2-induced nociceptive transmission.     

Involvement of spinal neurokinins, excitatory amino acids, proinflammatory cytokines, nitric oxide and prostanoids in pain facilitation induced by Phoneutria nigriventer spider venom

The major local symptom of Phoneutria nigriventer envenomation is an intense pain, which can be controlled by infiltration with local anesthetics or by systemic treatment with opioid analgesics. Previous work showed that intraplantar (i.pl) injection of Phoneutria nigriventer venom in rats induces hyperalgesia, mediated peripherally by tachykinin and glutamate receptors. The present study examined the spinal mechanisms involved in pain-enhancing effect of this venom. Intraplantar injection of venom into rat hind paw induced hyperalgesia. This phenomenon was inhibited by intrathecal (i.t.) injection of tachykinin NK1 (GR 82334) or NK2 (GR 94800) receptor antagonists, a calcitonin gene-related peptide (CGRP) receptor antagonist (CGRP8-37) and N-methyl-D-aspartate (NMDA; MK 801 and AP-5), non-NMDA ionotropic (CNQX), or metabotropic (AIDA and MPEP) glutamate receptor antagonists, suggesting the involvement of spinal neurokinins and excitatory amino acids. The role of proinflammatory cytokines, nitric oxide (NO), and prostanoids in spinally mediated pain facilitation was also investigated. Pharmacological blockade of tumour necrosis factor-alpha (TNFalpha) or interleukin-1beta (IL-1beta) reduced the hyperalgesic response to venom. Intrathecal injection of L-N6-(1-iminoethyl)lysine (L-NIL), but not of 7-nitroindazole (7-NI), inhibited hyperalgesia induced by the venom, indicating that NO, generated by the activity of the inducible form of nitric oxide synthase, also mediates this phenomenon. Furthermore, indomethacin, an inhibitor of cyclooxigenases (COX), or celecoxib, a selective inhibitor of COX-2, abolished venom-induced hyperalgesia, suggesting the involvement of spinal prostanoids in this effect. These data indicate that the spinal mechanisms of pain facilitation induced by Phoneutria nigriventer venom involves a plethora of mediators that may cooperate in the genesis of venom-induced central sensitization.     

Dexmedetomidine prevents remifentanil-induced postoperative hyperalgesia and decreases spinal tyrosine phosphorylation of N-methyl-d-aspartate receptor 2B subunit

Numerous studies have demonstrated that prolonged opioid exposure can enhance pain sensitivity that presents as opioid-induced hyperalgesia (OIH). Activation of spinal α2-adrenergic receptor may play an important role in the development of OIH. Dexmedetomidine is an α2-adrenergic agonist that has been shown to synergize with opioids. The aim of this study was to investigate the antihyperalgesia effects of dexmedetomidine on remifentanil-induced postinfusion hyperalgesia in a rat model of incision pain. We also evaluated whether the antihyperalgesic effects of dexmedetomidine were associated with suppression of NMDAR excitability, as measured by a reduction in spinal cord NR2B phosphorylation. Dexmedetomidine (12.5 μg/kg, 25 μg/kg, 50 μg/kg) was administered subcutaneously 30 min before plantar incision. Pretreatment with dexmedetomidine significantly decreased remifentanil-induced hyperalgesia, as indicated by increased paw withdrawal latencies and thresholds to thermal and mechanical stimulation respectively. Correlated with the pain behavior changes, Western blotting experiments also revealed that dexmedetomidine could decrease NR2B subunit phosphorylation (Tyr1472 site) in the dorsal horn, which was upregulated after remifentanil infusion. These results suggest that dexmedetomidine can efficiently alleviate OIH and it may be an effective novel option for the treatment of OIH. Our data also provide evidence that dexmedetomidine's anti-hyperalgesic effect may depend on its ability to modulate spinal cord NMDAR activation via suppression of NR2B phosphorylation.     

Effects of aging on hyperalgesia and spinal dynorphin expression in rats with peripheral inflammation

The aging process is associated with various morphological and biochemical changes in the nervous system that may affect the processing of noxious inputs. This study showed greater hyperalgesia and up-regulation of spinal dynorphin (DYN) expression in aging than in young adult rats during CFA-induced peripheral inflammation. These data indicate that nociception is regulated differently in aging individuals, a fact that should be considered when selecting treatment strategies for aging populations with persistent pain.     

Effects of pre-emptively administered nociceptin on the development of thermal hyperalgesia induced by two models of experimental mononeuropathy in the rat

Pre-emptive analgesia is thought to be produced by the prevention of spinal facilitation evoked by nociceptive input to the spinal cord. Opioid receptor-like 1 (ORL1) receptor agonist has been reported to inhibit the development of spinal facilitation. We investigated the effect of nociceptin, an ORL1 receptor agonist, on the development of thermal hyperalgesia and the expression of Fos-like immunoreactivity (Fos-LI) in the spinal dorsal horn induced by two neuropathic pain models, the chronic constriction injury model and the partial sciatic nerve injury model. Chronic constriction injury is created by placing four loosely tied ligatures around the right sciatic nerve. Partial sciatic nerve injury was created by tight ligation of one third to one half of the right sciatic nerve. All drugs were injected intrathecally 10 min before the nerve injury. The anti-hyperalgesic effect of drugs was evaluated by the measurement of the paw withdrawal latency (PWL) against thermal nociceptive stimulation. The PWLs of the injured paws were measured 7, 14 and 21 days after the nerve injury. Expression of Fos-LI was examined 2 h after the nerve injury. Intrathecal injection of nociceptin significantly delayed the development of thermal hyperalgesia and decreased the expression of Fos-LI induced by chronic constriction injury, but not that induced by partial sciatic nerve injury. These data indicate that pre-emptive administration of nociceptin might be one strategy for the prevention of the development of neuropathic pain.     

Combination of fasudil and celecoxib promotes the recovery of injured spinal cord in rats better than celecoxib or fasudil alone

Resistance mechanisms of rho-associated kinase (ROCK) inhibitors are associated with the enhanced expression of cyclooxygenase-2 (COX-2). The therapeutic effects of ROCK on nervous system diseases might be enhanced by COX-2 inhibitors. This study investigated the synergistic effect of the combined use of the ROCK inhibitor fasudil and a COX-2 inhibitor celecoxib on spinal cord injury in a rat model established by transecting the right half of the spinal cord at T₁₁. Rat models were orally administrated with celecoxib (20 mg/kg) and/or intramuscularly with fasudil (10 mg/kg) for 2 weeks. Results demonstrated that the combined use of celecoxib and fasudil significantly decreased COX-2 and Rho kinase II expression surrounding the lesion site in rats with spinal cord injury, improved the pathomorphology of the injured spinal cord, and promoted the recovery of motor function. Moreover, the effects of the drug combination were better than celecoxib or fasudil alone. This study demonstrated that the combined use of fasudil and celecoxib synergistically enhanced the functional recovery of injured spinal cord in rats.     

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

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

A parametric study of electroacupuncture on persistent hyperalgesia and Fos protein expression in rats

We previously reported the anti-hyperalgesia of electroacupuncture (EA) on persistent inflammatory pain in an unrestrained, unsedated, and conscious rat model. Using this model, induced by injecting complete Freund's adjuvant (CFA) into one hind paw, we systematically evaluated the anti-hyperalgesia of EA stimulation parameters (frequency, intensity, treatment duration, and pulse width). We assessed hyperalgesia by paw withdrawal latency (PWL) to a noxious thermal stimulus and found that 10- and 100-Hz EA frequencies at a current intensity of 3 mA produced the greatest anti-hyperalgesia, when compared to other parameters. Both frequencies significantly increased PWL in the early phases of hyperalgesia (2.5 and 24 h; p < 0.05), and 10 Hz EA also significantly increased PWL in later phases (5 to 7 days; p < 0.05). A sufficient but tolerable intensity of 3 mA was more effective than lower intensities (1-2 mA). A 20-min treatment produced better anti-hyperalgesia than longer and shorter (10 and 30 min) treatments. Acupoint specificity study demonstrated that GB30 produced significant EA anti-hyperalgesia, while Waiguan (TE5) and sham points, an abdominal point and a point at the opposite aspect of GB30, did not. The spinal Fos protein expression study demonstrated that the optimal EA selectively suppressed Fos expression in superficial laminae (I/II) and activated it in deeper laminae (III/IV) of the spinal dorsal horn. The results suggest that the EA anti-hyperalgesia is parameter-dependent and point-specific, and they provide important information for designing further clinical acupuncture research on persistent inflammatory pain.     

Spinal orexin-1 receptors mediate anti-hyperalgesic effects of intrathecally-administered orexins in diabetic neuropathic pain model rats

Orexin-A and orexin-B are endogenous ligands of orexin receptors that contain orexin-1 and orexin-2. Activation of the orexinergic system can produce antinociceptive effects in acute inflammatory, mono-neuropathic, and postoperative pain animal models, though the effects of orexins on diabetic neuropathic pain have not been previously investigated. In this study, we studied the anti-hyperalgesic effects of intrathecally administered orexins in a streptozotocin-induced diabetic rat. First, dose-dependent effects were investigated by measuring hind paw withdrawal thresholds in response to noxious-heat and punctate stimuli, after which orexin levels in the cerebrospinal fluid of diabetic rats were measured and compared with those of normal rats using a radioimmunoassay method. The functional role of spinal orexin-1 receptors with the anti-hyperalgesic effects of orexins was also investigated using intrathecal pretreatment with SB-334867, a selective orexin-1 receptor antagonist. Intrathecally administered orexins produced an antinociceptive effect in diabetic rats, however, not in normal rats, though the orexin levels in the cerebrospinal fluid of diabetic rats were similar to those in normal rats. In addition, the anti-hyperalgesic effects of orexins were significantly inhibited by pretreatment with SB-334867. These findings demonstrate that the anti-hyperalgesic effects of orexins in diabetic rats are unlikely due to any direct effect by the supplement on decreased endogenous orexins in the cerebrospinal fluid and that orexin-1 receptors in the spinal cord may be involved in the modulation of nociceptive transmission in diabetic neuropathy. We conclude that the spinal orexinergic system may be a possible target for elucidating the mechanisms of diabetes-induced hyperalgesia.