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.     

Involvement of opioid receptors in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation

Our previous study showed that electroacupuncture (EA) significantly attenuated inflammatory hyperalgesia. It has also been reported that EA analgesia in uninjured animals is mediated by mu and delta opioid receptors at 2-15 Hz and by kappa opioid receptor at 100 Hz. Because persistent pain changes neural response to external stimulation, we hypothesized that (1) the mechanisms of EA anti-hyperalgesia may be different under conditions of persistent pain and that (2) combining EA with a sub-effective dose of morphine could enhance EA anti-hyperalgesia. Hyperalgesia, decreased paw withdrawal latency (PWL) to a noxious thermal stimulus, was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into the hind paws of rats. Selective antagonists against mu (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2, CTOP), delta (naltrinodole, NTI) and kappa (nor-binaltorphimine, BNI) opioid receptors were administered intrathecally 10 min before each of two EA treatments at acupoint Huantiao (GB30), one immediately post and the other 2 h post-CFA. Morphine was given (i.p.) 40 min before the second EA treatment. PWL was measured before and 2.5 and 5 h post-CFA. Both 10 and 100 Hz EA-produced anti-hyperalgesia were blocked spinally by mu- and delta- but not kappa-receptor antagonists. EA combined with a sub-threshold dose of morphine (2.5 mg/kg) enhanced anti-hyperalgesia additively (10 Hz EA) or synergistically (100 Hz EA) compared to that produced by each component alone. These results suggest selective involvement of mu and delta, but not kappa, receptors in EA-produced anti-hyperalgesia in rats. A combined EA and opioid drug protocol may provide an improved treatment strategy for inflammatory pain.     

Involvement of nociceptin/orphanin FQ and its receptor in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (NOP receptor), has been demonstrated to be involved in many physiological and pathological functions including pain regulation. In the present study, the involvement of N/OFQ-NOP receptor system in electroacupuncture (EA)-produced anti-hyperalgesia was investigated in rats with peripheral inflammation. Intrathecal (i.t.) administration of N/OFQ (15 nmol) or EA at acupoints GB30 and GB34 could significantly attenuate hyperalgesia which was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into one hindpaw of rats, manifesting as decreased paw withdrawal latency (PWL) to the noxious thermal stimulus. The anti-nociceptive effect of N/OFQ or EA was significantly blocked by intrathecal injection of [Nphe(1)]nociceptin(1-13)NH(2) (20 nmol), a selective antagonist of the NOP receptor, indicating the NOP-receptor-mediated mechanism. Additionally, the combination of N/OFQ injection with EA treatment could enhance anti-hyperalgesia compared to that produced by each component alone. These findings suggested that the spinal N/OFQ-NOP system might be involved in EA analgesia, which may be one of the mechanisms underlying the anti-nociceptive effect of EA in rat's peripheral inflammatory pain.     

Kynurenic acid enhances electroacupuncture analgesia in normal and carrageenan-injected rats

The interaction between electroacupuncture (EA) and an intrathecally administered wide-spectrum excitatory amino acid (EAA) receptor(s) antagonist, kynurenic acid (KYNA) on carrageenan-induced thermal hyperalgesia and spinal Fos expression was investigated. Intrathecal (i.t.) injection of 0.1, 1, 10, and 100 nmol KYNA markedly and dose-dependently increased the latency of paw withdrawal (PWL) of the carrageenan-injected paw. While the PWLs of the non-injected and normal saline (NS)-injected paws were not obviously affected by application of KYNA at the doses tested. Intrathecal injection of 0.1 nmol KYNA significantly potentiated the anti-nociception induced by EA stimulation of contralateral 'Zu-San-Li' and 'Kun-Lun' acupoints either in the carrageenan- or NS-injected rats. Three hours after intraplantar (i.pl.) injection of carrageenan, the number of Fos-like immunoreactive (Fos-LI) neurons was significantly increased in all layers of ipsilateral spinal cord at L(4)-L(5) with the higher density in laminae I-II and V-VI. Intrathecally pre-administered KYNA (10 nmol) significantly reduced the total number of carrageenan-induced Fos-LI neurons with more apparent reduction in laminae I-II and IV-V. Pre-coapplication of 10 nmol KYNA and EA of bilateral 'Zu-San-Li' and 'Kun-Lun' acupoints, the numbers of carrageenan-induced Fos-LI neurons in laminae I-II and V-VI further reduced. The level of Fos expression in the spinal cord induced by carrageenan was significantly lower compared with that of i.t. injection of KYNA or EA alone. These results demonstrated that EAA receptor(s) antagonist could enhance EA-induced anti-nociception and anti-hyperalgesia.     

Additive anti-hyperalgesia of electroacupuncture and intrathecal antisense oligodeoxynucleotide to interleukin-1 receptor type I on carrageenan-induced inflammatory pain in rats

Accumulating evidence shows that spinal interleukin-1β (IL-1β) plays a critical role in inflammatory pain. Electroacupuncture (EA) can effectively attenuate inflammatory hyperalgesia both in clinical practices and experimental studies. However, little is known about the relationship between spinal IL-1β and EA analgesia. The present study was designed to evaluate the effects of EA and antisense oligodeoxynucleotide (ODN) to IL-1 receptor type I (IL-1RI) on carrageenan-induced thermal hyperalgesia and the expression of IL-1β as well as IL-1RI. It was demonstrated that carrageenan induced marked thermal hyperalgesia in the injected paw, hence making paw withdrawal latency (PWL) decrease to 3.47 ± 0.31 s at 180 min post-injection. Nevertheless, when EA was administered for 30 min at 180 min post-carrageenan injection, the PWLs were significantly increased between 10 and 90 min following the beginning of EA treatment and peaked at 30 min to 5.91 ± 0.61 s. And also EA partly reversed the elevation of IL-1β and IL-1RI expression induced by carrageenan. Down-regulation of IL-1RI expression by repeated intrathecal antisense ODN (50 μg/10 μl) significantly increased the mean PWL up to 5.75 ± 0.15 s in 180–300 min post-carrageenan injection. Additionally, when the combination of EA with antisense ODN was used, thermal hyperalgesia was further alleviated than EA or antisense ODN alone, with a maximum PWL of 7.66 ± 0.50 s at 30 min post the beginning of EA treatment. The results suggested an involvement of the spinal IL-1β/IL-1RI system in EA-induced anti-hyperalgesia in inflammatory pain.     

Disruption of glial function enhances electroacupuncture analgesia in arthritic rats

Activated glia play a major role in mediating behavioral hypersensitive state following peripheral inflammation. Electroacupuncture is well known to relieve persistent inflammatory pain. The present study was undertaken to examine whether fluorocitrate, a glial metabolic inhibitor, could synergize electroacupuncture antagonizing thermal hyperalgesia and mechanical allodynia evoked by ankle joint inflammation. Monoarthritis of rat ankle joint was induced by an intra-articular injection of Complete Freund's Adjuvant (CFA). The paw withdrawal latency (PWL) from a thermal stimulus and paw withdrawal threshold (PWT) from von Frey hairs were measured in awake rats. Intrathecal (i.t.) injection of 1 nmol fluorocitrate markedly suppressed monoarthritis-induced thermal hyperalgesia and mechanical allodynia. Unilateral electroacupuncture stimulation of "Huantiao" (GB30) and "Yanglingquan" (GB34) acupuncture points (100/2 Hz alternation, 1-2-3 mA) significantly elevated the PWLs and PWTs for 45 min after cessation of electroacupuncture in monoarthritic rats. Co-application of 0.1 or 1 nmol fluorocitrate with electroacupuncture significantly potentiated electroacupuncture analgesia, although 0.1 nmol fluorocitrate alone had no effect on PWLs and PWTs in monoarthritic rats. These results suggested that electroacupuncture and disrupting glial function could synergistically antagonize inflammatory pain, which might provide a potential strategy for the treatment of arthritic pain.     

Mu opioid receptor-containing neurons mediate electroacupuncture-produced anti-hyperalgesia in rats with hind paw inflammation

Previous studies showed that electroacupuncture (EA) significantly attenuates inflammatory hyperalgesia in a complete Freund's adjuvant (CFA)-induced inflammatory pain rat model. The present study demonstrates that pretreatment with Derm-sap, a selective toxin for neurons that contain mu opioid receptor (MOR), specifically decreases MOR and blocks EA anti-hyperalgesia. These data suggest that spinal MOR-containing neurons are involved in the processes by which EA produces anti-hyperalgesia.     

Effect of post-injury NMDA antagonist treatment on long-term Fos expression and hyperalgesia in a model of chronic neuropathic pain

Chronic constriction injury (CCI) of the sciatic nerve results in persistent mechanical hyperalgesia together with Fos protein expression in the lumbar spinal cord. We have examined the relationship between mechanical hyperalgesia and Fos expression within the lumbar spinal cord on days 14, 35 and 55 after either CCI or sham operation. To determine the role of NMDA receptor mechanisms in the maintenance of hyperalgesia and Fos expression, the NMDA antagonist MK-801 (0.3 mg kg-1 s.c.) was administered daily on days 28 to 34 after operation. CCI animals developed unilateral hind limb hyperalgesia that persisted unchanged from days 14 to 55 of the study. MK-801 treatment reduced hyperalgesia by 57% (p=0.02) on day 35 in CCI animals but did influence hyperalgesia at day 55. In the spinal cord, Fos positive cells were present bilaterally throughout laminae 3-10 at all time points examined in both CCI and sham group animals. Fos counts ipsilateral to the side of injury in laminae 3-10 correlated significantly with hyperalgesia scores in the CCI but not sham animals. MK-801 treatment resulted in a suppression of Fos expression in ipsilateral laminae 3-4 (p=0.0017) and laminae 5-10 (p=0.0026) of CCI animals on day 35. Fos expression in sham group animals was not inhibited by MK-801 treatment at day 35. These results indicate that Fos expression is maintained by differing mechanisms following nerve injury or sham operation. The functional consequences of Fos expression following nerve injury and sham operation are discussed.     

Territorial and extra-territorial distribution of Fos protein in the lumbar spinal dorsal horn neurons in rats with chronic constriction nerve injuries

This study aimed to examine the relationship between temporal and spatial expression patterns of Fos protein in the spinal dorsal horn neurons and thermal hyperalgesia behaviors in rats with chronic constriction injury (CCI) to the sciatic nerve. Our results demonstrated that Fos protein expression in the spinal dorsal horn neurons at L5 segment ipsilateral and contralateral to CCI of the sciatic nerve was significantly greater than in sham rats from days 10 to 30 postoperatively (PO 10d to 30d), and was concentrated on the injury (ipsilateral) side. Unlike the short-lived expression after tissue inflammation, laminae I to VI (especially laminae III/IV) displayed a persistent greater number of Fos-like immunoreactive (Fos-LI) neurons for at least 30 days after CCI of the sciatic nerve. After the increase in laminae III/IV, Fos-LI neurons tended to gradually increase in laminae I/II and V/VI at L5 segment from PO 2d to 30d, which were correlated with the heat hyperalgesia (48 degrees C) behaviors measured by paw withdrawal latency in CCI rats but not in sham rats. Interestingly, a persistent increase of Fos-LI neurons in laminae I to VI at L5 segment of the ipsilateral and contralateral sides and at the L1 segment that was out of the normal central terminations of the sciatic nerve suggested the probable presence of territorial and extra-territorial central sensitization or inadequate central nervous system (CNS) adaptive mechanisms. These findings may partly explain why abnormal pain sensations are sometimes distributed in a pattern that does not coincide with the territories of nerves or with the posterior roots of the peripheral nerve after injury.     

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.     

Effect of pre-emptive NMDA antagonist treatment on long-term Fos expression and hyperalgesia in a model of chronic neuropathic pain

The unilateral sciatic nerve chronic constriction injury (CCI) model of Bennett and Xie [G.J. Bennett, Y.-K. Xie, A peripheral neuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87-108] shows features of a neuropathic pain state. We examined mechanical hyperalgesia and Fos protein staining in the lumbar spinal cord 1, 7, 14 and 28 days after unilateral CCI to the sciatic nerve or sham operation. In addition, we examined the effect of the NMDA antagonist MK-801 (0.3 mg/kg s.c. administered 30 min prior to and 6 h following operation) on Fos expression and hyperalgesia at 28 days. CCI animals were hyperalgesic compared to the sham operated animals at 14 and 28 days post injury. MK-801 reduced hyperalgesia by 68% in CCI animals on day 28 (p=0.0001). In the spinal cord, Fos positive cells were present bilaterally in deeper laminae in both sham and CCI animals at all time points examined. Relatively few Fos positive cells were present in laminae 1-2 at any time point examined. At days 1 and 7, there were increased numbers of Fos positive cells ipsilaterally in the deeper laminae of the spinal cord in CCI animals compared to sham animals, but by 14 and 28 days Fos counts were similar in sham and CCI despite the obvious behavioural differences between the two groups. Fos counts ipsilateral to the injury in laminae 3-10 correlated with hyperalgesia scores in the CCI but not sham animals. Analysis at the 28-day time point showed that MK-801 differentially affected Fos expression: MK-801 significantly reduced the Fos count bilaterally in laminae 3-10 in the CCI but not in the sham group animals. These results indicate that Fos expression is initiated by different peripheral and central mechanisms following nerve injury or sham operation.     

Synergistic anti-hyperalgesia of electroacupuncture and low dose of celecoxib in monoarthritic rats: involvement of the cyclooxygenase activity in the spinal cord

Electroacupuncture (EA) can effectively control the exaggerated pain in humans with inflammatory disease and animals with experimental inflammatory pain. However, there have been few investigations on the effect of co-administration of EA and analgesics and the underlying synergistic mechanism. Using behavioral test, RT-PCR analysis, enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA), the present study demonstrated that (1) Unilateral intra-articular injection of complete Freund's adjuvant (CFA) produced a constant hyperalgesia and an up-regulation of the prostaglandin E(2) (PGE(2)) level as well as the tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 levels in the spinal cord; (2) Celecoxib, a selective inhibitor of cyclooxygenase-2 (COX-2), at a dose of 2, 10, and 20 mg/kg (twice daily, p.o.), presented a dose-dependent anti-hyperalgesic effect; (3) Repeated EA stimulation of ipsilateral 'Huan-Tiao' (GB30) and 'Yang-Ling-Quan' (GB34) acupoints significantly suppressed CFA-induced hyperalgesia, and markedly inhibited the CFA-induced increase of the level of PGE(2) as well as IL-1beta, IL-6, and TNF-alpha in the spinal cord; (4) EA combined with low dose of celecoxib (2 mg/kg, twice daily, p.o.) greatly enhanced the anti-hyperalgesic effects of EA, with a synergistic reversing effect on CFA-induced up-regulation of spinal PGE(2), but not on the IL-1beta, IL-6, or TNF-alpha. These data indicated that repeated EA combined with low dose of celecoxib produced synergistic anti-hyperalgesic effect in the CFA-induced monoarthritic rats, which could be made possible by regulating the activity of spinal COX, hence the spinal PGE(2) level. Thus, this combination may provide an effective strategy for pain management.     

Attenuation of mechanical but not thermal hyperalgesia by electroacupuncture with the involvement of opioids in rat model of chronic inflammatory pain

Opioid peptides have been proven effective in reducing the sign of hyperalgesia associated with inflammation. Electroacupuncture (EA) produces antinociception via release of endogenous opioid peptides in normal rats. Moreover, intrathecal injection of dynorphin has antinociceptive effect in rats. The present study was designed to examine whether EA has effect on the thermal and mechanical hyperalgesia in rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain. The results are the following: (1) single session of 100Hz EA (0.5-1.0-1.5 mA, 10 min for each intensity) at both Zusanli (ST 36) and Sanyinjiao acupoints (SP 6) significantly increased mechanical withdrawal threshold determined by von Frey filaments but not with thermal withdrawal latency that is determined by hot plate (52 +/- 0.2 degrees C); (2) 100 Hz EA applied twice a week for 4 weeks and showed a significant decrease in the mechanical hyperalgesia at the third and fourth week, with no effect on thermal hyperalgesia; (3) naloxone (20 mg kg(-1)) had the ability to reverse the inhibition of the mechanical hyperalgesia produced by a single session of EA. In conclusion, the present results indicate that a single or repetitive EA could reduce mechanical hyperalgesia, but not thermal hyperalgesia, in CFA-inflammatory pain rats, and the opioid system might be involved in these effects.     

Non-opioid-dependent anti-inflammatory effects of low frequency electroacupuncture

Low frequency electroacupuncture, which is commonly used in pain relief, is known to induce opioid-mediated analgesia. This study examined the contribution of the opioid system in mediating the anti-inflammatory effects of low frequency EA in a standard model of acute inflammation, the carrageenan-induced edema model. Carrageenan was injected in the hind paw of anesthetized rats and low frequency electroacupuncture was applied to acupoints equivalent to Zusanli (St 36) and Sanyinjiao (Sp 6) in humans just prior to the induction of inflammation in the ipsilateral leg. Induction of Fos protein, reflecting neuronal activation, was investigated in the spinal cord with immunohistochemistry. It was found that electroacupuncture strongly inhibited the carrageenan-induced edema by over 60%, and suppressed the associated Fos expression in the superficial laminae (I-II) of the ipsilateral dorsal horn by 50%. Neither the anti-edematous effect nor the suppression of Fos expression in the superficial spinal laminae was affected by intraperitoneal injection of the opioid antagonist naloxone. These results demonstrate that low frequency electroacupuncture is capable of inhibiting peripheral inflammation and the associated central neuronal activity via a non-opioid-dependent mechanism.     

Evidence for suppression of electroacupuncture on spinal glial activation and behavioral hypersensitivity in a rat model of monoarthritis

Our previous study demonstrated that single intrathecal (i.t.) application of fluorocitrate, a glial metabolic inhibitor, synergized electroacupuncture (EA) antagonizing behavioral hypersensitivity in complete Freund's adjuvant (CFA)-induced monoarthritic rat. To further investigate the relationship between spinal glial activation and EA analgesia, the present study examined the effects of multiple EA on spinal glial activation evoked by monoarthritis (MA). The results showed that (1) unilateral intra-articular injection of CFA produced a robust glial activation on the spinal cord, which was associated with the development and maintenance of behavioral hypersensitivity; (2) multiple EA stimulation of ipsilateral "Huantiao" (GB30) and "Yanglingquan" (GB34) acupoints or i.t. injection of fluorocitrate (1 nmol) significantly suppressed spinal glial activation; (3) inhibitory effects of EA on spinal glial activation and behavioral hypersensitivity were significantly enhanced when EA combined with fluorocitrate, indicating that disruption of glial function may potentiate EA analgesia in inflammatory pain states. These data suggested that analgesic effects of EA might be associated with its counter-regulation to spinal glial activation, and thereby provide a potential strategy for the treatment of arthritis.     

Electroacupuncture (EA) modulates the expression of NMDA receptors in primary sensory neurons in relation to hyperalgesia in rats

N-methyl-D-aspartate (NMDA) receptor on the central terminals of the dorsal root ganglion (DRG) appears to be playing an important role in the development of central sensitization related to persistent inflammatory pain. Acupuncture analgesia has been confirmed by numerous clinical observations and experimental studies to be a useful treatment to release different kinds of pains, including inflammatory pain and hyperalgesia. However, the underlying mechanisms of the analgesic effect of acupuncture are not fully understood. In the present study, using a rat model of inflammatory pain induced by complete Freund's adjuvant (CFA), we observed the effect of electroacupuncture (EA) on animal behavior with regard to pain and the expression of a subunit of NMDA receptor (NR1) and isolectin B4 (IB4) in the neurons of the lumbar DRG. Intraplantar injection of 50 microl CFA resulted in considerable changes in thermal hyperalgesia, edema of the hind paw and "foot-bend" score, beginning 5 h post-injection and persisting for a few days, after which a gradual recovery occurred. The changes were attenuated by EA treatment received on the ipsilateral "Huan Tiao" and "Yang Ling Quan" once a day from the first day post-injection of CFA. Using an immunofluorescence double staining, we found that the number of double-labeled cells to the total number of the IB4 and NR1-labeled neurons increased significantly on days 3 and 7 after CFA injection. The change was attenuated by EA treatment. These results suggest that EA affects the progress of experimental inflammatory pain by modulating the expression of NMDA receptors in primary sensory neurons, in particular, IB4-positive small neurons.     

Interleukin-1ra inhibits Fos expression and hyperalgesia in rats

It is known that interleukin-1beta facilitates pain, but the mechanisms of this are not understood. This study investigated the role of interleukin-1beta in the expression of Fos, a marker of neuronal activation, and hyperalgesia caused by injecting complete Freund's adjuvant into one hind paw of the rat. Interleukin-receptor antagonist (interleukin-1ra, 0.005 mg/rat) was given intrathecally twice, 24 h before complete Freund's adjuvant and immediately before complete Freund's adjuvant injection, to block interleukin-1beta action. Fos expression was measured 2 h after complete Freund's adjuvant injection. Paw withdrawal latency was used to assess hyperalgesia. The findings were that interleukin-1ra inhibited inflammation-induced Fos expression and hyperalgesia, which suggests that endogenous interleukin-1beta facilitates transmission of noxious messages at the spinal level by processes involving an enhanced Fos expression.     

Behavioural evidence for a bidirectional effect of systemic naloxone in a model of experimental neuropathy in the rat

In animal models of inflammatory pain, we have demonstrated that the opioid antagonist naloxone induces a paradoxical analgesic effect at very low systemic doses, and a hyperalgesic effect at high doses. We have therefore proposed, that opioid systems are modified in these animals with persistent pain. The aim of the present study was to investigate the activity of naloxone through another model of pain in the rat due to a peripheral neuropathy of the sciatic nerve. The neuropathy was created by 4 ligatures around the sciatic nerve. We analyzed the effects of i.v. naloxone (3 and 10 micrograms/kg, 1 mg/kg) on the vocalization thresholds to paw pressure 8 days after the sciatic ligation. Three and 10 micrograms/kg naloxone produced a significant paradoxical antinociceptive effect on responses from the affected paw (with a mean increase of about 50 and 30% of the preinjection values, respectively) and also from the non-affected paw, although the effect was less potent. By contrast, 1 mg/kg naloxone elicited a significant hyperalgesia on responses from the affected and non-affected paw. The effects of the microdoses, but not those of the high dose, were clearly related to the vocalization thresholds measured for each rat just before injection. This study clearly shows that naloxone induces bidirectional effects in a rat model of neuropathic pain, which contradicts the current statement that neuropathic pain is opioid-resistant. The present results also suggest that these effects are not related to inflammatory processes, and may be due to modifications of opioid systems in these animals 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.     

Electroacupuncture inhibits phosphorylation of spinal phosphatidylinositol 3-kinase/Akt in a carrageenan-induced inflammatory rat model

We investigated the changes of pain-related spinal signaling pathway after electroacupuncture (EA) stimulation in a carrageenan-induced rat model. EA stimulation (2Hz, 1mA) was needle-delivered for 30 min at acupoints corresponding to Zusanli and Sanyinjiao 3 h after carrageenan injection. Thermal and mechanical sensitivity of the hindpaw induced by carrageenan was strongly inhibited by EA stimulation. Phosphorylation of extracellular signal-regulated kinase, phosphatidylinositol 3-kinase (PI3K), Akt, and cAMP response element-binding protein (CREB) were examined in the L4-5 segments of the spinal cord by Western blot analysis 4h and 5h after carrageenan injection. Phosphorylation of Akt and especially PI3K were significantly induced by carrageenan-induction, but these expressions were markedly inhibited by EA stimulation. CREB phosphorylation showed a similar, but insignificant, pattern as like PI3k/Akt. Immunohistochemical analyses confirmed that phosphorylation of PI3K and Akt showed a similar pattern as Western blotting and were observed in most neurons and a few astrocytes. EA and PI3K inhibitor synergistically inhibited carrageenan-induced hyperalgesia. These results reveal that both neuronal PI3K and Akt may play an important role in EA-induced antinociception via inactivation in an inflammatory pain model.